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ROMANIAN JOURNAL OF INTERNAL MEDICINE Volume 49

No. 1, 2011

CONTENTS REVIEWS ADRIANA NICOLAU, R. TĂNĂSESCU, EUGENIA BĂLĂNESCU, P. BĂLĂNESCU, RUXANDRA PĂTRAŞCU, C. TĂNĂSESCU, Hepatitis C virus – mixed cryoglobulinemia – lymphoma relationship............................................... GH. GLUHOVSCHI, M. MODALCA, F. MĂRGINEANU, SILVIA VELCIOV, CRISTINA GLUHOVSCHI, F. BOB, LIGIA PETRICA, GH. BOZDOG, V. TRANDAFIRESCU, F. GĂDĂLEAN, Epidemiological data regarding Balkan endemic nephropathy in relationship with the Pliocene coal etiological hypothesis ......................................................... MIHAELA GĂMAN, ANA-MARIA VLĂDĂREANU, SÎNZIANA RADESI, The implications of revised WHO classification (2008) of chronic myeloid neoplasms .........................................................................................................

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ORIGINAL ARTICLES R. ROŞU, L. MUREŞAN, M. ANDRONACHE, DANA POP, C. POP, MARIA PUŞCHIŢĂ, ADINA MĂLAI, G. GUŞEŢU, D. ZDRENGHEA, Correlations between the surface ECG and the intracavitary electrocardiogram in typical atrial flutter................................................................................................................................................................................. CLAUDIA BUZAŞ, OLIMPIA CHIRA, TEODORA MOCAN, MONICA ACALOVSCHI, Comparative study of gallbladder motility in patients with chronic HCV hepatitits and with HCV cirrhosis ........................................................................ GIANINA MICU, FLORICA STĂNICEANU, SABINA ZURAC, ALEXANDRA BASTIAN, ELIZA GRĂMADĂ, CRISTIANA POPP, LUCIANA NICHITA, R. ANDREI, C. SOCOLIUC, ANCA ZAHARIA, C. LĂZĂROIU, R. MATEESCU, MĂDĂLINA MARINESCU, R. VOIOSU, The influence of Helicobacter pylori presence on the immunophenotype of inflammatory infiltrate in gastric diseases ..................................................................................... INIMIOARA MIHAELA COJOCARU, M. COJOCARU, GABRIELA MIU, VIOLETA ŞAPIRA, Study of interleukin-6 production in Alzheimer’s disease .................................................................................................................................... SILVIA VELCIOV, GH. GLUHOVSCHI, V. TRANDAFIRESCU, LIGIA PETRICA, GH. BOZDOG, CRISTINA GLUHOVSCHI, F. BOB, F. GĂDĂLEAN, M. BOBU, Specifics of the renal abscess in nephrology: observations of a clinic from a County Hospital in Western Romania..........................................................................................................

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CASE REPORTS VICTORIA ARAMĂ, DANIELA MUNTEANU, IOANA OLARU, MIHAELA RĂDULESCU, RALUCA MIHĂILESCU, ANA MARIA VLĂDĂREANU, MINODORA ONISÂI, ANAMARIA VINTILESCU, CAMELIA DOBRA, M. OLARIU, S.Ş. ARAMĂ, Reactivation of HBV infection in low grade lymphoma patient .........................................

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REVIEWS AND SYNTHESIS H. BĂLAN, ELENA POPESCU, GABRIELA ANGELESCU, Comparing different treatment schedules of Zomen (Zofenopril) ..... ANDREEA-CORINA ROPOTEANU, The level of emotional intelligence for patients with bronchial asthma and a group psychotherapy plan in 7 steps............................................................................................................................................

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LETTER TO THE EDITOR ANA MARIA VLĂDĂREANU, VERONICA VASILACHE, H. BUMBEA, MINODORA ONISÂI, Platelet dysfunction in acute leukemias and myelodysplastic syndromes.............................................................................................................. ROM. J. INTERN. MED., 2011, 49, 1, 1–96

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Hepatitis C Virus – Mixed Cryoglobulinemia – Lymphoma Relationship

ADRIANA NICOLAU1, R. TĂNĂSESCU²,3, EUGENIA BĂLĂNESCU1, P. BĂLĂNESCU1, RUXANDRA PĂTRAŞCU1,3, C. TĂNĂSESCU1,3 1

Internal Medicine, “Colentina” Clinical Hospital, Bucharest, RO 2 Neurology, “Colentina” Clinical Hospital, Bucharest, RO 3 University of Medicine and Pharmacy “Carol Davila”, Bucharest, RO

HCV (hepatitis C virus) chronic hepatitis has become one the most expensive diseases for public health systems all over the world in the past 10–20 years, a real epidemic, the second most frequent, after hepatitis B virus infection. Due to the complex manifestations, one may consider HCV infection as a “systemic” disease. Mixed cryoglobulinemia (MC) is the most common extrahepatic manifestation of HCV infection, but cryoglobulinemic vasculitis (CV) is considered to be relatively sparse although prevalence studies are needed. Presence of serum cryoglobulins is essential for MC diagnosis, but serum levels do not correlate with the disease activity or prognosis. MC can be defined as a B lymphocyte proliferation disease being characterized by polyclonal activation and antibody synthesis. Evolution to lymphoma should be considered continuous but also other infectious, environmental or genetic factors could be involved. The t (14.18) translocation and Bcl-2 activation in B lymphocytes, B cell-activating factor (BAFF), E2-CD81 interaction, immunoregulatory T CD4+CD25high+ lymphocytes and type III IFNs might play an important role in MC and lymphoma evolution in HCV patients. Key words: hepatitis C virus, mixed cryoglobulins, lymphoma, t (14.18), Bcl-2, BAFF, E2CD8, Tregs, type III IFNs.

HCV (hepatitis C virus) chronic hepatitis has become one of the most expensive diseases for public health systems all over the world, in the past 10– 20 years. We can say there is a real epidemic, with a prevalence of over 170 million, an incidence of 3.3% per year and responsible for over 12000 deaths/ year. Nearly 170 million people are already infected, meaning about 3% of world population, a number 5 times higher compared to HIV [1][2]. It was the major cause of posttransfusional hepatitis after 1990. When serological screening was introduced the incidence decreased significantly but still remains a problem for intravenous drug users. HCV infection is associated with autoimmune extrahepatic manifestations such as cryoglobulinemic vasculitis, nephritis, arthritis due to immunological imbalances that HCV can induce. A study group reported that 74% of patients with chronic hepatitis C had at least one extrahepatic manifestation, the most common being mixed cryoglobulinemia (MC– 40%) followed by arthritis or arthralgia (23%), paresthesia (17%), myalgia (15 %), pruritus (15%) and sicca syndrome (11%). Due to the complex manifestations, one may consider HCV infection as a “systemic” disease [3][4]. ROM. J. INTERN. MED., 2011, 49, 1, 3–10

MC is the most common extrahepatic manifestation of HCV. Recently it has been shown that most of the MC patients had also HCV infection. This correlation was found between 30 and 96% of cases, depending on the geographic area [3][5][6]. Some authors consider today that HCV infection can be excluded in MC patients only after the virus is not detected in the cryoprecipitate and/or tissue samples (skin, peripheral nerves, kidneys, lymphocytes). Its heterogenic composition can be assessed by cryoprecipitate analysis, which includes immunoelectrophoresis or immunofixation, or even more sensitive methods such as immunoblotting and twodimensional polyacrylamide gel electrophoresis. Molecular biology techniques (including VHCRNA via in-situ hybridization) and immunohistochemistry showed that HCV antigens are directly involved in cryoglobulinemic vasculitis (mediated by immune complexes) and its manifestations [6–9]. Thus, anti HCV antibodies and HCV-RNA are frequently detected in cryoprecipitate having concentrations of 30 to 1000 times higher than the serum levels, where cryoglobulins may remain undetected. So “essential cryoglobulinemia”term is used to a lesser extent and only after absence of immune and

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HCV-RNA detection in the cryoprecipitate [3][10]. Also, the fact that viral particles were detected by in-situ hybridization from nerve and muscle samples

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from HCV infected patients with peripheral neuropathy but without MC, raised the question if the virus has a direct cytopathic action [11][12].

Fig. 1. Etiopathogenesis of HCV syndrome, adapted with permission after Ferri et al., B-cells and mixed cryoglobulinemia Autoimmunity Reviews 7 (2007) 114–120. HCV AND CRYOGLOBULINS

Cryoglobulins are serum immunoglobulins that precipitate at temperatures below 37ºC and redissolve when the temperature rises. Cryoglobulins can be associated with hematological, infectious or rheumatologic diseases. The immunochemical composition divides cryoglobulins in three groups, all of them could be HCV associated: • type I (25%) monoclonal immunoglobulins (IgM more frequently) mainly associated with hematological malignancies (lymphoproliferative disorders, plasmocytoma, Waldenstrom macroglobulinemia, multiple myeloma, monoclonal gammopathy with undetermined significance). They are frequently asymptomatic or associated with clinical symptoms derived from rheological

changes (acrocyanosis, Raynaud syndrome, gangrene). • type II (25%) and III (50%) mixed cryoglobulinemia (MC)-usually polyclonal IgG with monoclonal IgM (type II) or polyclonal IgG with polyclonal IgM (type III). Monoclonal IgM has rheumatoid factor activity interacting with both F(ab2)2 IgG fragment or with Fc IgG fragment thus forming immune complexes. • type II/III (unknown prevalence) monoclonal or microheterogeneous IgM, identified by more specific and sensitive methods (immunoblotting, two-dimensional polyacrylamide gel electrophoresis).

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• some authors claim a continuous transition from pure polyclonal forms to some partial monoclonal forms due to a continuous clonal selection [13–15]. Cryoglobulins can change their immunochemical profile during chronic HCV infection. Type II MC is more stable in comparison to type III or type II/III. In some cases cryoglobulinemia can be asymptomatic or may form immune complexes that are deposited and can activate the complement cascade (pathogenic mechanism for cryoglobulinemic vasculitis-CV). According to Chapel Hill Consensus Conference classification CV affects small and medium size vessels by deposition of immune complexes with complement activation [6][7][16]. Another argument for a causal relationship between HCV-MC is the fact that HCV-MC prevalence depends on the dominant genotype and HCV incidence in different populations, and has the same evolutionary trends in a specific population. For example the prevalence of HCVCM increases in southern Europe, Africa and Asia, where the circulating genotypes and the HCV prevalence is growing but decreases in Italy and some Mediterranean countries, where significant reduction of HCV pool in the population was achieved by active detection and preventive measures [17]. Cryoglobulins are detected in 29–45% in HCV patients, type II being most frequent. There are studies showing higher prevalence in patients of certain ethnic group or from a particular geographic area (for example higher prevalence in southern Europe compared with northern Europe or North America [18–23]. HCV-CM prevalence is higher with a particular genotype for example, Japanese genotype 1b when compared to Egyptian’s genotype 4 [24]. HCV treatment improves CV, suggesting a cause-effect relationship between HCV and MC [25–27]. CV represents the most frequent extrahepatic manifestation. It is characterized by the difficulty of the therapeutic approach coming from the absence of a standardized protocol that is implemented for this specific group of patients. The disease is considered to be relatively sparse, but the prevalence studies are not confident due to the clinical polymorphism and the dispersed addressability to different medical specialties [4][6][10][22][28][29]. Increase of HCV prevalence in the world will determine an increase in the prevalence of secondary MC, particularly in undeveloped countries with high HCV prevalence [4]. Serum cryoglobulins are

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essential for MC diagnosis but serum levels do not correlate with the disease activity or prognosis [6]. Sansonno et al. determined intralymphocytic viral load in patients with HCV infection with or without MC. Intracellular viral load was higher in patients with MC. These data explain the associated low viral loads in CV [25]. An abruptly cryoglobulin level decline and their peripheral disappearance (sometimes associated with higher C4 serum levels) may be associated with incipient signs proliferation malignant B [30]. HCV, MC AND B CELLS

There is still debate whether MC is the consequence of the concomitant intervention of viral, genetic and environmental factors. HCV is hepatotropic and also lymphotropic, suggested by the active viral replication in marrow and peripheral lymphocytes [31][32]. Moreover, t (14.18) translocation and Bcl-2 activation (anti apoptotic molecule) in B lymphocytes are observed in HCV infected patients (for instance t (14.18) approximately 37% –particularly in MC type II patients). They seem to be responsible for CD5+ B cell clonal expansion in the blood, liver, blood and marrow, for the increased polyclonal IgM production (with rheumatoid factor activity), for autoantibodies synthesis – including here cryoglobulins – and for lymphoproliferation [5][27][33][34]. CD5+ B cells oligoclonal prolixferation seems to be the most important characteristic of MC. Actually MC can be defined as a B lymphocyte proliferation disease being characterized by polyclonal activation and antibody synthesis (Fig. 2). B cell-activating factor (BAFF) is a recently discovered TNF- α member playing a key role in B lymphocyte survival [35]. BAFF changes have been associated with autoimmune diseases such as Sjogren syndrome, systemic lupus erythematosus and rheumatoid arthritis. Recent studies showed higher BAFF serum levels in patients with chronic VHC infection with or without MC [36–38]. Different BAFF mutations were described (– 871C/T ) and are associated with monocyte BAFF mRNA increase in rheumatoid arthritis and lymphoproliferative diseases [39][40]. Giani et al. demonstrated that this BAFF promoter mutation could play a pathogenic role in MC by activating the immune system and promoting lymphoproliferation. In this case, along with other gene mutations, one may speak about a specific cryoglobulinemic genotype [41].

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Fig. 2. B cell lymphoma evolution during HCV chronic infection, adapted after Ferri C. et al., B-cells and mixed cryoglobulinemia. Autoimmunity Reviews 7 (2007) 114–120 (abbreviations: MLDUS – Monoclonal Lymphoproliferative Disorders of Undetermined Significance, B – B lymphocyte).

E2 antigen binding on CD81 expressed by B lymphocytes could represent the chronic stimulus for the continuous, polyclonal activation thus increasing CD5+ lymphocyte number and peripheral autoantibody production in HCV infection. Evolution to lymphoma should be considered continuous but also other infectious, environmental or genetic factors could be implicated. For instance, Charles et al. described that MC patients with HCV infection also had CD27+ CD21lowk-IgM+IgDlow lymphocyte population expansion [42]. Vallat et al. also identified clone B lymphocyte expansion in chronic HCV patients blood and liver, positively correlated with the age of patients, duration of the HCV infection, serum cryoglobulins levels, symptoms and clinical severity. A significant percentage (25%) developed neoplasia [27]. Kronenberger et al. studied CD81 expression variations during HCV infection treatment. Flow-cytometry analysis of CD81 and CD8+, CD4+, CD19+and CD56+ during interferon and ribavirine therapy revealed T CD8+ lymphocytes downregulation , important NK CD56+ cells downregulation, transient B CD5+ lymphocyte upregulation and weak and delayed T CD4+ lymphocyte downregulation. No correlation between viral load and CD81 expression was determined. After the treatment, CD81+CD8+T lymphocytes remained decreased but in non-

responders the number started to increase along with CD56+ NK cells. It was suggested that CD81 variations during antiviral treatment are interferon related and do not correlate with HCV RNA.The mechanism is still unclear, and variations in lymphocyte subclasses are described. CD81 decrease after interferon treatment could induce a decrease in the lymphocyte HCV replication and thus of the immune associated phenomena [43]. IL-6 and IL-8. HCV B cell activation can also be achieved by means of other than E2-CD81 interaction. For example, HCV core protein can stimulate IL-6 and IL-8 synthesis by interaction of a specific NS3 region with TLR2. Serum IL-6 levels were higher in chronic HCV infected patients compared to healthy control, became normal after infection eradication by antiviral treatment. Antonelli et al. compared IL-6 and TNF-α levels in patients with and without MC. IL-6 and TNF-α serum levels were higher in MC patients when compared with patients without MC and negative controls [44]. These data suggest that IL6 reflects the HCV immune stimulation. IL-6 is a strong stimulus for B lymphocyte maturation and development. Significantly elevated IL-6 levels were found in patients with HCV and MC when compared with the patients without MC stressing the importance of TLR2 in the HCV related lymphoproliferative

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manifestations. TLR2 implication in other lymphoproliferative disorders has already been studied, for example in MALT after chronic Helicobacter pylori infection [45]. These data may reveal new therapeutic targets in MC and nonHodgkin lymphoma in HCV patients. T regulatory lymphocytes(Treg). MC development in chronic HCV infected patients seems to be due to some host particularity rather than aminoacid changes in viral particles (like the first hypervariable sequence (HVR1)) [46]. The fact that T lymphocytes are present in the inflammatory infiltrate, associated with antibody and autoantibody synthesis and some HLA specific genotypes in CV asociated with CVH, suggest that autoimmune phenomena are implicated in pathogenesis of CV[47, 48]. Immunoregulatory T CD4+CD25high+ lymphocytes play a key role in the physiologic control over autoimmune phenomena. Treg’s deficient mice infused with T lymphocytes developed autoimmunity [49], and Treg infusion in these mice determined inhibition of these phenomena [50]. Some FoxP3 (specific marker of natural and adaptive/ induced Treg) mutations could induce severe and fatal autoimmune disease due to its associated Treg deficiency [51]. Researchers focused upon their implication in diabetes mellitus, systemic lupus erythematosus, rheumatoid arthritis, asthma, inflammatory bowel disease, multiple sclerosis, hoping that cellular therapies using Foxp3 positive cells may, one day, help overcome these diseases [52–56]. Programmed Death 1, or PD-1, is a Type I membrane protein, a member of the extended CD28/CTLA-4 family of T cell regulators [57]. Many studies indicate that PD-1 and its ligands negatively regulate immune responses. First, PD-1 knockout mice develop lupus-like glomerulonephritis and dilated cardiomyopathy on the C57BL/6 and BALB/c backgrounds, respectively [58]. In vitro, treatment of anti-CD3 stimulated T cells with PD-L1-Ig results in reduced T cell proliferation and IFN-γ secretion [59]. Reduced T cell proliferation correlated with attenuated IL-2 secretion, which can be rescued by addition of cross-linking antiCD28 antibodies or exogenous IL-2. Together, these data suggest that PD-1 negatively regulates T cell responses. Blocking PD-1 in HCV lymphoma patients determine a decrease in the inhibitory Treg activity, suppressing T cell responses, thus PD-1 seems to play an important role in Treg function. Future studies are necessary to evaluate its implication in HCV lymphoma [60]. Treg depletion in symptomatic CV patients seems to play an important role in CV HCV associated pathogenesis. The

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number of Treg cells is severely decreased before the interferon therapy. Interestingly there was no difference in the peripheral Treg number in asymptomatic MC patients when compared to healthy controls [61]. Responder patients have a significant increase in peripheral Treg number compared to the non-responders where there were no changes during interferon therapy. This correlation suggests an important role of Treg depletion in CV pathogenesis. Tregs play a dual role, on the one hand, they limit autoimmune phenomena and, on the other hand, they decrease viral clearance [61][62]. There is also the possibility that Treg cells are recruited at the inflammation sites determining peripheral depletion but a recent study in rheumatoid arthritis showed that Treg accumulation in the synovial fluid does not associate with peripheral depletion. Future long prospective studies should focus upon the triggers of Treg deficiency in CV. Treg depletion can promote T CD4+ stimulatory function on autoantibody synthesis. In vitro studies showed that Th2 lymphocytes from MC patients are oligoclonally expansioned upon IL-2 stimulation, thus suggesting a larger antigen activated Th2 pool in these patients [61]. Additionally, hypergammaglobulinemia and autoantibody secretion in mice is the result of virus specific CD4+ T cells and HCV infected B lymphocyte interaction [63]. IL-29 (IFN-λ1), Il28A (IFN-λ2) si IL28B (IFN-λ3), type III IFN or IFN lambda-members of a newly discovered class II cytokine family, induced by class I IFN and by viral infection, have a demonstrated antiviral action, similar with type I IFN family [64–67]. Between IL-28A and IL-29 there is a 81% structural similarity. IL-28 and IL29 receptors have 2 polypeptide chains- one common chain present in most class II cytokines receptors (IL-10Rβ) and one specific orphan chain, IL-28Rα [66]. Although this receptor is widely expressed (hepatocytes, pneumocytes, myocardial cells) it is less expressed in hematopoietic cells thus future IFNλ therapies would have less hematologic side effects. Type II IFN receptor is mainly expressed by B lymphocytes. T lymphocytes, NK cells and other myeloid derived cells (monocytes, neutrophils) express only IFNAR2 receptor for type I IFN. Therefore, IL-29 induces STAT-1 phosphorylation in B lymphocytes but not in T and NK cells hence IFNλ therapy would have less systemic immune effects compared to type I IFN therapy [68]. The serological marker for IL-29 activation seems to be represented by β2-microglobulin (B2M), an extracellular component of

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class I major histocompatibility complex (MHC I). MHC I presents intracellular antigens to T CD8+ lymphocytes, its expression being enhanced by type I IFN with serum β2M levels increase. Serum β 2M levels increase also via IL-29 stimulation in a dose-dependent manner [69].Recent studies focused upon a point IL-28 mutation – rs minor 8099917 allele – which is associated with non-responsive therapeutic behavior especially in genotype 1 or 4 HCV chronic infection. Future studies should focus upon type III IFN and autoimmune phenomena in HCV chronic infection [65, 67], and whether their mutations might contribute to the continuum CVHMC-lymphoma.

6 CONCLUSION

Chronic HCV infection should be considered as a continuous evolution from asymptomatic to lymphoma. Any MC stage (from asymptomatic to CV) should be considered as a prelymphomatous disorder. In this review we presented only a few recent discoveries that try to decipher the mechanisms of transition that predispose or induce autoimmunity or advanced malignancy in chronic HCV infected patients. Nevertheless, current technological progress will give us, in a not too distant future, the opportunity to precociously select individuals at high risk.

Hepatita cronică C a devenit în ultimii 10–20 de ani o problemă de sănătate publică în toată lumea evoluând ca o reală epidemie, a doua ca frecvenţă după cea cu virus hepatitic B. Datorită manifestărilor clince complexe infecţia cronică cu virus hepatic C poate fi considerată o boală sistemică. Crioglobulinemia mixtă (CM) este una din cele mai frecvente manifestări extrahepatice ale hepatitei cu virus C, dar vasculita crioglobulinemică are o prevalenţă relativ scăzută deşi sunt încă necesare studii epidemiologice. Prezenţa crioglobulinelor serice este obligatorie pentru diagnosticul CM, dar criocritul nu se corelează cu manifestările clinice şi nici cu prognosticul bolii. CM poate fi definită ca un status de proliferare limfocitară B, caracterizată prin activare policlonală şi sinteză de anticorpi. Evoluţia spre limfom poate fi considerată astfel un continuum, dar alţi factori infecţioşi, de mediu sau genetici ar putea fi implicaţi. Translocaţia t(14;18) şi activarea bcl-2 în limfocitele B, BAFF,interacţiunea dintre E2 şi CD81, limfocitele T, reglatorii CD4+CD25+ şi interferonii de tip III ar putea juca un rol important în evoluţia CM către limfom. Corresponding author: Adriana Nicolau MD 19–21, Ştefan cel Mare Street, Colentina Clinical Hospital, E-mail [email protected]

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41. PETRARCA A., MONTI M., LAFFI G., ZIGNEGO A.L., Can BAFF promoter polymorphism be a predisposing condition for HCV-related mixed cryoglobulinemia? Blood, 2008; 10(112):4353–4354. 42. CHARLES E.D., GREEN R.M., MARUKIAN S. et al., Clonal expansion of immunoglobulin M +CD27+ B cells in HCVassociated mixed cryoglobulinemia, Blood. 2008; 111(3):1344–1356. 43. KRONENBERGER B., HERRMANN E., HOFMANN P. et al., Dynamics of CD81 expression on lymphocyte subsets during interferon-based antiviral treatment of patients with chronic hepatitis CJ. Leukoc.Biol. 2006; 80:298–308. 44. ANTONELLI A., FERRI C., FERRARI S.M. et al., High interleukin-6 and tumor necrosis factor-alpha serum levels in hepatitis C infection associated or not with mixed cryoglobulinemia. Clin Rheumatol. 2009; 60(12):3841–7. 45. FELDMANN G., NISCHALKE H.J., NATTERMANNET J. et al., Induction of Interleukin-6 by Hepatitis C Virus Core Protein in Hepatitis C Associated Mixed Cryoglobulinemia and B-Cell Non Hodgkin’s Lymphoma, Clin Cancer Res 2006; 12(15): 4491–4498. 46. BIANCHETTIN G., BONACCINI C. et al., Analysis of hepatitis C virus hypervariable region 1 sequence in cryoglobulinemic patients and associated controls, J. Virol 2007; 81(9):4564–71. 47. LENZI C., FRISONI M., MANTOVANI V. et al., Haplotype HLA-B8-DR3 confers susceptibility to hepatitis C virus-related mixed cryoglobulinemia. Blood.1998; 91:2062–2066. 48. 48. CACOUB P., RENOU C., KERR G. et al., Influence of HLA-DR phenotype on the risk of hepatitis C virus-associated mixed cryoglobulinemia. Arthritis Rheum. 2001; 44:2118–2124. 49. SAKAGUCHI S., SAKAGUCHI N., ASANO M., ITOH M.,TODA M., Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995; 155:1151–1164. 50. SALOMON B., LENSCHOW D-J., RHEE L. et al., B7/CD28 costimulation is essential for the homeostasis of the CD4 + CD25 + immunoregulatory T cells that control autoimmune diabetes. Immunity 2000; 12:431–440. 51. BENNETT C-L., CHRISTIE J., RAMSDELL F. et al., The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3.Nat Genet. 2001; 27:20–21. 52. CAO D., MALMSTROM V., BAECHER-ALLAN C., HAFLER D,KLARESKOG L., TROLLMO C., Isolation and functional characterization of regulatory CD25+CD4+ T cells from the target organ of patients with rheumatoid arthritis. Eur J Immunol. 2003; 33:215–223. 53. PUTMAN A-L., VENDRAME F., GOTTLIEB P-A., Characterization of CD4+CD25hi T cells in human type 1 diabetes. Clin Immunol. 2003; S1:177a. 54. KUKREJA A., COST G., MARKER J. et al., Multiple immuno-regulatory defects in type-1 diabetes. J ClinInvest. 2002; 109: 131–140. 55. VIGLIETTA V., BAECHER-ALLAN C-M., HAFLER D-A., CD4+CD25+ regulatory T cells have reduced function in patients with multiple sclerosis.Clin Immunol. 2003; S1:175a. 56. CRISPIN J-C., MARTINEZ A., ALCOCER-VARELA J., CD4+CD25+ T cells are decreased in patients with systemic lupus erythematosus. Clin Immunol. 2003; S1:177a. 57. ISHIDA Y., AGATA Y., SHIBAHARA K., HONJO T., Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 1992; 11(11):3887–95. 58. NISHIMURA H., NOSE M., HIAI H., MINATO N., HONJO T., Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999; 11(2):141–51. 59. FREEMAN G.J., LONG A.J., IWAI Y. et al., Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000; 192(7):1027–34. 60. LEI N., CHENG J., YING Z. et al., PD-1 modulates regulatory T cells and suppresses T-cell responses in HCV-associated lymphoma. Immunology and cell biology. 2010 epub ahead of print. 61. BOYER O., SAADOUN D., ABRIOL J., CD4/CD25 regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis. Blood. 2004; 103:3428–3430. 62. LANDAU D.A., ROSENZWAJG M., SAADOUN D. et al., Correlation of Clinical and Virologic Responses to Antiviral Treatment and Regulatory T Cell Evolution in Patients With Hepatitis C Virus–Induced Mixed Cryoglobulinemia Vasculitis, Arthritis & Rheumatism, 2008; 58(9), 2897–2907. 63. HUNZIKER L., RECHER M., MAPHERSON A-J. et al., Hypergammaglobulinemia and autoantibody induction mechanisms in viral infections. Nat Immunol.2003; 4:343–349. 64. ANK N., WEST H., BARTHOLDY C. et al., Lambda Interferon (IFN-λ), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo. Journal of Virology. 2006; 80(9):4501–9. 65. RAUCH A., KUTALIK Z., DESCOMBES P. et al., Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study. 2010; 138(4):1338–45. 66. SHEPPARD P., KINDSVOGEL W., XU W. et al., IL-28, IL-29 and their class II cytokine receptor IL-28R. Nat Immunol. 2003 Jan; 4(1):63–8. 67. SUPPIAH V., MOLDOVAN M., AHLESTIEL G. et al., IL28B is associated with response to chronic hepatitis C interferon-α and ribavirin therapy. Nature Genetics, 2009; 41(10):1100–4. 68. SAMUEL C.E., Antiviral actions of interferons. Clinical Microbiology Reviews, Oct 2001, 14(4):778–809. 69. ZHOU F., Molecular Mechanism of IFN-gamma to Up-Regulate MHC Class I Antigen Processing and Presentation, International Reviews of Immunology 2009; 28(3,4), 239–260. Received February 17, 2011

Epidemiological Data Regarding Balkan Endemic Nephropathy in Relationship with the Pliocene Coal Etiological Hypothesis GH. GLUHOVSCHI, M. MODALCA*, F. MĂRGINEANU*, SILVIA VELCIOV, CRISTINA GLUHOVSCHI, F. BOB, LIGIA PETRICA, GH. BOZDOG, V. TRANDAFIRESCU, F. GĂDĂLEAN Nephrology Dept., University of Medicine and Pharmacy Timişoara, Romania *Renamed Dialysis Center, Drobeta Turnu Severin, Romania

Balkan Endemic nephropathy (BEN) is a tubulointerstitial disease of unknown etiology signaled in a limited geographical area. In the neighbourhood of endemic villages are coal deposits from the Pliocene, that contain toxic substances that by mobilizing groundwater can leach in water sources used by the inhabitants. In the present paper the possible impact of the coal from Pliocene on people that worked many years in mines in the endemic County Mehedinti, Romania, and who lived in this area are analysed. The risk of toxicity of coal was theoretically increased in miners because they consumed frequently water from mine springs that came from coal layers, while at home water from wells could have been contaminated by toxic substances from coal. It has been found that only 5 of the 96 patients with BEN were under dialysis program in 2008. Also out of 34 former miners only 3 had GFR < 60 ml/min/1.73 sqm, and only one with creatinine of 3 mg/dl had GFR < 30ml/min/1.73 sqm. The mean GFR in the 34 miners was: 94.13 ± 26.58 ml/min/1.73 sqm. We analysed GFR and proteinuria in persons from the endemic zone from 2 types of villages: some with mining activity presently (Husnicioara) others where presently there are no mining activities (Hinova, Bistrita, Livezile). We also analysed comparatively 2 non-endemic localities near the endemic focus: Drobeta Turnu Severin (without mining activity) and Motru with mining activity where different coal deposits are (non-Pliocene). Data were provided from the family doctors databases. The GFR was lower in the inhabitants from the endemic villages Bistrita and Hinova than in the investigated inhabitants from the non-endemic town Drobeta Turnu Severin (p = 0.008 and p = 0.0004 respectively). Inhabitants from the endemic village Husnicioara (Pliocene coal mine still functioning) had a higher GFR than inhabitants from Drobeta Turnu Severin and higher than inhabitants from the endemic village Livezile (mine closed 10 years ago): p = 0.0055 and p = 0.001 respectively, but a lower than the investigated inhabitants from the non-endemic town Motru (where a non-Pliocene coal mine is functioning): p < 0.001. Proteinuria was present in 8.03% of the inhabitants from the endemic village Bistrita and in 7.4% of the inhabitants from the endemic village Hinova. In the non-endemic town Drobeta Turnu Severin, proteinuria was present in 7.08% of the investigated inhabitants. Proteinuria was present in 0.78% of the investigated inhabitants of the non-endemic town Motru (where a non-Pliocene coal mine is functioning) and 2.5% of the inhabitants of the endemic village Husnicioara (Pliocene coal mine still functioning). Our paper does not show any relationship between exposure to Pliocene coal and the etiology of BEN.

Balkan Endemic Nephropathy (BEN) is a disease encountered in Southeastern Europe. It has been recorded in five countries: Romania, Bulgaria, Serbia, Croatia, and Bosnia, all in the Balkan region. The disease is endemic in the zone where it has been recorded, hence the name BEN. In the BEN zone, lignite deposits are present, which have a different structure from other types of lignite. Lignite deposits in the BEN zone, according to Feder, are dominated by tertiary sediments, including Pliocene lignite deposits, that are a source of many potentially toxic organic compounds [1]. ROM. J. INTERN. MED., 2011, 49, 1, 11–24

In the neighborhood of the villages from the BEN area that are usually disposed in deep valleys surrounded by hills there are lignite deposits from the Pliocene, a fact that prompted the opening of underground or surface mines. The vicinity of coal deposits originating in the Pliocene generated the hypothesis of the involvement of toxic substances originating from these reserves in BEN etiology [1]. The hypothesis sustains that Pliocene lignite hydrocarbons (polycyclic aromatic hydrocarbons, aromatic amines, and others) from coal deposits in the BEN area are leaching into the groundwater.

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Toxic substances in Pliocene coal dissolve easily in water. During times of rain, they are taken and spilled into groundwater. In this way they could contaminate the drinking water in springs and wells and could exert nephrotoxic and carcinogenic actions [1, 2]. In the endemic zone in Romania, many mines that were once opened have been closed after many years of operation, the most recent one, 10 years ago, in the village of Livezile. Presently in the endemic zone, there is a single mine of reduced capacity in the village Husnicioara. One of the purposes of this paper is represented by an observation of a visit made to the BEN area, when a patient under dialysis in the Hemodialysis Center of Turnu Severin, who was accompanying us, told us the following: he and other persons in the area worked in mines in the neighborhood of endemic villages. When asked what type of water they drank, he told us that, because of the heat inside the mines, they drank as much as 10 liters of water per day. Because this large amount could not be brought from the surface, he and others preferred to drink water directly from the mine, water that appeared as springs between the coal layers. Few miners brought water from the surface. At present, the drinking water is delivered by mining authorities. Because the patient as well as other miners used the water filtered between coal layers for many years, and they still had opportunities to drink contaminated water from the wells after retirement while living in the endemic area, it was considered plausible that toxic compounds from coal could participate in the etiology of BEN. Thus, regarding the hypothesis of the involvement of coal from the Pliocene in the etiology of BEN, the following has to be remembered: miners, before working in mines, lived and used drinking water originating from the endemic zone that could have been contaminated by toxic substances from coal that reached groundwater. In the period when they worked in mines, after coming home, they used the same water for drinking and dishwashing. After retirement, which is usually at a much younger age than other employees (usually 45), they continued to live in the endemic zone, consuming water of the same origin. It is presumed that in case coal from Pliocene has an important role in producing BEN, the miners being exposed to larger doses of toxic substances from the Pliocene coal should present BEN more frequently.

2

Those who came from non-endemic villages have left the zone. They are not the subject of the present paper. In these conditions and in the case of double contamination, i.e., from drinking water directly from springs between coal layers and from drinking water outside the workplace and after retirement, persons who work in mines could have an increased intake of toxic substances coming from coal. Thus, in the hypothesis that the toxicity of substances released from Pliocene coal forms the basis of BEN, the disease should be more frequent among miners. It should also be more frequent in the villages where mines function. OBJECTIVES OF THE PAPER

Therefore, we decided, as our first aim, to analyze the frequency of cases of BEN in dialysis programs coming from miners compared to nonminers. The second objective was to analyze glomerular filtration rate GFR from persons that worked in mines for variable periods of time to see how many of them have elements of chronic kidney disease, BEN. This is the aim of the present study, which should be confirmed through epidemiological observations. The third objective was to analyze GFR in BEN villages where mines existed, now closed, and in villages with BEN without coal mines, as well as in persons who live in villages where a coal mine is still functioning. The GFR was also analyzed in a general practice in the town of Drobeta Turnu Severin, a town without BEN, but in the vicinity of the BEN area, as well as in a mining town in the vicinity but where there is another type of coal, not Pliocene, involved in the etiology of the disease. We believe the analysis of the data obtained permits an estimation of the possible involvement of toxic substances from the Pliocene coal in the etiology of BEN. In performing this work we benefited from screening investigations performed in the whole country, including the BEN area, by the Ministry of Health. A similar action to that performed in Romania has been organized in Japan [3]. We tried to delineate the possible involvement of some toxic substances that originate from coal mines in the generation of BEN, through the direct action on the people who worked in mines and,

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indirectly, through the water from the wells from the villages in the endemic zone. Therefore, we assessed the period in which they worked in mines (underground or surface) and the period when they lived in the area and could have consumed water from fountains. This observation does not exclude a complementary role of other factors along with toxic substances in coal. The three proposed objectives can designate an epidemiological frame that refers to a possible involvement of toxic substances from the Pliocene coal in the etiology of BEN. Because there are several important arguments regarding the involvement of Aristolochia clematitis and aristolochic acid in the etiopathogenesis of the disease, without excluding a potential plurifactorial aspect, we analyzed the use of this plant as a therapeutic remedy in the persons investigated. We also analyzed the presence of urothelium tumors due to the fact that BEN is frequently associated with these. The main objective is the presentation of epidemiological data about the relationship between BEN and potential exposure to toxic substances originating from Pliocene coal. MATERIALS AND METHODS

The frequency of BEN cases under dialysis, in the Dialysis centers from Drobeta Turnu Severin, has been studied. The frequency of BEN cases in dialysis programs coming from miners compared to nonminers has been analysed. We analyzed GFR and proteinuria in persons from the endemic region from two types of settlements, some with mining activity in the present and others where presently there is no mining activity. The endemic villages where no coal mine is currently functioning were Hinova, Bistriţa, and Livezile. It should be mentioned that in Livezile, a coal mine functioned in the past (10 years before) but is now closed. The endemic village with a still-functioning coal mine was represented by Husnicioara. Data from these villages have been provided by general practitioners from these settlements. They come from their files. In Drobeta Turnu Severin, where no mine is functioning, all patients came from a general practitioner, servicing a part of the population. In

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Motru, where an important non-Pliocene coal mine is present, information was obtained from a general practitioner, servicing also a part of the population. Data from these villages have been provided by general practitioners from these settlements. Existing data from the family doctors, from the studied villages, have been analysed. They provided these data regarding the existent investtigations from their databases for the year 1998. These data have 2 origins: – From the adult persons from the endemic zone, that presented for consultation, and where performed currently due to the fact that there is an endemic zone: serum creatinine, urea and proteinuria. Also a complete anamnesis was performed with great importance to data specific to BEN: BEN in families, urothelium tumours, data regarding relatives, neighbours. – Other data provided by family doctors are with regard to existing ones after investtigations performed in the area, in the case of screening of the population from the BEN zone, performed by the Ministry of Health. As mentioned above, in 2008 the Ministry of Public Health undertook a screening regarding the general health status of the population. The screening has bperformed by family doctors based on the action of the Health Ministry, which sustained these actions financially. The screening had the purpose of discovering the main diseases encountered in practice: ex diabetes mellitus and hypertension, as well as other diseases. Patients had to complete a survey, and additionally the following were assessed: complete blood count, fasting serum glucose, cholesterol, triglycerides, AST, ALT, urinalysis, urine sediment and proteinuria. Proteinuria was measured using a dipstick analysis. In patients over 50, serum creatinine was also measured. Serum creatinine was determined using the Jaffe method. The eGFR was estimated using the MDRD 4 formula. It is worth mentioning that proteinuria was assessed concomitantly because it is an important element in the early diagnosis of BEN. In the endemic zone data were accumulated regarding the epidemiological situation of the renal diseases, including BEN. These investigations permit an assessment of the endemic region at present. It is worth mentioning that proteinuria is an important element in the early diagnosis of BEN. In the studied villages from the BEN area the number of inhabitants over 50 has been established, the number of persons over 18, the number of miners compared

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to the number of persons over 18, the number of dialysed patients from every investigated village miners and non-miners. Thirty four persons coming from the BEN region who had worked in mines were investigated for GFR, proteinuria, and the period when they worked in surface mines/underground. These persons had lived in the endemic region all their lives. At the same time, we investigated whether these persons had used therapeutic remedies containing Aristolochia clematitis. As mentioned before, BEN is considered by some authors an aristolochic nephropathy [20]; other authors assert that the etiology of BEN is multifactorial [32]. We analyzed the prevalence of miners among dialyzed BEN patients as compared to non-BEN dialyzed patients who did not work in mines. It has to be mentioned that the age of retirement was lower in miners as compared to other population groups, due to hard working conditions (usually 45 years). Miners from the BEN region are exposed to toxic substances originating from coal, by drinking water from springs and wells adjacent to the mines as well as from their households. It is to be noted that there is considerable hardship in performing epidemiological investtigations in the endemic region. Persons from the BEN region are reluctant to undergo medical investigations, given the mentality that a person with BEN is ostracized by the society. Therefore, some persons refused to participate in the screening conducted by the Ministry of Health, even when they were prompted by their general practitioner at the request of the nephrologist. During the course of this paper we will use the term “miner” also for those who are currently retired but used to be miners. RESULTS

The results are presented in the four Tables. Table I. GFR and proteinuria in persons coming from the endemic zone and the control villages (Motru: with coal mine, without BEN; and Drobeta Turnu Severin: no coal mine or BEN) Table II. Miners in a hemodialysis program in the dialysis centers of Drobeta Turnu Severin. Table III and IV. Persons who worked in mines from the endemic zone, settled in BEN villages. Table V. Comparative assessment of glomerular filtration rate in persons over 50 from endemic villages Bistriţa, Hinova, Livezile, Husnicioara with persons from a non-endemic zone (Drobeta Turnu Severin)

4

Table 6. Ratio miners/ adult population in the studied villages/ towns. In 2000, 139 patients (36.69% BEN) were under dialysis treatment in the two dialysis centers in Drobeta Turnu Severin: one at the County Hospital of Drobeta Turnu Severin and another at the Railways Hospital of Drobeta Turnu Severin. During the years 2001–2009, the number of dialyzed patients in the two centers increased: in 2008, 187 patients were dialyzed, of whom 96 (51.34%) had BEN. At the end of 2009, 174 patients were under dialysis, of whom 96 (52.87%) had BEN. Only five miners were dialyzed, representing 5.21% of the total number of BEN patients dialysed in 2008. None of the dialyzed patients presented urothelium tumors, nor did any of the 34 former miners from the endemic zone. The presence of urothelium tumors was not revealed in any of the examined persons from the localities studied. Regarding dialyzed patients from endemic localities, we found the following: In Hinova village there were 4 dialyzed patients out of 1049 inhabitants, representing 1 dialyzed patient per 262 inhabitants, none of them being a dialyzed miner (in this village there is no miner among the investigated persons). In the village Bistrita there were 8 dialyzed patients out of 1352 inhabitants representing 1 dialyzed patient per 169 inhabitants. Out of the 8 dialyzed patients one was a miner. In the village Livezile, where 10 years before this study mining activities had been performed there were 2 dialyzed patients out of 1060 inhabitants, a rate of 1 per 530 inhabitants, none of them miner. In the commune Livezile that comprises 5 villages including the village Livezile, center of commune, 1091 inhabitants there are 11 persons under dialysis, that is one patient out of 99 inhabitants. In the village Husnicioara, where mining activity was still performed, a single patient was dialyzed out of 326 inhabitants. In the commune Husnicioara (1700 inhabitants), that comprises the village Husnicioara together with other villages there are 4 persons under dialysis, that represents one dialyzed patient out of 420 inhabitants, and no miner dialyzed. Out of the 34 miners, represented by persons who worked in mines from the endemic zone settled in BEN villages, that were analysed regarding other parameters as well, regarding mining activity both in underground and/or surface mines, as well as aristolochic acid containing

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remedies consumption, we found out that only 3 persons showed GFR under 60ml/min, and only one with GFR below 30 ml/min. The miners worked in mines for a mean period of 11.85 ± 4.94 years. Pearson’s correlation coefficient between the period of years worked in mines and the GFR was R = 0.211, p = 0.184. 6002 persons that represent the population of the investigated villages, of whom 509 were miners. From the population of the investigated villages without miners only 38 out of 5499 (6.04%) are under dialysis. From the 509 miners only one is under dialysis representing 0.19%.

15

The present paper aimed to analyze, based on epidemiological studies, the relationship between the presence in the endemic zone of Pliocene coal and the etiology of BEN. Therefore, we first analyzed the current situation of BEN in Mehedinti county, Romania, the main BEN focus. Romania also harbored another small focus of BEN, in Caraş-Severin county, but no new cases of BEN have been reported there in recent years. This study shows that BEN continues to represent a public health problem in the Mehedinti endemic focus.

important percentage of the dialysis patients, approximately 50%. At the same time, we did not observe a tendency of BEN to decrease. The main hypothesis concerning BEN at present is that this disease is caused by aristolochic acid, as endemic nephropathy considered an aristolochic nephropathy. According to the aristolochic acid hypothesis in the etiology of BEN, Aristolochia clematitis seeds from wheat crops would contaminate the wheat seeds. Flour prepared from wheat would thus be contaminated, and its consumption over many years would produce a cumulative toxic effect, leading to BEN [4, 5]. If the hypothesis that BEN is produced by Aristolochia clematitis holds true, the number of BEN patients should be decreasing, all the more so because herbicides and modern milling procedures that remove the Aristolochia clematitis seeds are being used. The contamination of wheat used in bread production has declined, which should have led to a reduction of patients, which was not the case. Due to the fact that we could not demonstrate a tendency of BEN to diminish in spite of the measures undertaken to avoid contamination by Aristolochia clematitis seeds, we considered it useful to analyze the potential involvement of other etiological factors, such as toxic substances from Pliocene coal. Therefore, we analyzed the frequency of miners with BEN from the endemic zone.

Dialysed patients from Dialysis Centers from the endemic zone

The epidemiological situation of BEN in other countries where BEN is present

A modality for determining the epidemiological situation of BEN in the Mehedinti focus is the analysis of dialyzed patients. This is due to the fact that, at present, there is no clear record of these patients because they avoid medical services for this disease until the advanced stages. Actually, even in the countries where this disease is endemic, epidemiological data refer mainly to dialyzed patients. In 2000 in the two dialysis centers in Drobeta Turnu Severin that serve Mehedinti county, which contains the main focus of BEN in Romania, 139 patients were undergoing dialysis, 51 of them BEN patients. In 2008, 187 patients were undergoing dialysis, 96 of them BEN patients. In 2009, 174 patients were undergoing dialysis, 92 of them BEN patients. If we consider that the BEN focus covers a quarter of the county’s surface, it becomes clear that patients from the endemic focus represent an

In Bulgaria, a decline in BEN has been observed after 1984, partially due to the migration of the population [6]. In Serbia, studies performed by Cukuranovic regarding hemodialysis patients in 8 dialysis centers indicate that the incidence of BEN cases has decreased [7]. Djukanovic et al. report that the incidence of BEN among patients who started dialysis in three endemic regions in Serbia is stable [8]. Bukvic et al. have found the occurrence of new cases in families in whom cases of BEN have previously been found, indicating that BEN is still present in the Kolubara region of Serbia [9]. In Croatia, Miletic-Medved et al., in an epidemiological survey in spring 2005, found that the prevalence of BEN in Croatia is approximately the same as it was in previous decades [10]. In Bosnia, Imamovic et al. evaluated the epidemiological status of BEN patients on renal replacement therapy from 2003 to 2005. He reports that the trend of BEN patients was stable during this period

DISCUSSION

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[11]. The disease is present in elderly persons. Bukvic currently considers BEN to be a disease of the elderly with a good prognosis [12]. THE MINERS WITH BEN UNDER DIALYSIS

We found that of the dialyzed BEN cases, there were five miners, representing 5.21% of the dialyzed BEN patients in 2008. If toxic substances contained in Pliocene coal are an important etiological factor in producing BEN, the disease should have been more frequent among miners who were most exposed to these substances: – By consuming water from mine springs from the coal layers during their work in mines. – By consuming water from wells from their households, contaminated by toxic substances leaching into the groundwater from Pliocene coal layers. It has to be remarked that the incidence of dialyzed persons from endemic villages studied that contain 6002 persons of which 509 miners it has been found out that if not counting the miners from the total population we have a remaining number of 5493 persons. Of these 38 are dialyzed representing 0.6% of the population. Of the 509 miners only one is dialyzed representing 0.19% of them. It can be observed that the prevalence of dialyzed persons among miners is lower than in the general population of the investigated villages. Because a possible association of the toxicity of Aristolochia clematitis with that of the toxic substances in coal was present in only one of five persons, this does not indicate an important role for therapeutic remedies in the production of disease. None of the five miners undergoing renal replacement therapy showed urothelium tumors. Only one miner under chronic dialysis used Aristolochia clematitis as a therapeutic. It is known that Aristolochia clematitis consumption is associated with the occurrence of urothelium tumors. Assessing GFR in BEN settlements with mines as compared to BEN settlements without mines, along with the assessment of GFR in two nonendemic cities without mines Another method to assess whether persons in the endemic region are at increased risk of

6

developing BEN was the assessment of kidney function by measuring GFR in settlements where mines are present, which could contain increased groundwater contamination, compared to settlements where coal layers are absent. Our observations show that in persons over 50 originating from endemic villages Bistrita and Hinova, GFR was significantly lower than in persons from nonendemic town Drobeta Turnu Severin. This could be related with the endemicity of the area, where the two villages are situated. In none of these villages mining activity is present. At the same time, GFR in persons from the endemic village Livezile was slightly higher, although not significantly, than the GFR in persons from Drobeta Turnu Severin. It is to be noted that in the endemic village Livezile, conditions exist for the contamination of groundwater because a mine had functioned there until 10 years prior. Furthermore, GFR in persons from Husnicioara, where a mine containing Pliocene coal was functioning, was higher than the GFR from the endemic village Livezile, where the mine had been closed for 10 years and endemic villages without mines (Bistriţa and Hinova). At the same time, GFR in persons from Husnicioara was significantly higher than GFR in persons from the non-endemic town of Drobeta Turnu Severin. If toxic substances from Pliocene coal play an important role in BEN, greater renal function impairment would be expected in the village of Husnicioara, where a mine was functioning. However, it is hard to incriminate the contamination of groundwater by toxic substances in the decreased GFR of persons living in the endemic area. On the other hand, it has been observed that GFR in the endemic village of Husnicioara (where a mine with Pliocene coal was functioning) was lower than the GFR from the non-endemic city of Motru, where a mine containing non-Pliocene coal was functioning. It has to be mentioned that in the etiology of BEN toxic substances from Pliocene coal are incriminated. Presently there are studies that mention that estimating GFR in a population based study by using MDRD or Cockroft-Gault equations is limited [13]. We consider that in front of the difficulties of assessing patients from the BEN zone from Mehedinti, obtaining supplemental data is an acceptable solution. Melamed et al. consider that although eGFR determining is only one tool nephro-

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Epidemiological Balkan endemic nephropathy data

logists need to educate patients and their primary care colleagues in the use of this tool [14]. We consider that this tool used in the BEN area can take us to epidemiological observations in the BEN focus, using data obtained from general practitioners regarding the investigation of patients, that are addressing to them from the BEN area, as well as data resulting from other investigations, respectively the screening of the population from the BEN zone and the epidemiological survey performed by the Ministry of Health, that was the first important populational study in the BEN area in Romania where the incidence of BEN is is not well known, the single data are those provided by Dialysis centers that are in the area. Assessing GFR in persons living in the endemic region who had worked in mines We measured GFR in a group of persons living in the endemic region who had worked in mines (surface or underground). This is important because of possible exposure to toxic substances from coal. Out of 34 former miners from the BEN region, 16 (39.22%) had a GFR < 90 ml/min, while 3 had GFR below 60 ml/min (7.3%). Only one former miner had a GFR below 30 ml/min. Taking into account that these former miners experienced double exposure to toxic substances originating from Pliocene coal by means of drinking large amounts of water from springs coming from the coal layers as well as from wells in their households, we would have expected a higher degree of renal functional impairment, which proved not to be the case. The question is raised whether toxic substances from Pliocene coal, from deposits from the surrounding hills, were indeed mobilized, and, if so, to what extent. We did not find a relationship between the duration of mining activity (surface or underground) and GFR. None of the former miners in whom GFR was estimated showed urothelium tumors, which indicates the lack of a carcinogenic effect of substances originating from Pliocene coal. The association with the use of Aristolochia clematitis remedies, that contains aristolochic acid involved in urothelium tumors etiology, did not reveal in our patients the presence of urothelium tumors. It should be mentioned that the referral of patients suspected of having BEN to the family

17

doctor is influenced by the fear of being diagnosed with this disease and the ostracizing effect this might have. The family doctors have no clear evidence of these persons and of their actual and former occupation. The reliable data were obtained during the screening performed by the Ministry of Health, but this was also limited due to the refusal of some part of the population to participate in the screening. Therefore, the number of dialyzed patients seems to better reflect the disease burden. Due to the same reasons we undertook a study regarding GFR and proteinuria of 34 former miners from the endemic region. The number of miners that presented for assessment of GFR and proteinuria at the general practitioner was low. In the village of Livezile wherefrom the most miners come (31 of them) we had problems with the referral of 20 persons who came to the doctor but refused to perform the required investigations. Because obtaining data from patients from the endemic zone is difficult, we compared endemic miner localities with endemic localities without mines, and as a control group we used the population from a non-endemic region with and another without mines. We could not demonstrate a relationship between mining activity and BEN. From our study, we can conclude that, at the moment, the hypothesis regarding the etiology of BEN in association with Pliocene coal present in endemic zones cannot be supported. If the increased intake of water from the mine springs after it had passed coal layers had mobilized toxic substances, the proportion of former miners afflicted with BEN would have been higher, in dialysis centers and among the investigated persons. BEN is frequently associated with urothelium tumors [15]. If toxic substances from Pliocene coal had exerted a carcinogenic effect, we would have detected in the former miners urothelium tumors, but we did not. Many toxic organic compounds derived from coal exert toxic and carcinogenic effects. Polycyclic aromatic hydrocarbons (PAH) may have a carcinogenic potential in the urothelium [16]. Thus, according to Ghanem et al., many PAH can cause DNA adducts and initiate carcinogenesis [17]. Bonassi et al. have reported bladder cancer in those exposed occupationally to PAH [18]. Similar observations have been made by Mastrangelo et al. [19].

18

Gh. Gluhovschi et al.

For a better understanding of the possible involvement of toxic substances from Pliocene coal in BEN, we consider it useful to present them. We also consider it necessary to mention the studies performed on drinking water from wells in the BEN zone. On the other hand, the incidence of patients with BEN with urothelium tumors who present in Dialysis Centers in Turnu Severin is not decreasing [15]. All of these findings could suggest the existence of another etiological factor in the main endemic focus. We mentioned earlier that the most important similarities with BEN are presented by aristolochic nephropathy, the model being Chinese herbs nephropathy. Most authors favor the aristolochic hypothesis of BEN etiology and of urothelium tumor production [20, 21]. In a recently published paper we found that therapeutic remedies based on aristolochic acid do not seem to play a major role in BEN etiology [22]. Today in hypotheses of the etiology of BEN, the involvement of aristolochic acid is predominant, the disease being considered by some as an aristolochic nephropathy [20, 23]. At the basis of this concept stand numerous arguments regarding the resemblance of BEN to Chinese herbs nephropathy produced by plants of the aristolochic group (Aristolochia fangchi) [24, 25]. Moreover, DNA adducts of aristolochic acid have been found in the kidneys of persons from the BEN area. The hypothesis of the involvement of Aristolochia clematitis in BEN mentions the use of bread prepared from flour infected with Aristolochia clematitis seeds. This could lead, in time, to the production of aristolochic nephropathy. The disease manifests itself as a chronic renal failure with long evolution, associated frequently with urothelium tumors [21]. In BEN etiology hypotheses, numerous factors besides aristolochic acid are also involved, among them compounds originating from Pliocene coal. These toxic compounds could dissolve in groundwater, draining into the wells containing drinking water and water used for cooking. Other factors have been implicated in BEN, such as mycotoxins, especially ochratoxin A, slow viruses (e.g., coronaviruses), other viruses (hantavirus), heavy metals, selenium deficiency, and others [26–31]. Genetic factors are also considered of some importance, due to the familial character of the disease. This fact has led to the hypothesis of a plurifactorial etiology of BEN [32].

8

If the aristolochic acid hypothesis were correct, measures performed for herbicidation, as well as the withdrawal of aristolochia seeds when flour is prepared in the BEN area, should lead to a decrease of the incidence of this disease. This was not revealed by our observations. This was the second reason why we explored the exposure to other agents that could be involved in the etiology of BEN. Toxic products from Pliocene coal and studies performed in the drinking water from the endemic zone One of the hypotheses mentioned earlier is the possibility of the intervention of some toxic hydrocarbons that originate from the Pliocene coal. In the BEN area, there are lignite reserves with a different structure that were formed in the Pliocene period. These differ from other forms of lignite. The hypothesis of the toxicity of Pliocene hydrocarbons holds that water from BEN-area coal deposits leaches into the groundwater deposits, allowing toxic substances to contaminate wells in nearby villages. People who use the well/spring water are then exposed to the coal-derived toxic organic compounds [33, 34]. The main toxic compounds involved are aromatic hydrocarbons. According to Orem, other leachates involved are cycloalkanes/alkenes, steranic structures, monoaromatic and polyaromatic terpenes, polycyclic aromatic hydrocarbons, aromatic amines, and N-, S-, and O-containing heterocyclic compounds. Many of these compounds have attached oxygen-based functional groups, and some of them contain heterocyclic nitrogen or amino groups, structural features that could make them nephrotoxic and carcinogenic [35]. Voice et al., by analyzing PAH concentration in a large number of patients from endemic and non-endemic villages in Bulgaria, found that all PAH levels were low, with none exceeding the drinking water standard for benzo-[a]-pyrene, the most toxic PAH. Studies regarding the detection of dissolved organic compounds that leach from coal have failed to detect dissolved organic compounds. Voice et al. found no basis to connect PAH or other unknown organic compounds to the etiology of BEN. They suggest that evidence in support of the Pliocene lignite hypothesis is limited to the special circumstances originally proposed [36].

9

Epidemiological Balkan endemic nephropathy data

Goldberg examined water from Bulgaria and the former Yugoslavian countries using excitationemission matrix fluorescence spectroscopy and identified organic substances, including a high concentration of fumic acid. Following are the main pros and cons regarding the involvement of toxic substances from Pliocene coal in BEN etiology, and we will present our data in relationship with this hypothesis. Arguments and counterarguments regarding the hypothesis of coal involvement in the etiology of BEN To analyze the results from our study in relationship with observations from the literature, we considered it useful to present arguments that support the hypothesis of the involvement of Pliocene coal in the etiology of BEN as well as the counterarguments. Arguments in favor of the involvement of Pliocene hydrocarbons could be represented by: – In the vicinity of most villages with BEN, lignite deposits originating from the Pliocene are present. – Numerous mines have been opened in the area. The most representative surface mining facility is in Serbia, near Lazarevic. In Romania many mines have been closed. – In the water from wells in the endemic area, toxic compounds have been found [34]. – In the USA numerous cases of neoplasia in the areas surrounding coal reserves, similar to the endemic zone, have been found [33]. This hypothesis could explain why in some households BEN is present, while in others it is not: perhaps groundwater does not equally contaminate all wells used by the population. It could also explain the presence in a family of many cases of BEN. The reduced concentration of toxic compounds of the PAH type recorded in the water from the wells in the endemic area could be related to the time necessary for the development of the disease and to the fact that it is necessary to live many years in the BEN area. Counterarguments against this hypothesis include: – Deposits of lignite have not been found in the vicinity of all BEN villages. – It is not explained why in the same household not all members develop the disease, e.g., sometimes the husband does and the wife does not.

19

– It cannot explain the ethnic character; the Roma population does not develop the disease.[22]. – The quantities of PAH in the water supplies are small, according to some authors. A possible cumulative effect could not be demonstrated [36]. Our own observations revealed: – The number of former miners under hemodialysis treatment was only five. – Only 3 of the 34 miners (8.85%) presented GFR < 60 ml/min. If toxic substances from coal play an important role in the etiology of BEN, the disease should be more frequent among miners. They, just as the population from endemic villages, consumed water from wells that could have been contaminated by the toxins that leached from rain water and later into groundwater. Moreover, these miners consumed water from the springs from the mines that could have incorporated toxic substances from coal. Thus, the number of BEN patients should be larger. In the endemic village Husnicioara, where a coal mine was functioning at the time of the study, the mean GFR of the population was greater compared to other endemic villages without mines (Hinova, Bistriţa, Livezile). If mining were involved in the release of toxic substances from coal with the leakage in the groundwater, this could influence the renal function of these persons. Thus, GFR should be lower in the village of Husnicioara. Regarding the ratio of dialyzed patients/ number of inhabitants from endemic villages, we found a larger ratio in villages where no mines were located (Hinova and Bistrita) compared to Husnicioara, where a mine was functioning, and to Livezile, where a mine used to function 10 years ago. Not even in this case could we remark that the presence of mining facilities could influence the presence of BEN. In the examined former miners, urothelium tumors were not found. BEN is frequently associated with urothelium tumors. In these cases the involvement of toxic substances from coal in the etiology of BEN could not be proven. In conclusion, our epidemiological observations do not support the toxic hypothesis regarding the relationship between BEN and the lignite compounds. The hypothesis of aristolochic acid involvement has more arguments mentioned by many authors in its favor, but we cannot exclude a plurietiological participation, which might include toxic compounds from Pliocene coal.

20

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10

Table I GFR and proteinuria in persons coming from the endemic zone and the control villages (Motru: with coal mine, without BEN; and Drobeta Turnu Severin: no coal mine or BEN)

Table II Miners in a hemodialysis program in the dialysis centers of Drobeta Turnu Severin

11

Epidemiological Balkan endemic nephropathy data

Table III Persons who worked in mines from the endemic zone, settled in BEN villages

Table IV Persons who worked in mines from the endemic zone, settled in BEN villages

21

22

Gh. Gluhovschi et al.

12

Table V Comparative assessment of glomerular filtration rate in persons over 50 from endemic villages Bistrita, Hinova, Livezile, Husnicioara with persons from a non-endemic zone (Drobeta Turnu Severin)

Table VI Ratio miners/adult population in the studied villages/towns

Number of inhabitants HUSNICIOARA COMMUNE HUSNICIOARA VILLAGE LIVEZILE COMMUNE WITH 5 VILLAGES LIVEZILE VILLAGE IZV. ANESTILOR VILLAGE VALEA IZVORULUI VILLAGE PETRIS VILLAGE IZVORELU DE JOS VILLAGE MOTRU TOWN (patients from one family doctor) HINOVA VILLAGE BISTRITA VILLAGE

1700 326 1901 1060 261 190 120 270 2466 1049 1352

Number of inhabitants over 50 patients Persons over 18 years 1275 = 75% 245 = 75% 782 = 41.13% 449 = 42.35% 101 = 38.69% 80 = 42.10% 54 = 45% 98 = 36.29% 366 = 14.84% 335 = 31.93% 429 = 31.73%

Number of miners compared to persons over 18 years

Number of BEN dialysed Number patients (nonof miners miners)

Number of BEN dialysed patients (miners)

1425

30%

427

4

0



30%

98

1

0

1358 775

5.37% 4.51%

73 35

11 2

0 0

194

11.85%

23

3

0

129 88

4.65% 10.22%

6 9

4 0

0 0

172

0.00%

0

2

0

1978 780 1079

4.95% 0 1.76%

98 0 19

4 8

0 1

13

Epidemiological Balkan endemic nephropathy data

23

Nefropatia endemică balcanică (NEB) este o afecţiune tubulo-interstiţială de etiologie necunoscută semnalată într-o arie limitată. În vecinătatea satelor endemice sunt depozite de cărbune din Pliocen care conţin substanţe toxice care prin intermediul apei pot ajunge în sursele de apă ale locuitorilor. În lucrarea prezentă impactul posibil al cărbunelui din pliocen asupra populaţiei care a lucrat ani de zile în minele din focarul endemic din Judeţul Mehedinţi, România, sau au trăit în această zonă. Riscul toxicităţii cărbunelui este teoretic crescut la mineri deoarece au consumat frecvent apă din izvoarele din mină, iar acasă au consumat apă din fântânile posibil contaminate. S-a constatat că doar 5 din cei 96 pacienţi cu NEB au fost dializaţi în 2008. De asemenea, dintre cei 34 foşti mineri doar 3 au avut Rata de Filtrare Glomerulară (RFG) < 60 ml/min//1,73 mp.s.c., si doar unul cu creatinină de 3 mg/dl avea RFG < 30 ml/min//1,73 mp.s.c. Rata medie de Filtrare Glomerulara la cei 34 mineri a fost: 94,13 ± 26,58 ml/min/1,73 mp.s.c. Am analizat RFG şi proteinuria la persoane din zona endemică din două tipuri de sate: unele cu activitate minieră în prezent (Husnicioara), altele unde nu este activitate minieră în prezent (Hinova, Bistriţa, Livezile). Am analizat, de asemenea, comparativ, două localităţi non-endemice din apropierea focarului endemic: Drobeta Turnu Severin (fără activitate minieră) şi Motru cu activitate minieră, dar cu cărbune diferit de cel din pliocen. Datele au fost obţinute de la medicii de familie, din baza lor de date. RFG a fost mai mică la locuitorii din satele endemice Bistriţa şi Hinova decât la locuitorii investigaţi din oraşul non-endemic Drobeta Turnu Severin (p = 0,008 şi respectiv p = 0,0004). Locuitorii din satul endemic Husnicioara (unde o mină cu cărbune din Pliocen este încă funcţională) au a vut o RFG mai mare decât locuitorii investigaţi din Drobeta Turnu Severin şi mai mare decât locuitorii din satul endemic Livezile (mina închisă cu 10 ani în urmă) : p = 0,0055 şi respectiv p = 0,001, dar o RFG mai mică decât locuitorii investigaţi din oraşul minier non-endemic Motru (unde funcţionează o mină ce extrage cărbune non-Pliocenic : p < 0,001. Proteinuria a fost prezentă la 8,03% din locuitorii din satul endemic Bistriţa şi 7,4% din locuitorii din satul endemic Hinova. În oraşul non-endemic Drobeta Turnu Severin proteinuria a fost prezentă la 7,08% din locuitorii investigaţi. Proteinuria a fost prezentă la 0,78% din locuitorii investigaţi din oraşul nonendemic Motru (unde o mină de cărbune non-Pliocenic este funcţională) şi la 2,5% din locuitorii din satul endemic Husnicioara (unde o mină de cărbune din Pliocen este încă funcţională). Lucrarea noastră nu arată nici o relaţie între expunerea la cărbunele din Pliocen şi etiologia NEB. Corresponding author: Prof. Dr. Gh. Gluhovschi Nephrology Department, University of Medicine and Pharmacy, Timişoara E-mail: ggluhovschi @yahoo.com REFERENCES 1. 2. 3. 4. 5. 6. 7.

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SHANKAR A., LEE K.E., KLEIN B.E.K., MUNTNER P., BRAZY P.C., GRUICKSSHANKS K.J., NIETO F.J., DANFORTH L.G., SCHUBERT C.R., TSAI M.Y., KLEIN R., Estimating glomerular filtration rate in a population-based study. Vasc. Health Risk Manag. 2010, 6, 619–627. MELAMED M.L., BAUER C., HOSTETTER T.H., eGFR: is it ready for early identification of CKD. Clin J Am Soc Nephrol. 2008, 3, 1569–1572. GLUHOVSCHI G., MARGINEANU F., VELCIOV S., GLUHOVSCHI C., BOB F., PETRICA L., BOZDOG G., TRANDAFIRESCU V., MODALCA M., 50 years of Balkan Endemic Nephropathy in Romania. Some aspects of the endemic focus in the Mehedinti county. Clin Nephrol 2010 in press. PESCH B., HEARTING J., RANFT U., KLIMPEL A., DELSCHLAGEL B., SCHILL W. and the MURC Study Group. Occupational risk factors for urothelial carcinoma: agent-specific results from a case-control study in Germany. Internat J Epidemiol, 2000, 29, 238–247. GHANEM M., BATTELLI M., LAW B.F., CASTRANOVA V., KASHON M.L., NATH J., HUBBA A.F., Coal dust alters betanephthoflavone-induced aryl hydrocarbon receptor nuclear translocation in alveolar type II cells. Part Fibre Toxicol 2009, 6:21. BONASSI S., MERLO F., PEARCE N., PUNTONI R., Bladder cancer and occupational exposure to polycyclic aromatic hydrocarbons. Int J Cancer 1989, 44, 4, 648–651. MASTRANGELO G., FADDA E., MARZIA V., Polycyclic aromatic hydrocarbons and cancer in man. Environmental Health Perspectives 1996, 107, 10, 1166–1170. GROLLMAN A.P., JELAKOVIC B., Role of environmental toxins in endemic (Balkan) nephropathy. J. Am. Soc Nephrol, 2007, 18, 2817–2823. STEFANOVIC V., RADOVANOVIC Z., Balkan Endemic Nephropathy and associated urothelial cancer. Nat Clin Pract Urol 2008, 5(2), 105–112. GLUHOVSCHI G., MARGINEANU F., KAYCSA A., VELCIOV S., GLUHOVSCHI C., BOB F., PETRICA L., BOZDOG G., DUMITRU S., OLOSZ E., MODALCA M., Therapeutic remedies based on Aristolochia clematitis in the main foci of Balkan Endemic Nephropathy in Romania. Nephron Clin Pract. 2010, 116(1), c36–c46. HRANJEC T., KOVAC A., KOS J., MAO W., CHEN J.J., GROLLMAN A.P., JELAKOVIC B., Endemic nephropathy: the case of chronic poisoning by aristolochia. Croat. Med J 2005, 46, 1, 116–125. COSYNS I.P., JADOUL M., SQUIFFET I.P., DE PLAEN I.F., FERLIGA D., Van YPERSELE De STRIHOU C., Chinese herb nephropathy. A clue to Balkan endemic nephropathy. Kidney Int 1994, 45, 1680–1688. De JONGE H., VANRENTERGHEM Y., Aristolochic acid, the common culprit of Chinese herbs nephropathy and Balkan Endemic Nephropathy. Nephrol Dial transplant 2008, 23, 39–41. PFOHL-LESKOWICZ A., TOZLOVANU M., MANDERVILLE D., PERAICA M., CASTEGNARO M., STEFANOVIC V., New molecular and field evidences for the implication of mycotoxins but not aristolochic acid and urinary tumor. Mol. Nutr. Food Res 2007, 51, 9, 1131–1146. GLUHOVSCHI G., DAMINESCU L., CHETRINESCU N., MARGINEANU F., The study of aflatoxin B1 and Ochratoxin A in the blood of patients with endemic nephropathy, of patients with chronic renal diseases coming from localities with Balkan Endemic Nephropathy (article in Romanian). Nefrologia 2003, 8, 20–21, 63–66. UZELAC-KESEROVIC B., SPASIC P., BOJANIC N., DIMITRIEVIC J., LAKO B., LESPANOVIC Z., KULJIC-KAPULICA N., VASIC D., APOSTOLOV K., Isolation of a coronavirus from kidney biopsies of endemic Balkan Nephropathy patients. Nephron 1999, 81, 141–145. GLUHOVSCHI G., ROSCA A., MARGINEANU F., Hantavirus – specific IgM and IgG in Balkan Endemic Nephropathy and chronic renal disease. Facta Universitatis series Medicine and Biology, Nis, 2002, 9,1, 76–78. KARMAUS W., DIMITROV P., SIMEONOV V., TSOLOVA S., BONEV A., GEORGIEVA R., Metals and kidney markers in adult offspring of endemic nephropathy patients and controls: a two year follow-up study. Envir Health, 2008, 3, 7–11. BARCELOUX D.G., Selenium. J. Toxicol. Clin. Toxicol. 1999, 37, 2, 145–172. TONCHEVA D., DIMITROV T., STOIANOVA D., Etiology of Balkan Endemic Nephropathy: a multifactorial disease. Europ J Epidemiol. 1998, 14, 389–394. OREM W., TATU C., PAVLOVIC N., BUNNELL J., LERCH H., PAUNESCU V., ORDODI V., FLORES D., CORUM M., BATES A., Health effects of toxic organic substances from coal: toward “pandemic nephropathy”. Ambio. 2007, 36, 1, 98–102. OREM W.H., TATU C.A., LERCH H.E., MAHARAJ V.M., PAVLOVIC N., PAUNESCU V., DUMITRASCU V., Identification and environmental significance of the organic compounds in water supplies associated with a Balkan Endemic Nephropathy region in Romania. 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Received December 7, 2010

The Implications of Revised WHO Classification (2008) of Chronic Myeloid Neoplasms MIHAELA GĂMAN, ANA-MARIA VLĂDĂREANU, SÎNZIANA RADESI Department of Hematology, Emergency Universitary Hospital, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

Myeloid disorders constitute a subgroup of hematological malignancies of myeloid lineage. In the revised 2008 WHO classification system, the nomenclature for myeloproliferative entities has been changed from “chronic myeloproliferative diseases” to “myeloproliferative neoplasms”, the definition criteria has changed, and and the MDS/MPN has emerged as a well defined entity the subgroup formerly known as “myelodysplastic/myeloproliferative diseases” has changed to “myelodysplastic/myeloproliferative neoplasms”. The World Health Organization classification of myeloid neoplasms relies on the association of clinical, morphologic, immunophenotypic and genetic diagnostic features. The current review highlights these changes and also provides diagnostic algorithms that are tailored to routine clinical practice. Key words: revised, WHO classification, myelodysplastic syndroms, myeloproliferative neoplasms, myelodysplastic/myeloproliferative neoplasms.

Chronic myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) represent malignant disorders, with an incidence of 6–10 cases/100.000 people/year and > 20 cases/ 100.000 people over 70 years /year and a prevalence that increases continuously [1]. The myelodysplastic syndromes (MDS) are a group of hematologic disorders characterized by ineffective clonal hematopoiesis, morphological dysplasia, peripheral blood cytopenias and progressive bone marrow failure [2][3]. The FAB classification [4] known for more than 20 years classified MDS into five categories: refractory anemia(RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-T) and chronic myelomonocytic leukemia (CMML). In 2001 a group of pathologists and clinicians working under the World Health Organization (WHO) [5–8] modified this classification by lowering the percentage of blasts required to make the diagnosis of AML from 30% to 20%, eliminating RAEBT category, including new categories refractory cytopenia with multilineage dysplasia (RCMD) and “5q-syndrome” and reclassifing chronic myelomonocytic leukemia (CMML) from a subcategory of MDS to a subcategory of myelodysplastic/ myeloproliferative disorder. This classification has recently been revised, as advances in molecular ROM. J. INTERN. MED., 2011, 49, 1, 25–30

biology, immunology, cytology and genetics are being used in the diagnosis, subgrouping and prediction of prognosis for MDS-WHO classification of tumors of haematopoietic and lymphoid tissues 2008. The updated classification of MDS is now available as working document (Table I) [9][10]. The myeloproliferative diseases (MPDs) are characterized by clonal expansion of malignant hematopoietic stem cells that retain the capacity to differentiate into mature blood cells. The 2001 World Health Organization (WHO) classification of hematopoietic tumors [8] presents chronic myeloproliferative diseases (CMPDs) as a subgroup of myeloid neoplasms, that includes the four classic myeloproliferative disorders (MPDs)chronic myelogenous leukemia, polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF)-and chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia/ hypereosinophilic syndrome (CEL/HES) and “CMPD, unclassifiable” [11]. In 2005, the discovery of the JAK2 V617F mutation provided evidence that suggested a common pathogenesis for the Philadelphia Chromosome negative MPDs [12–15] and has raised the prospect of genetic classification and molecular diagnosis in these disorders [16][17]. Then, the discovery of MPLW515L/K gene mutation [18] [19] and of JAK2 exon 12 mutation [20–22] was also reported. The presence of any of these

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molecular abnormalities represents a major diagnostic criterion in the revised classification of myeloid neoplasms of the World Health Organization published in 2008 (Table II). The term “CMPDs” was replaced by “myeloproliferative neoplasms (MPNs)”, reflecting the underlying clonal genetic changes of this group of diseases. The MPN category now includes mast cell disease (MCD) and myeloid neoplasms with molecularly characterized clonal eosinophilia “Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB and FGFR1” [23][24] are now assembled into a new category of their own. Although MDS and MPN appear to have entirely different pathophysiological mechanisms, the existence of overlapping morphological features including hypercellularity, mild/nonspecific dysplastic changes and variable bone marrow fibrosis is well established. The unitary approach to these ethiopathogenic entities is based on the following: 1. The common pathogenic pathway, in both situations, is a primary stem-cell defect. Also, their evolution to acute leukemia, as end event, is mentioned in both cases, but it has a variable rate. 2. The presence of common clinical and biological elements, like predisposition to thrombosis and hemorrhage due to morphologic and functional platelet anomalies, represents an important key. 3. The WHO classification created a new category named myelodysplastic/myeloproliferative neoplasms (9), that includes the disorders characterized by both dysplastic and proliferative features (Table III). The myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are clonal myeloid neoplasms characterized by the simultaneous presence of both myelodysplastic and myeloproliferative features, which prevent them from being classified as either myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN). The etiology of MDS/ MPN is not known. The incidence of MDS/MPD varies widely, ranging from approximately 3 per 100,000 individuals older than 60 years annually for CMML to 0.13 per 100,000 children from birth to 14 years annually for JMML [25]. Data concerning the incidence of aCML, a recently defined

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entity, are not available. The incidence of MDS/ MPD-UC is unknown. The three best-defined entities within the MDS/MPN group are: chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML) and juvenile myelomonocytic leukemia (JMML). Less well defined is a group of MDS/MPN, Unclassifiable (MDS/MPN-UC) [26], that shows features of both myeloproliferative neoplasm and myelodysplastic disease but does not meet the criteria for any of the other MDS/MPN entities. Within this group, the provisional entity RA with ringed sideroblasts and marked thrombocytosis (RARS-T) has a particular significance. Clinically, RARS-T patients present myelodysplastic features, but also have marked thrombocytosis and to some extent myelofibrosis [27][28]. RARS-T is defined by < 5% marrow blasts, > 15% ringed sideroblasts and a persistent platelet count of > 600 ×109/L. In the 2008 proposal, the cut-off value for thrombocytosis was decreased to 450 ×109/L (29). It has recently been described that around half of RARST patients, along with a small subset of other MDS and mixed myelodysplastic/myeloproliferative disorders, carry the JAK2 mutation [30–33] and less commonly, the MPL W515K/L mutation. Chromosomal abnormalities are frequent in myeloid malignancies, but in most cases of MDS/ MPN underlying molecular lesions are unknown or they remain poorly characterized. Mutations of the ten-eleven translocation 2 (TET2) gene are frequently observed in these disorders, approximately 1/4 of patients diagnosed with CMML, a CML and RARS-T. Other mutations like AML1/RUNX1, and N- or K-RAS genes have been reported with low prevalence in MDS/MPN [34][35]. The contribution of these oncogenic events during MDS/MPN pathogenesis regarding clinical behavior, prognosis and treatment response remains to be identified. In conclusion, this review highlights changes in the classification system of myeloid tumors as advances in molecular biology, immunology, cytology, and genetics are increasingly being used in the diagnosis, subgrouping, staging, and prediction of prognosis for these myeloid neoplasms.

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Table I Proposal for updated WHO classification of myelodysplastic syndromes 2008 Disease Bone marrow findings Refractory cytopenias with unilineage dysplasia (RCUD): 1.Refractory anemia (RA) 2.Refractory neutropenia (RN) 3.Refractory thrombocytopenia (RT) Refractory anemia with ringed sideroblasts (RARS)

Blood findings Bone marrow findings Unicytopenia or bicy- Unilineage dysplasia; ≥10% of the cells of the affected lineage topenia* are dysplastic No or rare blasts (5% (BM) Mastocytosis Cutaneous mastocytosis Indolent systemic mastocytosis Systemic mastocytosis with associated clonal haematological non-mast-cell-lineage disease Aggressive Systemic Mastocytosis Mast cell leukemia Mast cell sarcoma Extracutaneous mastocytoma Myeloproliferative neoplasm, unclassifiable Fail to meet the criteria for any of the myeloproliferative neoplasms (MPN, U) Essential thrombocythaemia (ET)

Table III Proposal for updated WHO classification of myelodysplastic/myeloproliferative neoplasms [9] Disease Blood findings Bone marrow findings Molecular basis Dysplasia in one/more myeloid NRAS or KRAS mutations in 1/3 PB monocytosis < 1 ×109 /L CMML No BCR/ABL-1 fusion gene lineage* of patients (uncertain significance < 20% blasts Blasts include myeloblasts, regarding prognosis) monoblasts, promonocytes. TET2 mutations in up to 40% of No rearrangement of PDGFRA or patients RUNX (formerly AML1) PDGFRB mutations in 40% of patients with high WBC CBL mutations in about 10% of patients (35) JAK2 (V617F) found occasional Leukocytosis, Neutrophilia Neutrophil dysplasia with or with- NRAS or KRAS mutations (34) in aCML Neutrophil precursors>10% of leukocytes out other dysplastic lineages about one 1/3 of patients (uncertain Blasts < 20% < 20% blasts significance regarding prognosis) No BCR-ABL1 fusion gene TET2 mutations are found in about No rearrangement of PDGFRA or PDGFRB one (34) Minimal basophilia CBL mutations in about 10% of Mo 1 ×109 /L blasts, monoblasts, promonocytes < 20% blasts Usually WBC > 10 ×109 /L < 20% blasts MDS/MPN MixedMDS and MPN features Mixed MDS and MPN features No prior diagnosis of MDS or MPN < 20% blasts No history of recent growth factor or cytotoxic therapy to explain MDS or MPN features No BCR-ABL1 fusion gene or rearrangements of PDGFRA or PDGFRB † RARS-T Persistent thrombocytosis > 450 ×109 /L Morphologic features of RARS; TET2 mutations in about one (provisional Anemia ≥15% of erythroid precursors are fourth patients (34) entity) BCR-ABL1 negative ringed sideroblasts JAK2 and/or MPL mutation in Cases with t(3; 3)(q21; q26), inv(3)(q21q26), Abnormal megakaryocytes similar 60–80% cases and isolated del (5q) are excluded to those observed in BCR-ABL1– negative MPN ** Hellström-Lindberg and Cazzola, Hematology, 2008 (1): 52 * If myelodysplasia is minimal or absent, CMML can still be diagnosed if the other requirements are met and there is an acquired clonal cytogenetic or molecular genetic abnormality present in the hematopoietic cells, or the monocytosis has persisted for at least 3 months and all other causes of monocytosis have been excluded. † Provisional entity. In WHO classification from 2002 the cut-off for platelet count was 600 ×109 /L. In the WHO 2008 classification the cut-off value is 450 ×109/L, to be in line with the classification of essential thrombocythemia. Around 50% of cases with RARS-T carry the JAK2 mutation, but this is not a diagnostic criterion.

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Afecţiunile mieloproliferative cronice reprezintă un subgrup aparte al neoplaziilor maligne. In cadrul clasificării World Health Organization 2008 au fost modificate următoarele: nomenclatura sindroamelor mieloproliferative cronice în „neoplazii mieloproliferative”, criteriile de diagnostic ale acestora, iar subgrupul neoplazii mielodisplazice/mieloproliferative cronice reprezintă o entitate separată. Clasificarea actuală WHO cuprinde aspecte clinice, morfologice, imunofenotipice şi genetice, scopul acestei lucrari fiind sublinierea modificarilor survenite şi prezentarea unui algoritm de diagnostic util în practica clinică curentă. Corresponding author: Mihaela Găman, MD Department of Hematology, Emergency Universitary Hospital 169, Splaiul Independenţei, 050098, Bucharest, Romania E-mail: [email protected]

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ORIGINAL ARTICLES

Correlations between the Surface ECG and the Intracavitary Electrocardiogram in Typical Atrial Flutter R. ROŞU1, L. MUREŞAN1, M. ANDRONACHE2, DANA POP1, C. POP3, MARIA PUŞCHIŢĂ4, ADINA MĂLAI1, G. GUŞEŢU1, D. ZDRENGHEA1 1

SCU Cluj-Napoca, Cardiology – Rehabilitation Hospital, Cluj-Napoca, Romania 2 CHU Nancy Brabois, Department of Electrophysiology, Nancy Cedex, France 3 “Constantin Opriş” Baia Mare Emergency County Hospital, Baia Mare, Romania 4 Arad County Hospital, Arad, Romania

Typical atrial flutter (cavo-tricuspid isthmus-dependent) has as an electrophysiological substrate a macro-reentry circuit localized in the right atrium. Depending on the right atrial depolarization sequence, the rotation of the macro-reentry circuit can be counterclockwise (with an inferior to superior activation of the right atrium free wall and superior to inferior activation of the interatrial septum), characterized by negative F waves in inferior leads (DII, DIII, aVF) and V6, and positive in V1 on the surface electrogram (ECG), or clockwise (with a superior to inferior activation of the right atrium free wall and inferior to superior activation of the interatrial septum) characterized by positive F waves in inferior leads (DII, DIII, aVF) and V6, and negative in V1. Nevertheless, it is considered that for the diagnosis of the typical or atypical nature of this arrhythmia, the surface ECG has limited value. The purpose of this study was to compare the relationship between the flutter rotation sequence determined by the intracavitary electrogram and the morphology of the F waves on the surface ECG. Methods. The study included 387 patients admitted to the Cardiology – Rehabilitation Hospital from Cluj-Napoca between January 2007 and May 2010, diagnosed with typical atrial flutter during an electrophysiological study. Using the intracavitary electrograms the flutter rotation sequence was determined (clockwise or counterclockwise). The F waves’ aspect on the surface ECG in leads DII, DIII, aVF, aVL, V1 and V6 was then analyzed. Results. One hundred and fifty two patients (39.3%) were diagnosed with clockwise atrial flutter and 235 patients (60.7%) with counterclockwise atrial flutter. The positive predictive value (PPV) of negative F waves in inferior leads and positive in V1 was, in the case of counterclockwise atrial flutter 98%; the negative predictive value (NPV) was 79%; sensitivity (Se) was 83% and specificity (Sp) was 97%. For typical clockwise atrial flutter, the PPV of the positive F waves in the inferior leads and negative in V1 was 94% (p