aDivision of Infectious Diseases, Center for the Study of Emerging and Re-emerging Pathogens and School of Public Health, and bThe Division of Gastroenterology, The University of Texas, Houston Medical School, Houston, TX, USA; cThe Division of Infectious Diseases, and dDepartment of Immunology, Baylor College of Medicine, Houston, TX, USA.
Grant Support: US Environmental Protection Agency (#CR-819814 and #CR-824759), National Institutes of Health R01 AI 41735-01 and RR-02558 and the Food and Drug Administration (FD-U-001621-01).
Received: 18 October 2000; accepted: 30 January 2001.
Chronic cryptosporidiosis in AIDS patients was formerly a debilitating illness that frequently resulted in death . With effective antiretroviral therapy, many patients experience complete a resolution of symptoms and suppression , or elimination of the parasite [3,4]. Although a previous study demonstrated an increase in CD4 T cell numbers in the colonic mucosa of AIDS patients after short-term combination therapy , little information exists on the effects of antiretroviral combination therapy on local cytokine production by the gastrointestinal tract in AIDS after highly active antiretroviral therapy (HAART). To our knowledge no data exist regarding the local intestinal cytokines associated with the control of opportunistic infection or after immuno-reconstitution in AIDS.
We previously demonstrated that the resolution of Cryptosporidium parvum infection in immunocompetent adults is associated with the production of IL-15. In contrast, infection in sensitized individuals is controlled by the production of γ-IFN . The objective of this study is to describe the local cytokine response in the intestines in AIDS patients with chronic cryptosporidiosis, and to document the changes in cytokine profiles noted when cryptosporidiosis resolves in response to HAART.
Seven AIDS patients with AIDS, chronic diarrhea, cryptosporidiosis, and C. parvum oocysts in their stool participated in the study. A baseline evaluation included a medical examination, the determination of circulating CD4 T cell numbers and HIV viral load. The baseline mean CD4 T cell count was 25 ± 28 cells (range 5–75) and the mean viral load was 5.82 (log). All patients were antiretroviral experienced. At baseline, two patients were receiving monotherapy with stavudine (biopsied before the availability of protease inhibitors), two patient were receiving combination regimens but with antiviral failure, and two had been heavily pretreated and were off antiviral therapy at the time of enrolment.
Subjects were asked to provide 24 h stool collections for oocyst quantitation as well as to undergo endoscopy with jejunal biopsy. On the basis of previous antiviral exposure, the antiretroviral regimens were modified. Patients were started on azithromycin 600–1200 mg a day and paromomycin 2 g a day (in two to four doses) . Four to 24 weeks later, patients were reassessed for response. In those who demonstrated clinical improvement, the endoscopy was repeated and stools examined for oocysts. Intestinal biopsy specimens were immediately fixed in diethyl pyrocarbonate-treated paraformaldehyde (60 min, room temperature), washed in diethyl pyrocarbonate-treated phosphate-buffered saline, and stored in 70% ethanol until sectioning. Cytokine responses were assessed by in-situ hybridization with [35S]-labelled riboprobes for IFNγ, transforming growth factor beta (TGFβ), IL-15, and IL-4 using methods previously described [6,8]. TGFβ was the only cytokine identified in the baseline biopsies and was found in two out of six patients studied.
Out of the seven patients, three resolved their diarrhea, and one improved but did not resolve the infection after modifications in antiviral therapy as outlined in Table 1. In the three responders, the CD4 T cell counts increased a mean of 85 cells (range 57–140), and the viral load decreased by a median of 1.41 log (range 0.24–3.04). Patient five demonstrated an increase in the IL-15 signal at week 4, which was followed by increases in IFNγ and IL-4 at week 12. Patient six demonstrated IL-15 weakly at week 24. Finally, patient 7 demonstrated increases in both IFNγ and IL-4 at week 16. Patient 4 demonstrated some clinical improvement at week 4, and was found to have messenger RNA for IFNγ, IL-15, TGFβ and IL-4.
These data suggest that several intestinal cytokines are expressed in response to chronic cryptosporidiosis in patients who are receiving antiviral therapy. TGFβ stimulates B cells to switch to the production of IgA. It also participates in the restoration of damaged epithelium. Its expression in chronic infection is consistent with our previous observation of expression in normal volunteers shedding oocysts, and probably reflects a response to counteract ongoing injury. By contrast, mRNA for either IL-15 or IFNγ and IL-4 could be identified in biopsies from all three patients who had resolved cryptosporidiosis in response to antiretroviral therapy. In previous studies, we noted IFNγ and IL-4 as part of the response of sensitized HIV-negative normal volunteers to C. parvum, whereas the expression of IL-15 was associated with the response in naive volunteers. The numbers of patients and biopsies studied here is insufficient to determine the timing. However, the sequential expression in subject 5 suggests that perhaps AIDS patients develop a local naive and then a memory response. For now, we can conclude that in AIDS patients who are undergoing immune reconstitution, intestinal cytokine expression increases the associated with the resolution or improvement of Cryptosporidium infection. The patterns of cytokines expressed are similar to those noted in self-limited infection in normal volunteers. There is thus evidence of local as well as systemic immune reconstitution.
Pablo C. Okhuysena
Maria T. Nguyena
Esteban C. Nanninia
Dorothy E. Lewisd
Cynthia L. Chappella
A. Clinton White Jrcd
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