Roles of Salmonella enterica serovar Typhimurium encoded Peptidase N during systemic infection of Ifnγ(-/-) mice.

Pathogen encoded peptidases are known to be important during infection; however, their roles in modulating host responses in immunocompromised individuals are not well studied. The roles of S. typhimurium (WT) encoded Peptidase N (PepN), a major aminopeptidase and sole M1 family member, was studied in mice lacking Interferon-γ (IFNγ), a cytokine important for immunity. S. typhimurium lacking pepN (ΔpepN) displays enhanced colony forming units (CFU) compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ(-/-) mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ΔpepN. Concomitantly, reintroduction of pepN in ΔpepN (ΔpepN/pepN) reduces CFU, demonstrating pepN-dependence. Interestingly, expression of a catalytically inactive PepN (ΔpepN/E298A) also lowers CFU, demonstrating that the decrease in CFU is independent of the catalytic activity of PepN. In addition, three distinct differences are observed between infection of C57BL/6 and Ifnγ(-/-) mice: First, serum amounts of TNFα and IL1β post infection are significantly lower in Ifnγ(-/-) mice. Second, histological analysis of C57BL/6 mice reveals that damage in spleen and liver upon infection with WT or ΔpepN is greater compared to ΔpepN/pepN or ΔpepN/E298A. On the other hand, Ifnγ(-/-) mice are highly susceptible to organ damage by all strains of S. typhimurium used in this study. Finally, greater survival of C57BL/6, but not Ifnγ(-/-) mice, is observed upon infection with ΔpepN/pepN or ΔpepN/E298A. Overall, the roles of the host encoded IFNγ during infection with S. typhimurium strains with varying degrees of virulence are highlighted.

ATP release and autocrine signaling through P2X4 receptors regulate gamma delta T cell activation

Purinergic signaling plays a key role in a variety of physiological functions, including regulation of immune responses. Conventional alpha beta T cells release ATP upon TCR cross-linking; ATP binds to purinergic receptors expressed by these cells and triggers T cell activation in an autocrine and paracrine manner. Here, we studied whether similar purinergic signaling pathways also operate in the ``unconventional'' gamma delta T lymphocytes. We observed that gamma delta T cells purified from peripheral human blood rapidly release ATP upon in vitro stimulation with anti-CD3/CD28-coated beads or IPP. Pretreatment of gamma delta T cells with (10)panx-1, CBX, or Bf A reversed the stimulation-induced increase in extracellular ATP concentration, indicating that panx-1, connexin hemichannels, and vesicular exocytosis contribute to the controlled release of cellular ATP. Blockade of ATP release with (10)panx-1 inhibited Ca2+ signaling in response to TCR stimulation. qPCR revealed that gamma delta T cells predominantly express purinergic receptor subtypes A2a, P2X1, P2X4, P2X7, and P2Y11. We found that pharmacological inhibition of P2X4 receptors with TNP-ATP inhibited transcriptional up-regulation of TNF-alpha and IFN-gamma in gamma delta T cells stimulated with anti-CD3/CD28-coated beads or IPP. Our data thus indicate that purinergic signaling via P2X4 receptors plays an important role in orchestrating the functional response of circulating human gamma delta T cells. J. Leukoc. Biol. 92: 787-794; 2012.

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/ mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2(-/-) mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnf alpha and Ifn gamma in sera are not significantly affected, Nos2(-/-) mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.

Extracts of Strawberry Fruits Induce Intrinsic Pathway of Apoptosis in Breast Cancer Cells and Inhibits Tumor Progression in Mice

Background: The consumption of berry fruits, including strawberries, has been suggested to have beneficial effects against oxidative stress mediated diseases. Berries contain multiple phenolic compounds and secondary metabolites that contribute to their biological properties. Methodology/Principal Findings: Current study investigates the anticancer activity of the methanolic extract of strawberry (MESB) fruits in leukaemia (CEM) and breast cancer (T47D) cell lines ex vivo, and its cancer therapeutic and chemopreventive potential in mice models. Results of MTT, trypan blue and LDH assays suggested that MESB can induce cytotoxicity in cancer cells, irrespective of origin, in a concentration-and time-dependent manner. Treatment of mice bearing breast adenocarcinoma with MESB blocked the proliferation of tumor cells in a time-dependent manner and resulted in extended life span. Histological and immunohistochemical studies suggest that MESB treatment affected tumor cell proliferation by activating apoptosis and did not result in any side effects. Finally, we show that MESB can induce intrinsic pathway of apoptosis by activating p73 in breast cancer cells, when tumor suppressor gene p53 is mutated. Conclusions/Significance: The present study reveals that strawberry fruits possess both cancer preventive and therapeutic values and we discuss the mechanism by which it is achieved.

Studies on reactivation of regressing bonnet monkey corpus luteum on day 1 of menses: A pilot study

Studies on functional characteristics of the regressing primate corpus luteum (CL) to luteotrophic stimulus on day 1 of the non-fertile menstrual cycle are scarce. Recombinant human luteinizing hormone (rhLH) (20 IU/Kg BW; n = 10) or human chorionic gonadotropin (hCG) (180 IU; n = 6) were administered intravenously to female bonnet monkeys on day 1 of menses. Exogenous treatment of rhLH or hCG caused a significant increase in circulating progesterone (P4) levels 2-4 hours post treatment (P < 0.05). Lutectomy prior to onset of menses confirmed that CL is the site of the increased P4 concentrations. Increased levels of phosphorylated P44/42 MAPK, MKK3/6 activation and concomitant histological changes were observed within 4 hours in CL of monkeys receiving hCG treatment. The results from this study demonstrate the acute progesterone synthesizing capacity of regressing monkey CL after LH or hCG challenge. This has potential implications for interpreting the steroidogenic response after gonadotropin stimulation tests in the early follicular phase of the normal ovulatory and anovulatory women undergoing controlled ovarian stimulation protocols as part of assisted reproductive technology (ART) and in women with polycystic ovarian syndrome.

In vitro cytotoxicity and in vivo osseointergration properties of compression-molded HDPE-HA-Al2O3 hybrid biocomposites

The aim of this study was to investigate the in vivo biocompatibility in terms of healing of long segmental bone defect in rabbit model as well as in vitro cytotoxicity of eluates of compression-molded High density polyethylene (HDPE)hydroxyapatite (HA)-aluminum oxide (Al2O3) composite-based implant material. Based on the physical property in terms of modulus and strength properties, as reported in our recent publication, HDPE-40 wt % HA and HDPE-20 wt % HA-20 wt % Al2O3 hybrid composites were used for biocompatibility assessment. Osteoblasts cells were cultured in conditioned media, which contains varying amount of composite eluate (0.01, 0.1, and 1.0 wt %). In vitro, the eluates did not exhibit any significant negative impact on proliferation, mineralization or on morphology of human osteoblast cells. In vivo, the histological assessment revealed neobone formation at the bone/implant interface, characterized by the presence of osteoid and osteoblasts. The observation of osteoclastic activity indicates the process of bone remodeling. No inflammation to any noticeable extent was observed at the implantation site. Overall, the combination of in vitro and in vivo results are suggestive of potential biomedical application of compression-molded HDPE- 20 wt % HA- 20 wt % Al2O3 composites to heal long segmental bone defects without causing any toxicity of bone cells.

In vitro and in vivo characterization of designed immunogens derived from the CD-helix of the stem of influenza hemagglutinin

The conserved stem domain of influenza virus hemagglutinin (HA) is a target for broadly neutralizing antibodies and a potential vaccine antigen for induction of hetero-subtypic protection. The epitope of 12D1, a previously reported bnAb neutralizing several H3 subtype influenza strains, was putatively mapped to residues 76-106 of the CD-helix, also referred to as long alpha helix (LAH) of the HA stem. A peptide derivative consisting of wt-LAH residues 76-130 conjugated to keyhole limpet hemocyanin was previously shown to confer robust protection in mice against challenge with influenza strains of subtypes H3, H1, and H5 which motivated the present study. We report the design of multiple peptide derivatives of LAH with or without heterologous trimerization sequences and show that several of these are better folded than wt-LAH. However, in contrast to the previous study immunization of mice with wt-LAH resulted in negligible protection against a lethal homologous virus challenge, while some of the newly designed immunogens could confer weak protection. Combined with structural analysis of HA, our data suggest that in addition to LAH, other regions of HA are likely to significantly contribute to the epitope for 12D1 and will be required to elicit robust protection. In addition, a dynamic, flexible conformation of isolated LAH peptide may be required for eliciting a functional anti-viral response. Proteins 2013; 81:1759-1775. (c) 2013 Wiley Periodicals, Inc.

Early osseointegration of a strontium containing glass ceramic in a rabbit model

The most important property of a bone cement or a bone substitute in load bearing orthopaedic implants is good integration with host bone with reduced bone resorption and increased bone regeneration at the implant interface. Long term implantation of metal-based joint replacements often results in corrosion and particle release, initiating chronic inflammation leading onto osteoporosis of host bone. An alternative solution is the coating of metal implants with hydroxyapatite (HA) or bioglass or the use of bulk bioglass or HA-based composites. In the above perspective, the present study reports the in vivo biocompatibility and bone healing of the strontium (Sr)-stabilized bulk glass ceramics with the nominal composition of 4.5SiO(2)-3Al(2)O(3)-1.5P(2)O(5)-3SrO-2SrF(2) during short term implantation of up to 12 weeks in rabbit animal model. The progression of healing and bone regeneration was qualitatively and quantitatively assessed using fluorescence microscopy, histological analysis and micro-computed tomography. The overall assessment of the present study establishes that the investigated glass ceramic is biocompatible in vivo with regards to local effects after short term implantation in rabbit animal model. Excellent healing was observed, which is comparable to that seen in response to a commercially available implant of HA-based bioglass alone. (C) 2013 Elsevier Ltd. All rights reserved.

Effects of elastase and collagenase on the nonlinearity and anisotropy of porcine aorta

We use enzymatic manipulation methods to investigate the individual and combined roles of elastin and collagen on arterial mechanics. Porcine aortic tissues were treated for differing amounts of time using enzymes elastase and collagenase to cause degradation in substrate proteins elastin and collagen and obtain variable tissue architecture. We use equibiaxial mechanical tests to quantify the material properties of control and enzyme treated tissues and histological methods to visualize the underlying tissue microstructure in arterial tissues. Our results show that collagenase treated tissues were more compliant in the longitudinal direction as compared to control tissues. Collagenase treatment also caused a decrease in the tissue nonlinearity as compared to the control samples in the study. A one hour collagenase treatment was sufficient to cause fragmentation and degradation of the adventitial collagen. In contrast, elastase treatment leads to significantly stiffer tissue response associated with fragmented and incomplete elastin networks in the tissue. Thus, elastin in arterial walls distributes tensile stresses whereas collagen serves to reinforce the vessel wall in the circumferential direction and also contributes to tissue anisotropy. A microstructurally motivated strain energy function based on circumferentially oriented medial fibers and helically oriented collagen fibers in the adventitia is useful in describing these experimental results.

Terminalia paniculata bark extract attenuates non-alcoholic fatty liver via down regulation of fatty acid synthase in high fat diet-fed obese rats

Background: This study was performed to understand the possible therapeutic activity of Terminalia paniculata ethanolic extract (TPEE) on non alcoholic fatty liver in rats fed with high fat diet. Methods: Thirty six SD rats were divided into 6 groups (n = 6): Normal control (NC), high fat diet (HFD), remaining four groups were fed on HFD along with different doses of TPEE (100,150 and 200 mg/kg b.wt) or orlistat, for ten weeks. Liver tissue was homogenized and analyzed for lipid profiles, activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content. Further, the expression levels of FAS and AMPK-1 alpha were also studied in addition to histopathology examination of liver tissue in all the groups. Results: HFD significantly increased hepatic liver total cholesterol (TC), triglycerides (TG), free fatty acids (FFA) and MDA but decreased the activities of SOD and CAT which were subsequently reversed by supplementation with TPEE in a dose-dependent manner. In addition, TPEE administration significantly down regulated hepatic mRNA expression of FAS but up regulated AMPK-1 alpha compared to HFD alone fed group. Furthermore, western blot analysis of FAS has clearly demonstrated decreased expression of FAS in HFD + TPEE (200 mg/kg b. wt) treated group when compared to HFD group at protein level. Conclusions: Our biochemical studies on hepatic lipid profiles and antioxidant enzyme activities supported by histological and expression studies suggest a potential therapeutic role for TPEE in regulating obesity through FAS.

In vitro osteogenic cell proliferation, mineralization, and in vivo osseointegration of injection molded high-density polyethylene-based hybrid composites in rabbit animal model

The present work reports the biocompatibility property of injection molded HDPE-HA-Al2O3 hybrid composites. In vitro cytocompatibility results reveal that osteogenic cell viability and bone mineralization are favorably supported in a statistically significant manner on HDPE-20% HA-20% Al2O3 composite, in comparison to HDPE-40 wt.% HA or HDPE-40 wt.% Al2O3. The difference in cytocompatibility property is explained in terms of difference in substrate wettability/surface energy and importantly, both the cell proliferation at 7 days or bone mineralization at 21 days on HDPE-20% HA-20% Al2O3 composite are either comparable or better than sintered HA. The progressive healing of cylindrical femoral bone defects in rabbit animal model was assessed by implantation experiments over 1, 4 and 12 weeks. Based on the histological analysis as well as histomorphometrical evaluation, a better efficacy of HDPE-20% HA-20% Al2O3 over high-density polyethylene (HDPE) for bone regeneration and neobone formation at host bone-implant interface was established. Taken together, the present study unequivocally establishes that despite the presence of 20% Al2O3, HDPE-based hybrid composites are as biocompatible as HA in vitro or better than HDPE in vivo.

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.

Methylation Silencing of ULK2, an Autophagy Gene, Is Essential for Astrocyte Transformation and Tumor Growth

Glioblastoma (GBM) is the most aggressive type of brain tumor and shows very poor prognosis. Here, using genome-wide methylation analysis, we show that G-CIMP+ and G-CIMP-subtypes enrich distinct classes of biological processes. One of the hypermethylated genes in GBM, ULK2, an upstream autophagy inducer, was found to be down-regulated in GBM. Promoter hypermethylation of ULK2 was confirmed by bisulfite sequencing. GBM and glioma cell lines had low levels of ULK2 transcripts, which could be reversed upon methylation inhibitor treatment. ULK2 promoter methylation and transcript levels showed significant negative correlation. Ectopic overexpression of ULK2-induced autophagy, which further enhanced upon nutrient starvation or temozolomide chemotherapy. ULK2 also inhibited the growth of glioma cells, which required autophagy induction as kinase mutant of ULK2 failed to induce autophagy and inhibit growth. Furthermore, ULK2 induced autophagy and inhibited growth in Ras-transformed immortalized Baby Mouse Kidney (iBMK) ATG5(+/+) but not in autophagy-deficient ATG5(-/-) cells. Growth inhibition due to ULK2 induced high levels of autophagy under starvation or chemotherapy utilized apoptotic cell death but not at low levels of autophagy. Growth inhibition by ULK2 also appears to involve catalase degradation and reactive oxygen species generation. ULK2 overexpression inhibited anchorage independent growth, inhibited astrocyte transformation in vitro and tumor growth in vivo. Of all autophagy genes, we found ULK2 and its homologue ULK1 were only down-regulated in all grades of glioma. Thus these results altogether suggest that inhibition of autophagy by ULK1/2 down-regulation is essential for glioma development.

Evaluation of anti-obesity activities of ethanolic extract of Terminalia paniculata bark on high fat diet-induced obese rats

Background: The prevalence and severity of obesity and associated co-morbidities are rapidly increasing across the world. Natural products-based drug intervention has been proposed as one of the crucial strategies for management of obesity ailments. This study was designed to investigate the anti-obesity activities of ethanolic extract of Terminalia paniculata bark (TPEE) on high fat diet-induced obese rats. Methods: LC-MS/MS analysis was done for ethanolic extract of T. paniculata bark. Male Sprague-Dawley (SD) rats were randomly divided into six groups of six each, normal diet fed (NC), high fat diet-fed (HFD), HFD+ orlistat (standard drug control) administered, and remaining three groups were fed with HFD + TPEE in different doses (100,150 and 200 mg/kg b. wt). For induction of obesity rats were initially fed with HFD for 9 weeks, then, (TPEE) was supplemented along with HFD for 42 days. Changes in body weight, body composition, blood glucose, insulin, tissue and serum lipid profiles, atherogenic index, liver markers, and expression of adipogenesis-related genes such as leptin, adiponectin, FAS, PPARgamma, AMPK-1alpha and SREBP-1c, were studied in experimental rats. Also, histopathological examination of adipose tissue was carried out. Results: Supplementation of TPEE reduced significantly (P < 0.05) body weight, total fat, fat percentage, atherogenic index, blood glucose, insulin, lipid profiles and liver markers in HFD-fed groups, in a dose-dependent manner. The expression of adipogenesis-related genes such as Leptin, FAS, PPARgamma, and SREBP-1c were down regulated while Adiponectin and AMPK-1alpha were up regulated in TPEE + HFD-fed rats. Furthermore, histopathological examination of adipose tissue revealed the alleviating effect of TPEE which is evident by reduced size of adipocytes. Conclusions: Together, the biochemical, histological and molecular studies unambiguously demonstrate the potential anti adipogenic and anti obesity activities of TPEE promoting it as a formidable candidate to develop anti obesity drug.

A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented Delta mimG: Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant Msm Rv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-kappa B, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.