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Mounting evidence points to alterations in mitochondrial metabolism in renal cell carcinoma (RCC). However, the mechanisms that regulate the TCA cycle in RCC remain uncharacterized. Here, we demonstrate that loss of TCA cycle enzyme expression is retained in RCC metastatic tissues. Moreover, proteomic analysis demonstrates that reduced TCA cycle enzyme expression is far more pronounced in RCC relative to other tumor types. Loss of TCA cycle enzyme expression is correlated with reduced expression of the transcription factor PGC-1α, which is also lost in RCC tissues. PGC-1α reexpression in RCC cells restores the expression of TCA cycle enzymes in vitro and in vivo and leads to enhanced glucose carbon incorporation into TCA cycle intermediates. Mechanistically, TGF-β signaling, in concert with histone deacetylase 7 (HDAC7), suppresses TCA cycle enzyme expression. Our studies show that pharmacologic inhibition of TGF-β restores the expression of TCA cycle enzymes and suppresses tumor growth in an orthotopic model of RCC. Taken together, this investigation reveals a potentially novel role for the TGF-β/HDAC7 axis in global suppression of TCA cycle enzymes in RCC and provides insight into the molecular basis of altered mitochondrial metabolism in this malignancy.

The transcriptional events that promote invasive and metastatic phenotypes in renal cell carcinoma (RCC) remain poorly understood. Here we report that the decreased expression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1α) and the increased expression of several genes encoding collagen family members are associated with RCC tumor progression. PGC1α restoration attenuates invasive phenotypes and suppresses tumor progression in vivo. In contrast, collagens produced by RCC cells promote invasive and migratory phenotypes. PGC1α restoration suppresses the expression of collagens and tumor phenotypes via the induction of miR-29a. Furthermore, decreased collagens via the PGC1α/miR-29a axis suppresses collagen-mediated activation of discoidin domain receptor 1 (DDR1)/ERK signaling. In turn, the suppression of collagen/DDR1 signaling by PGC1α leads to decreased levels of the known EMT regulators SNAIL1 and 2. Collectively, our results demonstrate a novel role for PGC1α in the regulation of proinvasive SNAIL proteins., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier B.V.)

Human neonates are at significantly greater risk of serious infection than immunocompetent adults. In particular, very low birth weight infants in the neonatal intensive care nursery are at high risk of developing life-threatening bacterial and fungal infections. Recent studies have identified Th17 cells as critical mediators of immunity to bacterial and fungal infections at epithelial barriers. Little is known, however, about the ontogeny of Th17-cell responses in humans. The frequency of serious bacterial infections in preterm infants and the importance of Th17 cells in providing protection against such infections in animal studies prompted us to study Th17-cell development in human neonates. Naïve CD4(+) T cells from extremely preterm infants, term infants, and adults were assayed for their capacity to develop into Th17 effector cells. Surprisingly, Th17-cell capacity was inversely related to developmental age. Neonates expressed higher levels of IL-23R, RORγt, and STAT3 prior to activation and showed a significant Th17-cell bias after activation. In contrast, adult cells expressed more TBX21 with a corresponding Th1-cell bias. CD161 expression on Th17-cell precursors was also developmentally regulated. Our results suggest there is significant developmental regulation of CD4(+) effector lineages with a strong bias toward Th17-cell development early in life., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: All human immunodeficiency virus (HIV-1) uses a host tRNALys,3 as the primer for reverse transcription. The tRNALys,3 is bound to a region on the HIV-1 genome, the primer-binding site (PBS), that is complementary to the 18 terminal nucleotides of tRNALys,3. How HIV-1 selects the tRNA from the intracellular milieu is unresolved., Results: HIV-1 tRNA primer selection has been investigated using viruses in which the primer-binding site (PBS) and a sequence within U5 were altered so as to be complementary to tRNAMet, tRNAPro or tRNAIle. Analysis of the replication of these viruses in human peripheral blood mononuclear cells (PBMC) revealed preferences for the selection of certain tRNAs. HIV-1 with the PBS altered to be complementary to tRNAMet, with and without the additional mutation in U5 to be complementary to the anticodon of tRNAMet, stably maintains the PBS complementary to tRNAMet following extended in vitro culture in PBMC. In contrast, viruses with either the PBS or PBS and U5 mutated to be complementary to tRNAIle were unstable during in vitro replication in PBMC and reverted to utilize tRNALys,3. Viruses with the PBS altered to be complementary to tRNAPro replicated in PBMC but reverted to use tRNALys,3; viruses with mutations in both the U5 and PBS complementary to tRNAPro maintained this PBS, yet replicated poorly in PBMC., Conclusion: The results of these studies demonstrate that HIV-1 has preferences for selection of certain tRNAs for high-level replication in PBMC.

The initiation of HIV-1 reverse transcription occurs at an 18-nucleotide sequence in the viral genome designated as the primer binding site (PBS), which is complementary to the 3' terminal nucleotides of tRNA(Lys,3). Since the PBS is highly conserved among all infectious HIV-1, it represents an attractive target for the development of new therapeutics to inhibit viral replication. In this study, we have evaluated three approaches using small interfering RNA (siRNAs) targeted to the PBS for the capacity to inhibit HIV-1 replication. In the first, transfection of a 21-nucleotide siRNA complementary to the PBS into cells inhibited production of HIV-1 following infection. Control siRNAs of the same length complementary to HIV-1 gag mRNA or to gfp mRNA decreased the production of virus or had no effect on virus replication, respectively. Analysis of the PBS of integrated proviruses derived from viruses that ultimately grew in cultures transfected with siRNA all contained wild-type PBS sequence, demonstrating that HIV-1 did not mutate to escape inhibition by siRNA. In the second approach, hairpin siRNA targeted to the wild-type PBS were expressed using an adeno-associated virus (AAV) vector. HIV-1 replication was inhibited in cells infected with AAV encoding the siRNA to the wild-type PBS, but not in cells infected with AAV encoding an siRNA of the same length targeted to an irrelevant PBS. Finally, studies from this laboratory have shown that alteration of the PBS to be complementary to tRNAHis results in the production of infectious virus that rapidly reverts to utilize tRNALys,3 following in vitro culture. A proviral genome containing a PBS complementary to tRNAHis that encodes an siRNA molecule complementary to the wild-type PBS under control of a U6 promoter within the nef gene was as infectious as the parent HIV-1 genome containing no insert in nef. The virus with the PBS only complementary to tRNAHis reverted to use tRNALys,3, coincident with rapid virus growth, while the virus encoding siRNA grew slower than the virus without siRNA and maintained the PBS complementary to tRNAHis longer in culture. At later times of infection, viruses with the PBS complementary to tRNAHis and the siRNA exhibited a rapid increase in p24 antigen in the culture. Analysis of the PBS revealed that it was now complementary to tRNALys,3. Analysis of the gene encoding the siRNA revealed that the reversion of the PBS coincided with the deletion of the gene encoding siRNA. The results of these studies show that siRNA targeted to the PBS of HIV-1 can inhibit virus replication, supporting the concept that HIV-1 has evolved a strong preference to select tRNALys,3 for high-level replication and establishing the PBS and primer selection as a potential target for new therapeutics.

The replication in human peripheral blood mononuclear cells (PBMC) of unique HIV-1 that select tRNA(His) or tRNA(Lys1,2) for reverse transcription was compared to the wild-type virus that uses tRNA(Lys,3). HIV-1 with only the primer-binding site (PBS) changed to be complementary to these alternative tRNAs initially replicated more slowly than the wild-type virus in PBMC, although all viruses eventually reached equivalent growth as measured by p24 antigen. Viruses with only a PBS complementary to the 3' terminal 18 nucleotides of tRNA(His) or tRNA(Lys1,2) reverted to use tRNA(Lys3). HIV-1 with mutations in the U5-PBS to allow selection of tRNA(His) and tRNA(Lys1,2) following long-term growth in SupT1 cells were also evaluated for growth and PBS stability following replication in PBMC. Although both viruses initially grew slower than wild type, they maintained a PBS complementary to the starting tRNA and did not revert to the wild-type PBS after long-term culture in PBMC. Analysis of the U5-PBS regions following long-term culture in PBMC also revealed few changes from the starting sequences. The virus that stably used tRNA(His) was less infectious than the wild type. In contrast, the virus that stably used tRNA(Lys1,2) evolved to be as infectious as wild-type virus following extended culture in PBMC. The results of these studies highlight the impact of the host cell on the tRNA primer selection process and subsequent infectivity of HIV-1.

Daclizumab and mycophenolate mofetil (MMF) decrease the incidence of acute allograft rejection. This double-blind, randomized, placebo-controlled trial was performed primarily to assess the pharmacokinetics of MMF in an immunosuppressive regimen incorporating daclizumab. At five centers, 75 renal transplant recipients were randomized 2:1 to receive either daclizumab 1 mg/kg or placebo pre-transplantation and every other week, for a total of five doses. All patients received cyclosporine, steroids, and MMF. Levels of mycophenolic acid (MPA), its glucuronide metabolite, and daclizumab were measured after dosing on days 28 and 56. Safety parameters evaluated included: adverse events, laboratory abnormalities, infections, patient/graft survival, incidence of lymphoproliferative disorders, and incidence of acute rejection at 12 months. The concomitant administration of daclizumab and MMF had no effect on the pharmacokinetics of MPA: AUC(0-8) values (microg h/mL +/- SD) on day 28 were 30.1 +/- 13.3 for daclizumab-treat patients vs. 31.1 +/- 12.4 for placebo and on day 56, 37.7 +/- 18.2 for daclizumab-treated patients vs. 35.7 +/- 14.0 for placebo. Adverse events were similar between the two groups. Acute rejection at 12 months occurred in 14% of patients receiving daclizumab and 20% of patients receiving placebo. The coadministration of daclizumab did not result in a pharmacokinetic interaction with MPA, the active metabolite of MMF.

Different T cell subsets may play different roles in allorecognition and allograft rejection. It has been suggested that CD8 T cells can only initiate rejection with help from CD4 T cells. Since CD8 T cells may have different requirements for allorecognition and for costimulation, it is important to clarify the role of CD8 cells in rejection. We examined the role of CD8 cells in allorecognition using a TCR transgenic mouse transplantation model. In our study, CD8 cells were able to recognize alloantigens and reject allografts in the absence of help from CD4 T cells. Furthermore our study provides a model to study the mechanisms of CD8-mediated allograft rejection. It may be important in the future, to consider that CD8 T cells may need to be targeted independently of CD4 T cells in strategies used to prevent rejection and induce tolerance.

Cyclosporine (CsA) has been implicated in both acute and chronic graft dysfunction. The addition of humanized IL-2 receptor antibody daclizumab (DZB) to CsA-based immunosuppression decreases the rate of acute renal transplant rejection. Therefore, 45 patients were evaluated in an immunosuppressive protocol that included DZB, mycophenolate mofetil (MMF), and steroids without CsA. This was a prospective, nonrandomized, open-label trial of the efficacy and safety of the treatment. DZB was given intravenously at 2 mg/kg before transplantation and then at 1 mg/kg every 2 wk for four doses, MMF was given orally at 3 g/d, and methylprednisolone/prednisone was given at 7 mg/kg per day and tapered to 15 mg/d at 6 mo. CsA was added to the regimen when patients developed acute rejection episodes or adverse effects to steroids or MMF; 49% of patients were spared CsA maintenance. Patients without CsA had lower serum creatinine at 6 mo and needed fewer medications to control BP. Incidence of biopsyproven rejections was 31% and occurred early (median, 10 d). These rejection episodes occurred earlier in cadaver transplants (median, 7 d) and later in living donor transplants (median, 62 days). Acute rejections occurred at a higher frequency (46% versus 34%) and earlier (6.5 versus 15 d) in patients with delayed graft function compared with patients without delayed graft function. Most of the rejections were moderate and easily reversible. The actuarial 1-yr graft survival was 95% with 100% patient survival.

Adenoviral vectors, encoding genes for cell surface antigens or receptors, have been used to induce their high level expression on tumor cells in vitro and in vivo. These induced antigens and receptors can then be targeted with radiolabeled antibodies or peptides for potential radiotherapeutic applications. The purpose of this study was to determine a dosing schema of an adenoviral vector encoding the human somatostatin receptor subtype 2 (AdCMVhSSTr2) for achieving the highest tumor localization of [(111)In]-DTPA-D-Phe1-octreotide, which binds to this receptor, in a human ovarian cancer model as a prelude to future therapy studies. AdCMVhSSTr2 was produced and used to induce hSSTr2 on A427 human nonsmall cell lung cancer cells and on SKOV3.ipl human ovarian cancer cells in vitro, as demonstrated by competitive binding assays using [125I]-Tyr1-somatostatin and [(111)In]-DTPA-D-Phe1-octreotide. Mice bearing i.p. SKOV3.ip1 tumors administered 1 x 10(9) plaque-forming units of AdCMVhSSTr2 i.p. 5 days after tumor cell inoculation, followed by an i.p. injection of [(111)In]-DTPA-D-Phe1-octreotide 2 days later, showed a range of 15.3-60.4% median injected dose/gram (ID/g) in tumor at 4 h after injection compared with 3.5% ID/g when [125I]-Tyr1-somatostatin was administered and 0.3% ID/g when the negative control peptide [125I]-mIP-bombesin was administered. Mice administered a control adenoviral vector encoding the gastrin-releasing peptide receptor did not have tumor localization of [(111)In]-DTPA-D-Phe1-octreotide (<1.6% ID/g), demonstrating specificity of [(111)In]-DTPA-D-Phe1-octreotide for the AdCMVhSSTr2 induced tumor cells. In another set of experiments, the tumor localization of [(111)In]-DTPA-D-Phe1-octreotide was not different 1, 2, or 4 days after AdCMVhSSTr2 injection (31.8, 37.7, and 40.7% ID/g, respectively; P = 0.88), indicating that multiple injections of radiolabeled peptide can be administered with equivalent uptake over a 4-day period. [(111)In]-DTPA-D-Phe1-octreotide tumor localization in animals administered AdCMVhSSTr2 on consecutive days or 2 days apart was 22.4% ID/g and 53.2% ID/g, respectively (P = 0.009) when [(111)In]-DTPA-D-Phe1-octreotide was given 1 day after the second AdCMVhSSTr2 injection. There was no difference in [(111)In]-DTPA-D-Phe1-octreotide localization after a single AdCMVhSSTr2 injection (40.7% ID/g) or two injections of AdCMVhSSTr2 given 1 (45.9% ID/g) or 2 (53.2% ID/g) days apart, where [(111)In]-DTPA-D-Phe1-octreotide was given in each case 4 days after the first AdCMVhSSTr2 injection (P = 0.65). Therefore, two AdCMVhSSTr2 injections did not increase [(111)In]-DTPA-D-Phe1-octreotide tumor localization compared with one injection, which eliminates concerns about an immune response to a second dose of AdCMVhSSTr2. This will be the basis for a therapeutic protocol with multiple administrations of an octreotide analogue labeled with a therapeutic radioisotope.

Interleukin 12 is a heterodimeric cytokine involved in the regulation of natural killer cell and T lymphocyte responses. In previous studies, we found that IL-12 induces proliferation of T cells only after co-stimulation with lectin, alloantigen, or anti-CD3 antibody. The IL-2-mediated proliferation of long-term T cell lines generated in this fashion is typically insensitive to the immunosuppressive agent, cyclosporine but sensitive to rapamycin. In this study, we examined the effect of cyclosporine and rapamycin on T cells responsive to IL-12. For long-term cultured T cell lines stimulated with phytohemagglutinin, alloantigen, or solid-phase anti-CD3 antibody, rapamycin blocked IL-12-induced proliferation to background levels. Culture in cyclosporine produced minimal inhibition of IL-12-induced T cell proliferation. Freshly isolated CD3+ cells did not proliferate in response to IL-12, nor did culture of these cells in IL-12 lead to upregulation of IL-2 receptor. These data suggest that the effect of IL-12, an important growth regulator for activated T lymphocytes, may involve late cellular activation events.

CdG, the carbocyclic analog of 2'-deoxyguanosine, is active against herpes, hepatitis B, and human cytomegaloviruses. We have studied the interaction of the tritiated enantiomers of CdG with the herpes simplex virus type 1-specific thymidine kinase (HSV-1 TK) and have examined their metabolism in uninfected and HSV-1-infected cells. D- and L-CdG were equally effective competitive inhibitors of the phosphorylation of thymidine (dThd) by the partially purified HSV-1 TK (Ki values were 2.1 and 3.4 microM, respectively) and were also equal as substrates (Km values were 17 and 26 microM, respectively, and Vmax values of the enantiomers were equal and about 50% greater than the Vmax for dThd). The partially purified enzyme preparation, which contained cellular nucleotide kinase activities (pyruvate kinase also was present in the assay medium), converted D-CdG almost exclusively to the triphosphate and L-CdG almost exclusively to the monophosphate. Similarly, in virus-infected cells the D-enantiomer was converted predominantly to the triphosphate and the L-enantiomer predominantly to the monophosphate. In uninfected cells the results were qualitatively similar. In CEM cells deoxycytidine (dCyd) kinase (EC 2.7.1.74) seemed to be the enzyme principally responsible for the phosphorylation of both enantiomers, as shown by competition studies. Thus, both the HSV-1 TK and cellular dCyd kinase (of CEM cells) showed no selectivity for the enantiomers of CdG. This lack of enantiomeric specificity has obvious implications for the design of inhibitors of both viral proliferation and cellular metabolism.

1. The NEOB is a very large organ procurement organization (OPO) recovering multiple organs and tissues on behalf of 15 transplant centers serving 11.5 million people scattered over 6 states. 2. The database maintained by the New England Organ Bank (NEOB) demonstrates a changing pattern of donors within our region. Trauma is decreasing as a cause of death and the age of donors is steadily increasing. 3. A flexible system for the allocation and distribution of kidneys is described. This system emphasizes waiting time as the primary criterion for allocation. This emphasis has not disadvantaged the highly sensitized patient and is equitable for minority recipients. The use of the longest waiting unsensitized patient to assign donors to transplant centers maintains the patient-based nature of the system, while allowing transplant centers to recover kidneys for their own patients and reducing ischemic time. The system is highly adaptable to a variety of local situations. 4. Extensive public education and research projects not feasible for small organizations are made possible through the resources available to a large OPO.

Background: There is a high prevalence of liver disease among the recipients of organs from donors with antibodies to hepatitis C virus (HCV). We undertook a study to determine the frequency of persistent HCV infection, as indicated by the presence of HCV RNA, among both cadaveric organ donors positive for antibodies to HCV (anti-HCV) and the recipients or organs from these donors., Methods: Serum samples from donors and recipients were tested for HCV RNA with the reverse transcriptase polymerase chain reaction, with use of primers from the 5' untranslated region of the HCV genome, and for anti-HCV with the first-generation enzyme-linked immunosorbent assay (ELISA) and two second-generation tests., Results: HCV RNA was detected in 9 of the 11 organ donors (82 percent) with a positive first-generation ELISA for anti-HCV. Among the organ recipients, the prevalence of HCV RNA increased after transplantation: 7 of 26 patients (27 percent) had positive samples before transplantation, as compared with 23 of 24 patients (96 percent) after transplantation (P less than 0.001). Among 13 recipients who were HCV RNA-negative before receiving organs from the nine HCV RNA-positive donors, HCV infection was detected in all 13 after transplantation, and anti-HCV developed in 8 (62 percent). On the basis of a positive test for HCV RNA, the maximal sensitivity of the three anti-HCV tests was 57 percent (positive in 4 of 7 patients with end-stage organ failure) before transplantation and 70 percent (positive in 16 of 23 patients) after transplantation., Conclusions: Nearly all the recipients of organs from anti-HCV-positive donors become infected with HCV. The current tests for anti-HCV antibodies underestimate the incidence of transmission and the prevalence of HCV infection among immunosuppressed organ recipients.

Background: Liver disease is a frequent and major complication after organ transplantation. We sought to determine whether hepatitis C virus (HCV) is transmitted by organ transplantation and whether it causes post-transplantation liver disease., Methods: Serum samples from all cadaver organ donors to the New England Organ Bank between 1986 and 1990 were screened retrospectively for antibodies to HCV (anti-HCV) by enzyme-linked immunosorbent assay (ELISA). We reviewed the hospital records of all recipients of organs from anti-HCV-positive donors for evidence of liver disease. Serum samples from recipients obtained before transplantation and during follow-up were analyzed for anti-HCV., Results: Of 716 organ donors, 13 (1.8 percent) were positive for anti-HCV. Their organs (19 kidneys, 6 hearts, and 4 livers) went to 29 recipients. Non-A, non-B hepatitis developed after transplantation in 14 of the 29 (48 percent), for a prevalence 7.4 times the 6.5 percent prevalence after transplantation from untested donors that was previously reported by two institutions in the organ bank (P less than 0.0001). The liver disease began a mean of 3.8 months after transplantation and became chronic in 12 patients; the other 2 had subfulminant hepatic failure. Liver disease was more frequent in the patients who had received antilymphocyte preparations (P = 0.04). HCV was the cause of the post-transplantation liver disease in 12 of the 13 recipients (92 percent) for whom serum samples were available. Anti-HCV was detected by ELISA in eight and enzyme immunoassay in one; in three others, HCV RNA was detected by polymerase chain reaction in serum samples obtained after transplantation., Conclusions: Organ transplantation can transmit hepatitis C. This raises serious questions about the continued acceptance of organs from donors positive for anti-HCV.

Recent advances in knowledge of the structure of the T-cell receptor and of major histocompatibility complex (MHC) molecules have increased our understanding of the nature of their interaction in the immune response. Nevertheless, it remains unclear how the T-cell receptor recognizes foreign MHC molecules in the process of graft rejection. One approach to this problem is to characterize the alloreactivity of a given T-cell receptor. We have chosen to take this approach in vivo by examining patterns of rejection of vascularized heart allografts in transgenic mice carrying a rearranged T-cell receptor-beta-chain gene, in which essentially all alpha beta T cells bear the rearranged gene product. Heterotopic heart allografts were performed in transgene-positive and transgene-negative recipients. The data show that transgene-positive mice will reject fully allogeneic grafts of three different haplotypes after a modest delay, but will not reject grafts from F1 mice that bear H-2 antigens from these same haplotypes and from the recipient strain. Transgene-negative animals reject all grafts promptly. These results suggest that the restricted T-cell receptor repertoire expressed by transgene-positive recipients affects their ability to respond to an alloantigen as expressed on a vascularized graft and that this response is influenced by the presence of self-MHC molecules on the graft.

Cyclosporine has been used as primary immunosuppression in renal allograft recipients in our unit for the past decade. The overall clinical experience and long-term effects of the agent are reviewed. There were 461 consecutive recipients of kidney grafts; 379 received grafts from cadaver donors (CAD) and 82 from living related donors (LRD). Four separate clinical protocols were used sequentially using progressively decreasing doses of CyA; azathioprine was added in group 4 recipients of LRD grafts, and in patients receiving secondary CAD grafts (group 5). The patient mortality rate was less than 5%, with sepsis being the prime contributor. The majority of kidney grafts were lost within the first 2 months after operation; those that never functioned were found almost invariably to have been irreversibly rejected. During the subsequent years of follow-up, attrition of CAD grafts was significantly greater than LRD grafts. In contrast, the attrition rate of primary and secondary CAD grafts was the same after the first 3 months, emphasizing the importance of early immunologic graft destruction. Primary nonfunction occurred in 49% of CAD kidneys and 17% of LRD grafts; however 71% of initially nonfunctioning LRD grafts never functioned at all compared to 34% of CAD grafts, the majority of such organs undergoing fulminate rejection. Individual graft loss after 1 year was almost inevitably due to chronic rejection; there were no differences in long-term allograft function among the treatment groups. Although CyA has improved overall results of kidney transplantation, chronic rejection remains a major unresolved problem.

Patient entry is now complete in a prospective trial of anti-Tac, a murine IgG2a monoclonal antibody directed against the p55 chain of the human IL-2 receptor, for the prevention of renal allograft rejection. Recipients of primary cadaver allografts were randomized to receive either anti-Tac (20 mg q.d. x 10 days beginning POD 1) plus low-dose CsA (4 mg/kg/day), azathioprine (2 mg/kg/day), and prednisone (30 mg q.d.), or conventional triple therapy with CsA (8 mg/kg/day), azathioprine, and prednisone. Forty patients were entered in each group, with current followup from 6 to 26 months. The results show a significant reduction in early rejection episodes in the anti-Tac-treated patients. During the 10-day treatment, 5 of 40 anti-Tac patients had rejection episodes, compared with 21 of 40 control patients (P less than 0.001). Anti-Tac significantly delayed the time to the first rejection (12.5 +/- 6.3 vs. 7.6 +/- 6.7 days) (P less than 0.05). Despite these effects, there were no differences in either actual or actuarial graft or patient survival between the two groups. Pneumonia, primarily CMV, developed in 5 treated and 4 control patients. In patients with functioning grafts mean serum creatinine at 3 months was 1.8 +/- 0.7 in the anti-Tac group and 2.0 +/- 0.8 in the control group (P = NS); at 12 months the values were 2.3 +/- 1.5 and 1.8 +/- 0.5, respectively (P = NS). The peak expression of IL-2 receptors on circulating T-cells was significantly lower in anti-Tac patients (15.1 +/- 3.6%) than in controls (21.9 +/- 4.5%) (P less than 0.05). Seven of 10 patients tested to date developed antimouse immunoglobulin antibodies, with antiidiotype shown in 6. These antibodies do not preclude subsequent treatment with OKT3. Five patients in this and previous anti-Tac protocols have received OKT3 for acute rejection despite known pretreatment antimouse antibodies, with resolution of rejection in all cases.

The ability of RO 23-6457, a retinoid compound with marked in vitro immunosuppressive properties, to prolong vascularized allografts was examined in several in vivo transplantation models. In the murine heterotopic heart model, efficacy was shown in two H-2 incompatible strain combinations, with indefinite graft survival at some doses. In the rat heterotopic heart model, oral administration prolonged Wistar-Furth grafts in Lewis hosts an average of 1 week, with no long-term survivors at a variety of doses. Given subcutaneously, grafts were further prolonged, but the compound proved toxic. In a bilaterally nephrectomized renal transplant model in cynomolgus monkeys treated intravenously at a dose of 2 mg/kg/day, host survival was prolonged to 18, 32, 33, and 74 days, compared with 11, 11, 12, 13, and 26 days in untreated controls (P less than 0.05 by rank-sum testing). The three shorter surviving recipients died from anorexia and weight loss with normal renal function, while the longest survivor rejected its kidney when exhaustion of iv sites precluded further treatment. The toxic effects of the compound resemble the syndrome of hypervitaminosis A. RO 23-6457 will prolong graft survival as a single agent, justifying further preclinical testing and efforts to reduce toxicity.

Secretory IgA is the dominant immunoglobulin produced in the small intestine and one important component of the local defense against dietary and infectious agents present in the gut lumen. The effect of small intestine transplantation on total production of sIgA and on the response to a newly presented antigen, cholera toxin, was determined in a rat segmental heterotopic intestinal transplant model. Lewis x Brown Norway F1 (LBNF1) allografts in Lewis hosts made normal amounts of sIgA, when compared with LBNF1 Thiry-Vella loops or LBNF1 isografts. In contrast, the allografts failed to make a significant specific sIgA response when immunized with cholera toxin at days 0 and 7 following transplantation. This failure was not the result of surgical manipulation, as isografts made normal amounts of specific sIgA directed against cholera toxin. Cyclosporine immunosuppression delayed, but did not prevent, the secretion of specific antibody in isografts. This failure to respond to a new antigen may have important implications for the safety of small bowel transplantation.

Between February 1984 and May 1988, 55 patients underwent orthotopic cardiac transplantation at the Brigham and Women's Hospital, Boston, Mass. Basic immunosuppression was accomplished with steroid and cyclosporine therapies. Twelve patients suffered 14 major complications, including perforated ulcer in 3 patients; pancreatitis in 3 patients; pneumatosis coli in 2 patients; and cholecystitis, colonic necrosis, appendicitis, incarcerated umbilical hernia, pancreatic abscess, and toxic epidermal necrolysis in 1 patient each. Aggressive management of the patients included laparotomy in all but 2 patients with mild pancreatitis and the patient with toxic epidermal necrolysis, who was treated as a patient with a severe burn. In all of the patients, there was a resolution of these complications, except in one 59-year-old man with fatal hemorrhagic pancreatitis. Eleven of the 14 complications occurred during the initial hospitalization. The fatal case of pancreatitis was 1 of 5 (9%) operative mortalities in the entire series. Fifty operative survivors have been followed up for an average of 19 months, with four late deaths (8%) related to rejection. The actuarial probability of survival in patients discharged from the hospital was 90% at 12, 24, and 48 months.

We assessed the structural and functional evolution of small intestinal transplant rejection in a rat model by use of 1-micron section, electron microscopic, and in vitro electrophysiologic techniques to study jejunal mucosa 3, 6, and 9 d posttransplantation. The earliest structural abnormalities detected in jejunal loops transplanted from Lewis X Brown Norway F1 hybrids into Lewis rats occurred within 3 d posttransplantation and consisted of focal endothelial cell injury of the microvasculature and focal injury of crypt epithelial cells. Both alterations were associated with adjacent infiltration of large lymphoid cells, and both markedly progressed and became rather diffuse over the following 6 d. In contrast, villus absorptive cells were not markedly altered in structure until the 9th postoperative day. As compared with host jejuna, allograft jejunal epithelium demonstrated multiple functional abnormalities. Transepithelial resistance declined progressively by days 6 and 9 (both P less than 0.05), although baseline transepithelial spontaneous potential difference was only affected at day 9 (P less than 0.01). Stimulated absorption by allograft jejuna, as assessed by measuring electrical response to mucosal glucose, was not significantly diminished until day 9 (P less than 0.05). In contrast, stimulated secretion assessed by measurement of electrical response to serosal theophylline was diminished by day 6 (P less than .01). These data suggest that the earliest epithelial injury during rejection, as judged both structurally and functionally, occurs in the crypt and is paralleled by endothelial injury at the level of the microvasculature. Thus, the primary targets for rejection are most likely endothelial cells and crypt epithelial cells. In contrast, structural and functional impairment of villus epithelium is detectable only at substantially later times during rejection and are most likely secondary processes related to either ischemia produced by microvascular injury or decreased epithelial regenerative ability secondary to crypt injury. Last, we show that the detrimental structural and functional sequellae of jejunal transplantation across the major histocompatibility complex in this model is strikingly ameliorated with cyclosporine therapy.

Although hypertension appears not infrequently among recipients of kidney transplants, renal artery stenosis is relatively rare as a causative factor. A 23-year experience of patients receiving kidney grafts at the Brigham and Women's Hospital was reviewed to ascertain the incidence of renal artery stenosis and its surgical management. Risk factors leading to the condition and selection of patients for operation are emphasized. The incidence of arterial stenosis severe enough to require operation was 2.7% of 914 kidney transplants; the overall incidence in these patients is unknown, although operated patients comprise about one-half of those undergoing arteriography to diagnose hypertension. The mean time for development of the condition was 21.4 months from date of engraftment. A successful outcome as measured by fall in blood pressure and/or serum creatinine was achieved in 14 of 21 patients (67%) in whom surgical repair of the effected artery was undertaken. Reparative surgery was unsuccessful in seven patients, although hypertension was improved in one of these individuals following transplant nephrectomy. Surgery was never undertaken in four patients because of chronic rejection noted on biopsy. There was no mortality. Operative repair should be offered to patients with renal artery stenosis leading to unmanageable hypertension or renal dysfunction, but withheld from those with documented chronic rejection regardless of major arterial compromise.

Fifty seven recipients of renal allografts initially treated with CsA and low-dose prednisone were switched to azathioprine and low-dose steroids. Ten had prolonged (greater than 28 days) allograft nonfunction after transplantation (group 1), 8 had ongoing, poorly controlled rejection (group 2), and 39 had stable functioning grafts (group 3). With a mean follow-up period of 5 +/- 3 months after conversion, 50 grafts remained functional including 6 of 10 in group 1, 6 of 8 in group 2, and 38 of 39 in group 3. Thirty-seven (65%) had improved function, 12 (21%) had stable function, and 8 (14%) experienced declining renal function. Three of these latter 8 patients required reinstitution of CsA therapy. There were 20 episodes of acute rejection in 18 patients; one graft lost function because of acute rejection unresponsive to therapy. Reasons for the 6 other graft losses were persistent primary nonfunction in 3 patients from group 1, untreated rejection in 2 patients who had multiple prior rejection episodes while on CsA, and chronic rejection in one patient. Although renal function has improved or stabilized in the majority (86%) of individuals changed to azathioprine therapy, there was substantial risk of acute rejection (32%) complicating this procedure. Patients most likely to benefit from conversion to azathioprine therapy are those with prolonged graft nonfunction after transplantation and those with serum creatinines greater than 2.0 mg/dl.

Nineteen patients with acute rejection of a renal allograft were treated with the monoclonal antibody anti-T12, directed against a determinant present on all post-thymic T cells. Seven patients had a good response, four had an equivocal response, and eight failed to respond. Histologic studies demonstrated that the good responders had primarily cellular rejection. The nonresponders included 4 patients with moderate-to-severe humoral rejection, one patient with an inadequate dose of antibody, one patient who withdrew before completing the study, and one patient with late end-stage rejection. All eleven patients with good or equivocal responses have functioning kidneys in a follow-up of 1-15 months (mean 7 months). Only one patient has had a subsequent acute rejection episode, which responded to a steroid pulse. No significant complications of anti-T12 therapy occurred.

We have reviewed the results of patient survival and transplant function of the last 100 recipients of renal allografts treated with cyclosporine (CyA) plus low-dose steroids since November 1981; the follow-up varies between 3 months and 2 years. A group of 56 individuals transplanted between January 1980 and October 1982 and immunosuppressed with azathioprine (Aza) and steroids were used as comparison. There were five deaths among 100 patients treated with CyA and two among 56 treated with Aza. There were, however, marked differences in allograft function. Using actuarial curves, 2-year allograft survival from 24 living, related, one haplotype matched donors was 83%, as compared to an unsatisfactory 60% graft function among 24 nonrandomized, comparable, Aza-treated recipients. The 2-year actuarial survival of 76 allografts from cadaver donors was 76%; that of 36 grafts in patients treated with Aza, 48%. Interestingly, function of first cadaver allografts was 84% at 2 years, far better (p less than 0.002) than cadaver graft function (58%) in patients who had been previously transplanted; these latter results are comparable to Aza-treated cadaver recipients. Side effects and complications of this difficult drug, as well as its benefits, have been stressed in this article.

We undertook a prospective randomized trial to examine whether an intravenous cytomegalovirus (CMV) immune globulin would prevent primary CMV disease in renal-transplant recipients. Fifty-nine CMV-seronegative patients who received kidneys from donors who had antibodies against CMV were assigned to receive either intravenous CMV immune globulin or no treatment. The immune globulin was administered in multiple doses over the first four months after transplantation. The incidence of virologically confirmed CMV-associated syndromes was reduced from 60 percent in controls to 21 percent in recipients of CMV immune globulin (P less than 0.01). Fungal or parasitic superinfections were not seen in globulin recipients but occurred in 20 percent of controls (P = 0.05). Only 4 percent of globulin recipients had marked leukopenia (reflecting serious CMV disease), as compared with 37 percent of the controls (P less than 0.01). There was a concomitant but not statistically significant reduction in the incidence of CMV pneumonia (17 percent of controls as compared with 4 percent of globulin recipients). A significant reduction in serious CMV-associated disease was observed even when patients were stratified according to therapy for transplant rejection (P = 0.04). We observed no effect of immune globulin on rates of viral isolation or seroconversion, suggesting that treated patients often harbored the virus but that clinically evident disease was much less likely to develop in them. We conclude that CMV immune globulin provides effective prophylaxis in renal-transplant recipients at risk for primary CMV disease.

As previously reported, acute cyclosporine-induced nephrotoxicity is characterized by a decline in glomerular filtration rate and a selective intrarenal production of the vasoconstrictor thromboxane (TxA2), but not vasodilator prostaglandin E2 (PGE2), or prostacyclin (PGI2), cyclooxygenase metabolites. Fish oils (FO), that are rich in n-3 polyunsaturated fatty acids have a high affinity for cyclooxygenase but serve as poor substrate inhibit TxA2 synthesis. We have shown that when FO replaces olive oil (OO) as the vehicle for CsA, CsA-induced nephrotoxicity and increased TxA2 synthesis are obviated in rodent models. In this study, we demonstrate that the FO vehicle for CsA does not compromise CsA's immunosuppressive properties as deduced from studies of a delayed-type hypersensitivity (DTH) model in BALB/c mice and in a rat heart transplant model. In fact, concurrent FO administration with CsA actually enhances immunosuppression. A dose of CsA incapable of blunting DTH when injected in OO was suppressive when given in FO. Administration of as little as 0.05 ml of FO vehicle potentiated the suppressive action of CsA. In addition, nonconcurrent dietary supplementation of FO in animals receiving CsA caused an increase in the immunosuppressive action of CsA in DTH. FO alone reduced DTH as compared with OO, but was far less effective than CsA plus FO. Furthermore, doses of CsA (5 mg/kg/day or 1.5 mg/kg/day), which are subtherapeutic when administered with OO, prolonged engraftment of Lewis recipients of Lewis x Brown-Norway F1 hearts when CsA was solubilized with FO. These studies indicate that concurrent administration of CsA and FO potentiates the activity of CsA and thus increases its therapeutic index. Thus, CsA plus FO is potentially a safe, potent antirejection therapy worthy of clinical testing, especially insofar as FO prevents CsA-induced acute nephrotoxicity in the rodent.

The ability of proton NMR relaxation times to detect cardiac allograft rejection was studied in an inbred rat heterotopic cardiac transplantation model. Hearts from 25 Lewis X Brown Norway F1 hybrid rats were anastomosed to the abdominal aorta and vena cava of Lewis recipients; 25 Lewis donor hearts served as isograft controls. Groups of five allografts and five isografts were harvested daily between two and six days post-transplant. The relaxation times T1 and T2 of the transplanted hearts were determined in vitro with a 10 MHz spectrometer. T1 and T2 values in allografts did not differ significantly from those in isografts at days 2 and 3 post-transplant. However, at days 4, 5, and 6 T1 and T2 of the allografts were significantly prolonged. This finding correlated with an elevation in tissue water content and the onset of rejection as determined histologically. An additional 21 allografts, treated with cyclosporine, were studied in the same way from four to more than 100 days post-transplant. T1 and T2 values of these treated allografts did not change significantly during the observation period and were similar to the relaxation values obtained in the isografts at days 2 to 6. These data suggest that serial measurements of myocardial T1 and T2 may be useful in detecting acute cardiac allograft rejection and monitoring the effect of antirejection treatment.

The use of percutaneous nephrostomy with or without placement of a ureteric stent has been of increasing aid in treating both acute and chronic ureteral obstruction in recipients of renal allografts. Eight patients who underwent transplantation, had ureteric obstruction develop and were treated by this means are reported herein. The technique is standard and can be performed in an arteriography suite. Once a catheter is inserted into the allograft collecting system, a nephrostomy tube or a stent, or both, is easily inserted. Three of the patients had acute ureteric obstruction secondary to clot forming after needle biopsy of the transplant kidney. Percutaneous nephrostomy tubes were passed, the renal pelvis and ureter irrigated and the clots lysed. Operation was unnecessary. In five patients with declining renal function secondary to chronic obstruction, percutaneous nephrostomy placement allowed return of renal function to base line levels, while placement of a stent through the ureter into the bladder facilitated location of the ureter at operation for ureteral reimplantation. The technique of percutaneous nephrostomy is simple, convenient and allows patient recovery preoperatively. It has become part of our standard armamentarium in transplant recipients.

In an effort to produce specific immunosuppression through the targeting of those lymphocytes expressing cell surface interleukin 2 receptors in response to an allograft, the anti-human IL-2 receptor monoclonal antibody anti-Tac was administered to cynomolgus monkeys receiving renal transplants. The data demonstrate that anti-Tac produces a significant delay in renal allograft rejection and prolongs host survival in cynomolgus monkeys. Though higher doses of anti-Tac produce modest delays in rejection, there was a surprising finding of greatly prolonged survival in three of five monkeys treated with much lower doses of anti-Tac. Anti-Tac was not shown to be synergistic with cyclosporine in this model. Animals treated with anti-Tac developed high titers of antibodies against the murine monoclonal antibody after 6-8 days of treatment, associated with the disappearance of plasma anti-Tac staining of activated lymphocytes as measured by flow cytometry. The data confirm the utility of the IL-2 receptor as a target for immunosuppressive therapy, and suggest that investigations of dosage and of methods to reduce the immunogenicity of anti-IL-2 receptor agents may be beneficial.