romanian journal of internal medicine

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ROMANIAN JOURNAL OF INTERNAL MEDICINE Volume 48

No. 4, 2010

CONTENTS REVIEWS CARMEN GINGHINĂ, RALUCA MIHALACHE, B.A. POPESCU, RUXANDRA JURCUŢ, Restrictive cardiomyopathy – an outdated concept? ........................................................................................................................................................ GIANINA MICU, FLORICA STĂNICEANU, SABINA ZURAC, ALEXANDRA BASTIAN, ELIZA GRĂMADĂ, LUCIANA NICHITA, CRISTIANA POPP, LIANA STICLARU, R. ANDREI, C. SOCOLIUC, Carcinogenesis and infection with Helicobacter pylori .................................................................................................................................................. MIHAELA BÎCU, MARIA MOŢA, N.M. PANDURU, CORINA GRĂUNŢEANU, E. MOŢA, Oxidative stress in diabetic kidney disease .................................................................................................................................................................. CORINA GRĂUNŢEANU, E. MOŢA, MARIA MOŢA, N.M. PANDURU, MIHAELA BÎCU, IULIA VLADU, Cardiovascular risk in patients with diabetic kidney disease......................................................................................................................

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ORIGINAL ARTICLES LAURA POANTĂ, ANCA CERGHIZAN, DANA POP, Blood pressure pattern and heart rate variability in normotensive patients with type 2 diabetes mellitus................................................................................................................................ VIOLETA ŞAPIRA, INIMIOARA MIHAELA COJOCARU, GABRIELA LILIOS, M. GRIGORIAN, M. COJOCARU, Study of endothelin-1 in acute ischemic stroke ................................................................................................................. ALEXANDRINA LIZICA DUMITRESCU, CARMEN TOMA, VIORICA LASCU, Relationship of humour with oral health status and behaviours ........................................................................................................................................................ M. POROJAN, SIMONA COSTIN, LAURA POANTĂ, ANCA CERGHIZAN, DANA POP, D.L. DUMITRAŞCU, Autonomic neuropathy and plasma catecholamine in patients with diabetes mellitus .........................................................................

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CASE REPORTS DENISE CARMEN MIHAELA ZAHIU, M.RIMBAŞ, ADRIANA NICOLAU, SABINA ZURAC, M.R. VOIOSU, Ankylosing spondylitis or Crohn’s disease? Case report and review of the literature...................................................... LUCIA AGOŞTON-COLDEA, L.C. MOCAN, TEODORA MOCAN, SILVIA LUPU, Right renal artery occlusion as a complication of fibromuscular dysplasia...........................................................................................................................

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POINTS OF VIEW H. BĂLAN, At the dawn of a new era in treating angina pectoris, or just another antianginal drug? Some considerations about ranolazine ................................................................................................................................................................ I.B. IAMNADESCU, LILIANA DIACONESCU, Stress vulnerability in patients with drug allergy. Psychological aspects revealed from some personal studies................................................................................................................................. ALEXANDRA BASTIAN, H.H. GOEBEL, Protein aggregation in inclusion body myositis, a sporadic form among protein aggregate myopathies, and in myofibrillar myopathies. A comparative study ..................................................................

ROM. J. INTERN. MED., 2010, 48, 4, 291–385

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REVIEWS

Restrictive Cardiomyopathy – an Outdated Concept? CARMEN GINGHINĂ1,2, RALUCA MIHALACHE2, B.A. POPESCU1,2, RUXANDRA JURCUŢ1,2 1 Department of Cardiology, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Department of Cardiology, “Prof. Dr. C.C. Iliescu” Institute of Emergency for Cardiovascular Diseases, Bucharest, Romania

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Restrictive cardiomyopathy is probably the least common of all cardiomyopathies, with a nonspecific clinical presentation and a frequently unknown cause. The concept of RCM has changed tremendously over time. Today it includes a large panel of disorders characterized by a nonhypertrophied, non-dilated cardiac phenotype and a restrictive ventricular filling pattern. Several unsuccessful attempts to define and classify cardiomyopathies have been made, but they all proved problematic due to the contradiction in terms and the overlap between classical patterns. Advances in disease pathology, genomics and molecular biology are emerging as the framework of a new revolutionary classification system, focused on the dynamic interaction between genotype and phenotype. In this context, RCM is evolving as a self-contained hemodynamic and pathophysiological concept, although questionable due to its uncertain practical utility. Key words: restrictive cardiomyopathy, restrictive ventricular filling pattern.

DEFINITION AND CLASSIFICATION

Owing to the increasing number of pathologies that are associated with a restrictive ventricular filling pattern, the definition of restrictive cardiomyopathy (RCM) has always been a challenging matter. Often, RCM is defined as an abnormal diastolic ventricular function in which increased stiffness of the myocardium causes precipitous rises in ventricular pressure with only small increases in volume. Restrictive ventricular physiology occurs in the presence of normal or reduced diastolic volumes (with single or biventricular involvement), normal or reduced systolic volumes and normal or increased ventricular wall thickness. Systolic function is characteristically preserved, but not entirely normal [1]. Finding the official place of RCM in the spectrum of cardiovascular pathology proves to be a difficult task, especially because of the spectacular transformations undergone by the concept and the classification system of cardiomyopathies in general and of RCM in particular. In the 1980 report of the WHO/ISFC task force, RCM was acknowledged as one of the three major cardiomyopathies (defined as “heart muscle diseases of unknown cause” in order to differentiate them from specific heart muscle diseases, of known cause). Back then, RCM was divided into two separate forms: with or without obliteration, including endomyocardial fibrosis and Löffler cardiomyopathy. Many of the entities that today are recognized as ROM. J. INTERN. MED., 2010, 48, 4, 293–298

part of the RCM pattern (e.g. metabolic disorders, infiltrative and familial storage diseases, muscular dystrophies, etc.) [2] were known as specific heart muscle diseases. Fifteen years later, cardiomyopathies were redefined as diseases of the myocardium associated with cardiac dysfunction, five types of cardiomyopathies being described, according to the dominant pathophysiology or to the etiological and pathogenetic factors. Still, the classification introduced the term specific cardiomyopathies in order to designate heart muscle diseases associated with specific cardiac or systemic disorders, previously known as specific heart muscle diseases [3]. Over the years, in accordance with the rapid evolution of molecular genetics in cardiology and dramatic advances in diagnosis and knowledge of causation, many disease definitions have become outdated and several other classification frameworks emerged. Thus, in 2006 the American Heart Association expert consensus panel introduced a new genomic and molecular oriented classification system intended to facilitate the interactions among the clinical and research communities. According to the broad definition, cardiomyopathies are associated with failure of myocardial performance, which may be mechanical (diastolic or systolic dysfunction) or a primary electrical disease prone to life-threatening arrhythmias. For the first time, recently described ion channelopathies were de-

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signated as primary cardiomyopathies, despite the fact that there are no macroscopic abnormalities identifiable by either conventional imaging or myocardial biopsy. The panel recommends that cardiomyopathies can be most effectively classified as primary (solely or predominantly confined to heart muscle): genetic, mixed (genetic and non-genetic), acquired; and secondary (myocardial involvement as part of a large number and variety of generalized systemic disorders). The classification distinguishes between the primary restrictive non-hypertrophied cardiomyopathy as a primary mixed cardiomyopathy and secondary cardiomyopathies that may share a restrictive phenotype (infiltrative, storage, endomyocardial, inflammatory, craniofacial, neuromuscular, toxic disorders, etc.) [4]. More recently, the 2008 European Society of Cardiology position statement regarding the classifycation of cardiomyopathies supported the use of a clinically oriented classification system according to ventricular morphology and function. Each of the five phenotypes of cardiomyopathies are subclassified into familial and non-familial forms in order to raise awareness of the genetic causes of heart muscle dysfunction and to offer a pertinent framework for further investigations. According to the new classification system, RCM may be either familial (predominantly associated with autosomal dominant inheritance, e.g. troponin I or desmin mutations, or rarely with autosomal recessive or Xlinked inheritance) or non-familial (e.g. amyloidosis, scleroderma, endomyocardial fibrosis, carcinoid heart disease, etc.) [1]. However, an inevitable limitation of any classification is the considerable overlap encountered between categories into which diseases have been segregated. The traditional classification of “hypertrophic-dilated-restrictive cardiomyopathies” has major limitations, leading to confusion by mixing anatomic designations (ie hypertrophic and dilated) with a functional one (i.e. restrictive) [4]. While distinct types of cardiomyopathies may share the same anatomy and type of ventricular filling pattern, other entities may segregate as distinct phenotypes. For example, hypertrophic cardiomyopathy (HCM) and infiltrative and storage cardiomyopathies are both characterized by often substantially increased left ventricular wall thickness, in the absence of ventricular dilatation, and by restriction to diastolic filling [4]. At the same time, there are several case reports that emphasize the overlap between the restrictive and the hypertrophic pattern in patients with Noonan syndrome [5]. Up to 20% of patients with Noonan syndrome have a cardiomyopathy

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with clinical, echocardiographic, hemodynamic and histopathological features indistinguishable from the cardiac phenotype of HCM, while others display a restrictive pattern [6]. Although myocyte hypertrophy is found in both conditions, RCM is not associated with macroscopic hypertrophy or histological myocyte disarray [7]. Similarly, Watson or Leopard syndrome, certain troponin I mutations, Fabry disease, etc. may share either of the two patterns. It is expected that genetic insights into these disorders will soon offer a plausible explanation of the genotype–phenotype interaction. Furthermore, some diseases have a dynamic expression, progressing, through continuous remodeling, from one pattern to another during their natural clinical course. HCM, amyloidosis and other infiltrative conditions may evolve from a nondilated (often hyperdynamic) state with ventricular stiffness to a dilated form with systolic dysfunction and failure. As new cardiomyopathies are being defined by means of genomics and proteomics and understanding of pathophysiology is evolving, the dilatedhypertrophic-restrictive classification pattern appears unable to satisfy the new requirements [4]. A thorough review of the medical literature of the last decades highlights important transformations undergone by the concept of RCM. At first, primary RCM raised as an independent hemodynamic entity resembling constrictive pericarditis. Most of the efforts, focused on the discrimination between the two conditions, often require exploratory thoracotomy and surgical biopsy. Histological findings revealed a “non-tropical” form of endomyocardial fibrosis, suggesting that eosinophilia may play a leading role in the genesis of RCM [8]. Later research introduced the term idiopathic RCM as a rare and poorly characterized entity, carrying a variable prognosis and showing a poor response to treatment, that should not be confused or equated with other diseases resulting in restrictive hemodynamics [9] [10]. Extensive reports were dedicated to various systemic disorders that share a restrictive ventricular filling pattern, often known as secondary RCM. Advances in cardiovascular imaging [11] and histopathology led to the characterization of a growing number of disorders that may manifest as RCM during their clinical course: infiltrative disorders (familial or non-familial amyloidosis [12], sarcoidois), storage disorders (hemochromatosis, Fabry disease [13], glycogen storage diseases), pseudoxanthoma elasticum [14], carcinoid syndrome, etc. Drugs like anthracyclines, methysergide, busulfan proved to be cardiotoxic causing endomyocardial fibrosis and a restrictive ventricular filling pattern [15]. More recently, genetic molecular testing in patients with

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apparent idiopathic RCM discovered several mutations in the genes encoding sarcomere proteins (e.g. troponin, actin)[16]. DIAGNOSTIC APPROACHES

Beyond controversies over definition and nomenclature, there are several definable pathophysiological, hemodynamic and echocardiographic features that outline the concept of RCM. Biatrial enlargement and normal sized ventricles with normal or increased ventricular wall thickness are the characteristic morphological features of RCM. Microscopic examination may reveal myocardial fibrosis, infiltration, endomyocardial scarring and myocyte hypertrophy (especially in the idiopathic form), as well as specific findings, particular to secondary RCM [17]. Diastolic dysfunction is a definable feature of all forms of RCM. Small sized ( 200 msec), reduced filling fraction in the first third of the diastole, increased (>30%) atrial contribution to ventricular filling are often encountered in patients with RCM [23] (Fig. 2). Endomyocardial biopsy is extremely valuable in the identification of secondary RCM (infiltrative heart disease, hypereosinophilic syndrome) and in the differential diagnosis with pericardial disease. Surgical exploration is rarely needed today [17]. PROGNOSIS RCM carries a variable prognosis dependent on etiology. Most often, especially in the case of amyloidosis, it responds poorly to medical or surgical treatment, invariably progressing to death. Apart from the etiological treatment (e.g. iron removal in hemochromatosis, enzyme replacement therapy in Fabry disease), the management of idiopathic RCM is directed at reducing pulmonary and systemic congestion by carefully decreasing filling pressure with diuretics, controlling heart rate to allow adequate filling time, maintaining atrial contraction, correcting atrioventricular conduction disturbances with permanent pacing, if needed, and avoiding anemia, nutritional deficiency, calcium overload and electrolyte imbalance. In addition, patients with atrial fibrillation should be considered for chronic anticoagulation, to decrease the risk of thromboembolism. Experience with cardiac transplantation in RCM is limited [9] [10]. Unlike adults, idiopathic RCM in children evolves with an accelerated progression to heart failure and severe pulmonary hypertension. Medical treatment of these patients has not shown any significant long term benefit and transplantation appears the only realistic treatment option [24]. CONCLUSION Despite its shortcomings (mainly regarding its questionable practical utility and polymorphic

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presentation), RCM remains a self-contained hemoimportance of applying Bayesian principles at all dynamic pattern. The presence of dilated atria and phases of diagnostic evaluation and the consideration non-hypertrophied, non-dilated ventricles in patients of the clinical context are critical steps when with congestive heart failure should be more interpreting results of diagnostic testing. Given the commonly recognized as a separate disease entity difficulties encountered in its assesment, RCM was and should raise the suspicion of RCM, although and continues to be an issue of clinical debate and this is only just the beginning of a long and comscientific research and above all a tough challenge plicated etiologically oriented algorithm. Still, the to any cardiologist. __________________________________________________________________ Cardiomiopatia restrictivǎ este probabil cea mai rarǎ cardiomiopatie, având o prezentare clinicǎ nespecificǎ şi adeseori cauzǎ necunoscutǎ. Conceptul de cardiomiopatie restrictivǎ s-a schimbat fundamental de-a lungul timpului. Astǎzi include o gamǎ largǎ de boli caracterizate printr-un fenotip cardiac non-hipertrofic, non-dilatativ şi un profil restrictiv al umplerii ventriculare. Deşi au existat numeroase tentative de clasificare şi definire a cardiomiopatiilor, toate s-au dovedit problematice din cauza contradicţiei în termeni şi a suprapunerii tipurilor clasice de cardiomiopatie. Progresele în întelegerea patologiei, genomicii şi biologiei moleculare vin sǎ punǎ bazele unui sistem nou, revoluţionar de clasificare, bazat pe interacţiunea dinamicǎ dintre genotip şi fenotip. În acest context, cardiomiopatia restrictivǎ evolueazǎ ca un concept hemodinamic şi fiziopatologic de sine stǎtǎtor, a cǎrei utilitate clinicǎ rǎmâne însǎ incertǎ. __________________________________________________________________ Corresponding author: Carmen Ginghină, MD, Professor “Prof.C.C.Iliescu” Institute of Cardiovascular Disease 285,Sos.Fundeni, Bucharest, Romania E-mail: [email protected]

REFERENCES 1.

ELLIOT P., ANDERSSON B., ARBUSTINI E., BILINSKA Z., CECCHI F., CHARRON P. et al., Classification of the cardiomyopathies: a position statement from the European Society of Cardiology Working Group on myocardial and pericardial diseases. Eur. Heart J., 2008; 29:270–6. 2. BRANDENBURG R., CHAZOV E., CHERIAN G., FALASE A., GROSGOGEAT Y., KAWAI C. et al., Report of the WHO/ISFC Task Force on the Definition and Classification of Cardiomyopathies. Br Heart J., 1980; 44: 672–673. 3. NORDET P., MARTIN I., GYARFAS I., GOODWIN J., THIENE G., OLSEN E.M. et al., Report of the 1995 WHO/ ISFC task force on the definition and classifiaction of cardiomyopathies. Circulation, 1996;93:841–42. 4. MARON B., TOWBIN J., THIENE G., ANTZELEVITCH C., CORRADO D., ARNETT D. et al., Contemporary Definitions and Classification of the Cardiomyopathies, An American Heart Association Scientific Statement From the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation, 2006; 113: 1807–16. 5. HIROTA Y., SHIMIZU G., KITA Y., NAKAYAMA Y., SUWA M., KAWAMURA K. et al., Spectrum of restrictive cardiomyopathy; report of the national survey in Japan. Am.Heart J., 1990; 120: 188–94. 6. SHARLAND M., BURCH M., MCKENNA W.M., PATON M.A., A clinical study of Noonan syndrome. Arch.Dis.Child., 1992; 67: 178–83. 7. KATRITSIS D., WILMSHURST P.T., WENDON J.A., DAVIES M.J., WEBB-PEPLOE M.M., Primary restrictive cardiomyopathy: clinical and pathological characteristics. J.Am.Coll.Cardiol., 1991; 18: 1230–5. 8. CHEW C., ZIADY G., RAPHAEL J., NELLEN M., OAKLEY C.P., Primary restrictive cardiomyopathy. Non-tropical endomyocardial fibrosis and hypereosinophilic heart disease. British Heart Journal., 1977; 39: 399–413. 9. AMMASH N., SEWARD J., BAILEY K., EDWARDS W., TAJIK A., Clinical Profile and Outcome of Idiopathic Restrictive Cardiomyopathy. Circulation, 2000; 101; 2490–96. 10. SIEGEL R., SHAH P., FISHBEIN M.C., Idiopathic restrictive cardiomyopathy. Circulation, 1984; 70: 165–9.

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11. LIMA J., DESAI M., Cardiovascular Magnetic Resonance Imaging: Current and Emerging Applications, Journal of the American College of Cardiology. 2004, 44. 12. FALK R., Diagnosis and Management of the Cardiac Amyloidoses. Circulation, 2005; 112; 2047–2060. 13. NAGUEH S., Fabry disease, Heart, 2003; 89: 819–20. 14. CHALLENOR V., CONWAY N., MONRO J., The surgical treatment of restrictive cardiomyopathy in Pseudoxantoma elasticum, Br.Heart J., 1988; 59: 266–9. 15. MORTENSEN S., OLSEN H., BAANDRUPT U., Chronic anthracycline cardiotoxicity: haemodynamic and histopathological manifestations suggesting a restrictive endomyocardial disease. Br.Heart J., 1986; 55: 274–82. 16. KASKI J., SYRRIS P., BURCH M., TOME ESTEBAN M., FENTON M., CHRISTIANSEN M. et al., Idiopathic restrictive cardiomyopathy in children is caused by mutations in cardiac sarcomere protein genes. Heart, 2008; 94: 1478–84. 17. HARE J.M., The dilated, restrictive and infiltrative cardiomyopathies. In: Braunwald’s Heart Disease: A Textbook of Cardiovascular Medicine, 8th edition, Saunders Elsevier, 2008: 1739–62. 18. HOIT B.D., GUPTA S., Restrictive, obliterative and infiltrative cardiomyopathies. In: Hurst’s 12th edition The Heart, McGrawHill Medical 2008. 19. GUADALAHARA J., VERA-DELGADO A., GASPAR-HERNANDEZ J., GALVAN-MONTIEL O., HUERTA-HERNANDEZ D., Echocardiographic Aspects of Restrictive Cardiomyopathy. Echocardiography, 1998: 297–311. 20. BABUIN L., ALEGRIA J., OH J. et al., Brain Natriuretic Peptide Levels in Constrictive Pericarditis and Restrictive Cardiomyopathy. J. Am.Coll.Cardiol., 2006; 47: 1489–1491. 21. OTTO C., Cardiomyopathies, hypertensive and pulmonary heart disease. In: Textbook of clinical echocardiography. 4th edition, Saunders Elsevier, 2009:212–41. 22. GINGHINA C., POPESCU B., JURCUT R., Esential in ecocardiografie. Editura Medicala Antaeus, 2005:151–75. 23. GINGHINA C., APETREI E., JOVIN G., STANESCU D., MARINESCU M., DRAGOMIR D., Can radionuclide angiography offer a coherent interpretation of various entities included in restrictive cardiomyopathy? Journal of Nuclear Cardiology, 2001, 8. 24. FENTON M., CHUBB H., MCMAHON A. et al., Heart and heart-lung transplantation for idiopathic restrictive cardiomyopathy in children. Heart, 2006, 92: 85–9. Received October 29, 2010

Carcinogenesis and Infection with Helicobacter pylori GIANINA MICU1, FLORICA STĂNICEANU1,2, SABINA ZURAC1,2, ALEXANDRA BASTIAN1, ELIZA GRAMADĂ1, LUCIANA NICHITA1,2, CRISTIANA POPP1, LIANA STICLARU1, R. ANDREI1, C. SOCOLIUC1 1

“Colentina” Clinical Hospital, Department of Pathology, Bucharest, Romania 2 “Carol Davila” University of Medicine, Bucharest, Romania

It was accepted several years ago that, in the carcinogenesis process of human cancers, biologic agents, especially the viruses, are playing an etiologic role. This is the case of lymphomas (retroviruses), hepatocarcinoma (hepatic viruses) and cervical carcinoma (papilloma viruses). Helicobacter pylori is the first bacteria recognized as a first class carcinogen for gastric cancer. Nevertheless, comparing with the most validated human carcinogens, the activity of H. pylori is very little studied. As a consequence, at this moment, in its case, explanation of carcinogenesis mechanism is more or less hypothetical. Key words: Helicobacter pylori, CagA, VacA, carcinogenesis, epithelial premalignant lesions, lymphoid malignities.

THE EPIDEMIOLOGICAL PERSPECTIVE

In the last fifty years we witnessed a significant decrease of gastric cancer incidence in the developed countries population. An important contribution to this decrease was brought by the Japanese school of gastroenterology [1] [3] by healing of early gastric cancers in over 90% cases. Even so, at a world level, this terrible disease continues to be a frontrunner among cancer-caused deaths (2nd place) and it is considered the world’s 14th cause of mortality [1]. Until H. pylori (Fig. 1) to become known as a direct carcinogen [2], the association of the H. pylori gastric infection with different gastric lesions was considered a facilitating (and not inductive) factor for carcinogenesis through the induction of intestinal metaplasia, glandular atrophy and hypochlorhydria followed by the accumulation of N-nitrous carcinogenic components, through the free radicals-generating inflammatory reaction and excessive cellular proliferation. Afterwards it was demonstrated that bacteria not only favour, but also produce malign modifycations at the level of the gastric mucosa: both histological variants of antral gastric adenocarcinoma (the intestinal type and the diffuse type, according to the Lauren classification) and the gastric lymphoma, without being implicated in eso-cardial junction cancers. Cardial lesions appear in patients with a significant gastro-oesophageal reflux, and in this case H. pylori infection can have a protective role [4]. ROM. J. INTERN. MED., 2010, 48, 4, 299–306

Recently, sero-epidemiological studies approximate that at least 70% of diagnosed gastric carcinomas have Helicobacter pylori as a determining cause [3] [4], and the serologic presence of HP antibodies was demonstrated at least ten years before the disease was diagnosed. The arguments belong to descriptive epidemiology that presents a geographical distribution [3][5] of the gastric cancer overlapped by that of Helicobacter pylori infection. In countries with a high presence of gastric cancer, the incidence of Helicobacter pylori infection is also high. These observations are also sustained by the conclusions of the epidemiological studies that show a high frequency of the gastric cancer in patients that belong to economically disadvantaged classes, subjects that also proved to have a significantly high incidence of the infection [1] [6]. Epidemiological paradoxes From the researchers in epidemiology point of view there are still several unsolved paradoxes: – Why, even if H. Pylori infection affects about half of the planet’s population, only very few subjects develop a gastric cancer? [7] Explanations for this situation are probably in the role of genetic and diet factors. – On the other hand, even if H. pylori infection has a relatively even distribution in both sexes, why does the gastric cancer affect mainly men? [4] [7]

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– How come there are areas where the infection’s prevalence is very high (around 100%), but the gastric cancer’s prevalence is almost null? [1] [5] [7]. But, with or without clarifying the epidemiological paradoxes that, one fact remains certain: the risk of developing gastric cancer by a person with H. pylori infection is under 1% and seems to depend on the interaction between the virulence factors of the infecting bacteria strain and the host’s genetically-determined immune response [8]. The implications of H. pylori in carcinogenesis are sustained by epidemiological studies as well as by animal models and researches made for clarifying the molecular mechanisms involved in gastric carcinogenesis. Most gastric cancers are preceded by premalignant lesions that evolve for decades. Atrophic gastritis perturbs the secretion of gastric acid by increasing the pH, allowing gastric colonization with anaerobe bacteria. These bacteria produce reductase, implicated in the formation of N-nitroso carcinogenic components [8] [9]. PATHOGENIC MECHANISMS OF INDUCTION IN GASTRIC CARCINOGENESIS

H. pylori induces carcinogenesis both directly through his virulence factors, as well as indirectly through the induction of the inflammatory response from the host [7–9]. A. DIRECT FACTORS – VIRULENCE FACTORS OF HELICOBACTER PYLORI

The direct carcinogenetic action of Helicobacter pylori: Helicobacter pylori’s virulence factors are produced due to its endowment with a series of structural factors or the bacteria’s secretion products, among which the most significant are urease, phospholipase A, proteolytic enzymes, adhesins – common to all Helicobacter pylori strains–, as well as the cag cytotoxin, present in 60–70% of the strains and the vacuolisant protein vacA, present in 60–65% of Helicobacter pylori strains [10] [11]. Helicobacter pylori genome is heterogenic, with some strains playing a more significant role in developing malignity. This gene group bears the name of pathogen islet CagA (PAI) and is made of 31 genes. While the positive CagA types are proved to imply a high risk of gastric cancer in the Western population, in Asian population this correlation is poorly supported. The vacuolisant cytotoxin vacA is responsible for the lesion of epithelial cells associated with carcinogenesis –

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genotypes vacA s1 and vacA m1 have a high malignnant potential. The carcinogenetic action of both CagA and vacA was expressed experimentally through their inoculation in Mongolian gorillas, which determined intestinal metaplasia and gastric cancer. On the other hand, the development of B cells gastric lymphoma was recently associated with virulent forms of H. pylori, such as HopZ [11–13]. Oxidative stress. Gastritis is associated with the increase in production of nitric acid (NO). The nitroso components are recognized as gastric carcinogens in experimental milieus. Among the host’s response factors to Helicobacter pylori infection, interleukin 1 and the necrotic tumoral factor (TNF–A–308) present a high risk for gastric cancer. B. INDIRECT FACTORS – CHRONIC INFECTION AND ITS CONSEQUENCES ON GASTRIC MUCOSA CELLS

The inflammation of the gastric mucosa infected with H. pylori implicates cytokines, gamma interferon, TN-α, IL-1b, IL-6, IL-8, IL-12 and IL-17. The expression of cytokines is secondary to the activation of the transcriptional factor NF-kB, activation induced in epithelial cells through the translocation of the cagA gene. The intensity of the gastric inflammation depends on the host’s hereditary factors [12] [13]. Some H. pylori strains induce a severe inflammatory reaction while others are not at all accompanied by inflammation. This difference of the inflammation degree is parallel to the balance of pro- and anti-inflammatory cytokines. H. pylori is capable of modulating the cytokines’ response. The bacteria’s genomic recombination seems to play a very important role in perpetuating the bacteria in spite of the inflammatory response. The bacteria can survive about 24 h the macrophages’ phagosomes, the bacteria’s lipopolysaccharides inhibiting the macrophages’ apoptosis. On the other hand, the vacuolisant toxin vacA permits the survival in an acid milieu through the formation of intercellular vacuoles containing ammonia which is freed when the bacteria come into contact with the cell’s apical pole. Thus, the bacteria’s virulence factors allow it to survive in spite of host’s immune response [14]. I. CARCINOGENESIS ON THE EPITHELIAL LINE THE CARCINOGENIC CASCADE

The carcinogenic cascade triggered by H. pylori infection, as it was proposed by Correa et al in

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1975, goes through several important stages [1] [11] [17]: fundic and corporeal infection with Helicobacter pylori → chronic gastritis → glandular atrophy → intestinal metaplasia → dysplasia → adenocarcinoma PREMALIGNANT LESIONS ON THE EPITHELIAL LINE

The initial lesion is constituted by chronic gastritis together with the decrease of peptic acid secretion and of the intragastric ascorbic acid concentration, a substance recognised as having protective role against cancer (Fig. 2). The absence of H. pylori in the areas of intestinal metaplasia, areas where the neoplastic transformation originates, suggests a distant carcinogenic influence through the bacteria’s products, as well as through the inflammatory response generated by the infection [9] [10] [19]. Intestinal metaplasia associated to cancer is the incomplete type, either 2 or 3, and it is distributed diffusely, antro-fundic, or along the lesser curvature, from the cardia to the pylorus [18] – Fig. 3. As far as the atrophy associated with cancer is concerned, recent studies confirm that it can be continuous or multifocal, located most often antral and fundic. German researchers have put forward the hypothesis that the gastric cancer risk is higher in subjects with fundic-located gastritis if the level of activity is equal to the antral one. Japanese studies confirm the fact that the predominance of gastritis at the gastric fundus, as well as severe atrophy and intestinal metaplasia are risk factors for gastric cancer [17] [19]. Atrophy is defined by a glandular depletion, probably a consequence of a fault in the replacement of cells through apoptosis (Fig. 3). The histopathologic examination highlights the abundance of inflammatory cells and apoptotic epithelial cells at the level of the neck glands, which is precisely where there are epithelial strain cells. The multi factorial analysis done on patients with adenocarcinoma, duodenal ulcer or gastritis indicates the association between genotype s1 and m1 cag and the density of the inflammatory infiltrate, the mucosa’s degree of atrophy and the type of intestinal metaplasia. A role in the appearance of the gastric mucosa atrophy seems to be also played by the auto-antibodies through the activation of the local immune system and the induction of a cellular mediated reaction followed by the destruction of parietal cells by cytotoxic lymphocytes [18–20].

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Apoptosis. Among the carcinogenesis mechanisms there is also the maladjustment of the apoptotic ways. Apoptosis is a form of geneticallyprogrammed cellular death in view of regulating the number of epithelial cells of the digestive tract. Both the presence of inflammatory mediators and the secretion product of H. pylori can intervene directly in the enzyme cascade that forms the base of the molecular mechanisms of the apoptosis [19]. As a consequence, modifications of the cellular turnover appear. The ways through which H. pylori can induce the acceleration of the apoptosis’ rhythm in the gastric epithelial cells belong to two main categories: a) Direct pathway, through the bacteria’s virulence factors, especially cagA, cagE and the vacuolisant cytotoxin vacA, which is confirmed by the absence of an accelerated apoptosis in infections with H. pylori strains with the above-mentioned virulence factors. b) Indirect pathway, via the inflammation’s mediators: the gamma interferon (IFN-γ) and TNF-α amplify the apoptosis induced by H. pylori through a mechanism that implies the over-expression of the Fas receptor at the level of gastric epithelial cells. This increases the susceptibility of gastric epithelial cells towards T-cells. Apoptosis is accelerated by a series of bacterial elements: ammonia, urease, ceramide [7] [18]. In cell cultures the over expression of Bak (correspondent of the Bcl-2 protein, inductor of the apoptosis) was also noticed. At the present moment there are two theories regarding the significance of apoptosis in H. pylori infection. According to the first one, the induction of apoptosis stimulates the cellular proliferation, explaining the hyper prolixferative response of the host epithelium associated to the infection. The second theory affirms that, on the contrary, apoptosis could be the answer to epithelial hyper proliferation in view of preventing tissue hypertrophy [23]. In vivo the apoptosis’ mechanisms depend significantly on the level of pro inflammatory cytokines (IL-8, IL-6, gamma interferon and TN-α). These induce apoptosis through the induction of synthetase nitrogen monoxide. NO has a degrading effect on the DNA, the increase in the production of NO can determine irreversible lesions of the genome and the appearance of pro carcinogenic mutations. The free radicals and NO induce the expression of the P53 protein, that repairs DNA’s lesions or produces cells’ apoptosis [20] [22].

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Epithelial proliferation – Fig. 4. In the gastric mucosa infected with H. pylori there is a concomitance between the accelerated apoptotic process and the cellular proliferation. When the balance between these two opposed effects processes is broken in favour of the proliferative factors, the evolution leads to cancer. Cellular proliferation is secondary to the inflammation induced by the H. pylori bacteria. The central role in its production seems to be played by cyclooxygenase Cox-2 and nitrogen monoxide (NO), both expressed at an increased level in gastric adenocarcinoma. Excessive cellular proliferation decreases significantly after the eradication of H. pylori infection [20]. On the other hand, one must note its increase in the absence of a parallel increase of the apoptosis, of genetic (genetic mutations) or epigenetic (modifycations of the gene’s expression) alterations. Genetic modifications seem to be more precocious in the diffuse-type cancer than in the intestinal type, even if in the latter’s case precancerous lesions such as intestinal metaplasia can be described. Somatic mutations of gene E-cadherine or a hypermethylation of one of these gene’s promoters are characteristic to the diffuse type of adenocarcinoma, while for the intestinal type the mutations frequently affect gene p53. Mutations of the APC and betacatenina genes are much rarer. All these genetic modifications can be induced by the oxidative stress produced by H. pylori [20] [22] [24]. H. pylori interferes in angiogenesis through the induction of a vascular factor for endothelial growth – A (VEGF – A). Other growth factors induced through H. pylori’s virulence include the epidermal growth factor (EGF), the heparin growth factor (EGF-like) and amphiregulin. Through the CagA protein it also activates the c-Met growth factor. There are numerous studies that describe cellular and genetic modifications in malignant gastric cells: the affection of the intercellular adhesion owing to the mutations E-cadherin, alpha and betacatenins, as well as to the increase in the activity of telomerase and the instability of microsatellites. The most common genetic abnormalities are connected to p53, as well as to the activation of the oncogenes c-Med and Her2/Neu, while the K-raz mutations occur less and less frequently. It was demonstrated that some of these genetic modifycations can appear even before intestinal metaplasia is installed [20] [25]. Intraepithelial neoplasia (“dysplasia”) represents a renewal and tissue-development process; it is frequently associated to chronic gastritis and can recede under treatment. It appears at the level

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of the normal gastric mucosa and is signalled by a foveolar hyper-proliferation and/or intestinal metaplasia. Cytoarchitectural alterations start at the level of the glands’ neck, where glands appear grouped in small “packages”. It can be plane/polypoid/ depressed from a macroscopic point of view, with a microscopic tubular/tubulo-villous/ villous or papillary pattern [20–22] [25] – Fig. 5. Microscopic criteria of considering the gastric intraepithelial neoplasia are: – structural disorganisation: deformation of the crypts, the appearance of epithelial intraluminal buds, the relation between the epithelial tissue and the conjunctive one, modified in favour of the former. – the presence of cellular atypia, predominantly nuclear: polymorphism, hyperchromasia, loss of nuclear polarity, stratification, presence of an increased number of mitoses. – anomalies of differentiation: modification of the secretion, increase of the number of non-differentiated cells. According to these criteria two types of intraepithelial neoplasia can be described, comprising the three degrees of epithelial dysplasia formerly described: low degree intraepithelial neoplasia (light and medium epithelial dysplasia) and high degree intraepithelial neoplasia (severe epithelial dysplasia); the cases which lack of criteria for a certain definition are classified in the indefinite intraepithelial neoplasia category (OMS) [1] [25]. In the low degree intraepithelial neoplasia, the mucosa’s architecture is slightly modified, and it presents tubular ramified/ budded structures, with elongated crypts, cystic dilatations, glands covered by large-size, low-Muncie columnar cells; pseudo stratified vesiculous round-ovoid nuclei. In the case of high degree intraepithelial neoplasia visible architectural distortions appear; the tubes gain an irregular, ramified shape; the glandules become crowded and one can identify visible cellular atypical situations; there is no stromal invasion; the mucus secretion is either minimal or absent; the nuclei become pseudo stratified, pleomorphic, hyperchromatic, cigar-shaped with prominent, amphophilous nucleoli. THE PROGRESSION OF INTRAEPITHELIAL NEOPLASIA TOWARDS CARCINOMA

Over 80% of intraepithelial neoplasia cases progress towards invasion. The carcinoma diagnosis is imposed when the tumour invades lamina propria (intramucous carcinoma) or the muscularis mucosae [24] [27].

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N.B.: the association of extensive lesions of intestinal metaplasia with lesions of intraepithelial neoplasia in the presence of the sulphomucinesecreting phenotype has a high risk of evolving towards carcinoma – Figs. 6, 7. PHYSIOPATHOLOGICAL AND CLINIC MODIFICATIONS: GASTRITIS, HYPOCHLORHYDRIA AND THE RISK OF ADENOCARCINOMA

The chronic infection with H. pylori can lead in time to the appearance of pan gastritis when inflammatory lesions at the level of the fundic mucosa lead to the decrease of the acid secretion. From a physiopathological point of view, hypochlorhydria is partly caused by the decrease in the secretion of histamines by the ECL cells; on the other hand, the parietal cells’ acid secretion is inhibited by TN-α and IL1β [26]. H. pylori infection induces an immune reaction from the organism, whose first stage is the alteration of gastric epithelium through the presence of the bacteria in the mucosa that covers cells’ apical pole. The consequence is secretion of numerous chemotactic factors, of cytokines and the stimulation of lymphocytes. Even if sometimes it is spontaneously eliminated by local defence mechanisms, in most cases the infection continues to persist. In time, it appears a local inflammatory reaction that provokes the acceleration of the cellular turn-over and, sometimes, genomic lesions that can lead to cancer if lesions of the DNA-repair mechanisms appear. Chronic inflammation can also induce hypochlorhydria in patients with a pre inflammatory genotype of interleukins. Convergence of bacterial virulence factors with the host’s immune response can generate varied diseases, from ulcer to the atrophy of the mucosa and afterwards the development of cancer [26] [27]. ERADICATION OF H. PYLORI AND PREVENTION OF GASTRIC CANCER

The major problem with gastric cancer prevention strategies derives at this moment from the fact that we do not know exactly at which stage gastritis, atrophy, intestinal metaplasia or intraepithelial neoplasia become irreversible. Random, prospective studies and placebo (like the one

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applied by Wong and his team) suggest the fact that the eradication of H. pylori reduces the incidence of gastric cancer only in patients that did not present gastric atrophy and/or intestinal metaplasia. But even in the case where these lesions’ point of no return has been surpassed, eradication of H. pylori seems to stop their evolution. The regression of precancerous lesions is thought 66% possible for patients that become H. pylori negative after treatment and only 14% possible for patients that stay positive after treatment [1] [26]. According to some studies, non-atrophic gastritis is completely reversible after the eradication of the H. pylori infection. Others, in contrast [19] [28], have demonstrated that both atrophic gastritis and intestinal metaplasia could be reversible, even if these studies have noticed a decrease in the incidence of gastric cancer; this was though related to the decrease of cellular proliferation after eradication of active infection [27]. Some researchers [20] [26] mentioned the existence of a stage of pre-atrophic gastritis in which the parietal epithelial cells have disappeared, but the glandular architecture was conserved and the strain cells were also not affected. At least theoretically, at this stage the lesions are reversible. To conclude, the real amplitude of the role played by H. pylori’s eradication in the prevention of gastric cancer remains still a subject for study. As long as after the eradication of H. pylori precancerous lesions stop evolving and, sometimes, they even regress, the practical decision imposes itself: the infection’s treatment must be applied even without the evidence of pre neoplasia modifications. Another important, but theoretical idea, is that of the existence of the point of no-return, beyond which bacteria-induced genetic modifications make atrophy and intestinal metaplasia irreversible, in spite of the elimination of the carcinogen agent (H. pylori) [25–27]. REVERSIBILITY OF PRECANCEROUS LESIONS

At the present moment there are still few studies concerning the role that the infection’s eradication can have in the prevention of the gastric cancer through the reversibility of pre neoplasia modifications: atrophic gastritis, intestinal metaplasia and the intraepithelial neoplasia of different degrees [16] [25] [26].

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As far as the malign transformation on a lymphoid line is concerned, the pathogenic sequence described above at this moment is the following: Gastric infection with Helicobacter pylori→ lymphoid hyperplasia → clone abnormalities at the level of B lymphoid population → low degree MALT lymphoma dependent on the Helicobacter pylori infection’s level→ (possibly via the t translocation (1;14) → low degree MALT lymphoma independent of the level of Helicobacter pylori infection→ (possibly via the p53 mutation) → high degree MALT lymphoma. EPIDEMIOLOGY

Epidemiological studies, as well as the detection of the H. pylori infection in most gastric lymphomas have proved the tight connection between these and the bacteria. The regression and even healing of some lymphomas following the antibiotic treatment aimed at H. pylori infection also come in support of this idea [1] [21]. The carcinogenetic steps in this case begin with the activation of T cells in the presence of the chronic H. pylori infection, starting towards cells that further on activate the population of polyclonal B lymphocytes. In time, a proliferation of monoclonal B cells with the possible accumulation of genetic mutations takes place. Because the lymphoma appears in the lymphoid tissue associated to the mucosa (MALT), they are called MALT-oms. B-cells that proliferate come from the lymphoid follicle’s peripheral area, which explains this tumour’s other name, that of marginal area lymphoma [1] [21] [24]. Even if the first studies concerning the low degree MALT lymphoma launched the idea of the presence of Helicobacter pylori infection among the etiologic factors up to 98%, more recent studies keep the bacteria in the fore-group, but decrease its incidence at 62–77%. It was proven that lymphoma is preceded by a H. pylori infection, but there are still controversies concerning this theme, especially because a series of serious studies have published partially contradicting results. For example, some studies mention the association between high degree lesions and positive cagA strains and the nonassociation of this strain with low degree lymphoma [21].

6 THE MALT LYMPHOMA CONCEPT

The term MALT was proposed by Isaacson et al. for the immune system’s components developed at the level of the gastrointestinal tract’s mucosa; these contain lymph ganglions (which in ileum form the Payer paches), the lymphocyte and plasmocytes in lamina propria and the intraepithelial lymphocytes. These immune components of the MALT system have distinct morph functional traits, as well as the lymphoma developed from them (MALT). It is considered that, in order to develop a tumour from this tissue, an important role is played by H. pylori, the latter’s eradication leading to the lymphoma’s remission [1] [28]. Between the lymphoid follicles there are variable sized lymphoid cells, without mitosis, frequent immunoblasts, post-capillary venules and sometimes plasmocytes – Fig. 8. The specific immunohistochemical markers are: CD19, CD20, CD21, CD35, bcl-2, sometimes CD43. LYMPHOID HYPERPLASIA

Lymphoid hyperplasia, named until recently “pseudo-lymphoma”, represents a reactive condition that appears frequently in association with ulcerations/ gastric erosions and who is accompanied by an extensive fibrosis and a vascular proliferation. At a microscopic level, one can describe the presence of reactive germinal centres in a polymorph inflammatory population (including mature lymphocytes and plasmocytes). Initially, the pseudo-lymphoma was considered a benign reactive inflammatory process. Later on, it was recognised as a pre-malignant lesion. At the present moment, thanks to data provided by immunohistochemical studies and molecular biology, that can make the difference between monoclonal (neoplasia) and polyclonal (reactive) lymph proliferations, the term of pseudo-lymphoma is not longer used [1] [23]. MAIN CHARACTERISTICS OF MALT GASTRIC LYMPHOMA

Over 95% of gastric lymphoma is non-Hodgkin type. Most of them are B cells lymphoma, T cells being reported fewer than 8%.

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Lesions appear at the level of the mucosa’s junction with the submucosa, making difficult at this stage a diagnosis through endoscopic biopsy. Also difficult is the differentiation of the low-degree malignity lymphoma from benign inflammatory infiltrates at the level of endoscopic biopsies. In early or borderline cases there can be significant confusions with follicular gastritis. In these cases it is necessary to have a immunohistochemical and molecular confirmation of the monoclonality of B cells [1] [21] [23]. THE TUMORIGENIC ROLE OF H. PYLORI IN MALT-TYPE GASTRIC LYMPHOMA

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TUMOUR CELLS E RESPONSE OF TO H. PYLORI INFECTION

Tumour B cells are not directly stimulated by the bacteria. H. pylori stimulate the intratumoral T cells that, in their turn, favour the proliferation of tumour cells. It was demonstrated that the same patient’s spleen T cells do not respond to H. pylori, which implies that the population of T cells responsive to the bacteria is a local one. This is one of the explanations for the fact that the MALT gastric lymphoma at least initially remains localized, the lymphoma being dependent on the activated T cells present in great number in H. pylori produced gastritis [24]. It is necessary for the progenitors of the malign B cells clone to have certain properties, a possible genetic alteration or the ability to recognise antigens that allow their uncontrolled proliferation in the presence of T cells. The MALT lymphomatous cells present a genetic instability, genetic anomalies and respond to a variety of auto antigens. In 2000, De Jong proposed a model of oncogenesis for the MALT-type gastric lymphoma. H. pylori → chronic gastritis → gastric lymphoma Non H type MALT, low malignity → gastric lymphoma Non H type MALT, high malignity. If the passage from chronic gastritis to the low degree MALT lymphoma is regulated through immunological processes, the passage to the high degree lymphoma is achieved through an autonomous proliferation [23] [26].

It was demonstrated (Isaacson et al. 1984, Wyatt et al. 1988, Worth Erspool, 1991) that chronic H. pylori infection determines the stimulation of the lymphoid tissue in the gastric mucosa. The presence of lymphoid follicles at this level is pathognomonic to the long-term infection with H. pylori; the appearances of lymphoepithelial lesions definitely mark the development of a MALT lymphoma. The chronic infection with Helicobacter pylori determines the recruitment of B and T cells in gastric mucosa as an immune response. The proliferation of B cells is secondary to the specific activation of T cells by the bacteria and cytokines. Because gene alterations of the malign clone are not sufficient for insuring autonomy in relation to cellular death, the MALT lymphoma can regress the anti-Helicobacter pylori treatment. In time, the malignant clone can accumulate varied gene alterations (t (1; 14)), inactivation of the p53 gene REGRESSION OF THE MALT GASTRIC LYMPHOMA or the p16 gene) that determine acquiring of an AFTER ERADICATION OF H. PYLORI autonomous proliferation capacity and/or apoptosis inhibition. As a consequence, the low degree MALT Antibiotic therapy for the MALT gastric lymphoma that would have responded to the lymphoma is efficient only in low degree lymphoma antibiotic treatment become high degree lymphoma and the extension is limited to the mucosa and/or that do not respond to the antibiotic treatment the submucosa [23] [25] [28]. anymore and tend to lead to metastasis. __________________________________________________________________ În procesul carcinogenezei cancerelor umane au fost acceptaţi de mulţi ani agenţi biologici cu rol etiologic, în special virusurile. Acesta este cazul limfoamelor (retrovirusurile), hepatocarcinomului (virusurile hepatitice) şi cancerului de col uterin (papiloma virusurile). Helicobacter pylori este prima bacterie recunoscută ca şi carcinogen de clasa I, fiind demonstrat epidemiologic ca agent cauzal pentru cancerul gastric. Totuşi, prin comparaţie cu cei mai mulţi carcinogeni umani validaţi, acţiunea carcinogenetică a H. pylori este încă puţin experimentată. În consecinţă, la momentul prezent, în cazul său, explicarea mecanismelor carcinogenezei este încă mai mult sau mai puţin una ipotetică. __________________________________________________________________

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Correponding author: Gianina Micu Colentina Clinical Hospital, Department of Pathology 19–21 Şos. Ştefan cel Mare, Bucharest E-mail: [email protected]

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

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Jr., Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer, 2010; 10:403–14. BARROS R., CAMILO V., PEREIRA B., FREUND J.N,. DAVID L., ALMEIDA R., Pathophysiology of intestinal metaplasia of the stomach: emphasis on CDX2 regulation. Biochem Soc Trans., 2010; 38:358–63. HONG S., LEE H.J., KIM S.J., Hahm K.B., Connection between inflammation and carcinogenesis in gastrointestinal tract: Focus on TGF-β signaling. World J Gastroenterol., 2010; 16:2080–2093. LAGNER M., MACHADO R.S., PATRICIO F.R., KAWAKAMI E., Evaluation of gastric histology in children and adolescents with Helicobacter pylori gastritis using the Update Sydney System. Arq Gastroenterol., 2009; 46:328–32. Correa P., The gastric precancerous process. Cancer Surv, 2:437–450, 1983. LIU D., HE Q., LIU C., Correlations among Helicobacter pylori infection and the expression of cyclooxygenase-2 and vascular endothelial growth factor in gastric mucosa with intestinal metaplasia or dysplasia. J Gastroenterol Hepatol., 2010; 25:795–9. TANIH N.F., MCcMILLAN M., NAIDOO N., NDIP L.M., WEAVER L.T., NDIP R.N., Prevalence of Helicobacter pylorivacA,cagA and iceA genotypes in South African patients with upper gastrointestinal diseases. Acta Trop., 2010 Jun 4 [Epub ahead of print]. MIENDJE DEYI V.Y., VANDERPAS J., BONTEMS P. et al., Marching cohort of Helicobacter pylori infection over two decades (1988–2007): combined effects of secular trend and population migration. Epidemiol Infect., 2010 Jun 7:1–9 [Epub ahead of print]. SENTHILKUMAR C., NIRANJALI S., JAYANTHI V., RAMESH T., DEVARAJ H., Molecular and histological evaluation of tumor necrosis factor-alpha expression in Helicobacter pylori-mediated gastric carcinogenesis. J Cancer Res Clin Oncol., 2010 May 29 [Epub ahead of print]. ADAMU M.A., WECK M.N., ROTHENBACHER D., BRENNER H., Incidence and risk factors for the development of chronic atrophic gastritis: Five year follow-up of a population based cohort study. Int J Cancer, 2010 May 25 [Epub ahead of print]. PARSONNET J., HANSEN S., RODRIQUEZ B.S., et al., Helicobacter pylori infection and gastric lymphoma. N Engl J Med., 330:1267–1271, 1994. COMPARE D., ROCCO A., NARDONE G., Risk factors in gastric cancer. Eur Rev Med Pharmacol Sci., 2010; 14:302–8. GIULIANI A., SPADA .S, CORONA M. et al., Cancer precursor lesions in intact stomach Helicobacter pylori gastritis and in resected stomach gastritis. J Exp Clin Cancer Res., 2003; 22:371–8. KIM Y.J., CHUNG J.W., LEE S.J., CHOI K.S., KIM J.H., HAHM K.B., Progression from chronic atrophic gastritis to gastric cancer; tangle, toggle, tackle with Korea red ginseng. J Clin Biochem Nutr., 2010; 46:195–204. ANDERSON L.A., MURPHY S.J., JOHNSTON B.T. et al., Relationship between Helicobacter pylori infection and gastric atrophy and the stages of the oesophageal inflammation, metaplasia, adenocarcinoma sequence: results from the FINBAR casecontrol study. Gut., 2007. 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SHIN C.M., KIM N., JUNG Y. et al., Role of Helicobacter pylori infection in aberrant DNA methylation along multistep gastric carcinogenesis. Cancer Sci., 2010 Feb 18 [Epub ahead of print]. KOSUNEN T.U., PUKKALA E., SAMA S. et al., Gastric cancers in finnish patients after cure of Helicobacter pylori infection: a cohort study. Int J Cancer., 2010 Mar 22 [Epub ahead of print].

Received July 24, 2010

Oxidative Stress in Diabetic Kidney Disease MIHAELA BÎCU1, MARIA MOŢA2, N. M. PANDURU5, CORINA GRĂUNŢEANU4 , E. MOŢA3 1

County Clinical Emergency Hospital of Craiova, Clinic of Diabetes, Nutrition and Metabolic Diseases, Craiova, Romania 2 University of Medicine and Pharmacy Craiova, Department of Diabetes, Nutrition and Metabolic Diseases 3 “Carol Davila” University of Medicine and Pharmacy, Bucharest, Department of Physiopathology 4 County Clinical Emergency Hospital of Craiova, Clinic of Nephrology 5 University of Medicine and Pharmacy Craiova, Department of Nephrology Diabetic Kidney Disease (DKD) represents a worldwide public health problem, due to its ever growing incidence and high costs connected to the imposed therapies regarding substitution of kidney functions. DKD includes all the anatomical, clinical and functional alterations that occur at kidney level in a patient with Diabetes Mellitus (DM), as a result of numerous metabolic and haemodynamic factors at the level of kidney microcirculation, based on a polygenous genetical polymorphism that generates an individual susceptibility for this complication. DKD is found in 20–40% of DM patients and it represents the main cause of chronic kidney disease. In DKD pathogeny, an important part is played by the oxidative stress determined by hyperglycemia. Among the mechanisms by which hyperglycemia may affect the kidney we may enumerate polyol pathway activation, C protein-kinase activation (PKC), non-enzymatic protein glycosylation. Out of the highly reactive molecules involved in the oxidative stress of DKD, an important role is attributed to •O2–, •NO and ONOO.– The role of oxidative stress played in DKD pathogeny is also supported by the promising results of some antioxidant therapies in DKD: AGE inhibitors (pyridorin, 2,3 diamino-phenazine, bromo-phenylacetic thiazolium, aminoguanidine/pimagedine), diacylglycerol pathway inhibitors (vitamin E, thiamine, benfotiamine, aminoguanidine), PKC inhibitors (ruboxistaurin), transketolase activators (thiamine and benfotiamine). Key words: oxidative stress, diabetic kidney disease, hyperglycaemia.

Diabetic kidney disease (DKD) includes all the anatomical, clinical and functional alterations that occur at kidney level in a patient with Diabetes Mellitus (DM), as a result of numerous metabolic and haemodynamic factors at the level of kidney microcirculation, having as basis a polygenous genetical polymorphism that generates an individual susceptibility for this complication [1–7]. DKD is found in 20–40% of DM patients and represents the main cause of chronic kidney disease (CKD) [6] [7]. In USA, approximately 50% of patients included in therapies regarding the substitution of kidney functions (TSKF) have DM and CKD in terminal stage [7]. In Europe, 13.1% of CKD cases in the 5th stage that have been treated through haemodialysis are due to DKD, and 4% in Romania [7] [8]. In type 1 DM, DKD incidence increases alongside DM duration, reaching 2–3%/year after 13–20 years of evolution, and after 20 years, the incidence decreases to 0.5%/year [7] [9]. The DKD prevalence in type 2 DM is approximately 40%, with high variability of literature data, data concerned with genetical inheritance, lifestyle, ethnicity, etc. [7]. ROM. J. INTERN. MED., 2010, 48, 4, 307–312

Afro-American, Mexican-American, Indian-American and Polynesian patients present the highest risk in developing DKD and also a rapid progression of DKD to a terminal stage [7] [10]. In DKD pathogeny, an important role is played by the oxidative stress determined by hyperglycemia [11–14]. Oxidative stress may be defined as the imbalance between the production of antioxidants and free-radicals, as a result either of antioxidants decrease or of free-radicals increase, or of both mechanisms, with potential consequences over the emergence of oxidative lesions [14]. Oxidative stress represents the excessive formation of highly reactive molecules as reactive oxygen species (ROS) and reactive nytrogen species (RNS) [11] [15][16]. ROS include free-radicals: • O2– (superoxide), •OH (hydroxyl), •RO2 (peroxyl), • HRO2– (hydroperoxyl) and nonradicals: H2O2 (hydrogen peroxide), HClO (hydrochlorous acid). RNS include free-radicals: •NO (nitric oxide), •NO2– (nitrogen dioxide) and nonradicals: ONOO– (peroxynitrite), HNO2 (nitrous oxide), RONOO (alkyl peroxynitrates) [11] [15] [17]. Relatively recent

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researches study the implication of sulphur reactive species in oxidative stress [14]. Among the mechanisms by which hyperglycemia may affect the kidney we may enumerate polyol pathway activation, C protein-kinase activation (PKC), protein non-enzymatic glycosylation. Polyol (sorbitol) pathway. Through the polyol pathway, glucose excess may be metabolized into sorbitol and fructose by aldose reductase (AR) and sorbitol dehydrogenase (SDH). Polyol pathway seems to intervene in DKD pathogenesis, especially through the interference with the formation of advanced glycosylation end-products (AGE) out of fructose. At kidney level, AR is found in glomerular podocytes, in distal contort tubes and in Henle loop. In rat induced diabetes, there appears the increase of polyol pathway agents, decrease of myoinositol tissular levels and increase of intracellular osmolarity, some phenomena that precede proteinuria [7] [12–14] [18–20]. Polyol pathway determines an endothelial dysfunction (ED) by 3 mechanisms: – high sorbitol accumulation increases osmotic stress [18]; – increase of NADH/NAD+ cytosolic ratio, as a result of a redox imbalance similar to that of tissular hypoxia, also called hyperglycemic pseudohypoxia [22]; – accumulation of triose-phosphates stimulates the formation of methylglyoxal (MG), the most active AGE predecessor, thus increasing the oxidative stress [7] [18]. PKC pathway. PKC activation is accomplished by de novo synthesis of diacylglycerol (DAG), through acylation of glycerol-3-phosphate (GP) into phosphatidic acid (PA). In the cells with low AR activity (like the endothelial cells), DAG is de novo synthesized from glycolysis intermediates, dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA-3-P) [23]. PKC is stimulated in DM by polyol pathway, angiotensin II (Ang II), activation of NAD(P)H oxidase [24] [25] [26–32]. PKC activity is high in the glomerules of DM patients, having the following effects: – direct or indirect alterations of vascular permeability (through the vascular endothelial growth factor – VEGF) [33]; – alterations of blood flow by decreasing endothelial nitric oxide synthesis (eNOS) activity and/or by increasing endothelin-1 (ET-1) synthesis [34]; – thickening of glomerular basement membrane (GBM) through TGF-β (transforming growth

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factor-β), which mediates the growth of type IV collagen and fibronectin synthesis [18]; – fibrinolysis modifications through the plasmatic growth of PAI-1 (plasminogen activator inhibitor-1) [18]; – increase of oxidative stress by activating NADPH oxidase [18]; – alterations of the receptors of some hormones and growth factors (pathway of activating TGF-β synthesis); – alterations of ionic channels activity; – alterations of intracellular pH levels [14]. In diabetic animals, oral administration of LY333531 – a selective inhibitor of isoform βII of PKC – ameliorates the growth of glomerular filtration rate and the accelerated expansion of glomerular mesangium, thus partially correcting albumin urinary excretion (AUE) [12] [14] [18]. AGE pathway (Non-enzymatic glycosylation) consists in the non-enzymatic reaction of glucose and dicarbonyl compounds (MG, glyoxal and 3-deoxyglucosone) with basic aminoacids (lysine and arginine) [7] [13] [14] [18] [19] [21] [35]. AGE may grow through glucose self-oxidation to glyoxal, Amadori products formation and their decomposition into 3-deoxyglucosone and also GA-3P glycolise formation, decomposing into MG. AGE are accumulated into glomerules and kidney tubes only in DM patients. In diabetic animals, the AGE accumulation in the kidneys determines protenuria, proliferation of mesangial cells and GBM thickening [7] [36]. AGE acts through AGE-R1, AGE-R2, AGER3 receptors (RAGE), and also by receptor-independent pathways [7] [14] [37]. As a result of AGE – RAGE interaction there are activated the serine/ threonine kinases family, PKC and other important transcription pathways: nuclear factor – kappa B (NF-kB) and mitogen activated protein-kinases (MAPK) [7] [36]. At a cellular level, the AGE – RAGE interaction induces the synthesis and release of cytokines: TGF-1, platelets-derived growth factor (PDGF), insulin-like growth factor (IGF), increasing the production of collagen IV, laminin and fibronectin, [7] [14] [36]. Synthesizing, the AGEs role in DKD is the following: ↑AGE in GBM, ↑vascular permeability, GBM thickening, ↑production of mesangial matrix with glomerular hypertrophy and glomerulosclerosis [14]. Hyperglycaemia may increase ROS by activating NAPDH oxidase or by inactivating antioxidant enzymes, like SOD (superoxide dismutase), catalase, or eNOS (Fig. 1) [18] [19].

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Fig. 1. – Mechanisms of oxidative stress in DKD (modified after C.G. Schalkwijk, R.B. Jadidi). AGE advanced glycosylation end-products; NADP+ nicotinamide adenine dinucleotide phosphate; NADPH reduced form of NADP+; SOD superoxide dismutase; eNOS endothelial nitric oxide synthase; AR aldoreductase; SDH sorbitol dehydrogenase; G-6-P glucose-6-phosphate; F-6-P fructose-6-phosphate; F-1,6 bi-P fructose 1,6 biphosphate; NAD+ nicotinamide adenine dinucleotide; NADH reduced form of NAD+; DHAP dihydroxyacetone phosphate; GA-3-P glyceraldehyde-3-phosphate; GADPH glyceraldehyde-3-phosphate dehydrogenase; PARP Poly (ADP-ribose) polymerase; ROS reactive oxygen species; • O2– superoxide; Acyl CoA – Acyl Coenzyme A; GP glycerol-3-phosphate; PA phosphatidic acid; DAG dyacyl glycerol; PKC – C protein kinase; MG metylglioxal. AN IMPORTANT ROLE IN OXIDATIVE STRESS OF DKD IS PLAYED BY •O2–, •NO AND ONOO–

O2– is generated through the reduction of oxygen, by various means: NAD(P)H oxidase, xantin-oxidase, cyclooxygenase, eNOS, mitochondrial transport chain [11] [38–41]. Under normal conditions, •O2– is rapidly eliminated through antioxidant defense mechanisms: •O2– is transformed into H2O2 by manganese superoxide dismutase (Mn-SOD) at mitochondrial level and by copper superoxide dismutase (Cu-SOD) at cytosolic level [11] [38]; H2O2 may be transformed into H2O and O2 by glutathione peroxidase (GSH-Px) at mitochondrial level or by catalase at lysosomal level; also, H2O2 may be converted into •OH, in the presence of Fe or Cu [18]. •O2– may activate the AGE pathway, the polyol pathway and PKC, involved in DKD pathogeny. •O2– and H2O2 activate stress signaling mechanisms, like NF-kB, p38-MAPK and STAT-JAK (Signal Transducer and Activator of Transcription – Janus kinase), thus determining •

the migration and proliferation of vascular smooth muscular cells [11]. • NO is produced normally from L-arginine by eNOS; it has a vasodilatation, antiplatelet, antiproliferative and antiinflammatory effect [11] [41], inhibits platelets and leukocytes adhesion to vascular endothelium, regulates cytokines expression VCAM-1 (vascular cell adhesion molecule-1) and MCP-1 (monocyte chemotactic protein-1) [42]. The main beneficial effects of NO in DKD are: antagonizing Ang II effects; increasing capillary flow; inhibiting mesangial proliferation; decreasing endothelial permeability for albumin; reducing oxidative stress in endothelial cells [7]. In the first stages of experimental DM, the NO synthesis increases and its vasodilatation effect seems to be modulated by VEGF; in advanced stages of DM, the NO production decreases, thus determining the alteration of vasodilatation response [7]. ONOO– results in the reaction of •NO with • O2–; cytotoxic ONOO– favours lipid peroxidation

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– AGE inhibitors: pyridorin, 2,3 diamino[15], alters the functions of biological membranes phenazine, bromo-phenacetylthiasonium, [11] [16] [39–42], determines DNA alteration, de• aminoguanidine (pimagedine – prevents creases NO bioavailability, oxidizes tetrahydroincrease of genic expression of TGF β1) biopterin (BH4) – an important cofactor for NOS, [14] [46] thus resulting in uncoupled eNOS, and producing • – diacylglycerol pathway inhibitors – vitamin O2– instead of •NO [16]. E, thiamine, benfotiamine, aminoguanidine Oxidative stress represents a cause for ED [46] in DKD [14]. In DKD, ROS directly determines – PKC inhibitors – ruboxistaurin, vitamin E ED (through peroxidation of lipidic membranes, [14] [46] [47] NF-kB activation and interference with NO avail– Aldosereductase inhibitors – sorbinil – ability) and indirectly (through growth factors and clinical studies in progress cytokines): TGF-β, TNF-α (tumor necrosis factor– Inhibitors of matrix accumulation by transα), IGF-1, EGF (epidermal growth factor) [18] [43] formation factors, modified heparingluco[44]. Micro- and macroalbuminuria are associated samine, sulodexide (reducing micro- and with ED both in type 1 DM and in type 2 DM as macroalbuminuria in type 1 and type 2 DM) well [11] [45]. [46] The role of oxidative stress played in DKD – Transketolase activators (thiamine, benfotiamine) in high doses prevent DKD pathogeny is also supported by the promising development [47] [48]. results of some antioxidant therapies in DKD: __________________________________________________________________ Boala renală diabetică (BRD) reprezintă o problemă de sănătate publică la nivel mondial, prin incidenţa sa în continuă creştere şi prin costurile ridicate ale terapiilor de substituţie a funcţiilor renale pe care le impune. BRD include toate modificările anatomo-clinice şi funcţionale ce apar la nivel renal la un pacient cu diabet zaharat (DZ), fiind rezultatul interacţiunii a numeroşi factori metabolici şi hemodinamici la nivelul microcirculaţiei renale, având ca substrat un polimorfism genetic de tip poligenic, ce generează o susceptibilitate individuală pentru această complicaţie. BRD apare la 20–40% dintre pacienţii cu DZ şi este principala cauză a bolii cronice de rinichi. În patogenia BRD un rol important îl are stresul oxidativ determinat de hiperglicemie. Printre mecanismele prin care hiperglicemia poate afecta rinichiul se numără activarea căii poliol, activarea protein kinazei C (PKC), glicozilarea non-enzimatică a proteinelor. Dintre moleculele înalt reactive implicate în stresul oxidativ din BRD, un rol important este atribuit •O2–, •NO şi ONOO.– Rolul stresului oxidativ în patogenia BRD este susţinut şi de rezultatele promiţătoare ale unor terapii antioxidante în BRD: inhibitorii AGE (pyridorin, 2,3 diaminofenazina, bromura de fenacetilthiazonium, aminoguanidina/pimagedine), inhibitorii căii diacilglicerol (vitamina E, tiamina, benfotiamina, aminoguanidina), inhibitorii PKC (ruboxistaurina), activatorii transketolazei (tiamina şi benfotiamina). __________________________________________________________________ Corresponding author: Professor Maria Moţa, MD, PhD Clinical Centre of Diabetes, Nutrition & Metabolic Diseases 1, Tabaci Str., Craiova, Romania E-mail: [email protected]

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Cardiovascular Risk in Patients with Diabetic Kidney Disease CORINA GRĂUNŢANU1, E. MOŢA1, MARIA MOŢA1, M.N. PANDURU2, MIHAELA BÎCU1, IULIA VLADU1 2

1 University of Medicine and Pharmacy, Craiova, Romania “N.C.Paulescu” National Institute for Diabetes, Nutrition, and Metabolic Diseases, Bucharest, Romania

In the past decades, chronic kidney disease has become a public health problem all over the world. Both the incidence and the prevalence are continually increasing. Diabetic nephropathy is, by far, the most frequent cause of CKD, with a prevalence of 40% in patients with end-stage renal disease (ESRD). Present studies have shown the fact that microalbuminuria and chronic kidney disease are independently associated to a high risk of cardiovascular events, as well as to a high mortality rate of all causes and also of cardiovascular cause, both in the general population and also in patients with high risk or an already present cardiovascular disease. There is a permanent association between the level of urinary albumin excretion and the risk for cardiovascular disease, macroalbuminuria and clinical nephropathy being associated to a higher risk for cardiovascular events than microalbuminuria. Due to the importance of clinical data and low cost, microalbuminuria and glomerular filtration rate should be introduced in the clinical practice for the evaluation of cardiovascular risk, especially in the patients with previously known heart disease. An early identification of the factors that determine the emergence and progression of diabetes complications is essential, in order to reduce the cardiovascular mortality and morbidity. Key words: diabetic kidney disease, cardiovascular disease, microalbuminuria, chronic kidney disease.

Present studies have shown the fact that microalbuminuria and chronic kidney disease are independently associated to a high risk of cardiovascular events, as well as to a high mortality rate of all causes and also of cardiovascular cause, both in the general population and also in patients with high risk or an already present cardiovascular disease. DEFINITIONS OF MICROALBUMINURIA AND CHRONIC KIDNEY DISEASE (CKD)

Microalbuminuria as a predicting factor for diabetic nephropathy in patients with type 1 diabetes was first described in 1982 by Viberti et al. [1], and since then, the definition of albuminuria has been permanently readjusted in order to include all the possible determination methods for urinary albumin excretion. Currently, microlbuminuria is defined as the urinary albumin excretion between 30 and 300 mg/ day if measured in a 24 hrs urine collection, 20– 200 µg/min if measured in a timed urine collection or 30–300 mg/g if there is measured the urinary albumin/ creatinine ratio in a spot urine collection. Values under these limits are considered to be ROM. J. INTERN. MED., 2010, 48, 4, 313–319

normal and those above reflect the presence of macroalbuminuria or clinical proteinuria [2]. Chronic kidney disease is defined as the kidney damage confirmed by kidney biopsy or by markers of damage, or as the decline of glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, for a period longer than 3 months. Decreased kidney function starts at GFR < 89 mL/min and it is considered to be pronounced if the eGFR is < 60mL/min, which corresponds to 1.5 mg/dL of creatinine in men and 1.3 mg/dL in women [3–5]. 25–30 years ago, diabetic nephropathy was identified with Kimmelstiel-Willson nodular glomerulosclerosis. In time, there was proven that all types of kidney lesions overlap in each patient and, depending on the genetic susceptibility, they may lead to the progression towards chronic kidney disease. Because various authors associate the notion of diabetic nephropathy with the old concept, there should be more and more suggested the adoption of a new terminology, like diabetic kidney disease (DKD), which includes all the lesions that appear at kidney level in a patient with diabetes mellitus.

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PREVALENCE OF CHRONIC KIDNEY DISEASE AND DIABETIC NEPHROPATHY (DN)

In the past decades, chronic kidney disease has become a public health problem all over the world. In the National Health and Nutrition Examination Survey (NHANES) III (1988–1994), the prevalence of CKD in adult population was of 11% in the USA, approximately 20 million of American adults suffering from CKD, out of which 8 million having GFR < 60 mL/min/1.73 m2. Both the incidence and the prevalence are continually increasing. Diabetic nephropathy is, by far, the most frequent cause of CKD, with a prevalence of 40% in patients with end-stage renal disease (ESRD). The microalbuminuria incidence in patients with type 1 diabetes was of 12.6% over a period of 7.3 years in the European Diabetes (EURODIAB) Prospective Complications Study Group [6] and of 33% in a study performed over a period of 18 months in Denmark [7]. In patients with type 2 diabetes, the microalbuminuria incidence was of 2.0% per year, with a prevalence of 25% after 10 years after diagnosis in the U.K. Prospective Diabetes Study (UKPDS). The proteinuria prevalence is between 15 and 40% in patients with type 1 diabetes, with a high incidence 15–20 years after diagnosing diabetes, while in patients with type 2 diabetes, the proteinuria prevalence varies more, between 5 and 20% [7–9]. NATURAL PROGRESSION OF DIABETIC KIDNEY DISEASE (DKD)

Primary studies highlighted a high prevalence of microalbuminuria in patients suffering from diabetes, but recent studies did not confirm these outcomes. These prevalence variations may be attributed to differences regarding age, race, blood pressure (BP), or DKD stages in the studied population, as well as to the determination methods of microalbuminuria. The incidence of microalbuminuria is of 2.0% per year since the diagnosis, the progression of microalbuminuria to macroalbuminuria is of 2.8% per year and from macroalbuminuria to high values of seric creatinine is of 2.3% per year. In the UKPDS Study, after 10 years since the diagnosis, microalbuminuria was present in 24.9% of the patients, macroalbuminuria or clinical proteinuria in 5.3% of the patients and seric creatinine was high in 0.8% of the patients [8].

2

The progression rate of diabetic nephropathy as well as the cardiovascular risk seem to be lower in patients with a good glycaemic and blood pressure control [10]. The Diabetes Control and Complication Trial (DCCT) performed on 1.441 volunteers with type 1 diabetes compared the effects of a standard control versus the intensive glycemic control and it showed that a well-balanced metabolism slows down the emergence and progression of kidney and eye damage. The Epidemiology of Diabetes Intervention and Complications Study (EDIC) has shown that an intensive glycemic control reduced the risk for cardiovascular events with 42% and death from cardiovascular causes with 57% [11] [12]. A poor glycemic control is a major risk factor for microalbuminuria, while the progression to advanced stages of diabetic kidney disease is influenced by high blood pressure, dyslipidaemia and genetic factors [13]. Recently, there has been brought into discussion the HbA1C variability and the risk for diabetes complications. Theoretically, a variable glycemic profile may determine a high risk for diabetes complications by the increase of oxidative stress. In the FinnDiane Study, performed on 2107 patients with type 1 diabetes, the HbA1C variability was associated to the progression of chronic kidney disease (HR 1.92) and to the presence of cardiovascular events (HR 1.98) [14]. The impact of lipids in the progression of diabetic kidney disease was studied within the same prospective study (FinnDiane). High values of triacylglycerol, apolipoprotein (Apo) B, ApoA-II and HDL3-cholesterol predict microalbuminuria. The progression to macroalbuminuria was associated to triacylglycerol and apolipoprotein (Apo) B [15]. Also, there has recently been noticed that high seric levels of uric acid in patients recently diagnosed with type 1 diabetes are associated to an increased risk for later development of diabetic nephropathy [16]. MICROALBUMINURIA AND THE CARDIOVASCULAR RISK

The first in reporting an association between microalbuminuria and cardiovascular disease was Yudkin et al. [17]. Since then, various studies performed either on the general population or on high risk patients have reported an association between microalbuminuria and traditional cardiovascular risk factors (age, high blood pressure,

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obesity, smoking, cholesterol, LDL-cholesterol, high triglycerides, low HDL-cholesterol), as well as other new risk factors (insulin-resistance, endothelial dysfunction, oxidative stress) [18–27]. Lipoprotein subclasses, measured by NMR spectroscopy increase the prediction for coronary heart disease in patients with type 1 diabetes [28]. That is why the risk factors for atherosclerosis may also be risk factors for diabetic kidney disease: oxidative stress, circulant immune complexes containing LDL oxidation, adhesion molecules play an important part in the progression of diabetic nephropathy [29]. Systemic inflammation markers and cellular adhesion molecules are high since the early stages of diabetic kidney disease, before the onset of the end-stage renal disease. High levels of C-reactive protein (CRP) may favour lipid aterogenity and may induce the expression of cellular adhesion molecules (ICAM-1) [30]. Moreover, there has been suggested that microalbuminuria is an independent risk factor for morbidity and mortality of cardiovascular cause, both in patients with and without diabetes. Further on, there were performed prospective studies that confirmed the independent association between microalbuminuria and cardiovascular or total cause mortality [31–34]. Also, the post-hoc analysis of long-term clinical trials points out the association between albuminuria and cardiovascular risk. In the Heart Outcomes Prevention Evaluation (HOPE) Study, performed on patients with a history of cardiovascular disease or diabetes mellitus, microalbuminuria was correlated to major cardiovascular events (relative risk – RR 1.83), to all-cause mortality ((RR 2.09) and to hospitalization for congestive heart ((RR 3.23), the relative risk being similar in patients with or without diabetes. The risk for developing cardiovascular events started at the level of urinary albumin excretion under the cut-off value for microalbuminuria and has gradually increased. For each growth of urinary albumin excretion of 0.4 mg/mmol, the risk for cardiovascular events increased by 5.9% [35]. In a subpopulation of the NHANES II study, examined between 1976 and 1980 and monitored on a period of 16 years, the hazard rate for cardiovascular and all-cause mortality was of 1.57 and 1.64 for patients with proteinuria between 30– 299 mg/dL, and 1.77, 2.00 respectively, in patients with urinary protein excretion of 300 mg/dL in comparison to the patients with levels below 30 mg/ dL [36].

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The results of the European Prospective Investigation into Cancer and Nutrition Study, Norfolk, UK (the EPIC-Norfolk Study) support the information above – after a monitoring period of 6.2–7.2 years the presence of albuminuria was independently associated with a significant greater risk of 36% for incident CHD, 49% for stroke, 103% for CV mortality and 48% for all-cause mortality. The important fact is that the risk for cardiovascular events was higher in the presence of macroalbuminuria [37]. In a recent analysis of the New ONTARGET Study, the doubling of albuminuria after 2 years, observed in 28% of the participants, was associated to a dramatic mortality growth (50%) (HR 1.47; p 60 mL/min/1.73m2. This fact did not correlate with the ejection fraction of the left ventricle, which is an independent predictor for cardiovascular risk [63]. Due to the importance of clinical data and low cost, microalbuminuria and the rate of glomerular

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filtration should be introduced in the clinical practice for the evaluation of cardiovascular risk, especially in the patients with previously known heart disease. An early identification of the factors that determine the emergence and progression of diabetes complications is essential, in order to reduce the cardiovascular mortality and morbidity.

317

For a better understanding regarding diabetes mellitus and its complications, further clinical studies are necessary. At present, there are current clinical trials that study therapies preventing the adverse effects of hyperglycaemia: advanced glycation end products inhibitors, protein kinase C inhibitors and aldose reductase inhibitors.

Fig. 1. – Cardiovascular mortality rate associated to eRFG, Am J Epidemiol., 2008, 167:1226–1234.

___________________________________________________________________ În ultimele decenii boala cronică de rinichi (BCR) a devenit o problemă de sănătate publică în toată lumea. Incidenţa şi prevalenţa este în continuă creştere. Nefropatia diabetică este, de departe, cea mai frecventă cauză a BCR cu o prevalenţă de aproximativ 40% la pacienţii cu BCR în stadiul final. Studiile efectuate până în prezent au evidenţiat faptul că microalbuminuria şi boala cronică de rinichi sunt independent asociate cu un risc crescut de evenimente cardiovasculare, precum şi cu o mortalitate crescută de toate cauzele şi de cauză cardiovasculară, atât în populaţia generală, cât şi la pacienţii cu factori de risc sau cu boală cardiovasculară deja prezentă. Există o asociere permanentă între nivelul excreţiei urinare de albumină şi riscul de boală cardiovasculară, macroalbuminuria şi proteinuria clinic manifestă sunt asociate cu un risc mai mare de evenimente cardiovasculare decât microalbuminuria. Datorită importanţei lor clinice şi costului scăzut, microalbuminuria şi rata filtrării glomerulare ar trebui introduse în practica clinică pentru evaluarea riscului cardiovascular, în special la pacienţii cu boală cardiacă cunoscută. Este esenţială identificarea precoce a factorilor care determină apariţia şi progresia complicaţiilor diabetului pentru a reduce mortalitatea şi morbiditatea cardiovasculară. ___________________________________________________________________ Corresponding author: Dr. Grăunţanu Corina, MD Clinical Emergeny Hospital Craiova, Romania, Nephrology Clinic 1, Tabaci Str., Craiova, Romania E-mail: [email protected]

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CHATURVEDI N., BANDINELLI S., MANGILI R., PENNO G., ROTTIERS R.E., FULLER J.H., Microalbuminuria in type 1 diabetes: rates, risk factors and glycemic threshold. Kidney Int., 2001, 60:219–227. HOVIND P., TARNOW L., ROSSING P., JENSEN B.R., GRAAE M. et al., Predictors of the development of microalbuminuria and macroalbuminuria in patients with type 1 diabetes: inception cohort study. BMJ 328:1105–1108, 2004. ADLER A.I., STEVENS R.J., MANLEY S.E., BILOUS R.W., CULL C.A., HOLMAN R.R., Development and progression of nephropathy in type 2 diabetes: the United Kingdom Prospective Diabetes Study (UKPDS 64). Kidney Int, 2003, 63:225–232. WONG T.Y., SHANKAR A., KLEIN R., KLEIN B.E., Retinal vessel diameters and the incidence of gross proteinuria and renal insufficiency in people with type 1 diabetes. Diabetes, 2004, 53:179–184. ADLER S.G., SALANT D.J., An outline of essential topics in glomerular pathophysiology, diagnosis, and treatment for nephrology trainees. 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HILLEGE H.L., JANSSEN W.M., BAK A.A. et al., Microalbuminuria is common, also in a nondiabetic, nonhypertensive population, and an independent indicator of cardiovascular risk factors and cardiovascular morbidity. J Intern Med, 2001, 249:519–526. KIM YI, KIM C.H., CHOI C.S. et al., Microalbuminuria is associated with the insulin resistance syndrome independent of hypertension and type 2 diabetes in the Korean population. Diabetes Res Clin Pract., 2001, 52:145–152. ROMUNDSTAD S., HOLMEN J., HALLAN H., KVENILD K., KRUGER O., MIDTHJELL K., Microalbuminuria, cardiovascular disease and risk factors in a nondiabetic/nonhypertensive population. The Nord-Trondelag Health Study (HUNT, 1995–97), Norway. J Intern Med., 2002, 252:164–172. YUYUN M.F., KHAW K.T., LUBEN R. et al., Microalbuminuria, cardiovascular risk factors and cardiovascular morbidity in a British population: the EPIC-Norfolk population-based study. Eur J Cardiovasc Prev Rehabil, 2004, 11:207–213. TSIOUFIS C., DIMITRIADIS K., CHATZIS D. et al., Relation of microalbuminuria to adiponectin and augmented C-reactive protein levels in men with essential hypertension. Am J Cardiol., 2005, 6:946–951. KISTORP C., RAYMOND I., PEDERSEN F., GUSTAFSSON F., FABER J., HILDEBRANDT P., N-terminal pro-brain natriuretic peptide, C-reactive protein, and urinary albumin levels as predictors of mortality and cardiovascular events in older adults. JAMA, 2005, 293:1609–1616. DE COSMO S., MINENNA A., LUDOVICO O. et al., Increased urinary albumin excretion, insulin resistance, and related cardiovascular risk factors in patients with type 2 diabetes: evidence of a sex specific association. Diabetes Care, 2005, 28:910–915. SOEDAMAH-MUTHU S.S., CHANG Y.F., OTVOS J., EVANS R.W., ORCHARD T.J., Pittsburgh Epidemiology of Diabetes Complications S. Lipoprotein subclass measurements by nuclear magnetic resonance spectroscopy improve the prediction of coronary artery disease in Type 1 diabetes. A prospective report from the Pittsburgh Epidemiology of Diabetes Complications Study. Diabetologia, 2003, 46:674–682. ORCHARD T.J., VIRELLA G., FORREST K.Y., EVANS R.W., BECKER D.J., LOPES-VIRELLA M.F., Antibodies to oxidized LDL predict coronary artery disease in type 1 diabetes: a nested case-control study from the Pittsburgh Epidemiology of Diabetes Complications Study. Diabetes, 1999, 48:1454–1458. CRAIG L., NELSON, CONNIE S., KARSCHIMK U.S., GEORGE DRAGICEVIC et al., Systemic and vascular inflammation is elevated in early IgA and type 1 diabetic nephropathies and relates to vascular disease risk factors and renal function. Nephrol Dial Transplant (2005), 20:2420–2426.

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ORIGINAL ARTICLES

Blood Pressure Pattern and Heart Rate Variability in Normotensive Patients with Type 2 Diabetes Mellitus LAURA POANTĂ1, ANCA CERGHIZAN2, DANA POP3 1

“Iuliu Haţieganu” University of Medicine and Pharmacy, Second Department of Internal Medicine, Cluj-Napoca, Romania 2 County Hospital Cluj, Diabetes Center, Cluj-Napoca 3 “Iuliu Haţieganu” University of Medicine and Pharmacy, Hospital of Cardiac Reabilitation, Cluj-Napoca, Romania

Ambulatory blood pressure monitoring (ABPM) has shown that almost one third of presumed normotensive patients with type 2 diabetes mellitus have instead masked hypertension. There is also a relationship between cardiovascular autonomic neuropathy and blood pressure patterns even in normotensive patients with diabetes mellitus. The aim of the present study was to analyze the blood pressure patterns in type 2 diabetic patients without any history of hypertension and to establish the connection between heart rate variability parameters, ultrasound parameters and ABPM parameters. Material and methods. Fifty-two subjects with type 2 diabetes, aged 59 (±6), were consecutively recruited at the Internal Medicine Department of the County Hospital in Cluj. Informed consent was obtained from all participants. A control group of 47 subjects, age and sex matched, was also analyzed. Results. More than half of the patients had a non-dipping pattern, despite the fact that they are considered normotensive patients. Heart rate variability parameters are lower in the non-dipping group, but the difference is significant only for vagal activity. Left ventricle is thicker in non-dipping group. The mean age of the non-dipping group (61.23 ±2.02 years) was significantly higher than the age of the dipping group (55.11 ±3.88 years) (p angina. Angina, as other symptomps of CVD, can be considered as what can be called “the tip of the iceberg” that is a summum of risk factors (dyslipidaemia, smoking, obesity, age, etc.) and subclinical organ damage. The ACC/AHA Guidelines on stable angina, released in 2002 [3], stated that the basis of medical management remains the pharmacological approach (with the exception of a few high risk situations), that includes what can be called: life saving drugs; drugs maintaining/ameliorating the quality of life, by relieving angina related discomfort. ROM. J. INTERN. MED., 2010, 48, 4, 361–369

Among patients with a history of angina, the frequency of exacerbations is the most important determinant of quality of life [4]. The recent ESC Congress in Stockholm, Sweden offered an overview about the benefic capacity of a novel substance, a late Na+ inhibitor of offering anti-ischemic efficacy in subjects suffering from stable angina (either in monotherapy, or in combination therapy). The clinical proofs are consistent, summarizing the results obtained in the following trials (their presentation in detail will follow): MARISA: chronic angina (n = 191)[5]; CARISA: chronic angina (n = 829)[5], ERICA: chronic angina (n = 565) [6], ROLE: chronic angina (n = 746)[7], MERLIN – TIMI – 36 Non – STEMI ACS (n = 6.560) [8] [9], in a consistent number of patients. In the first place, it is worth mentioning that the severity of angina is in a direct and close relationship with the mortality, conclusion that was highlighted by using the Seattle Angina Questionnaire (SAQ) [10] – item disease-specific health status measure for patients with coronary artery disease. The different predictor variables in SAQ were the scores for: physical limitation; angina stability; angina frequency; quality of life, and the primary outcome was the 1-year all cause mortality and a secondary one: hospitalization for an ACS. The 1-year mortality rate ranged from 2% in patients with minimal physical limitation to over 12% in those with severe physical limitation.

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The principal two aims of the management of stable angina pectoris are: 1) To improve prognosis by preventing myocardial infarction and death; 2) To diminish/abolish the symptoms. The armamentarium considered to play an active role in the conservative management of stable angina is composed by: anti-thrombotic drugs, statins, angiotensin converting enzyme inhibitors (ACEI), beta-blockers (BB), nitrates, calcium channel blockers/ calcium antagonists (CCB/CA), potassium channel offerers, sinus node inhibitors, metabolic agents. On top of that came the new therapeutical agent that represents the subject of this review: a late sodium channel blocker, ranolazine. First, why it is so important to have new therapeutic agent in chronic angina: because the incidence and prevalence of patients with angina is anticipated to increase in the coming decades, due to evident and increasing cardio-vascular burden: the aging of the population, the “tsunamy” of obesity/“diabesity”, the increasing use of lifeprolonging therapies. Ranolazine, a piperazine derivative, the first member of a new class of drugs (that have antiischemic effects through antagonism of the late phase of the inward sodium current, significantly increased in myocardial ischemic and also significantly contributing to calcium overload [11–13], received the approval for the following benefits that she proved to generate: improved exercise performance; reduced angina frequency; reduced use of sublingual nitroglycerin. MERLIN-TIMI 36 trial [8] [9] enrolled 6.560 patients, of which 3.565 (54%) had a history of prior chronic angina (the mean duration of the angina was 5.2 years), the majority of patients were in Canadian Cardiovascular Society Class (CCSC) class 2 (41%), 32% reporting were severe anginal symptoms (a non–ST–segment elevation ACS). Study patients with an ACS had at least 1 indicator of moderate to high risk of death on recurrent ischemic events. First, Ranolazine was tested as monotherapy in patients with exercise – limiting angina and significant ST-segment depression between 3 and 9 minutes, on an exercise tolerance test (ETT), using the Bruce protocol [14] [15]. Also, Ranolazine was tested in combination with other antianginal agents in patients with the same ETT criteria [16] or in patients with at least 3 episodes of angina/week (although they used the maximal dose of amlodipine) [17].

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NB. On the basis of this evidence (the results on over 1.500 patients of this randomized, doubleblind, placebo-controlled trial MERLIN-TIMI 36), ranolazine is an effective antianginal and antiischemic agent, but it is not useful as a diseasemodifying secondary preventive therapy or for prophylaxis of recurrent angina in asymptomatic patients stabilized after an ACS. Active medications for secondary prevention were used in a high proportion: aspirin 95%; betablockers 89%; statins 78%. More than one third of the patients had a history of prior revascularization (448 had a percutaneous coronary intervention – PCI) in the prior 24 months, of which 315 in the past 12 months before study coronary angiography was undertaken in 49% of patients. The results of the COURAGE (Clinical Outcomes Utilizing Revascularization and Aggressive Drug Evaluation) [18] which showed no difference between aggressive secondary preventive pharmacotherapy plus PCI compared with optimal medical therapy is reasonable for most patients with stable CAD and that medical therapy and PCI are important complementary options for managing angina. But, even with aggressive pharmacotherapy and revascularization, approximatively 25% of patients with chronic angina continue to experience attacks with contemporary therapy [19] [20]. In addition, use of traditional antianginal agents is limited in some patients by side effects. The median duration of follow-up was 350 days. The mean number of antianginal agents administered at hospital discharge to patients with prior angina was 1.9 (with no difference between treatment groups). Over the entire duration of follow-up, the mean number of antianginals used / patient was 2.9 (67.5% of the patients were treated with 2 or more medication). Efficacy of ranolazine as antianginal therapy The primary end-point (cardiovascular death, AMI or recurrent ischemia) was lower in patients treated with ranolazine compared with placebo (25.2% vs. 29.4%; p < 0.017); this difference is due only to the impact of ranolazine on recurrent ischemia. One should note that ranolazine had no effect on the risk of cardiovascular death or AMI in the patients with prior angina. But, it is worth mentioning that ranolazine significantly reduced the incidence of each of the major end points, with respect to its antianginal

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Another anginal drug?

efficacy: reduction of the incidence of recurrent ischemia (HR: 0.78%; 95% CI; p < 0.002); worsening angina (HR = 0.77; 95% CI; p < 0.048); intensifycation of antianginal therapy (HR = 0.77; 95% CI; p < 0.005). Ranolazine improved severe recurrent ischemia in patients with prior angina [defined as ischemia associated with new electrocardiographic changes or leading to hospital stay or revascularization (11.9% vs 14.4%; HR = 0.81; 95% CI; p < 0.026)]. In patients without prior angina there was no detectable benefit of ranolazine, with respect to recurrent ischemia (HR = 1.03; 95% CI; p < 0.83). And, finally, this effect of ranolazine on the primary end point and recurrent ischemia was consistent in patients with prior angina who were treated with an early invasive management strategy (n = 1.184; HR = 0.75; 95% CI; p < 0.013 and HR = 0.71; 95% CI; p < 0.015, respectively). Ranolazine slightly reduced also the number of traditional antianginal drugs (2.8 vs. 2.9; p < 0.045). In an analysis conducted to evaluate the chronic effect of ranolazine after the first 30 days, ranolazine reduced the incidence of recurrent ischemia (HR = 0.80; 95% CI; p < 0.015), reduced worsening angina (HR = 0.76; 95% CI; p < 0.044), reduced the intensification of other antianginal therapy (HR = 0.77; 95% CI; p < 0.007). At 8 months (or the final visit, if sooner), ranolazine significantly improved all metrics of exercise performance on ETT or bicycle exercise testing: exercise duration (514 ± 7s vs. 482 ± 7s) (p < 0.002), the time to onset of angina (508 ± 7s vs. 477 ± 7s) (p < 0.002), the time to onset of 1-mm ST-segment depression (509 ± 7s vs. 479 ± 7s) (p < 0.003). Among patients undergoing treadmill testing (n = 1.459) (the primary study assessment in prior studies of ranolazine) the mean difference in exercise duration compared with placebo was 44 s (589 ± 10s vs. 545 ± 10s, p < 0.001). There was no significant impact of ranolazine on exercise duration (p < 0.14) or time to ischemia on exercise testing (p < 0.17) in those without a history of stable angina, consistent with the anterior mentioned lack of effect on recurrent ischemia in patients without prior angina [8] [9] [17]. Safety and tolerability. Ranolazine was generally welltolerated in patients with prior angina. The few mentioned adverse effects were: dizziness (12.4% vs. 7.4%), nausea (9.7% vs. 6.1%), constipation (8.5% vs. 3.3%).

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In 8.1% patients ranolazine was discontinued due to an adverse effect (vs. 4.1% in the placebo group) (p < 0.001). The dose of ranolazine had to be decreased in: 190 (10.6%) patients for renal dysfunction; 10 (0.6%) patients for persistant prolongation of the corrected QT interval; 154 (8.6%) patients for adverse events; 8 patients for unknown cause. There was no difference in the incidence of the major safety end points in patients with prior angina treated with ranolazine vs. placebo. Death from any cause did not differ between treatment groups (HR = 1.01; 95% CI; p < 0.96). No significant increase in frequency of symptomatic documented arrhythmias; clinically significant arrhythmias on Holter evaluation were significantly lower (73.9% vs. 83.1%; p < 0.0001). There were no differences regarding the incidence of discontinuation of ranolazine for an adverse effect in those treated with calcium antagonists (diltiazem and verapamil have inhibitory effect on the clearance of ranolazine). Ranolazine was effective as an antianginal, reducing the incidence of recurrent ischemia by 22% with a corresponding 24% reduction in the incidence of worsening angina and improvement in exercise performance on treadmill testing. No evident effect on: the incidence of cardiovascular death or MI, outcomes or symptoms in patients with CAD without chronic angina, although its efficacy as an antianginal drug is clear and significant. Ranolazine is an effective antianginal and antiischemic agent but there were no benefits considering a disease-modifying secondary preventive therapy or the prophylaxis of recurrent angina in asymptomatic patients stabilized after an acute coronary syndrome (ACS). But, we must remember that the frequency of exacerbations is the most important determinant of quality of life [4]. That’s why the guidelines from the American Heart Association and American College of Cardiology are stating clearly the dual goals for the management of patients with chronic CAD: the secondary prevention of cardiovascular death and AMI and the amelioration of angina [3]. The tablets should be swollen without chewing or dividing them. The absorption is not influenced by the meals. The antianginal action of ranolazine may be related to partial inhibition of fatty acid oxidation [21] [22], which can produce anti-ischemic effects

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without depressing hemodynamic function, but it was later proved that this effect is obtained only at much higher serum levels than the ones clinically used. Inhibition of fatty acid oxidation reciprocally increases glucose oxidation which generates more adenosine triphosphate for each molecule of oxygen consumption [23]. This shift may reduce myocardial oxygen supply needed to support a given level of coronary flow, resulting that ischemia should be less likely. The Monotherapy Assessment of Ranolazine in Stable Angina (MARISA) trial was the first placebo-controlled trial to establish the antianginal and anti-ischemic effects of ranolazine monotherapy, demonstrating increased exercise tolerance and prolonged times to exercise-induced angina and ischemic ST-segment depression with twice-daily ranolazine doses ranging from 500 mg to 1.500 mg [5]. The main tendency in treating angina pectoris, because myocardial oxygen extraction is maximal at rest, was to minimize O2 demand (preload, afterload, heart rate, myocardial contractility). By its capacity of reducing myocardial O2 demand by a novel mechanism, ranolazine is effective to reduce manifestations of ischemia and angina, and raised new hopes for having benefic effects in the management in left ventricular dysfunction (especially diastolic dysfunction), arrhythmias (especially chronic atrial fibrillation). The actual data are indicating that the real mechanism of action is represented by the inhibition of the late inward sodium channel (late INa) [1] [6] [11] [12], which remains opened (pathologically) in many situations, allowing to many adverse stimuli to influence myocardium. In normal situations, the inward sodium channels rapidly inactivate after a short activation, remaining closed during the plateau phase of the action potential. Following electrical activation other ion channels open; among them, calcium channels: the calcium ions that enter the cell during the plateau phase will trigger the release of large quantities of calcium ions from the endoplasmic reticulum. In many pathological situations that can be considered myocardial insults: myocardial ischemia, ventricular hypertrophy, oxidative stress the late sodium channels fail to inactivate/to close or fail to reopen, so that the sodium ions continue to enter the cell, generating an intracellular overload of sodium, and thus leading to significant electro-

4

physiological disturbances: in contractility, in metabolic environment. The balance of sodium/calcium is disturbed in such a manner that the elevation of the intracellular sodium concentration will promote an increased exchange of intracellular sodium for extracellular calcium through the Na+/Ca++ exchanger mechanism, the initial sodium overload (at microscopic level the condition that will expose permanently the contractile elements: actin and myosin, to the calcium ions, that mean a continuous tonic contracture. A continuous tonic contracture is the functional basis for an increased diastolic stiffness, associated with an abnormal elevation in myocardial contractile work, which is leading to exactly the opposite that is desired by any antianginal treatment: increased oxygen consumption and the compression of the vascular space during diastole [20] [22]. This late one effect determines a reduction in myocardial blood flow, thus supplementarily reducing it. By reducing the late sodium influx (in a concentration, voltage- and frequency-dependent manner) ranolazine has the capacity to prevent the intracellular sodium overload and the subsequent calcium overload, a cascade of benefic events that promotes: the facilitation of diastolic relaxation, the preservation of the myocardial blood flow during ischemia and reperfusion, the reduction of oxygen consumption, the restauration of the electrical stability. At therapeutic plasmatic concentrations ranolazine has not any significant effect on the cardiovascular performances of the non-ischemic myocytes. Ranolazine is an active piperazine derivative available in oral and intravenous forms. Its major metabolic biotransformation is through the cytochrome P4503A4-mediated pathway [23], an important clue to remember, because drugs like ketoconazole (diltiazem, verapamil, macrolide antibiotics, HIV protease inhibitors, grapefruit juice), which inhibit the Cyp3A isoenzymes increase ranolazine levels (from 2.5 to 4.5 times). It is also important to mention that ranolazine increases digoxin levels. Ranolazine showed a dose-related benefic effect in myocardial ischemia at submaximal and maximal exercise in patients who had stable coronary artery disease attributable to an improvement in myocardial blood flow without significantly influencing heart rate or blood pressure.

5

Another anginal drug?

Does that mean a new era in antianginal treatment or just a useful complementary help, by reducing the O2 demand? For answering this important question, studies have been made using both immediate-release formulation and sustained-release formulation. The anti-ischemic effect of ranolazine appeared without a significant change in myocardial O2 demand; in comparison with placebo it was noted: a significant reduction of anginal episodes, a significant reduction of nitroglycerin use, a significant improvement in exercise duration and time to exercise-induced myocardial ischemia. MARISA (the Monotherapy Assessment of Ranolazine in Stable Angina) trial, using the sustained-release formulation in 500 mg twice a day, 1.000 mg twice a day, 1.500 mg twice a day versus placebo in 191 patients [5] showed a significant, dose-dependent increase in: exercise duration (maximal increase: 56 seconds), exercise time to angina, exercise time to 1-mm ST segment depression compared to placebo (maximal increase 69 seconds). CARISA (Combination Assessment of Ranolazine in Stable Angina) trial highlighted the supplementary benefit of adding ranolazine to a combination of antianginal drugs (atenolol 50 mg every day, diltiazem 180 mg every day or amlodipine 5 mg every day [5] in 823 patients). The addition of ranolazine was followed by: a significant reduction in antianginal frequency and nitroglycerin consumption, an increase in exercise duration (115.6 seconds above baseline), an increase of the period of time to angina, an increase of the period of time to 1-mm ST segment depression compared to placebo (also at trough, as at peak drug effect), without a significant difference between the two doses used: 750 mg twice a day and 1.000 mg twice a day. The benefic, above-mentioned effects were not connected to any change in blood pressure or heart rate. The most common dose-related adverse effects were constipation, dizziness, nausea and asthenia (that appeared in less than 7% of the ranolazinetreated patients, compared to less than 1% of the placebo-treated subjects). The higher doses (up to 2.000 mg twice a day) can be followed by postural hypotension and syncope (probably due to alpha 1-adrenergic receptor blocking activities) [24]. The diabetics in CARISA trial showed a significant (by an unknown mechanism) improvement in HbA1c [5].

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ERICA trial (Efiicacy of Ranolazine in Chronic Angina) [6] investigated the antianginal effects of ranolazine vs. placebo in 565 patients with persistent angina symptoms despite maximal dosing (10 mg daily) of amlodipine. And, what should be especially noted is that those patients, who had more frequent angina per week (over 4.5 episodes) had a much more marked beneficial response to ranolazine addition to baseline treatment, expressed in: angina frequency, nitroglycerin consumption, seattle Angina Questionnaire. The MERLIN-TIMI-36 trial studied the effects of ranolazine (first administered by intravenous infusion, followed by oral ranolazine) in patients who had a non-ST segment elevation acute coronary syndrome (ACS) [8] [9] – a total of 6.560 patients who had either unstable angina or NSTEMI (enrolled within 48 hours of ischemic symptoms and were treated, as mentioned with ranolazine, first by i.v. infusion, after oral administration, that were followed for a median of 348 days). The primary efficacy end-point was a composite of: cardio-vascular death, myocardial infarction, recurrent ischemia; no difference was noted between the two treatment groups, with the exception of recurrent ischemia. Concerning this effect, a trend toward the reduction of recurrent ischemic complications (13.9% in the ranolazine-treated patients vs. 16.1% in the placebo-treated group) (HR = 0.75; p = 0.03). Besides the above-mentioned long-term effect of the treatment with ranolazine: the worsening angina by: at least one Canadian Cardiovascular Society class requiring intensification of medical therapy, less frequent escalation in antianginal medication, improvement in anginal frequency using the SAQ, another benefic longterm effect of ranolazine was the prevention of atrial and ventricular arrhythmias in this class of high-risk subject (with non-ST elevation ACS) [9]. In a substudy analysis ranolazine was associated with a significant reduction in the composite primary end-point in the high-risk subgroup of patients (characterized by elevated levels of brain natriuretic peptide [BNP]: greater than 80 pg/ml (p = 0.009), showing no beneficial effect in those considered low-risk (those with a normal BNP value). Such a large trial demonstrated convincingly the safety of ranolazine: no difference in mortality in those treated with ranolazine vs. placebo; no

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difference in the incidence of sudden death; no difference in the incidence of symptomatic documented arrhythmias. Discontinuation of treatment because an adverse effect occurred in 28% in the ranolazine group and in 22% in the placebo group (p < 0.01), the most frequent adverse effects, occurring in more than 4% were: dizziness (13% vs. 7%), nausea (9% vs. 6%), syncope (3.3% vs. 2.3%) in placebo in placebo (p = 0.01). Regarding the electrophysiological effects and antiarrhythmic properties of ranolazine, although its effects: inhibition of the late INa and the late ICa are modest (5–10 miliseconds) regarding the duration of the action potential, this means the shortening or normalization of a prolonged action potential (due to ischemia or to exposure to arrhythmogenic compounds). His minimal effect on prolongation of QT interval is considered as another source of potential effects: In drug-induced “torsades des pointes”, in reduction in the incidence of early after depolarization, to not exacerbate the transmural dispersion of repolarization (associated with ischemic and exposure to arrhythmogenic drugs). In all the trials conducted until now there were very rare cases of torsdes des pointes, each time the same incidence as in the placebo group. Although not used as an antiarrhythmic drug, his efficacy in this domain was proven in the MERLIN-TIMI 36, where 97% of the 6.560 patients with an ACS were contiuously ECGmonitored, so it was possible to make a very solid analysis. The results of this analysis showed that ranolazine was associated with a significant reduction in ventricular arrhythmias (with no effect on sustained ventricular tachycardia greater than 30 seconds); a reduction of the incidence of supraventricular arrhythmias; a trend toward reducing the incidence of new-onset atrial fibrillation (p = 0.08); a very significant reduction of the ventricular tachycardias lasting 8 beats or more in the high-risk patients: those who had prior heart failure, reduced left ventricular function, prolonged QTc interval at baseline and a high [25–27] TIMI risk score; and with a reduction of the incidence of bradycardia less than 45 bpm for at least 4 beats. Although it is not clear if these antiarrhythmic effects are secondary to prevention of ischemia-associated arrhythmias or from a primary antiarrhythmic effect (maybe related to its effect on Na and Ca ions channels.

6

The actual data show promising results of ranolazine in the management of diastolic LV dysfunction, especially associated with ischemic cardiomyopathy and left ventricular hypertrophy (LVH) (conditions associated with abnormal late INa and secondary Na and Ca overload, associated with oxidative stress). Hayashida et al. [28], by intravenous administration of ranolazine in 15 patients with a previous transmural MI in whom were demonstrated ischemic or infarcted segments observed a significantly increased regional peak filling rate and the regional wall lengthening in the ischemic segments (p < 0.05). The echo Doppler analysis of the left ventricular filling dynamics [29] showed an improved peak filling rate. In patients with documented genetic defect in the late sodium channel, the hereditary long QT syndrome LQT3-delta KPQ, intravenous administration of ranolazine managed to shorten the prolonged QTc and to improve the associated diastolic dysfunction. ROLE trial (Ranolazine Open Label Experience) [7] studied the safety and tolerability of ranelazine (n = 746). The ROLE program involved patients who completed MARISA and CARISA trial, so all ROLE participants were having severe functional limitations due to angina. All the subjects had severe functional impairment from angina and adverse events (AE) reporting was performed periodically. Investigators could titrate to optimal ranolazine dosages between 500 and 1.000 mg b.i.d. on the basis of clinical responses at up to 6 initial weekly visits. Patients who experienced recurrent angina or adverse events (AE) during the maintenance phase could be titrated to higher or lower drug doses. The optimal addition of other antianginal effects was permitted. Safety and tolerability assessments included vital signs, electrocardiograms (ECGs), AE evaluation, with physical examination and laboratory analyses (hematology, chemistry and plasma ranolazine concentrations) were conducted at baseline and all maintainance-phase visits; urinanalysis and serum lipids were assessed at baseline and every 6 months during maintenance. All the reasons for study drug discontinuation were recorded. Every new drug is considered, at the beginning, with hopeful feelings and some concerns, the last ones being justified by the relatively recent cases of mibefradil and nesiritide (agents that received

7

Another anginal drug?

regulatory approval for the symptomatic relief that they offered, and were subsequently withdrawn/ kept under strict observation after the release of safety data. The results of this observational trial can be summarized as follows: 23.2% of the patients did not complete 2 years of therapy, but only less than one half (9.7%) stopped the ranolazine administration due to AE. Predictive modeling showed a significantly increased likelihood of AE-discontinuation only with the variable represented by an age of over 64 years. Some previous data that showed improvement of the left ventricular function in animals [9] [17] are, possibly, sustaining the good tolerance of ranolazine in those with CHF. Their mortality (all of them, with a mean Duke Treadmill Score [TDS] [30] of – 14.4 was considered at high risk (category characterized by TDS score < –10) was compared with the Duke Treadmill Score (DTS) predictive model and other contemporary cohorts of high-risk CHD patients (during a mean follow-up of 2.82 years). Although the open-label methodology of this trial is not a perfect tool for a rigorous analysis in this sense, the data offered by this trial are consistent: the mean DTS would imply a poor prognosis and a yearly mortality estimate above 5%; at the end of the study the 2 year mortality of 5.6%, with a yearly mortality of approximately one-half the DTS predicted rate, a very supportive data in favor of ranolazine. It is also useful to mention that these data are an answer regarding the very negative impact of the QTc interval prolongation (a mean of 2.4 msec), a previous study regarding the Bazzett’s corrected interval [31] showed a prolongation of less than 10 msec (and, it is worth mentioning that is now demonstrated [32] that QTc interval prolongation alone is not sufficient to cause torsades des pointes, it is mandatory that induction of early after depolarizations and an increase in transmural dispersion be present. Finally, there were no investigator-ordered discontinuations related to QTc interval prolongation or cases of torsade des pointes [33]. A populational analysis of data from patients and healthy volunteers demonstrated that the increase of the QTc duration as function of the plasmatic concentration was estimated to 2.4 msec at 1.000 ng/ml, that means an increase of 2–7 msec over the interval of the plasmatic concentration of ranolazine in doses ranging from 500 mg twice a day to 1.000 mg twice a day. This prolongation of QTc must be cautiously considered when ranolazine is intended to be administered in: patients

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with an inherited prolongation of QTc, in patients with acquired QT prolongation, or in patients that are concomitantly treated with QTc prolongating-drugs: some antihistaminic drugs (terfenadine, astemizole, mizolastine), some antiarrhythmic drugs (quinidine, disopiramide, procainamide), tricyclic antidepressive drugs (imipramine, amitryptiline, doxepine). It is also mandatory to carefully establish the dosing in: congestive heart failure, renal failure, hepatic failure, third age subjects, those with reduced weight; it is not recommended to administer ranolazine in children and adolescents. Regarding the AE 72 patients (9.7%) they discontinued ranolazine due to AE, only the age of over 64 years being highly predictive for high withdrawal rates. Mean QTc interval was prolonged by 2.4 msec (without any treatment discontinuation due to this prolongation) and with no torsades des pointes reported. The total mortality (by extending observations to all patients in the double-blind trials (preceding the ROLE program) was 2.8% compared with > 5% as predicted by DTS. Long-term safety and tolerability of ranolazine in patients with chronic stable angina seems favorable without indication of increased long-term cardiac mortality compared with reference populations. So, there are solid arguments sustaining that ranolazine is well tolerated in high-risk CHD patients and survival analysis suggests that symptommatic improvements attributable to ranolazine are important, compared to the absence of an increase of the mortality. As conclusions: • myocardial ischaemia is associated with a pathological increase of the late cardiac Na+ current (INa); • this increase in late INa causes Ca2+ overload, generating increased diastolic wall tension and, respectively, a worsening of ischaemia; • by these effects, the anti-anginal/antiischaemic effects are not secondary to any modification in the haemodynamic parameters; • the well-demonstrated improvement in exercise performance (MARISA, CARISA, MERLINTIMI 36), the significant decrease in weekly attacks and in nitrates consumption (CARISA, ERICA) and the benefic effects on angina/ischaemia (MERLIN-TIMI 36) are solid arguments for this new and well-tolerated anti-anginal agent, that can, possibly, have also benefic antiarrhythmic properties.

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8

Ranolazine este un medicament nou, recent aprobat de Food and Drug Administration (FDA) pentru tratamentul pacienţilor cu angină pectorală stabilă refractară la terapia antianginoasă convenţională. Ranolazine s-a dovedit a fi eficace atât ca monoterapie, cât şi în combinaţie cu alte principii terapeutice. Această trecere în revistă este inspirată de prezentarea pe care figuri legendare ale cardiologiei contemporane, precum Braunwald, Komajda, Camm au făcut-o recent acestui medicament la ultimul Congres al Societăţii Europene de Cardiologie, desfşurat la Stockholm, în Suedia, anul acesta.

Corrresponding author: H. Bălan Medical Clinic, Clinical Emergency Hospital Ilfov County, 49–51, Bucharest, Basarabia Blvd. e-mail: [email protected]

REFERENCES 1. 2. 3.

4. 5. 6. 7. 8.

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11. 12. 13. 14. 15.

Chronic stable angina, the tip of the iceberg: walk on with a new innovative drug. ESC Congress 2010 – Symposium Chairpersons: E. Braunwald, M. Komajda – 30 August, Stockholmsmässan. KERN M.J., Coronary Blood Flow and Myocardial Ischemia in “Braunwald’s Heart Disease” A Textbook of Cardiovacular Medicine, 7th ed., Elsevier Saunders 2005, p. 113–1128. GIBBONS R.J., ABRAMS., CHATTERJEE K. et al., ACC/AHA 2002 guideline update for the management of patients with chronic stable angina-summary article: a report of the American College of Cardiology/American Heart Association Task force on practice guidelines (Committee on the Management of Patients With Chronic Stable Angina), J.Am.Coll.Cardiol., 2003, 41, 159–68. BEINART S.C., SALES A.E., SPERTUS J.A. et al., Impact of angina burden and other factors on treatment satisfaction after acute coronary syndromes. Am.Heart J., 2003, 146, 646–52. STONE P.H., CHAITMAN B.R., KOREN A. et al., Effects of ranolazine as monotherapy and combination therapy on rate pressure product at rest and during exercise: results from the MARISA and CARISA trials. Circulation, 2006, 114 (Suppl II): II–715. STONE P.H., GRATSIANSKY N.A., BLOKHIN A. et al., Antianginal efficacy of ranolazine when added to treatment with amlodipine: the ERICA (Efficacy of Ranolazine in Chronic Angina) trial. J.Am.Coll.Cardiol., 2006., 48, 566–75. KOREN M.J., CRAGER M.R., SWEEENEY M., Long-Term Safety of a Novel Antianginal Agent in Patients with Severe Chronic Stable Angina – The Ranolazine Open Label Experience (ROLE). JACC, 2007, 49, 1027–1034. MORROW D.A., SCIRICA B.M., KARWATOWSKA-PROKOPCZUK E., et al., Effects of ranolazine on recurrent cardiovascular events in patients with non-ST-elevation acute coronary syndromes: the MERLIN-TIMI 36 randomized trial. JAMA 2007, 297, 1775–83. SCIRICA B.M., MORROW D.A., HOD H. et al., Effect of ranolazine, an antianginal agent with novel electrophysiological properties, on the incidence of arrhythmias in patients with non ST-segment elevation acute coronary syndrome: results from the Metabolic Efficiency With Ranolazine for Less Ischemia in Non ST-Elevation Acute Coronary Syndrome Thrombolysis in Myocardial Inferction 36 (MERLIN-TIMI 36) randomized controlled trial. Circulation, 2007, 116, 1647–52. BRAUNWALD E., ANTMAN E.M., BEASLEY J.W. et al., ACC/AHA 2002 guideline update for the management of patients with unstable angina and non-ST-segment elevation myocardial infarction – summary article: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee on the Management of Patients With Unstable Angina). J.Am.Coll.Cardiol., 2002, 40, 1366–74. CHAITMAN B.R., Ranolazine for the treatment of chronic angina and potential use in other cardiovascular conditions. Circulation, 2006, 113, 2462–72. ANTZELEVITCH C., BELARDINELLI L., ZYGMUNT A.C. et al., Electrophysiological effects of ranolazine, a novel antianginal agent with antiarrhythmic properties. Circulation, 2004, 110, 904–10. BELARDINELLI L., SHRYOCK J.C., FRASER H., The mechanism of ranolazine action to reduce ischemia-induced diastolic dysfunction. Eur.Heart J., 2006, 8, A10–3. CHAITMAN B.R., SKETTINO S.L., PARKER J.O. et al., Antiischemic effects and long-term survival during ranolazine monotherapy in patients with chronic severe angina. J.Am.Coll.Cardiol., 2004, 43, 1375–82. CHAITMAN B.R., PEPINE C.J., PARKER J.O. et al., Effects of ranolazine with atenolol, amlodipine, or diltiazem on exercise tolerance and angina frequency in patients with severe chronic angina: a randomized controlled trial. JAMA, 2004, 291, 309–16.

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16. STONE P.H., GRATSIANSKY N.A., BLOKHIN A. et al., Antianginal efficacy of ranolazine when added to treatment with amlodipine: the ERICA (Efficacy of Ranolazine in Chronic Angina) trial. J.Am.Coll.Cardiol., 2006, 48, 566–75. 17. MORROW D.A., SCIRICA B.M., KARWATOWSKA-PROKOPCZUK E. et al., Effects of ranolazine on recurrent cardiovascular events in patients with non-ST elevation acute coronary syndromes: the MERLIN-TIMI 36 randomized trial. JAMA, 2007, 297, 1775–83. 18. BODEN W.E., O’ROURKE R.A., TEO K.K. et al., Optimal medical therapy with or without PCI for stable coronary disease. N.Engl.J.Med., 2007, 356, 1503–16. 19. HOLUBKOV R., LASKEY W.K., HAVILAND A. et al., Angina 1 year after percutaneous coronary intervention: a report from the NHLBI Dynamic Registry. Am. Heart J., 2002, 144, 826–33. 20. SERRUYS P.W., UNGER F., SOUSA J.E. et al., Comparison of coronary-artery bypass surgery and stenting for the treatment of multivessel disease. N.Engl.J.Med., 2001, 344, 1117–24. 21. CLARKE B., WYATT K.M., MCCORMACK J.G., Ranolazine increases active pyruvate dehydrogenase in perfused rat hearts: evidence for an indirect mechanism . J.Mol.Cell.Cardiol., 1996, 28, 341–350. 22. McCORMACK J.G., BARR R.L., WOLFF A.A., LOPASCHUK G.D., Ranolazine stimulates glucose oxidation in normoxic, ischemic, and reperfused ischemic rat hearts . Circulation, 1996, 93, 135–142. 23. WOLFF A.A., ROTMENSCH H.H., STANLEY W.C., FERRARI R., Metabolic approaches to the treatment of ischemic heart disease: the clinicians’ perspective. Heart Fail.Rev., 2002, 7, 187–203. 24. THOMAS E.J., BRENNAN T.A., Incidence and types of preventable adverse events in elderly patients: population based review of medical records. BMJ, 2000, 320, 741–4. 25. ARNOLD S.V., MORROW D.A., WANG A. et al., Effects of ranolazine on disease-specific health status and quality of life: results from the MERLIN-TIMI 36 randomized trial [abstract no. 1024–54]. J.Am.Coll.Cardiol., 2008, 51 (Suppl. A): A215. 26. MEGA J.L., HOCHMAN J.S., SCIRICA B.M. et al., Anti-ischemic effects of ranolazine in women: results from the randomized, placebo-controlled MERLIN-TIMI 36 trial [abstract no. 2451]. Circulation, 2007, 116 (16 Suppl. II: 539. 27. WILSON S.R., MORROW D.A., SCIRICA B.M. et al., Efficacy and safety of ranolazine in chronic angina: observations from the randomized, double-blind, placebo-controlled MERLIN-TIMI 36 trial [abstract no. 1031–45]. J.Am.Coll.Cardiol., 2008, 51, (1 Suppl. A: A225. 28. HAYASHIDA W., VAN EYLL C., ROUSSEAU M.F. et al., Effects of ranolazine on left ventricular regional diastolic function in patients with ischemic heart disease. Cardiovasc.Drugs Ther., 1994, 8, 741–7. 29. ROUSSEAU M.F., COCCO G., BOUVY T. et al., Effects of a novel metabolic modulator, ranolazine, on exercise tolerance and left ventricular filling dynamics in patients with angina pectoris. Circulation, 1992, 86 (Suppl I): I–714. 30. MARK D.B., HLATKY M.A., HARRELL F.E. Jr. et al., Exercise treadmill score for predicting prognosis in coronary artery disease. Ann.Intern.Med., 1987, 106, 793–800. 31. CHAITMAN B.R., PEPINO C.J., PARKER J.D. et al., Effects of ranolazine with atenolol, amlodipine, or diltiazem on exercise tolerance and angina frequency in patients with severe chronic angina: a randomized controlled trial. JAMA, 2004, 291, 309–16. 32. BELARDINELLI L., ANTZELEVITCH C., VOS M.A., Assessing predictors of drug-induced torsade des pointes. Trends Pharmacol.Sci., 2003, 24, 619–25. 33. MOSS A., ZAREBA W.Z, SCHWARZ K.Q. et al., Ranolazine shortens repolarization and improves myocardial relaxation in patients with type-3 long QT syndrome. J.Am.Coll.Cardiol., 2008, 51 (Suppl A):A15. Received September 18, 2010

Stress Vulnerability in Patients with Drug Allergy – Psychological Aspects Revealed from some Personal Studies I.B. IAMANDESCU, LILIANA DIACONESCU “Carol Davila” University of Medicine and Pharmacy, Department of Medical Psychology, Bucharest, Romania

Previous studies revealed the fact that patients with allergic-type reactions to drugs display very frequently psychical disturbances, possibly in relation to the large amount of stress perceived by them. Some of these patients with psychiatric comorbidity show a disproportionate reaction, manifested as extensive psychosomatic disturbances, mimicking the psychic and somatic symptoms of a previous anaphylactic or anaphylactoid reaction, when a new drug or even when placebo preparations are given. The neurotic symptoms, very frequently encountered in patients with allergictype reactions to drugs (including asthma patients) appear to be secondarily-induced by the anxious experience of the drug-provoked accident. The vulnerability to psychic stress, together with many life stressors events may represent a potential risk for developing drug allergy. Their main problem is the risk of repeating the allergic accidents, especially in patients with various comorbidity which need medication. Key words: drug allergy, psychotomatic disease.

Belonging to allergic diseases, drug allergy is considered a psychosomatic disease with multifactorial etiology with a psychogenic component binding [1]. In iatrogenic pathology, patients with allergic reactions to drugs are a specific subpopulation both in terms of allergic mechanisms of allergic reactions to medicines and of the site of these reactions – skin and mucous membranes (especially respiratory and digestive) as tissues with a barrier role. Together with allergic reactions to drugs can be considered a fairly large group of “pseudoallergic” reactions included under the term “nonallergic hypersensitivity” [2] whose mechanism does not “appeal” to allergic antibodies or to the sensitized cells, but other “non-allergic” mechanisms (e.g., cyclooxygenase inhibition by NSAIDs, direct action on mast cell as polymixine). These nonallergic mechanisms “use” the effector link of allergic reaction represented by mast cell degranulation and other blood cells, as eosinophils, resulting in the release of allergic mediators (histamine, leukotriene, PAF, etc.). Therefore, since both allergic reactions and non-allergic reactions to drugs have same causes, one of us (Iamandescu) grouped both types of reactions as “allergic-type reactions to drugs (medicines)” (AtRD). Since the psychological effects of these reactions are the same, the references to drug allergy presented in this article will actually refer AtRD term, which is less used in the literature. ROM. J. INTERN. MED., 2010, 48, 4, 371–375

Psychological vulnerability to stress may be regarded as a particular feature of individuals which respond in an easy manner to psychological stress in a wide range of stress agents [3] and appears as an element favoring psychosomatic disease in which an organ vulnerability is associated [4]. Meanwhile, any psychosomatic disease generates somato-psychic disorders, which are enhanced by this type of personality with an increased vulnerability (especially in the field of affective-emotional) [5]. We can speak about a double vulnerability: psychological and physical. In selected cases, may consider the presence of a constitutional vulnerability to stress (generated by inducing genes of such vulnerabilities, which coexist with genes bearing immunological characteristics of future allergic). But no matter whether we speak about an acquired or constitutional vulnerability, patients who experienced an allergic reaction to medication, especially if it was severe (anaphylactic shock, glottis edema), may submit a series of psychological symptoms, arising from the allergic reaction recurrence [6] [7]: – a high degree of anxiety in the event of an illness requiring medical treatment; – fear of a new allergic accident, resulting in anxious expectation of possible allergic reactions; – disease phobia and drug phobia especially to drugs; – (in surgery) assumption of increased suffering due to fear of developing allergy after anesthesia;

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– extrapolation of allergic reactions from certain drugs to all drugs; – excessive neuro-vegetative reactions (headache, dizziness, palpitations, tachycardia) at the administration of medicines, and some-times even in placebo-prepared (in which case we speak about nocebo effect). These symptoms may be a clinical expression of a state of extreme anxiety. Personality type and psychic symptoms of patients with allergic reactions to drugs Two sub-populations can be distinguished, from the viewpoint of personality features, corresponding to the two clinical-pathogenic groups defined above [8]: 1) Patients with pure allergy or with pseudoallergy to drugs (group A) From the 79 patients that we investigated, 55.7% had T values over 70 percentile on the Hs (Hipocondria), D (Depression) and Hy (Hysteria) scales, constituting together the so-called “neurotic triad”. Also, a record of events on the Holmes and Rahe scale showed that 82.3% of these patients had a total score over 300 points, with regard to the summation of psycho-traumatizing events occurred in the last 6 months preceding the allergic-type reactions to drugs.

2) Patients with psychosomatic disturbances – mimicking drug allergy – on repeated administration of drugs (other than the initial ones, that had triggered allergic-type reactions) (group B). This subgroup included 40 patients with characteristics indicating coexistence of a true neurosis, both by the large number of neurotic symptoms (> 5) found in almost each patient (90% of all cases), and by the high values of T levels on the neurotic triad scales as evaluated with the MMPI Questionnaire (96% of all cases). All the patients had been submitted to psychical stress, and had scores above 300 points on the Holmes and Rahe Scale over the last 6 months that had preceded the first episode of allergic-type intolerance to certain drugs. Concluding on these relationships mentioned above the following can be inferred: • The permeability factor for the onset of allergic-type reactions to drugs, as well as of neurotic disturbances secondary to these reactions was the overall vulnerability to stress of most of the patients, as demonstrated with the psychological MMPI test, which showed values characteristic for

2

neurotic patients(the neurotic triad: Hs + Hy + D) in 55.7% of the cases in the first group (A) (with lower neurotic disturbances), and in 90% of the patients in the second group (B) (with chronic neurotic disturbances and “noisy” psychosomatic reactions to placebo testing). • In the vast majority of patients with allergic-type reactions to drug – 82.3% of the 79 patients with exclusive allergic or pseudo-allergic reactions to drugs group and in 100% of the 40 patients with initial allergic reactions followed by psychosomatic disturbances to placebo administration – the presence of major stresses was noted, before the onset of first allergic-type manifesttations, and these stresses were later exacerbated by the psychologicallytraumatizing experience of drug-induced accidents, including the fear for their possible recurrence. • Neurotic symptoms evidenced by us in patients with allergic-type reactions to drugs (considered at present to be “neurotic disturbances of personality”) were present in these patients, either in an isolated form of 1–2 symptoms (for instance: anxiety and depression), with a transient evolution, or as “true neurotic syndromes” with a chronic evolution. To conclude (Table I): • Patients with allergic-type reactions to drugs display very frequently psychical disturbances, possibly in relation to the large amount of stress perceived by them; • Many of these patients are neurotic and a large part of them show a disproportionate reaction, manifested as extensive psychosomatic disturbances, mimicking the psychic and somatic symptoms of a previous anaphylactic or anaphylactoid reaction, when a new drug or even when placebo preparations are given; • The neurotic symptoms, very frequently encountered in patients with allergic-type reactions to drugs (including asthma patients), appear to be secondarily-induced by the anxious experience of the drug-provoked accident. This authentic somato-psychic reaction to drug allergy can be conditioned in some patients (see group B), but it achieves this secondary “neurotic state” only when certain personality features already exist (that can even reflect personality

3

Stress vulnerability and drug allergy

disturbances!), making these patients highly vulnerable to psychical stress; • The vulnerability to psychic stress, together with many life stressors events may repre-

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sent a potential risk for developing drug allergy in a predisposed patient, but this hypothesis – emerged from our studies – has still to be demonstrated. Table I

Psychotic parameters 1. Stresors of life events

Subpopulations of patients with drug induced symptoms Allergy or pseudo-allergy Mimicking allergy (Group A) (Group B) ↑ ↑ ↑ ↑ ↑ ↑ ↑

2. High vulnerability to stress (MMPI) 3. Neurotic symptoms - absent - few (< 5) - many (> 5) (real neurotic patients)

Depression and anxiety Allergic patients have a high rate of anxiety disorders and/or depression [9] [10]. Furthermore, studies on patients with affective and anxiety disorders [11] showed a high prevalence of allergic reactions. In these patients a higher percentage of affective disorders, anxiety, depression and panic have been present. Anxiety is characterized by excessive anxiety and vegetative hyperexcitation associated with distorted perceptions of symptoms, leading to an increased number and severity of complaints/symptoms [12] [13] and avoidant behaviour [14]. Somato-psychic recoil In addition to somatic or psychological terrain favorable to allergic reactions, a definite contribution to the perpetuation of an allergy is somatopsychic recoil [9]. In this regard, the doctor should discover the individual representations of allergic reaction to medication which are related to issues such as [15]: – a correct identification of allergy; – identifying the consequences (physical, emotional, social, economic); – the patient knowledge about the real causes of allergy; – the control of drug allergy (the extent to which the patient identifies the allergic reaction as a problem that can be controlled). If drug allergy has or has not vital risk, patients with allergic-type reactions will use adaptive strategies, such as [15]:

↑ ↑

↑ ↑ ↑ ↑

↑ ↑ ↑ ↑ ↑ ↑

0 ↑ ↑ ↑ ↑ ↑

– reorganization of the relationship with others; – reorganization of the self image; – affective and behavioral regression; – emotional reactions (anxiety, depression); – problem-centered coping (e.g., analyzing, resolving/minimizing the situation) or emotioncentered coping (e.g. denying, resignation, fatalism). We conducted a recent study (2009) in order to highlight some psychodiagnostic and experimental aspects in drug allergy. MATERIAL AND METHOD

In the study 30 subjects with drug allergy and 30 healthy subjects were included. In both groups of subjects psychological tests were applied: • Anxiety and Depression Scale – HADS [16]. It is a brief self-report questionnaire (14 items) which assesses anxiety (HADS-A) and depression (HADS-D) as two distinct dimensions in non-psychiatric populations. It has been used widely in clinical settings where anxiety and depression can co-occur with physical pathology. • Perceived Stress Scale [17]. It is a psychological instrument (10 items) which measures a global perception of stress. The questions ask about feelings and thoughts during the last month. Items were designed to tap how unpredictable, uncontrollable and overloaded respondents find their lives. • Stress Vulnerability Scale [18]. It is a self report questionnaire (20 items) which

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measures how vulnerable is someone to stress. It refers to a number of factors that affect one’s vulnerability to stress – among them are eating and sleeping habits, caffeine and alcohol intake, and how people express their emotions. Considering the finding that emotional stimuli modify the sudor secretion [19], the subjects were submitted to a musical-test diagnostic (MTD, Iamandescu) including 3 sets of music as a psychosomatic stimulus that induces changes in the moisture of skin which was measured with the Multi Skin Test Center MC 750.

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RESULTS (shown in Table II)

Levels of anxiety, depression and perceived stress were significantly elevated in patients with drug allergy compared with the control group. Increased scores at the stress vulnerability (with values between 32 and 65 points) correlated with high scores on perceived stress scale (.408, p < 0.01). Sudor secretion was increased in patients with drug allergy after hearing each music fragment, but especially after the sad music pieces (perceived as a distressing stimulus).

Table II

Anxiety Depression Perceived stress Stress vulnerability

Patients with drug allergy 9.25 5.72 41.55 38.97

Healthy subjects 6.73 3.80 28.80 31.46

Considering those issues is useful and necessary a psychosomatic approach of patients with drug allergy, an approach that focuses on items like: 1. High levels of anxiety and depression that – the evolution of allergy, although disconpatients have presented underline the somatic-psychic tinuous and sometimes with the possibility of recoil and psychological impact of allergic reactions. avoiding allergic episodes remains often unpre2. High scores on scales of perceived stress dictable; and vulnerability to stress indicate that these – the risk of occurrence of episodes which patients are characterized by a particular psychocan endanger life (shock, glottis edema); logical profile, with personality traits that imply the – personality traits dominated by the presence existence of a psychological vulnerability to stress. of anxiety, depression; 3. Higher value of perceived stress and – psychological stress vulnerability that entails vulnerability to stress in patients with drug allergy which can maximize the role of stressful life (comparing with healthy subjects) may indicate context and enhance the appearance of somatothat these patients would have limited capacity to psychic recoil with anticipatory anxiety and phobia cope with stressful events), generated either by towards drugs and any condition that requires acquired or constitutional vulnerability. treatment, the feeling of helplessness and hope4. The increasing of sweat secretion level lessness or neuro-vegetative reactions that mimic especially after the sad music passage (as a psychoan allergic reaction; – the utility of psychotherapeutic interventions, somatic stimulus) is an objective proof of the at least supportive or relaxant therapies. influence of psychic stress. ___________________________________________________________________ DISCUSSION

Studiile precedente au relevat faptul că pacienţii cu reacţii de tip alergic la medicamente prezintă tulburări psihice foarte frecvent, eventual în relaţie cu încărcătura de stres perceput. Unii dintre aceşti pacienţi cu comorbiditate psihiatrică au o reacţie disproporţionată, manifestată prin tulburări psihosomatice extinse, ce pot mima simptome psihice şi somatice ale unei reacţii anafilactice sau anafilactoide anterioare, atunci când este administrat un nou medicament sau chiar un preparat placebo. Simptomele nevrotice, foarte frecvent întâlnite la pacienţii cu reacţii de tip alergic la medicamente (inclusiv la pacienţii cu astm), par a fi induse în mod secundar şi de experienţa anxioasă a unui accident provocat de medicamente.

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Vulnerabilitatea la stres psihic, împreună cu evenimentele stresante de viaţă, poate reprezenta un risc potenţial pentru apariţia reacţiilor alergice la medicamente. Problema principală este reprezentată de riscul repetării accidentelor alergice, în special la pacienţii cu diferite comorbidităţi şi care necesită medicaţie. ___________________________________________________________________ Corresponding author: Liliana Diaconescu Email: [email protected]

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

IAMANDESCU I.B., CHIVU A., Alergia medicamentoasă, In: I.B. Iamandescu (ed), Psihologie Medicală- Psihosomatică Generală şi Aplicată, Editura Infomedica, 2009, Bucureşti. JOHANSSON S.G.O., BIEBER T., DAHL R. et al., Revised nomenclature for allergy for global use: Report of the Nomenclature Review Committee of the World Allergy Organization, October 2003. J Allergy Clin Immunol, 2004, 113:832–836. IAMANDESCU I.B., LUBAN-PLOZZA B., Stresul psihic – placă turnantă a Medicinii Psihosociale, In: B. Luban-Plozza and I.B. Iamandescu (edited by), Dimensiunea psihosocială a Practicii Medicale, Editura Infomedica, 2003, Bucureşti. IAMANDESCU I.B., Stresul psihic din perspectivă psihologică şi psihosomatică, Editura Infomedica, 2002, Bucureşti. IAMANDESCU I.B., Stresul psihic şi bolile interne, Editura All, 1993, Bucureşti. IAMANDESCU I.B., Elemente de psihosomatică alergologică, In: I.B. Iamandescu (edited by), Elemente de psihosomatică generală şi aplicată, Editura Infomedica, 1999, Bucureşti. DIACONESCU L., IAMANDESCU I.B., Allergic-type reactions to drugs, In: I.B. Iamandescu (edited by), Psychoneuroallergology (2nd ed.), 2007, Bucureşti, Editura Medicală Amaltea. IAMANDESCU I.B., POPA-VELEA O., Neurotic symptomatology in allergic and in non allergic asthma patients, Allergy, 1995, 50, 26, 310. STAUDER A., KOVACS M., Anxiety symptoms in allergic patients: identification and risk factors, Psychosomatic medicine, 2003, 65, 816–823. ZIFFRA M.S., GOLLAN J.K., Management of the psychologically complicated patient, In: L.C. Grammer & P.A. Greenberger (eds), Patterson’s Allergic Diseases, 7th edition, Lippincott Williams & Wilkins, 2009, p. 662–670. KOVACS M., ARATO M., The prevalence of allergic diseases in affective and anxiety disorders, European Neuropsychopharmacology, 1997, 7, suppl. 2, S273–S274. KOVACS M., STAUDER A., SZEDMAK S. (2003), Severity of allergic complaints: the importance of depressed mood, Journal of Psychosoamtic Research, 54, 549–557. AFFLEK G., APTER A., TENNEN H., REISINE S., BARROWS E. et al., Mood states associated with transitory changes in asthma symptoms and expiratory peak flow, Psychosomatic medicine, 2000, 62, 61–68. YELLOWLEES P.M., KALUCY R.S., Psychobiological aspects of asthma and the consequent research implications, Chest, 1990, 97, 628–634. DIACONESCU L., Implicaţii psihologice ale tratamentului farmacologic, In: Popa-Velea (edited by), Diaconescu, L., Necula Cioca, I., Psihologie medicală, Bucureşti, Editura Universitară “Carol Davila”, 2006. ZIGMOND R., SNAITH S., Hospital Anxiety and Depression Scale. Acta Psychiatr. Scand., 1983, 67:361–370. COHEN S., WILLIAMSON G., Perceived stress in a probability sample of the United States, In: S. Spacapam & S. Oskamp (eds.), The social psychology of health: Claremont Symposium on applied social psychology. Newbury Park, CA: Sage. MILLER L., SMITH A.D.D., Stress Vulnerability Scale, University of California, Berkeley Wellness Letter, August, 1985. KAMEI T., TSUDA T., KITAGAWA S., NAITOH K., NAKASHIMA K., OHHASHI T., Physical stimuli and emotional stress-induced sweat secretions in the human palm and forehead; International Cyber Congress on Analytical BioSciences, 1998, no. 1, JAPON (21/08/1997), vol. 365, no. 1–3 (334 p.) (16 ref.), p. 319–326.

Received September 25, 2010

Protein Aggregation in Inclusion Body Myositis, a Sporadic Form Among Protein Aggregate Myopathies, and in Myofibrillar Myopathies – a Comparative Study ALEXANDRA BASTIAN1, H.H. GOEBEL2 1

“Colentina” University Hospital, Department of Pathology, Bucharest, Romania Johannes Gutenberg University Clinical Hospital, Department of Neuropathology, Mainz ,Germany

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Protein aggregation has been identified in muscle fibres and, thus, in certain neuromuscular disorders. There are certain similarities between IBM and DRM: midlife or late-onset clinical symptoms, apparently of both sporadic and genetic background, morphologically autophagocytosis by vacuole formation, which is frequent in IBM though rare in DRM, and presence of tubulofilamentous aggregates, which is almost regular in IBM but scantily found in DRM as β-amyloid components have been identified as accruing proteins, both in IBM and DRM. Previous studies pointed to the hypothesis that clear morphological borders between the two types of diseases – hereditary inclusion body myopathies/myositis and desmin-related myopathies may not exist. Therefore, we analysed and morphologically characterised the spectrum of proteins accumulating in both types of disorders in order to compare them and more clearly define similarities and dissimilarities between these two different groups of protein aggregate myopathies. Previous studies [7] showed that there is an overlap among some of the proteins accruing in these diseases , but there might also be differences in that a large number of proteins found aggregated in desmin-related myopathies had not yet been described in IBM. The aim of describing the comparative protein profiles is to give more insights into the mechanism of protein aggregation within muscle fibres. Material & Methods. We studied diagnostic muscle biopsies from 10 sIMB patients and 6 MM patients with histological, histochemical, enzyme histochemical, ultrastructural and immunohistochemical techniques using a large number of antibodies. Results. We noticed a partial overlap of protein expression in the two cohorts of patients for sarcomeric, chaperone and mostly for cytoskeletal proteins. In both of the cohorts, the nuclear proteins were absent in the cytoplasmic bodies. A different pattern of immunolabelling was noted for transsarcolemmal proteins, constantly enhanced in the inclusion bodies in MM, but never found in IBM, except for δ-sarcoglycan, dysferlin and caveolin. Conclusions. The partial overlap among some of the proteins accruing in these diseases raise the hypothesis that clear nosological borders between s-IBM and MM may not always exist. There are also dissimilarities in the pattern of protein aggregation, suggesting that other additional factors are involved in the pathogenesis. Keywords: myofibrillar myopathies, desmin-related myopathies (DRM), inclusion body myositis (IBM), rimmed vacuoles.

Sporadic inclusion body myositis (s-IBM) is an inflammatory myopathy, morphologically characterized by varying degrees of inflammatory infiltrates and rimmed vacuoles with or without inclusion bodies made by abnormal aggregates of proteins in muscle fibres. Myofibrillar myopathies (MM)/ desmin-related myopathies (DRM), a subgroup in the family of protein aggregates myopathies, are a clinically and genetically heterogeneous group of muscular diseases marked by abnormal accumulation of proteins in muscle fibres. We compared the spectrum of proteins accumulating in both types of disorders. We studied diagnostic muscle biopsy specimens from ten unrelated patients with s-IBM. In all ROM. J. INTERN. MED., 2010, 48, 4, 377–384

the cases, the diagnosis was established following combined clinical and morphopathological criteria [1–3]. Three patients were women and seven were men. The age of onset varied from 40 to 75 years. Symptoms at the time of diagnosis consisted of slowly progressing proximal and distal muscle weakness and wasting with depressed or absent tendon reflexes especially in the lower limbs. The pattern of muscle weakness was variable, but mostly distal and asymmetric. In all of the cases the skeletal muscle biopsies were characterized by variable numbers of atrophic or normally sized fibers containing one or more rimmed vacuoles, often associated with a nucleus and best seen by modified Gömöri trichrome stain and muscle fibers

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with cytoplasmic inclusions. The vacuolated muscle fibres were ultramorphologically characterised by intracytoplasmic and, though less frequently, also intranuclear, tubulofilaments with diameters of around 16–21 nm which appeared as paired helical filaments, a morphologic hallmark of IBM. Other common findings at electron microscopic level were myelin-like bodies (membranous whorls) of varying size and shape disrupting the myofibrils and aggregates of mitochondria and occasionaly honeycomb structures. A constant feature at light microscopy level were varying degrees of inflammatory infiltrates consisting of large numbers of autoaggressive CD8 + T lymphocytes and CD68 + macrophages, the latter reacting histochemically for acid phosphatase, surrounding and partially invading non-necrotic muscle fibres, while other regions of the muscle fiber appeared intact. Muscle fibers showed upregulation of MHC class I antigen (major histocompatibility complex). Endomysial inflammatory infiltrates, in the form of linear distribution of cells among muscle fibers or as larger collections around muscle fibers, were mostly T lymphocytes around vessels and mainly macrophages near and within muscle fibres. Variation in myofiber diameters, endomysial fibrosis, occasional necrotic and regenerating fibers, angulated atrophic, rarely grouped muscle fibers and ragged red and cytochrome – C-oxidase – negative fibers were further myopathic features. The second cohort of patients with the diagnosis of myofibrillar myopathy (MM) established after examination of the muscle biopsies by light and electron microscopy included six cases. Two of these were brothers, aged 52 and 42 years. The older brother noticed increased muscle weakness first in the left and then in the right leg, followed by marked gait disturbances and distal paresis of peroneal distribution. In the last six months he had also noted weakness in his left hand, and in the past two months weakness in his right hand. At the time of examination he had conspicuous distal muscle atrophy in both legs and arms, asymmetric deep tendon reflexes, decreased knee jerks on the left and absent Achilles tendon reflexes on both sides. He also had cardiac arrythmia with atrial fibrillation and considerably enlarged atria and a history of two myocardial infarcts at the ages 44 and 46 years. His brother, 10 years younger, developed restrictive cardiomyopathy at the age of 20 years, which required cardiac transplantation at the age of 41 years. Their sister and an uncle were said to have had myocardial infarcts.

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We studied biopsied skeletal muscle specimens of the older brother obtained from gastrocnemius and deltoid muscles, as well as from his heart tissue and also from the explanted heart of his younger brother. In the skeletal muscle tissues, the pathological features consisted in variation in fibre size, considerable increase in internally located nuclei and muscle fibres showing irregular densities within the sarcoplasm, in subsarcolemmal or centrally located areas, as eosinophilic masses on hematoxylin-eosin stain and dark-blue on modified Gömöri trichrome stain, often devoid of oxidative and ATPase enzyme activities. Phagocytosis, necrosis and basophilia of muscle fibres were absent, as well as COX-negative fibres and inflammatory infiltrates. One ragged red fibre was noted in the deltoid muscle specimen. There was no upregulation of MHC-I on muscle fibres. Immunohistochemically, desmin and other proteins were focally expressed within numerous muscle fibres. Electron microscopy of deltoid and gastrocnemius muscle tissues revealed granulofilamentous material beneath the sarcolemma as well as among the muscle fibrils. In both muscles autophagic vacuoles contained some pseudomyelin lamellae and debris as well as – deposited as aggregates – tubulofilaments of the helicalfilament type. The brother’s explanted heart muscle showed variation in muscle cell diameters and numerous densities within muscle fibres which, by immunohistochemistry, contained desmin and numerous other proteins. By electron microscopy, these patches within cardiac myocytes were composed of granular and filamentous material or electron dense material, very similar to the granulofilamentous material within skeletal muscle fibres. Molecular analysis based on DNA extracted from nucleated blood cells revealed a heterozygous novel GAG-GAC mutation in exon 3 of the desmin gene at 2q35 chromosome, resulting in amino acid change Glu245Asp in the desmin molecule, confirmed the morphological diagnosis of desminopathy. This case, published by us in 2005 [6], enlarged the spectrum of known mutations in the desmin gene, but also the molecular spectrum of desmin-related myopathies as well as genotype-morphotype correlations. The third case of MM was a 67-year old man with proximal and distal muscle atrophy of the lower limbs, difficulty climbing stairs and getting out a chair for the past few years, and a cardiomyopathy with an ejection fraction reduced at 25%. He was clinically diagnosed with a distal myopathy of the Markesbery-Griggs-Udd type. His five year-

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younger brother had a ten to twelve year history of slowly progressive weakness first in the feet, subsequently involving his distal upper extremities and carried the diagnosis of s-IBM. On the muscle biopsy, he had numerous rimmed vacuoles, but no endomysial inflammation and no invasion of nonnecrotic muscle fibres. The muscle biopsies of our patient, from quadriceps and triceps suralis, showed numerous cytoplasmic inclusion bodies containing desmin and other proteins, necrotic and angular atrophic fibres and many rimmed vacuoles. Patient 4 was a 42 year-old man with a rigidspine syndrome, muscle weakness when climbing stairs or walking long distances, myopathic EMG and elevated CK level. He had a younger brother also affected. The biopsy from biceps brachialis muscle revealed numerous cytoplasmic bodies and autophagic-rimmed vacuoles with tubulofilamentous aggregates. The cytoplasmic bodies stained positive with anti-desmin antibodies and others. The molecular analysis showed no mutations in desmin, αB-Crystallin or Selenoprotein N1 genes. The fifth patient was a woman aged 62 years with weakness in her lower limbs for two years, a myopathic EMG and elevated CK level. Her biopsy muscle specimen from triceps suralis muscle showed atrophic and hypertrophic fibres, increased number of internal nuclei and areas of condensation of the sarcoplasm indicating myofibrillary myopathy, reacting with numerous antibodies. Patient 6 was a 74 year-old woman clinically diagnosed with a proximal myopathy of the lower limbs. Her skeletal muscle biopsy from quadriceps femoris muscle revealed abnormal deposits in the muscle fibres, located mainly subsarcolemmaly, but also in the center of the fibres, as well as lobulated muscle fibres, numerous core-targetoid lesions and rare ragged red fibres. By electron microscopy, she had areas of Z-disk streaming and granulofilamentous material between the myofibrils in her muscle biopsy tissue specimen. RESULTS EXPRESSION OF TRANSSARCOLEMMAL PROTEINS

In eight of the ten patients with s-IBM we found normal subsarcolemmal staining for dystrophin, only two cases showing mild sarcoplasmic increase as granular deposits. In all the cases with MM we found ectopic cytoplasmic expression of dystrophin

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in structurally abnormal fibres, that clearly indicate dystrophin as a useful immunocytochemical marker for abnormal regions in MM. In normal adult muscle, utrophin is located at the neuromuscular synapse and myotendinous junctions, where it participates in post-synaptic membrane maintenance and acetylcholine receptor clustering. In four of our six patients with MM, utrophin was found aggregated within cytoplasmic bodies, as well as along the membrane in some of the regenerated fibres. Only four of our ten s-IBM cases showed areas of increased DRP 2 expression along the sarcolemmal surface, but utrophin was not found aggregated in the cytoplasm of the muscle fibres. We studied the profiles of four members of the sarcoglycans complex, as part of the dystrophin-based membrane cytoskeleton of muscle fibre. Its normal function being the stabilisation of the transmembrane β-dystroglycan protein with dystrophin, we were interested in their pattern in the two types of diseases.We found varriable expression of the four sarcoglycans in the MM cases, more consistent, among them, for δ-sarcoglycan in the sarcoplasmic inclusions and all the cases showed normal sarcolemmal expression. In the s-IBM cohort α, β and γ sarcoglycans were never found accruing in inclusion bodies or elsewhere in the sarcoplasm of vacuolated or normal appearing fibres, but were constantly positive along the sarcolemmal surface. Surprisingly, δ sarcoglycan was expressed not only at the periphery of the fibres, but in seven of the ten cases also in cytoplasmic bodies and at the rim of vacuoles as granular deposits Laminin α 2 (merosin), major component of the myofibre basal lamina, interacts in normal muscle fibre with the plasma membrane and mediates interactions between the basal lamina and the endomysial connective tissue. In our patients with s-IBM, the two isoforms of merosin were never found aggregated elsewhere than in the normal location at the basal lamina. On the contrary, we found abnormal accumulation of merosin within the muscle fibres in five of the six cases of MM. The expression of α and β dystroglycans, that bind dystrophin intracellularly and laminin extracellularly, thus forming a critical link between the extracellular matrix and the cytoskeleton, was found normal in all our studied patients from the sIBM lot. In five of the six cases of MM, abnormal accumulations of α and β dystroglycans were detected in cytoplasmic bodies within muscle fibres.

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Collagen 6, component of the extracellular matrix, with its important role in anchoring basal lamina to the endomysial connective tissue, was normally expressed in all our s-IBM cases, as well as in all cases with MM. In two of the latter group of patients we found several additional foci of immunopositivity in the sarcoplasmic inclusions. Normal expression of β and γ laminins was found in all s-IBM specimens; two of the six patients with MM showed limited areas of abnormal accumulation in the sarcoplasm. The study of dysferlin showed in all cases, from both cohorts, apart from normal immunostain on the plasma membrane, constant increased expression. In muscle fibres of patients with s-IBM, dysferlin was expressed in small and large vacuolated fibres, appearing as granular deposits bordering vacuoles, as diffuse accumulation in the sarcoplasm and even inside the vacuoles. Immunoreactivity of dysferlin was observed as positive subsarcolemmal aggregates and intracytoplasmic inclusions in all MM cases. Caveolin, a major protein of plasmalemmal microdomain caveolae, is a proven intracellular transporter of cholesterol, thus influencing its homeostasis. In IBM muscle fibres, caveolin immunoreactive inclusions were observed in vacuolated fibres as plaque-like deposits or diffuse accumulation, but were also noticed in regenerated and necrotic fibres. In the muscle fibres of the MM cohort, caveolin showed focal increased immunostain under the sarcolemma and in centrally located areas in all the cases. The expression of n NOS (neural nitric oxide synthase) in the muscle fibres was found increased in all patients from MM cohort as part of the cytoplasmic bodies and in eight of ten patients with IBM as granular or compact foci of immunopositivity in the cytoplasm of vacuolated muscle fibres, more pronounced at the rim of the vacuoles. EXPRESSION OF NUCLEAR PROTEINS

Emerin is a nuclear membrane protein which is missing or defective in Emery-Dreifuss muscular dystrophy (EDMD). It is one member of a family of lamina-associated proteins which includes LAP1, LAP2 and lamin B receptor (LBR). Lamins are nuclear intermediate filaments, which form a network-like structure underneath the nuclear membrane, the nuclear lamina, as well as complexes in the nuclear interior. Lamins associate with

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numerous proteins in the inner nuclear membrane, including emerin, and lamina-associated polypeptides. In our study, nuclear membrane proteins were never found expressed in muscle fibres of patients with IBM in other location than perinuclear. Only one patient of the MM cohort had additional focal increased emerin expression in cytoplasmic deposits of his cardiomyocytes, but these were not stained with antibodies against lamins A/C. EXPRESSION OF THE SARCOMERIC PROTEINS

Actin, the major component of thin myofilaments, has been previously found in lesions of desminopathies by some investigators [14]. In our study, actin was found accumulated in cytoplasmic bodies in all cases with MM and in nine of ten cases with s-IBM as diffuse accumulation in the cytoplasm, but more consistently expressed at the border areas of the vacuoles. α actinin is a physiological protein component of the Z-bands, cross-linking actin filaments. Electron microscopic findings showed excessive involvement of Z bands and so of α actinin in the hyaline structures of desminopathies, but in another extensive study this was only occasionally expressed by immunohistochemistry [14]. All our MM cases showed consistent expression of α actinin in the sarcoplasmic inclusions, simmilar to the positive immunostaining found in all ten IBM cases in vacuolated muscle fibres, mostly at the periphery of vacuoles and as patchy cytoplasmic deposits. In our comparative study we were especially interested in the expression of myotilin as a recently discovered Z-disk-associated key protein localized along the sarcolemmal membrane and within I bands that control sarcomere assembly, cross-links actin filaments and binds to α actinin and γ-filamin. Recently, mutations in myotilin were found in myofibrillar myopathy [4]. An extensive study on 63 patients of MM described, for the first time in the literature, increased myotilin expression in 90% of the fibres that were abnormal in trichrome-stained sections and suggested that myotilin is the most reliable immunocytochemical marker for abnormal fibre regions in MM [4]. In fact, all our muscle biopsy fibres from MM patients expressed myotilin in the sarcoplasmic inclusions, as all the cases with IBM had increased myotilin immunostain in the cytoplasm of vacuolated fibres in the vicinity of vacuoles and even in normal appearing fibres as irregular areas of deposition.

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Protein aggregation in inclusion body myositis EXPRESSION OF CYTOSKELETAL PROTEINS

Desmin is located in mature skeletal muscle between the subsarcolemmal region and the nuclear membrane, associated with lamin B and around the myofibrillar Z discs, encircling and interconnecting myofibrils at this level, thus aligning myofibrils and linking them to nuclei, to the plasma membrane, especially in the region of the costameres and to cytoplasmic organelles such as mitochondria. In the heart, desmin is increased in Purkinje fibres, as a major component, and at the level of intercalated discs. In our immunohistochemical study we found in each muscle specimen of patients with MM a markedly increased expression of desmin in intrasarcoplasmic deposits, ultrastructurally with a granulofilamentous aspect. The accumulation of desmin in IBM is still controversial in the literature; desmin has been shown to accumulate abnormally, among other proteins [7]. Other study found normal expression of desmin in hypertrophic and normal sized muscle fibres in all patient biopsies [16]. Our study showed in all patients strong expression of desmin, more obvious at the rim of vacuoles, as diffuse or patchy accumulation in the cytoplasm of vacuolated and even normal appearing muscle fibres, as well as in small regenerated fibres. Plectin is a highly conserved and ubiquitously expressed intermediate filament – associated protein concentrated at sites of mechanical stress, such as the hemidesmosomes in skin, the Z disk of skeletal muscle fibers and the intercalated disks in cardiac muscle cells. Plectin is also normally associated with the sarcolemma, the postsynaptic membrane, the nuclear membrane, and the intermyofibrillar network of skeletal muscle. Plectin is known to associate with vimentin, integrin, desmin, lamin B, myosin II, vimentin and actin. Plectin is also associated with mitochondria and is important in the localization of intracytoplasmic organelles. We analysed the expression of plectin in three MM cases and we found in all of them increased immunostain in abnormal fibres. All the patients with sIBM had conspicuous plectin deposits in inclusion bodies of vacuolated muscle fibres, as well as in focal accumulations in nonvacuolated fibres. Vimentin, a member of the intermediate filament family, is absent in healthy mature muscle fibres, being expressed only in developing muscle, colocalized with desmin, and in regenerating muscle fibres. We found increased expression of vimentin in five of the six MM cases in the

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sarcoplasmic inclusions as well as in regenerated fibres. Nine of ten patients with IBM also showed increased expression of vimentin in small and degenerated fibres, but also in vacuolated fibres. EXPRESSION OF CHAPERONE PROTEINS

αB-crystallin is a member of the small heat shock protein family , which exerts a role as molecular chaperones by binding unfolded or denatured proteins, suppressing irreversible protein aggregation and consecutive cell damage, their essential role in neuromuscular disorders being corroborated by the observation that a mutation of the human αB-crystallin gene causes an autosomal dominant myofibrillar myopathy morphologically characterized by αB-crystallin and desmin accumulation and granulofilamentous material by electron microscopy. We found strong expression of αBcrystallin in sarcoplasmic inclusions in all MM cases, colocalized with desmin, as well as in all IBM cases, in the vacuolated muscle fibres at the border of vacuoles and in inclusion bodies, but also in normal appearing fibres, as previously suggested. The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. Abnormalities in ubiquitinmediated processes have been shown to cause pathological conditions. In our study we observed accumulation of ubiquitin in all cases with MM, two of them showing strong aggregation in the cytoplasmic bodies, in the other the increase was only mild. Muscle biopsy specimens from IBM patients also revealed enhanced expression of ubiquitin in vacuolated fibres and, focally, also in nonvacuolated ones. These findings indicate that muscle fibres contained undesirable protein material targeted for non-lysosomal degradation, but the mild ubiquitin positivity in some of the cases suggest operation of an alternative pathway of protein degradation. Heat shock proteins play an important role in protein-protein interactions, including folding and assisting in establishing proper protein conformation, and prevention of inappropriate protein aggregation. Heat shock proteins are synthesized under different stress conditions and act as molecular chaperones for protein molecules. We found increased expression of HSP 72/73 in all our MM cases as well as in all IBM muscle biopsy specimens. In the first group, HSP 72/73 was mainly found at

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subsarcolemmal and central sites in muscle fibres; in the latter group, HSP 72/73 was enhanced in degenerated and regenerated muscle fibres and in inflammatory infiltrates invading muscle fibres, as well as in vacuolated fibres, more conspicuous around vacuoles. DISCUSSION

Our study shows, as previously expected, some similarities and partial overlaps in protein expression between MM and IBM, but also some dissimilarities. The mechanisms responsible for formation of the multiprotein inclusions in IBM muscle are not understood, although the histopathology of s-IBM has been well described, but it seems that unfolding and misfolding of proteins probably play a role, as well as the cellular aging that promotes accumulation of abnormal proteins and slows degradation of normal and abnormal proteins. Only a minority of MM cases were, up to now, shown to be caused by mutations in desmin, alpha B-crystallin, myotilin and selenoprotein N1 genes, suggesting that the majority of them are due to yet unidentified gene defects or are non-genetic at all, also requiring further mutational analyses of other genes such as for paranemin, synemin and syncoilin. On the contrary, IBM is a sporadic disease. Both disorders share accumulation of different types of proteins, indicating a partially common pathogenesis. Accumulation of extrasarcomeric cytoskeletal proteins was a common feature in both diseases. We found increased expression of desmin, alpha B crystallin and plectin in all the muscle biopsies, showing a marked disturbance of filamentous intermyofibrillar cytoskeleton, with its important role in structural and functional maintenance of striated muscle fibres in response to stress. Thus, we amplified the findings of other investigators [9] who described desmin and alpha B crystallin accumulation in both MM/DRM and IBM. We found accretion of plectin, with its essential role in the proper spacing, stabilization and subcellular attachement of intermediate filaments. We also confirmed the occurrence of increased immunomarcation of alpha B crystallin in abnormal vacuolated muscle fibres, as well as in the normal appearing fibres in IBM, as described by Banwell et al., [9]. Heat shock protein 72/73, a “stress marker”, was also found overexpressed in both disorders.

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We observed a similar expression of sarcomeric proteins in both diseases, as actin and alpha actinin aggregates. Concerning the expression of myotilin, a recently discovered protein that, when mutated, causes MM, we found in all the cases increased accumulation in the cytoplasm of vacuolated fibres in IBM patients, as well as in the sarcoplasmic inclusions in our DRM/MM specimens, thus supporting the suggestion that myotilin is the most reliable immunocytochemical marker for abnormal regions in the muscle fibres in MM [4]. Our study shows additional abnormal myotilin immunomarcation in the vacuolated muscle fibres in IBM. Only normal immunolabelling of nuclear membrane proteins was encountered in both diseases, but there are data that these proteins may be found aggregated in sarcoplasmic bodies in MM. Further studies may eventually correlate these accretions with a particular genetic profile. The pattern of transsarcolemmal protein immunolabelling showed, in our studied cases, consistent dissimilarities. We found prominent dystrophin colocalisation with desmin aggregates in MM cases, our IBM cases always showing a normal immunolabelling at the sarcolemmal level. The same pattern was found for alpha and beta dystroglycans. The sarcoglycans alpha, beta and gamma were always normally expressed in the muscle fibres of IBM patients, but occasionally aggregated in the cytoplasmic bodies in MM. Delta sarcoglycan was markedly increased in MM specimens, and occasionally also in IBM specimens. Merosin showed normal expression in IBM, but was frequently found coaggregated with desmin in MM cases, as were beta and gamma laminin. Dysferlin and caveolin were overexpressed in both MM and IBM cases. These observations point to a more severe impairment of the sarcolemmal architecture in MM than in IBM. CONCLUSIONS

1. There is considerable aggregation of proteins in both IBM and DRM. 2. A large number of same proteins accrue in both groups of conditions. 3. In each of the two groups, however, there are proteins aggregated only in one group, not in the other. 4. In both groups of disorders, impairment in extralysosomal protein degradation is an important pathogenetic principle, high-

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lighted by the common involvement of the no such mutant proteins have been identified in h-IBM. chaperone protein α-B crystallin and by While s-IBM is considered a sporadic and the involvement in both groups of disorders acquired condition, among patients with DRM, a of proteins engaged in the ubiquitin pronon-hereditary form has also been suggested but, teasomal degradative pathway of proteins perhaps, will be more difficult to prove as a fair While DRM consist of both desminopathies, number of genes in DRM may indicate that there i.e. desmin-accumulating myopathies, and other are still unidentified genes involved as well and, forms related to mutations in other genes such as thereby, recognizing true acquired DRM most selenoprotein N1, and myotilin, suggesting that exclusively by exclusion of any hereditary form. mutant proteins form part of the protein aggregates, __________________________________________________________________ Miozita cu incluzii- forma sporadica(s-IBM) este o miopatie inflamatorie caracterizată morfologic prin variate grade de infiltrate inflamatorii si prezenţa de vacuole tivite cu sau fără corpi de incluzie compuşi din agregate anormale de proteine în fibrele musculare. Miopatiile miofibrilare/miopatiile legate de desmină, un subgroup în familia miopatiilor cu aggregate proteice, reprezintă un grup de afecţiuni musculare clinic şi genetic heterogene caracterizate prin prezenţa de acumulări proteice anormale în fibrele musculare. Am comparat spectrul proteinelor acumulate în ambele tipuri de afecţiuni. __________________________________________________________________ Acknowledgment: A.B. was supported by a fellowship of the European Neurological Society (ENS). Corresponding author: Alexandra Bastian “Colentina” Clinical Hospital, 19, Şos. Ştefan cel Mare, Bucharest E-mail: [email protected]

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NEW BOOKS

Dilemmas and Certainties in a Cardiology Textbook Although time passing, technological developments, the apparent exit from the Gutenberg galaxy and entry in an area of visual dominance could have pushed away the written pages – medical literature continues to occupy a central place on the scale of values of the medical world. Through their own education – doctors perceive the textbooks as possible keys for their professional existence. In this context the newly published Romanian monograph “Textbook of Cardiology” edited by Carmen Ginghină (Romanian Academy Publishing House) poses a particular interest. The book brings together elements of classical structure with a modern approach. In a classic, almost ultra-conservative approach, the anatomy and physiology of the cardio-vascular system is included together with the presentation of the detailed clinical evaluation of the cardiac patient, the paraclinical evaluation, the presentation of cardiovascular diseases and the description of cardiac involvement in other pathological conditions. Classic is also the constant findings of definitions (detailed) and the presence of short “dictionary of terms”at the beginning of the chapters with subjects more distant to the clinical cardiologist. The modern view is represented by the addopted approach (e.g., anatomical images are shown along with images obtained through echocardiography, computed tomography, magnetic resonance imaging, coronary angiography), the presence of chapters focusing on genetics (for cardiologists!) and epidemiology of cardiovascular diseases (with data from Romania), the attention towards cardiac biomarkers, the chapter dedicated to the choice of method of evaluation. Modern is also the continuous reference to the guidelines – predominantly the European ones and frequently the American ones (as unique recent guidelines for some subjects or difference of opinions from the European ones) or even national guidelines (Canadian, British) when they are of particular interest. The most consistent part of the book includes the description of the main cardiovascular diseases; it includes up to date information and reference data (well-known monographs, articles) filtered through the experience of the authors. This section contains 51 case reports (short ones – only one page) with comments that complete the topic in an attractive manner. All cases are “experienced” by the authors and the atmosphere of “real life” is a plus for the publication. It is a book full of life which includes dilemmas and certainties. Dilemmas are inherent to the trends of modern cardiology to promote guided, preformed medical decisions. The Greek term dilemma (the main source for the current international word) is composed of di –“two or twice” and lemma – “premise”. However, in cardiology – and the book illustrates this – we are facing more than two possibilities. Dilemmas arise in the book not only as a matter of logic and reasoning but also as an act of reflection in particular cases. Certainties derive from the immutable truths of clinical cardiology tested during time, from the certitude of modern laboratory data and the confidence in medical common sense. The monograph is clearly written, deliberately ordered and the explanations are often accompanied by drawings and diagrams that facilitate the understanding. The “visible” authors of this book are mainly cardiologists from the Cardiology Clinic of the Institute for Emergency in Cardiovascular Disorders “Prof. C.C. Iliescu”, most of them with academic background, and in addition a distinguished anatomist and experts in medical imaging. Other cardiologists, radiologists, surgeons, pathologists are “non-visible” contributors on the list of authors, but cited in the reference list of each chapter . The book continues the tradition of Cardiology Clinic ASCAR/FUNDENI for which the publication of textbooks and manuals appears a “modus vivendi” promoted over the time by all the mentors. In fact Professor Carmen Ginghină says in the preface of the book: “We have followed the message of our mentors: spoken language can be brilliant but short-lived in the communication of scientific ideas, while the written page implies performance and requires rigour and depth of understanding from both author and reader”. Prof. dr. Tiberiu Nanea ROM. J. INTERN. MED., 2010, 48, 4, 385