Hyperimmunoglobulin Transfer Factor R.
WILLIAM RAYMOND
E Syndrome: Response and Ascorbic Acid Therapy’
FRIEDENBERG,?
L.
HANSEN,
J. RAY C.
JAMES AND
MARX.
March
JR..
HASELB~
Departments of Hematology, Allergy, and Infectious Medicine. and the Marshfield Medical Foundation. Inc., Mwshfield. Received
to
Marshfield Clinic,. Wisconsirr 54449
7. I978
man with hyperimmunoThis report presents the case history of a 19-year-old globulin E syndrome and the successful treatment with transfer factor and ascorbic acid. The patient presented with a marked defect in the ability of his lymphocytes to respond to mitogens, in skin test response to DNCB challenge and other recall antigens, and a variety of in vitro granulocyte function defects. The presence of a serum inhibitor to lymphocyte and granulocyte function was found. This individual had multiple episodes of mucocutaneous candidiasis and staphylococcal infections. Clinical improvement was evident after transfer factor and ascorbic acid treatment. Many of the in vitro funcrions also returned to normal.
INTRODUCTION
Patients with the hyperimmunoglobulin E (HIE) syndrome usually have: (i) recurrent severe bacterial or fungal infections: (ii) clinical manifestations of allergic disease; (iii) hyperimmunoglobulinemia E; and (iv) defective neutrophil chemotaxis (1). Some patients with this syndrome have associated cellular immune defects and chronic mucocutaneous candidiasis (2, 3) and others have all of the manifestations of the syndrome except for clinical evidence of allergic disease (4). Other patients have recurrent staphylococcal abscesses associated with defective neutrophil chemotaxis and allergic rhinitis in the absence of hyperimmunoglobulinemia E (5). Inhibitors of chemotaxis have not been found in this syndrome (1) although they have been described in association with other similar diseases (6- 14). The following case report describes a patient with typical characteristics of the HIE syndrome. except for the absence of clinical manifestations of allergic disease and a demonstrable serum inhibitor against both the chemotactic response of granulocytes and mitogenic response of lymphocytes. Transfer factor has been reported to improve mucocutaneous candidiasis (15-20) and ascorbic acid has improved impaired chemotaxis in patients with the Chediak-Higashi syndrome (21). This patient responded to transfer factor and ascorbic acid with improvement in both in \,ifro granulocyte and lymphocyte function, as well as clinical improvement.
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1979
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and the Marshfield
Dr. William Marshfield.
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Medical
Foundation. Marshfield
Inc. Medical
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Case Report
D.W., a 29-year-old man, was admitted to the hospital for dental surgery. A review of his medical history showed repeated hospitalization and failure to thrive since 5 months of age. By 3 years of age this patient had developed recurrent infections including Candida albicans pharyngitis, superficial and deep skin abscesses induced by Staphylococcus aureus, staphylococcal abscesses of the neck, and a diffuse skin infection with hepatosplenomegaly. At 3 years he was hospitalized for a staphylococcal pneumonitis; at 6 years with another staphylococcal abscess of the buttock. By age 16 he had required multiple admissions for orthopedic procedures to correct bilateral osteochondrosis of the medial aspect of the proximal tibia1 epiphysis. He was also found to have progressive pulmonary fibrosis and cystic pulmonary abscesses. At 21 years of age, this patient had another staphylococcal infection of extensive hemangiomata of the buttocks (chronic botryomycosis). For the next 8 years he had recurrent problems with abscesses in his mouth, frequently culturing Staphylococcus aureas and Candida al&cans, requiring multiple drainage procedures and subsequently grafts to close ulcers. Six months prior to admission he developed a rash in his pubic area, biopsy of which revealed nonspecific dermatitis from which grew Staphylococcus aureus.
On admission, routine laboratory workup demonstrated a white cell count of 10,200 mm3 with 62% segmented neutrophils, 1% bands, 4% eosinophils, 6% monocytes, and 25% lymphocytes. The hemoglobin was 13.9 g% and the hematocrit 38.5. The following tests were all found to be normal: calcium, phosphorus, fasting blood sugar, urinalysis, cholesterol, bilirubin, alkaline phosphatase, LDH, SGOT, and T+ The IgG was 1280 IU, the IgA 534 IU, IgM 130 IU, and the IgE 4437 IU. The secretory IgA obtained from saliva was 7.6 mg/ml (normal = >3.0 mgiml). The blood type of the patient is 0 positive with an anti-B titer of 1:4 and an anti-A of 1:8. The RAST tests are summarized in Table 1. Total hemolytic complement levels (CH,,), as well as C3 and C4 levels, were normal. A 24-hr skin window using the Senn technique revealed a
RAST
TO COMMON
TABLE 1 RESPIRATORY
ALLERGENS 2” 3 1 0 0 I 2 3 2 1 0
Timothy Common ragweed Maple Oak Cat Epithelium Dog Epithelium Cladosporium herbarum Alternaria tenuis House dust Dermatophagoides pteryonyssihus Dermatophagoides farinae o Graded
response
for the RAST
on a scale of 0 to 4 +. A value
2 1 is indicative
of a positive
test.
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LYMPHOCYTE
a)
PHA --_ “PWM . . . . . . . . . . ‘,‘CON A4_-
FUNCTION
&Z* 9.
0,
/-c
m-
A’ +--4
1.L 1.0-
WBC Lymphocytes-
_ _
TB--7F = Transfer
Factor
ASzAscorbic
Acid
>
‘-
‘,.I
/
mo100
o---*-------.’
n,z&
1 pi?/77 TF TF
4 p&7
TF
ts,i,n
6&l
AS
Phagocytic Index
Migration Index
NBT Unrtim. NET
_
Stim.---
TF=Tronsfer AS:Ascorbic
Fact< Acid
TF TF TF AS response before and after therapy. The response of the lymphocyte system to transfer factor (TF) therapy is depicted (IA). The results of the T and B cells are expressed as the number of T or B cells/mm3. The WBC count is expressed as the number of cells X 104/mm3, and the lymphocytes as the number of cells x 103/mm3. The results of the lymphocyte transformation studies with PHA, PWM, and Con A are expressed as the stimulation index X 102. Granulocyte function (1B) in response to ascorbic acid treatment is expressed as a percentage of the control values. The controls were normal individuals studied. FIG.
1. In
viva
and
in virrc~
cellular
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HIE
blood granulocyte clearance (BGC) of 2.7 ml/cm‘?24 hr and a total leukocyte mobilization (TLM) of 22.0 x 106/cm2/24 hr. The in vitro and in vivo granulocyte and lymphocyte results are summarized in Fig. 1. There was a decrease in the relative and absolute number of T cells, and lymphocyte transformation response to mitogens was markedly impaired. Baseline nitroblue tetrazolium (NBT) reduction is above normal, but there was a poor response to stimulation with endotoxin. The phagocytic and bactericidal indices were normal, but the chemotactic response was markedly impaired. Table 2 demonstrates that there was an intrinsic granulocyte defect, but that the patient’s serum was also capable of suppressing normal granulocyte chemotaxis. Random mobility of granulocytes appeared normal throughout the course of illness. Skin tests including PPD, mumps, histoplasmin, streptokinase-streptodornase (SK-SK), Candida, and DNCB were negative at 24 and 48 hr. He did have an immediate hypersensitivity reaction to the Candida skin test at a dilution of 1:500 and a higher dose was not given. Ten days later, on rechallenge with DNCB, no response was noted. A sweat test revealed 20 meq/liter of chloride. Baseline febrile agglutinins were all less than 1:20 except for Salmonella OB which was 1:40. Ten days after vaccination with typhoid 1.5 cc IM there was no change in the titers. Two days after rechallenge with DNCB the patient was given his first injection of transfer factor from a donor who was skin test positive to histoplasmin, SK-SD, mumps, and Candida. Forty-eight hours later the patient developed a marked reaction at the site of the initial DNCB application with marked erythema and formation of vesicles.Ten days after the first injection of transfer factor, all the above skin tests were repeated and there was slight erythema without any induration only with histoplasmin (PPD, mumps, SK-SD, TABLE SERUM
INHIBITORS
2
OFCELLULAR
FUNCTION
Lymphocyte response
Pretherapy Post-therapy
PHA
Con A
PWM
47” 97
49 -
71 -
Chemotactic granulocyte response Bacterial factor Pretherapy Patient cells Control cells Post-therapy Patient cells Control cells
35* 274 -
a Percentage of control values. b Percentage of control cells similarly stimulated.
Zymosan-NHS
Zymosan patient serum
43 -
40 47
57 -
44 22
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and Candida remained negative). Four days later the patient was given a second injection of transfer factor from the same donor. In the following weeks he noticed improvement in the rash in his pubic area which became much less indurated and pruritic. The oral thrush completely disappeared.Two weeks later he received his third injection of transfer factor. By February 22. 1977, he had shown a dramatic improvement in his lymphocyte function (Fig. 1). but no improvement in his granulocyte function. No further improvement in the skin rash was noted. He was begun on ascorbic acid 1.0 g orally three times a day. The results of his in \,itro and in rvi~o granulocyte and lymphocyte studies 2.5 and 6.5 months later are summarized in Fig. 1. There was a marked improvement in lymphocyte and granulocyte function. Although chemotaxis of granulocytes with bacterial factor was better than normal, zymosan induced chemotaxis was still subnormal. His IgE was now 4650 IUiml and skin window studies revealed a BGC of 2.4 ml/cm?24 hr and a TLM of 17.1 x lO”icmY24 hr, which are not significantly improved. His pubic rash was almost completely gone. Lymphocyte Function Tests The lymphocyte system was assessed by the method previously outlined from this laboratory (22). Briefly, lymphocyte subpopulations were identified after separation using a density gradient Ficoll-Hypaque column centrifuged at 400g. Sheep red blood cell rosetting (E-RFC) was used to identify T cells, while complement receptors (EAC-RFC) and surface immunoglobulins identified B cells. Percentages from a normal population were as follows: E-RFC = 50-75%: EAC-RFC = lo-34%; IgG = 5-20%; IgM =3-150/o; and IgA = O-4%. Lymphocyte function was assessed by in vitro lymphocyte transformation to phytohemagglutinin, concanavalin A, and pokeweed mitogen. Lymphocyte transformation has been previously described in detail (22) and was performed as described. The results are expressed as the stimulation index (SI) or ratio of counts in the cultures with mitogen to the counts in control cultures. Greater than twofold stimulation by antigen was considered a positive test. The presence of a serum inhibitor to lymphocyte function was determined using purified cell populations. Lymphocytes were obtained from the patient and from a normal donor and separated on Ficoll-Hypaque gradients as described above. The cells were washed in RPM1 1640 and resuspended in RPM1 1640 containing 10% serum. Normal human serum was used as a control. The cells were incubated for 2-6 days in varying concentrations of mitogens. The peak SI was calcuated as above and recorded. In Vivo Granulocyte Function Tests The capability of granulocytes to migrate to a site of inflammation was measured by the technique outlined by Senn and Jungi (23). A plastic chamber was placed over an area of abraded skin and filled with fresh autologous serum. The chambers were glued in place with collodion. The contents of the chambers were collected after 24 hr. The number of cells. type, and viability were determined routinely. The following parameters were calculated on the basis of cell counts: TLM = cumulative number of leukocytes/cm2/24 hr, leukocyte mobilization rate (LMR) = average leukocytes/cmVhr, and BGC = TLM/cir-
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culating neutrophilsml of blood. Values obtained in 14 normal individuals are TLM = $9.5 + 8.7 (mean ? SE), LMR = 3.7 + 0.3, and BGC = 31.8 + 4.5. In Vitro Granulocyte Function Tests The functional integrity of the granulocyte system was assessed by a battery of tests including NBT, chemotaxis in response to bacteria-derived factor(s) and zymosan-induced C5a, random mobility, phagocytic and bactericidal capabilities, and chemiluminescence. These granulocytes were separated by sedimentation at lg for 45 min at 37°C. The cell-rich plasma was then layered on a Ficoll-Hypaque gradient and centrifuged for 10 min at 4OOg. The sedimented cells were 95% pure granulocytes. The cells were washed in Hank’s balanced salt solution (HBSS) and resuspended to appropriate concentrations. NBT Reduction The model utilized for NBT dye reduction is that described by Park et al. (24). Venous blood was collected with 10 units of heparin/ml. The blood was incubated with 0.2% NBT in normal saline at 37°C for 15 min. Blood smears are prepared, stained with Wright’s stain, and a 200-cell differential was made noting the number of cells which have the reduced formazan deposits within the cytoplasm. These cells are easily recognized as PMNs with large irregular dark amorphous masses within the confines of the cytoplasm. A portion of whole blood was preincubated with 1 mg/ml bacterial lipopolysaccharide (LPS) (Difco, Detroit, Mich.) for 15 min at 37°C and the NBT test was repeated as above. Normal individuals routinely showed values ~20% in the unstimulated cultures and >30% in the LPS stimulated cultures. Chemotaxis The chemotaxis response was determined as outlined in detail by Ward (25). The chemotactic chambers (Bellco Glass, Vineland, N.J.) are prepared using S-pm porosity SMWP 02500 Millipore filters. The cells are cultured in RPM1 1640 buffered with bicarbonate at pH 7.3. Chemotactic factors used included a bacterial culture filtrate of Escherichia coli, zymosan-activated normal human serum, and the appropriate controls. The chambers are incubated at 37°C for 3 hr. The filters are carefully removed from the chambers, fixed in absolute propanol, stained with Wright’s stain, and finally cleared in xylene. The filters are placed on conventional glass slides with mounting media and observed at 25x magnification. A minimum of 10 fields are viewed and the number of cells migrating completely through the filter are counted. The results are expressed as the ratio of the average number of cells per 25x field compared to unstimulated controls. The presence of serum inhibitors of chemotaxis was determined by replacing the normal human serum pool with the patient’s serum and repeating the test on cells from normal donors. Leukocyte Random Mobility The ability of leukocytes to randomly migrate from a capillary tube in a culture chamber is measured by the technique outlined by Miller (26). The technique involves incubation of 1 x lo7 PMNs in RPM1 1640 for 24 hr. The
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degree of migration from this capillary tube is measured by planimetry at 4 and 24 hr. The results are expressed as the area of the cells migrating out of this capillary tube. Phagocytic and Bactericidal Activities The ability of the granulocytes to ingest and kill bacteria was studied by the method outlined by Weir (27). An 18-hr culture of Staphylococcus aureus. strain 502A, (ATCC, Bethesda, Md.) was incubated with 1 x lo7 granulocytes in a ratio of bacteria to granulocytes of 1. For phagocytic indexes, aliquots of the bacteria-cell mixture were sampled at 30, 60, and 120 min. The cells are centrifuged at 400g and the number of bacteria remaining in the supernatant was calculated based on viable plate counts. Bactericidal abilities of these granulocytes were determined by mixing cultures of Staphylococcus aureus and granulocytes in a ratio of 1: 1 for 15 min at 37°C. Excess bacteria were removed by washing. Aliquots were then taken at 30, 60, 90, and 120 min. The number of bacteria contained within the granulocytes was determined after lysing the washed granulocytes by viable plate counts. The results of these two assays were expressed as the phagocytic index (PI) and bactericidal index (KI). PI and KI are calculated by comparing the log number of bacteria at 0 and 120 min. Normal regression lines can be plotted. Chemiluminescence The ability of granulocytes to generate photons of visible light after stimulation was measured by a modification of the method of Allen (28). Polymorphonuclear leukocytes were separated as described and resuspended to 1 x lo7 PMN/ml in HBSS. The cells were incubated at 37°C with 1 mg of zymosan previously opsonized with normal human serum by incubation at 37°C for 1 hr. The emission of light was measured in a Packard Tri-Carb scintillation counter with coincidence off for 0.2 min at 1% gain. Duplicate counts were obtained and the samples immediately returned to the water bath. Repetitive counts were made every 10 min for 2 hr. The results are expressed as the counts per minute/IO’ granulocytes at the peak of the curve. Cells from normal individuals yield a mean ? SE of 7111 +- 528. Complement Profile Total hemolytic complement activity was determined as described by Campbell er al. (29). Individual complement components were determined either as a titration of hemolytic activity or as the concentration of protein by radial immunodiffusion. Hemolytic activity was determined by commercially available kits (Cordis Lab, Inc., Miami, Fla.) and performed as directed. Protein determinations were obtained by radial immunodiffusion using commercially prepared immunoplates (Behring, Somerville, N.J.). Transfer Factor Preparation Transfer factor was prepared as a dialyzable extractable, DNase resistant extract of peripheral blood mononuclear cells after the procedure outlined by Spitler (30). One unit of peripheral blood was collected in CPD and the leukocytes separated by sedimentation with 10 ml 5% Dextran 200. One unit
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of transfer factor was defined as the amount of extractable material derived from 5 x lOa lymphocytes. The donor was selected because of strong positive reaction to candidin skin test allergen and an in vitro stimulation of lymphocytes with Candidu afbicans extract. He was also positive to histoplasmin, SK-SD, and mumps, but his response to DNCB is unknown. DISCUSSION
This patient was typical of the HIE syndrome. He had recurrent severe bacterial infections with staphylococcus uureus, hyperimmunoglobulinemia E, and defective neutrophil chemotaxis. He did not, however, manifest any clinical signs of allergic disease. Patients with HIE have previously been reported who lack the manifestations of allergic disease (4). It is of interest that he did have positive RAST tests and immediate hypersensitivity to Candida antigen. The patient is also atypical in that a serum inhibitor against both granulocyte chemotaxis and lymphocyte function was readily detectable. However, a serum inhibitor was not his only defect. An intrinsic cellular defect in both lymphocytes and granulocytes was demonstrated (Table 2). Many diseases have been associated with impaired chemotaxis (31). Serum inhibitors of chemotaxis have been infrequently described (6, 7, 10, 32, 33). An inhibitor was found in this patient’s serum but was not characterized further. Impaired cellular immunity has been found in mucocutaneous candidiasis associated with impaired lymphocyte transformation and MIF production with Candida antigen (34-41). Serum inhibitors of cellular immunity, specifically directed against T cells have been reported in Hodgkin’s disease (42), as well as other conditions (43), but not in mucocutaneous candidiasis or the HIE syndrome. This patient demonstrated an inhibitor against lymphocyte transformation with multiple antigens. The short stature of this patient is of interest because of previous reports of immunological deficiency syndromes associated with short-limbed dwarfism and ectodermal dysplasia (44-46). However, this patient had no evidence of achondroplasia, ectodermal dysplasia, or endocrinopathy. It is most likely that his short stature is based on his chronic illness as a child. Transfer factor has been used successfully to treat mucocutaneous candidiasis (15-20). However, in a patient with abnormal granulocyte chemotaxis associated with mucocutaneous candidiasis, Gallin found that multiple doses of transfer factor failed to correct her chemotactic defect and suppressed cellular immune responses (47). Snyderman et al. (39) found that a patient with chronic mucocutaneous candidiasis who had defective mononuclear leukocyte chemotaxis improved with transfer factor. Our patient improved with transfer factor by: (i) improved in vitro lymphocyte function; (ii) improved response to DNCB testing; and (iii) clinical improvement in the degree of oral thrush. Ascorbic acid has been shown to improve granulocyte chemotaxis in both normal people and patients with Chediak-Higashi syndrome (21). Since there had been little improvement in granulocyte function with transfer factor, ascorbic acid was given. There was a dramatic and progressive improvement in this patient’s chronic pubic skin rash and further improvement in the in vitro tests
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of lymphocyte and granulocyte function, although the skin window results were unchanged. Levamisole has been used to treat patients with the HIE syndrome (48). Chemotaxis of neutrophils and mononuclear cells from patients with the HIE syndrome were stimulated by levamisole both in vir~ and in ~+ro. Two of these patients also had mucocutaneous candidiasis and impaired lymphocyte function. Although in \litro testing improved, there was no evaluation of clinical improvement in these four patients. The initial clinical and laboratory improvement in this patient following transfer factor was intensified with the addition of ascorbic acid. The improvement in lymphocyte responsiveness in this patient correlated with the clinical improvement in the oral thrush and was temporally related to the treatment with transfer factor. The improvement in granulocyte function correlated with complete clearing of the oral thrush and improvement in the pubic rash and was temporally related to the treatment with ascorbic acid. It seems unlikely that the improvement in granulocyte function was due to a delayed effect of the transfer factor rather than to the ascorbic acid. The ascorbic acid may have contributed to the improved in vitro lymphocyte and granulocyte function by increasing levels of 3’-5’-guanosine monophosphate (cyclic GMP) in granulocytes (49-54) as well as in lymphocytes (55). Effective treatment of the HIE syndrome would improve the quality of life of these individuals. Bale ef 01. (56) have described a fatal histiocytic lymphoma in a child with the HIE syndrome. If transfer factor and ascorbic acid prove useful in other patients with the HIE syndrome, fatal lymphoreticular malignancies in these patients, as in others with immunodeficiency syndromes, might be prevented. ACKNOWLEDGMENTS The authors would Amy Witte and Howard
like to express Rand. and Alice
their sincere gratitude Stargardt for preparation
for the technical of this manuscript.
assistance
of
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15. Schulkind, M. L., Adler, W. H., Altemeier, W. A., III, and Ayoub, E. M., Cell, Immunol, 3, 606, 1972. 16. Wybran, J., Levin, A. S., Spitler, L. E., and Fudenberg, H. H., N. Engl. .I. Med. 288,710, 1973. 17. Potter, H., Rosenfeld, S., and Dressler, D., Ann. Intern. Med. 81, 838, 1974. 18. Lawrence, H. W.. N. Engl. J. Med. 283, 411, 1970. 19. Fudenberg, H. H., Levin. A. S., Spitler, L. E., Sybran, J., and Byers, V., Hosp. prac. 9, 95, 1974. 20. Littmen, B. H., Rocklin, R. E., Parkman, R., and David, J. R., In “Transfer Factor: Basic Properties and Clinical Applications“ (M. S. Ascher, A. A. Gotlieb, and C. H. Kirkpatrick, Eds.), p. 495, Academic Press, New York, 1976. 21. Boxer, L. A., Watanabe, A. M., Rister, M., Besch, H. R., Jr., Allen, J., and Baehner, R. L., N. Engl. J. Med. 295, 1041. 1976. 22. Hansen. R. L.. Marx Jr., J. J., Ptacek, L., and Roberts, R. C., Amer. J. Dis. Child. 131, 518, 1977. 23. Senn, H. J. and Jungi, W. F., Semin. Hematol. 12, 27, 1975. 24. Park, P. H., Fikrig, S. M., and Smithwick, E. M., Lancer 2, 532, 1968. 25. Ward, P. A., In “Manual of Clinical Immunology” (N. R. Rose and H. Friedman, Eds.), Amer. Sot. for Microbial., Washington, D.C., 1976. 26. Miller, M. E., Semin. Hematol. 12, 59, 1975. 27. Weir, D. M., “Handbook of Experimental Immunology,” Blackwell, Oxford, 1973. 28. Allen, R. C., Stjemholm, R. L., Reed, M. A., Harper, T. B., III, Gupta, S., Steele, R. H., and Waring, W. W.. J. Infect. Dis. 136, 510, 1977. 29. Campbell, D. H., Garvey, J. S., Cremer, N. E., and Sursdorf, D. H., “Methods in hm.mo]ogy,” Benjamin, New York, 1964. 30. Spitler, L. E., Levin. A. S., and Fudenberg, H. H., In “Clinical Immunobiology” (F. H. Bach and R. A. Good, Eds.1. Vol. 2, p. 153, Academic Press, New York, 1974. 31. Miller, M. E., Semin. Hematol. 12, 59, 1975. 32. Berenberg, J. L., and Ward, P. A., J. Clin. Invest. 52, 1200, 1973. 33. Ward, P. A., In “The Granulocyte: Function and Clinical Utilization” (T. J. Greenw& and G. A. Jamieson, Eds.), p. 77, Liss, New York, 1977. 34. Marmor, M. F., and Barnett, E. V., Amer. J. Med. 44, 979, 1968. 35. Hermans, P. E., Ulrich, J. A., and Markowitz, H., Amer. J. Med. 47, 503, 1969. 36. Valdimarsson, H., Riches, H. R. C., Holt, L., and Hobbs, J. R., Lancer 1, 1259, 1970. 37. Goldberg, L. S., Bluestone, R., Barnett, E. V.. and Landau, J. W., C/in. Exp. Zmmunol. 8, 37, 1971. 38. Kirkpatrick, C. H., Rich, R. R., and Bennett, J. E., Ann. Intern. Med. 74, 955, 1971. 39. Snyderman, R., Altman, L. C., Frankel, A., and Blaese, R. M., Ann. Intern. Med. 78,509, 1973. 40. Clark, R. A., Root, R. K., Kimball, H. R., and Kirkpatrick, C. H., Ann. Intern. Med. 78, 515, 1973. 41. Quie, P. G., and Chilgren, R. A., Semin. Hematol. 8, 227, 1971. 42. Fuks, Z., Strober, S., and Kaplan, H. S., N. Engl. J. Med. 295, 1273, 1976. 43. Fitzgerald, M. G., and Hosking, C. S., Immunology 30, 33, 1976. 44. Clinicopathological Conference, &it. Med. J. 2, 1371, 1966. 45. Gratti. R. A., Platt, N., Pomerance, H. H., Hong, R., Langer, L. G., Kay, H. E. M., and Good, R. A., Pediatrics 75, 675. 1969. 46. Lux. S. E., Johnston, R. B., August, C. S., Say, B., Penchaszadeh, V. B., Rosen, F. S., and McKusick, V. A., N. Engl. J. Med. 282, 231, 1970. 47. Gallin, J. I., In “The Phagocytic Cell in Host Resistance” (J. A. Bellanti and D. H. Dayton, Eds.). p. 227, Raven Press, New York, 1975. 48. Wright, D. G., Kirkpatrick, C. H., and Gallin, J. I., J. Clin. Invest. 59, 941, 1977. 49. Sandier, J. A., Gallin, J. I., and Vaughan. M., J. Cell Biol. 67, 480, 1975. 50. Goetzl, E. J., Wasserman, S. I., Gigli, I., and Austen, K. F., J. C/in. Znvesr. 53, 813, 1974. 51. Zurier, R. B., Weissmann, G., Hoffstein, S., Kammerman, S., and Tai, H. H., J. Clin. Invest. 53, 297, 1974.
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52. Estensen, R. D., Hill, H. R.. Quie, P. G., Hogan, N., and Goldberg, 245, 458. 1973. 53. Sandler, J. A., Clyman. R. I., Manfaniello, V. C., and Vaughan, M., 1975. 54. Boxer, L. A.. Rister. M.. Allen, J. M., and Baehner, R. L.. Blood 49, 55. Strom, T. B., Lundin. A. P., III. and Carpenter, C. B.. In “Progress (R. S. Schwartz, Eds.), Vol. 3, p. 115, Grune & Stratton, New York, 56. Bale, J. F.. Jr., Wilson, J. F.. and Hill. H. R., Cancer 39, 2386, 1977.
N. D.. Narure (London) J.
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Invest. 55, 431.
9. 1977. in Clinical Immunology” 1977.
WILLIAM RAYMOND
E Syndrome: Response and Ascorbic Acid Therapy’
FRIEDENBERG,?
L.
HANSEN,
J. RAY C.
JAMES AND
MARX.
March
JR..
HASELB~
Departments of Hematology, Allergy, and Infectious Medicine. and the Marshfield Medical Foundation. Inc., Mwshfield. Received
to
Marshfield Clinic,. Wisconsirr 54449
7. I978
man with hyperimmunoThis report presents the case history of a 19-year-old globulin E syndrome and the successful treatment with transfer factor and ascorbic acid. The patient presented with a marked defect in the ability of his lymphocytes to respond to mitogens, in skin test response to DNCB challenge and other recall antigens, and a variety of in vitro granulocyte function defects. The presence of a serum inhibitor to lymphocyte and granulocyte function was found. This individual had multiple episodes of mucocutaneous candidiasis and staphylococcal infections. Clinical improvement was evident after transfer factor and ascorbic acid treatment. Many of the in vitro funcrions also returned to normal.
INTRODUCTION
Patients with the hyperimmunoglobulin E (HIE) syndrome usually have: (i) recurrent severe bacterial or fungal infections: (ii) clinical manifestations of allergic disease; (iii) hyperimmunoglobulinemia E; and (iv) defective neutrophil chemotaxis (1). Some patients with this syndrome have associated cellular immune defects and chronic mucocutaneous candidiasis (2, 3) and others have all of the manifestations of the syndrome except for clinical evidence of allergic disease (4). Other patients have recurrent staphylococcal abscesses associated with defective neutrophil chemotaxis and allergic rhinitis in the absence of hyperimmunoglobulinemia E (5). Inhibitors of chemotaxis have not been found in this syndrome (1) although they have been described in association with other similar diseases (6- 14). The following case report describes a patient with typical characteristics of the HIE syndrome. except for the absence of clinical manifestations of allergic disease and a demonstrable serum inhibitor against both the chemotactic response of granulocytes and mitogenic response of lymphocytes. Transfer factor has been reported to improve mucocutaneous candidiasis (15-20) and ascorbic acid has improved impaired chemotaxis in patients with the Chediak-Higashi syndrome (21). This patient responded to transfer factor and ascorbic acid with improvement in both in \,ifro granulocyte and lymphocyte function, as well as clinical improvement.
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Clinic
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i”>
1979
of reproduction
by
-11$01.00/O Academic m
any
Press.
Inc
form
rewr~ed
and the Marshfield
Dr. William Marshfield.
R. Friedenberg. Wise. 54449.
Medical
Foundation. Marshfield
Inc. Medical
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Case Report
D.W., a 29-year-old man, was admitted to the hospital for dental surgery. A review of his medical history showed repeated hospitalization and failure to thrive since 5 months of age. By 3 years of age this patient had developed recurrent infections including Candida albicans pharyngitis, superficial and deep skin abscesses induced by Staphylococcus aureus, staphylococcal abscesses of the neck, and a diffuse skin infection with hepatosplenomegaly. At 3 years he was hospitalized for a staphylococcal pneumonitis; at 6 years with another staphylococcal abscess of the buttock. By age 16 he had required multiple admissions for orthopedic procedures to correct bilateral osteochondrosis of the medial aspect of the proximal tibia1 epiphysis. He was also found to have progressive pulmonary fibrosis and cystic pulmonary abscesses. At 21 years of age, this patient had another staphylococcal infection of extensive hemangiomata of the buttocks (chronic botryomycosis). For the next 8 years he had recurrent problems with abscesses in his mouth, frequently culturing Staphylococcus aureas and Candida al&cans, requiring multiple drainage procedures and subsequently grafts to close ulcers. Six months prior to admission he developed a rash in his pubic area, biopsy of which revealed nonspecific dermatitis from which grew Staphylococcus aureus.
On admission, routine laboratory workup demonstrated a white cell count of 10,200 mm3 with 62% segmented neutrophils, 1% bands, 4% eosinophils, 6% monocytes, and 25% lymphocytes. The hemoglobin was 13.9 g% and the hematocrit 38.5. The following tests were all found to be normal: calcium, phosphorus, fasting blood sugar, urinalysis, cholesterol, bilirubin, alkaline phosphatase, LDH, SGOT, and T+ The IgG was 1280 IU, the IgA 534 IU, IgM 130 IU, and the IgE 4437 IU. The secretory IgA obtained from saliva was 7.6 mg/ml (normal = >3.0 mgiml). The blood type of the patient is 0 positive with an anti-B titer of 1:4 and an anti-A of 1:8. The RAST tests are summarized in Table 1. Total hemolytic complement levels (CH,,), as well as C3 and C4 levels, were normal. A 24-hr skin window using the Senn technique revealed a
RAST
TO COMMON
TABLE 1 RESPIRATORY
ALLERGENS 2” 3 1 0 0 I 2 3 2 1 0
Timothy Common ragweed Maple Oak Cat Epithelium Dog Epithelium Cladosporium herbarum Alternaria tenuis House dust Dermatophagoides pteryonyssihus Dermatophagoides farinae o Graded
response
for the RAST
on a scale of 0 to 4 +. A value
2 1 is indicative
of a positive
test.
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LYMPHOCYTE
a)
PHA --_ “PWM . . . . . . . . . . ‘,‘CON A4_-
FUNCTION
&Z* 9.
0,
/-c
m-
A’ +--4
1.L 1.0-
WBC Lymphocytes-
_ _
TB--7F = Transfer
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Acid
>
‘-
‘,.I
/
mo100
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n,z&
1 pi?/77 TF TF
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6&l
AS
Phagocytic Index
Migration Index
NBT Unrtim. NET
_
Stim.---
TF=Tronsfer AS:Ascorbic
Fact< Acid
TF TF TF AS response before and after therapy. The response of the lymphocyte system to transfer factor (TF) therapy is depicted (IA). The results of the T and B cells are expressed as the number of T or B cells/mm3. The WBC count is expressed as the number of cells X 104/mm3, and the lymphocytes as the number of cells x 103/mm3. The results of the lymphocyte transformation studies with PHA, PWM, and Con A are expressed as the stimulation index X 102. Granulocyte function (1B) in response to ascorbic acid treatment is expressed as a percentage of the control values. The controls were normal individuals studied. FIG.
1. In
viva
and
in virrc~
cellular
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HIE
blood granulocyte clearance (BGC) of 2.7 ml/cm‘?24 hr and a total leukocyte mobilization (TLM) of 22.0 x 106/cm2/24 hr. The in vitro and in vivo granulocyte and lymphocyte results are summarized in Fig. 1. There was a decrease in the relative and absolute number of T cells, and lymphocyte transformation response to mitogens was markedly impaired. Baseline nitroblue tetrazolium (NBT) reduction is above normal, but there was a poor response to stimulation with endotoxin. The phagocytic and bactericidal indices were normal, but the chemotactic response was markedly impaired. Table 2 demonstrates that there was an intrinsic granulocyte defect, but that the patient’s serum was also capable of suppressing normal granulocyte chemotaxis. Random mobility of granulocytes appeared normal throughout the course of illness. Skin tests including PPD, mumps, histoplasmin, streptokinase-streptodornase (SK-SK), Candida, and DNCB were negative at 24 and 48 hr. He did have an immediate hypersensitivity reaction to the Candida skin test at a dilution of 1:500 and a higher dose was not given. Ten days later, on rechallenge with DNCB, no response was noted. A sweat test revealed 20 meq/liter of chloride. Baseline febrile agglutinins were all less than 1:20 except for Salmonella OB which was 1:40. Ten days after vaccination with typhoid 1.5 cc IM there was no change in the titers. Two days after rechallenge with DNCB the patient was given his first injection of transfer factor from a donor who was skin test positive to histoplasmin, SK-SD, mumps, and Candida. Forty-eight hours later the patient developed a marked reaction at the site of the initial DNCB application with marked erythema and formation of vesicles.Ten days after the first injection of transfer factor, all the above skin tests were repeated and there was slight erythema without any induration only with histoplasmin (PPD, mumps, SK-SD, TABLE SERUM
INHIBITORS
2
OFCELLULAR
FUNCTION
Lymphocyte response
Pretherapy Post-therapy
PHA
Con A
PWM
47” 97
49 -
71 -
Chemotactic granulocyte response Bacterial factor Pretherapy Patient cells Control cells Post-therapy Patient cells Control cells
35* 274 -
a Percentage of control values. b Percentage of control cells similarly stimulated.
Zymosan-NHS
Zymosan patient serum
43 -
40 47
57 -
44 22
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and Candida remained negative). Four days later the patient was given a second injection of transfer factor from the same donor. In the following weeks he noticed improvement in the rash in his pubic area which became much less indurated and pruritic. The oral thrush completely disappeared.Two weeks later he received his third injection of transfer factor. By February 22. 1977, he had shown a dramatic improvement in his lymphocyte function (Fig. 1). but no improvement in his granulocyte function. No further improvement in the skin rash was noted. He was begun on ascorbic acid 1.0 g orally three times a day. The results of his in \,itro and in rvi~o granulocyte and lymphocyte studies 2.5 and 6.5 months later are summarized in Fig. 1. There was a marked improvement in lymphocyte and granulocyte function. Although chemotaxis of granulocytes with bacterial factor was better than normal, zymosan induced chemotaxis was still subnormal. His IgE was now 4650 IUiml and skin window studies revealed a BGC of 2.4 ml/cm?24 hr and a TLM of 17.1 x lO”icmY24 hr, which are not significantly improved. His pubic rash was almost completely gone. Lymphocyte Function Tests The lymphocyte system was assessed by the method previously outlined from this laboratory (22). Briefly, lymphocyte subpopulations were identified after separation using a density gradient Ficoll-Hypaque column centrifuged at 400g. Sheep red blood cell rosetting (E-RFC) was used to identify T cells, while complement receptors (EAC-RFC) and surface immunoglobulins identified B cells. Percentages from a normal population were as follows: E-RFC = 50-75%: EAC-RFC = lo-34%; IgG = 5-20%; IgM =3-150/o; and IgA = O-4%. Lymphocyte function was assessed by in vitro lymphocyte transformation to phytohemagglutinin, concanavalin A, and pokeweed mitogen. Lymphocyte transformation has been previously described in detail (22) and was performed as described. The results are expressed as the stimulation index (SI) or ratio of counts in the cultures with mitogen to the counts in control cultures. Greater than twofold stimulation by antigen was considered a positive test. The presence of a serum inhibitor to lymphocyte function was determined using purified cell populations. Lymphocytes were obtained from the patient and from a normal donor and separated on Ficoll-Hypaque gradients as described above. The cells were washed in RPM1 1640 and resuspended in RPM1 1640 containing 10% serum. Normal human serum was used as a control. The cells were incubated for 2-6 days in varying concentrations of mitogens. The peak SI was calcuated as above and recorded. In Vivo Granulocyte Function Tests The capability of granulocytes to migrate to a site of inflammation was measured by the technique outlined by Senn and Jungi (23). A plastic chamber was placed over an area of abraded skin and filled with fresh autologous serum. The chambers were glued in place with collodion. The contents of the chambers were collected after 24 hr. The number of cells. type, and viability were determined routinely. The following parameters were calculated on the basis of cell counts: TLM = cumulative number of leukocytes/cm2/24 hr, leukocyte mobilization rate (LMR) = average leukocytes/cmVhr, and BGC = TLM/cir-
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culating neutrophilsml of blood. Values obtained in 14 normal individuals are TLM = $9.5 + 8.7 (mean ? SE), LMR = 3.7 + 0.3, and BGC = 31.8 + 4.5. In Vitro Granulocyte Function Tests The functional integrity of the granulocyte system was assessed by a battery of tests including NBT, chemotaxis in response to bacteria-derived factor(s) and zymosan-induced C5a, random mobility, phagocytic and bactericidal capabilities, and chemiluminescence. These granulocytes were separated by sedimentation at lg for 45 min at 37°C. The cell-rich plasma was then layered on a Ficoll-Hypaque gradient and centrifuged for 10 min at 4OOg. The sedimented cells were 95% pure granulocytes. The cells were washed in Hank’s balanced salt solution (HBSS) and resuspended to appropriate concentrations. NBT Reduction The model utilized for NBT dye reduction is that described by Park et al. (24). Venous blood was collected with 10 units of heparin/ml. The blood was incubated with 0.2% NBT in normal saline at 37°C for 15 min. Blood smears are prepared, stained with Wright’s stain, and a 200-cell differential was made noting the number of cells which have the reduced formazan deposits within the cytoplasm. These cells are easily recognized as PMNs with large irregular dark amorphous masses within the confines of the cytoplasm. A portion of whole blood was preincubated with 1 mg/ml bacterial lipopolysaccharide (LPS) (Difco, Detroit, Mich.) for 15 min at 37°C and the NBT test was repeated as above. Normal individuals routinely showed values ~20% in the unstimulated cultures and >30% in the LPS stimulated cultures. Chemotaxis The chemotaxis response was determined as outlined in detail by Ward (25). The chemotactic chambers (Bellco Glass, Vineland, N.J.) are prepared using S-pm porosity SMWP 02500 Millipore filters. The cells are cultured in RPM1 1640 buffered with bicarbonate at pH 7.3. Chemotactic factors used included a bacterial culture filtrate of Escherichia coli, zymosan-activated normal human serum, and the appropriate controls. The chambers are incubated at 37°C for 3 hr. The filters are carefully removed from the chambers, fixed in absolute propanol, stained with Wright’s stain, and finally cleared in xylene. The filters are placed on conventional glass slides with mounting media and observed at 25x magnification. A minimum of 10 fields are viewed and the number of cells migrating completely through the filter are counted. The results are expressed as the ratio of the average number of cells per 25x field compared to unstimulated controls. The presence of serum inhibitors of chemotaxis was determined by replacing the normal human serum pool with the patient’s serum and repeating the test on cells from normal donors. Leukocyte Random Mobility The ability of leukocytes to randomly migrate from a capillary tube in a culture chamber is measured by the technique outlined by Miller (26). The technique involves incubation of 1 x lo7 PMNs in RPM1 1640 for 24 hr. The
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degree of migration from this capillary tube is measured by planimetry at 4 and 24 hr. The results are expressed as the area of the cells migrating out of this capillary tube. Phagocytic and Bactericidal Activities The ability of the granulocytes to ingest and kill bacteria was studied by the method outlined by Weir (27). An 18-hr culture of Staphylococcus aureus. strain 502A, (ATCC, Bethesda, Md.) was incubated with 1 x lo7 granulocytes in a ratio of bacteria to granulocytes of 1. For phagocytic indexes, aliquots of the bacteria-cell mixture were sampled at 30, 60, and 120 min. The cells are centrifuged at 400g and the number of bacteria remaining in the supernatant was calculated based on viable plate counts. Bactericidal abilities of these granulocytes were determined by mixing cultures of Staphylococcus aureus and granulocytes in a ratio of 1: 1 for 15 min at 37°C. Excess bacteria were removed by washing. Aliquots were then taken at 30, 60, 90, and 120 min. The number of bacteria contained within the granulocytes was determined after lysing the washed granulocytes by viable plate counts. The results of these two assays were expressed as the phagocytic index (PI) and bactericidal index (KI). PI and KI are calculated by comparing the log number of bacteria at 0 and 120 min. Normal regression lines can be plotted. Chemiluminescence The ability of granulocytes to generate photons of visible light after stimulation was measured by a modification of the method of Allen (28). Polymorphonuclear leukocytes were separated as described and resuspended to 1 x lo7 PMN/ml in HBSS. The cells were incubated at 37°C with 1 mg of zymosan previously opsonized with normal human serum by incubation at 37°C for 1 hr. The emission of light was measured in a Packard Tri-Carb scintillation counter with coincidence off for 0.2 min at 1% gain. Duplicate counts were obtained and the samples immediately returned to the water bath. Repetitive counts were made every 10 min for 2 hr. The results are expressed as the counts per minute/IO’ granulocytes at the peak of the curve. Cells from normal individuals yield a mean ? SE of 7111 +- 528. Complement Profile Total hemolytic complement activity was determined as described by Campbell er al. (29). Individual complement components were determined either as a titration of hemolytic activity or as the concentration of protein by radial immunodiffusion. Hemolytic activity was determined by commercially available kits (Cordis Lab, Inc., Miami, Fla.) and performed as directed. Protein determinations were obtained by radial immunodiffusion using commercially prepared immunoplates (Behring, Somerville, N.J.). Transfer Factor Preparation Transfer factor was prepared as a dialyzable extractable, DNase resistant extract of peripheral blood mononuclear cells after the procedure outlined by Spitler (30). One unit of peripheral blood was collected in CPD and the leukocytes separated by sedimentation with 10 ml 5% Dextran 200. One unit
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of transfer factor was defined as the amount of extractable material derived from 5 x lOa lymphocytes. The donor was selected because of strong positive reaction to candidin skin test allergen and an in vitro stimulation of lymphocytes with Candidu afbicans extract. He was also positive to histoplasmin, SK-SD, and mumps, but his response to DNCB is unknown. DISCUSSION
This patient was typical of the HIE syndrome. He had recurrent severe bacterial infections with staphylococcus uureus, hyperimmunoglobulinemia E, and defective neutrophil chemotaxis. He did not, however, manifest any clinical signs of allergic disease. Patients with HIE have previously been reported who lack the manifestations of allergic disease (4). It is of interest that he did have positive RAST tests and immediate hypersensitivity to Candida antigen. The patient is also atypical in that a serum inhibitor against both granulocyte chemotaxis and lymphocyte function was readily detectable. However, a serum inhibitor was not his only defect. An intrinsic cellular defect in both lymphocytes and granulocytes was demonstrated (Table 2). Many diseases have been associated with impaired chemotaxis (31). Serum inhibitors of chemotaxis have been infrequently described (6, 7, 10, 32, 33). An inhibitor was found in this patient’s serum but was not characterized further. Impaired cellular immunity has been found in mucocutaneous candidiasis associated with impaired lymphocyte transformation and MIF production with Candida antigen (34-41). Serum inhibitors of cellular immunity, specifically directed against T cells have been reported in Hodgkin’s disease (42), as well as other conditions (43), but not in mucocutaneous candidiasis or the HIE syndrome. This patient demonstrated an inhibitor against lymphocyte transformation with multiple antigens. The short stature of this patient is of interest because of previous reports of immunological deficiency syndromes associated with short-limbed dwarfism and ectodermal dysplasia (44-46). However, this patient had no evidence of achondroplasia, ectodermal dysplasia, or endocrinopathy. It is most likely that his short stature is based on his chronic illness as a child. Transfer factor has been used successfully to treat mucocutaneous candidiasis (15-20). However, in a patient with abnormal granulocyte chemotaxis associated with mucocutaneous candidiasis, Gallin found that multiple doses of transfer factor failed to correct her chemotactic defect and suppressed cellular immune responses (47). Snyderman et al. (39) found that a patient with chronic mucocutaneous candidiasis who had defective mononuclear leukocyte chemotaxis improved with transfer factor. Our patient improved with transfer factor by: (i) improved in vitro lymphocyte function; (ii) improved response to DNCB testing; and (iii) clinical improvement in the degree of oral thrush. Ascorbic acid has been shown to improve granulocyte chemotaxis in both normal people and patients with Chediak-Higashi syndrome (21). Since there had been little improvement in granulocyte function with transfer factor, ascorbic acid was given. There was a dramatic and progressive improvement in this patient’s chronic pubic skin rash and further improvement in the in vitro tests
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of lymphocyte and granulocyte function, although the skin window results were unchanged. Levamisole has been used to treat patients with the HIE syndrome (48). Chemotaxis of neutrophils and mononuclear cells from patients with the HIE syndrome were stimulated by levamisole both in vir~ and in ~+ro. Two of these patients also had mucocutaneous candidiasis and impaired lymphocyte function. Although in \litro testing improved, there was no evaluation of clinical improvement in these four patients. The initial clinical and laboratory improvement in this patient following transfer factor was intensified with the addition of ascorbic acid. The improvement in lymphocyte responsiveness in this patient correlated with the clinical improvement in the oral thrush and was temporally related to the treatment with transfer factor. The improvement in granulocyte function correlated with complete clearing of the oral thrush and improvement in the pubic rash and was temporally related to the treatment with ascorbic acid. It seems unlikely that the improvement in granulocyte function was due to a delayed effect of the transfer factor rather than to the ascorbic acid. The ascorbic acid may have contributed to the improved in vitro lymphocyte and granulocyte function by increasing levels of 3’-5’-guanosine monophosphate (cyclic GMP) in granulocytes (49-54) as well as in lymphocytes (55). Effective treatment of the HIE syndrome would improve the quality of life of these individuals. Bale ef 01. (56) have described a fatal histiocytic lymphoma in a child with the HIE syndrome. If transfer factor and ascorbic acid prove useful in other patients with the HIE syndrome, fatal lymphoreticular malignancies in these patients, as in others with immunodeficiency syndromes, might be prevented. ACKNOWLEDGMENTS The authors would Amy Witte and Howard
like to express Rand. and Alice
their sincere gratitude Stargardt for preparation
for the technical of this manuscript.
assistance
of
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