Neutrophil Lactoferrin Content in Viral Infections

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4. 5. 6. 7. 8. 9. 10.

of oral urea administration on red cell survival in sickle cell disease. Am J Med Sci 1972;264:283-287. Berk PD, Blaschke TF, Scharschmidt BF, Waggoner JG, Berlin NI. A new approach to quantitation of the various sources of bilirubin in man. J Lab Clin Med 1976;87:767-780. Bertles JF, Dobler J. Reversible and irreversible sickling: a distinction by electron microscopy. Blood 1969;33:884-898. Cline MJ, Berlin NI. An evaluation of DF32P and 5,Cr as methods of measuring red cell lifespan in man. Blood 1963;22:459-465. Coburn RF, Williams WJ, Forster RE. Effect of erythrocyte destruction on carbon monoxide production in man. J Clin Invest 1964;43:1098-1103. Coburn RF, Williams WF, Kahn SB. Endogenous carbon monoxide production in patients with hemolytic anemia. J Clin Invest 1966;45:460-468. Collison HA, Rodkey FL, O'Neal JD. Determination of carbon monoxide in blood by gas chromatography. Clin Chem 1968;14:162-171. Coltman CA Jr, Dudley GM III. The relationship between endogenous carbon monoxide production and total heme mass in normal and abnormal subjects. Am J Med Sci 1969,238:374385. Gillette PN, Manning JM, Cerami A. Increased survival of sickle cell erythrocytes after treatment in vitro with sodium cyanate. Proc Natl Acad Sci USA 1971;68:2791-2793.

12. Kambam JR, Chen LH, Hyman SA. Effect of short-term smoking halt on carbonxyhemoglobin levels and Pj 0 values. Anesth Analg 1986;65:1186-1188. 13. May A, Bellingham AJ, Huehns ER, Beaven GH. Effect of cyanate on sickling. Lancet 1972;1:658-661. 14. McCurdy PR. DF32P and 51Cr for measurement of red cell life span in abnormal hemoglobin syndromes. Blood 1969;33:214-222. 15. McCurdy PR, Sherman AS. Irreversibly sickled cells and red cell survival in sickle cell anemia: a study with both DF32P and "Cr. Am J Med 1978;64:253-258. 16. Milner PF, Charache S. Life span of carbamylated red cells in sickle cell anemia. J Clin Invest 1973;52:3161 -3171. 17. Padilla F, Bromberg PA, Jensen WN. The sickle-unsickle cycle: a cause of cell fragmentation leading to permanently deformed cells. Blood 1973;41:653-660. 18. Pearson HA, Noyes WD. Failure of phenothiazimes in sickle cell anemia. JAMA 1967;199:91-92. 19. Rodgers GP, Schechter AN, Noguchi CT, Klein HG, Nienhuis AW, Bonner RF. Periodic microcirculatory flow in patients with sickle cell disease. N Engl J Med 1984;311:1534-1538. 20. Serjeant GR, Serjeant BF, Milner PF. The irreversibly sickled cell: a determinant of hemolysis in sickle cell anemia. Br J Haematol 1969;17:527-533.

Neutrophil Lactoferrin Content in Viral Infections ROY D. BAYNES, M.MED, WERNER R. BEZWODA, PH.D., AND NAZMA MANSOOR, B.Sc.

In an attempt to elucidate the previously observed decrease in plasma lactoferrin-neutrophil ratio in subjects with acute viral infections, a study of the neutrophil lactoferrin content in such infections was undertaken. With the use of an immunoperoxidase stain for lactoferrin, neutrophils in viral illness were shown to have reduced lactoferrin content (mean score 97.9 ± SD (38.0] per 100 neutrophils) as compared with normal subjects (mean score 196.4 ± SD [3.6] per 100 neutrophils) (t = 7.69; P < 0.0005). This suggests an acquired defect of neutrophil lactoferrin synthesis in viral infection. Thisfindingis of importance when seen against the well-recognized increased risk of bacterial superinfection in subjects who have recently had a viral infection. (Key words: Neutrophil; Lactoferrin; Viral illness) Am J Clin Pathol 1988;89:225-228 DATA PREVIOUSLY REPORTED from this laboratory have demonstrated that the plasma lactoferrin concentration is reduced in subjects with acute viral illnesses.2 The reduced plasma concentration of lactofer-

Department of Hematology and Oncology and Department of Medicine, University of the Witwatersrand Medical School, Johannesburg, South Africa

rin in patients with viral infection occurred despite the fact that total neutrophil counts in these subjects were comparable to those of normal persons. There was thus a significant reduction in the lactoferrin-neutrophil ratio in subjects with viral illness. It was speculated that the mechanisms responsible might include an acquired defect of lactoferrin synthesis by neutrophils, defective or inhibited degranulation, or increased lactoferrin clearance. The current study was undertaken in an attempt to clarify the answer to this question. Subjects and Methods

Received March 11, 1987; received revised manuscript and accepted for publication June 8, 1987. Supported by a grant from the Medical Research Council of South Africa. Address reprint requests to Dr. Bezwoda: Department of Medicine, University of the Witwatersrand Medical School, York Road, Parktown, 2193, Johannesburg, South Africa.

Twenty-six young adults with viral illnesses were studied together with nine normal young adults. The viral diagnoses were based on clinical presentation and positive serologic test results. They included chickenpox (11 subjects), measles (3 subjects), rubella (8 subjects), Ebstein-Barr virus infection (2 subjects), and 1 subject each

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FIG. 1. Scatterplot of neutrophil lactoferrin content based on the score obtained on 100 neutrophils per smear. Score: absent = 0; positive = 1; strong positive = 2.

with hepatitis A and hepatitis B. Each subject was evaluated during the acute phase of illness. Freshly prepared blood smears were made on glass slides, air dried, and then fixed for 2 minutes in methanol. The authors and other laboratory staff members were the control subjects. They were tested at a time when they were feeling in good health with no clinical evidence of intercurrent viral infection. The studies had been approved by the Committee for Research on Human Subjects of the University of the Witwatersrand. Immunoperoxidase staining was performed with a polyclonal monospecific rabbit antilactoferrin antibody prepared in this laboratory by previously reported methods.3 Smears were rehydrated by sequential passage through graded alcohol solutions (absolute, 90%, 70%) and water. After being rinsed in phosphate-buffered saline (PBS), the slides were immersed in a 2.5% hydrogen peroxide solution to quench nonspecific peroxidase activity. After being rinsed again in PBS, the slides were flooded with nonimmune goat serum and incubated at room temperature. After again being rinsed in PBS, the slides were incubated with a 1:2,000 dilution of a 2 g/L antilactoferrin antibody in PBS. Thereafter, the slides were again rinsed and incubated with horseradish peroxidase-conjugated goat antirabbit IgG (1:4,000 dilution). After being rinsed again, the slides were immersed in a solution of 3,3' diaminobenzidine. The slide was then slowly washed, lightly counterstained with Meyer's hematoxylin, washed in water, immersed in Scott's tap water substitute, dehydrated, cleared in xylene, and mounted. Each batch of samples incorporated a nega-

tive control (a smear processed normally except for the ommission of treatment with an antilactoferrin antibody), and each batch incorporated both normal subjects and those with viral illness. The method of slide preparation—namely preparation of a smear, followed by air drying and fixation in methanol for 2 minutes— resulted in a nuclear localization pattern of the stained lactoferrin.7 The stained smears were then examined microscopically. A scoring system was employed that scored a cell with negative findings as 0, a cell with positive findings as 1, and a cell with strongly positive findings as 2. One hundred neutrophils per slide were scored without the examiners' prior knowledge of the diagnosis. In all cases the negative control was accurately identified blind, thus showing adequate quenching of endogenous peroxidase activity. In addition, only neutrophils were noted to stain positively. Results The results of the neutrophil lactoferrin scores based on 100 neutrophils per slide are summarized on a scatterplot in Figure 1. The mean score for neutrophils in normal subjects was 196.4 ± 3.6 per 100 neutrophils. The comparable score in subjects with viral illness was significantly different at 97.9 ± 38.0 per 100 neutrophils (t = 7.69, P < 0.0005). Interestingly, the two subjects with viral illness who had a high lactoferrin score both had Ebstein-Barr viral infections. A representative example of normal and viral illness neutrophil staining for lactoferrin is shown in Figure 2. Discussion In a previous study from this laboratory, it has been demonstrated that plasma lactoferrin content levels are abnormally low in patients with viral infection. Further confirmation of abnormal lactoferrin concentrations in viral illness has come from the demonstration that cerebrospinal fluid lactoferrin concentration is reduced in subjects with viral infections of the central nervous system.10 From the current study it would seem that the reduced plasma concentrations of lactoferrin observed in some subjects with viral illness are associated with a reduced neutrophil lactoferrin content. These findings suggest that some viral illnesses result in an acquired defect of neutrophil lactoferrin synthesis. Additional studies of lactoferrin mRNA content in granulocyte precursors in normal subjects and those with viral illness are indicated to establish whether this is occurring at a transcriptional level. These findings, however, may be true only of certain viral infections. A number of observations suggest that


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FIG. 2. Photomicrograph showing normal lactoferrin staining in a neutrophil (left) and that obtained with neutrophils in subjects with viral illness (right) (X1,000). O

cases have frequently exhibited an abnormal predisposition to bacterial infection. The current study suggests that certain viral infections may well be another cause of acquired lactoferrin deficiency. References 1. Abramson JS, Parce JW, Lewis JC, et al. Characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses. Blood 1984;64:131-138. 2. Baynes RD, Bezwoda WR, Khan Q, Mansoor N. Plasma lactoferrin content: differential effect of steroid administration and infective illnesses: lack of effect of ambient temperature at which specimens are collected. Scand J Haematol 1986;37:353-359. 3. Bezwoda WR, Baynes RD, Khan Q, Mansoor N. Enzyme linked immunosorbent assay for lactoferrin. Plasma and tissue measurements. Clin Chim Acta 1985;151:61-69. 4. Birgens H. The biological significance of lactoferrin in haematology. Scand J Haematol 1984;33:225-230. 5. Boxer LA, Coates TD, Haak RA, Wolach JB, Hoffstein S, Baehner RL. Lactoferrin deficiency associated with altered granulocyte function. N Engl J Med 1982;307:404-410. 6. Breton-Gorius J, Mason DY, Buriot D, Vilde J-L, Griscelli C. Lactoferrin deficiency as a consequence of a lack of specific granules in neutrophils from a patient with recurrent infections. Am J Clin Pathol 1980;99:413-419. 7. Briggs RC, Montiel MM, Wojtkowiak Z. Nuclear localization of lactoferrin in the human granulocyte: artefact incurred by slide preparation. J Histochem Cytochem 1983;31:1152-1162. 8. Bullen JJ, Rogers HJ, Leigh L. Iron binding proteins in milk and resistance to Escherichia coli infection in infants. Br Med J 1972;1:69-75. 9. Gallin JI, Fletcher MP, Seligmann BE, Hoffstein S, Cehrs K, Mouriessa N. Human neutrophil-specific granule deficiency: a model to assess the role of neutrophil-specific granules in the evolution of the inflammatory response. Blood 1982;59:1317— 1329. 10. Hallgren R, Terent A, Venge P. Lactoferrin, lysozyme and B2-microglobulin levels in cerebrospinal fluid: differential indices of CNS inflammation. Inflammation 1982;6:291-304. 11. Hanson LA, Andersson B, Carlsson B, et al. Defence of mucous membranes by antibodies, receptor analogues and nonspecific host factors. Infection 1984; 12:111-115.


in some instances defective release of lactoferrin may be caused by viral infection. First, Abramson and coworkers have reported that in vitro degranulation is abnormally reduced in neutrophils experimentally exposed to influenza virus.1 It has also been demonstrated that in vitro infection of mononuclear leukocytes with influenza and respiratory syncytial viruses results in the production of significant amounts of anti-interleukin-1 activity by macrophages in vitro.16 Release of anti-interleukin-1 activity would be expected to impair neutrophil degranulation,12 resulting in low plasma levels but elevated neutrophil levels of lactoferrin. Another observation that suggests a heterogeneous neutrophil response to viral infection is that two patients with clinically proven viral infection in this study appeared to have a normal content of neutrophil lactoferrin. Both of these patients had Ebstein-Barr virus infection. Unfortunately, no patients with proven influenza virus or respiratory syncytial virus infection were available for study. It should be noted that the vast majority of subjects studied had viral infections associated with skin exanthemata. Determination of whether these alterations in neutrophil lactoferrin are peculiar to these viruses or reflect a wider spectrum of viral disease requires additional evaluation. One additional point may be emphasized. The finding of a reduced neutrophil content of lactoferrin together with reduced plasma concentrations in viral illness may be of relevance for the host defense mechanisms. Bacterial infection and superinfection have been noted as frequent complications of viral illness. Lactoferrin has been shown to have a number of functions in host defense against bacterial infection.4-8,13"1518 Congenital569 and acquired1117 defects of lactoferrin production by neutrophils have previously been reported. The reported

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12. KJempner MS, Dinarello CA, Gallin JJ. Human leukocyte pyrogen induces release of specific granule contents from human neutrophils. J Clin Invest 1978;61:1330-1336. 13. Lima MF, Kierszenbaum F. Lactoferrin effects on phagocytic cell function. 1. Increased uptake and killing of an intracellular parasite by murine macrophages and human monocytes. J Immunol 1985;134:4176-4183. 14. Oseas R, Yang HH, Baehner RL, Boxer LA. Lactoferrin: a promoter of polymorphonuclear leukocyte adhesiveness. Blood 1981;57:939-945. 15. Reiter B. The biological significance of lactoferrin. International Journal of Tissue Reactions 1983;5:87-96.

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16. Roberts NJ, Prill AH, Mann TN. Interleukin-1 and interleukin-1 inhibitor production by human macrophages exposed to influenza virus or respiratory syncytial virus: respiratory syncytial virus is a potent inducer of inhibitory activity. J Exp Med 1986;163:511-519. 17. Venge P, Foucard T, Henrickson J, Hakansson L, Kreuger A. Serum levels of lactoferrin, lysozyme and myeloperoxidase in normal, infection-prone and leukemic children. Clin Chim Acta 1984;136:121-130. 18. Vercellotti GM, van Asbeck S, Jacobs HS. Oxygen-radical-induced erythrocyte hemolysis by neutrophils: a critical role of iron and lactoferrin. J Clin Invest 1985;76:956-962.

Evaluation of a Latex Agglutination Test for Diagnosis of Clostridium d\W\c\\e-Associated Colitis

Current methods for diagnosis of Clostridium difficile-associated colitis (CAC) based on detection of cytotoxin B by a tissue culture assay (TCA) require technical expertise and up to 48 hours incubation. Recently, a latex agglutination (LA) test (Marion Laboratories) for rapid diagnosis of CAC has become available. Although early evaluations have been favorable, new evidence suggests that the LA reagent binds a soluble bacterial antigen that is not unique to toxigenic strains of C difficile. The authors examined 201 stools received for CAC testing by LA and a reference TCA and investigated discrepant results. They obtained 29 LA(+)/TCA(+) and 155 L A ( - ) / TCA(-) results. Eleven patients had LA(+)/TCA(-) results and 6 had LA(—)/TCA(+) results. The sensitivity and specificity of the LA were 83% and 93%, respectively, compared with TCA. The predictive values of positive and negative results obtained with the LA were 72% and 96%, respectively. Concentrated broth supernatants and live suspensions of three C. difficile isolates with LA(+)/TCA(—) results were tested in a rabbit ileal loop assay. All failed to demonstrate ability to produce an enterotoxin. The authors conclude that the LA method is suitable for rapid screening, but LA(+) results require confirmation by testing with other methods. (Key words: Clostridium difficile colitis; Antibiotic-associated diarrhea; Antibiotic-associated pseudomembranous colitis; Latex agglutination test; Clostridium difficile toxin) Am J Clin Pathol 1988;89:228-233

ANTIBIOTIC-ASSOCIATED colitis resulting from toxigenic strains of Clostridium difficile is a potentially Received May 5, 1987; received revised manuscript and accepted for publication June 17, 1987. Presented in part at the 26th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, Louisiana, 1986. * Current address: Montefiore Hospital and Medical Center, Bronx, New York. Address reprint requests to Dr. DeGirolami: Department of Clinical Pathology, New England Deaconess Hospital, 185 Pilgrim Road, Boston, Massachusetts 02215.

Department of Pathology, New England Deaconess Hospital and Department of Infectious Disease, Children's Hospital Medical Center, Boston, Massachusetts

life-threatening illness that demands prompt recognition and appropriate medical intervention. Although fulminant pseudomembranous colitis is often apparent clinically, laboratory testing is essential for diagnosis of most cases of Clostridium-associated colitis (CAC). Numerous laboratory techniques for detection of pathogenic strains of C. difficile have been devised,30,37 but the ideal test still awaits development. Pathogenic strains of C. difficile are generally believed to produce both enterotoxin (toxin A) and cytotoxin (toxin B), whereas nonpathogenic colonizers secrete neither toxin.21'25 Therefore, demonstration of either toxin should secure the diagnosis of CAC. Methods based on recognition of the characteristic cytopathic effect of toxin B in tissue culture assay (TCA) are of proven clinical utility,410 but require technical expertise and up to 48 hours incubation. Some workers have suggested that detection of toxin B by TCA may lack sensitivity,29 and several studies have documented that toxin B titers do not correlate consistently with severity of clinical disease.3'4,7'13 Although the relative contributions of toxin A, toxin B, and a motility factor17 to human disease are unknown, toxin A appears responsible for much of the intestinal pathologic findings observed in experimental animal models.2'22"24 The pathogenic significance of C. difficile strains isolated from toxin-negative stools of pa-


MARK E. SHERMAN, M.D.,* PAOLA C. DEGIROLAMI, M.D., GRACE M. THORNE, PH.D. JUDITH KIMBER, MT(ASCP), AND KAREN EICHELBERGER, MT(ASCP)