1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 Insulin-Producing Intestinal K Cells Protect Nonobese Diabetic Mice From Autoimmune Diabetes Majid Mojibian,* Ada W. Y. Lam,* Yukihiro Fujita, Ali Asadi, Guntram A. Grassl, Peter Dickie, Rusung Tan, Anthony T. Cheung, and Timothy J. Kieffer

Background: Neuropsychiatric disorders during HIV/AIDS are common although the contribution of HIV-1 infection within the brain, and in particular individual HIV-1 proteins, to the development of these brain disorders is unknown. Herein, an in vivo transgenic mouse model was generated in which the HIV-1 Nef protein was expressed in microglia cells, permitting investigation of neurobehavioral phenotypes and associated cellular and molecular properties.Methods: Transgenic (Tg) mice that expressed full length HIV-1 nef under the control of the c-fms promoter and wildtype (Wt) littermates were investigated using different measures of neurobehavioral performance including locomotory, forced swim (FST), elevated plus maze (EPM) and T-maze tests. Host gene and transgene expression were assessed by RT-PCR, immunoblotting, enzymatic activity and immunohistochemistry. Biogenic amine levels were measured by HPLC with electrochemical detection.Results: Tg animals exhibited Nef expression in brain microglia and cultured macrophages. Tg males displayed hyperactive behaviors including augmented locomotor activity, decreased immobility in the FST and increased open-arm EPM exploration compared to Wt littermates (p < 0.05). Tg animals showed increased CCL2 expression with concurrent IFN-alpha suppression in striatum compared with Wt littermates (p < 0.05). Dopamine levels, MAO activity and the dopamine transporter (DAT) expression were reduced in the striatum of Tg animals (p < 0.05).Conclusions: HIV-1 Nef expression in microglia induced CCL2 expression together with disrupting striatal dopaminergic transmission, resulting in hyperactive behaviors which are observed in mania and other psychiatric comorbidities among HIV-infected persons. These findings emphasize the selective effects of individual viral proteins in the brain and their participation in neuropathogenesis. (C) 2014 Published by Elsevier Inc.

BACKGROUND & AIMS:Type 1 diabetes is caused by an aberrant response against pancreatic β cells. Intestinal K cells are glucose-responsive endocrine cells that might be engineered to secrete insulin. We generated diabetes-prone non-obese diabetic (NOD) mice that express insulin, via a transgene, in K cells. We assessed the effects on immunogenicity and diabetes development.METHODS:Diabetes incidence and glucose homeostasis were assessed in NOD mice that expressed mouse preproinsulin II from a transgene in K cells and nontransgenic NOD mice (controls); pancreas and duodenum tissues were collected and analyzed by histology. We evaluated T cell responses to insulin, levels of circulating autoantibodies against insulin, and the percentage of circulating antigen-specific T cells. Inflammation of mesenteric and pancreatic lymph node cells was also evaluated.RESULTS:The transgenic mice tended to have lower blood levels of glucose than control mice, associated with increased plasma levels of immunoreactive insulin and proinsulin. Fewer transgenic mice developed diabetes than controls. In analyses of pancreas and intestine tissues from the transgenic mice, insulin-producing K cells were not affected by the immune response and the mice had reduced destruction of endogenous β cells. Fewer transgenic mice were positive for insulin autoantibodies compared with controls. Cells isolated from mesenteric lymph nodes of the transgenic mice had significantly lower rates of proliferation and T cells from transgenic mice tended to secrete lower levels of inflammatory cytokines than from controls. At 15 weeks, transgenic mice had fewer peripheral CD8(+) T cells specific for NRP-V7 than control mice.CONCLUSIONS:NOD mice with intestinal K cells engineered to express insulin have reduced blood levels of glucose, are less likely to develop diabetes, and have reduced immunity against pancreatic β cells compared with control NOD mice. This approach might be developed to treat patients with type 1 diabetes.

MicroRNAs (miRNAs) are small noncoding RNA molecules, which are known to regulate gene expression in physiological and pathological conditions. miRNA profiling was performed using brain tissue from patients with HIV encephalitis (HIVE), a neuroinflammatory/degenerative disorder caused by HIV infection of the brain. Microarray analysis showed differential expression of multiple miRNAs in HIVE compared to control brains. Target prediction and gene ontology enrichment analysis disclosed targeting of several gene families/biological processes by differentially expressed miRNAs (DEMs), with cell death-related genes, including caspase-6, showing a bias toward down-regulated DEMs. Consistent with the miRNA data, HIVE brains exhibited higher levels of caspase-6 transcripts compared with control patients. Immunohistochemical analysis showed localization of the cleaved form of caspase-6 in astrocytes in HIVE brain sections. Exposure of cultured human primary astrocytes to HIV viral protein R (Vpr) induced p53 up-regulation, loss of mitochondrial membrane potential, and caspase-6 activation followed by cell injury. Transgenic mice, expressing Vpr in microglial cells, demonstrated astrocyte apoptosis in brain, which was associated with caspase-6 activation and neurobehavioral abnormalities. Overall, these data point to previously unrecognized alterations in miRNA profile in the brain during HIV infection, which contribute to cell death through dysregulation of cell death machinery.

The granzyme B gene is activated upon cytotoxic T cell stimulation and the protein is a key inducer of apoptosis in target cells. Previous studies have identified important proximal regulatory regions but these proved insufficient to drive expression in vivo. We identified a DNase1 hypersensitive site (HS2) 3.9kb upstream of the transcription start site that was present in stimulated but not resting CD8+ cells. The CTL line CTLL R8 was stably transfected with GFP reporter constructs and showed consistently higher fluorescence values when HS2 was included. In transgenic mice the presence of the relevant region of DNA resulted in inducible, CTL-specific transcription of the transgene in all transgenic founder lines analyzed. Deletion of HS2 resulted in a 10-fold reduction in expression. This is the first report of a major distal regulatory element in the control of granzyme B transcription.

Despite the introduction of highly active antiretroviral therapy, dementia caused by human immunodeficiency virus-1 (HIV-1) infection remains a devastating and common neurological disorder. Although the mechanisms governing neurodegeneration during HIV-1 infection remain uncertain, the HIV-1 accessory protein, viral protein R (Vpr), has been proposed as a neurotoxic protein. Herein, we report that Vpr protein and transcript were present in the brains of HIV-infected persons. Moreover, soluble Vpr caused neuronal apoptosis, involving cytochrome c extravasation, p53 induction, and activation of caspase-9 while exerting a depressive effect on whole-cell currents in neurons (p < 0.05), which was inhibited by iberiotoxin. Vpr-activated glial cells secreted neurotoxins in a concentration-dependent manner (p < 0.001). Transgenic (Tg) mice expressing Vpr in brain monocytoid cells displayed the transgene principally in the basal ganglia (p < 0.05) and cerebral cortex (p < 0.01) compared with hindbrain expression. Vpr was released from cultured transgenic macrophages, which was cytotoxic to neurons and was blocked by anti-Vpr antibody (p < 0.05). Neuronal injury was observed in Tg animals compared with wild-type littermates, chiefly affecting GAD65 (p < 0.01) and vesicular acetylcholine transferase (p < 0.001) immunopositive neuronal populations in the basal ganglia. There was also a loss of subcortical synaptophysin (p < 0.001) immunoreactivity as well as an increase in activated caspase-3, which was accompanied by a hyperexcitable neurobehavioral phenotype (p < 0.05). Thus, HIV-1 Vpr caused neuronal death through convergent pathogenic mechanisms with ensuing in vivo neurodegeneration, yielding new insights into the mechanisms by which HIV-1 injures the nervous system.

In humans, spontaneous autoimmune attack against cardiomyocytes often leads to idiopathic dilated cardiomyopathy (IDCM) and life-threatening heart failure. HLA-DQ8 transgenic IAb knockout NOD mice (NOD.DQ8/Ab(0); DQA1*0301, DQB1*0302) develop spontaneous anticardiomyocyte autoimmunity with pathology very similar to human IDCM, but why the heart is targeted is unknown. In the present study, we first investigated whether NOD/Ab(0) mice transgenic for a different DQ allele, DQ6, (DQA1*0102, DQB1*0602) would also develop myocarditis. NOD.DQ6/Ab(0) animals showed no cardiac pathology, implying that DQ8 is specifically required for the myocarditis phenotype. To further characterize the cellular immune mechanisms, we established crosses of our NOD.DQ8/Ab(0) animals with Rag1 knockout (Rag1(0)), Ig H chain knockout (IgH(0)), and beta(2)-microglobulin knockout (beta(2)m(0)) lines. Adoptive transfer of purified CD4 T cells from NOD.DQ8/Ab(0) mice with complete heart block (an indication of advanced myocarditis) into younger NOD.DQ8/Ab(0) Rag1(0) animals induced cardiac pathology in all recipients, whereas adoptive transfer of purified CD8 T cells or B lymphocytes had no effect. Despite the absence of B lymphocytes, NOD.DQ8/Ab(0)IgH(0) animals still developed complete heart block, whereas NOD.DQ8/Ab(0)beta(2)m(0) mice (which lack CD8 T cells) failed to develop any cardiac pathology. CD8 T cells (and possibly NK cells) seem to be necessary to initiate disease,whereas once initiated, CD4 T cells alone can orchestrate the cardiac pathology, likely through their capacity to recruit and activate macrophages. Understanding the cellular immune mechanisms causing spontaneous myocarditis/IDCM in this relevant animal model will facilitate the development and testing of new therapies for this devastating disease.

The progression of immune responses is generally associated with an increase in the overall avidity of antigen-specific T cell populations for peptide-MHC. This is thought to result from preferential expansion of high-avidity clonotypes at the expense of their low-avidity counterparts. Since T cell antigen-receptor genes do not mutate, it is puzzling that high-avidity clonotypes do not predominate from the outset. Here we provide a developmental basis for this phenomenon in the context of autoimmunity. We have carried out comprehensive studies of the diabetogenic CD8 T cell population that targets residues 206-214 of the beta cell antigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP(206-214)) and undergoes avidity maturation as disease progresses. We find that the succession of IGRP(206-214)-specific clonotypes with increasing avidities during the progression of islet inflammation to overt diabetes in nonobese diabetic mice is fueled by autoimmune inflammation but opposed by systemic tolerance. As expected, naive high-avidity IGRP(206-214)-specific T cells respond more efficiently to antigen and are significantly more diabetogenic than their intermediate- or low-avidity counterparts. However, central and peripheral tolerance selectively limit the contribution of these high-avidity T cells to the earliest stages of disease without abrogating their ability to progressively accumulate in inflamed islets and kill beta cells. These results illustrate the way in which incomplete deletion of autoreactive T cell populations of relatively high avidity can contribute to the development of pathogenic autoimmunity in the periphery.

Clinical and morphologic features of human immunodeficiency virus (HIV)-associated nephropathy (HIVAN), such as proteinuria, sclerosing glomerulopathy, tubular degeneration, and interstitial disease, have been modeled in mice bearing an HIV proviral transgene rendered noninfectious through a deletion in gag/pol. Exploring the genetic basis of HIVAN, HIV transgenic mice bearing mutations in either or both of the accessory genes nef and vpr were created. Proteinuria and focal glomerulosclerosis (FGS) only developed in mice with an intact vpr gene. Transgenic mice bearing a simplified proviral DNA (encoding only Tat and Vpr) developed renal disease characterized by FGS in which Vpr protein was localized to glomerular and tubular epithelia by immunohistochemistry. The dual transgenic progeny of HIV[Tat/Vpr] mice bred to HIV[ΔVpr] proviral transgenic mice displayed a more severe nephropathy with no apparent increase in Vpr expression, implying that multiple viral genes contribute to HIVAN. However, the unique contribution of macrophage-specific Vpr expression in the development of glomerular disease was underscored by the induction of FGS in multiple murine lines bearing a c-fms/vpr transgene.

Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286–300 (p286) of GAD65. These mice have GAD65-specific CD4+ T cells, as shown by staining with an I-Ag7(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, and IL-10 when stimulated in vitro with GAD65 peptide 286–300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4+ T cells, or p286-tetramer+CD4+ Tcells, from GAD65 286–300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286–300-specific T cells have disease protective capacity and are not pathogenic.

HIV transgenic mice bearing multiple copies of a noninfectious (Delta gag/pol) proviral DNA were tested for the systemic production of nitric oxide (NO). Serum levels of NO metabolites were reduced about 50% in HIV transgenic mice compared with nontransgenic sibling mice. This difference persisted when NO production was induced with peritoneal injections of bacterial endotoxin (LPS). Peritoneal inflammatory macrophages, but not resident peritoneal macrophages, derived from HIV-1 transgenic mice and activated in vitro with LPS and IFN-T (or tumor necrosis factor a and IFN-gamma) also produced about 50% less NO than did macrophages harvested from nontransgenic litter-mates. Isogenic, transgenic mice bearing mutated nef or vpr genes had normal serum levels of NO metabolites and their macrophages produced normal levels of NO when stimulated. An explanation for the reduced NO response of HIV[Vpr+Nef+] macrophages was not apparent from measured levels of iNOS expression, viral gene expression, or arginase activity in activated macrophages. Inhibition of nitric oxide synthase (NOS) isoforms with L-NAME or aminoguanidine blocked time-dependent increases in HIV gene expression in activated macrophages cultured ex vivo. Inhibition with L-NAME occurred despite high levels of NO generated by iNOS, and exogenously supplied NO induced HIV gene expression only weakly, suggesting that cNOS had the greater influence on proviral gene induction. This system is presented as a model of HIV-1 proviral gene expression and dysfunction in macrophages.

The expression of calreticulin, a Ca(2+)-binding chaperone of the endoplasmic reticulum, is elevated in the embryonic heart, and because of impaired cardiac development, knockout of the Calreticulin gene is lethal during embryogenesis. The elevated expression is downregulated after birth. Here we have investigated the physiological consequences of continued high expression of calreticulin in the postnatal heart, by producing transgenic mice that overexpress the protein in the heart. These transgenic animals exhibit decreased systolic function and inward I(Ca,L), low levels of connexin43 and connexin40, sinus bradycardia, and prolonged atrioventricular (AV) node conduction followed by complete heart block and sudden death. We conclude that postnatal downregulation of calreticulin is essential in the development of the cardiac conductive system, in particular in the sinus and AV nodes, when an inward Ca(2+) current is required for activation. This work identifies a novel pathway of events, leading to complete heart block and sudden cardiac death, which involves high expression of calreticulin in the heart.

The Ras signaling pathway plays a critical role in thymopoiesis and T cell activation, but the mechanism of Ras regulation is controversial. At least one mode of Ras regulation in T cells involves the messenger diacylglycerol (DAG). RasGRP, a Ras activator with a DAG-binding C1 domain, is expressed in T cells and thymocytes. Here we show that thymi of RasGRP-null mutant mice have approximately normal numbers of immature thymocytes but a marked deficiency of mature, single-positive (CD4+CD8− and CD4−CD8+) thymocytes. In Ras signaling and proliferation assays, mutant thymocytes showed a complete lack of response to DAG analogs or T cell receptor (TCR) stimulation by antibodies. Thus, TCR and DAG are linked through RasGRP to Ras signaling.

BACKGROUNDHuman immunodeficiency virus (HIV)-associated nephropathy is characterized by focal segmental glomerulosclerosis and microcystic tubular dilation. We have previously described a mouse transgenic for a Deltagag-pol HIV-1 genome, which develops glomerulosclerosis, cutaneous papillomas, and cataracts.METHODSWe developed mice transgenic for a Deltagag-pol-nef HIV genome in order to investigate the role of the nef gene in these phenotypes.RESULTSOne transgenic line, X5, expressed HIV mRNA in kidney and consistently manifested focal segmental glomerulosclerosis and tubular dilation by six weeks of age. Northern analysis indicated that renal transgene expression was higher in the Deltagag-pol-nef mice compared with the Deltagag-pol mice. In situ hybridization and immunostaining demonstrated HIV RNA and protein expression within the glomerular epithelial cells and tubular epithelial cells. These cell types showed histologic evidence of toxicity, including vacuolation and detachment from basement membrane, and exhibited increased rates of apoptosis. These data suggest that the renal disease seen in the Deltagag-pol-nef transgenic mouse may be caused by the expression of HIV genes within renal epithelial cells, that this expression may induce cellular toxicity, including apoptosis, and that nef is not required for the induction of renal disease. We have previously described mice bearing the nef gene, which do not manifest renal disease. In further experiments, Deltagag-pol-nef mice were bred with nef mice; these dual-transgenic mice developed renal disease that generally resembled that seen in Deltagag-pol-nef mice, but with somewhat more severe glomerulosclerosis and less severe tubulointerstitial injury.RESULTSThe results of these transgenic studies suggest that the role of nef is complex and may act both to reduce transgene expression and to potentiate glomerular injury induced by other HIV-1 gene products.

Transgenic mice bearing HIV-1 proviral DNA deleted in the gag/pol region (HIVd1443 mice) model a chronic, nonproductive form of viral gene expression in various cell types including macrophages. They display a disease phenotype that includes HIV-associated nephropathy (HIVAN), congenital cataracts, papillomatosis, and growth failure. The role of HIV-1 Nef in viral gene regulation and the development of disease was explored in mice bearing an isogenic HIV transgene in which nef was mutated by frameshift mutation. Like its Nef(+) counterpart, HIVd1443[Nef(-)] mice expressed HIV gene products in the skin, muscle, kidney, and peritoneal macrophages. While these mice did not develop cataracts, papillomatous skin lesions, or display any apparent growth defect, they did develop HIVAN. Nef expression was introduced to HIVd1443[Nef(-)] mice through breeding to mice bearing an HIV LTR-linked nef transgene. Nef-complemented HIVd1443[Nef(-)] mice had reduced levels of viral gene products in the muscle and kidney. In contrast, HIV gene expression in the skin of these mice remained high and papillomatous lesions emerged that were more severe than those on wildtype HIVd1443 mice. Still, Nef had a negative effect on LPS-induced viral gene expression in visibly normal skin. In comparisons of peritoneal macrophages, viral RNA expression was significantly reduced in resident macrophages of Nef(+) mice. HIV inflammatory macrophages expressed viral genes and displayed an altered FAGS profile. In particular, Nef+ populations were marked by an increased proportion of F4/80(med)/Mac-1(-) cells as well as fewer Mac-1 cells and reduced F4/80 staining. This HIV proviral transgenic model has demonstrated the capacity of HIV-1 Nef to contribute to HIV cytopathicity by altering cellular maturation and viral gene expression in vivo.

Calreticulin is a ubiquitous Ca2+ binding protein, located in the endoplasmic reticulum lumen, which has been implicated in many diverse functions including: regulation of intracellular Ca2+ homeostasis, chaperone activity, steroid-mediated gene regulation, and cell adhesion. To understand the physiological function of calreticulin we used gene targeting to create a knockout mouse for calreticulin. Mice homozygous for the calreticulin gene disruption developed omphalocele (failure of absorption of the umbilical hernia) and showed a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Transgenic mice expressing a green fluorescent protein reporter gene under the control of the calreticulin promoter were used to show that the calreticulin gene is highly activated in the cardiovascular system during the early stages of cardiac development. Calreticulin protein is also highly expressed in the developing heart, but it is only a minor component of the mature heart. Bradykinin-induced Ca2+ release by the InsP3-dependent pathway was inhibited in crt−/− cells, suggesting that calreticulin plays a role in Ca2+ homeostasis. Calreticulin-deficient cells also exhibited impaired nuclear import of nuclear factor of activated T cell (NF-AT3) transcription factor indicating that calreticulin plays a role in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway.