Manikkam Suthanthiran

Stimulation of transforming growth factor‐β1 transcription by cyclosporine

In searching for a candidate mechanism for the immunosuppressive as well as fibrogenic consequences of cyclosporine usage, we have explored the hypothesis that cyclosporine stimulates transcription of transforming growth factor‐β 1 (TGF‐β 1), a multifunctional cytokine endowed with immunosuppressive and fibrogenic properties. Our results demonstrate that cyclosporine (i) stimulates TGF‐β 1 promoter‐dependent transcription of chloramphenicol acetyl transferase gene in transiently transfected human A‐549 cells, (ii) stimulates the synthesis of TGF‐β 1 RNA transcripts in human T cells, and (iii) permits the expression/emergence of DNA regulatory proteins (retinoblastoma control factor‐1 (RCF‐1) and RCF‐2) that bind and regulate TGF‐β 1 promoter activity. Our studies demonstrate for the first time that cyclosporine stimulates TGF‐β 1 gene transcription and suggest a novel mechanism of action of cyclosporine.

Clinical Application of Molecular Biology: A Study of Allograft Rejection With Polymerase Chain Reaction

This article explores the clinical usefulness of reverse transcriptase-polymerase chain reaction in organ graft recipients. In this study, reverse transcriptase-polymerase chain reaction was used to identify intrarenal expression of cytotoxic attack molecules (granzyme B and perforin) and immunoregulatory cytokines (IL-2, IL-4, IL-10, IFN-gamma, and TGF-beta 1) in human renal allograft biopsies. The biopsies (n = 127) were classified using the Banff criteria, and intrarenal gene expression was correlated with the histologic diagnosis. Molecular analyses revealed that intragraft display of mRNA encoding granzyme B, IL-10, or IL-2 is a correlate of acute rejection, and intrarenal expression of TGF beta 1 mRNA is a correlate of chronic rejection. In addition to demonstrating differential and highly selective intragraft gene expression during rejection, these data suggest that therapeutic strategies directed at the molecular correlates of rejection might refine existing antirejection strategies.

Independent Risk Factors for Urinary Tract Infection and for Subsequent Bacteremia or Acute Cellular Rejection: A Single Center Report of 1166 Kidney Allograft Recipients

Background Urinary tract infection (UTI) is a frequent, serious complication in kidney allograft recipients. Methods We reviewed the records of 1166 kidney allograft recipients who received their allografts at our institution between January 2005 and December 2010 and determined the incidence of UTI during the first 3 months after transplantation (early UTI). We used Cox proportional hazards models to determine the risk factors for early UTI and whether early UTI was an independent risk factor for subsequent bacteremia or acute cellular rejection (ACR). Results UTI, defined as 105 or more bacterial colony-forming units/mL urine, developed in 247 (21%) of the 1166 recipients. Independent risk factors for the first episode of UTI were female gender (hazard ratio [HR], 2.9; 95% confidence intervals [CI], 2.2–3.7; P<0.001), prolonged use of Foley catheter (HR, 3.9; 95% CI, 2.8–5.4; P <0.001), ureteral stent (HR, 1.4; 95% CI, 1.1–1.8; P=0.01), age (HR, 1.1; 95% CI, 1.0–1.2; P=0.03), and delayed graft function (HR, 1.4; 95% CI, 1.0–1.9; P=0.06). Trimethoprim/sulfamethoxazole prophylaxis was associated with a reduced risk of UTI (HR, 0.6; 95% CI, 0.3–0.9; P=0.02). UTI was an independent risk factor for subsequent bacteremia (HR, 2.4; 95% CI, 1.2–4.8; P=0.01). Untreated UTI, but not treated UTI, was associated with an increased risk of ACR (HR, 2.8; 95% CI, 1.3–6.2; P=0.01). Conclusions Female gender, prolonged use of Foley catheter, ureteral stent, age, and delayed graft function are independent risk factors for early UTI. UTI is independently associated with the development of bacteremia, and untreated UTI is associated with subsequent ACR.

Circulating transforming growth factor-β1 levels and the risk for kidney disease in African Americans

Transforming growth factor-β1 (TGF-β1) is well known to induce progression of experimental renal disease. Here we determined whether there is an association between serum levels of TGF-β1 and the risk factors for progression of clinically relevant renal disorders in 186 black and 147 white adults none of whom had kidney disease or diabetes. Serum TGF-β1 protein levels were positively and significantly associated with plasma renin activity along with the systolic and diastolic blood pressure in blacks but not whites after controlling for age, gender and body mass index. These TGF-β1 protein levels were also significantly associated with body mass index and metabolic syndrome and more predictive of microalbuminuria in blacks than in whites. The differential association between TGF-β1 and renal disease risk factors in blacks and whites suggests an explanation for the excess burden of end-stage renal disease in the black population but this requires validation in an independent cohort. Whether these findings show that it is the circulating levels of TGF-β1 that contributes to renal disease progression or reflects other unmeasured factors will need to be tested in longitudinal studies.

Immunobiology and immunopharmacology of organ allograft rejection

Much has been learned regarding immunobiological mechanisms responsible for the rejection of histoincompatible allografts. There has also been considerable progress in our understanding of mechanisms responsible for tolerance. The new knowledge gained regarding graft destructive alloimmunity process and the mechanisms of action of immunosuppressants have resulted in solid organ graft survival rates that are in excess of 80% at one year posttransplantation. The principles of tolerance mechanisms are yet to be successfully applied in the clinic. In this review, molecular and cellular mechanisms of action of clinically useful immunosuppressive drugs are reviewed from the perspective of regulation of the anti-allograft repertory.

Adoptive immunotherapy for stage IV renal cell carcinoma: A novel protocol utilizing periodate and interleukin-2-activated autologous leukocytes and continuous infusions of low-dose interleukin-2

Abstract In a pilot study involving 13 patients with advanced stage IV renal cell carcinoma, anti-tumor effects and toxicity of a novel form of adoptive immunotherapy were determined. The protocol utilizes infusions of autologous mononuclear leukocytes treated with the oxidizing mitogen sodium periodate (IO 4 − ) and cultured in medium containing human recombinant interleukin-2 (IL-2), and continuous infusions of low-dose IL-2 (mean ± SD dose=39.5 ± 8.6 × 10 3 U/kg/24 hours). Leukocytes (5 to 10 × 10 9 ) were removed by leukapheresis three times per week, mononuclear cells were separated, activated with IO 4 − and cultured in medium containing IL-2 (500 U/ml) for 48 to 72 hours. The cells were re-infused following the next leukapheresis procedure. IL-2 was administered five days per week. Treatment was continued for two three-week cycles. An increase in peripheral blood mononuclear cells bearing the natural killer cell (NK) surface marker, Leu 11, an increase in NK- and antibody-dependent cell-mediated cytotoxicity, and a slight increase in spontaneous cytotoxicity for non-NK targets were noted. Regressions (more than 50 percent decrease in tumor mass) of pulmonary, liver, bone, or soft tissue metastases were induced in six patients. Severe fluid retention did not develop in any patient and no patient required treatment in the intensive care unit. Five of the patients who showed a response have experienced a relapse at 5.2 ± 1.0 (mean ± SD) months. These observations indicate that IO 4 − /IL-2-activated killer cells plus continuous infusions of low-dose IL-2 can result in regression of metastatic renal cell carcinoma.

Which way for drug-mediated immunosuppression?

The basic immunosuppressive protocol involves the use of multiple drugs, each directed at a discrete site in the T-cell activation cascade. The prevailing paradigm regarding the mechanisms of action of immunosuppressants is that they function to prevent allograft rejection by preventing cell activation, proliferation and/or cytokine production. A new hypothesis is that some of the immunosuppressants might function by stimulating the expression of immunosuppressive molecules and/or cells.

MECHANISMS AND MANAGEMENT OF ACUTE RENAL ALLOGRAFT REJECTION

We used RT-PCR for the molecular characterization of human renal graft rejection. The studies showed that intragraft display of mRNA encoding cytotoxic attack molecule granzyme B, and immunoregulatory cytokines IL-10 or IL-2 are correlates of acute rejection, and intrarenal expression of TGF-1 mRNA, of chronic rejection. The current immunosuppressive protocol involves the use of multiple drugs, each directed at a discrete site in the T-cell activation cascade and each with distinct side effects. The immunosuppressants can be classified as inhibitors of: transcription (CsA, tacrolimus); nucleotide synthesis (azathioprine, mycophenolate mofetil, and mizoribine); growth factor signal transduction (sirolimus); and differentiation (DSG). Polyclonal antibodies and monoclonal antibodies directed at cell surface proteins are quite effective as induction therapy or anti-rejection drugs.

Molecular Correlates of Human Renal Allograft Rejection

In one of thecytotoxic pathways, granzyme B, a serine protease requiredfor the induction of rapid DNA fragmentation, and per-forin, a pore-forming protein homologous to complementcomponents, collaborate to induce lysis of target cells. Inthe other cytotoxic pathway, the death gene Fas is ligatedand crosslinked by the death factor, Fas ligand, and thisresults in programmed cell death (apoptosis) of Fas-anti-gen-bearing cells.Our molecular analysis of human renal allograft biopsyspecimens demonstrated that intragraft expression of gran-zyme B mRNA is a significant correlate of acute but not ofchronic rejection (Tables 1 and 2). Our finding that intra-renal expression of granzyme B mRNA is a correlate ofacute rejection of human renal allografts is in agreementwith the elegant studies of Lipman et al

A phase II clinical trial of adoptive immunotherapy for advanced renal cell carcinoma using mitogen-activated autologous leukocytes and continuous infusion interleukin-2.

Forty patients with metastatic, recurrent, or unresectable renal cell carcinoma were entered into a study of the therapeutic efficacy of adoptive immunotherapy using periodate (IO4-) and interleukin-2 (IL2)-activated autologous leukocytes and continuous infusion low-dose IL2. Patient survival was also examined. The first 15 consecutive patients were enrolled in protocol A without an IL2 priming phase and the following 25 patients were entered in protocol B where a 5-day priming phase was initiated before leukapheresis. A maintenance regimen consisted of either 3 x 10(6) units of recombinant interferon-alpha (rIFN-alpha), three times per week only or together with leukapheresis and infusion of IO4-/IL2-activated cells and 2 days of continuous infusion IL2 per month. Thirty-four patients completed the protocol treatment. Four patients were removed from the study owing to rapid tumor progression and two patients died while receiving treatment. The clinical response rate was 22%: two patients had a complete response and five patients had a partial response. Among the 25 patients who had no clinical response, 11 patients had either a mixed response or stabilization. Neither response, response duration, nor site response correlated with the total dose of IL2 administered or the number of activated killer cells infused. Patients who received maintenance therapy had longer survival times than patients who did not receive such therapy. All toxicity and side effects associated with IL2 treatment were transient and resolved after discontinuation of the drug. Patients on maintenance therapy tolerated both rIFN-alpha and monthly infusions of activated killer cells and IL2 well. This study confirms the concept of adoptive immunotherapy as a new treatment approach for advanced renal cell carcinoma and suggests that maintenance therapy may prolong survival time.