In vitro modelling of rat mucosal mast cell function in Trichinella spiralis infection

Intestinal infection with the parasitic nematode, Trichinella spiralis, provides a robust context for the study of mucosal mast cell function. In rats, mucosal mast cells are exposed to parasites during the earliest stage of infection, affording an opportunity for mast cells to contribute to an innate response to infection. During secondary infection, degranulation of rat mucosal mast cells coincides with expulsion of challenge larvae from the intestine. The goal of this study was to evaluate the rat bone marrow‐derived mast cells (BMMC) and the rat basophilic leukaemia cell line (RBL‐2H3) as models for mucosal mast cells, using parasite glycoproteins and antibody reagents that have been tested extensively in rats in vivo. We found that BMMC displayed a more robust mucosal phenotype. Although T. spiralis glycoproteins bound to mast cell surfaces in the absence of antibodies, they did not stimulate degranulation, nor did they inhibit degranulation triggered by immune complexes. Parasite glycoproteins complexed with specific monoclonal IgGs provoked release of rat mast cell protease II (RMCPII) and β‐hexosaminidase from both cell types in a manner that replicated results observed previously in passively immunized rats. Our results document that RBL‐2H3 cells and BMMC model rat mucosal mast cells in the contexts of innate and adaptive responses to T. spiralis.     

The prevalence and causes of chronic diarrhea in patients with celiac sprue treated with a gluten-free diet

BACKGROUND & AIMS: The majority of patients with celiac sprue experience diarrhea before diagnosis. There have been no studies of the prevalence or causes of chronic diarrhea in these patients after treatment with a gluten-free diet. METHODS: Seventy-eight patients with celiac sprue (59 women and 19 men) treated with a gluten-free diet for at least 12 months were surveyed about their bowel habits. Those with chronic diarrhea, defined as passage of loose stools three or more times per week for 6 months, underwent an extensive diagnostic evaluation to determine its cause. RESULTS: Sixty-two of the 78 patients (79%) experienced diarrhea before treatment, and 13 (17%) had chronic diarrhea (of lesser severity) after treatment. The causes of diarrhea in 11 patients consenting to this study were microscopic colitis, steatorrhea secondary to exocrine pancreatic insufficiency, dietary lactose or fructose malabsorption, anal sphincter dysfunction causing fecal incontinence, and the irritable bowel syndrome. Only 1 patient had antigliadin antibodies detected in serum or small intestinal villous atrophy. CONCLUSIONS: After treatment of celiac sprue with a gluten-free diet, chronic diarrhea persists in a substantial percentage of patients. Although ongoing gluten ingestion is one possible cause, other causes may be more frequent. Therefore, diagnostic investigation of diarrhea in celiac sprue after treatment seems warranted. (Gastroenterology 1997 Jun;112(6):1830-8)     

Thrombolytic therapy with tissue plasminogen activator or streptokinase induces transient thrombin activity

We have determined the plasma level of fibrinopeptide A as a specific index of thrombin activity during the infusion of a thrombolytic agent in patients with acute myocardial infarction. Peripheral venous plasma levels of fibrinopeptide A increased following the initiation of thrombolytic therapy from 2.7 nmol/L to a peak of 13.0 nmol/L at 30 minutes with streptokinase and from 1.1 nmol/L to a peak of 10.7 nmol/L at 90 minutes with tissue plasminogen activator. The amount of fibrinogen converted to fibrin I was determined by integration of the plasma level of fibrinopeptide A over time. The amount of fibrin I formed over the five-hour period from the start of drug infusion was approximately 10 mg/dL in response to either streptokinase or recombinant tissue plasminogen activator. We conclude that activation of coagulation occurs in response to thrombolytic therapy despite heparin administration. This thrombin action, though transient, would be sufficient to cause rethrombosis if localized and incompletely opposed by fibrinolytic activity.     

Black sexuality, social construction, and research targeting 'The Down Low' ('The DL')

PURPOSE: The purpose of this commentary is to explain how social constructions of black sexuality are relevant to research targeting black sexual behavior and the ostensibly new and race-specific phenomenon known as "the Down Low" (the DL). The term "the DL" is widely used to refer to black men publicly presenting as heterosexual while secretly having sex with other men and presumably spreading human immunodeficiency virus and acquired immune deficiency syndrome (HIV/AIDS) to unsuspecting women. METHODS: We briefly review lay and public health literature from 1998 to 2004 about the DL, describe existing social constructions of black sexuality, discuss two implications for epidemiologic research, and offer recommendations to guide future research. RESULTS: The lifestyle referenced by the term the DL is neither new nor limited to blacks, and sufficient data linking it to HIV/AIDS disparities currently are lacking. Common perceptions about the DL reflect social constructions of black sexuality as generally excessive, deviant, diseased, and predatory. Research targeting black sexual behavior that ignores these constructions may unwittingly reinforce them. CONCLUSIONS: Unaddressed social constructions of black sexuality have implications for epidemiologic research targeting black sexual behavior. Explicit examination of these concerns is necessary to eliminate fundamental causes of health disparities.     

Correction of both immunodeficiency and hypoparathyroidism by thymus transplantation in complete DiGeorge syndrome

Combined immune deficiency due to athymia in patients with complete DiGeorge syndrome can be corrected by allogeneic thymus transplantation. Hypoparathyroidism is a frequent concomitant clinical problem in these patients, which persists after thymus transplantation. Cotransplantation of allogeneic thymus and parental parathyroid tissue has been attempted but does not achieve durable correction of the patients' hypoparathyroidism due to parathyroid graft rejection. Surprisingly, we observed correction of hypoparathyroidism in one patient after thymus transplantation. Immunohistochemical analysis and fluorescence in situ hybridization confirmed the presence of allogeneic parathyroid tissue in the patient's thymus transplant biopsy. Despite a lack of HLA‐matching between thymus donor and recipient, the reconstituted immune system displays tolerance toward the thymus donor. Therefore we expect this patient's hypoparathyroidism to be permanently cured. It is recognised that ectopic parathyroid tissue is not infrequently found in the thymus. If such thymuses could be identified, we propose that their use would offer a compelling approach to achieving lasting correction of both immunodeficiency and hypoparathyroidism.     

T cell transduction and suicide with an enhanced mutant thymidine kinase

Retroviral transfer of Herpes simplex virus thymidine kinase to T cells has been used to confer sensitivity to the antiviral agent ganciclovir. This has allowed therapeutic approaches to be developed in which T cells mediating graft-versus-host disease after bone marrow transplantation can be selectively eliminated by the administration of ganciclovir. Although the strategy has been shown to be generally successful in early clinical trials, there are concerns about possible resistance to ganciclovir and the risk of myelosuppressive side-effects at the doses required to induce T cell suicide. We have incorporated the enhanced mutant HSV-TKSR39 into retroviral vectors tailored to exhibit high levels of expression in T cells and have used protocols optimized for the transduction and selection of primary lymphocytes. We demonstrate that leukemic and primary T cells can be efficiently transduced and highly enriched under conditions that should be readily adaptable for clinical use. T cells carrying HSV-TKSR39 were inhibited by exposure to ganciclovir at concentrations an order of magnitude below those required for wild-type HSV-TK. The less toxic agent aciclovir also eliminated T cells transduced with HSV-TKSR39 (but not HSV-TK), underlining the increased therapeutic potential of the mutant suicide gene system in the bone marrow transplantation setting.     

Local administration of an adeno-associated viral vector expressing IL-10 reduces monocyte infiltration and subsequent photoreceptor damage during experimental autoimmune uveitis.

Autoimmune posterior uveitis is a chronic, potentially blinding inflammatory disease of the eye. It is commonly treated with immunosuppressive drugs that have adverse long-term effects. Advances in gene transfer techniques have enabled long-term, stable transduction of retinal cells following subretinal injection with adeno-associated viral (AAV) vectors. Here we report for the first time that subretinal injection of rAAV-2 encoding murine IL-10 into the retina of C57BL/6 mice significantly decreases the median experimental autoimmune uveitis (EAU) disease severity. This protection is shown to be due to a decrease in the number and activation status of infiltrating monocytes during EAU, as determined by costimulatory molecule expression and nitrotyrosine detection. No differences within splenocyte proliferative responses or serum antibody levels were detected, emphasizing the potential of gene therapy strategies in ameliorating autoimmune responses in local microenvironments without unwanted systemic effects.     

Altered BCR and TLR signals promote enhanced positive selection of autoreactive transitional B cells in Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder frequently associated with systemic autoimmunity, including autoantibody-mediated cytopenias. WAS protein (WASp)–deficient B cells have increased B cell receptor (BCR) and Toll-like receptor (TLR) signaling, suggesting that these pathways might impact establishment of the mature, naive BCR repertoire. To directly investigate this possibility, we evaluated naive B cell specificity and composition in WASp-deficient mice and WAS subjects (n = 12). High-throughput sequencing and single-cell cloning analysis of the BCR repertoire revealed altered heavy chain usage and enrichment for low-affinity self-reactive specificities in murine marginal zone and human naive B cells. Although negative selection mechanisms including deletion, anergy, and receptor editing were relatively unperturbed, WASp-deficient transitional B cells showed enhanced proliferation in vivo mediated by antigen- and Myd88-dependent signals. Finally, using both BCR sequencing and cell surface analysis with a monoclonal antibody recognizing an intrinsically autoreactive heavy chain, we show enrichment in self-reactive cells specifically at the transitional to naive mature B cell stage in WAS subjects. Our combined data support a model wherein modest alterations in B cell–intrinsic, BCR, and TLR signals in WAS, and likely other autoimmune disorders, are sufficient to alter B cell tolerance via positive selection of self-reactive transitional B cells.Development of the adaptive immune system requires selection of antigen receptors to establish a diverse but self-tolerant lymphocyte repertoire. Mechanisms to prevent selection of autoreactive B lymphocytes include clonal deletion, anergy, and receptor editing (Nemazee, 2006; Meffre and Wardemann, 2008). Alternatively, a growing body of literature also suggests that antigen-dependent positive selection of transitional B cells can occur via increased survival and/or clonal expansion (Hayakawa et al., 1999; Levine et al., 2000; Gaudin et al., 2004; Meyer-Bahlburg et al., 2008; Zikherman et al., 2012). These negative and positive selection mechanisms function in concert to shape the mature naive B cell repertoire.Positive selection of transitional B cells is regulated by tonic B cell receptor (BCR) signaling (Stadanlick et al., 2008), signaling via the cytokine B cell–activating factor (BAFF; Stadanlick and Cancro, 2008), and T cell help via CD40L-CD40 signaling (Schwartz et al., 2014) to promote cell survival. Positive selection may help to select BCR specificities that maintain key homeostatic functions, including apoptotic cell clearance or conserved pathogen recognition (Grönwall and Silverman, 2014). Although positive selection can be beneficial for these important immune functions, enhanced positive selection of autoreactive BCRs, through incompletely defined mechanisms, is also predicted to occur in autoimmune-prone settings; this process is likely to lead to an enrichment in BCR specificities that may facilitate detrimental immune responses (Groom et al., 2002; Wang and Clarke, 2003; Eilat and Wabl, 2012).In addition to BCR specificity, emerging data suggest a role for TLR signals in modulation of B cell selection. Previous data have shown that TLR signaling adapters, including MyD88, IRAK-4, and UNC93b, may operate in conjunction with the BCR to facilitate negative selection of autoreactive B cells (Isnardi et al., 2008). In contrast to promoting negative selection in immature B cells, dual signals mediated via the BCR and TLR pathways in mature B cells (Leadbetter et al., 2002; Groom et al., 2007; Silver et al., 2007; Rawlings et al., 2012) markedly enhance B cell activation and may directly initiate humoral autoimmunity. In this latter setting, loss in B cell tolerance occurs via generation of self-reactive, germinal center responses, leading ultimately to production of class-switched pathogenic autoantibodies (Jackson et al., 2015). Notably, although these combined data implicate TLR/MyD88 signals in both early and late B cell tolerance checkpoints, a potential role of BCR and/or TLR engagement in transitional B cell positive selection into the naive mature B cell compartment has not been defined.Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency that results from mutations within the gene encoding the WAS protein (WASp), a key multiadapter protein linking a broad range of receptor signaling effectors to the actin cytoskeleton. This complex disorder is characterized by multiple alterations in hematopoietic cell surface receptor signal transduction, cell trafficking, and lineage- and developmental subset–specific homeostasis. Notably, up to 70% of WAS patients exhibit autoimmunity, including autoantibody-mediated cytopenias and organ-specific disease (Notarangelo and Ochs, 2003; Ochs and Thrasher, 2006; Bosticardo et al., 2009). In previous work, we have shown that WASp deficiency modestly enhances both BCR and TLR signaling in naive B cells (Becker-Herman et al., 2011). Furthermore, we and others have demonstrated that B cell–intrinsic WASp deficiency is sufficient to alter B cell tolerance and can promote production of class-switched autoantibodies and autoantibody-mediated autoimmune disease (Becker-Herman et al., 2011; Recher et al., 2012). This break in tolerance is associated with spontaneous GC formation and requires both BCR and TLR/MyD88 signaling (Becker-Herman et al., 2011; Jackson et al., 2014).In this study, we hypothesized that increased BCR and TLR signaling in WASp-deficient B cells may also impact establishment of the mature, naive BCR repertoire. In partial support of this idea, previous studies have revealed evidence for skewing of heavy chain usage in both class-switched and bulk naive peripheral blood B cells isolated from WAS subjects (Castiello et al., 2014; O’Connell et al., 2014; Simon et al., 2014). Here, we present a detailed analysis of the impact of WASp deficiency on the selection of the naive B cell repertoire in mice and humans. Our combined data support a model wherein altered BCR and TLR signaling orchestrates increased positive selection of transitional B cells expressing low-affinity self-reactive BCRs, leading to their enrichment within the naive B cell compartment. As multiple human autoimmune disorders are associated with genetic changes potentially impacting B lineage signaling function, our findings have important implications with respect to understanding events that promote altered B cell selection in both WAS subjects and in other autoimmune-prone individuals.