Belatacept-Resistant Rejection Is Associated With CD28+ Memory CD8 T Cells.

Recently, newer therapies have been designed to more specifically target rejection in an effort to improve efficacy and limit unwanted toxicity. Belatacept, a CD28-CD80/86 specific reagent, is associated with superior patient survival and graft function compared with traditional therapy, but its adoption as a mainstay immunosuppressive therapy has been tempered by increased rejection rates. It is essential that the underlying mechanisms associated with this rejection be elucidated before belatacept is more widely used. To that end, we designed a study in a nonhuman primate kidney transplant model where animals were treated with either a belatacept- or a tacrolimus-based immunosuppressive regimen. Interestingly, we found that elevated pretransplant frequencies of CD28+ CD8+ TEMRA cells are associated with rejection on belatacept but not tacrolimus treatment. Further analysis showed that the CD28+ CD8+ TEMRA cells rapidly lose CD28 expression after transplant in those animals that go on to reject with the allograft infiltrate being predominantly CD28- . These data suggest that CD28+ memory T cells may be resistant to belatacept, capable of further differentiation including loss of CD28 expression while maintaining effector function. The unique signaling requirements of CD28+ memory T cells provide opportunities for the development of targeted therapies, which may synergize with belatacept to prevent costimulation-independent rejection.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Adaptive immunity rather than viral cytopathology mediates polyomavirus-associated nephropathy in mice.

Nephropathy associated with BK polyomavirus causes kidney allograft dysfunction and failure. Understanding the pathogenesis of polyomavirus-associated allograft nephropathy (PVAN) is hampered by the species specificity of Polyomaviridae family members. Using a mouse polyomavirus (MPyV) kidney transplant model, we investigated clinically relevant variables that may contribute to PVAN. We found that the timing and source (i.e. donor vs. recipient) of MPyV infection and the titer of the viral inoculum have significant effects on the extent of allograft injury, with acute infection of the recipient by high-titer MPyV inoculums producing the most profound PVAN. In contrast, altering the degree of MHC matching or increasing ischemia/reperfusion injury by prolonging the cold ischemic time of the allograft did not affect the severity of PVAN. Survival correlated positively with serum creatinine levels, but not with viral loads in the kidney allograft. Using splenectomized alymphoplasia mice, which are unable to mount primary adaptive immune responses, we further demonstrate that persistent high viral loads in the kidney are not sufficient to cause advanced PVAN. These findings suggest that the mechanism of PVAN in mice is not a direct consequence of viral cytopathology, but rather involves interplay between viral infection and the recipient antidonor immune response.

Identification and transcriptional mapping of genes encoded at the IR/Us junction of equine herpesvirus type 1.

Two open reading frames (ORFs) encoded at the inverted repeat unique short (Us) junction of the Short (S) region of the equine herpesvirus type 1 genome were identified by DNA sequencing of a 2876 base pair (bp) genomic segment, and transcripts encoding these ORFs were characterized by Northern blot, S1 nuclease, and primer extension analyses. These studies also established the size of each inverted repeat to be 12,768 nucleotides (nts). The IR6 ORF (816 bp), mapping at nts 12,317-11,502 of the S region, is the last gene completely encoded within each inverted repeat and encodes a predicted 30.1-kDa protein of 272 amino acids, which does not exhibit homology to other alphaherpesvirus proteins. IR6 is expressed as an early transcript of 1.2 kb which is detected initially at 1.5 hr p.i. and up to 12 hr p.i. The transcription initiation and termination sites of IR6 were mapped by primer extension and S1 nuclease analyses to nts 12,465 and 11,408, respectively. The first ORF encoded within the Us segment (909 bp; EUS1), mapping at nts 13,397-12,489, encodes a predicted 33.5-kDa protein of 303 amino acids that exhibits 29% identity to the US2 protein of herpes simplex virus 1. EUS1 is expressed as a 2.3-kb mRNA of the gamma-1 class, as its synthesis begins prior to viral DNA replication at 4 hr p.i. but is retarded by phosphonoacetic acid, an inhibitor of viral DNA replication. The Tci and Tct sites of EUS1 were mapped by S1 nuclease analyses to nts 13,637 and 11,408, respectively. Interestingly, this termination site is also utilized by three late mRNAs of 5.8, 3.8, and 1.7 kb which originate within the Us and overlap the IR6 mRNA encoded in the terminal inverted repeat (TR) of the prototype genomic isomer. EUS1 is 3' coterminal with IR6 in the inverted repeat, whereas, the 5.8, 3.8, and 1.7 kb transcripts are 3' coterminal with IR6 of the TR.

Albuterol and spacer-induced atrial fibrillation.

Albuterol used with a spacer device which induced atrial fibrillation is described. Inhaled sympathomimetics have been extensively studied for the treatment of asthma and have generally been found to be safe from cardiac arrhythmias. A review of the literature is presented.

Spacer-induced atrial fibrillation.

Inhaled sympathomimetics, studied extensively for treatment of asthma, have been found to be safe from cardiac arrhythmias. We discuss a case report of albuterol used with a spacer device that induced atrial fibrillation. We review relevant literature.

Synthesis and disposition of 14C-labelled gentian violet in F344 rats and B6C3F1 mice.

The synthesis of [phenyl-U-14C]gentian violet from [U-14C]benzene is described. The 14C-labelled dye was administered by gavage to groups of male and female F344 rats which were killed at 2, 4, 14, 24 or 36 hr after the single dose. Radioactivity was measured in urine, and determined in faeces, liver, kidney, fatty tissue, gonads and muscle by combustion analysis. Residues were maximal at 4 hr in liver, kidney, muscle and gonads, and in fat they reached a plateau after 24 hr. Depletion half-lives for male and female livers were 14.5 and 17.0 hr, respectively. The 14C-labelled dye was also administered in multiple doses by gavage to both sexes of F344 rats and B6C3F1 hybrid mice for 7 days. The highest residue level was found in fatty tissue of females of both species, with a highly significant sex difference (P less than 0.01). Significant sex differences were also noted for residue levels in kidney and muscle tissue from both species and in mouse liver. Bile collected from cannulated rats contained 5.7-6.4% of a single oral dose of the dye. The results suggest that gentian violet is absorbed from the gastro-intestinal tract to a greater extent than has been reported for other triphenylmethane dyes.

Strain differences in the response of the mouse to diethylstilbestrol.

BALB/c StCrlfC3Hf/Nctr, C57BL/6/, C57BL/6 X BALB/c F1 hybrid (B6CF1), and monohybrid-cross offspring from the breeding of B6CF1 mice were examined with respect to uterine, vaginal, and thymus responses to diethylstilbestrol (DES). About 400 mice of each genetic population were used. Weanling mice were fed DES at dietary concentrations of 2.5 to 1,000 ppb (microgram/kg feed) for 6 days and were killed by cervical dislocation about 20 hr after removal of the feed. C57BL/6, B6CF1, and the monohybrid-cross offspring did not differ in the uterine-weight response to DES, but the slope of the dose-response line was shallower for the BALB/c than for the other strains. Dietary DES concentrations of 250 ppb or more inhibited the uterotrophic response in all populations. Vaginal cornification occurred at lower concentrations of DES in the C57BL/6 strain than in the B6CF1 animals. BALB/c and monohybrid-cross offspring were indistinguishable from each other in their vaginal response to Des and were less sensitive to DES than the other mouse populations. The use of ethanol or corn oil as the solvent for mixing DES into the diet had no apparent effect on the uterine weight or vaginal response in any of the mice. DES depressed thymus weight in a dose-related fashion at dietary concentrations of 100 ppb and above in all genetic populations.

Mast cell neoplasia in mice.

Gross and histopathologic findings were reported for mast cell neoplasia in 2 female BALB/cStCrIBR mice. Microscopic findings included neoplastic mast cells in the liver, spleen, kidney and lymph nodes. Neoplastic mast cells were present in the blood smear of one of the animals.