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Immunologic abnormalities in patients with hemophilia A
Immunologic abnormalities in patients with hemophilia A To assess the immunologic status of healthy persons with hemophilia ,4, we performed studies of T cell immunity in 21 patients, 10 given only cryoprecipitate and 11 given factor VIII concentrate. Patients in the factor VIII group had significantly decreased helper/suppressor T cell ratios. Both groups had diminished mononuclear cell response to phytohemagglutinin and normal mixed lymphocyte culture, compared with controls. Abnormalities in T cell number or function did not correlate with the presence of antibody to cytomegalovirus, Epstein-Barr virus, or hepatitis B. Physicians caring for patients with hemophilia ,4 should realize that asymptomatic individuals may have early evidence of immunodeficiency. (J PEDIATI~ 103:692, 1983)
Peggy S. Weintrub, M.D., Marion A. Koerper, M.D., Joseph E. Addiego, Jr., M.D., W. Lawrence Drew, M.D., Ph.D., Evelyn T. Lennette, Ph.D., Richard Miner, B.A., Morton J. Cowan, M.D., and Arthur J. Ammann, M.D. S a n F r a n c i s c o a n d O a k l a n d , Calif.
THREE PATIENTS WITH HEMOPHILIA A, receiving factor VIII concentrate, have been reported who developed Pneumocystis carinii pneumonia and acquired immunodeficiency syndrome? Infections with opportunistic pathogens have not previously been recognized in this population, and therefore concerns have been raised that therapy might result in AIDS. In the current epidemic of AIDS in homosexual men, Haitians, and drug addicts, studies of the immune system have shown abnormalities in these groups, including cutaneous anergy, lymphopenia, decreased numbers of T cells, reduced helper/suppressor T cell ratios, and depressed proliferative responses of peripheral blood mononuclear cells to mitogens, antigens, and allogeneic cells. 2-4 Apparently healthy homosexuals also have evidence of immunologic dysregulation, but to a lesser degree
From the Divisions of Pediatric Immunology-Rheumatology and Hematology-Oncology, University of California, San Francisco, the Department of Hematology-Oncology, Children's Hospital Medical Center, and the Department of Pathology and Laboratory Medicine. Mt. Zion Hospital and Medical Center. Supported by grants from the National Hemophilia Foundation, Judith Graham Pool Post-Graduate Rd'~e~rch Fellowship, Immunology Research Foundation, Clinical Research Center Grant RRO1271-O1, and the John Kerner Fund, Mt. Zion Hospital and Medical Center. Reprint requests: Arthur J. Ammann, M.D., Pediatric lmmunology-Rheumatology, M-679, University of California, San Francisco, San Francisco, CA 94143.
692
TheJournalofPEDIATRICS
than those with clinical evidence of disease? ,6 To determine whether apparently healthy patients with hemophilia A have laboratory evidence of AIDS, we performed studies of T cell immunity in 21 patients.
See related article, 103:18, 1983.
AIDS Acquired immune deficiency syndrome EBNA Epstein-Barr nuclear antigen EBV Epstein-Barr virus Hb~Ag ,,Hepatitis B surface antigen HBSS Hanks Balanced Salts Solution HBV Hepatitis B virus MLC Mixed lymphocyte culture PBMC Peripheral blood mononuclear cells VCA Viral capsid antigen
,METHODS Patients were from the hematology clinics of the University of California, San Francisco, and the Children's Hospital Medical Center, Oakland, California. Twentyone healthy prepubescent or heterosexual males with hemophilia A were evaluated. The patients were divided into two groups: those given only cryoprecipitate (10 patients) and those given factor VIII concentrate (11 patients), many of whom had received prior cryoprecipi-
Volume 103 Number 5
Immunologic abnormalities in hemophilia A
693
Table I. T cell numbers in patients with hemophilia A
Group Control Cryoprecipitate Factor VIII
TCR (%)
OKT4 (helper) (%)
OKT8 (suppressor) (%)
OKT4/8 (helper/suppressor)
67.5 + 7.6 52.4 + 14.2 65.0 _+ 8.3
54.2 + 4.6 49.8 _+ 9.1 44.5 _+ 9.8
28.0 _-!- 4.0 24.2 _+ 3.4 33.8 + 13.6
2.1 _+ 0.5 2.1 _+ 0.5 1,6 + 0.7
Values are mean + SD.
tote therapy. Patients in the cryoprecipitate group ranged in age from 10 months to 18 years (mean 5.5 years), and those in the factor VIII group from 3 to 21 years (mean 12.5 years). Controls were healthy, heterosexual men and women. A complete blood count with differential was obtained in 20 of the 21 patients and sent to the clinical laboratory to determine absolute lymphocyte counts; T cell numbers were determined by sheep red blood cell rosetting techniques. Preparation of peripheral blood mononuelear cells. Heparinized blood was diluted in phosphate-buffered saline solution and centrifuged over Ficoll-Hypaque (SG 1.077). The PBMCs at the interface were washed three times with Hanks Balanced Salt Solution and resuspended to the desired concentration. Subset enumeration. Indirect immunoflu0rescence was used to measure OKT4 (helper) and OKT8 (supressor) positive lymphocytes. Briefly, l06 PBMCs in 0.1 ml HBSS were added to 10 /~g monoclonal antibody (OKT4 or OKT8; Orthodiagnostics, Raritan, N J) and incubated on ice for 45 minutes. Cells were washed three times with phosphate-buffered saline solution containing 2% bovine serum albumin and 0.01% sodium azide. Then fluoresceinlabeled goat anti-mouse IgG (Meloy Laboratories, Springfield, VA) was added and the cells incubated on ice for an additional 45 minutes. The cells were washed three times, and the percent of positive staining lymphocytes, determined by size and morphologic characteristics, were counted with a Zeiss phase-contrast microscope with epifluorescence. A minimum of 200 cells per patient were counted; PBMCs from one or more controls were counted each time. Phytohemagglutinin response. The proliferative response to PHA was measured using a microculture technique, previously described: Briefly, PBMCs were resuspended to 5 )< 104 cells/ml in RPMI 1640 medium supplemented with 15% heat-inactivated pooled human AB plasma, 20 mmol L-glutamine, 25 mmol HEPES buffer, and 100 ~t~/ml penicillin and streptomycin. Cultures were set up in five replicates in round-bottom microculture plates (Linbro Scientific, McLean, VA), and PHA in final concentrations of 0, 2, and 5 ug/ml, and were cultured for 7 days in a
Table II. PBMC function in patients with hemophilia A Group Control Cryoprecipitate Factor VIII
PHA (epm)
MLC (cpm)
41,873 _+ 11,963 23,277 _+ 7,933 (P = 0.001)* 21,537 _+ 7,493 (P = 0.001)*
16,108 _+ 6,953 12,039 + 1,646 (NS) 12,483 _+ 6,573 (NS)
Values are mean_+SD. *Patient groupvs controlgroup.
humidified 95% air/5% CO2 atmosphere. One day prior to harvesting, 3H-methyl thymidine (New England Nuclear, Boston, MA) was added. The cells were harvested with an automated multiple-sample harvester onto glass fiber filters, and the radioactive counts per minute measured with a liquid scintillation counter (LS7000; Beckman Instruments, Chicago, IL). The PHA response was defined as the maximum of the mean counts per minute resulting from the two PHA concentrations tested. PBMCs from at least one normal control were tested with each run. Allogeneie response. PBMCs from patients were used as responder cells, and radiated (3000 R) PBMCs from healthy donors as stimulator cells in a one-way mixed lymphocyte culture. Cultures with 2 • 105 cells each of stimulator and responder cells in supplemented RPMI 1640 medium as described above were set up in triplicate in flat-bottom microculture plates, and incubated for 6 days in a humidified 95% air/5% CO2 atmosphere. Each well was pulsed with 3H-thymidine 489 hours prior to harvesting and counting, as described above. PBMCs from at least one control were tested with each run. Viral antibo6y titers. Cytomegalovirus antibodies were measured using a nuclear antieomplement immunofluorescence test that used as antigen the isolated nuclei of virus-infected fibroblasts, as described elsewhere) Measurements of antibodies to Epstein-Barr virus capsid antigen, to D and R components of the early antigen complex were done by indirect immunofluorescence procedures described by Henle et al. 9 Antibodies to EB nuclear antigen were assayed by methods of Reedman and Klein, ~~ with guinea pig complement and fluorescein-labeled rabbit anti-guinea pig C substituted for the human counterparts.
694
Weintrub et al.
Hepatitis B surface antigen and antibody to Hb~Ag were detected by standard radioimmunoassay. Statistics. Differences in T cell numbers and subsets were calculated using the Student two-tailed unpaired t test. Difference in mean P H A and MLC were evaluated with the Mann-Whitney U test. RESULTS Absolute lymphocyte counts were normal in all patients. In patients given cryoprecipitate, the mean T cell percentages were significantly decreased, compared with control values (P = 0.005); the mean helper/suppressor ratio was no different from that in controls (Table I). In patients given factor VIII, the T cell percentages were similar to those in controls. The helper/suppressor ratio was significantly decreased (P = 0.04) as a result of a decreased percentage of helper cells (P = 0.003); the difference in suppressor cells was statistically insignificant. Results of PBMC functional studies, PHA, and MLC are shown in Table II. In both cryoprecipitate and factor VIII groups, the mean response to P H A was significantly decreased. The mean M L C response in both groups was similar to control values. Viral titers to cytomegalovirus (IgM and IgG) and Epstein-Barr virus (anti-VCA, anti-EA, and anti-EBNA) were determined for 18 of the 21 patients. Seven patients had evidence of past CMV infection; in one patient, titers suggested active or recent infection (IgM 1:32, IgG 1 : 512). Five patients had antibody titers to EBV indicative of past infection, and two patients had elevated anti-VCA with low anti-EBNA, a pattern that has been shown to represent either recent or past infection with recent immunosuppression. H The number of patients with CMV and EBV positive titers was similar in the two groups. All 21 patients were evaluated for the presence of Hb~Ag and for antibody to Hb~Ab. All patients in the factor VIII group were HbsAg negative and Hb~Ab positive. All of the 11 patients in the cryoprecipitate group were HbsAb negative, and 10 of the 11 were HbsAg negative. The presence of antibody to HBV, CMV, or EBV did not correlate with alterations in T cell numbers or PBMC function, with one exception: both patients with EBV antibody titers indicating ~ecent infection or immunosuppression had at least one significant T cell abnormality (one patient had 18% T cell rosettes and 30% helper cells, the other had a helper/ suppressor ratio of 0.4). DISCUSSION Our results show that asymptomatic patients with hemophilia A have altered immunologic function. Our patients given cryoprecipitate have decreased percentages of T cell rosettes, and those given factor VIII have depressed
The Journal of Pediatrics November 1983
helper/suppressor ratios; both groups have diminished PBMC responses to PHA. There is no clear explanation of why the two groups have different mean T cell numbers and subsets but similar functional responses. The T cell abnormalities in our group of patients are not as severe as those in patients with hemophilia and AIDS. The three hemophilic patients reported with P. carinii pneumonia all had lymphopenia, and in the two patients tested, mitogen responses were absent? It is possible that the abnormalities in our patients are progressive and that further reduction in their immunologic function may result in increased numbers of opportunistic infections. The cause of AIDS is not known. Drug abuse, homosexuality, and sexual promiscuity, common in other patient populations with AIDS, were not present in our patients. One possible cause, which is most consistent with epidemiologic findings in AIDS, is an immunosuppressive infectious agent. Homosexual men have increased exposure to numerous viral infections (particularly HBV, CMV, and EBV, 11"14all of which have immunosuppressive effects) 5-t7 Factor VIII concentrate, used in one group of patients, is a lyophilized pooled-donor product and carries a high risk of hepatitis B transmission; for this reason, all patients receiving this therapy have antibody to Hb~Ag. Cryoprecipitate is a single-donor blood product. It is likely that, through transfused blood products, patients with hemophilia A have increased exposure to other viruses as well. TM Although viral infections do result in an abnormal helper/ suppressor cell ratio, cell proportions return to normal several weeks to months after acute infection) 9'2~We did not find a correlation between abnormal studies and evidence of past CMV, HBV, or EBV infection. It has been postulated that the abnormal distribution of helper and suppressor cells is a result of chronic immune complex formation. Some patients with AIDS have been reported to have elevated circulating immune complexes, z.4 Patients with hemophilia A who are receiving factor VIII concentrate therapy~ are exposed to repeated doses of hepatitis B, which results in chronic immune complex formation3 ~ This could result in immunologic dysregulation by covering receptor sites on T cells, stimulating anti-idiotypic antibodies (with stimulation of suppressor cells or other factors), or by adhering to T cells and causing selective removal of T cell subsets in the spleen. Other antigens present in concentrate or precipitate, such as histocompatibility or protein antigens, might also result in chronic immune complex formation or production of antibody to autoantigens, resulting in immunologic dysregulation. The clinical status and the degree of immunologic suppression are different in our patients and in those reported with P. carinii pneumonia. This finding suggests
Volume 103 Number 5
Immunologic abnormalities in hemophilia A
either a spectrum of immunologic dysregulation in the patients or progressive immunologic attrition. Sequential studies of immunologic function should be performed in patients with newly diagnosed or established hemophilia A. We have shown that clinically healthy patients with hemophilia A given cryoprecipitate or factor VIII have decreased T cell percentages and helper/Suppressor ratios, respectively. Both patient groups have diminished proliferative iesponses to the mitoge n P H A . Past infection with C M V , EBV, or H B V did not correlate with abnormal studies. A d d i t i o n a l evaluation of the cause and natural history of these abnormalities i s needed, because this information could influence future therapeutic regimens used in hemophilia. It is important for clinicians caring for patients with hemophilia +to realize that asymptomatic individuals may have early evidence of immunodeficiency.
defect in selective IgA deficiency using a microculture method for PHA stimulation and limiting dilution. Clin Immunol Immunopathol 17:595, 1980. Mintz L, Miner RC, Yeager AS: Anticomplement immunofluorescence test that uses isolated fibroblast nuclei for detection of antibodies to human cytomegalovirus. J Clin Microbiol 12:562, 1980. Henle W, Henle G, Horwitz CA: Infectious mononucleosis and Epstein-Barr virus associated malignancies. In Lennette EH, Schmidt N J, editors: Diagnostic procedures for viral, rickettsial and chlamydia infections, ed 5. Washington, DC, 1979, American Public Health Association, pp 441-470. Reedman BM, Klein G: Cellular localization of Epstein-Barr (EBV) associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int J Cancer 1i:499, i973. Henle W, Henle G: Consequences of persistent Epstein-Barr virus'infections. Cold Spring Harbor Conf Cell Prolif 7:3, 1980. Drew WL, Mintz L, Miner RC, Sands M, Ketterer B: Prevalence of cytomegalovirus infection in homosexual men. J Infect Dis 143:188, 1981. Abrams DI: Lymphoproliferative diseases in homosexual males. In Purtilo DT, editor: Immune deficiency and cancer: Epstein-Barr virus and lymphoproliferative malignancies. New York, 1983, Plenum Press. Dietzman DE, Harnisch JP, Ray CG, Alexander ER, Holmes KK: Hepatitis B surface antigen (HB~Ag) and antibody to HB~Ag: Prevalence in homosexual and heterosexual men. JAMA 238:2625, 1977. Mengi R J, Niederman JC, Kelleher JE, et al: Depression of cell-mediated immunity during acute infectious mononucleosis. N Engl J Med 291:1149, 1974. Rinaldo CR, Walter WP, Richter BS, Black PH, Hirsch MS: Mechanisms of immunosuppression in cytomegaloviral mononucleosis. J Infect Dis 141:488, 1980. Thomas HC, Brown D, Labrooy J, Epstein O: T cell subsets in autoimmune and HBV-induced chronic liver disease, HBs antigen carriers with normal histology and primary biliary cirrhosis: A review of the abnormalities and the effects of treatment. J Clin Immunol 2:575, 1982. Gomperts ED, Lazerson J, Berg D, Lockhart D, SergisDeavenporte E: Hepatocellular enzyme patterns and hepatitis B virus exposure in multitransfused young and very young hemophilia patients. Am J Hematol 11:55, 1981. Rubin RH, Carney R T, Schooley RB, et al: The effect of infection on T lymphocyte subpopulations: A preliminary report. Int J Immunopharmacol 3:307, 1981. Reinherz EL, O'Brien C, Rosenthal P, Schlossman SF: The cellular basis for viral-induced immunodeficiency: Analysis by monoclonal antibodies. J Immunol 125:!269 , 1980. Anh-Tuan N, Novak E, Hollan S: Hepatitis B surface antigen circulating immune complexes (HBsAg-CiCs) in patientswith bleeding disorders. Vox Sang 40:12, 1981.
ADDENDUM Since submission of the manuscript, an additional 13 patients with A I D S and hemophilia have been reported to the Centers for Disease Control, bringing the total to 16. We thank Nenita Arias, Patricia Daedalus, R.N., Susan Karp, R.N., M.S., and Carol Dahlstrom for assistance.
8.
9.
10.
11.
I2.
13.
14.
15.
REFERENCES 1. Pneumocystis carinii pneumonia among persons with hemophilia A: MMWR 31:365, 1982. 2. Gottlieb MD, Schroff R, Schanker HM, Weisman JD, Fan PT, Wolf RA, Saxon A: Pneumocystis carini'i pneumonia and mueosal candidiasis in previously healthy homosexual men. N Engl J Med 305:1425, 1981. 3. Masur HM, Michelis MA, Green IB, et al: An outbreak of community-acquired Pneumocystis carinii pneumonia: Initial manifestation of cellular immune dysfunction. N Engl J Med 305:1431, 1981. 4. Siegal FP, Lopez C, Hammer GS, et al: Severe acquired itnmunodeficiency in male homosexuals manifested by chronic perianal ulcerative herpes simplex lesions. N Engl J Med 305:1439, 1981. 5. Stahl RE, Friedman-Kien A, Dubin R, Marmor M, ZollaPazner S: Immunologic abnormalities in homosexual men: Relation to Kaposi's sarcoma. Am J Med 73:171, 1982. 6. KornfeldH, Vande Stouwe RA, Lange M, Reddy MM, Grieco MH: T-lymphocyte subpopulations in homosexual men. N Engl J Med 307:729, 1982. 7. Cowan M J, Fujiwara P, Ammann A J: Cellular immune
16.
17.
18.
19.
20.
21.
695
Peggy S. Weintrub, M.D., Marion A. Koerper, M.D., Joseph E. Addiego, Jr., M.D., W. Lawrence Drew, M.D., Ph.D., Evelyn T. Lennette, Ph.D., Richard Miner, B.A., Morton J. Cowan, M.D., and Arthur J. Ammann, M.D. S a n F r a n c i s c o a n d O a k l a n d , Calif.
THREE PATIENTS WITH HEMOPHILIA A, receiving factor VIII concentrate, have been reported who developed Pneumocystis carinii pneumonia and acquired immunodeficiency syndrome? Infections with opportunistic pathogens have not previously been recognized in this population, and therefore concerns have been raised that therapy might result in AIDS. In the current epidemic of AIDS in homosexual men, Haitians, and drug addicts, studies of the immune system have shown abnormalities in these groups, including cutaneous anergy, lymphopenia, decreased numbers of T cells, reduced helper/suppressor T cell ratios, and depressed proliferative responses of peripheral blood mononuclear cells to mitogens, antigens, and allogeneic cells. 2-4 Apparently healthy homosexuals also have evidence of immunologic dysregulation, but to a lesser degree
From the Divisions of Pediatric Immunology-Rheumatology and Hematology-Oncology, University of California, San Francisco, the Department of Hematology-Oncology, Children's Hospital Medical Center, and the Department of Pathology and Laboratory Medicine. Mt. Zion Hospital and Medical Center. Supported by grants from the National Hemophilia Foundation, Judith Graham Pool Post-Graduate Rd'~e~rch Fellowship, Immunology Research Foundation, Clinical Research Center Grant RRO1271-O1, and the John Kerner Fund, Mt. Zion Hospital and Medical Center. Reprint requests: Arthur J. Ammann, M.D., Pediatric lmmunology-Rheumatology, M-679, University of California, San Francisco, San Francisco, CA 94143.
692
TheJournalofPEDIATRICS
than those with clinical evidence of disease? ,6 To determine whether apparently healthy patients with hemophilia A have laboratory evidence of AIDS, we performed studies of T cell immunity in 21 patients.
See related article, 103:18, 1983.
AIDS Acquired immune deficiency syndrome EBNA Epstein-Barr nuclear antigen EBV Epstein-Barr virus Hb~Ag ,,Hepatitis B surface antigen HBSS Hanks Balanced Salts Solution HBV Hepatitis B virus MLC Mixed lymphocyte culture PBMC Peripheral blood mononuclear cells VCA Viral capsid antigen
,METHODS Patients were from the hematology clinics of the University of California, San Francisco, and the Children's Hospital Medical Center, Oakland, California. Twentyone healthy prepubescent or heterosexual males with hemophilia A were evaluated. The patients were divided into two groups: those given only cryoprecipitate (10 patients) and those given factor VIII concentrate (11 patients), many of whom had received prior cryoprecipi-
Volume 103 Number 5
Immunologic abnormalities in hemophilia A
693
Table I. T cell numbers in patients with hemophilia A
Group Control Cryoprecipitate Factor VIII
TCR (%)
OKT4 (helper) (%)
OKT8 (suppressor) (%)
OKT4/8 (helper/suppressor)
67.5 + 7.6 52.4 + 14.2 65.0 _+ 8.3
54.2 + 4.6 49.8 _+ 9.1 44.5 _+ 9.8
28.0 _-!- 4.0 24.2 _+ 3.4 33.8 + 13.6
2.1 _+ 0.5 2.1 _+ 0.5 1,6 + 0.7
Values are mean + SD.
tote therapy. Patients in the cryoprecipitate group ranged in age from 10 months to 18 years (mean 5.5 years), and those in the factor VIII group from 3 to 21 years (mean 12.5 years). Controls were healthy, heterosexual men and women. A complete blood count with differential was obtained in 20 of the 21 patients and sent to the clinical laboratory to determine absolute lymphocyte counts; T cell numbers were determined by sheep red blood cell rosetting techniques. Preparation of peripheral blood mononuelear cells. Heparinized blood was diluted in phosphate-buffered saline solution and centrifuged over Ficoll-Hypaque (SG 1.077). The PBMCs at the interface were washed three times with Hanks Balanced Salt Solution and resuspended to the desired concentration. Subset enumeration. Indirect immunoflu0rescence was used to measure OKT4 (helper) and OKT8 (supressor) positive lymphocytes. Briefly, l06 PBMCs in 0.1 ml HBSS were added to 10 /~g monoclonal antibody (OKT4 or OKT8; Orthodiagnostics, Raritan, N J) and incubated on ice for 45 minutes. Cells were washed three times with phosphate-buffered saline solution containing 2% bovine serum albumin and 0.01% sodium azide. Then fluoresceinlabeled goat anti-mouse IgG (Meloy Laboratories, Springfield, VA) was added and the cells incubated on ice for an additional 45 minutes. The cells were washed three times, and the percent of positive staining lymphocytes, determined by size and morphologic characteristics, were counted with a Zeiss phase-contrast microscope with epifluorescence. A minimum of 200 cells per patient were counted; PBMCs from one or more controls were counted each time. Phytohemagglutinin response. The proliferative response to PHA was measured using a microculture technique, previously described: Briefly, PBMCs were resuspended to 5 )< 104 cells/ml in RPMI 1640 medium supplemented with 15% heat-inactivated pooled human AB plasma, 20 mmol L-glutamine, 25 mmol HEPES buffer, and 100 ~t~/ml penicillin and streptomycin. Cultures were set up in five replicates in round-bottom microculture plates (Linbro Scientific, McLean, VA), and PHA in final concentrations of 0, 2, and 5 ug/ml, and were cultured for 7 days in a
Table II. PBMC function in patients with hemophilia A Group Control Cryoprecipitate Factor VIII
PHA (epm)
MLC (cpm)
41,873 _+ 11,963 23,277 _+ 7,933 (P = 0.001)* 21,537 _+ 7,493 (P = 0.001)*
16,108 _+ 6,953 12,039 + 1,646 (NS) 12,483 _+ 6,573 (NS)
Values are mean_+SD. *Patient groupvs controlgroup.
humidified 95% air/5% CO2 atmosphere. One day prior to harvesting, 3H-methyl thymidine (New England Nuclear, Boston, MA) was added. The cells were harvested with an automated multiple-sample harvester onto glass fiber filters, and the radioactive counts per minute measured with a liquid scintillation counter (LS7000; Beckman Instruments, Chicago, IL). The PHA response was defined as the maximum of the mean counts per minute resulting from the two PHA concentrations tested. PBMCs from at least one normal control were tested with each run. Allogeneie response. PBMCs from patients were used as responder cells, and radiated (3000 R) PBMCs from healthy donors as stimulator cells in a one-way mixed lymphocyte culture. Cultures with 2 • 105 cells each of stimulator and responder cells in supplemented RPMI 1640 medium as described above were set up in triplicate in flat-bottom microculture plates, and incubated for 6 days in a humidified 95% air/5% CO2 atmosphere. Each well was pulsed with 3H-thymidine 489 hours prior to harvesting and counting, as described above. PBMCs from at least one control were tested with each run. Viral antibo6y titers. Cytomegalovirus antibodies were measured using a nuclear antieomplement immunofluorescence test that used as antigen the isolated nuclei of virus-infected fibroblasts, as described elsewhere) Measurements of antibodies to Epstein-Barr virus capsid antigen, to D and R components of the early antigen complex were done by indirect immunofluorescence procedures described by Henle et al. 9 Antibodies to EB nuclear antigen were assayed by methods of Reedman and Klein, ~~ with guinea pig complement and fluorescein-labeled rabbit anti-guinea pig C substituted for the human counterparts.
694
Weintrub et al.
Hepatitis B surface antigen and antibody to Hb~Ag were detected by standard radioimmunoassay. Statistics. Differences in T cell numbers and subsets were calculated using the Student two-tailed unpaired t test. Difference in mean P H A and MLC were evaluated with the Mann-Whitney U test. RESULTS Absolute lymphocyte counts were normal in all patients. In patients given cryoprecipitate, the mean T cell percentages were significantly decreased, compared with control values (P = 0.005); the mean helper/suppressor ratio was no different from that in controls (Table I). In patients given factor VIII, the T cell percentages were similar to those in controls. The helper/suppressor ratio was significantly decreased (P = 0.04) as a result of a decreased percentage of helper cells (P = 0.003); the difference in suppressor cells was statistically insignificant. Results of PBMC functional studies, PHA, and MLC are shown in Table II. In both cryoprecipitate and factor VIII groups, the mean response to P H A was significantly decreased. The mean M L C response in both groups was similar to control values. Viral titers to cytomegalovirus (IgM and IgG) and Epstein-Barr virus (anti-VCA, anti-EA, and anti-EBNA) were determined for 18 of the 21 patients. Seven patients had evidence of past CMV infection; in one patient, titers suggested active or recent infection (IgM 1:32, IgG 1 : 512). Five patients had antibody titers to EBV indicative of past infection, and two patients had elevated anti-VCA with low anti-EBNA, a pattern that has been shown to represent either recent or past infection with recent immunosuppression. H The number of patients with CMV and EBV positive titers was similar in the two groups. All 21 patients were evaluated for the presence of Hb~Ag and for antibody to Hb~Ab. All patients in the factor VIII group were HbsAg negative and Hb~Ab positive. All of the 11 patients in the cryoprecipitate group were HbsAb negative, and 10 of the 11 were HbsAg negative. The presence of antibody to HBV, CMV, or EBV did not correlate with alterations in T cell numbers or PBMC function, with one exception: both patients with EBV antibody titers indicating ~ecent infection or immunosuppression had at least one significant T cell abnormality (one patient had 18% T cell rosettes and 30% helper cells, the other had a helper/ suppressor ratio of 0.4). DISCUSSION Our results show that asymptomatic patients with hemophilia A have altered immunologic function. Our patients given cryoprecipitate have decreased percentages of T cell rosettes, and those given factor VIII have depressed
The Journal of Pediatrics November 1983
helper/suppressor ratios; both groups have diminished PBMC responses to PHA. There is no clear explanation of why the two groups have different mean T cell numbers and subsets but similar functional responses. The T cell abnormalities in our group of patients are not as severe as those in patients with hemophilia and AIDS. The three hemophilic patients reported with P. carinii pneumonia all had lymphopenia, and in the two patients tested, mitogen responses were absent? It is possible that the abnormalities in our patients are progressive and that further reduction in their immunologic function may result in increased numbers of opportunistic infections. The cause of AIDS is not known. Drug abuse, homosexuality, and sexual promiscuity, common in other patient populations with AIDS, were not present in our patients. One possible cause, which is most consistent with epidemiologic findings in AIDS, is an immunosuppressive infectious agent. Homosexual men have increased exposure to numerous viral infections (particularly HBV, CMV, and EBV, 11"14all of which have immunosuppressive effects) 5-t7 Factor VIII concentrate, used in one group of patients, is a lyophilized pooled-donor product and carries a high risk of hepatitis B transmission; for this reason, all patients receiving this therapy have antibody to Hb~Ag. Cryoprecipitate is a single-donor blood product. It is likely that, through transfused blood products, patients with hemophilia A have increased exposure to other viruses as well. TM Although viral infections do result in an abnormal helper/ suppressor cell ratio, cell proportions return to normal several weeks to months after acute infection) 9'2~We did not find a correlation between abnormal studies and evidence of past CMV, HBV, or EBV infection. It has been postulated that the abnormal distribution of helper and suppressor cells is a result of chronic immune complex formation. Some patients with AIDS have been reported to have elevated circulating immune complexes, z.4 Patients with hemophilia A who are receiving factor VIII concentrate therapy~ are exposed to repeated doses of hepatitis B, which results in chronic immune complex formation3 ~ This could result in immunologic dysregulation by covering receptor sites on T cells, stimulating anti-idiotypic antibodies (with stimulation of suppressor cells or other factors), or by adhering to T cells and causing selective removal of T cell subsets in the spleen. Other antigens present in concentrate or precipitate, such as histocompatibility or protein antigens, might also result in chronic immune complex formation or production of antibody to autoantigens, resulting in immunologic dysregulation. The clinical status and the degree of immunologic suppression are different in our patients and in those reported with P. carinii pneumonia. This finding suggests
Volume 103 Number 5
Immunologic abnormalities in hemophilia A
either a spectrum of immunologic dysregulation in the patients or progressive immunologic attrition. Sequential studies of immunologic function should be performed in patients with newly diagnosed or established hemophilia A. We have shown that clinically healthy patients with hemophilia A given cryoprecipitate or factor VIII have decreased T cell percentages and helper/Suppressor ratios, respectively. Both patient groups have diminished proliferative iesponses to the mitoge n P H A . Past infection with C M V , EBV, or H B V did not correlate with abnormal studies. A d d i t i o n a l evaluation of the cause and natural history of these abnormalities i s needed, because this information could influence future therapeutic regimens used in hemophilia. It is important for clinicians caring for patients with hemophilia +to realize that asymptomatic individuals may have early evidence of immunodeficiency.
defect in selective IgA deficiency using a microculture method for PHA stimulation and limiting dilution. Clin Immunol Immunopathol 17:595, 1980. Mintz L, Miner RC, Yeager AS: Anticomplement immunofluorescence test that uses isolated fibroblast nuclei for detection of antibodies to human cytomegalovirus. J Clin Microbiol 12:562, 1980. Henle W, Henle G, Horwitz CA: Infectious mononucleosis and Epstein-Barr virus associated malignancies. In Lennette EH, Schmidt N J, editors: Diagnostic procedures for viral, rickettsial and chlamydia infections, ed 5. Washington, DC, 1979, American Public Health Association, pp 441-470. Reedman BM, Klein G: Cellular localization of Epstein-Barr (EBV) associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int J Cancer 1i:499, i973. Henle W, Henle G: Consequences of persistent Epstein-Barr virus'infections. Cold Spring Harbor Conf Cell Prolif 7:3, 1980. Drew WL, Mintz L, Miner RC, Sands M, Ketterer B: Prevalence of cytomegalovirus infection in homosexual men. J Infect Dis 143:188, 1981. Abrams DI: Lymphoproliferative diseases in homosexual males. In Purtilo DT, editor: Immune deficiency and cancer: Epstein-Barr virus and lymphoproliferative malignancies. New York, 1983, Plenum Press. Dietzman DE, Harnisch JP, Ray CG, Alexander ER, Holmes KK: Hepatitis B surface antigen (HB~Ag) and antibody to HB~Ag: Prevalence in homosexual and heterosexual men. JAMA 238:2625, 1977. Mengi R J, Niederman JC, Kelleher JE, et al: Depression of cell-mediated immunity during acute infectious mononucleosis. N Engl J Med 291:1149, 1974. Rinaldo CR, Walter WP, Richter BS, Black PH, Hirsch MS: Mechanisms of immunosuppression in cytomegaloviral mononucleosis. J Infect Dis 141:488, 1980. Thomas HC, Brown D, Labrooy J, Epstein O: T cell subsets in autoimmune and HBV-induced chronic liver disease, HBs antigen carriers with normal histology and primary biliary cirrhosis: A review of the abnormalities and the effects of treatment. J Clin Immunol 2:575, 1982. Gomperts ED, Lazerson J, Berg D, Lockhart D, SergisDeavenporte E: Hepatocellular enzyme patterns and hepatitis B virus exposure in multitransfused young and very young hemophilia patients. Am J Hematol 11:55, 1981. Rubin RH, Carney R T, Schooley RB, et al: The effect of infection on T lymphocyte subpopulations: A preliminary report. Int J Immunopharmacol 3:307, 1981. Reinherz EL, O'Brien C, Rosenthal P, Schlossman SF: The cellular basis for viral-induced immunodeficiency: Analysis by monoclonal antibodies. J Immunol 125:!269 , 1980. Anh-Tuan N, Novak E, Hollan S: Hepatitis B surface antigen circulating immune complexes (HBsAg-CiCs) in patientswith bleeding disorders. Vox Sang 40:12, 1981.
ADDENDUM Since submission of the manuscript, an additional 13 patients with A I D S and hemophilia have been reported to the Centers for Disease Control, bringing the total to 16. We thank Nenita Arias, Patricia Daedalus, R.N., Susan Karp, R.N., M.S., and Carol Dahlstrom for assistance.
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