Anti-Insulin B Cells Are Poised for Antigen Presentation in Type 1 Diabetes.

Early breaches in B cell tolerance are central to type 1 diabetes progression in mouse and man. Conventional BCR transgenic mouse models (VH125.Tg NOD) reveal the power of B cell specificity to drive disease as APCs. However, in conventional fixed IgM models, comprehensive assessment of B cell development is limited. To provide more accurate insight into the developmental and functional fates of anti-insulin B cells, we generated a new NOD model (VH125SDNOD) in which anti-insulin VDJH125 is targeted to the IgH chain locus to generate a small (1-2%) population of class switch-competent insulin-binding B cells. Tracking of this rare population in a polyclonal repertoire reveals that anti-insulin B cells are preferentially skewed into marginal zone and late transitional subsets known to have increased sensitivity to proinflammatory signals. Additionally, IL-10 production, characteristic of regulatory B cell subsets, is increased. In contrast to conventional models, class switch-competent anti-insulin B cells proliferate normally in response to mitogenic stimuli but remain functionally silent for insulin autoantibody production. Diabetes development is accelerated, which demonstrates the power of anti-insulin B cells to exacerbate disease without differentiation into Ab-forming or plasma cells. Autoreactive T cell responses in VH125SDNOD mice are not restricted to insulin autoantigens, as evidenced by increased IFN-γ production to a broad array of diabetes-associated epitopes. Together, these results independently validate the pathogenic role of anti-insulin B cells in type 1 diabetes, underscore their diverse developmental fates, and demonstrate the pathologic potential of coupling a critical ß cell specificity to predominantly proinflammatory Ag-presenting B cell subsets.

Long-Term Outcomes of Elagolix in Women With Endometriosis: Results From Two Extension Studies.

OBJECTIVE: To evaluate the efficacy and safety of elagolix, an oral, nonpeptide gonadotropin-releasing hormone antagonist, over 12 months in women with endometriosis-associated pain. METHODS: Elaris Endometriosis (EM)-III and -IV were extension studies that evaluated an additional 6 months of treatment after two 6-month, double-blind, placebo-controlled phase 3 trials (12 continuous treatment months) with two elagolix doses (150 mg once daily and 200 mg twice daily). Coprimary efficacy endpoints were the proportion of responders (clinically meaningful pain reduction and stable or decreased rescue analgesic use) based on average monthly dysmenorrhea and nonmenstrual pelvic pain scores. Safety assessments included adverse events, clinical laboratory tests, and endometrial and bone mineral density assessments. The power of Elaris EM-III and -IV was based on the comparison to placebo in Elaris EM-I and -II with an expected 25% dropout rate. RESULTS: Between December 28, 2012, and October 31, 2014 (Elaris EM-III), and between May 27, 2014, and January 6, 2016 (Elaris EM-IV), 569 participants were enrolled. After 12 months of treatment, Elaris EM-III responder rates for dysmenorrhea were 52.1% at 150 mg once daily (Elaris EM-IV 550.8%) and 78.2% at 200 mg twice daily (Elaris EMIV 575.9%). Elaris EM-III nonmenstrual pelvic pain responder rates were 67.5% at 150 mg once daily (Elaris EM-IV 566.4%) and 69.1% at 200 mg twice daily (Elaris EM-IV 567.2%)."After 12 months of treatment, Elaris EM-III dyspareunia responder rates were 45.2% at 150 mg once daily (Elaris EM-IV=45.9%) and 60.0% at 200 mg twice daily (Elaris EM-IV=58.1%). Hot flush was the most common adverse event. Decreases from baseline in bone mineral density and increases from baseline in lipids were observed after 12 months of treatment. There were no adverse endometrial findings. CONCLUSION: Long-term elagolix treatment provided sustained reductions in dysmenorrhea, nonmenstrual pelvic pain, and dyspareunia. The safety was consistent with reduced estrogen levels and no new safety concerns were associated with long-term elagolix use. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT01760954 and NCT02143713.

B Cell-Intrinsic mTORC1 Promotes Germinal Center-Defining Transcription Factor Gene Expression, Somatic Hypermutation, and Memory B Cell Generation in Humoral Immunity.

B lymphocytes migrate among varied microenvironmental niches during diversification, selection, and conversion to memory or Ab-secreting plasma cells. Aspects of the nutrient milieu differ within these lymphoid microenvironments and can influence signaling molecules such as the mechanistic target of rapamycin (mTOR). However, much remains to be elucidated as to the B cell-intrinsic functions of nutrient-sensing signal transducers that modulate B cell differentiation or Ab affinity. We now show that the amino acid-sensing mTOR complex 1 (mTORC1) is vital for induction of Bcl6-a key transcriptional regulator of the germinal center (GC) fate-in activated B lymphocytes. Accordingly, disruption of mTORC1 after B cell development and activation led to reduced populations of Ag-specific memory B cells as well as plasma cells and GC B cells. In addition, induction of the germ line transcript that guides activation-induced deaminase in selection of the IgG1 H chain region during class switching required mTORC1. Expression of the somatic mutator activation-induced deaminase was reduced by a lack of mTORC1 in B cells, whereas point mutation frequencies in Ag-specific GC-phenotype B cells were only halved. These effects culminated in a B cell-intrinsic defect that impacted an antiviral Ab response and drastically impaired generation of high-affinity IgG1. Collectively, these data establish that mTORC1 governs critical B cell-intrinsic mechanisms essential for establishment of GC differentiation and effective Ab production.

Somatic mosaicism of an intragenic FANCB duplication in both fibroblast and peripheral blood cells observed in a Fanconi anemia patient leads to milder phenotype.

BACKGROUND: Fanconi anemia (FA) is a rare disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer. Patients harboring X-linked FANCB pathogenic variants usually present with severe congenital malformations resembling VACTERL syndrome with hydrocephalus. METHODS: We employed the diepoxybutane (DEB) test for FA diagnosis, arrayCGH for detection of duplication, targeted capture and next-gen sequencing for defining the duplication breakpoint, PacBio sequencing of full-length FANCB aberrant transcript, FANCD2 ubiquitination and foci formation assays for the evaluation of FANCB protein function by viral transduction of FANCB-null cells with lentiviral FANCB WT and mutant expression constructs, and droplet digital PCR for quantitation of the duplication in the genomic DNA and cDNA. RESULTS: We describe here an FA-B patient with a mild phenotype. The DEB diagnostic test for FA revealed somatic mosaicism. We identified a 9154 bp intragenic duplication in FANCB, covering the first coding exon 3 and the flanking regions. A four bp homology (GTAG) present at both ends of the breakpoint is consistent with microhomology-mediated duplication mechanism. The duplicated allele gives rise to an aberrant transcript containing exon 3 duplication, predicted to introduce a stop codon in FANCB protein (p.A319*). Duplication levels in the peripheral blood DNA declined from 93% to 7.9% in the span of eleven years. Moreover, the patient fibroblasts have shown 8% of wild-type (WT) allele and his carrier mother showed higher than expected levels of WT allele (79% vs. 50%) in peripheral blood, suggesting that the duplication was highly unstable. CONCLUSION: Unlike sequence point variants, intragenic duplications are difficult to precisely define, accurately quantify, and may be very unstable, challenging the proper diagnosis. The reversion of genomic duplication to the WT allele results in somatic mosaicism and may explain the relatively milder phenotype displayed by the FA-B patient described here.

Assessing the spectrum of germline variation in Fanconi anemia genes among patients with head and neck carcinoma before age 50.

BACKGROUND: Patients with Fanconi anemia (FA) have an increased risk for head and neck squamous cell carcinoma (HNSCC). The authors sought to determine the prevalence of undiagnosed FA and FA carriers among patients with HNSCC as well as an age cutoff for FA genetic screening. METHODS: Germline DNA samples from 417 patients with HNSCC aged <50 years were screened for sequence variants by targeted next-generation sequencing of the entire length of 16 FA genes. RESULTS: The sequence revealed 194 FA gene variants in 185 patients (44%). The variant spectrum was comprised of 183 nonsynonymous point mutations, 9 indels, 1 large deletion, and 1 synonymous variant that was predicted to effect splicing. One hundred eight patients (26%) had at least 1 rare variant that was predicted to be damaging, and 57 (14%) had at least 1 rare variant that was predicted to be damaging and had been previously reported. Fifteen patients carried 2 rare variants or an X-linked variant in an FA gene. Overall, an age cutoff for FA screening was not identified among young patients with HNSCC, because there were no significant differences in mutation rates when patients were stratified by age, tumor site, ethnicity, smoking status, or human papillomavirus status. However, an increased burden, or mutation load, of FA gene variants was observed in carriers of the genes FA complementation group D2 (FANCD2), FANCE, and FANCL in the HNSCC patient cohort relative to the 1000 Genomes population. CONCLUSIONS: FA germline functional variants offer a novel area of study in HNSCC tumorigenesis. FANCE and FANCL, which are components of the core complex, are known to be responsible for the recruitment and ubiquitination, respectively, of FANCD2, a critical step in the FA DNA repair pathway. In the current cohort, the increased mutation load of FANCD2, FANCE, and FANCL variants among younger patients with HNSCC indicates the importance of the FA pathway in HNSCC. Cancer 2017;123:3943-54. © 2017 American Cancer Society.

Treatment of Endometriosis-Associated Pain with Elagolix, an Oral GnRH Antagonist.

BACKGROUND: Endometriosis is a chronic, estrogen-dependent condition that causes dysmenorrhea and pelvic pain. Elagolix, an oral, nonpeptide, gonadotropin-releasing hormone (GnRH) antagonist, produced partial to nearly full estrogen suppression in previous studies. METHODS: We performed two similar, double-blind, randomized, 6-month phase 3 trials (Elaris Endometriosis I and II [EM-I and EM-II]) to evaluate the effects of two doses of elagolix - 150 mg once daily (lower-dose group) and 200 mg twice daily (higher-dose group) - as compared with placebo in women with surgically diagnosed endometriosis and moderate or severe endometriosis-associated pain. The two primary efficacy end points were the proportion of women who had a clinical response with respect to dysmenorrhea and the proportion who had a clinical response with respect to nonmenstrual pelvic pain at 3 months. Each of these end points was measured as a clinically meaningful reduction in the pain score and a decreased or stable use of rescue analgesic agents, as recorded in a daily electronic diary. RESULTS: A total of 872 women underwent randomization in Elaris EM-I and 817 in Elaris EM-II; of these women, 653 (74.9%) and 632 (77.4%), respectively, completed the intervention. At 3 months, a significantly greater proportion of women who received each elagolix dose met the clinical response criteria for the two primary end points than did those who received placebo. In Elaris EM-I, the percentage of women who had a clinical response with respect to dysmenorrhea was 46.4% in the lower-dose elagolix group and 75.8% in the higher-dose elagolix group, as compared with 19.6% in the placebo group; in Elaris EM-II, the corresponding percentages were 43.4% and 72.4%, as compared with 22.7% (P<0.001 for all comparisons). In Elaris EM-I, the percentage of women who had a clinical response with respect to nonmenstrual pelvic pain was 50.4% in the lower-dose elagolix group and 54.5% in the higher-dose elagolix group, as compared with 36.5% in the placebo group (P<0.001 for all comparisons); in Elaris EM-II, the corresponding percentages were 49.8% and 57.8%, as compared with 36.5% (P=0.003 and P<0.001, respectively). The responses with respect to dysmenorrhea and nonmenstrual pelvic pain were sustained at 6 months. Women who received elagolix had higher rates of hot flushes (mostly mild or moderate), higher levels of serum lipids, and greater decreases from baseline in bone mineral density than did those who received placebo; there were no adverse endometrial findings. CONCLUSIONS: Both higher and lower doses of elagolix were effective in improving dysmenorrhea and nonmenstrual pelvic pain during a 6-month period in women with endometriosis-associated pain. The two doses of elagolix were associated with hypoestrogenic adverse effects. (Funded by AbbVie; Elaris EM-I and EM-II ClinicalTrials.gov numbers, NCT01620528 and NCT01931670 .).

Germinal centre hypoxia and regulation of antibody qualities by a hypoxia response system.

Germinal centres (GCs) promote humoral immunity and vaccine efficacy. In GCs, antigen-activated B cells proliferate, express high-affinity antibodies, promote antibody class switching, and yield B cell memory. Whereas the cytokine milieu has long been known to regulate effector functions that include the choice of immunoglobulin class, both cell-autonomous and extrinsic metabolic programming have emerged as modulators of T-cell-mediated immunity. Here we show in mice that GC light zones are hypoxic, and that low oxygen tension () alters B cell physiology and function. In addition to reduced proliferation and increased B cell death, low impairs antibody class switching to the pro-inflammatory IgG2c antibody isotype by limiting the expression of activation-induced cytosine deaminase (AID). Hypoxia induces HIF transcription factors by restricting the activity of prolyl hydroxyl dioxygenase enzymes, which hydroxylate HIF-1α and HIF-2α to destabilize HIF by binding the von Hippel-Landau tumour suppressor protein (pVHL). B-cell-specific depletion of pVHL leads to constitutive HIF stabilization, decreases antigen-specific GC B cells and undermines the generation of high-affinity IgG, switching to IgG2c, early memory B cells, and recall antibody responses. HIF induction can reprogram metabolic and growth factor gene expression. Sustained hypoxia or HIF induction by pVHL deficiency inhibits mTOR complex 1 (mTORC1) activity in B lymphoblasts, and mTORC1-haploinsufficient B cells have reduced clonal expansion, AID expression, and capacities to yield IgG2c and high-affinity antibodies. Thus, the normal physiology of GCs involves regional variegation of hypoxia, and HIF-dependent oxygen sensing regulates vital functions of B cells. We propose that the restriction of oxygen in lymphoid organs, which can be altered in pathophysiological states, modulates humoral immunity.

Class-switched anti-insulin antibodies originate from unconventional antigen presentation in multiple lymphoid sites.

Autoantibodies to insulin are a harbinger of autoimmunity in type 1 diabetes in humans and in non-obese diabetic mice. To understand the genesis of these autoantibodies, we investigated the interactions of insulin-specific T and B lymphocytes using T cell and B cell receptor transgenic mice. We found spontaneous anti-insulin germinal center (GC) formation throughout lymphoid tissues with GC B cells binding insulin. Moreover, because of the nature of the insulin epitope recognized by the T cells, it was evident that GC B cells presented a broader repertoire of insulin epitopes. Such broader recognition was reproduced by activating naive B cells ex vivo with a combination of CD40 ligand and interleukin 4. Thus, insulin immunoreactivity extends beyond the pancreatic lymph node-islets of Langerhans axis and indicates that circulating insulin, despite its very low levels, can have an influence on diabetogenesis.

Reversing Tolerance in Isotype Switch-Competent Anti-Insulin B Lymphocytes.

Autoreactive B lymphocytes that escape central tolerance and mature in the periphery are a liability for developing autoimmunity. IgG insulin autoantibodies that predict type 1 diabetes and complicate insulin therapies indicate that mechanisms for tolerance to insulin are flawed. To examine peripheral tolerance in anti-insulin B cells, we generated C57BL/6 mice that harbor anti-insulin VDJH-125 site directed to the native IgH locus (VH125(SD)). Class switch-competent anti-insulin B cells fail to produce IgG Abs following T cell-dependent immunization of VH125(SD) mice with heterologous insulin, and they exhibit markedly impaired proliferation to anti-CD40 plus insulin in vitro. In contrast, costimulation with LPS plus insulin drives robust anti-insulin B cell proliferation. Furthermore, VH125(SD) mice produce both IgM and IgG2a anti-insulin Abs following immunization with insulin conjugated to type 1 T cell-independent Brucella abortus ring test Ag (BRT). Anti-insulin B cells undergo clonal expansion in vivo and emerge as IgM(+) and IgM(-) GL7(+)Fas(+) germinal center (GC) B cells following immunization with insulin-BRT, but not BRT alone. Analysis of Igκ genes in VH125(SD) mice immunized with insulin-BRT reveals that anti-insulin Vκ from the preimmune repertoire is selected into GCs. These data demonstrate that class switch-competent anti-insulin B cells remain functionally silent in T cell-dependent immune responses, yet these B cells are vulnerable to reversal of anergy following combined BCR/TLR engagement that promotes Ag-specific GC responses and Ab production. Environmental factors that lead to infection and inflammation could play a critical yet underappreciated role in driving loss of tolerance and promoting autoimmune disease.

NFATc2 (NFAT1) assists BCR-mediated anergy in anti-insulin B cells.

NFAT transcription factors play critical roles in both the activation and repression of T and B lymphocyte responses. To understand the role of NFATc2 (NFAT1) in the maintenance of tolerance for anti-insulin B cells, functionally inactive NFATc2 (NFATc2(-/-)) was introduced into C57BL/6 mice that harbor anergic anti-insulin 125Tg B cells. The production and peripheral maturation of anti-insulin B cells into follicular and marginal zone subsets was not altered by the absence of functional NFATc2. Surface B cell receptor expression levels, important for tonic signaling and altered by anergy, were not altered in any spleen B cell subset. The levels of anti-insulin antibodies were not different in 125Tg/B6/NFATc2(-/-) mice and the anti-insulin response remained silenced following T cell dependent immunization. However, studies addressing in vitro proliferation reveal the anergic state of 125Tg B cells is relieved in 125Tg/B6/NFATc2(-/-) B cells in response to BCR stimulation. In contrast, anergy is not released in 125Tg/B6/NFATc2(-/-) B cells following stimulation with anti-CD40. The relief of anergy to BCR stimulation in 125Tg/B6/NFATc2(-/-) B cells is associated with increased transcription of both NFATc1 and NFATc3 while expression of these NFATs does not change in anti-IgM stimulated 125Tg/B6/NFATc2(+/+) B cells. The data suggest that NFATc2 plays a subtle and selective role in maintaining anergy for BCR stimulation by repressing the transcription of other NFAT family members.

Estrogen receptor α polymorphism in a species with alternative behavioral phenotypes.

The evolution of behavior relies on changes at the level of the genome; yet the ability to attribute a behavioral change to a specific, naturally occurring genetic change is rare in vertebrates. In the white-throated sparrow (Zonotrichia albicollis), a chromosomal polymorphism (ZAL2/2(m)) is known to segregate with a behavioral phenotype. Individuals with the ZAL2(m) haplotype engage in more territorial aggression and less parental behavior than individuals without it. These behaviors are thought to be mediated by sensitivity to sex steroids, and the chromosomal rearrangement underlying the polymorphism has captured a prime candidate gene: estrogen receptor 1 (ESR1), which encodes estrogen receptor α (ERα). We therefore hypothesized that the behavioral effects of the ZAL2(m) rearrangement are mediated by polymorphism in ESR1. We report here that (i) the ESR1 promoter region contains fixed polymorphisms distinguishing the ZAL2(m) and ZAL2 alleles; (ii); those polymorphisms regulate transcription efficiency in vitro and therefore potentially do the same in vivo (iii); the local expression of ERα in the brain depends strongly on genotype in a free-living population; and (iv) ERα expression in the medial amygdala and medial preoptic area may fully mediate the effects of genotype on territorial aggression and parenting, respectively. Thus, our study provides a rare glimpse of how a chromosomal polymorphism has affected the brain and social behavior in a vertebrate. Our results suggest that in this species, differentiation of ESR1 has played a causal role in the evolution of phenotypes with alternative life-history strategies.

B lymphocyte "original sin" in the bone marrow enhances islet autoreactivity in type 1 diabetes-prone nonobese diabetic mice.

Effective central tolerance is required to control the large extent of autoreactivity normally present in the developing B cell repertoire. Insulin-reactive B cells are required for type 1 diabetes in the NOD mouse, because engineered mice lacking this population are protected from disease. The Cg-Tg(Igh-6/Igh-V125)2Jwt/JwtJ (VH125Tg) model is used to define this population, which is found with increased frequency in the periphery of NOD mice versus nonautoimmune C57BL/6 VH125Tg mice; however, the ontogeny of this disparity is unknown. To better understand the origins of these pernicious B cells, anti-insulin B cells were tracked during development in the polyclonal repertoire of VH125Tg mice. An increased proportion of insulin-binding B cells is apparent in NOD mice at the earliest point of Ag commitment in the bone marrow. Two predominant L chains were identified in B cells that bind heterologous insulin. Interestingly, Vκ4-57-1 polymorphisms that confer a CDR3 Pro-Pro motif enhance self-reactivity in VH125Tg/NOD mice. Despite binding circulating autoantigen in vivo, anti-insulin B cells transition from the parenchyma to the sinusoids in the bone marrow of NOD mice and enter the periphery unimpeded. Anti-insulin B cells expand at the site of autoimmune attack in the pancreas and correlate with increased numbers of IFN-γ-producing cells in the repertoire. These data identify the failure to cull autoreactive B cells in the bone marrow as the primary source of anti-insulin B cells in NOD mice and suggest that dysregulation of central tolerance permits their escape into the periphery to promote disease.

Tolerant anti-insulin B cells are effective APCs.

Autoreactive B lymphocytes that are not culled by central tolerance in the bone marrow frequently enter the peripheral repertoire in a state of functional impairment, termed anergy. These cells are recognized as a liability for autoimmunity, but their contribution to disease is not well understood. Insulin-specific 125Tg B cells support T cell-mediated type 1 diabetes in NOD mice, despite being anergic to B cell mitogens and T cell-dependent immunization. Using this model, the potential of anergic, autoreactive B cells to present Ag and activate T cells was investigated. The data show that 1) insulin is captured and rapidly internalized by 125Tg BCRs, 2) these Ag-exposed B cells are competent to activate both experienced and naive CD4(+) T cells, 3) anergic 125Tg B cells are more efficient than naive B cells at activating T cells when Ag is limiting, and 4) 125Tg B cells are competent to generate low-affinity insulin B chain epitopes necessary for activation of diabetogenic anti-insulin BDC12-4.1 T cells, indicating the pathological relevance of anergic B cells in type 1 diabetes. Thus, phenotypically tolerant B cells that are retained in the repertoire may promote autoimmunity by driving activation and expansion of autoaggressive T cells via Ag presentation.

Autoreactive preplasma cells break tolerance in the absence of regulation by dendritic cells and macrophages.

The ability to induce Ab responses to pathogens while maintaining the quiescence of autoreactive cells is an important aspect of immune tolerance. During activation of TLR4, dendritic cells (DCs) and macrophages (MFs) repress autoantibody production through their secretion of IL-6 and soluble CD40L (sCD40L). These soluble mediators selectively repress B cells chronically exposed to Ag, but not naive cells, suggesting a means to maintain tolerance during TLR4 stimulation, yet allow immunity. In this study, we identify TNF-α as a third repressive factor, which together with IL-6 and CD40L account for nearly all the repression conferred by DCs and MFs. Similar to IL-6 and sCD40L, TNF-α did not alter B cell proliferation or survival. Instead, it reduced the number of Ab-secreting cells. To address whether the soluble mediators secreted by DCs and MFs functioned in vivo, we generated mice lacking IL-6, CD40L, and TNF-α. Compared to wild-type mice, these mice showed prolonged anti-nuclear Ab responses following TLR4 stimulation. Furthermore, adoptive transfer of autoreactive B cells into chimeric IL-6(-/-) × CD40L(-/-) × TNF-α(-/-) mice showed that preplasma cells secreted autoantibodies independent of germinal center formation or extrafollicular foci. These data indicate that in the absence of genetic predisposition to autoimmunity, loss of endogenous IL-6, CD40L, and TNF-α promotes autoantibody secretion during TLR4 stimulation.

Follicular B cell trafficking within the spleen actively restricts humoral immune responses.

Follicular (FO) and marginal zone (MZ) B cells are maintained in distinct locations within the spleen, but the genetic basis for this separation is still enigmatic. We now report that B cell sequestration requires lineage-specific regulation of migratory receptors by the transcription factor Klf2. Moreover, using gene-targeted mice we show that altered splenic B cell migration confers a significant in vivo gain-of-function phenotype to FO B cells, including the ability to quickly respond to MZ-associated antigens and pathogens in a T cell-dependent manner. This work demonstrates that in wild-type animals, naive FO B cells are actively removed from the MZ, thus restricting their capacity to respond to blood-borne pathogens.

Reduced diabetes in btk-deficient nonobese diabetic mice and restoration of diabetes with provision of an anti-insulin IgH chain transgene.

Type 1 diabetes results from T cell-mediated destruction of insulin-producing beta cells. Although elimination of B lymphocytes has proven successful at preventing disease, modulation of B cell function as a means to prevent type 1 diabetes has not been investigated. The development, fate, and function of B lymphocytes depend upon BCR signaling, which is mediated in part by Bruton's tyrosine kinase (BTK). When introduced into NOD mice, btk deficiency only modestly reduces B cell numbers, but dramatically protects against diabetes. In NOD, btk deficiency mirrors changes in B cell subsets seen in other strains, but also improves B cell-related tolerance, as indicated by failure to generate insulin autoantibodies. Introduction of an anti-insulin BCR H chain transgene restores diabetes in btk-deficient NOD mice, indicating that btk-deficient B cells are functionally capable of promoting autoimmune diabetes if they have a critical autoimmune specificity. This suggests that the disease-protective effect of btk deficiency may reflect a lack of autoreactive specificities in the B cell repertoire. Thus, signaling via BTK can be modulated to improve B cell tolerance, and prevent T cell-mediated autoimmune diabetes.

Thermal safety of Er:YAG and Er,Cr:YSGG lasers in hard tissue removal.

OBJECTIVE: The aim of this study was to compare the thermal safety of Er:YAG and Er,Cr:YSGG lasers with conventional multi-use and single-use diamond burs. BACKGROUND DATA: Thermal effect of tooth preparation is mostly evaluated through the pulp chamber because it is difficult to measure the temperature of the preparation surface. A new in vitro method was introduced to simultaneously evaluate the heat increase of the preparation surface together with the pulp chamber. METHODS: Six laser and bur instrument groups were used to make standardized preparations on buccal surfaces of 60 intact third molars. The preparations removed an equal volume of hard tissue from each tooth (4 mm occluso-gingival x 8 mm mesial-distal x 1.6 mm bucco-lingual). The teeth also included tunnel preparations from the opposite (lingual) surface, exposing the pulpal axial wall (axial dentin wall in contact with the pulp chamber from the preparation surface site). An infrared thermal camera was positioned to capture the preparation surface in direct vision, while the pulpal axial wall was indirectly reflected to the thermal camera via a minimal-energy-loss mirror. Data from both surfaces were analyzed statistically using Nested Least Squares Analysis. RESULTS: The laser groups generated significantly lower heat compared to bur groups on the preparation surfaces. In contrast, both lasers generated greater pulpal heat increase, and the Er:YAG laser group showed significance (p < 0.0001). CONCLUSIONS: Lasers produced less heat on the preparation surface but more on the pulpal axial wall. However the temperature rise was less than the 5.5 degrees C threshold margin of safety.

Functional silencing is initiated and maintained in immature anti-insulin B cells.

Mechanisms of B cell tolerance act during development in the bone marrow and periphery to eliminate or restrict autoreactive clones to prevent autoimmune disease. B cells in the spleens of mice that harbor anti-insulin BCR transgenes (125Tg) are maintained in a functionally silenced or anergic state by endogenous hormone, but it is not clear when and where anergy is induced. An in vitro bone marrow culture system was therefore used to probe whether small protein hormones, a critical class of autoantigens, could interact with the BCR to induce anergy early during B cell development. Upon exposure to insulin, anti-insulin (125Tg) immature B cells show similar hallmarks of anergy as those observed in mature splenic B cells. These include BCR down-regulation, impaired proliferative responses to anti-CD40, and diminished calcium mobilization upon stimulation with BCR-dependent and independent stimuli. Inhibition of calcineurin also results in reduced immature B cell proliferation in a similar manner, suggesting a potential mechanism through which reduced intracellular calcium mobilization may be altering cellular proliferation. Signs of impairment appear after short-term exposure to insulin, which are reversible upon Ag withdrawal. This suggests that a high degree of functional plasticity is maintained at this stage and that constant Ag engagement is required to maintain functional inactivation. These findings indicate that tolerance observed in mature, splenic 125Tg B cells is initiated by insulin in the developing B cell compartment and thus highlight an important therapeutic window for the prevention of insulin autoimmunity.

Impaired intracellular calcium mobilization and NFATc1 availability in tolerant anti-insulin B cells.

B lymphocytes that recognize soluble self-Ags are routinely found in normal individuals in a functionally inactive or anergic state. Current models indicate that this tolerant state is maintained by interactions with self-Ags that uncouple the BCR from downstream signaling pathways and increase levels of free calcium. Contrary to this expectation, B cells that harbor anti-insulin Ig transgenes (125Tg) are maintained in a tolerant state even though free calcium levels remain normal and tyrosine kinase substrate phosphorylation is preserved following BCR stimulation. Under basal conditions, intracellular levels of inositol 1,4,5-trisphosphate are increased and NFATc1 levels are reduced in 125Tg B cells. The 125Tg B cells are markedly impaired in their ability to mobilize calcium upon stimulation with ionomycin, and BCR-induced calcium mobilization from internal stores is decreased. In contrast, poisoning intracellular calcium pumps with thapsigargin increases calcium mobilization in 125Tg B cells. Changes in calcium signaling are accompanied by a failure of 125Tg B cells to translocate NFATc1 into the nucleus following stimulation with either anti-IgM or ionomycin. Thus, disassociation of BCR from multiple signaling pathways is not essential for maintaining tolerance in anti-insulin 125Tg B cells. Rather, BCRs that are occupied by autologous insulin deliver signals that induce changes in intracellular calcium mobilization and maintain tolerance by preventing activation of key transcription factors such as NFAT.

The gene of retroviral origin Syncytin 1 is specific to hominoids and is inactive in Old World monkeys.

Syncytin 1 is one of the best known examples of recent acquisition of a new gene from an endogenous retrovirus (HERV) in the human genome and has been implicated in placental physiology. Within primates, Syncytin 1 is conserved in all hominoids but has not been characterized in Old World monkeys (OWMs). In this study, we investigated the status of Syncytin 1 in 14 hominoid and OWM species. We show that although the HERV-W provirus responsible for the origin of this gene was present in the genome of the most recent common ancestor of hominoids and OWMs, Syncytin 1 is inactive in OWMs. In addition, we were able to determine that the evolution of Syncytin 1 in hominoids involved an accumulation of amino acid changes and showed signatures of both positive and purifying selection. Our results indicate that Syncytin 1 is indeed a hominoid-specific gene and illustrate the complex and dynamic process associated with the origin of new genes.