Differential pairing of transmembrane domain GxxxG dimerization motifs defines two HLA-DR MHC class II conformers.

MHC class II molecules function to present exogenous antigen-derived peptides to CD4 T cells to both drive T cell activation and to provide signals back into the class II antigen-presenting cell. Previous work established the presence of multiple GxxxG dimerization motifs within the transmembrane domains of MHC class II α and ß chains across a wide range of species and revealed a role for differential GxxxG motif pairing in the formation of two discrete mouse class II conformers with distinct functional properties (i.e., M1-and M2-paired I-Ak class II). Biochemical and mutagenesis studies detailed herein extend this model to human class II by identifying an anti-HLA-DR mAb (Tü36) that selectively binds M1-paired HLA-DR molecules. Analysis of the HLA-DR allele reactivity of the Tü36 mAb helped define other HLA-DR residues involved in mAb binding. In silico modeling of both TM domain interactions and whole protein structure is consistent with the outcome of biochemical/mutagenesis studies and provides insight into the possible structural differences between the two HLA-DR conformers. Cholesterol depletion studies indicate a role for cholesterol-rich membrane domains in the formation/maintenance of Tü36 mAb reactive DR molecules. Finally, phylogenetic analysis of the amino acid sequences of Tü36-reactive HLA-DR ß chains reveals a unique pattern of both Tü36 mAb reactivity and key amino acid polymorphisms. In total, these studies bring the paradigm M1/M2-paired MHC class II molecules to the human HLA-DR molecule and suggest that the functional differences between these conformers defined in mouse class II extend to the human immune system.

The new kidney allocation system does not equally advantage all very high cPRA candidates - A single center analysis.

The new UNOS kidney allocation system awards very high points to candidates with cPRA 99% and 100%, and allows for national sharing for cPRA 100% candidates. We sought to determine the effect of this new kidney allocation system on candidates who are very highly sensitized (90-98% cPRA) but not eligible for very high points or national sharing by examining offers to these candidates for 5months pre-implementation and two consecutive 5month periods post-implementation and comparing them to cPRA⩾99% candidates. We found that the cPRA⩾99% candidates received significantly more offers and transplants after implementation, while offers and transplants to the 90-98% candidates decreased. A slight adjustment to the allocation system may be needed to provide more equitable distribution of kidneys to all high cPRA candidates.

CIITA enhances HIV-1 attachment to CD4+ T cells leading to enhanced infection and cell depletion.

Activated CD4(+) T cells are more susceptible to HIV infection than resting T cells; the reason for this remains unresolved. Induction of CIITA and subsequent expression of the MHC class II isotype HLA-DR are hallmarks of CD4(+) T cell activation; therefore, we investigated the role of CIITA expression in T cells during HIV infection. CIITA-expressing SupT1 cells display enhanced virion attachment in a gp160/CD4-dependent manner, which results in increased HIV infection, virus release, and T cell depletion. Although increased attachment and infection of T cells correlated with HLA-DR surface expression, Ab blocking, transient expression of HLA-DR without CIITA, and short hairpin RNA knockdown demonstrate that HLA-DR does not directly enhance susceptibility of CIITA-expressing cells to HIV infection. Further analysis of the remaining MHC class II isotypes, HLA-DP and HLA-DQ, MHC class I isotypes, HLA-A, HLA-B, and HLA-C, and the class II Ag presentation genes, invariant chain and HLA-DM, demonstrate that these proteins likely do not contribute to CIITA enhancement of HIV infection. Finally, we demonstrate that in activated primary CD4(+) T cells as HLA-DR/CIITA expression increases there is a corresponding increase in virion attachment. Overall, this work suggests that induction of CIITA expression upon CD4(+) T cell activation contributes to enhanced attachment, infection, virus release, and cell death through an undefined CIITA transcription product that may serve as a new antiviral target.

Select phytochemicals suppress human T-lymphocytes and mouse splenocytes suggesting their use in autoimmunity and transplantation.

We have considered a novel "rational" gene targeting approach for treating pathologies whose genetic bases are defined using select phytochemicals. We reason that one such potential application of this approach would be conditions requiring immunosuppression such as autoimmune disease and transplantation, where the genetic target is clearly defined; i.e., interleukin-2 and associated T-cell activation. Therefore, we hypothesized that select phytochemicals can suppress T-lymphocyte proliferation both in vitro and in vivo. The immunosuppressive effects of berry extract, curcumin, quercetin, sulforaphane, epigallocatechin gallate (EGCG), resveratrol, alpha-tocopherol, vitamin C and sucrose were tested on anti-CD3 plus anti-CD28-activated primary human T-lymphocytes in culture. Curcumin, sulforaphane, quercetin, berry extract and EGCG all significantly inhibited T-cell proliferation, and this effect was not due to toxicity. IL-2 production was also reduced by these agents, implicating this important T-cell cytokine in proliferation suppression. Except for berry extract, these same agents also inhibited mouse splenic T-cell proliferation and IL-2 production. Subsequent in vivo studies revealed that quercetin (but not sulforaphane) modestly suppressed mouse splenocyte proliferation following supplementation of BALB/c mice diets. This effect was especially prominent if corrected for the loss of supplement "recall" as observed in cultured T-cells. These results suggest the potential use of these select phytochemicals for treating autoimmune and transplant patients, and support our strategy of using select phytochemicals to treat genetically-defined pathologies, an approach that we believe is simple, healthy, and cost-effective.

Redox response of the endogenous calcineurin inhibitor Adapt 78.

Adapt 78 (DSCR 1/calcipressin/MCIP 1) is a potent natural inhibitor of calcineurin, an important intracellular phosphatase that mediates many cellular responses to calcium. We previously reported two major cytosolic isoforms (1 and 4) of Adapt 78, and that isoform 4 is an oxidative and calcium stress-response protein. Using a higher cell culture density and new antibody, we again observed that both major isoforms localized to the cytosol, but a significant level of isoform 4 (but not isoform 1) was also detected in the nucleus where it was present in the non-soluble region and not associated with RNA. Exposure of cells to hydrogen peroxide led to the significant loss of isoform 4 from the nucleus with a moderate increase in cytosolic localization. The change in isoform 4 phosphorylation state in response to oxidative stress, characterized by a loss of the lesser (hypo) phosphorylated Adapt 78, was not due to accelerated degradation, although general Adapt 78 degradation was proteosome mediated. Finally, stimulation of Jurkat and primary T-lymphocyte signaling led to isoform 4 induction. This induction was BAPTA, diphenylene iodonium, and N-acetylcysteine inhibitable, and accompanied by induction of the classic immune response mediator and calcineurin-pathway-stimulated interleukin-2. These studies reveal new redox-related activities for Adapt 78 isoform 4, which may contribute to its known calcineurin-regulating and cytoprotective activities, and further suggest that Adapt 78 plays a role in basic T-cell response.

The impact of obesity in renal transplantation: an analysis of paired cadaver kidneys.

BACKGROUND: Previous studies indicate that obesity is a risk factor in renal transplantation. However, these analyses did not control for variable donor factors that may strongly influence outcome. To control for donor variables such as age, cause of death, procurement techniques, preservation methods, cold ischaemia time and implantation technique, we analysed patient and graft survival in recipients of paired kidneys, derived from the same procurement procedure, preserved in the same manner, subjected to similar cold ischaemia time and implanted by the same surgical team. Between June 1992 and August 1999, 28 procurement procedures provided kidneys which were transplanted into one obese and one non-obese recipient. Body mass index (BMI) was calculated as kg/m2. Recipients were classified as obese (BMI > 30) or non-obese (BMI < 30). Immunotherapy for all recipients consisted of a triple therapy regimen of cyclosporine or prograf, azathioprine or cellcept, and prednisone. Patients with delayed graft function (DGF), defined as the need for dialysis within 72 h of the transplant procedure, were treated with anti-thymocyte globulin (ATG) or thymoglobulin (TMG) induction for 5-7 d. The rate of DGF (7.1 versus 10.7%) and acute rejection (39.3 versus 35.7%) were similar in the obese and non-obese recipient groups. Patient survival was similar at 1, 3 and 5 yr in both groups. In addition, graft survival was similar at 1 yr. However, a trend toward decreased medium-term graft survival, which reached significance at 5 yr, was observed in the obese group. Furthermore, mean serum creatinine at 1 yr was higher in the obese group (2.0) compared with the non-obese group (1, 4) (p=0.12). This analysis of paired cadaver kidneys indicated that obesity is not a risk factor for DGF, acute rejection, and 1-yr graft survival. However, a decreased medium- and long-term graft survival trend, which reached statistical significance at 5 yr, was observed in obese recipients.