Tumor necrosis factor family member LIGHT acts with IL-1ß and TGF-ß to promote airway remodeling during rhinovirus infection.

BACKGROUND: Rhinovirus (RV) can exacerbate allergen-driven asthma. However, it has been suggested that serial infections with RV may also lead to asthma-like features in childhood without prior allergen exposure. AIM: We sought to test the effects of RV infection in the absence of allergen challenge on lung tissue remodeling and to understand whether RV induced factors in common with allergen that promote remodeling. METHODS: We infected C57BL/6 mice multiple times with RV in the absence or presence of allergen to assess airway remodeling. We used knockout mice and blocking reagents to determine the participation of LIGHT (TNFSF14), as well as IL-1ß and TGF-ß, each previously shown to contribute to lung remodeling driven by allergen. RESULTS: Recurrent RV infection without allergen challenge induced an increase in peribronchial smooth muscle mass and subepithelial fibrosis. Rhinovirus (RV) induced LIGHT expression in mouse lungs after infection, and alveolar epithelial cells and neutrophils were found to be potential sources of LIGHT. Accordingly, LIGHT-deficient mice, or mice where LIGHT was neutralized, displayed reduced smooth muscle mass and lung fibrosis. Recurrent RV infection also exacerbated the airway remodeling response to house dust mite allergen, and this was significantly reduced in LIGHT-deficient mice. Furthermore, neutralizing IL-1ß or TGF-ß also limited subepithelial fibrosis and/or smooth muscle thickness induced by RV. CONCLUSION: Rhinovirus can promote airway remodeling in the absence of allergen through upregulating common factors that also contribute to allergen-associated airway remodeling.

Lessons Learned: Early Termination of a Randomized Trial of Calcineurin Inhibitor and Corticosteroid Avoidance Using Belatacept.

The intent of this National Institutes of Health-sponsored study was to compare a belatacept-based immunosuppressive regimen with a maintenance regimen of tacrolimus and mycophenolate. Nineteen primary, Epstein-Barr virus-immune renal transplant recipients with a negative cross-match were randomized to one of three groups. All patient groups received perioperative steroids and maintenance mycophenolate mofetil. Patients in groups 1 and 2 were induced with alemtuzumab and maintained on tacrolimus or belatacept, respectively. Patients in group 3 were induced with basiliximab, received 3 mo of tacrolimus, and maintained on belatacept. There was one death with a functioning allograft due to endocarditis (group 1). There were three graft losses due to vascular thrombosis (all group 2) and one graft loss due to glomerular disease (group 1). Biopsy-proven acute cellular rejection was more frequent in the belatacept-treated groups, with 10 treated episodes in seven participants compared with one episode in group 1; however, estimated GFR was similar between groups at week 52. There were no episodes of posttransplant lymphoproliferative disorder or opportunistic infections in any group. Protocol enrollment was halted prematurely because of a high rate of serious adverse events. Such negative outcomes pose challenges to clinical investigators, who ultimately must weigh the risks and benefits in randomized trials.

The allo- and viral-specific immunosuppressive effect of belatacept, but not tacrolimus, attenuates with progressive T cell maturation.

Tacrolimus impairs allo- and viral-specific T cell responses. Belatacept, a costimulation-based alternative to tacrolimus, has emerged with a paradoxical picture of less complete control of alloimmunity with concomitant impaired viral immunity limited to viral-naïve patients. To reconcile these signatures, bulk population and purified memory and naïve lymphocytes from cytomegalovirus (CMV)-seropositive (n=10) and CMV-seronegative (n=10) volunteers were studied using flow cytometry, interrogating proliferation (carboxyfluorescein succinimidyl ester dilution) and function (intracellular cytokine staining) in response to alloantigens or CMV-pp-65 peptides. As anticipated, T cells from CMV-experienced, but not naïve, individuals responded to pp-65 with a small percentage of their repertoire (<2.5%) consisting predominantly of mature, polyfunctional (expressing interferon gamma, tumor necrosis factor alpha and IL-2) T effector memory cells. Both CMV naïve and experienced individuals responded similarly to alloantigen with a substantially larger percentage of the repertoire (up to 48.2%) containing proportionately fewer polyfunctional cells. Tacrolimus completely inhibited responses of CMV- and allo-specific T cells regardless of their maturation. However, belatacept's effects were decreasingly evident in increasingly matured cells, with minimal effect on viral-specific triple cytokine producers and CD28-negative allo-specific cells. These data indicate that belatacept's immunosuppressive effect, unlike tacrolimus's, wanes on progressively developed effector responses, and may explain the observed clinical effects of belatacept.

Fecal microbiota transplantation for refractory Clostridium difficile colitis in solid organ transplant recipients.

Fecal microbiota transplantation (FMT) has been shown to be safe and efficacious in individuals with refractory Clostridium difficile. It has not been widely studied in individuals with immunosuppression due to concerns about infectious complications. We describe two solid organ transplant recipients, one lung and one renal, in this case report that both had resolution of their diarrhea caused by C. difficile after FMT. Both recipients required two FMTs to achieve resolution of their symptoms and neither had infectious complications. Immunosuppressed individuals are at high risk for acquisition of C. difficile and close monitoring for infectious complications after FMT is necessary, but should not preclude its use in patients with refractory disease due to C. difficile. Sequential FMT may be used to achieve cure in these patients with damaged microbiota from antibiotic use and immunosuppression.

High CTLA-4 expression on Th17 cells results in increased sensitivity to CTLA-4 coinhibition and resistance to belatacept.

The CD28/cytotoxic T-lymphocyte antigen 4 (CTLA-4)blocker belatacept selectively inhibits alloreactive T cell responses but is associated with a high incidence of acute rejection following renal transplantation,which led us to investigate the etiology of belatacept­resistant graft rejection. T cells can differentiate into functionally distinct subsets of memory T cellsthat collectively enable protection against diverse classes of pathogens and can cross-react with allogeneicantigen and mediate graft rejection. T helper 17(Th17) cells are a pro-inflammatory CD4+ lineage that provides immunity to pathogens and are pathogenic in autoimmune disease. We found that T helper 1 (Th1)and Th17 memory compartments contained a similar frequency of divided cells following allogeneic stimulation.Compared to Th1 cells, Th17 memory cells expressed significantly higher levels of the coinhibitory molecule CTLA-4. Stimulation in the presence of belatacept inhibited Th1 responses but augmented Th17 cells due to greater sensitivity to coinhibition by CTLA-4. Th17 cells from renal transplant recipients were resistant to ex vivo CD28/CTLA-4 blockade with belatacept, and an elevated frequency of Th17 memory cells was associated with acute rejection during belatacept therapy. These data highlight important differences in costimulatory and coinhibitory requirements of CD4+ memory subsets, and demonstrate that the heterogeneity of pathogen-derived memory has implications for immunomodulation strategies.

Renal transplantation using belatacept without maintenance steroids or calcineurin inhibitors.

Kidney transplantation remains limited by toxicities of calcineurin inhibitors (CNIs) and steroids. Belatacept is a less toxic CNI alternative, but existing regimens rely on steroids and have higher rejection rates. Experimentally, donor bone marrow and sirolimus promote belatacept's efficacy. To investigate a belatacept-based regimen without CNIs or steroids, we transplanted recipients of live donor kidneys using alemtuzumab induction, monthly belatacept and daily sirolimus. Patients were randomized 1:1 to receive unfractionated donor bone marrow. After 1 year, patients were allowed to wean from sirolimus. Patients were followed clinically and with surveillance biopsies. Twenty patients were transplanted, all successfully. Mean creatinine (estimated GFR) was 1.10 ± 0.07 mg/dL (89 ± 3.56 mL/min) and 1.13 ± 0.07 mg/dL (and 88 ± 3.48 mL/min) at 12 and 36 months, respectively. Excellent results were achieved irrespective of bone marrow infusion. Ten patients elected oral immunosuppressant weaning, seven of whom were maintained rejection-free on monotherapy belatacept. Those failing to wean were successfully maintained on belatacept-based regimens supplemented by oral immunosuppression. Seven patients declined immunosuppressant weaning and three patients were denied weaning for associated medical conditions; all remained rejection-free. Belatacept and sirolimus effectively prevent kidney allograft rejection without CNIs or steroids when used following alemtuzumab induction. Selected, immunologically low-risk patients can be maintained solely on once monthly intravenous belatacept.

Belatacept and sirolimus prolong nonhuman primate renal allograft survival without a requirement for memory T cell depletion.

Belatacept is an inhibitor of CD28/B7 costimulation that is clinically indicated as a calcineurin inhibitor (CNI) alternative in combination with mycophenolate mofetil and steroids after renal transplantation. We sought to develop a clinically translatable, nonlymphocyte depleting, belatacept-based regimen that could obviate the need for both CNIs and steroids. Thus, based on murine data showing synergy between costimulation blockade and mTOR inhibition, we studied rhesus monkeys undergoing MHC-mismatched renal allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on costimulation blockade-resistant rejection, some animals also received CD2 blockade with alefacept (LFA3-Ig). Belatacept and sirolimus therapy successfully prevented rejection in all animals. Tolerance was not induced, as animals rejected after withdrawal of therapy. The regimen did not deplete T cells. Alefecept did not add a survival benefit to the optimized belatacept and sirolimus regimen, despite causing an intended depletion of memory T cells, and caused a marked reduction in regulatory T cells. Furthermore, alefacept-treated animals had a significantly increased incidence of CMV reactivation, suggesting that this combination overly compromised protective immunity. These data support belatacept and sirolimus as a clinically translatable, nondepleting, CNI-free, steroid-sparing immunomodulatory regimen that promotes sustained rejection-free allograft survival after renal transplantation.

Inhibition of αvß6 promotes acute renal allograft rejection in nonhuman primates.

The integrin αvß6 activates latent transforming growth factor-ß (TGF-ß) within the kidney and may be a target for the prevention of chronic allograft fibrosis after kidney transplantation. However, TGF-ß also has known immunosuppressive properties that are exploited by calcineurin inhibitors (CNIs); thus, the net benefit of αvß6 inhibition remains undetermined. To assess the acute impact of interference with αvß6 on acute rejection, we tested a humanized αvß6-specific monoclonal antibody (STX-100) in a randomized, double-blinded, placebo-controlled nonhuman primate renal transplantation study to evaluate whether αvß6 blockade alters the risk of acute rejection during CNI-based immunosuppression. Rhesus monkeys underwent renal allotransplantation under standard CNI-based maintenance immunosuppression; 10 biopsy-confirmed rejection-free animals were randomized to receive weekly STX-100 or placebo. Animals treated with STX-100 experienced significantly decreased rejection-free survival compared to placebo animals (p = 0.049). Immunohistochemical analysis confirmed αvß6 ligand presence, and αvß6 staining intensity was lower in STX-100-treated animals (p = 0.055), indicating an apparent blockade effect of STX-100. LAP, LTBP-1 and TGF-ß were all decreased in animals that rejected on STX-100 compared to those that rejected on standard immunosuppression alone, suggesting a relevant effect of αvß6 blockade on local TGF-ß. These data caution against the use of αvß6 blockade to achieve TGF-ß inhibition in kidney transplantation.

Selective targeting of human alloresponsive CD8+ effector memory T cells based on CD2 expression.

Costimulation blockade (CoB), specifically CD28/B7 inhibition with belatacept, is an emerging clinical replacement for calcineurin inhibitor-based immunosuppression in allotransplantation. However, there is accumulating evidence that belatacept incompletely controls alloreactive T cells that lose CD28 expression during terminal differentiation. We have recently shown that the CD2-specific fusion protein alefacept controls costimulation blockade-resistant allograft rejection in nonhuman primates. Here, we have investigated the relationship between human alloreactive T cells, costimulation blockade sensitivity and CD2 expression to determine whether these findings warrant potential clinical translation. Using polychromatic flow cytometry, we found that CD8(+) effector memory T cells are distinctly high CD2 and low CD28 expressors. Alloresponsive CD8(+) CD2(hi) CD28(-) T cells contained the highest proportion of cells with polyfunctional cytokine (IFNγ, TNF and IL-2) and cytotoxic effector molecule (CD107a and granzyme B) expression capability. Treatment with belatacept in vitro incompletely attenuated allospecific proliferation, but alefacept inhibited belatacept-resistant proliferation. These results suggest that highly alloreactive effector T cells exert their late stage functions without reliance on ongoing CD28/B7 costimulation. Their high CD2 expression increases their susceptibility to alefacept. These studies combined with in vivo nonhuman primate data provide a rationale for translation of an immunosuppression regimen pairing alefacept and belatacept to human renal transplantation.

Potassium channel openers exhibit cross-tolerance with morphine in two experimental models of pain.

OBJECTIVE: The study was performed to assess the effect of potassium channel openers on morphine tolerance and vice-versa. METHODS: Swiss albino mice of either gender weighing between 25-30 g were used for the study The study assesses the effect of potassium channel openers (cromakalim, diazoxide and minoxidil) on morphine tolerance and vice-versa, using formalin and tail-flick tests. RESULTS: The antinociceptive effect of cromakalim and minoxidil was significantly reduced when administered to morphine-tolerant mice, in both the behavioural tests. However reduced analgesic effect of diazoxide was observed on morphine-tolerance in the formalin test but not in the tail-flick test. Tolerance was observed when morphine was administered to animals chronically treated with any of the potassium channel openers. The same effect was observed when morphine was injected into a group treated with a combination of morphine and any of the potassium channel openers. CONCLUSIONS: This study, therefore, suggests that both morphine and potassium channel openers are cross-tolerant. However such interaction occurs at the level of potassium channels rather than at the level of receptors.

Choline supplementation reduces oxidative stress in mouse model of allergic airway disease.

BACKGROUND: Asthma is a multi-factorial inflammatory disease associated with increased oxidative stress and altered antioxidant defences. We have evaluated the effect of choline on oxidative stress in a mouse model of airway disease. MATERIALS AND METHODS: Balb/c mice were sensitised with 100 microg of ovalbumin on days 0 and 14, and challenged with aerosolized ovalbumin on days 25-27. Mice were administered 1 mg kg(-1) of choline via oral gavage or intranasal route on days 14-27. Mice were also administered 100 mg kg(-1) of alpha-lipoic acid as standard antioxidant. Total cell counts, eosinophils and eosinophil peroxidase (EPO) activity were determined in bronchoalveolar lavage (BAL) fluid. Reactive oxygen species (ROS), lipid peroxidation and isoprostanes levels were measured in BAL fluid. IL-13 and tumour necrosis factor-alpha (TNF-alpha) levels were also measured in BAL fluid and spleen cell culture supernatant. Nuclear factor kappaB (NFkappaB) p65 protein expression was measured after last ovalbumin challenge in nuclear and cytosolic extracts of lungs. RESULTS: Compared with ovalbumin-challenged mice, choline and alpha-lipoic acid treated mice had significantly reduced eosinophilic infiltration and EPO activity in BAL fluid. Choline and alpha-lipoic acid treatment reduced ROS production and isoprostanes level significantly in BAL fluid and thus suppressed oxidative stress. Choline and alpha-lipoic acid administration by either route decreased lipid peroxidation levels and down regulated NFkappaB activity. Further, choline and/or alpha-lipoic acid treatment suppressed TNF-alpha level significantly as compared with that of ovalbumin-challenged mice. CONCLUSIONS: Choline administration reduces oxidative stress possibly by modulating the redox status of the cell and inhibits inflammatory response in a mouse model.

A systematic review of commercial weight loss programmes' effect on glycemic outcomes among overweight and obese adults with and without type 2 diabetes mellitus.

OBJECTIVE: We examined the glycemic benefits of commercial weight loss programmes as compared with control/education or counselling among overweight and obese adults with and without type 2 diabetes mellitus (T2DM). METHODS: We searched MEDLINE, Cochrane Database of Systematic Reviews, and references cited by individual programmes. We included randomized controlled trials of ≥12 weeks duration. Two reviewers extracted information on study design, population characteristics, interventions, and mean changes in haemoglobin A1c and glucose. RESULTS: We included 18 randomized controlled trials. Few trials occurred among individuals with T2DM. In this population, Jenny Craig reduced A1c at least 0.4% more than counselling at 12 months, Nutrisystem significantly reduced A1c 0.3% more than counselling at 6 months, and OPTIFAST reduced A1c 0.3% more than counselling at 6 months. Among individuals without T2DM, few studies evaluated glycemic outcomes, and when reported, most did not show substantial reductions. DISCUSSION: Few trials have examined whether commercial weight loss programmes result in glycemic benefits for their participants, particularly among overweight and obese individuals without T2DM. Jenny Craig, Nutrisystem and OPTIFAST show promising glycemic lowering benefits for patients with T2DM, although additional studies are needed to confirm these conclusions. © 2016 World Obesity.

Immunocytochemical localization of the beta(3) subunit of the gamma-aminobutyric acid(A) receptor in the rat brain.

A novel anti-beta(3) subunit-specific GABA(A) receptor (GABA(A)R) antibody has been prepared by immunizing a rabbit with a bacterial fusion protein of the large intracellular loop of the beta(3) subunit. The antiserum immunoprecipitated the solubilized GABA(A) receptor. The anti-beta(3) antibody was affinity purified on immobilized beta(3) large intracellular loop peptide. In immunoblots, the purified antibody reacted with a 57 KDa peptide. Immunocytochemistry with the affinity-purified antibody has revealed the localization of the beta(3) subunit in the rat brain. A comparative study with the immunocytochemical distribution of the beta(2) subunit has also been performed. There are areas of the brain and cell types where the distribution of beta(2) and beta(3) overlap (i.e., cerebral cortex, cerebellum,and most layers of the olfactory bulb). There are also clear differences in the expression of beta(3) and beta(2) in other brain areas and cell types. Thus, high beta(3) but low or no beta(2) expression was observed in the corpus striatum and in granule cells of the olfactory bulb. In the hippocampus the expression of beta(3) was considerably higher than that of beta(2), but some hippocampal interneurons showed high expression of beta(2). High beta(2) but little or no expression of beta(3) was observed in thalamic nuclei, substantia nigra, globus pallidus, inferior colliculus and the short axon cells of the olfactory bulb.

Baclofen induces catatonia in rats.

Baclofen (10 and 20 mg/kg, i.p.) induced catatonia in rats within 10 min of its administration and the effect lasted for 3 hr. Muscimol (100 ng i.c.v. or 1 mg/kg, i.p.) as well as GABA (5 micrograms i.c.v.) potentiated the effect without producing any effect per se. Bicuculline, bromocriptine and scopolamine failed to modify the catatonia induced by baclofen, thereby ruling out the involvement of GABAA receptors, dopaminergic and cholinergic mechanisms. However, GABAB receptor antagonists, such as homotaurine and delta-amino-n-valeric acid, reversed the catatonia induced by baclofen in rats. Since baclofen is known to bind to a subpopulation of GABA receptors (bicuculline-insensitive) and baclofen-induced catatonia was susceptible to reversal by homotaurine and delta-amino-n-valeric acid, it is suggested that this effect could be mediated through GABAB receptors.

Unsulfated and sulfated neurosteroids differentially modulate the binding characteristics of various radioligands of GABA(A) receptors following chronic ethanol administration.

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) inhibited the binding of [(3)H]flunitrazepam (2 nM), [(3)H]muscimol (5 nM) and 4 nM [(35)S]t-butylbicyclophosphorothionate [(35)S]TBPS in the rat cerebellum as well as cerebral cortex. DHEAS-induced inhibition of binding of these radioligands (62% to 100%) was more pronounced as compared to that in the case of DHEA (5% to 31%). DHEAS, unlike DHEA, inhibited [(3)H]flunitrazepam binding significantly to a lesser extent in the cerebellum of ethanol-dependent rats as compared to the control group (I(max):82+/-1vs.92+/-2%, p<0.005). However, DHEA, unlike DHEAS, inhibited [(35)S]TBPS binding to a greater extent in the ethanol-dependent rat cerebellum as compared to the control group (I(max):31+/-2vs.19+/-2%, p<0.005). Furthermore, DHEA was more potent in inhibiting [(35)S]TBPS binding in the cerebellum (IC(50):55+/-5 vs. 74+/-7 microM, p<0.05) and cerebral cortex (IC(50):26+/-4vs.64+/-9 microM, p<0.05) of ethanol-dependent rats as compared to the control group. These observations indicate that unsulfated and sulfated androstane-steroids modulate the GABA(A) receptors in the control as well as the ethanol-dependent rats differentially, and also suggest that the androstane-steroid binding sites associated with the GABA(A) receptors play an important role during ethanol dependence.

The alpha 4 subunit of the GABAA receptors from rat brain and retina.

A novel anti-alpha 4 antibody has been used for the purification and characterization of the alpha 4-containing GABAA receptors in the rat brain and for studying the immunocytochemical distribution of the alpha 4 subunit peptide in rat brain and retina. The anti-alpha 4 antibody recognized a 66 kDa peptide in brain membranes and immunoprecipitated 10-28% of the brain GABAA receptors in various brain regions as determined by [3H]muscimol binding. The highest immunoprecipitation values were obtained in the thalamus and the lowest in the cerebellum. Surprisingly, the receptors immunoprecipitated by anti-alpha 4 showed little or no diazepam-insensitive or diazepam-sensitive [3H]Ro15-4513 binding sites in any brain region. In the cerebellum, where 25% of the [3H]Ro15-4513 binding is diazepam-insensitive, much of the latter was immunoprecipitated by an anti-alpha 6 antibody but not by the anti-alpha 4 antibody. Immunoblots of immunoaffinity-purified GABAA receptors from the cerebral cortex on immobilized anti-alpha 4 revealed molecular colocalization of alpha 4 and gamma 2. However, the absence of significant benzodiazepine binding in these GABAA receptors suggests that the assembly of the alpha 4 and gamma 2 subunits in the cerebral cortex and in other brain regions is such that they do not normally form diazepam-insensitive [3H]Ro15-4513 binding sites. This result contrasts with the presence of diazepam-insensitive [3H]Ro15-4513 binding sites in the GABAA receptors expressed in heterologous systems resulting from the combination of alpha 4, gamma 2 and beta 2 subunits. Immunocytochemistry has revealed the abundance of alpha 4 peptide immunoreactivity in the thalamus and dentate gyrus (mainly in the hilar neurons and the inner third of the granule cell layer). The alpha 4 immunoreactivity is also present in the external plexiform layer of the olfactory bulb and in all layers of the neocortex and pyriform cortex. In the retina, alpha 4 is concentrated on ganglion cells (including some giant ganglion cells), the inner plexiform layer and to a lesser extent in the outer plexiform layer.