Association of variants in CDKN2A/2B and CDKAL1 genes with gestational insulin sensitivity and disposition in pregnant Han Chinese women.

AIMS/INTRODUCTION: Variants in cell cycle regulation genes, CDKAL1 and CDKN2A/2B, have been suggested to be associated with type 2 diabetes, and also play a role in insulin procession in non-diabetic European individuals. Rs7754580 in CDKAL1 and rs7020996 in CDKN2A/2B were found to be associated with gestational diabetes in Chinese individuals. In order to understand the metabolism mechanism of greatly upregulated maternal insulin signaling during pregnancy and the pathogenesis of gestational diabetes, we investigated the impact of rs7754580 and rs7020996 on gestational insulin regulation and procession. MATERIALS AND METHODS: We recruited 1,146 unrelated, non-diabetic, pregnant Han Chinese women (age 28.5 ± 4.1 years, body mass index 21.4 ± 2.6 kg/m(2)), and gave them oral glucose tolerance tests. The indices of insulin sensitivity, insulin disposition, insulin release and proinsulin to insulin conversion were calculated. Rs7754580 in the CDKAL1 gene and rs7020996 in the CDKN2A/2B gene were genotyped. Under an additive model, we analyzed the associations between the variants and gestational insulin indices using logistic regression. RESULTS: By adjusting for maternal age, body mass index and the related interactions, CDKAL1 rs7754580 risk allele C was detected to be associated with increased insulin sensitivity (P = 0.011), decreased insulin disposition (P = 0.0002) and 2-h proinsulin conversion (P = 0.017). CDKN2A/2B rs7020996 risk allele T was found to be related to decreased insulin sensitivity (P = 0.002) and increased insulin disposition (P = 0.0001). CONCLUSIONS: The study showed that cell cycle regulating genes might have a distinctive effect on gestational insulin sensitivity, ß-cell function and proinsulin conversion in pregnant Han Chinese women.

Familial multinodular goiter syndrome with papillary thyroid carcinomas: mutational analysis of the associated genes in 5 cases from 1 Chinese family.

BACKGROUND: Familial papillary thyroid cancer (fPTC) is recognized as a distinct entity only recently and no fPTC predisposing genes have been identified. Several potential regions and susceptibility loci for sporadic PTC have been reported. We aimed to evaluate the role of the reported susceptibility loci and potential risk genomic region in a Chinese familial multinodular goiter (fMNG) with PTC family. METHODS: We sequenced the related risk genomic regions and analyzed the known PTC susceptibility loci in the Chinese family members who consented to join the study. These loci included (1) the point mutations of the BRAF and RET; (2) the possible susceptibility loci to sporadic PTC; and (3) the suggested potential fMNG syndrome with PTC risk region. RESULTS: The members showed no mutations in the common susceptible BRAF and RET genomic region, although contained several different heterozygous alleles in the RET introns. All the members were homozygous for PTC risk alleles of rs966423 (C) at chromosome 2q35, rs2910164 (C) at chromosome 5q24 and rs2439302 (G) at chromosome 8p12; while carried no risk allele of rs4733616 (T) at chromosome 8q24, rs965513 (A) or rs1867277 (A) at chromosome 9q22 which were associated with radiation-related PTC. The frequency of the risk allele of rs944289 (T) but not that of rs116909374 (T) at chromosome 14q13 was increased in the MNG or PTC family members. CONCLUSIONS: Our work provided additional evidence to the genetic predisposition to a Chinese familial form of MNG with PTC. The family members carried quite a few risk alleles found in sporadic PTC; particularly, homozygous rs944289 (T) at chromosome 14q13 which was previously shown to be linked to a form of fMNG with PTC. Moreover, the genetic determinants of radiation-related PTC were not presented in this family.

Immunity against a therapeutic xenoprotein/Fc construct delivered by gene transfer is reduced through binding to the inhibitory receptor FcγRIIb.

BACKGROUND: Therapeutic xenoproteins are immunogenic and can induce neutralizing antibodies. When delivered by intramuscular injection of a plasmid vector, this mimics classical DNA vaccination. To demonstrate this, we chose Exendin-4 (Ex4), which is a glucagon-like peptide-1 mimetic xenoprotein in clinical use for treating type 2 diabetes. We constructed an Ex4 and mouse immunoglobulin (Ig)G1-Fc fusion fragment (Ex4/Fc), and hypothesized that it would have minimal immunogenicity as a result of its capacity to bind the inhibitory Fc receptor FcγRIIb expressed by B lymphocytes. METHODS: Plasmid vectors encoding Ex4/Fc constructs, with wild-type or mutant Fc, were injected intramuscularly into mice, and local electroporation was applied to enhance gene transfer. Gene transfer was performed in both wild-type and FcγRIIb knockout mice. Antibody production was detected in serum by an enzyme-linked immunosorbent assay. RESULTS: Recombinant Ex4/Fc bound only to B cells expressing FcγRIIb. This binding was dependent on a motif in the Fc region, which we mutated to abolish binding (Ex4/Fcmut). Ex4 antibody was detected in mice treated with Ex4, as well as Ex4/Fcmut, but not in those treated with Ex4/Fc. Thus, wild-type Fc was associated with reduced immunogenicity. To confirm this was related to the presence of inhibitory Fc receptors, we also performed experiments in FcγRIIb-null mice. Mice lacking this receptor produced antibodies against all Ex4 constructs, including the wild-type Fc (Ex4/Fc). CONCLUSIONS: The present study shows that inhibitory FcγRIIb receptors interacting with the wild-type IgG1-Fc reduce immunity against Ex4/Fc, suggesting an approach for reducing the immunogenicity of therapeutic proteins in the context of gene therapy.

T helper17 cells are sufficient but not necessary to induce acute graft-versus-host disease.

T helper (Th)1 cells were considered responsible for the induction of graft-versus-host disease (GVHD), but recently the concept has been challenged. Th17 cells play a critical role in mediating autoimmune diseases, but their role in the pathogenesis of GVHD remains unclear. Herein we compare the ability of in vitro generated Th1 and Th17 cells from C57BL/6 mice to induce GVHD in lethally irradiated BALB/c recipients. Allogeneic Th17 cells had superior expansion and infiltration capabilities in GVHD target organs, which correlated with their increased pathogenicity when compared with naïve or Th1 controls. Th17 cells caused no pathology in the syngeneic recipients, indicating that antigen-activation was required for their pathogenicity. Polarized Th17 cells could not maintain their phenotype in vivo as they produced a significant amount of interferon (IFN)-gamma after being transplanted into allogeneic recipients; however, IFN-gamma was not required for Th17 cell-induced GVHD. Further, we evaluated the pathogenesis of Th17 cells in GVHD by using polyclonal nonprimed CD4T cells in a clinically relevant allogeneic bone marrow transplantation (BMT) setting. We found that disruption of Th17-differentiation alone by targeting RORgammat (Th17-specific transcription factor) had no significant effect on GVHD development. We conclude that Th17 cells are sufficient but not necessary to induce GVHD.

Beta2 integrins separate graft-versus-host disease and graft-versus-leukemia effects.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation. Migration of donor-derived T cells into GVHD target organs plays an essential role in the development of GVHD. beta2 integrins are critically important for leukocyte extravasation through vascular endothelia and for T-cell activation. We asked whether CD18-deficient T cells would induce less GVHD while sparing the graft-versus-leukemia (GVL) effect. In murine allogeneic bone marrow transplantation models, we found that recipients of CD18-/- donor T cells had significantly less GVHD morbidity and mortality compared with recipients of wild-type (WT) donor T cells. Analysis of alloreactivity showed that CD18-/- and WT T cells had comparable activation, expansion, and cytokine production in vivo. Reduced GVHD was associated with a significant decrease in donor T-cell infiltration of recipient intestine and with an overall decrease in pathologic scores in intestine and liver. Finally, we found that the in vivo GVL effect of CD18-/- donor T cells was largely preserved, because mortality of the recipients who received transplants of CD18-/- T cells plus tumor cells was greatly delayed or prevented. Our data suggest that strategies to target beta2 integrin have clinical potential to alleviate or prevent GVHD while sparing GVL activity.

Opposing effects of ICOS on graft-versus-host disease mediated by CD4 and CD8 T cells.

ICOS, a CD28 family member expressed on activated CD4(+) and CD8(+) T cells, plays important roles in T cell activation and effector function. Here we studied the role of ICOS in graft-vs-host disease (GVHD) mediated by CD4(+) or CD8(+) T cells in allogeneic bone marrow transplantation. In comparison of wild-type and ICOS-deficient T cells, we found that recipients of ICOS(-/-) CD4(+) T cells exhibited significantly less GVHD morbidity and delayed mortality. ICOS(-/-) CD4(+) T cells had no defect in expansion, but expressed significantly less Fas ligand and produced significantly lower levels of IFN-gamma and TNF-alpha. Thus, ICOS(-/-) CD4(+) T cells were impaired in effector functions that lead to GVHD. In contrast, recipients of ICOS(-/-) CD8(+) T cells exhibited significantly enhanced GVHD morbidity and accelerated mortality. In the absence of ICOS signaling, either using ICOS-deficient donors or ICOS ligand-deficient recipients, the levels of expansion and Tc1 cytokine production of CD8(+) T cells were significantly increased. The level of expansion was inversely correlated with the level of apoptosis, suggesting that increased ability of ICOS(-/-) CD8(+) T cells to induce GVHD resulted from the enhanced survival and expansion of those cells. Our findings indicate that ICOS has paradoxical effects on the regulation of alloreactive CD4(+) and CD8(+) T cells in GVHD.

Donor CD8+ T cells facilitate induction of chimerism and tolerance without GVHD in autoimmune NOD mice conditioned with anti-CD3 mAb.

Prevention of autoimmune diabetes and induction of islet transplantation tolerance in nonobese diabetic (NOD) mice can be reached by induction of mixed chimerism via bone marrow transplantation (BMT), but this procedure requires total body irradiation (TBI) conditioning of the recipients. The toxicity of radiation and potential for graft-versus-host disease (GVHD) prevents its clinical application. Donor CD8+ T cells play a critical role in facilitation of engraftment but also contribute to induction of GVHD in TBI-conditioned recipients. Here, we showed that high doses of donor CD8+ T cells in combination with bone marrow (BM) cells induced mixed chimerism without GVHD in NOD recipients conditioned with anti-CD3 monoclonal antibody (mAb). The prevention of GVHD in those recipients was associated with low-level production of inflammatory cytokines (ie, tumor necrosis factor alpha [TNF-alpha]), high-level production of anti-inflammatory cytokines (ie, interleukin 4 [IL-4] and IL-10), and confining of the donor CD8+ T-cell expansion to lymphohematopoietic tissues. The chimeric NOD recipients showed donor-specific tolerance and reversal of insulitis. These results demonstrate that donor CD8+ T-cell-mediated facilitation of engraftment can be separated from GVHD in nonirradiated recipients. This regimen may have potential application in the treatment of autoimmune disorders as well as induction of transplantation tolerance.