Combined analysis of cytokine genotype polymorphism and the level of expression with allograft function in African-American renal transplant patients.

Cytokine gene polymorphism and expression levels were evaluated in a group of African-American patients who had undergone renal transplantation. It was hypothesized that possession of specific cytokine alleles might be influential in predisposing the recipient to allograft rejection. Thus, we sought to establish a relationship between cytokine gene polymorphism, the levels of cytokine expression, and the outcome of allograft function. Cytokine genotypes and mRNA transcript levels of IL-2, TNF-alpha, TGF-beta1, IL-10, IL-6 and IFN-gamma were determined using peripheral blood cells. Genomic DNA samples from 77 transplant recipients and 77 controls were tested by a multiplex PCR with specific primers for the above cytokines. The frequency distributions of cytokines were analyzed in respect to the clinical characterization, including delayed graft function (DGF), rejection episodes (REs) and stable graft function (SGF). The mRNA transcript level was tested both at pre- and early post-transplantation (day 1 and day 4) with primers for coding regions of the above cytokines in a RT-PCR assay. The majority of recipients with successful graft function were matched with their donors for only three out of the six HLA alleles. We have shown that the TGF-beta1 T/C G/G high producer and IFN-gamma T/A intermediate producer genotypes were associated with allograft rejection, whereas low IFN-gamma producer and high IL-10 producer genotypes were significantly protective of the allograft. There was some correlation between the TGF-beta1 high producer genotype and DGF, but it was not statistically significant. Overall, 77% of those who experienced REs carried the TGF-beta1 T/C G/G, high producer genotype as compared with 52% who experienced DGF, 39% with SGF (P<0.01, RR=2.0), and 27.3% of controls (P<0.003, RR=2.6). The IFN-gamma T/A intermediate producer genotype was found in 69.2% of patients with REs as compared with 26.8% of patients with SGF (P<0.008, RR=2.85). The IL-10, ATA/ATA low producer genotype was found in 38.5% of recipients with REs and 14.6% of recipients without REs (P<0.04, RR=0.53). Expression levels of mRNA transcript were correlated with genotype data, except for the TGF-beta1 high producer genotype where there was no significant difference between the level of mRNA transcript at pre- and post-transplantation. Low DRbeta1 and high DPbeta1 expression by recipient peripheral blood mononuclear cells before transplantation was associated with more SGF, whereas high DRbeta1 and low DPbeta1 expression at pretransplantation was associated with more REs (DRbeta1, P<0.001 and DPbeta1, P<0.05, respectively). We concluded that, dual analysis of cytokine genotype and expression levels by peripheral cells may be an important clue to understanding the contribution of the recipient's immune response to an allograft pre- and post-transplantation. Identification of peripheral markers diagnostic of rejection could allow advance anticipation of clinical outcome, and might reduce the need for tissue biopsy.

Photolytic destruction and polymeric resin decontamination of aqueous solutions of pharmaceuticals.

Amoxicillin, ampicillin, bleomycin, carmustine, cephalothin, dacarbazine, lomustine, metronidazole, norethindrone, streptozocin, sulfamethoxazole, and verapamil were completely degraded in solution, without the production of mutagenic residues, by photolysis using a medium-pressure mercury lamp in an all-quartz apparatus. A stream of air was passed through the solution and for amoxicillin, ampicillin, bleomycin, lomustine, metronidazole, and norethindrone it was necessary to add hydrogen peroxide. Dilute aqueous solutions of ampicillin, bleomycin, carmustine, cephalothin, lomustine, norethindrone, streptozocin, trimethoprim, and verapamil can be decontaminated using polymeric Amberlite resins.

Effect of exogenous heparin on anchorage-independent growth of fibroblasts induced by transforming cytokines.

Heparin at concentrations below 100 micrograms/ml stimulated anchorage-independent growth of NRK 49F (normal rat kidney fibroblasts, American type culture collection) rat fibroblasts at suboptimal cytokine concentrations but inhibited it at higher heparin concentrations regardless of the cytokine concentration. Heparin did not stimulate growth above that seen at optimal cytokine concentrations, suggesting that it alters the cellular response to the cytokines. These data suggest natural protein-glycosaminoglycan interactions play a role in modulating or mediating the actions of transforming cytokines and suggest they may play a role in acquisition of the transformed phenotype.

Protective effects of selenium on cadmium toxicity in rats: role of altered toxicokinetics and metallothionein.

Selenium prevents the toxicity of the carcinogenic metal cadmium through undefined mechanisms. In this study, we determined the effects of selenium on cadmium toxicokinetics and on the ability of cadmium to induce metallothionein, a metal-binding protein that is thought to confer tolerance to cadmium toxicity. To assess the acute protective effects of selenium, male Wistar (WF/NCr) rats were given selenium (as SeO2; 10 mumol/kg, sc) at -24, 0, and +24 h relative to cadmium (as CdCl2; 45 mumol/kg, sc). Over a 14-d period this dose of cadmium killed 6 out of 10 rats, while 100% of the cadmium-treated rats given concurrent selenium treatments survived. The acute increases in testicular weight that were seen with cadmium, indicative of edematous damage, were also prevented by concurrent selenium treatments. Further studies assessed the distribution and excretion of cadmium and its ability to induce metallothionein in rats given 40 mumol Cd/kg, sc, at time 0 and selenium (10 mumol/kg, sc) at -24 and 0 h. Selenium treatments enhanced cadmium accumulation at 24 h in the liver (23%), testes (145%), and epididymis (35%) but reduced renal accumulation by more than half. Urine samples, collected at 0-3, 3-6, and 6-24 h following cadmium administration, indicated a markedly reduced excretion of cadmium in selenium treated rats during all time periods. The synthesis of metallothionein was stimulated to a much lesser extent by cadmium in selenium-treated rat kidney (41% decrease) but was unaffected in liver. The levels of cadmium-binding proteins within the testes were markedly reduced by cadmium treatment, an effect unmodified by selenium treatments. These results suggest selenium prevents acute cadmium toxicity through a mechanism that does not involve induction of metallothionein and in spite of a markedly enhanced retention of cadmium.

Functional and structural characteristics of the glycosaminoglycans of the bladder luminal surface.

The glycosaminoglycan layer of bladder has been proposed to play a crucial role in protecting the bladder from harmful substances in urine. Rats were partially cystectomized to determine whether bladder glycosaminoglycans are routinely eluted from the bladder surface in detectable quantities. Cystectomy produced no detectable qualitative or quantitative changes in excreted GAG thereby showing that most urinary glycosaminoglycan originates in the kidney and not from the bladder. Damaging the glycosaminoglycan layer by a dilute acid wash, however, leads to a consistent decrease in the output of urinary GAG which recovers to normal at the same rate as the layer regenerates. This suggests that the newly exposed sites tightly bind urinary GAG. We suggest that such binding may be a component of the normal physiological defense mechanism of the bladder. The bladder glycosaminoglycan layer was isolated, dilute acid being used to elute ionically-bound material and brief trypsinization to elute intercalated proteoglycans from the luminal surface. The GAG from the luminal surface, which was present at a density of one chain per 50 nm.2 of bladder surface, was quite different in composition from that isolated from the whole bladder.