Efficacy and safety of vibegron for the treatment of irritable bowel syndrome in women: Results of a randomized, double-blind, placebo-controlled phase 2 trial.

BACKGROUND: Preclinical and clinical studies suggest that ß3 -adrenergic receptor activation may be a novel target for treating abdominal pain and gastrointestinal motility dysfunction in patients with irritable bowel syndrome (IBS). This proof-of-concept study evaluated the efficacy and safety of the ß3 -adrenergic agonist vibegron in treating IBS-related pain. METHODS: Adult women with predominant-diarrhea IBS (IBS-D) or with mixed diarrhea/constipation (IBS-M), diagnosed using Rome IV criteria, were randomized 1:1 to receive once-daily vibegron 75 mg or placebo for 12 weeks. The primary endpoint was the percentage of patients with IBS-D considered abdominal pain intensity (API) weekly responders, defined as ≥30% reduction from baseline at week 12 in mean weekly worst abdominal pain over 24 hours using the API score. Patients completed a pain diary at baseline and at weeks 2, 4, 8, and 12. Safety was assessed by adverse events (AEs) in the overall IBS population. KEY RESULTS: Of the 222 patients with IBS randomized (vibegron, N = 111; placebo, N = 111), 85% completed the trial. There was no significant difference in the percentage of patients with IBS-D (vibegron, N = 66; placebo, N = 63) considered API weekly responders with vibegron vs. placebo (p = 0.8222) after 12 weeks. The incidence of AEs was comparable between treatment groups (33.3% each), with equal rates of worsening IBS symptoms (2.7% each). CONCLUSIONS AND INFERENCES: In women with IBS-D, vibegron was not associated with significant improvement in the percentage of API weekly responders. Vibegron was generally safe and well tolerated and, in particular, did not worsen IBS symptoms vs. placebo.

Topical Quercetin and Resveratrol Protect the Ocular Surface in Experimental Dry Eye Disease.

Purpose: To determine the anti-inflammatory effect of quercetin (QCT), resveratrol (RES), and their combination in a dry eye disease (DED) model. Methods: 0.01% QCT, 0.1% RES, 0.01% QCT + 0.1% RES (QCT + RES) or vehicle were topically applied in a desiccating stress (DS) mice model. CD4+ T cells isolated from DS-exposed mice were transferred to athymic recipient mice. Corneal fluorescein staining, tear production, and tear cytokine levels were evaluated in DS-exposed mice, and conjunctival CD4+ T cell infiltration was evaluated in recipient mice. Results: QCT (p < 0.001) and QCT + RES (p < 0.05) reduced corneal staining in DS-exposed mice. IL-1α tear concentration was reduced by QCT, RES, and QCT + RES (p < 0.05, 0.01 and 0.01, respectively) compared to DS + vehicle mice. CD4+ T cells increased in recipients of DS-exposed mice (p < 0.05) and were lower in recipients of QCT- and RES-treated mice (p < 0.05). Conclusion: The anti-inflammatory effect of QCT, RES, and QCT + RES on DED-experimental model suggests that their topical application could be used for DED treatment.

Effect of desiccating environmental stress versus systemic muscarinic AChR blockade on dry eye immunopathogenesis.

PURPOSE: A majority of experimental data on dry eye disease (DED) immunopathogenesis have been derived from a murine model of DED that combines desiccating environmental stress with systemic muscarinic acetylcholine receptor (mAChR) inhibition. However, to our knowledge the effects of pharmacologic mAChR blockade on the pathogenesis of experimental DED have not been evaluated systemically. The purpose of our study was to investigate the differential effects of desiccating environmental stress and mAChR inhibition on the pathogenesis of DED. METHODS: DED was induced in female C57BL/6 mice by exposure to a desiccating environment in the controlled-environment chamber or to systemic scopolamine, or by performing extraorbital lacrimal gland excision. Clinical disease was assessed using corneal fluorescein staining (CFS) and the cotton thread test (CTT). Corneal CD11b(+) and conjunctival CD3(+) T-cell infiltration were evaluated by flow cytometry. T-cells from draining cervical lymph nodes (CLN) and distant inguinal lymph nodes (ILN) were analyzed for Th1, Th2, Th17, and Treg responses by flow cytometry and ELISA. RESULTS: Desiccating environmental stress and systemic mAChR blockade induced similar clinical signs of DED. However, desiccating environmental stress imparted higher conjunctival CD3(+) T-cell infiltration, and greater Th17-cell activity and Treg dysfunction than mAChR blockade, while mAChR blockade decreased tear secretion to a greater extent than desiccating environmental stress. Systemic mAChR blockade attenuated Th17 activity and enhanced Th2 and Treg responses without affecting Th1 activity. CONCLUSIONS: In vivo inhibition of mAChRs variably affects CD4(+) T-cell subsets, and desiccating environmental stress and systemic mAChR blockade induce DED through different primary pathogenic mechanisms.

Dry eye as a mucosal autoimmune disease.

Dry eye is a common ocular surface inflammatory disease that significantly affects quality of life. Dysfunction of the lacrimal function unit (LFU) alters tear composition and breaks ocular surface homeostasis, facilitating chronic inflammation and tissue damage. Accordingly, the most effective treatments to date are geared towards reducing inflammation and restoring normal tear film. The pathogenic role of CD4+ T cells is well known, and the field is rapidly realizing the complexity of other innate and adaptive immune factors involved in the development and progression of disease. The data support the hypothesis that dry eye is a localized autoimmune disease originating from an imbalance in the protective immunoregulatory and proinflammatory pathways of the ocular surface.

NK cells promote Th-17 mediated corneal barrier disruption in dry eye.

BACKGROUND: The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL). METHODOLOGY/PRINCIPAL FINDINGS: Here we characterized the IEL population prior to and after experimental desiccating stress (DS) for 5 or 10 days (DS5, DS10) and evaluated the effect of NK depletion on DS. The frequency of IELs in normal murine conjunctiva was CD3(+)CD103(+) (~22%), CD3(+)γδ(+) (~9.6%), CD3(+)NK(+) (2%), CD3(-)NK(+) (~4.4%), CD3(+)CD8α (~0.9%), and CD4 (~0.6%). Systemic depletion of NK cells prior and during DS led to a decrease in the frequency of total and activated DCs, a decrease in T helper-17(+) cells in the cervical lymph nodes and generation of less pathogenic CD4(+)T cells. B6.nude recipient mice of adoptively transferred CD4(+)T cells isolated from NK-depleted DS5 donor mice showed significantly less corneal barrier disruption, lower levels of IL-17A, CCL20 and MMP-3 in the cornea epithelia compared to recipients of control CD4(+)T cells. CONCLUSIONS/SIGNIFICANCE: Taken together, these results show that the NK IELs are involved in the acute immune response to desiccation-induced dry eye by activating DC, which in turn coordinate generation of the pathogenic Th-17 response.

Olig1 function is required for remyelination potential of transplanted neural progenitor cells in a model of viral-induced demyelination.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) resulting in cumulative neurologic deficits associated with progressive myelin loss. We have previously shown that transplantation of neural progenitor cells (NPCs) into mice persistently infected with the JHM strain of mouse hepatitis virus (JHMV) results in enhanced differentiation into oligodendrocyte progenitor cells (OPCs) that is associated with remyelination and axonal sparing. The current study examines the contributions of the transcription factor Olig1 on NPC differentiation and remyelination. Under defined conditions, NPCs preferentially differentiate into oligodendroglia whereas NPCs isolated from Olig1-deficient (Olig1-/-) mice exhibit enhanced differentiation into astrocytes. Transplantation of Olig1-/- and Olig1+/+ NPCs into JHMV-infected mice resulted in similar cell survival, proliferation, and selective migration to areas of demyelination. However, only recipients of wild type NPCs exhibited extensive remyelination compared to mice receiving Olig1-/- NPCs. In vivo characterization of NPCs revealed that Olig1+/+ NPCs preferentially differentiated into NG2-positive OPCs and formed processes expressing myelin basic protein that encircled axons. In contrast, the majority of transplanted Olig1-/- NPCs differentiated into GFAP-positive cells consistent with the astrocyte lineage. These results indicate that exogenous NPCs contribute to improved clinical and histological outcome and this is associated with remyelination by this donor population. Further, these findings reveal that Olig1function is required for the remyelination potential of NPCs after transplant, through specification and/or maintenance of oligodendroglial identity.

Autoantibodies contribute to the immunopathogenesis of experimental dry eye disease.

PURPOSE: The purpose of this study was to determine if autoantibodies play a role in the immunopathogenesis of experimental dry eye disease. METHODS: Dry eye was induced by exposing female C57BL/6 wild-type mice or hen egg lysozyme B-cell receptor transgenic mice to desiccating stress (subcutaneous scopolamine [0.5 mg/0.2 mL] 3 times a day, humidity < 40%, and sustained airflow) for 3 weeks, allowing sufficient time for a humoral immune response. Serum or purified IgG isolated from dry-eye mice or untreated controls was passively transferred to nude recipient mice, which were evaluated for ocular surface inflammation 3 days after transfer. To determine if complement activation contributed to serum-induced dry eye disease, cobra venom factor was used to deplete complement activity. RESULTS: Autoantibodies against kallikrein 13 were identified in serum from dry-eye mice, but were undetectable in untreated controls. Autoantibody-containing serum or purified IgG from dry-eye mice was sufficient to mediate complement-dependent ocular surface inflammation. Serum or purified IgG caused marked inflammatory burden and tissue damage within the ocular surface tissues, including elevated Gr1+ neutrophil infiltration and proinflammatory cytokines/chemokines associated with goblet cell loss. Moreover, complement C3b deposition was found within the ocular surface tissues of mice receiving dry-eye serum, but not in recipients of control serum. Functionally, complement depletion attenuated the ability to transfer dry-eye-specific serum or IgG-mediated disease. CONCLUSIONS: These data demonstrate for the first time a complement-dependent pathogenic role of dry-eye-specific autoantibodies, and suggest autoantibody deposition within the ocular surface tissues contributes to the predominantly T-cell-mediated immunopathogenesis of dry eye disease.

Ocular surface APCs are necessary for autoreactive T cell-mediated experimental autoimmune lacrimal keratoconjunctivitis.

As specialized sentinels between the innate and adaptive immune response, APCs are essential for activation of Ag-specific lymphocytes, pathogen clearance, and generation of immunological memory. The process is tightly regulated; however, excessive or atypical stimuli may ignite activation of APCs in a way that allows self-Ag presentation to autoreactive T cells in the context of the necessary costimulatory signals, ultimately resulting in autoimmunity. Studies in both animal models and patients suggest that dry eye is a chronic CD4(+) T cell-mediated ocular surface autoimmune-based inflammatory disease. Using a desiccating stress-induced mouse model of dry eye, we establish the fundamental role of APCs for both the generation and maintenance of ocular-specific autoreactive CD4(+) T cells. Subconjunctival administration of liposome-encapsulated clodronate efficiently diminished resident ocular surface APCs, inhibited the generation of autoreactive CD4(+) T cells, and blocked their ability to cause disease. APC-dependent CD4(+) T cell activation required intact draining cervical lymph nodes, as cervical lymphadenectomy also inhibited CD4(+) T cell-mediated dry eye disease. In addition, local depletion of peripheral conjunctival APCs blocked the ability of dry eye-specific CD4(+) T cells to accumulate within the ocular surface tissues, suggesting that fully primed and targeted dry eye-specific CD4(+) T cells require secondary activation by resident ocular surface APCs for maintenance and effector function. These data demonstrate that APCs are necessary for the initiation and development of experimental dry eye and support the standing hypothesis that dry eye is a self-Ag-driven autoimmune disease.

Surgical transplantation of mouse neural stem cells into the spinal cords of mice infected with neurotropic mouse hepatitis virus.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

Cell replacement therapies to promote remyelination in a viral model of demyelination.

Persistent infection of the central nervous system (CNS) of mice with the neuroadapted JHM strain of mouse hepatitis (MHV) is characterized by ongoing demyelination mediated by inflammatory T cells and macrophages that is similar both clinically and histologically with the human demyelinating disease multiple sclerosis (MS). Although extensive demyelination occurs in mice persistently infected with MHV there is only limited remyelination. Therefore, the MHV model of demyelination is a relevant model for studying disease and evaluating therapeutic approaches to protect cells of the oligodendrocyte lineage and promote remyelination. This concept is further highlighted as the etiology of MS remains enigmatic, but viruses have long been considered as potential triggering agents in initiating and/or maintaining MS symptoms. As such, understanding mechanisms associated with promoting repair within the CNS in the context of a persistent viral infection is critical given the possible viral etiology of MS. This review focuses on recent studies using either mouse neural stem cells (NSCs) or human oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem cell (hESC) to promote remyelination in mice persistently infected with MHV. In addition, the potential role for chemokines in positional migration of transplanted cells is addressed.

Endogenous remyelination is induced by transplant rejection in a viral model of multiple sclerosis.

Human embryonic stem cell-derived oligodendrocyte progenitors (OPCs) were transplanted into mice persistently infected with the neurotropic JHM strain of mouse hepatitis virus with established demyelination. Engrafted cells did not survive past 2 weeks following transplantation despite treatment with high dose cyclosporine A. While T cell infiltration into the CNS was dampened, elevated numbers of macrophage/microglia and endogenous OPCs were evident surrounding the implantation site and this was associated with increased remyelination. These data suggest that remyelination was initiated by the local response to xenograft transplantation. These findings illustrate the complexities of OPC transplantation into areas of robust immune-mediated pathology.

Mouse hepatitis virus infection of the CNS: a model for defense, disease, and repair.

Viral infection of the central nervous system (CNS) results in varied outcomes ranging from encephalitis, paralytic poliomyelitis or other serious consequences. One of the principal factors that directs the outcome of infection is the localized innate immune response, which is proceeded by the adaptive immune response against the invading viral pathogen. The role of the immune system is to contain and control the spread of virus within the CNS, and paradoxically, this response may also be pathological. Studies with a neurotropic murine coronavirus, mouse hepatitis virus (MHV) have provided important insights into how the immune system combats neuroinvasive viruses, and have identified molecular and cellular mechanisms contributing to chronic disease in persistently infected mice.

DRAK2 regulates memory T cell responses following murine coronavirus infection.

The contribution of DRAK2 [death-associated protein kinase (DAPK)-related apoptosis-inducing kinase 2] to anti-viral memory T cell responses following infection with mouse hepatitis virus (MHV) was examined. DRAK2 is a lymphoid-enriched serine/threonine kinase that is an important regulatory molecule involved in modulating T cell responses. Memory T cells derived from MHV-immunized Drak2(-/-) mice exhibited amplified proliferation and IFN-gamma secretion following stimulation with viral epitopes. Transfer of Drak2(-/-) memory T cells into Rag1(-/-) mice infected intracerebrally with MHV resulted in accelerated clearance of virus from the brain. Thus, DRAK2 may be a novel target for stimulating protective immunity to viral pathogens.

T cell antiviral effector function is not dependent on CXCL10 following murine coronavirus infection.

The chemokine CXCL10 is expressed within the CNS in response to intracerebral infection with mouse hepatitis virus (MHV). Blocking CXCL10 signaling results in increased mortality accompanied by reduced T cell infiltration and increased viral titers within the brain suggesting that CXCL10 functions in host defense by attracting T cells into the CNS. The present study was undertaken to extend our understanding of the functional role of CXCL10 in response to MHV infection given that CXCL10 signaling has been implicated in coordinating both effector T cell generation and trafficking. We show that MHV infection of CXCL10(+/+) or CXCL10(-/-) mice results in comparable levels of T cell activation and similar numbers of virus-specific CD4+ and CD8+ T cells. Subsequent analysis revealed no differences in T cell proliferation, IFN-gamma secretion by virus-specific T cells, or CD8+ T cell cytolytic activity. Analysis of chemokine receptor expression on CD4+ and CD8+ T cells obtained from MHV-immunized CXCL10(+/+) and CXCL10(-/-) mice revealed comparable levels of CXCR3 and CCR5, which are capable of responding to ligands CXCL10 and CCL5, respectively. Adoptive transfer of splenocytes acquired from MHV-immunized CXCL10(-/-) mice into MHV-infected RAG1(-/-) mice resulted in T cell infiltration into the CNS, reduced viral burden, and demyelination comparable to RAG1(-/-) recipients of immune CXCL10(+/+) splenocytes. Collectively, these data imply that CXCL10 functions primarily as a T cell chemoattractant and does not significantly influence T cell effector response following MHV infection.

Arginine-dependent gene regulation via the ArgR repressor is species specific in chlamydia.

Some, but not all, Chlamydia spp. are predicted to encode a homolog of ArgR, a master regulatory molecule that modulates arginine biosynthesis and catabolism in bacteria in response to intracellular arginine levels. While genes for arginine biosynthesis are apparently missing in Chlamydia, a putative arginine transport system encoded by glnP, glnQ, and artJ is present. We found that recombinant Chlamydia pneumoniae ArgR functions as an arginine-dependent aporepressor that bound specifically to operator sequences upstream of the glnPQ operon. ArgR was able to repress transcription in a promoter-specific manner that was dependent on the concentration of the corepressor l-arginine. We were able to locate ArgR operators upstream of glnPQ in C. pneumoniae and Chlamydophila caviae but not Chlamydia trachomatis, which corresponded to the predicted presence or absence of ArgR in these chlamydial species. Our findings indicate that only some members of the family Chlamydiaceae have an arginine-responsive mechanism of gene regulation that is predicted to control arginine uptake from the host cell. This is the first study to directly demonstrate a species-specific mechanism of transcriptional regulation in Chlamydia.

Mutational analysis of the Chlamydia trachomatis dnaK promoter defines the optimal -35 promoter element.

A long-standing question in the biology of the intracellular bacterium, Chlamydia, has been the structure of the promoter recognized by its RNA polymerase. The 'RNA polymerase sigma subunit paradox' refers to the difficulty reconciling the conservation between the RNA polymerases of Chlamydia and Escherichia coli, especially at the level of the promoter-recognition sigma subunit, with the general lack of homology between chlamydial promoters and the E.coli sigma(70) consensus promoter. While the -10 promoter element appears to be conserved between Chlamydia and E.coli, the structure of the chlamydial -35 promoter element has not been defined. We have investigated the structure of the -35 element of the Chlamydia trachomatis dnaK promoter by measuring the effects of single base pair substitutions on in vitro promoter activity. Most substitutions produced large decreases in promoter activity, which allowed us to define the optimal -35 sequence in the context of the dnaK promoter. We found that the optimal chlamydial -35 promoter sequence is identical to the E.coli sigma(70) consensus -35 promoter element (TTGACA). These results indicate that the optimal promoter specificities of the major form of chlamydial RNA polymerase and E.coli sigma(70) RNA polymerase are in fact highly conserved. A further implication of our results is that many chlamydial promoters have a suboptimal promoter structure. We hypothesize that these chlamydial promoters are intrinsically weak promoters that can be regulated during the chlamydial developmental cycle by additional transcription factors.