Evidence that beta cell death in the nonobese diabetic mouse is Fas independent.

Recent studies suggest that Fas expression on pancreatic beta cells may be important in the development of autoimmune diabetes in the nonobese diabetic (NOD) mouse. To address this, pancreatic islets from NOD mice were analyzed by flow cytometry to directly identify which cells express Fas and Fas ligand (FasL) ex vivo and after in vitro culture with cytokines. Fas expression was not detected on beta cells isolated from young (35 days) NOD mice. In vitro, incubation of NOD mouse islets with both IL-1 and IFN-gamma was required to achieve sufficient Fas expression and sensitivity for islets to be susceptible to lysis by soluble FasL. In islets isolated from older (>/=125 days) NOD mice, Fas expression was detected on a limited number of beta cells (1-5%). FasL was not detected on beta cells from either NOD or Fas-deficient MRLlpr/lpr islets. Also, both NOD and MRLlpr/lpr islets were equally susceptible to cytokine-induced cell death. This eliminates the possibility that cytokine-treated murine islet cells commit "suicide" due to simultaneous expression of Fas and FasL. Last, we show that NO is not required for cytokine-induced Fas expression and Fas-mediated apoptosis of islet cells. These findings indicate that beta cells can be killed by Fas-dependent cytotoxicity; however, our results raise further doubts about the clinical significance of Fas-mediated beta cell destruction because few Fas-positive cells were isolated immediately before the development of diabetes.     

NF-kappa B hyperactivation has differential effects on the APC function of nonobese diabetic mouse macrophages

Type 1 diabetes is characterized by a chronic inflammatory response resulting in the selective destruction of the insulin-producing beta cells. We have previously demonstrated that dendritic cells (DCs) prepared from nonobese diabetic (NOD) mice, a model for spontaneous type 1 diabetes, exhibit hyperactivation of NF-kappaB resulting in an increased capacity to secrete proinflammatory cytokines and stimulate T cells compared with DCs of nondiabetic strains of mice. In the current study, the activational status of NF-kappaB and its role in regulating the APC function of macrophages (Mphi) prepared from NOD, nonobese resistant (NOR), and BALB/c mice was investigated. Independent of the stimulus, splenic and bone marrow-derived Mphi prepared from NOD mice exhibited increased NF-kappaB activation relative to NOR and BALB/c Mphi. This hyperactivation was detected for different NF-kappaB complexes and correlated with increased IkappaBalpha degradation. Furthermore, increased NF-kappaB activation resulted in an enhanced capacity of NOD vs NOR or BALB/c Mphi to secrete IL-12(p70), TNF-alpha, and IL-1alpha, which was inhibited upon infection with an adenoviral recombinant encoding a modified form of IkappaBalpha. In contrast, elevated NF-kappaB activation had no significant effect on the capacity of NOD Mphi to stimulate CD4(+) or CD8(+) T cells in an Ag-specific manner. These results demonstrate that in addition to NOD DCs, NOD Mphi exhibit hyperactivation of NF-kappaB, which correlates with an increased ability to mediate a proinflammatory response. Furthermore, NF-kappaB influences Mphi APC function by regulating cytokine secretion but not T cell stimulation.     

T cell response to preproinsulin I and II in the nonobese diabetic mouse

Immunization against insulin, insulin B chain, or B chain peptide B(9-23) (preproinsulin peptide II(33-47)) prevents diabetes in the nonobese diabetic (NOD) mouse. Whether or not peptide II(33-47) is the only proinsulin determinant recognized by CD4 T cells remains unclear. Using two peptide libraries spanning the entire sequence of preproinsulin I and preproinsulin II, respectively, we identified T cells specific for four proinsulin epitopes within the islet cell infiltrate of prediabetic female NOD mice. These epitopes were among immunogenic epitopes to which a T cell response was detected after immunization of NOD mice with individual peptides in CFA. Immunogenic epitopes were found on both isoforms of insulin, especially proinsulin II, which is the isoform expressed in the thymus. The autoimmune response to proinsulin represented only part of the immune response to islet cells within the islet cell infiltrate in 15-wk-old NOD mice. This is the first systematic study of preproinsulin T cell epitopes in the NOD mouse model.     

Interkinetic nuclear migration is a broadly conserved feature of cell division in pseudostratified epithelia

Animal development requires tight integration between the processes of proliferative growth and epithelial morphogenesis, both of which play out at the level of individual cells. In this respect, not only must polarized epithelial cells assume complex morphologies, these distinct forms must be radically and repeatedly transformed to permit mitosis. A dramatic illustration of this integration between epithelial morphogenesis and cell proliferation is interkinetic nuclear migration (IKNM), wherein the nuclei of pseudostratified epithelial cells translocate to the apical epithelial surface to execute cell division. IKNM is widely considered a hallmark of pseudostratified vertebrate neuroepithelia, and prior investigations have proposed both actomyosin- and microtubule-dependent mechanisms for apical localization of the mitotic nucleus. Here, using comparative functional analysis in arthropod and cnidarian systems (Drosophila melanogaster and Nematostella vectensis), we show that actomyosin-dependent IKNM is likely to be a general feature of mitosis in pseudostratified epithelia throughout Eumetazoa. Furthermore, our studies suggest a mechanistic link between IKNM and the fundamental process of mitotic cell rounding.     

Induction of diabetes in nonobese diabetic mice by Th2 T cell clones from a TCR transgenic mouse

We have produced a panel of cloned T cell lines from the BDC-2.5 TCR transgenic (Tg) mouse that exhibit a Th2 cytokine phenotype in vitro but are highly diabetogenic in vivo. Unlike an earlier report in which T cells obtained from the Tg mouse were cultured for 1 wk under Th2-promoting conditions and were found to induce disease only in NOD.scid recipients, we found that long-term T cell clones with a fixed Th2 cytokine profile can transfer disease only to young nonobese diabetic (NOD) mice and never to NOD.scid recipients. Furthermore, the mechanism by which diabetes is transferred by a Tg Th2 T cell clone differs from that of the original CD4+ Th1 BDC-2.5 T cell clone made in this laboratory. Whereas the BDC-2.5 clone rapidly causes disease in NOD.scid recipients less than 2 wk old, the Tg Th2 T cell clones can do so only when cotransferred with other diabetogenic T cells, suggesting that the Th2 T cell requires the presence of host T cells for initiation of disease.     

Cutting edge: impaired transitional B cell production and selection in the nonobese diabetic mouse

Developing B cells undergo selection at multiple checkpoints to eliminate autoreactive clones. We analyzed B cell kinetics in the NOD mouse to establish whether these checkpoints are intact. Our results show that although bone marrow production is normal in NOD mice, transitional (TR) B cell production collapses at 3 wk of age, reflecting a lack of successful immature B cell migration to the periphery. This yields delayed establishment of the follicular pool and a lack of selection at the TR checkpoint, such that virtually all immature B cells that exit the bone marrow mature without further selection. These findings suggest that compromised TR B cell generation in NOD mice yields relaxed TR selection, affording autoreactive specificities access to mature pools.     

CD8+ T cell homing to the pancreas in the nonobese diabetic mouse is CD4+ T cell-dependent.

The adoptive transfer of type I diabetes in nonobese diabetic mice requires the contribution of both CD4+ and CD8+ T cells. To further elucidate the cellular pathway(s) of beta-cell destruction and the responsibility of each subset, high doses of committed T cells from diabetic mice purified to single subsets, were injected into syngeneic nonobese diabetic neonates. The recipients of single or mixed subsets were followed for clinical manifestations of diabetes and examined at 30 days of age for in situ lesions. None of the animals injected with either CD4+ or CD8+ T cells became overtly diabetic during the 30 days of observation whereas 8 of 23 mice inoculated with a mixture of the two subsets developed glycosuria and hyperglycemia. However, insulitis was found in 6 of the 13 mice injected with CD4+ T cells whereas only 1 of the 9 mice injected with CD8+ T cells showed marginal infiltration of the pancreas. The lesions initiated by CD4+ T cells alone were considerably less severe than those induced by the mixture of both subsets, corroborating the fact that overt disease did not occur in the former group. Together, these results suggest a distinct function for each diabetogenic T cell subset. CD4+ T cells, which have the capacity to home to the pancreas, promote in turn the influx of CD8+ effector T cells that do not by themselves accumulate in this organ. These results illustrate a novel form of T-T cell interactions leading to organ specific autoimmune lesions.     

Cytokine dysregulation induced by apoptotic cells is a shared characteristic of macrophages from nonobese diabetic and systemic lupus erythematosus-prone mice

Macrophages from nonobese diabetic (NOD) mice, which spontaneously develop type I diabetes, share a defect in elicited cytokine production with macrophages from multiple diverse strains of systemic lupus erythematosus (SLE)-prone mice. We have previously shown that, in SLE-prone mice, this defect is triggered by exposure to apoptotic cells. We report in this work that macrophages from prediseased NOD mice also respond abnormally to apoptotic cells, mimicking closely the apoptotic cell-dependent abnormality that we have observed in multiple SLE-prone strains. This defect is characterized by the underexpression of IL-1 beta and multiple other cytokines. In the presence of apoptotic cells or FBS, elicited expression of IL-1 beta by NOD macrophages is markedly reduced compared with that by macrophages from control mice, including three strains of mice that develop type II (nonautoimmune) diabetes. Given the increasing role of apoptotic cells in tolerance and autoimmunity, a macrophage defect triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. The concordance of this defect among so many diverse autoimmune-prone strains suggests that the genetic basis for this abnormality may constitute a permissive background for autoimmunity.     

Targeting of a T cell agonist peptide to lysosomes by DNA vaccination induces tolerance in the nonobese diabetic mouse

CD4 T cells are crucial effectors in the pathology of type 1 diabetes (T1D). Successful therapeutic interventions for prevention and cure of T1D in humans are still elusive. Recent research efforts have focused on the manipulation of T cells by treatment with DNA. In this paper, we studied the effects of a DNA treatment strategy designed to target antigenic peptides to the lysosomal compartment on a monospecific T cell population termed 2.5mi(+) T cells that shares reactivity with the diabetogenic T cell clone BDC-2.5 in the NOD mouse. MHC class II tetramer analysis showed that repeated administrations were necessary to expand 2.5mi(+) T cells in vivo. This expansion was independent of Ag presentation by B cells. A single peptide epitope was sufficient to induce protection against T1D, which was not due to Ag-specific T cell anergy. Typical Th2 cytokines such as IL-10 or IL-4 were undetectable in 2.5mi(+) T cells, arguing against a mechanism of immune deviation. Instead, the expanded 2.5mi(+) T cell population produced IFN-γ similar to 2.5mi(+) T cells from naive mice. Protection against T1D by DNA treatment was completely lost in NOD.CD28(-/-) mice which are largely deficient of natural regulatory T cells (Treg). Although Ag-specific Foxp3(+) Treg did not expand in response to DNA treatment, diabetes onset was delayed in Treg-reconstituted and DNA-treated NOD.SCID mice. These observations provide evidence for a Treg-mediated protective mechanism that is independent of the expansion or de novo generation of Ag-specific Treg.     

Down-regulation of blood-brain glucose transport in the hyperglycemic nonobese diabetic mouse

The intracarotid injection method has been utilized to examine blood-brain barrier (BBB) glucose transport in hyperglycemic (4–6 days) mice. In anesthetized mice, Brain Uptake Indices were measured over a range of glucose concentrations from 0.010–50 mmol/l; glucose uptake was found to be saturable and kinetically characterized. The maximal velocity (V_max) for glucose transport was 989±214 nmol·min^–1·g^–1· and the half-saturation constant estimated to be 5.80±1.38 mmol/l. The unsaturated Permeability Surface are product (PS) is=171+8 l·min.^–1·g^–1. A rabbit polyclonal antiserum to a synthetic peptide encoding the 13 C-terminal amino acids of the human erythrocyte glucose transporter immunocytochemically confirmed the presence of the GLUT1 isoform in non-obese diabetic (NOD) mouse brain capillary endothelia. These studies indicate that a down-regulation of BBB glucose transport occurs in these spontaneously hyperglycemic mice; both BBB glucose permeability (as indicated by PS product) and transporter maximal velocity are reduced (in comparison to normoglycemic CD-1 mice), but the half-saturation constant remains unchanged.     

NKG2D-RAE-1 receptor-ligand variation does not account for the NK cell defect in nonobese diabetic mice

NK cells from NOD mice induced with poly(I:C) in vivo exhibit low cytotoxicity against a range of target cells, but the genetic mechanisms controlling this defect are yet to be elucidated. Defects in the expression of NKG2D and its ligands, the RAE-1 molecules, have been hypothesized to contribute to the reduced NK function present in NOD mice. In this study, we show that segregation of the NK-mediated killing phenotype did not correlate with the NOD Raet1 haplotype and that the large alterations in NKG2D expression previously reported on NK cells expanded in vitro were not observed in primary, poly(I:C)-elicited NK cells in vivo. Additional studies indicate a complex genetic control of defective NOD NK cells including genes linked to the MHC and possibly those that are associated with an altered cytokine response to the TLR3-agonist poly(I:C).     

Metabolic flexibility is conserved in diabetic myotubes

The purpose of this study was to test the hypothesis that metabolic inflexibility is an intrinsic defect. Glucose and lipid oxidation were studied in human myotubes established from healthy lean and obese subjects and patients with type 2 diabetes (T2D). In lean myotubes, glucose oxidation is raised by increasing glucose concentrations (0-20 mmol/l) and acute insulin stimulation (P < 0.05), whereas it is inhibited by palmitate (PA). PA oxidation is raised by increasing PA concentrations (0-0.6 mmol/l), whereas 1.0 mmol/l PA inhibits its own oxidation (P < 0.05). Furthermore, PA oxidation is increased by acute insulin stimulation (P < 0.05) and inhibited by glucose. Even 0.05 mM PA and 2.5 mM glucose significantly reduce glucose and PA oxidation (P < 0.05), respectively. Glucose and PA oxidation are insulin-sensitive in myotubes established from lean (46% and 17% glucose and PA oxidation, respectively; P < 0.05 vs. basal), obese (31% and 14%; P < 0.05), and T2D (17% and 8%; P < 0.05) subjects. PA supplementation reduces both basal and insulin-stimulated glucose oxidation by 33-44% (P < 0.05), and myotubes are still insulin-sensitive in all three groups (P < 0.05). Therefore, the metabolic inflexibility described in obese and diabetic patients is not an intrinsic defect; rather, it is based on an extramuscular mechanism (i.e., the inability to vary extracellular fatty acid concentrations during insulin stimulation). Thus, skeletal muscles are metabolic-flexible per se.     

Plant growth enhancement is not a conserved feature in the Caulobacter genus

Aims

Species within the Caulobacter genus have been termed ‘hub species’ in the plant microbiome. To understand these interactions, we assessed the interactions between several Caulobacter strains and a common host plant.

Methods

We identified a set of 11 Caulobacter strains that range in genetic diversity and tested them for their ability to increase the growth of Arabidopsis thaliana. In addition, biochemical assays were employed to determine if these Caulobacter strains produce common plant growth promoting (PGP) biosynthates. To identify potential PGP-related genes, genomic analyses were performed to compare the genomes of PGP Caulobacter strains to those of non-PGP Caulobacter strains.

Results

For the PGP Caulobacter strains, we observed that common PGP biosynthates did not contribute to the observed Caulobacter-mediated plant growth stimulation. Genomic analyses suggested that the genomes of PGP strains maintain similar metabolic pathways compared to those of non-PGP strains, and that common genes related to PGP factors do not explain the PGP mechanisms for the Caulobacter strains we analyzed.

Conclusions

Plant growth enhancement is not a conserved feature in the Caulobacter genus, and some Caulobacter strains even inhibit plant growth. Moreover, common PGP factors do not fully explain Caulobacter-mediated plant growth enhancement.

     

Mycobacterium indicus pranii supernatant induces apoptotic cell death in mouse peritoneal macrophages in vitro

Mycobacterium indicus pranii (MIP), also known as Mw, is a saprophytic, non-pathogenic strain of Mycobacterium and is commercially available as a heat-killed vaccine for leprosy and recently tuberculosis (TB) as part of MDT. In this study we provide evidence that cell-free supernatant collected from original MIP suspension induces rapid and enhanced apoptosis in mouse peritoneal macrophages in vitro. It is demonstrated that the MIP cell-free supernatant induced apoptosis is mitochondria-mediated and caspase independent and involves mitochondrial translocation of Bax and subsequent release of AIF and cytochrome c from the mitochondria. Experiments with pharmacological inhibitors suggest a possible role of PKC in mitochondria-mediated apoptosis of macrophages.     

Viruses activate a genetically conserved cell death pathway in a unicellular organism

Given the importance of apoptosis in the pathogenesis of virus infections in mammals, we investigated the possibility that unicellular organisms also respond to viral pathogens by activating programmed cell death. The M1 and M2 killer viruses of Saccharomyces cerevisiae encode pore-forming toxins that were assumed to kill uninfected yeast cells by a nonprogrammed assault. However, we found that yeast persistently infected with these killer viruses induce a programmed suicide pathway in uninfected (nonself) yeast. The M1 virus-encoded K1 toxin is primarily but not solely responsible for triggering the death pathway. Cell death is mediated by the mitochondrial fission factor Dnm1/Drp1, the K+ channel Tok1, and the yeast metacaspase Yca1/Mca1 encoded by the target cell and conserved in mammals. In contrast, cell death is inhibited by yeast Fis1, a pore-forming outer mitochondrial membrane protein. This virus-host relationship in yeast resembles that of pathogenic human viruses that persist in their infected host cells but trigger programmed death of uninfected cells.     

An overhanging 3'' terminus is a conserved feature of telomeres.

The reactivity of single-stranded thymidines with osmium tetraoxide was used to demonstrate the existence of a terminal overhang of the G-rich strand of telomeres from two distantly related eucaryotes, the ciliated protozoan Tetrahymena spp. and the acellular slime mold Didymium spp. Conservation of a G-strand overhang at the molecular terminus of telomeres is consistent with our suggestion that an unusual DNA structure formed by the G-strand overhang is important for telomere function (E. Henderson, C. C. Hardin, S. K. Wolk, I. Tinoco Jr., and E. H. Blackburn, Cell 51:899-908, 1987).     

CD8+ T cell tolerance in nonobese diabetic mice is restored by insulin-dependent diabetes resistance alleles

Although candidate genes controlling autoimmune disease can now be identified, a major challenge that remains is defining the resulting cellular events mediated by each locus. In the current study we have used NOD-InsHA transgenic mice that express the influenza hemagglutinin (HA) as an islet Ag to compare the fate of HA-specific CD8+ T cells in diabetes susceptible NOD-InsHA mice with that observed in diabetes-resistant congenic mice having protective alleles at insulin-dependent diabetes (Idd) 3, Idd5.1, and Idd5.2 (Idd3/5 strain) or at Idd9.1, Idd9.2, and Idd9.3 (Idd9 strain). We demonstrate that protection from diabetes in each case is correlated with functional tolerance of endogenous islet-specific CD8+ T cells. However, by following the fate of naive, CFSE-labeled, islet Ag-specific CD8+ (HA-specific clone-4) or CD4+ (BDC2.5) T cells, we observed that tolerance is achieved differently in each protected strain. In Idd3/5 mice, tolerance occurs during the initial activation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes where CD25+ regulatory T cells (Tregs) effectively prevent their accumulation. In contrast, resistance alleles in Idd9 mice do not prevent the accumulation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes, indicating that tolerance occurs at a later checkpoint. These results underscore the variety of ways that autoimmunity can be prevented and identify the elimination of islet-specific CD8+ T cells as a common indicator of high-level protection.     

Adoptive T cell transfer of autoimmune nonobese diabetic mouse diabetes does not require recruitment of host B lymphocytes

The autoimmune nonobese diabetic mouse, a model of human juvenile type I diabetes mellitus, exhibits features of both B and T cell autoreactivity against insulin-producing cells. Using the neonatal cell transfer model of the disease, which we have described previously, we have shown that B cell suppression of newborn recipients by anti-mu treatment did not affect the transfer of diabetes by means of T cells. B cell-depleted, purified T cells from diabetic adults were injected into newborns treated with either IR-52, a control rat myeloma protein, or LOMM.9, a rat anti-mouse mu-chain mAb. Both groups developed diabetes over a similar time scale. Although the pancreases in both groups showed massive infiltration by T lymphocytes, B lymphocytes, presumably recruited in the host, were present in the IR-52-treated group, whereas they were absent in the LOMM.9-treated group. Anti-mu-treated diabetic animals showed substantial B cell suppression in vivo and in vitro when compared with IR-52-treated controls. These results suggest that B cell autoreactivity is a secondary phenomenon that is unimportant during the effector phase of diabetes in nonobese diabetic mice.     

Down syndrome cell adhesion molecule is conserved in mouse and highly expressed in the adult mouse brain

Down Syndrome (DS) is a major cause of mental retardation and is associated with characteristic well-defined although subtle brain abnormalities, many of which arise after birth, with particular defects in the cortex, hippocampus and cerebellum. The neural cell adhesion molecule DSCAM (Down syndrome cell adhesion molecule) maps to 21q22.2-->q22.3, a region associated with DS mental retardation, and is expressed largely in the neurons of the central and peripheral nervous systems during development. In order to evaluate the contribution of DSCAM to postnatal morphogenetic and cognitive processes, we have analyzed the expression of the mouse DSCAM homolog, Dscam, in the adult mouse brain from 1 through 21 months of age. We have found that Dscam is widely expressed in the brain throughout adult life, with strongest levels in the cortex, the mitral and granular layers of the olfactory bulb, the granule cells of the dentate gyrus and the pyramidal cells of the CA1, CA2 and CA3 regions, the ventroposterior lateral nuclei of the thalamus, and in the Purkinje cells of the cerebellum. Dscam is also expressed ventrally in the adult spinal cord. Given the homology of DSCAM to cell adhesion molecules involved in development and synaptic plasticity, and its demonstrated role in axon guidance, we propose that DSCAM overexpression contributes not only to the structural defects seen in these regions of the DS brain, but also to the defects of learning and memory seen in adults with DS.