A major histocompatibility complex class II restriction for BioBreeding/Worcester diabetes-inducing T cells

Inbred diabetes-prone (DP) BioBreeding/Worcester (BB/Wor) (RT1u) rats develop spontaneous autoimmune diabetes, which, like human insulin-dependent diabetes mellitus, is mediated by autoreactive T lymphocytes. Breeding studies have shown an absolute requirement for at least one copy of the major histocompatibility complex (MHC) RT1u haplotype for spontaneous diabetes expression. Concanavalin A-activated spleen cells from acutely diabetic DP rats adoptively transfer diabetes only to recipients that express at least one RT1u haplotype. To investigate the basis for the MHC requirement in BB/Wor autoimmunity, diabetes-inducing T cell lines were derived from the spleens of acutely diabetic DP rats. Upon activation in vitro with islet cells, the T cell lines adoptively transfer insulitis and diabetes into young DP recipients and non-diabetes-prone RT1 congenic rat strains that are class IIu. Recipients that are RT1u at only the class I A or C locus, but not at the class II B/D loci, do not develop diabetes after T cell transfer. The adoptive transfer of diabetes by Concanavalin A-activated diabetic DP spleen cells also requires that donor and recipient share class II B/Du gene products. Furthermore, the adoptive transfer of diabetes into MHC class IIu congenic rats is independent of the class I haplotype; i.e., it occurs in the presence of class I Aa Cu or Au Ca gene products. BB/Wor T cells can be activated in vitro for the transfer of diabetes with islet cell antigens and class II-positive but not class IIu-negative antigen-presenting cells. The inductive phase of BB diabetes is therefore MHC class II restricted, and this appears to operate at the level of interaction between inducing T cells and class IIu antigen-presenting cells. These results may explain the well-documented, but not yet understood, MHC class II genetic contribution to insulin-dependent diabetes mellitus pathogenesis, and they may facilitate the development of protocols designed to prevent diabetes onset in susceptible individuals

Barriers to Improving Primary Care of Depression: Perspectives of Medical Group Leaders

Using clinical trials, researchers have demonstrated effective methods for treating depression in primary care, but improvements based on these trials are not being implemented. This might be because these improvements require more systematic organizational changes than can be made by individual physicians. We interviewed 82 physicians and administrative leaders of 41 medical groups to learn what is preventing those organizational changes. The identified barriers to improving care included external contextual problems (reimbursement, scarce resources, and access to/communication with specialty mental health), individual attitudes (physician and patient resistance), and internal care process barriers (organizational and condition complexity, difficulty standardizing and measuring care). Although many of these barriers are challenging, we can overcome them by setting clear priorities for change and allocating adequate resources. We must improve primary care of depression if we are to reduce its enormous adverse social and economic impacts

Bitopic binding mode of an M1 muscarinic acetylcholine receptor agonist associated with adverse clinical trial outcomes

The realisation of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (M1 mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702, described previously as a potent M1 receptor allosteric agonist, which showed pro-cognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side-effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702 together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. We conclude that these properties, whilst imparting beneficial effects on learning and memory, are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data supports the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses

m-Calpain is required for preimplantation embryonic development in mice

BACKGROUND: μ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4(-/- )mice. RESULTS: To distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage. CONCLUSION: We conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that μ-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo

Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway

BACKGROUND: Angiogenesis is crucial to many physiological and pathological processes including development and cancer cell survival. Vascular endothelial growth factor-A (VEGFA) is the predominant mediator of angiogenesis in the VEGF family. During development, adverse environmental conditions like nutrient deprivation, hypoxia and increased protein secretion occur. IRE1alpha, PERK, and ATF6alpha, master regulators of the unfolded protein response (UPR), are activated under these conditions and are proposed to have a role in mediating angiogenesis. PRINCIPAL FINDINGS: Here we show that IRE1alpha, PERK, and ATF6alpha powerfully regulate VEGFA mRNA expression under various stress conditions. In Ire1alpha(-/-) and Perk(-/-) mouse embryonic fibroblasts and ATF6alpha-knockdown HepG2 cells, induction of VEGFA mRNA by endoplasmic reticulum stress is attenuated as compared to control cells. Embryonic lethality of Ire1alpha-/- mice is due to the lack of VEGFA induction in labyrinthine trophoblast cells of the developing placenta. Rescue of IRE1alpha and PERK in Ire1alpha(-/-) and Perk(-/-) cells respectively, prevents VEGFA mRNA attenuation. We further report that the induction of VEGFA by IRE1alpha, PERK and ATF6 involves activation of transcription factors, spliced-XBP-1, ATF4 and cleaved ATF6 respectively. CONCLUSIONS/SIGNIFICANCE: Our results reveal that the IRE1alpha-XBP-1, PERK-ATF4, and ATF6alpha pathways constitute novel upstream regulatory pathways of angiogenesis by modulating VEGF transcription. Activation of these pathways helps the rapidly growing cells to obtain sufficient nutrients and growth factors for their survival under the prevailing hostile environmental conditions. These results establish an important role of the UPR in angiogenesis

Sufficient conditions for two-dimensional localization by arbitrarily weak defects in periodic potentials with band gaps

We prove, via an elementary variational method, 1d and 2d localization within the band gaps of a periodic Schrodinger operator for any mostly negative or mostly positive defect potential, V, whose depth is not too great compared to the size of the gap. In a similar way, we also prove sufficient conditions for 1d and 2d localization below the ground state of such an operator. Furthermore, we extend our results to 1d and 2d localization in d dimensions; for example, a linear or planar defect in a 3d crystal. For the case of D-fold degenerate band edges, we also give sufficient conditions for localization of up to D states.Comment: 9 pages, 3 figure

The Armadillo Repeat Protein PF16 Is Essential for Flagellar Structure and Function in Plasmodium Male Gametes

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world\u27s population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite\u27s flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite