Immunobiology and long-term graft function in a transplant heterotopic renal rat model.

BACKGROUND: The Th1-Th2 paradigm proposes clonal expansion of Th2 lymphocytes as the basis of allograft tolerance. The Th2 cells have been found to be present in recipients with long-term allograft survival. However, the presence of Th2 cells and tolerance is not a uniform finding. Previously we have shown that pre-engraftment single dose rapamycin and a 7-d course of cyclosporin induce transplantation tolerance to 120 d. In the present study, we investigated the immunobiology of grafts in a long-term follow-up (>350 d). METHODS: Kidney allografts (n = 7), isografts (n = 5) and single nephrectomy (n = 3) groups were followed for 350 +/- 87 d. Heterotopic kidney transplant was performed by the same surgeon in the allograft group (ACI-Lewis) and the isograft group (Lewis-Lewis). The left kidney was removed in the single nephrectomy group. The allograft group was treated with pre-engraftment single dose rapamycin and a 7-d course of cyclosporin. A kidney biopsy was performed at midpoint time for histological study and tissue was frozen for measuring intragraft cytokine expression (IL-4, IL-10) in all animals. Prior to biopsy, serum blood urea nitrogen (BUN) and creatinine (Cr) levels were studied. Serum BUN, Cr levels, plus 24-h urinary protein (PRO) were measured prior to sacrifice. Randomly, four allograft rats received skin grafts (ACI, Lewis and Buffalo skin donors) after kidney biopsy. Skin grafts were studied for a mean of 6 weeks for signs of acceptance or rejection. Analysis of variance (ANOVA) with Tukey's test was used; p < 0.05 was considered statistically significant. RESULTS: The mean follow up was 352 +/- 87 d. BUN and Cr levels at biopsy time (mean 214 d) were not statistically different between the three groups (p = 0.19 and p = 0.66). At sacrifice (mean 352 d), BUN, Cr and PRO were statistically different between allograft and isograft groups (p = 0.013), and between allograft and single nephrectomy groups (p = 0.027). Functional and histological signs of graft loss occurred in three of seven (42.8%) of the allografts at 352 d. Using BANFF criteria, three allografts at biopsy time and seven allografts (100%) and four isografts (80%) at sacrifice time developed chronic histologic changes. Intragraft overexpression of IL-4 and IL-10 was seen at biopsy and sacrifice time in six of seven allografts and one of five isografts. All donor specific skin grafts (ACI-Lewis) on allografts were accepted and third party (Buffalo) donor skin grafts were rejected in all animals (>95% skin necrosis). CONCLUSIONS: This highly stringent, functional, renal transplant model yields 100% normal renal function as compared with isografts at 120 d follow-up. With the follow-up extended to 350 d, 43% of the allografts loose function and develop a chronic allograft histology despite a demonstrated intragraft Th2 cytokine dominance and donor specific skin graft acceptance.

Features of Chlamydia trachomatis and Neisseria gonorrhoeae infection in male Army recruits.

Non-health care-seeking male United States Army recruits were tested for Chlamydia trachomatis (n=2245) and Neisseria gonorrhoeae (n=884), using a urine ligase chain reaction test to determine prevalence and potential risk factors for infection. The prevalence of chlamydial infection was 5.3%. Black race, a new sex partner, a history of trichomonas, and the presence of symptoms were associated with chlamydial infection. The prevalence of N. gonorrhoeae infection was 0.6%. Only a reported history of or positive test for C. trachomatis was associated with gonorrheal infection. Of those testing positive for chlamydia, 14% reported symptoms versus 40% of those with gonorrhea. Younger age was not a predictor of either infection, as has been shown for women. A substantial number of male army recruits are infected with C. trachomatis, but few are infected with N. gonorrhoeae. Screening on the basis of symptoms alone would miss the majority of both infections.

Intragraft cytokine expression in tolerant rat renal allografts with rapamycin and cyclosporin immunosuppression.

The Th-1/Th-2 paradigm proposes clonal expansion of Th-2 lymphocytes as the basis of tolerance towards allografts. Intragraft cytokine expression was evaluated in a highly stringent model of renal transplantation. ACI and Lewis rats were used as donors and recipients, respectively, for heterotopic renal transplantation. Group A (n = 8) received a single dose of rapamycin and cyclosporin 12 h prior to engraftment, followed by 7 d of cyclosporin post-operatively. Isografts (Group B, n = 5) and control allografts (Group C, n = 4) received no immunosuppression. Sacrifice was performed after 120 d. Intragraft expression of IL-10, IL-4, and IFN-gamma was determined using qualitative reverse transcriptase-polymerase chain reaction (RT-PCR). All groups had functionally normal grafts at sacrifice, with 50% histological tolerance among Group A animals. No isografts showed evidence of cellular infiltrate, and all control allografts showed severe rejection. IL-10 was only detected in the tolerant animals (p < 0.001). Similarly, IL-4 was detected predominantly in the tolerant allografts (p < 0.05). IFN-gamma was only isolated in rejected allografts, whether treated or untreated (p < 0.001). We conclude that the expansion of Th-2 cells is associated with tolerance, while the expansion of Th-1 cell is associated with acute cellular rejection.

Intragraft cytokine expression and tolerance induction in rat renal allografts.

BACKGROUND: Intragraft cytokine expression was evaluated in a model of renal transplantation. ACI and Lewis rats were used as donors and recipients, respectively, for heterotopic renal transplantation. METHODS: Treated allograft rats (n=10) received a preoperative dose of rapamycin and cyclosporine, followed by 7 days of cyclosporine postoperatively. Isograft rats (n=5) and control allograft rats (n=4) received no immunosuppression. Sacrifice was performed after 120 days. Expression of interleukin (IL)-4, IL-10, and interferon-gamma (IFN-gamma) transcripts was determined with semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: All treated allograft rats had normal function with 50% histologic rejection. All isografts had normal function. IL-4 and IL-10 were in greater density in allografts with normal histology, whereas IFN-gamma was only seen in allografts with cellular rejection. No IL-10 was seen in isografts, but IL-4 was detected in 3/5 isografts. CONCLUSIONS: We conclude that the lymphocyte population's elaboration of IL-4 and IL-10 is associated with tolerance, whereas the production of IFN-gamma and absence of IL-4 is associated with histology suggestive of acute cellular rejection.

Flow cytometric analysis of chimerism in the rat tolerant to a renal allograft.

BACKGROUND: Chimerism, produced by the two-way migration of cells between graft and host, is a proposed mechanism by which tolerance occurs. The appearance of donor/recipient chimeras in tolerant ACI to Lewis rat heterotopic renal transplants was assessed in peripheral blood leukocytes using flow cytometry after staining with monoclonal antibodies. MATERIALS AND METHODS: ACI and Lewis rats were used as donor and recipient, respectively, after Rapamycin and Cyclosporin immunosuppression with or without donor blood or bone marrow transfusion. ACI and Lewis animals were also used for isograft and single-kidney controls. Animals were sacrificed at various time points after initial operation. Flow cytometry was performed on isolated peripheral blood leukocytes at sacrifice. Histologic and functional data were also obtained. The monoclonal antibody panel included RT1(a) (ACI, MHC I) combined with CD2, CD4, CD8, CD16, and CD25 or RT1(a,c) (bone marrow chimeras). RESULTS: RT1(a)+, CD8+ cells were transiently present in the peripheral blood leukocytes of Lewis recipients with the exception of allogeneic bone marrow recipients. No significant number of RT1(a)+, CD16+ ("dendritic" cell-line) chimeras was seen. Veto cells (RT1(a,c)+) were transiently present in the bone marrow recipients, but they did not lead to improved outcome. Furthermore, no correlation was made between histologic tolerance and any of these donor-derived cells. CONCLUSION: Donor/recipient chimerism, and the veto cell phenomenon are not operational tolerance mechanisms in this stringent model of ACI to Lewis rat renal transplantation.

The immunologic role of IFN-gamma in ACI to Lewis kidney transplantation.

BACKGROUND: One of the proposed mechanisms of tolerance induction is the Th-1/Th-2 paradigm. The Th-1 cell is proinflammatory, secreting IFN-gamma and IL-2. Conversely, the Th-2 cell is anti-inflammatory, secreting IL-4 and IL-10. In our earlier studies a shift toward Th-2 dominance was required for tolerance induction in this model. MATERIALS AND METHODS: ACI and Lewis rats were used as donors and recipients, respectively. Twelve hours prior to engraftment, rapamycin 1.5 mg/kg po and cyclosporin 10 mg/kg sc were given, followed by 5 mg/kg sc postop (days 1-7). Lewis rats were used as isografts. Functional allograft tolerance was induced consistently in 100% of the recipients with 50% of the allografts exhibiting normal histology beyond 120 days. Qualitative RT-PCR was performed on the grafts to determine IFN-gamma expression with beta-actin housekeeping gene as control. RESULTS: IFN-gamma was expressed in all untreated allografts (5/5) and all treated, yet rejecting, allografts (4/4). None of the isografts (0/5) or histologically tolerant allografts (0/4) expressed IFN-gamma. This distribution was statistically significant (P < 0.001, Fischer's exact test). CONCLUSION: Our findings support a shift from Th-2 to Th-1 predominance as the corollary mechanism responsible for preventing histologic tolerance.

Analysis of functional renal allograft tolerance with single-dose rapamycin based induction immunosuppression.

The induction of transplantation tolerance is one of the primary goals following solid organ transplantation. The combination of a single dose of rapamycin (RAPA) with a short course of cyclosporine (CsA) has been shown to induce transplantation tolerance in the nonfunctional rat heterotopic cardiac transplant model. The purpose of this study was to assess this effective induction protocol in a functional renal transplant model. Male ACI (RTl(a)) and Lewis (RT1(1)) rats were used as donor and recipients respectively. Allografts received a single RAPA dose of (1.5 mg/kg) combined with CsA (10 mg/kg) 12-14 hr prior to transplantation. CsA (5 mg/kg) was given daily on days +1 - +7. Untreated Lewis to Lewis isografts served as histological controls. Chimerism, assessed in recipient skin, and intragraft interleukin (IL) 10 expression was determined utilizing PCR and RT-PCR techniques respectively. Treated animals and isografts were sacrificed 120-130 days posttransplant for functional and histological evaluation. Allografts (n=9) were functionally tolerant with serum creatinine (0.77+/-0.1 vs. 0.88+/-0.1; P=0.275), blood urea nitrogen (37.6+/-4.6 vs. 23.3+/-1.9; P=0.123), and 24 hr protein excretion (27.0+/-4.4 vs. 17.9+/-5.2; P=0.131) similar to single kidney ACI controls. Histologically, 45% (4/9) allografts were indistinguishable from isografts with no evidence of rejection, and were considered immunologically tolerant. Donor/recipient chimerism was not detected. All immunologically tolerant allografts had evidence of intragraft IL-10 expression. Rejecting allografts and isografts did not express intragraft IL-10. This study confirms the efficacy of pre-engraftment single-dose RAPA combined with CsA in inducing true immunologic tolerance in this stringent functional renal transplant model. The expression of intragraft IL-10 in tolerant recipients suggests a Th-2 shift as the mechanism of tolerance in this model.

Effect of single-dose rapamycin-based immunosuppression on the development of cardiac allograft vasculopathy.

BACKGROUND: Cardiac allograft vasculopathy is the major cause of graft loss more than 1 year after transplantation. Daily rapamycin dosing has been shown to inhibit arterial intimal thickening caused by both alloimmune and mechanical injury. The combination of a single preoperative dose of rapamycin with a short (7 day) course of cyclosporine A has been shown to extend cardiac allograft survival, but its effects on the development of cardiac allograft vasculopathy has not been reported. METHODS: The ACI (RT1(a)) to Lewis (RT1(1)) heterotopic cardiac allograft model was used to assess the development of cardiac allograft vasculopathy and rejection. Treatment groups included nonimmunosuppressed control, cyclosporine A, cyclosporine A/donor-specific transfusion, and rapamycin/cyclosporine A. RESULTS: The addition of a single preoperative dose of rapamycin to a short course of cyclosporine A significantly reduced the prevalence of cardiac allograft vasculopathy in small (1.18 +/- 1.4 versus 0.05 +/- 0.3; p = 0.0001), medium (2.05 +/- 1.09 versus 0.26 +/- 0.62; p = 0.0001), and large (2.57 +/- 0.84 versus 1.43 +/- 1.2; p = 0.0008) vessels when compared with that in allografts treated with a single preoperative donor-specific transfusion and the same cyclosporine A schedule. Cardiac allograft vasculopathy did not develop in the nonimmunosuppressed control grafts or the group treated with cyclosporine A alone, because of the short survival times in these groups. In addition, there was a reduction of the rejection score in the rapamycin-treated allografts compared with that in the other treatment groups (4.0 +/- 0.0 versus 3.25 +/- 0.5; p = 0.0006). CONCLUSIONS: These results suggest that a single preoperative dose of rapamycin is efficacious in preventing the development of cardiac allograft vasculopathy, and continued immunosuppression with rapamycin may be unnecessary.

Cardiac allograft tolerance induction with limited immunosuppression.

We evaluated the efficacy and systemic toxicity of cyclosporine G, rapamycin, and cyclosporine A with multiple donor-specific blood transfusions in the stringent ACI to Lewis heterotopic cardiac transplant model. In addition, all animals received a 28-day post-operative course of cyclosporine A. Systemic toxicity was assessed by measuring recipient body weight at 30 and 60 days posttransplantation and at the time of graft rejection. Preengraftment cyclosporine G (10 mg/kg) resulted in a mean graft survival of 7.31 +/- 1.09 days (n=13; N.S. vs Control). A 10-day course of the novel immunosuppressant rapamycin (d4-14) combined with our standard 28-day cyclosporine A protocol resulted in a mean graft survival of 206.0 +/- 143.6 days (n=5; P < 0.05 vs Control). Furthermore, the rapamycin injection vehicle was found to have no intrinsic immunosuppressive activity or systemic toxicity. The addition of pre- (d-1) and post- (d7, 14, 21) engraftment donor-specific transfusion resulted in a mean allograft survival of 131.2 +/- 43.9 days. No significant difference in interval weight was observed in any of the experimental groups. We conclude that cyclosporine G is of little value in this experimental model. However, rapamycin combined with cyclosporine A provides effective immunosuppressive synergy without significant toxicity or the sensitization risk inherent in donor-specific blood transfusions.

Induction of long-term graft tolerance and donor/recipient chimerism.

We compared the effect of different immunosuppressive drug regimens on both mean and long-term allograft survival and the histologic appearance of donor/recipient chimeras in the ACI to Lewis rat heterotopic cardiac transplant model. Intraabdominal cardiac transplantation was performed in standard fashion and microchimerism was assessed using immunohistochemical staining of cut sections of recipient spleen and lymph nodes. All of the drug regimens studied resulted in excellent mean allograft survival. Furthermore, long-term (> 120 day) allograft survival was consistently observed in each group. In fact, many of the recipients continue to have functioning heterotopic grafts even as they approach the end of the natural life span of a Lewis rat. Mixed allogeneic chimerism, however, was not observed with 100% frequency despite the consistent induction of graft tolerance. This finding may be related to the immunosuppressive agents used or the sensitivity of the immunohistochemical assay. Therefore, the exact role, if any, of chimerism in the induction of graft tolerance remains unanswered.

Role of intragastric pressure, pH, and pepsin in gastric ulceration in the rat.

A method of continuous gastric perfusion with "artificial gastric juice" was used in a study of individual factors (intragastric pressure, pH, and pepsin) known to participate in the pathogenesis of peptic ulcers. This method allowed change of only one factor at a time, while the other two remained constant. The gastric mucosa of normal rats, fasted for 48 hr, was found to be resistant to the ulcerogenic effects of artificial gastric juice perfused through the stomach for 6 hr without increasing the intragastric pressure. Perfusion of hydrochloric acid (pH 1.3) under increasing pressure produced ulceration of the corpus as well as forestomach portion of the stomach. The degree of gastric ulceration paralleled increases in intragastric pressure, acidity, and pepsin proteolytic activity. Inhibition of pepsin activity by a pepsin inhibitor protected the gastric mucosa even at the very low pH of 1.3. These results demonstrate that under the experimental conditions used, hydrochloric acid alone in the absence of pepsin does not produce ulceration of the rat stomach.