NMDA Receptor Autoantibodies in Autoimmune Encephalitis Cause a Subunit-Specific Nanoscale Redistribution of NMDA Receptors.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder mediated by autoantibodies against the GluN1 subunit of the NMDAR. Patients' antibodies cause cross-linking and internalization of NMDAR, but the synaptic events leading to depletion of NMDAR are poorly understood. Using super-resolution microscopy, we studied the effects of the autoantibodies on the nanoscale distribution of NMDAR in cultured neurons.

Transcriptional regulation via TF-modifying enzymes: an integrative model-based analysis.

Transcription factor activity is largely regulated through post-translational modification. Here, we report the first integrative model of transcription that includes both interactions between transcription factors and promoters, and between transcription factors and modifying enzymes. Simulations indicate that our method is robust against noise.

{beta}-Cell secretory capacity and demand in recipients of islet, pancreas, and kidney transplants.

Context: beta-Cell secretory capacity, a measure of functional beta-cell mass, is often impaired in islet transplant recipients, likely because of a low engrafted beta-cell mass, although calcineurin inhibitor toxicity is often cited as the explanation.

Objective: We sought to determine whether use of the calcineurin inhibitor tacrolimus was associated with reduced beta-cell secretory capacity or with increased beta-cell secretory demand independent of engrafted islet mass.

Design And Participants: We compared metabolic measures in five intraportal islet recipients vs.

Effect of glucagon-like peptide-1 on beta- and alpha-cell function in isolated islet and whole pancreas transplant recipients.

Context: Glucose-dependent insulin secretion is often impaired after islet transplantation where reduced beta-cell secretory capacity indicates a low functional beta-cell mass.

Objective: We sought to determine whether glucagon-like peptide-1 (GLP-1) enhanced glucose-dependent insulin secretion and glucagon suppression in islet recipients, and whether GLP-1 effects were dependent on functional beta-cell mass by simultaneously studying recipients of whole pancreas transplants.

Setting: The study was performed in a clinical and translational research center.

Nephrotic syndrome after hematopoietic cell transplantation: do glomerular lesions represent renal graft-versus-host disease?

Glomerular disease associated with nephrotic syndrome has rarely been recognized as a distinct complication of allogeneic hematopoietic cell transplantation. Case reports in the English and Japanese literature since 1988 have described variable glomerular histology, comprising mainly membranous glomerulonephritis (MGN) in almost two thirds and minimal change disease (MCD) in nearly one quarter of patients. Review of the literature reveals a close temporal relationship between the development of nephrotic syndrome shortly after cessation of immunosuppression and the diagnosis of chronic graft-versus-host disease (GVHD).

An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach.

This protocol describes a method that we developed to adapt the tandem affinity purification (TAP) approach for use in mammalian cells. The protocol involves fusing a protein of interest with a tandem tag consisting of two FLAG tags (FF) followed by two protein-A immunoglobulin G (IgG) binding domains (ZZ). The protocol improves upon previously published TAP approaches by employing FLAG in place of calmodulin binding peptide (CBP) with resulting higher recovery during purification.

Characterization of sequences and mechanisms through which ISE/ISS-3 regulates FGFR2 splicing.

Alternative splicing of fibroblast growth factor receptor-2 (FGFR2) mutually exclusive exons IIIb and IIIc results in highly cell-type-specific expression of functionally distinct receptors, FGFR2-IIIb and FGFR2-IIIc. We previously identified an RNA cis-element, ISE/ISS-3, that enhanced exon IIIb splicing and silenced exon IIIc splicing. Here, we have performed comprehensive mutational analysis to define critical sequence motifs within this element that independently either enhance splicing of upstream exons or repress splicing of downstream exons.

Homeostatic T cell proliferation as a barrier to T cell tolerance.

The maintenance of T cell numbers in the periphery is mediated by distinct homeostatic mechanisms that ensure the proper representation of naïve and memory T cells. Homeostatic proliferation refers to the process by which T cells in lymphopenic hosts divide in the absence of cognate antigen to reconstitute the peripheral lymphoid compartment. During this process T cells acquire effector-memory like properties, including the ability to respond to low doses of antigen in the absence of CD28 costimulation.

From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy.

Nephropathy is a major complication of diabetes. Alterations of mesangial cells have traditionally been the focus of research in deciphering molecular mechanisms of diabetic nephropathy. Injury of podocytes, if recognized at all, has been considered a late consequence caused by increasing proteinuria rather than an event inciting diabetic nephropathy.

A novel intronic cis element, ISE/ISS-3, regulates rat fibroblast growth factor receptor 2 splicing through activation of an upstream exon and repression of a downstream exon containing a noncanonical branch point sequence.

Mutually exclusive splicing of fibroblast growth factor receptor 2 (FGFR2) exons IIIb and IIIc yields two receptor isoforms, FGFR2-IIIb and -IIIc, with distinctly different ligand binding properties. Several RNA cis elements in the intron (intron 8) separating these exons have been described that are required for splicing regulation. Using a heterologous splicing reporter, we have identified a new regulatory element in this intron that confers cell-type-specific inclusion of an unrelated exon that mirrors its ability to promote cell-type-specific inclusion of exon IIIb.

Vhlh gene deletion induces Hif-1-mediated cell death in thymocytes.

The von Hippel-Lindau gene product (pVHL) targets the alpha subunit of basic helix-loop-helix transcription factor hypoxia-inducible factor (HIF) for proteasomal degradation. Inactivation of pVhl in the mouse germ line results in embryonic lethality, indicating that tight control of Hif-mediated adaptive responses to hypoxia is required for normal development and tissue function. In order to investigate the role of pVhl in T-cell development, we generated mice with thymocyte-specific inactivation of Vhlh resulting in constitutive transcriptional activity of Hif-1, as well as mice with thymocyte-specific repression of Hif-1 in a wild-type and Vhlh-deficient background.

Heterologous immunity and homeostatic proliferation as barriers to tolerance.

The different threshold of activation for memory T cells compared to that of naïve T cells makes them resistant to immunomodulation, thus representing a barrier to tolerance. Recently it has been demonstrated that homeostatic proliferation and heterologous immunity represent two naturally occurring and distinct processes that can generate memory T cells. Homeostatic proliferation refers to the process by which, in a lymphodeficient host, normal T cells 'spontaneously' proliferate in response to self-MHC-peptide complexes.

Routes to transplant tolerance versus rejection; the role of cytokines.

The alloimmune response can be divided into specific junctures where critical decisions between tolerance and immunity are made which define the outcome of the transplant. At these "decision nodes" various cytokines direct alloresponsive T cells to develop either a proinflammatory response aimed at graft destruction or an immunoregulatory response facilitating graft acceptance. This review will focus on the role of these cytokines in influencing the progression of an alloimmune response leading ultimately to either allograft survival or rejection.

Reversibility of established diabetic glomerulopathy by anti-TGF-beta antibodies in db/db mice.

Treatment with a neutralizing anti-transforming growth factor-beta (TGF-beta) antibody can prevent the development of diabetic nephropathy in the db/db mouse, a model of type 2 diabetes. However, it is unknown whether anti-TGF-beta therapy can reverse the histological lesions of diabetic glomerulopathy once they are established. Diabetic db/db mice and their non-diabetic db/m littermates were allowed to grow until 16 weeks of age, by which time the db/db mice had developed glomerular basement membrane (GBM) thickening and mesangial matrix expansion.

Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells.

Activation of the transforming growth factor-beta (TGF-beta) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-beta signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-beta on fibronectin expression, using transient transfections of Smad expression vectors and TGF-beta-responsive reporter assays.

Involvement of the transforming growth factor-β system in the pathogenesis of diabetic nephropathy.

The manifestation of diabetic nephropathy may be a consequence of the actions of certain cytokines and growth factors. Prominent among them is transforming growth factor-β (TGF-β), which promotes renal cell hypertrophy and stimulates extracellular matrix accumulation, the two hallmarks of diabetic renal disease. In experimental and human diabetes mellitus, several reports describe overexpression of TGF-β in the glomeruli and tubulointerstitium.

Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes.

Unlabelled: Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes.

Background: The podocyte takes center stage in the pathogenesis of glomerular basement membrane (GBM) thickening and proteinuria in diabetic glomerulopathy. In part, GBM thickening may occur when the podocyte synthesizes increased amounts of collagen IV.

Diverse endogenous light chains contribute to basement membrane reactivity in nonautoimmune mice transgenic for an anti-laminin Ig heavy chain.

Basement membrane proteins are targeted in a variety of pathologic autoimmune responses, yet little is known regarding the origins and regulation of this subset of pathogenic lymphocytes. To examine the generation and fate of B cells reactive with a matrix autoantigen, nonautoimmune C57BL/6 mice were rendered transgenic for a nephrotropic lupus anti-laminin immunoglobulin (Ig) H chain, termed LamH-Cmu. We previously reported recovery of two distinct phenotypes among LamH-Cmu-transgenic mice: progeny of founders M6 and M29 contained abundant transgene-expressing B cells but little anti-laminin Ig, whereas spontaneous autoreactivity was readily recovered from the M7 lineage that expressed minimal B-cell mIgM.