Genetic and functional data identifying Cd101 as a type 1 diabetes (T1D) susceptibility gene in nonobese diabetic (NOD) mice.

Type 1 diabetes (T1D) is a chronic multi-factorial disorder characterized by the immune-mediated destruction of insulin-producing pancreatic beta cells. Variations at a large number of genes influence susceptibility to spontaneous autoimmune T1D in non-obese diabetic (NOD) mice, one of the most frequently studied animal models for human disease. The genetic analysis of these mice allowed the identification of many insulin-dependent diabetes (Idd) loci and candidate genes, one of them being Cd101. CD101 is a heavily glycosylated transmembrane molecule which exhibits negative-costimulatory functions and promotes regulatory T (Treg) function. It is abundantly expressed on subsets of lymphoid and myeloid cells, particularly within the gastrointestinal tract. We have recently reported that the genotype-dependent expression of CD101 correlates with a decreased susceptibility to T1D in NOD.B6 Idd10 congenic mice compared to parental NOD controls. Here we show that the knockout of CD101 within the introgressed B6-derived Idd10 region increased T1D frequency to that of the NOD strain. This loss of protection from T1D was paralleled by decreased Gr1-expressing myeloid cells and FoxP3+ Tregs and an enhanced accumulation of CD4-positive over CD8-positive T lymphocytes in pancreatic tissues. As compared to CD101+/+ NOD.B6 Idd10 donors, adoptive T cell transfers from CD101-/- NOD.B6 Idd10 mice increased T1D frequency in lymphopenic NOD scid and NOD.B6 Idd10 scid recipients. Increased T1D frequency correlated with a more rapid expansion of the transferred CD101-/- T cells and a lower proportion of recipient Gr1-expressing myeloid cells in the pancreatic lymph nodes. Fewer of the Gr1+ cells in the recipients receiving CD101-/- T cells expressed CD101 and the cells had lower levels of IL-10 and TGF-ß mRNA. Thus, our results connect the Cd101 haplotype-dependent protection from T1D to an anti-diabetogenic function of CD101-expressing Tregs and Gr1-positive myeloid cells and confirm the identity of Cd101 as Idd10.

Ptpn22 and Cd2 Variations Are Associated with Altered Protein Expression and Susceptibility to Type 1 Diabetes in Nonobese Diabetic Mice.

By congenic strain mapping using autoimmune NOD.C57BL/6J congenic mice, we demonstrated previously that the type 1 diabetes (T1D) protection associated with the insulin-dependent diabetes (Idd)10 locus on chromosome 3, originally identified by linkage analysis, was in fact due to three closely linked Idd loci: Idd10, Idd18.1, and Idd18.3. In this study, we define two additional Idd loci--Idd18.2 and Idd18.4--within the boundaries of this cluster of disease-associated genes. Idd18.2 is 1.31 Mb and contains 18 genes, including Ptpn22, which encodes a phosphatase that negatively regulates T and B cell signaling. The human ortholog of Ptpn22, PTPN22, is associated with numerous autoimmune diseases, including T1D. We, therefore, assessed Ptpn22 as a candidate for Idd18.2; resequencing of the NOD Ptpn22 allele revealed 183 single nucleotide polymorphisms with the C57BL/6J (B6) allele--6 exonic and 177 intronic. Functional studies showed higher expression of full-length Ptpn22 RNA and protein, and decreased TCR signaling in congenic strains with B6-derived Idd18.2 susceptibility alleles. The 953-kb Idd18.4 locus contains eight genes, including the candidate Cd2. The CD2 pathway is associated with the human autoimmune disease, multiple sclerosis, and mice with NOD-derived susceptibility alleles at Idd18.4 have lower CD2 expression on B cells. Furthermore, we observed that susceptibility alleles at Idd18.2 can mask the protection provided by Idd10/Cd101 or Idd18.1/Vav3 and Idd18.3. In summary, we describe two new T1D loci, Idd18.2 and Idd18.4, candidate genes within each region, and demonstrate the complex nature of genetic interactions underlying the development of T1D in the NOD mouse model.

High levels of self-reported prescription opioid use by HIV-positive individuals.

Prescription medication use (other than antiretroviral therapy (ART)) is highly prevalent among people living with HIV. Prescription medications may be used medically or non-medically: non-medical use includes using more medication than prescribed, using medication prescribed to someone else, or using medication for a purpose other than its prescribed use. During 12 weeks in 2014-2015, we characterized medical and non-medical prescription medication use among HIV-positive patients attending an academic medical center (n = 149) and a community clinic (n = 105). Separately for the past year and the past month, these 254 participants self-reported their use of prescription opioids, sedatives, stimulants, anti-anxiety medications, antipsychotic medications, and erectile dysfunction medications. Respondents were largely male (91%), aged 40 or older (61%), identified as gay or bisexual (79%), and were men who have sex with men (85%). ART use was nearly universal (95%). Nearly half (43%) of participants reported medical use of prescription opioids; 11% of the opioid use was reported as non-medical use. Anti-anxiety medication use was also frequent, and differed by site: 41% of community-clinic responders reported medical use of anti-anxiety medications compared to 23% of hospital clinic respondents who reported medical use. Prescription sedative use was also approximately twice as high among community-clinic participants, with medical use reported by 43% of respondents and non-medical use by 12%; in comparison, at the hospital clinic, sedative use was reported by 18% (medical) and 7% (non-medical) of participants. Stimulant use was rare in both sites. No demographic characteristic was significantly associated with medical or non-medical use of any prescription medication. The current focus of many studies on only non-medical prescription medication use not only underestimates the widespread exposure of HIV-positive individuals to these drugs, but may also underestimate potential adverse effects of prescription medications in this population.

Investigation of soluble and transmembrane CTLA-4 isoforms in serum and microvesicles.

Expression of the CTLA-4 gene is absolutely required for immune homeostasis, but aspects of its molecular nature remain undefined. In particular, the characterization of the soluble CTLA-4 (sCTLA-4) protein isoform generated by an alternatively spliced mRNA of CTLA4 lacking transmembrane-encoding exon 3 has been hindered by the difficulty in distinguishing it from the transmembrane isoform of CTLA-4, Tm-CTLA-4. In the current study, sCTLA-4 has been analyzed using novel mAbs and polyclonal Abs specific for its unique C-terminal amino acid sequence. We demonstrate that the sCTLA-4 protein is secreted at low levels following the activation of primary human CD4(+) T cells and is increased only rarely in the serum of autoimmune patients. Unexpectedly, during our studies aimed to define the kinetics of sCTLA-4 produced by activated human CD4(+) T cells, we discovered that Tm-CTLA-4 is associated with microvesicles produced by the activated cells. The functional roles of sCTLA-4 and microvesicle-associated Tm-CTLA-4 warrant further investigation, especially as they relate to the multiple mechanisms of action described for the more commonly studied cell-associated Tm-CTLA-4.

Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity.

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately twofold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we show that engineered haplodeficiency of Il2 gene expression not only reduces T cell IL-2 production by twofold but also mimics the autoimmune dysregulatory effects of the naturally occurring susceptibility alleles of Il2. Reduced IL-2 production achieved by either genetic mechanism correlates with reduced function of CD4(+) CD25(+) regulatory T cells, which are critical for maintaining immune homeostasis.

Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells.

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.

Identification of Cd101 as a susceptibility gene for Novosphingobium aromaticivorans-induced liver autoimmunity.

Environmental and genetic factors define the susceptibility of an individual to autoimmune disease. Although common genetic pathways affect general immunological tolerance mechanisms in autoimmunity, the effects of such genes could vary under distinct immune challenges within different tissues. In this study, we demonstrate this by observing that autoimmune type 1 diabetes-protective haplotypes at the insulin-dependent diabetes susceptibility region 10 (Idd10) introgressed from chromosome 3 of C57BL/6 (B6) and A/J mice onto the NOD background increase the severity of autoimmune primary biliary cirrhosis induced by infection with Novosphingobium aromaticivorans, a ubiquitous alphaproteobacterium, when compared with mice having the NOD and NOD.CAST Idd10 type 1 diabetes-susceptible haplotypes. Substantially increased liver pathology in mice having the B6 and A/J Idd10 haplotypes correlates with reduced expression of CD101 on dendritic cells, macrophages, and granulocytes following infection, delayed clearance of N. aromaticivorans, and the promotion of overzealous IFN-γ- and IL-17-dominated T cell responses essential for the adoptive transfer of liver lesions. CD101-knockout mice generated on the B6 background also exhibit substantially more severe N. aromaticivorans-induced liver disease correlating with increased IFN-γ and IL-17 responses compared with wild-type mice. These data strongly support the hypothesis that allelic variation of the Cd101 gene, located in the Idd10 region, alters the severity of liver autoimmunity induced by N. aromaticivorans.

Evidence that Cd101 is an autoimmune diabetes gene in nonobese diabetic mice.

We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.

Experience of a systematic approach to care and prevention of fragility fractures in New Zealand.

This narrative review describes efforts to improve the care and prevention of fragility fractures in New Zealand from 2012 to 2022. This includes development of clinical standards and registries to benchmark provision of care, and public awareness campaigns to promote a life-course approach to bone health. PURPOSE: This review describes the development and implementation of a systematic approach to care and prevention for New Zealanders with fragility fractures, and those at high risk of first fracture. Progression of existing initiatives and introduction of new initiatives are proposed for the period 2022 to 2030. METHODS: In 2012, Osteoporosis New Zealand developed and published a strategy with objectives relating to people who sustain hip and other fragility fractures, those at high risk of first fragility fracture or falls and all older people. The strategy also advocated formation of a national fragility fracture alliance to expedite change. RESULTS: In 2017, a previously informal national alliance was formalised under the Live Stronger for Longer programme, which includes stakeholder organisations from relevant sectors, including government, healthcare professionals, charities and the health system. Outputs of this alliance include development of Australian and New Zealand clinical guidelines, clinical standards and quality indicators and a bi-national registry that underpins efforts to improve hip fracture care. All 22 hospitals in New Zealand that operate on hip fracture patients currently submit data to the registry. An analogous approach is ongoing to improve secondary fracture prevention for people who sustain fragility fractures at other sites through nationwide access to Fracture Liaison Services. CONCLUSION: Widespread participation in national registries is enabling benchmarking against clinical standards as a means to improve the care of hip and other fragility fractures in New Zealand. An ongoing quality improvement programme is focused on eliminating unwarranted variation in delivery of secondary fracture prevention.

Expression of diabetes-associated genes by dendritic cells and CD4 T cells drives the loss of tolerance in nonobese diabetic mice.

In humans and NOD mice, defects in immune tolerance result in the spontaneous development of type-1-diabetes. Recent studies have ascribed a breakdown in tolerance to dysfunction in regulatory T cells that is secondary to reduced IL-2 production by T cells having the NOD diabetes susceptibility region insulin-dependent diabetes 3 (Idd3). In this study, we demonstrate a peripheral tolerance defect in the dendritic cells of NOD mice that is independent of regulatory T cells. NOD CD8 T cells specific for islet Ags fail to undergo deletion in the pancreatic lymph nodes. Deletion was promoted by expression of the protective alleles of both Idd3 (Il2) and Idd5 in dendritic cells. We further identify a second tolerance defect that involves endogenous CD4 T cell expression of the disease-promoting NOD alleles of these genetic regions. Pervasive insulitis can be reduced by expression of the Idd3 and Idd5 protective alleles by either the Ag-presenting cell or lymphocytes.

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).

Use of Antiretroviral Therapy During Pregnancy and Adverse Birth Outcomes Among Women Living With HIV-1 in Low- and Middle-Income Countries: A Systematic Review.

BACKGROUND: Worldwide, nearly 18 million women of reproductive age are living with HIV-1. Although increased access to antiretroviral therapy (ART) during pregnancy has significantly reduced HIV-1 mother-to-child transmission (MTCT), a similarly robust reduction in preterm birth (PTB) and low birthweight (LBW) among infants born to women living with HIV has not been observed. This study was designed to identify associations between classes of ART regimens and risk of PTB or LBW. SETTING: Low- and middle-income countries. METHODS: We conducted a systematic review of randomized and observational studies that assessed the effect of ART regimen on the risk of PTB (≤37 completed weeks of gestation) or LBW (<2500 g at birth) among pregnant women in low- and middle-income countries living with HIV-1. We searched Medline, COCHRANE, Web of Science, SCOPUS, and CPCI-S for included studies. RESULTS: When compared to monotherapy, both nonnucleoside reverse transcriptase inhibitor- and protease inhibitor-based regimens had a consistent, harmful association with LBW. There is mixed evidence suggesting both potential harm and potential benefit for most other regimens on risk of LBW and PTB, and the harmful or protective effects of certain regimens varies depending on the drug backbone. CONCLUSIONS: Although the benefits of ART during pregnancy for prevention of MTCT are undisputed, this systematic review indicates that ART regimens vary substantially in their association with LBW and PTB. Although challenging, optimization of ART regimens could simultaneously promote maternal health, prevent MTCT, and also minimize risks of PTB and LBW.

Natural genetic variants influencing type 1 diabetes in humans and in the NOD mouse.

The understanding of the genetic basis of type 1 diabetes and other autoimmune diseases and the application of that knowledge to their treatment, cure and eventual prevention has been a difficult goal to reach. Cumulative progress in both mouse and human are finally giving way to some successes and significant insights have been made in the last few years. Investigators have identified key immune tolerance-associated phenotypes in convincingly reliable ways that are regulated by specific diabetes-associated chromosomal intervals. The combination of positional genetics and functional studies is a powerful approach to the identification of downstream molecular events that are causal in disease aetiology. In the case of type 1 diabetes, the availability of several animal models, especially the NOD mouse, has complemented the efforts to localize human genes causing diabetes and has shown that some of the same genes and pathways are associated with autoimmunity in both species. There is also growing evidence that the initiation or progression of many autoimmune diseases is likely to be influenced by some of the same genes.

Type 1 diabetes genes and pathways shared by humans and NOD mice.

The identification of causative genes for the autoimmune disease type 1 diabetes (T1D) in humans and candidate genes in the NOD mouse has made significant progress in recent years. In addition to sharing structural aspects of the MHC class II molecules that confer susceptibility or resistance to T1D, genes and pathways contributing to autoimmune pathogenesis are held in common by the two species. There are data demonstrating a similar need to establish central tolerance to insulin. Gene variants for the interacting molecules IL2 and CD25, members of a pathway that is essential for immune homeostasis, are present in mice and humans, respectively. Variation of two molecules that negatively regulate T cells, CTLA-4 and the tyrosine phosphatase LYP/PEP, are associated with susceptibility to human and NOD T1D. These observations underscore the value of the NOD mouse model for mechanistic studies on human T1D-associated molecular and cellular pathways.

Obesity: impediment to postsurgical wound healing.

PURPOSE: To provide physicians and nurses with an overview of the impact of obesity on postoperative wound healing and how preplanning protocols can minimize skin and wound care problems in this patient population. TARGET AUDIENCE: This continuing education activity is intended for physicians and nurses with an interest in reducing skin and wound care problems in their patients who are obese. OBJECTIVES: After reading the article and taking the test, the participant will be able to: 1. Identify obesity-related changes in body systems and how these impede wound healing. 2. Identify complications of postoperative wound healing in obese patients and the assessments and intervention strategies that can reduce these complications. 3. Identify skin and wound care considerations for obese patients and the role of preplanning protocols in avoiding problems.

Obesity: impediment to wound healing.

Intrinsic and extrinsic factors affect wound healing. High risk factors for the obese patients include infection, seromas, anastomatic leaks, and incision dehiscence. Tissue perfusion is an issue of great concern and is a key factor in most assessments. Obesity adds another dimension to the needs of the patient and presents challenges to nurses. From routine evaluations to specialized assessments with attention to bariatric equipment needs, a thorough understanding of wound healing and potential problems of obese patients, and knowledge of interventions is needed. Nonjudgmental attitudes are imperative in planning care for the obese patients. Following a review of physiological needs and nursing interventions, a case study details one woman's surgical complications.

Fine mapping, gene content, comparative sequencing, and expression analyses support Ctla4 and Nramp1 as candidates for Idd5.1 and Idd5.2 in the nonobese diabetic mouse.

At least two loci that determine susceptibility to type 1 diabetes in the NOD mouse have been mapped to chromosome 1, Idd5.1 (insulin-dependent diabetes 5.1) and Idd5.2. In this study, using a series of novel NOD.B10 congenic strains, Idd5.1 has been defined to a 2.1-Mb region containing only four genes, Ctla4, Icos, Als2cr19, and Nrp2 (neuropilin-2), thereby excluding a major candidate gene, Cd28. Genomic sequence comparison of the two functional candidate genes, Ctla4 and Icos, from the B6 (resistant at Idd5.1) and the NOD (susceptible at Idd5.1) strains revealed 62 single nucleotide polymorphisms (SNPs), only two of which were in coding regions. One of these coding SNPs, base 77 of Ctla4 exon 2, is a synonymous SNP and has been correlated previously with type 1 diabetes susceptibility and differential expression of a CTLA-4 isoform. Additional expression studies in this work support the hypothesis that this SNP in exon 2 is the genetic variation causing the biological effects of Idd5.1. Analysis of additional congenic strains has also localized Idd5.2 to a small region (1.52 Mb) of chromosome 1, but in contrast to the Idd5.1 interval, Idd5.2 contains at least 45 genes. Notably, the Idd5.2 region still includes the functionally polymorphic Nramp1 gene. Future experiments to test the identity of Idd5.1 and Idd5.2 as Ctla4 and Nramp1, respectively, can now be justified using approaches to specifically alter or mimic the candidate causative SNPs.