Treatment of cryptosporidiosis with oral bovine transfer factor

CLINICAL

IMMUNOLOGY

AND

Treatment

44, 329-334

IMMUNOPATHOLOGY

(1987)

of Cryptosporidiosis with Oral Bovine Transfer Factor’

E. LOUIE, W. BORKOWSKY, P. H. KLESIUS,* T. B. HAYNES,” S. GORDON, S. BONK, AND H. S. LAWRENCE New

York

University

Medical Center, New York, Parasite Research Laboratory,

New York 10016, Auburn, Alabama

and *USDA 36830

ARS Animal

Cryptosporidia are intestinal protozoans long known to cause diarrhea in humans, especially those with acquired immune deficiency syndrome (AIDS). When transfer factor prepared from calves which possessed delayed-type hypersensitivity to Eimeria bovis was given to nonimmune calves and mice it conferred protection against clinical infection (coccidiosis). Recent studies with oral bovine transfer factor have shown that it can confer cell-mediated immunity to humans. Based on these findings we decided to treat eight AIDS patients suffering from Cryptosporidium-associated diarrhea with transfer factor prepared from calves immune to Cryptosporidium. Prior to treatment with transfer factor, three patients had been treated with spiramycin, one patient with a-difluoromethylornithine (DFMO), and one patient with furazolidone for greater than 1 month without clinical or laboratory improvement. Following administration of transfer factor, five or eight patients exhibited a decrease in the number of bowel movements and the development of formed stools. Cqptosporidium was eradicated from the stools of four patients but two of these patients subsequently relapsed and one patient continued to have diarrhea despite the absence of Cryptosporidium in the stool. One patient has been free of diarrhea and Cryptosporidium for 2 years after discontinuation of transfer factor therapy. 0 1987 Academic Press. Inc.

INTRODUCTION Cryptospovidium species are intestinal protozoa long known to cause disease in animals and recently found to cause diarrhea in humans ( l-3). In immunocompetent individuals the illness is self-limited; however, in the immunocompromised host such as patients with the acquired immune deficiency syndrome (AIDS), cryptosporidiosis is associated with a significant morbidity and mortality (4). The results of currently available therapy particularly in such AIDS patients with cryptosporidial infection have not proved very promising (1). However, transfer factor has been shown effective in the control of a similar parasite (Eimeria bovis) in infected cattle (5) and (Eimeriuferrisi) in mice (6). Additionally, recent studies have also shown that bovine transfer factor when given orally confers cell-mediated immunity to humans and mice (7, 8) and favorably affected the outcome in a patient with Epstein-Barr and cytomegalovirus (CMV) infection (9). These encouraging results in experimental animal models and in humans i Informed consent was obtained from the patients or their parents or guardians. Guidelines for human experimentation of the Institutional Review Board of NYU Medical Center were followed in conducting the clinical research. 329

0090-1229/87 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ET AL.

prompted an evaluation of the effects of immunotherapy with Cryptosporidiurnimmune bovine transfer factor in patients with AIDS and cryptosporidiosis. MATERIALS

AND METHODS

Selection of patients. Between May 1984 and June 1985, eight patients who were diagnosed as having cryptosporidiosis were treated orally with bovine transfer factor. All of these patients met the Centers for Disease Control criteria for the diagnosis of acquired immune deficiency syndrome and clinical criteria for diagnosis of cryptosporidiosis (10). The diagnosis of cryptosporidiosis was confirmed by examination of the stool using a modified acid fast stain or an auramine stain. Quantitative counts of Cryptosporidium oocysts were performed by using 1 cc of stool. A lambda loop of stool was then serially diluted with 0.85% saline until 100-200 oocysts could be counted. The number of oocysts per cubic centimeter of stool was then calculated. Preparation of dialysate with bovine anti-cryptosporidium activity (transfer factor). Calves are obtained on the day of birth and housed in individual pens. Two to 5 days after birth the calves are inoculated with 100,000 oocysts of Cryptosporidium per OS. The calves are then monitored daily and fecal samples are stained with auramine looking for the presence of Cryptosporidium. Only calves that develop resistance to Cryptosporidium are used as donors for the preparation

of lymphocyte cell extracts. Resistance coincides with the absence of oocysts in the stool and usually occurs when the calves are 3 to 4 weeks of age. The donor calves are then killed and the mesenteric, thoracic, submandibular, cervical, axillary, prefemoral, and popliteal lymph nodes are removed under sterile conditions. A single cell suspension is obtained by treating the lymph nodes with a cell blender in sterile Hanks’ balanced salt solution (HBSS) without sodium bicarbonate (pH 7.2). The bovine lymphocyte extract is prepared by adjusting the cell concentration to 7.5 x 10’ cells/cc and then incubated in HBSS for a period of 4 hr at 37°C in a shaker-type water bath. The pH of the culture fluid is allowed to drop during the incubation period (pH 5 to 6). The lymphocytes and culture fluids are separated by centrifugation (700g for 60 min). The culture fluid is then filtered to remove any unwanted cellular debris (Whatman qualitative filter paper). The filtered culture fluids are placed in a hollow fiber concentrator (Amicon DC 2, Danvers, MA) with a HlP30-43 hollow fiber filter (30,000 MW cutoff) and concentrated at a flow rate of 20 mYmin at 10 psi (0.7 kb/cm2). After reaching a concentration of *A of the original volume, the ultratiltrate representing the 30,000 MW or less fraction is lyophilized. The ultrafiltrate fraction is lyophilized and the resulting dry material is designated as bovine lymphocyte extract-30,000 molecular weight fraction. Approximately 1 x lo6 lymphocytes yield 2.0 mg dry weight of material. One unit is defined as the material derived from 5 x IO8 lymphocytes. The lymphocyte extract is dissolved in sterile, pyrogen-free deionized water and filter sterilized (0.22 pm filter) before use. Transfer factor regimen. All adult patients were given 1 unit of bovine transfer factor (5 x lo8 lymph node cell equivalents) weekly for at least 1% months. The pediatric patient was given 0.20 unit per week. The patient’s course was moni-

TREATMENT

331

OF CRYPTOSPORLDIOSIS

tored by determining serial weekly oocyst counts in the stool as well as recording the quantity and quality of their stool. Additionally, general symptoms and signs were followed such as appetite, weight gain, or weight loss. Patients were also tested for immunologic function by measuring lymphocyte phenotypes and their in vitro proliferative response to recall antigens and allogeneic stimuli. RESULTS Eight patients with cryptosporidiosis were treated with transfer factor prepared from calves immune to this parasite. The age, risk groups, AIDS-related infections, neoplasms, and T-cell ratios are summarized in Table 1. One patient (J.G.) was 27 months of age and the remaining seven patients were between 18 and 42 years of age. The risk factor for five patients was homosexuality. Two patients admitted to intravenous drug abuse and the child was born to a drug-abusing mother. In four patients, one of whom had Kaposi’s sarcoma, cryptosporidiosis was the only opportunistic infection. The remaining four patients had other opportunistic infections. All of the patients had marked reversal of their T-cell ratios. Prior to treatment with transfer factor three patients (D.R., J.N., and T.S.) had been treated with spiramycin, 1 patient (R.D.) with a-difluoromethylornithine (DFMO), and another (L.S.) with furazolidone for greater than 1 month without clinical or laboratory improvement. Following administration of transfer factor, five (J.G., L.S., D.R., T.S., and TABLE

Patient

Age

Risk group

J.G.

21 months

Parents IVDA

L.S.

18 years

IVDA

D.R. J.N.

24 years 24 years

IVDA Gay

T.S. R.D.

42 years 35 years

Gay Gay

S.G.

30 years

Gay

R.F.

29 years

Gay

1

Opportunistic

infection

Chronic lymphocytic interstitial pneumonia Recurrent pneumococcal sepsis Cryptosporidiosis > 1 month Candida esophagitis Pneumocystis carinii pneumonia Cryptosporidiosis >l month Cryptosporidiosis > 1 month Salmonella Cryptosporidiosis > 1 month Disseminated cytomegalovirus Cryptosporidiosis >l month Kaposi’s sarcoma Cryptosporidiosis > 1 month Pneumocystis carinii pneumonia Cryptosporidiosis > 1 month Cryptosporidiosis > 1 month

T-helper/ T-suppressor ratio 0.08

0.33

0.1 0.18 0.2 0.04 0.47

332

LOUIE

ET AL.

R.D.) of the eight patients exhibited a decrease in the number of bowel movements and the development of formed stools. Cryptosporidium was eradicated from the stools of four patients (J.G., L.S., D.R., and J.N.). In one patient (L.S.) Cryptosporidium was eradicated from the stool while on transfer factor and furazolidone but relapse with recurrence of cryptosporidiosis occurred 2 weeks after the transfer factor was discontinued while still on the furazolidone. Patient D.R. developed disseminated cytomegalovirus and expired. At the time of death he was noted to have recurrent cryptosporidiosis despite treatment with transfer factor. Patient J.N. continued to have diarrhea despite the detection of two negative stools for Cryptosporidium on two separate occasions. The etiology of the persistent diarrhea could not be determined. Patient T.S. had an initial clinical response but relapsed despite the continuance of transfer factor. DFMO was added to the transfer factor regimen without effect and the patient subsequently expired secondary to fungal sepsis. Patients S.G. and R.F. had no response to transfer factor (TF) and remained unresponsive following treatment with spiramycin. Despite the clinical and microbiological responses accompanying TF therapy, no significant changes in immunological parameters were documented. Of the eight patients who were enrolled in this study, five patients have expired (L.S., D.R., J.N., T.S., and R.F.) and two patients (R.D. and S.G.) were lost to follow-up. One patient (J.G.) is doing well since cessation of transfer factor therapy with no evidence of relapse after 2 years. These results are summarized in Fig. I. DISCUSSION Patients with the acquired immune deficiency syndrome are susceptible to infection with a variety of endogenous and exogenous microorganisms. These microorganisms include viruses, bacteria, fungi, and protozoan parasites. Cryptosporidium is a protozoan parasite that has been responsible for producing severe diarrhea in patients with AIDS. Although it produces self-limited disease in normal human hosts, it has produced an invariably chronic illness in patients with AIDS. To date currently available treatment is usually ineffective. There have been sporadic reports of remission in patients treated with furazolidone, spiramycin, and DFMO; however, in our experience, these are rarely seen. In view of the lack of effective therapy for cryptosporidiosis and the grave outcome of such infections in AIDS patients, we conducted a preliminary trial to evaluate the effects of therapy with oral administration of transfer factor prepared from calves immune to Cryptosporidium. Recent studies have shown that bovine transfer factor when given orally can confer cell-mediated immunity to humans and mice (8). Resistance to E. ferrisi had been previously induced in mice by a single prophylactic treatment of bovine transfer factor administered orally (7). In addition, when transfer factor prepared from calves which showed delayed hypersensitivity and cell-mediated immunity to E. bovis, a bovine pathogen related to Cryptosporidium, was given to nonimmune calves it conferred protection against clinical infection when the calves were challenged with E. bovis (5). The data obtained from these studies suggest that bovine transfer factor is (i) effective when

TREATMENT

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given orally; (ii) nontoxic in humans; and (iii) can confer cell-mediated immunity and resistance to challenge with Cryptosporidium. Five of the eight patients in the present study were treated with either spiramycin, DFMO, or furazolidone without a response. All of these patients (L.S., D.R., J.N., T.S., and R.D.) showed a clinical response and/or a microbiologic response after they were started on oral bovine transfer factor. In addition, one other patient (J.G.) showed a clinical and microbiologic response. These findings suggest that transfer factor may be effective in the treatment of cryptosporidiosis. Confirmation of this conclusion will have to await further controlled studies since five of the six patients who responded were followed for less than 5 months. Of interest was the finding that two of the patients (L.S. and D.R.) who showed a clinical and microbiological response relapsed following the development of another opportunistic infection (Pneumocystis carinii pneumonia or disseminated CMV), while one patient was still on transfer factor. This suggests that the transfer factor may not eradicate the infection but may boost the patient’s cellmediated immunity enough to prevent the Cryptosporidium from being clinically apparent. When another opportunistic infection occurs it may facilitate a recurrence of clinical infection. In addition, as with other infections that occur in pa-

334

LOUIE ET AL.

tients with AIDS, therapy with transfer factor may be necessary for an indeterminate period, although there is one patient who is still doing well 2 years after the transfer factor was discontinued. The patients in this study were monitored with CBC, SM&, SMA,, at least monthly without any notable changes in any of these parameters. In addition no adverse signs or symptoms or other evidence of toxicity that could be attributable to transfer factor were noted in the course of these studies. We conclude that oral bovine transfer factor may be effective in the treatment of cryptosporidiosis. In the absence of more known effective treatment for cryptosporidiosis a controlled study to evaluate the efficacy of oral bovine transfer factor is warranted. REFERENCES 1. Navin, T. R., and Juranek, D. D., Rev. Infect. Dis. 3, 313, 1984. 2. Nime, F. A., Burek, J. D.. Page, D. L., Holscher, M. A., and Yardley, J. H., Gasrroenterology 70, 592, 1976. 3. Tzipori, S., Microbial. Rev. 47, 84, 1983. 4. Wittner, M., Goldfarb, J., Vogl, S., and Tanowitz, H. B., Amer. .I. Med. Sci. 287, 47, 1984. 5. Klesius, P. H., and Kristensen, F., Clin. Immunol. Immunopathol. 7, 240, 1977. 6. Klesius, P. H., Qualin, D. F., Elsion, A. L., and Fudenberg, H. H., C/in. Zmmunol. Immunopathol. 10, 214, 1978. 7. Jeter, W. S., Kibler, R., Soli, T. C.. and Stephens, C. A. L., In “Immune Regulators in Transfer Factor” (A. Kahn, C. H. Kirkpatrick, and N. 0. Hill, Eds.), New York, 1979. 8. Klesius, P. H., Elston, A. L., Chambers, W. H.. and Fudenberg, H. H.. C/in. fmmunol. Zmmunopathol. 12, 143, 1979. 9. Jones, J. J., Jeter, W. S., Fulginiti, V. A., Minnich, L. L.. Pritchett, R. F., and Wedgwood, R. J., LancetO, 122, 1981. 10. Update on Acquired Immune Deficiency Syndrome (AIDS)-United States. MMWR 31, 507 and 513, 1982. Received January 20, 1987; accepted April 15, 1987