The Implant-Induced Foreign Body Response Is Limited by CD13-Dependent Regulation of Ubiquitination of Fusogenic Proteins.

Implanted medical devices, from artificial heart valves and arthroscopic joints to implantable sensors, often induce a foreign body response (FBR), a form of chronic inflammation resulting from the inflammatory reaction to a persistent foreign stimulus. The FBR is characterized by a subset of multinucleated giant cells (MGCs) formed by macrophage fusion, the foreign body giant cells (FBGCs), accompanied by inflammatory cytokines, matrix deposition, and eventually deleterious fibrotic implant encapsulation. Despite efforts to improve biocompatibility, implant-induced FBR persists, compromising the utility of devices and making efforts to control the FBR imperative for long-term function. Controlling macrophage fusion in FBGC formation presents a logical target to prevent implant failure, but the actual contribution of FBGCs to FBR-induced damage is controversial. CD13 is a molecular scaffold, and in vitro induction of CD13KO bone marrow progenitors generates many more MGCs than the wild type, suggesting that CD13 regulates macrophage fusion. In the mesh implant model of FBR, CD13KO mice produced significantly more peri-implant FBGCs with enhanced TGF-ß expression and increased collagen deposition versus the wild type. Prior to fusion, increased protrusion and microprotrusion formation accompanies hyperfusion in the absence of CD13. Expression of fusogenic proteins driving cell-cell fusion was aberrantly sustained at high levels in CD13KO MGCs, which we show is due to a novel CD13 function, to our knowledge, regulating ubiquitin/proteasomal protein degradation. We propose CD13 as a physiologic brake limiting aberrant macrophage fusion and the FBR, and it may be a novel therapeutic target to improve the success of implanted medical devices. Furthermore, our data directly implicate FBGCs in the detrimental fibrosis that characterizes the FBR.

A comprehensive profiling of quorum quenching by bacterial pigments identifies quorum sensing inhibition and antibiofilm action of prodigiosin against Acinetobacter baumannii.

Bacterial pigments represent a diverse group of secondary metabolites, which confer fitness advantages to the producers while residing in communities. The bioactive potential of such metabolites, including antimicrobial, anticancer, and immunomodulation, are being explored. Reckoning that a majority of such pigments are produced in response to quorum sensing (QS) mediated expression of biosynthetic gene clusters and, in turn, influence cell-cell communication, systemic profiling of the pigments for possible impact on QS appears crucial. A systemic screening of bacterial pigments for QS-inhibition combined with exploration of antibiofilm and antimicrobial action against Acinetobacter baumannii might offer viable alternatives to combat the priority pathogen. Major bacterial pigments are classified (clustered) based on their physicochemical properties, and representatives of the clusters are screened for QS inhibition. The screen highlighted prodigiosin as a potent quorum quencher, although its production from Serratia marcescens appeared to be QS-independent. In silico analysis indicated potential interactions between AbaI and AbaR, two major QS regulators in A. baumannii, and prodigiosin, which impaired biofilm formation, a major QS-dependent process in the bacteria. Prodigiosin augmented antibiotic action of ciprofloxacin against A. baumannii biofilms. Cell viability analysis revealed prodigiosin to be modestly cytotoxic against HEK293, a non-cancer human cell line. While developing dual-species biofilm, prodigiosin producer S. marcescens significantly impaired the fitness of A. baumannii. Enhanced susceptibility of A. baumannii toward colistin was also noted while growing in co-culture with S. marcescens. Antibiotic resistant isolates demonstrated varied responsiveness against prodigiosin, with two resistant strains demonstrating possible collateral sensitivity. Collectively, the results underpin the prospect of a prodigiosin-based therapeutic strategy in combating A. baumannii infection.

A structural and functional comparison between two recombinant human lubricin proteins: Recombinant human proteoglycan-4 (rhPRG4) vs ECF843.

Proteoglycan 4 (PRG4, lubricin) is a mucin-like glycoprotein present on the ocular surface that has both boundary lubricating and anti-inflammatory properties. Full-length recombinant human PRG4 (rhPRG4) has been shown to be clinically effective in improving signs and symptoms of dry eye disease (DED). In vitro, rhPRG4 has been shown to reduce inflammation-induced cytokine production and NFκB activity in corneal epithelial cells, as well as to bind to and inhibit MMP-9 activity. A different form of recombinant human lubricin (ECF843), produced from the same cell line as rhPRG4 but manufactured using a different process, was recently assessed in a DED clinical trial. However, ECF843 did not significantly improve signs or symptoms of DED compared to vehicle. Initial published characterization of ECF843 showed it had a smaller hydrodynamic diameter and was less negatively charged than native PRG4. Further examination of the structural and functional properties of ECF843 and rhPRG4 could contribute to the understanding of what led to their disparate clinical efficacy. Therefore, the objective of this study was to characterize and compare rhPRG4 and ECF843 in vitro, both biophysically and functionally. Hydrodynamic diameter and charge were measured by dynamic light scattering (DLS) and zeta potential, respectively. Size and molecular weight was determined for individual species by size exclusion chromatography (SEC) with in-line DLS and multi-angle light scattering (MALS). Bond structure was measured by Raman spectroscopy, and sedimentation properties were measured by analytical ultracentrifugation (AUC). Functionally, MMP-9 inhibition was measured using a commercial MMP-9 activity kit, coefficient of friction was measured using an established boundary lubrication test at a latex-glass interface, and collagen 1-binding ability was measured by quart crystal microbalance with dissipation (QCMD). Additionally, the ability of rhPRG4 and ECF843 to inhibit urate acid crystal formation and cell adhesion was assessed. ECF843 had a significantly smaller hydrodynamic diameter and was less negatively charged than rhPRG4, as assessed by DLS and zeta potential. Size was further explored with SEC-DLS-MALS, which indicated that while rhPRG4 had 3 main peaks, corresponding to monomer, dimer, and multimer as expected, ECF843 had 2 peaks that were similar in size and molecular weight compared to rhPRG4's monomer peak and a third peak that was significantly smaller in both size and molar mass than the corresponding peak of rhPRG4. Raman spectroscopy demonstrated that ECF843 had significantly more disulfide bonds, which are functionally determinant structures, relative to the carbon-carbon backbone compared to rhPRG4, and AUC indicated that ECF843 was more compact than rhPRG4. Functionally, ECF843 was significantly less effective at inhibiting MMP-9 activity and functioning as a boundary lubricant compared to rhPRG4, as well as being slower to bind to collagen 1. Additionally, ECF843 was significantly less effective at inhibiting urate acid crystal formation and at preventing cell adhesion. Collectively, these data demonstrate ECF843 and rhPRG4 are significantly different in both structure and function. Given that a protein's structure sets the foundation for its interactions with other molecules and tissues in vivo, which ultimately determine its function, these differences most likely contributed to the disparate DED clinical trial results.

PDGF inhibits BMP2-induced bone healing.

Bone regeneration depends on a pool of bone/cartilage stem/progenitor cells and signaling mechanisms regulating their differentiation. Using in vitro approach, we have shown that PDGF signaling through PDGFRß inhibits BMP2-induced osteogenesis, and significantly attenuates expression of BMP2 target genes. We evaluated outcomes of treatment with two anabolic agents, PDGF and BMP2 using different bone healing models. Targeted deletion of PDGFRß in αSMA osteoprogenitors, led to increased callus bone mass, resulting in improved biomechanical properties of fractures. In critical size bone defects BMP2 treatment increased proportion of osteoprogenitors, while the combined treatment of PDGF BB with BMP2 decreased progenitor number at the injury site. BMP2 treatment induced significant bone formation and increased number of osteoblasts, while in contrast combined treatment with PDGF BB decreased osteoblast numbers. This is in vivo study showing that PDGF inhibits BMP2-induced osteogenesis, but inhibiting PDGF signaling early in healing process does not improve BMP2-induced bone healing.

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells.

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the nuclear factor kappa B (NFκB) pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects tumor necrosis factor α (TNFα)-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells ± TNFα ± rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNFα-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleukins, and transcription factors. TNFα treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNFα and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NFκB activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

Proteoglycan 4 (PRG4) expression and function in dry eye associated inflammation.

Dry eye disease (DED) affects hundreds of millions of people worldwide. It is characterized by the production of inflammatory cytokines and chemokines as well as damaging matrix metalloproteinases (MMPs) at the ocular surface. While proteoglycan 4 (PRG4), a mucin-like glycoprotein present at the ocular surface, is most well known as a boundary lubricant that contributes to ocular surface integrity, it has been shown to blunt inflammation in various cell types, suggesting a dual mechanism of action. Recently, full-length recombinant human PRG4 (rhPRG4) has been shown to improve signs and symptoms of DED in humans. However, there remains a significant need for basic science research on rhPRG4's biological properties and its potential therapeutic mechanisms of action in treating DED. Therefore, the objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine whether exogenous rhPRG4 modulates cytokine and chemokine secretion in response to dry eye associated inflammation (TNFα and IL-1ß), explore interactions between rhPRG4 and MMP-9, and understand how experimental dry eye (EDE) in mice affects PRG4 expression. PRG4 secretion from hTCEpi cells was quantified by Western blot and expression visualized by immunocytochemistry. Cytokine/chemokine production was measured by ELISA and Luminex, while rhPRG4's effect on MMP-9 activity, binding, and expression was quantified using an MMP-9 inhibitor kit, surface plasmon resonance, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Finally, EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and by Western blot in lacrimal gland lysate. In vitro results demonstrate that hTCEpi cells synthesize and secrete PRG4, and PRG4 secretion is inhibited by TNFα and IL-1ß. In response to these pro-inflammatory stresses, exogenous rhPRG4 significantly reduced the stimulated production of IP-10, RANTES, ENA-78, GROα, MIP-3α, and MIG, and trended towards a reduction of MIP-1α and MIP-1ß. The hTCEpi cells were also able to internalize fluorescently-labelled rhPRG4, consistent with a mechanism of action that includes downstream biological signaling pathways. rhPRG4 was not digested by MMP-9, and it did not modulate MMP-9 gene expression in hTCEpi cells, but it was able to bind to MMP-9 and inhibited in vitro activity of exogenous MMP-9 in the presence of human tears. Finally, in vivo results demonstrate that EDE significantly decreased immunolocalization of PRG4 on the corneal epithelium and trended towards a reduction of PRG4 in lacrimal gland lysate. Collectively these results demonstrate rhPRG4 has anti-inflammatory properties on corneal epithelial cells, particularly as it relates to mitigating chemokine production, and is an inhibitor of MMP-9 activity, as well as that in vivo expression of PRG4 can be altered in preclinical models of DED. In conclusion, these findings contribute to our understanding of PRG4's immunomodulatory properties in the context of DED inflammation and provide the foundation and motivation for further mechanistic research of PRG4's properties on the ocular surface as well as expanding clinical evaluation of its ability as a multifunctional therapeutic agent to effectively provide relief to those who suffer from DED.

CD13 is a critical regulator of cell-cell fusion in osteoclastogenesis.

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell-cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.

Coronavirus Receptors as Immune Modulators.

The Coronaviridae family includes the seven known human coronaviruses (CoV) that cause mild to moderate respiratory infections (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1) as well as severe illness and death (MERS-CoV, SARS-CoV, SARS-CoV-2). Severe infections induce hyperinflammatory responses that are often intensified by host adaptive immune pathways to profoundly advance disease severity. Proinflammatory responses are triggered by CoV entry mediated by host cell surface receptors. Interestingly, five of the seven strains use three cell surface metallopeptidases (CD13, CD26, and ACE2) as receptors, whereas the others employ O-acetylated-sialic acid (a key feature of metallopeptidases) for entry. Why CoV evolved to use peptidases as their receptors is unknown, but the peptidase activities of the receptors are dispensable, suggesting the virus uses/benefits from other functions of these molecules. Indeed, these receptors participate in the immune modulatory pathways that contribute to the pathological hyperinflammatory response. This review will focus on the role of CoV receptors in modulating immune responses.

CD13 regulation of membrane recycling: implications for cancer dissemination.

Membrane recycling is critical to numerous cell functions and its dysregulation contributes to cancer and metastasis. We established that activation of the transmembrane molecule aminopeptidase N (ANPEP, also known as CD13) tethers the IQ motif containing, guanosine triphosphate hydrolase activating protein 1 (IQGAP1) scaffolding protein at the plasma membrane, thus stimulating the recycling regulator ADP-ribosylation factor 6 (ARF6) to ensure proper recycling of ß1-integrin and other membrane components impacting cell attachment.

CD13 deficiency leads to increased oxidative stress and larger atherosclerotic lesions.

BACKGROUND AND AIMS: Atherosclerosis is an inflammatory cardiovascular disorder characterized by accumulation of lipid-loaded macrophages in the intima. Prolonged accumulation leads to apoptosis of macrophages and eventually to progression of lesion development. Prevention of macrophage accumulation within the intima has been shown to reduce lesion formation. Since CD13 mediates trafficking of macrophages to sites of injury and repair, we tested the role of CD13 in atherosclerosis. METHODS: CD13+/+Ldlr-/- and CD13-/-Ldlr-/- (low density lipoprotein receptor) mice were fed basal or high fat diet (HFD) for 9, 12 and 15 weeks. Mice were euthanized and aortic roots along with innominate arteries were analyzed for atherosclerotic lesions. Cellular mechanisms were determined in vitro using CD13+/+ and CD13-/- bone marrow derived macrophages (BMDMs) incubated with highly oxidized low-density lipoprotein (oxLDL). RESULTS: At the 9 and 12 week time points, no differences were observed in the average lesion size, but at the 15 week time point, CD13-/-Ldlr-/- mice had larger lesions with exaggerated necrotic areas. CD13+/+ and CD13-/- macrophages endocytosed similar amounts of oxLDL, but CD13-/- macrophages generated higher amounts of oxidative stressors in comparison to CD13+/+ macrophages. This increased oxidative stress was due to increased nitric oxide production in oxLDL treated CD13-/- macrophages. Accumulated oxidative stress subsequently led to accelerated apoptosis and enhanced necrosis of oxLDL treated CD13-/- macrophages. CONCLUSIONS: Contrary to our prediction, CD13 deficiency led to larger atherosclerotic lesions with increased areas of necrosis. Mechanistically, CD13 deficiency led to increased nitric oxide production and consequently, greater oxidative stress.

CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, ß1 integrin recycling, and cell migration.

Cell attachment to the extracellular matrix (ECM) requires a balance between integrin internalization and recycling to the surface that is mediated by numerous proteins, emphasizing the complexity of these processes. Upon ligand binding in various cells, the ß1 integrin is internalized, traffics to early endosomes, and is returned to the plasma membrane through recycling endosomes. This trafficking process depends on the cyclical activation and inactivation of small guanosine triphosphatases (GTPases) by their specific guanine exchange factors (GEFs) and their GTPase-activating proteins (GAPs). In this study, we found that the cell surface antigen CD13, a multifunctional transmembrane molecule that regulates cell-cell adhesion and receptor-mediated endocytosis, also promoted cell migration and colocalized with ß1 integrin at sites of cell adhesion and at the leading edge. A lack of CD13 resulted in aberrant trafficking of internalized ß1 integrin to late endosomes and its ultimate degradation. Our data indicate that CD13 promoted ARF6 GTPase activity by positioning the ARF6-GEF EFA6 at the cell membrane. In migrating cells, a complex containing phosphorylated CD13, IQGAP1, GTP-bound (active) ARF6, and EFA6 at the leading edge promoted the ARF6 GTPase cycling and cell migration. Together, our findings uncover a role for CD13 in the fundamental cellular processes of receptor recycling, regulation of small GTPase activities, cell-ECM interactions, and cell migration.

Cell-intrinsic sphingosine kinase 2 promotes macrophage polarization and renal inflammation in response to unilateral ureteral obstruction.

Sphingosine Kinase-2 (Sphk2) is responsible for the production of the bioactive lipid Sphingosine-1 Phosphate, a key regulator of tissue repair. Here we address the in vivo significance of Sphingosine Kinase -2 in renal inflammation/fibrosis in response to unilateral ureteral obstruction using both genetic and pharmacological strategies. Obstructed kidneys of Sphk2-/- mice showed reduced renal damage and diminished levels of the renal injury markers TGFß1 and αSMA when compared to wild type controls. We found a consistently significant increase in anti-inflammatory (M2) macrophages in obstructed Sphk2-/- kidneys by flow cytometry and a decrease in mRNA levels of the inflammatory cytokines, MCP1, TNFα, CXCL1 and ILß1, suggesting an anti-inflammatory bias in the absence of Sphk2. Indeed, metabolic profiling showed that the pro-inflammatory glycolytic pathway is largely inactive in Sphk2-/- bone marrow-derived macrophages. Furthermore, treatment with the M2-promoting cytokines IL-4 or IL-13 demonstrated that macrophages lacking Sphk2 polarized more efficiently to the M2 phenotype than wild type cells. Bone marrow transplant studies indicated that expression of Sphk2-/- on either the hematopoietic or parenchymal cells did not fully rescue the pro-healing phenotype, confirming that both infiltrating M2-macrophages and the kidney microenvironment contribute to the damaging Sphk2 effects. Importantly, obstructed kidneys from mice treated with an Sphk2 inhibitor recapitulated findings in the genetic model. These results demonstrate that reducing Sphk2 activity by genetic or pharmacological manipulation markedly decreases inflammatory and fibrotic responses to obstruction, resulting in diminished renal injury and supporting Sphk2 as a novel driver of the pro-inflammatory macrophage phenotype.

Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.

Detection of hepatitis B virus infection: A systematic review.

AIM: To review published methods for detection of hepatitis B virus (HBV) infection. METHODS: A thorough search on Medline database was conducted to find original articles describing different methods or techniques of detection of HBV, which are published in English in last 10 years. Articles outlining methods of detection of mutants or drug resistance were excluded. Full texts and abstracts (if full text not available) were reviewed thoroughly. Manual search of references of retrieved articles were also done. We extracted data on different samples and techniques of detection of HBV, their sensitivity (Sn), specificity (Sp) and applicability. RESULTS: A total of 72 studies were reviewed. HBV was detected from dried blood/plasma spots, hepatocytes, ovarian tissue, cerumen, saliva, parotid tissue, renal tissue, oocytes and embryos, cholangiocarcinoma tissue, etc. Sensitivity of dried blood spot for detecting HBV was > 90% in all the studies. In case of seronegative patients, HBV DNA or serological markers have been detected from hepatocytes or renal tissue in many instances. Enzyme linked immunosorbent assay and Chemiluminescent immunoassay (CLIA) are most commonly used serological tests for detection. CLIA systems are also used for quantitation. Molecular techniques are used qualitatively as well as for quantitative detection. Among the molecular techniques version 2.0 of the CobasAmpliprep/CobasTaqMan assay and Abbott's real time polymerase chain reaction kit were found to be most sensitive with a lower detection limit of only 6.25 IU/mL and 1.48 IU/mL respectively. CONCLUSION: Serological and molecular assays are predominant and reliable methods for HBV detection. Automated systems are highly sensitive and quantify HBV DNA and serological markers for monitoring.

Association of Genital Chlamydia trachomatis Infection with Female Infer-tility, Study in a Tertiary Care Hospital in Eastern India.

BACKGROUND: Chlamydia trachomatis is recognized as one of the most common sexually transmitted pathogen in the world. 50-80% of infected females are asymptomatic. These untreated women are at risk of developing chronic sequelae leading to tubal pathology causing infertility. Infertility is defined as 1 year of unprotected intercourse without pregnancy. It may be primary or secondary. Aim : To find out the association of genital Chlamydia trachomatis infection with female infertility. Materials and Methodology : This case control study has been carried out in collaboration with R. G. Kar Medical College and Institute of Post Graduate Medical Education & Research, India, between July 2012 and June 2013. 40 infertile and 40 pregnant women were enrolled by purposive sampling as per inclusion and exclusion criteria. ELISA test was performed to detect serum IgG and IgA antibody against recombinant analogs of MOMP and 3 different PCR assays were done targeting MOMP and rRNA DNA from DNA extracted from first void urine. Results : IgG seropositivity was significantly higher (15% vs 0%, P=.0255) in cases than controls, though there was no significant difference in the proportion of IgA seropositivity among 2 groups (12.5% vs 2.5%, P=0.2007). Out of 80 samples 2 samples showed the production of amplicons with R1 - R2 primers. Only 1 sample gave positive result with production of amplicons with all the 3 primers used (R1 - R2, CT0005 - CT06 and JM15 - JM16). Conclusion : Persistent C. trachomatis infection must be recognized as a risk factor of infertility in this region of India. The low PCR positivity in FVU sample helps to conclude the diagnostic utility of serological tests in screening of infertile women.

Burden and Correlates of HIV among Men Who Have Sex with Men in West Bengal, India: Analysis of Sentinel Surveillance Data.

BACKGROUND: Little is known about the socio-behavioral risk factors for HIV acquisition among hard-to-reach men who have sex with men (MSM) population in India, particularly from the densely populated eastern part. Thus to measure the burden and correlates of HIV among MSM in West Bengal state of eastern India, a cross-sectional analysis of the national HIV Sentinel Surveillance (HSS) data was conducted. METHODS: In 2011, between July and September, involving all sentinel sites of the state, 1237 consenting MSM were anonymously interviewed and tested for HIV following national guidelines. Using a short, structured questionnaire, information was collected on socio-behavioral factors along with sexual practices and was analyzed to determine burden of HIV and the role of its socio-behavioral correlates on HIV acquisition. RESULTS: Among participants, mean age was 23.4 years, 44.55% were "Kothis" (usually receptive partner) and 25.1% admitted receiving money for sex with man. HIV sero-positivity was 5.09%. Using logistic regression method, for both bivariate and multivariate (with saturated model) analyses, transport-workers [adjusted odds ratio (AOR)=8.95, 95% confidence interval (95%CI): 1.09-73.71), large business-owners/self-employed (AOR=8.46, 95%CI: 1.25-57.49), small business-owners/cultivators (AOR=7.90, 95%CI: 1.67-37.38), those who visited the sentinel site for official purposes (AOR=7.60, 95%CI: 1.21-47.83) and paying money for having sex with men (AOR=3.03, 95%CI: 1.10-8.33) were strongly associated with higher HIV sero-positivity with than their counterparts. Using the parsimonious model for multivariate analysis, Kothis (AOR=4.64, 95%CI: 1.03-20.89), paying (AOR=2.96, 95%CI: 1.15-7.58) or receiving (AOR=2.06, 95%CI: 1.06-3.99) money for having sex with a man were associated with higher risk of HIV. CONCLUSIONS: Focused intervention targeting the high risk MSM subgroups including Kothis, transport-workers, business-owners/self-employed and those who exchanged money for having sex with men, seemed to be the need of the hour for preventing the spread of HIV infection within and from this understudied population.

CD13 restricts TLR4 endocytic signal transduction in inflammation.

Dysregulation of the innate immune response underlies numerous pathological conditions. The TLR4 is the prototypical sensor of infection or injury that orchestrates the innate response via sequential activation of both cell surface and endocytic signaling pathways that trigger distinct downstream consequences. CD14 binds and delivers LPS to TLR4 and has been identified as a positive regulator of TLR4 signal transduction. It is logical that negative regulators of this process also exist to maintain the critical balance required for fighting infection, healing damaged tissue, and resolving inflammation. We showed that CD13 negatively modulates receptor-mediated Ag uptake in dendritic cells to control T cell activation in adaptive immunity. In this study, we report that myeloid CD13 governs internalization of TLR4 and subsequent innate signaling cascades, activating IRF-3 independently of CD14. CD13 is cointernalized with TLR4, CD14, and dynamin into Rab5(+) early endosomes upon LPS treatment. Importantly, in response to TLR4 ligands HMGB1 and LPS, p-IRF-3 activation and transcription of its target genes are enhanced in CD13(KO) dendritic cells, whereas TLR4 surface signaling remains unaffected, resulting in a skewed inflammatory response. This finding is physiologically relevant as ischemic injury in vivo provoked identical TLR4 responses. Finally, CD13(KO) mice showed significantly enhanced IFNß-mediated signal transduction via JAK-STAT, escalating inducible NO synthase transcription levels and promoting accumulation of oxidative stress mediators and tissue injury. Mechanistically, inflammatory activation of macrophages upregulates CD13 expression and CD13 and TLR4 coimmunoprecipitate. Therefore, CD13 negatively regulates TLR4 signaling, thereby balancing the innate response by maintaining the inflammatory equilibrium critical to innate immune regulation.

Designing polymeric microparticulate drug delivery system for hydrophobic drug quercetin.

The aim of this study was to investigate pharmaceutical potentialities of a polymeric microparticulate drug delivery system for modulating the drug profile of poorly water-soluble quercetin. In this research work two cost effective polymers sodium alginate and chitosan were used for entrapping the model drug quercetin through ionic cross linking method. In vitro drug release, swelling index, drug entrapment efficiency, Fourier Transforms Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD) and Differential Scanning Calorimetric (DSC) studies were also done for physicochemical characterization of the formulations. Swelling index and drug release study were done at a pH of 1.2, 6.8 and 7.4 to evaluate the GI mimetic action which entails that the swelling and release of the all the Formulation1 (F1), Formulation2 (F2) and Formulation3 (F3) at pH 1.2 were minimal confirming the prevention of drug release in the acidic environment of stomach. Comparatively more sustained release was seen from the formulations F2 & F3 at pH 6.8 and pH 7.4 after 7 h of drug release profiling. Drug entrapment efficiency of the formulations shows in F1 (D:C:A = 2:5:30) was approximately 70% whereas the increase in chitosan concentration in F2 (D:C:A = 2:10:30) has shown an entrapment efficiency of 81%. But the comparative further increase of chitosan concentration in F3 (D:C:A = 2:15:30) has shown a entrapment of 80% which is not having any remarkable difference from F2. The FTIR analysis of drug, polymers and the formulations indicated the compatibility of the drug with the polymers. The smoothness of microspheres in F2 & F3 was confirmed by Scanning Electron Microscopy (SEM). However F1 microsphere has shown more irregular shape comparatively. The DSC studies indicated the absence of drug-polymer interaction in the microspheres. Our XRD studies have revealed that when pure drug exhibits crystalline structure with less dissolution profile, formulated microparticles can help us to obtain amorphous form of the same drug that is likely to have more dissolution property. The findings of the study suggest that the microsphere formulations were a promising carrier for quercetin delivery and can be considered as a favorable oral controlled release dosage form for hydrophobic drug quercetin.

Molecular mechanisms regulating CD13-mediated adhesion.

CD13/Aminopeptidase N is a transmembrane metalloproteinase that is expressed in many tissues where it regulates various cellular functions. In inflammation, CD13 is expressed on myeloid cells, is up-regulated on endothelial cells at sites of inflammation and mediates monocyte/endothelial adhesion by homotypic interactions. In animal models the lack of CD13 alters the profiles of infiltrating inflammatory cells at sites of ischaemic injury. Here, we found that CD13 expression is enriched specifically on the pro-inflammatory subset of monocytes, suggesting that CD13 may regulate trafficking and function of specific subsets of immune cells. To further dissect the mechanisms regulating CD13-dependent trafficking we used the murine model of thioglycollate-induced sterile peritonitis. Peritoneal monocytes, macrophages and dendritic cells were significantly decreased in inflammatory exudates from global CD13(KO) animals when compared with wild-type controls. Furthermore, adoptive transfer of wild-type and CD13(KO) primary myeloid cells, or wild-type myeloid cells pre-treated with CD13-blocking antibodies into thioglycollate-challenged wild-type recipients demonstrated fewer CD13(KO) or treated cells in the lavage, suggesting that CD13 expression confers a competitive advantage in trafficking. Similarly, both wild-type and CD13(KO) cells were reduced in infiltrates in CD13(KO) recipients, confirming that both monocytic and endothelial CD13 contribute to trafficking. Finally, murine monocyte cell lines expressing mouse/human chimeric CD13 molecules demonstrated that the C-terminal domain of the protein mediates CD13 adhesion. Therefore, this work verifies that the altered inflammatory trafficking in CD13(KO) mice is the result of aberrant myeloid cell subset trafficking and further defines the molecular mechanisms underlying this regulation.

CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle.

Mesenchymal stem cells (MSCs) are multipotent, tissue-resident cells that can facilitate tissue regeneration and thus, show great promise as potential therapeutic agents. Functional MSCs have been isolated and characterized from a wide array of adult tissues and are universally identified by the shared expression of a core panel of MSCs markers. One of these markers is the multifunctional cell surface peptidase CD13 that has been shown to be expressed on human and murine MSCs from many tissues. To investigate whether this universal expression indicates a functional role for CD13 in MSC biology we isolated, expanded and characterized MSCs from bone marrow of wild type (WT) and CD13(KO) mice. Characterization of these cells demonstrated that both WT and CD13(KO) MSCs expressed the full complement of MSC markers (CD29, CD44, CD49e, CD105, Sca1), showed comparable proliferation rates and were capable of differentiating toward the adipogenic and osteogenic lineages. However, MSCs lacking CD13 were unable to differentiate into vascular cells, consistent with our previous characterization of CD13 as an angiogenic regulator. Compared to WT MSCs, adhesion and migration on various extracellular matrices of CD13(KO) MSCs were significantly impaired, which correlated with decreased phospho-FAK levels and cytoskeletal alterations. Crosslinking human MSCs with activating CD13 antibodies increased cell adhesion to endothelial monolayers and induced FAK activation in a time dependent manner. In agreement with these in vitro data, intramuscular injection of CD13(KO) MSCs in a model of severe ischemic limb injury resulted in significantly poorer perfusion, decreased ambulation, increased necrosis and impaired vascularization compared to those receiving WT MSCs. This study suggests that CD13 regulates FAK activation to promote MSC adhesion and migration, thus, contributing to MSC-mediated tissue repair. CD13 may present a viable target to enhance the efficacy of mesenchymal stem cell therapies.