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Neutrophil extracellular traps (NETs) are implicated in host defense and inflammatory pathologies alike. A wide range of pathogen- and host-derived factors are known to induce NETs, yet the knowledge about specific receptor–ligand interactions in this response is limited. We previously reported that macrophage-inducible C-type lectin (Mincle) regulates NET formation. In this article, we identify glycosphingolipid β-glucosylceramide (β-GlcCer) as a specific NET-inducing ligand of Mincle. We found that purified β-GlcCer induced NETs in mouse primary neutrophils in vitro and in vivo, and this effect was abrogated in Mincle deficiency. Cell-free β-GlcCer accumulated in the lungs of pneumonic mice, which correlated with pulmonary NET formation in wild-type, but not in Mincle−/−, mice infected intranasally with Klebsiella pneumoniae. Although leukocyte infiltration by β-GlcCer administration in vivo did not require Mincle, NETs induced by this sphingolipid were important for bacterial clearance during Klebsiella infection. Mechanistically, β-GlcCer did not activate reactive oxygen species formation in neutrophils but required autophagy and glycolysis for NET formation, because ATG4 inhibitor NSC185058, as well as glycolysis inhibitor 2-deoxy-d-glucose, abrogated β-GlcCer–induced NETs. Forced autophagy activation by tamoxifen could overcome the inhibitory effect of glycolysis blockage on β-GlcCer–mediated NET formation, suggesting that autophagy activation is sufficient to induce NETs in response to this metabolite in the absence of glycolysis. Finally, β-GlcCer accumulated in the plasma of patients with systemic inflammatory response syndrome, and its levels correlated with the extent of systemic NET formation in these patients. Overall, our results posit β-GlcCer as a potent NET-inducing ligand of Mincle with diagnostic and therapeutic potential in inflammatory disease settings.

Background: Breast carcinoma is the most common malignancy in female worldwide, leading cause of death in women. Immunohistochemistry plays a very important role in the prognostication and treatment determination of breast carcinoma patients. Objective: The objective of the study was to analyze the immunohistochemical markers in invasive carcinoma of breast and to correlate the expression of hormonal receptors with age of the patient, tumor size, histological grade, and lymph node metastasis. Materials and Methods: The study was conducted on 88 infiltrating ductal breast carcinoma sample in a tertiary care hospital of Southern Assam for a period of 2 year (January2018-December 2019). Data including age, tumor size, and histologic grade and lymph node status retrieved from pathology department. Chi-square was used to determine the statistical significance between estrogen receptors and progesterone receptors (ER/PR) status human epidermal receptor growth factor 2 (HER2/neu) status along with their correlation with various clinicopathological parameters with respect to infiltrating ductal breast carcinoma. Result: The mean age of the patients was 56.6 years. We observed correlation between ER and PR expression with age, tumor size, and tumor grade. There was correlation between HER2/neu expression and age only. None of the markers showed correlation with lymph node involvement (P > 0.05). Conclusion: Our findings showed the importance of biomarkers (ER, PR, and HER2/neu) expression as prognostic factors for therapeutic decision.

The current study explored the bacteriological profile in the tonsillar core in patients undergoing tonsillectomy and compared it with tonsillar surface organisms. The antibiotic sensitivity and resistance patterns were also studied.This prospective and observational study was conducted in the Department of Otorhinolaryngology, Head and Neck surgery in a tertiary care institute, on a total of sixty-one patients clinically diagnosed as chronic tonsillitis and undergoing tonsillectomy. Tonsillar surface culture swabs and core tissue specimen were sent in separate vials. The culture isolates and their antibiotic sensitivity were analysed and compared.Bacterial growth was detected in 61% of the cases, in cultures from the surface or the core tissue or both. 50.8% specimens of core tissue versus 31.1% of tonsillar surface samples revealed bacterial growth. Of the 13 cases in which surface and core both had bacterial culture growth, the same pathogens were identified in 8 cases. Pseudomonas aeruginosa was the most commonly reported organism in both tonsillar core as well as surface swab cultures.Our study shows that surface swab culture is not always a reliable indicator of organisms present in core of tonsils in cases of chronic infection. The surface swabs did not always match the pathogens in the core. The higher prevalence and the variability of pathogenic organisms in the core tissue as compared to the surface suggests that a targeted antibiotic treatment based on surface culture swabs is unlikely to eradicate them.

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P

Macrophages/microglia with M2-activation phenotype are thought to play important anti-inflammatory and tissue reparative functions in the brain, yet the molecular bases of their functions in the CNS remain to be clearly defined. In a preclinical model of neurocysticercosis using brain infection with a parasite Mesocestoides corti, we previously reported the presence of large numbers of M2 cells in the CNS. In this study using female mice, we report that M2 macrophages in the parasite-infected brain display abundant galectin-3 expression. Disease severity was increased in Galectin-3-/- mice correlating with increased neurological defects, augmented cell death and, importantly, massive accumulation of neutrophils and M2 macrophages in the CNS of these mice. Because neutrophil clearance by efferocytosis is an important function of M2 macrophages, we investigated a possible role of galectin-3 in this process. Indeed, galectin-3-deficient M2 macrophages exhibited a defect in efferocytic clearance of neutrophils in vitro Furthermore, adoptive transfer of M2 macrophages from galectin-3-sufficient WT mice reduced neutrophilia in the CNS and ameliorated disease severity in parasite-infected Galectin-3-/- mice. Together, these results demonstrate, for the first time, a novel role of galectin-3 in M2 macrophage function in neutrophil turnover and resolution of inflammatory pathology in the CNS. This likely will have implications in neurocysticercosis and neuroinflammatory diseases.SIGNIFICANCE STATEMENT Macrophages/microglia with M1-activation phenotype are thought to promote CNS pathology, whereas M2-anti-inflammatory phenotype promote CNS repair. However, the mechanisms regulating M2 cell-protective functions in the CNS microenvironment are undefined. The current study reports that helminth infection of the brain induces an increased expression of galectin-3 in M2 macrophages accumulated in the CNS. Using multiple experimental models in vivo and in vitro, they show that galectin-3 in M2 macrophages functions to clear neutrophils accumulated in the CNS. Importantly, galectin-3 in M2 macrophages plays a central role in the containment of neuropathology and disease severity. These results provide a direct mechanistic evidence of the protective function of M2 macrophages in the CNS.

Neutrophil extracellular trap (NET) formation constitutes an important extracellular antimicrobial function of neutrophils that plays a protective role in bacterial pneumonia. Formation of reactive oxygen species (ROS) such as highly diffusible hydrogen peroxide (H(2)O(2)) is a hallmark of oxidative stress during inflammatory lung conditions including pneumonia. However, the impact of exogenous ROS on NET formation and the signaling pathway involved in the process is not completely understood. Here we demonstrate that the ROS-sensing, nonselective, calcium-permeable channel transient receptor potential melastatin 2 (TRPM2) is required for NET formation in response to exogenous H(2)O(2). This TRPM2-dependent H(2)O(2)-mediated NET formation involved components of autophagy and activation of AMPK and p38 MAPK, but not PI3K and AKT. Primary neutrophils from Trpm2(−)(/)(−) mice fail to activate this pathway with a block in NET release and a concomitant decrease in their antimicrobial capacity. Consequently, Trpm2(−)(/)(−) mice were highly susceptible to pneumonic infection with Klebsiella pneumoniae owing to an impaired NET formation and high bacterial burden despite increased neutrophil infiltration in their lungs. These results identify a key role of TRPM2 in regulating NET formation by exogenous ROS via AMPK/p38 activation and autophagy machinery, as well as a protective antimicrobial role of TRPM2 in pneumonic bacterial infection.—Tripathi, J. K., Sharma, A., Sukumaran, P., Sun, Y., Mishra, B. B., Singh, B. B., Sharma, J. Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.

Background Neutrophil extracellular traps (NETs) constitute antimicrobial function of neutrophils but have also been linked to perpetuation of inflammation. Despite this evident physiological relevance, mechanistic understanding of NET formation is poor. In this study, we examined the mechanism by which Mincle, a C-type lectin receptor, regulates NET formation. Methods NET formation, reactive oxygen species, autophagy activation and intracellular signaling pathways were analyzed in Mincle-sufficient and -deficient neutrophils stimulated in vitro with various stimuli and in vivo during Klebsiella infection. Results We found that Mincle mediates NET formation in response to several activation stimuli in vitro and in vivo during pneumoseptic infection with Klebsiella pneumoniae, indicating its regulatory role in NET formation. Mechanistically, we show that attenuated NET formation in Mincle-/- neutrophils correlates with an impaired autophagy activation in vitro and in vivo, whereas reactive oxygen species (ROS) formation in these neutrophils remained intact. The requirement of autophagy in Mincle-mediated NET formation was further supported by exogenous treatment with autophagy inducer tamoxifen, which rescued the NET formation defect in Mincle-/- neutrophils. Conclusions Our findings identify a previously unrecognized role of Mincle as a regulator of autophagy, which mediates NET formation without affecting ROS generation. Our study addresses a major challenge in the field by positing this pathway to be targeted for modulation of NETs while preserving ROS production, an important innate immune defense.

C-type lectin receptors (CLRs), the carbohydrate-recognizing molecules, orchestrate host immune response in homeostasis and in inflammation. In the present study we examined the function of macrophage galactose-type lectin-1 (MGL1), a mammalian CLR, in pneumonic sepsis, a deadly immune disorder frequently associated with a nonresolving hyperinflammation. In a murine model of pneumonic sepsis using pulmonary infection with Klebsiella pneumoniae, the expression of MGL1 was upregulated in the lungs of K. pneumoniae–infected mice, and the deficiency of this CLR in MGL1−/− mice resulted in significantly increased mortality to infection than in the MGL1-sufficient wild-type mice, despite a similar bacterial burden. The phagocytic cells from MGL1−/− mice did not exhibit any defects in bacterial uptake and intracellular killing and were fully competent in neutrophil extracellular trap formation, a recently identified extracellular killing modality of neutrophils. Instead, the increased susceptibility of MGL1−/− mice seemed to correlate with severe lung pathology, indicating that MGL1 is required for resolution of pulmonary inflammation. Indeed, the MGL1−/− mice exhibited a hyperinflammatory response, massive pulmonary neutrophilia, and an increase in neutrophil-associated immune mediators. Concomitantly, MGL1-deficient neutrophils exhibited an increased influx in pneumonic lungs of K. pneumoniae–infected mice. Taken together, these results show a previously undetermined role of MGL1 in controlling neutrophilia during pneumonic infection, thus playing an important role in resolution of inflammation. To our knowledge, this is the first study depicting a protective function of MGL1 in an acute pneumonic bacterial infection.

Antrochoanal polyp is a benign solitary sinonasal polyp arising within the maxillary sinus and extending into nasopharynx via sinus ostium. It is reported commonly in young adults mainly in 3rd to 5th decades. Antrochoanal polyp accounts 4% to 6% of sinonasal polyps in general population and approximately 33% in paediatric population. Here we are reporting a case of antrochoanal polyp in just six year child because of its relative rarity. We report a case of six year old female patient who presented with left sided nasal obstruction for 3 months. It was associated with nasal discharge. Anterior rhinoscopy showed presence of a single polypoidal mass filling left nasal cavity, posterior extent of the mass could not be assessed clinically due to small age of the patient. Non-contrast computed tomography of nose and paranasal sinuses was done to see the exact extent of the mass. The mass was removed completely by endoscopic approach and histopathologically confirmed as antrochoanal polyp. Antrochoanal polyp should be kept in differential diagnosis when a patient comes with unilateral nasal obstruction and discharge. Though it is commonly seen in young adults, rarely, children younger than 10 years of age can be affected. Complete endoscopic excision is necessary to avoid recurrence.

Sepsis resulting from acute pneumonic infections by Gram-negative bacteria is often characterized by dysfunction of innate immune components. Here we report a previously unrecognized innate protective function of SAP, an adaptor protein primarily reported in T cells, NK cells, and NKT cells, during acute pneumonic infection with Klebsiella pneumoniae (KPn). SAP-deficient mice were highly susceptible to this infection with elevated systemic bacterial spread and increased lung damage. While the overall influx of infiltrating cells in the lungs remained largely intact, increased mortality of SAP-deficient mice correlated with increased accumulation of large NK1.1+ cells harboring bacteria and an impairment of neutrophil extracellular trap formation in vivo during KPn pneumonia, which likely facilitated bacterial outgrowth. Neutrophils were found to express SAP; however, adoptive transfer experiment supported a neutrophil-extrinsic function of SAP in neutrophil extracellular trap formation. Collectively, these data present the first report depicting innate protective function of SAP in an acute pulmonary infection.

Summary Macrophage plasticity is essential for innate immunity, but in-depth signaling mechanism(s) regulating their functional phenotypes are ill-defined. Here we report that interferon (IFN) γ priming of naive macrophages induces store-mediated Ca2+ entry and inhibition of Ca2+ entry impairs polarization to M1 inflammatory phenotype. In vitro and in vivo functional analyses revealed ORAI1 to be a primary contributor to basal Ca2+ influx in macrophages, whereas IFNγ-induced Ca2+ influx was mediated by TRPC1. Deficiency of TRPC1 displayed abrogated IFNγ-induced M1 inflammatory mediators in macrophages. In a preclinical model of peritonitis by Klebsiella pneumoniae infection, macrophages showed increased Ca2+ influx, which was TRPC1 dependent. Macrophages from infected TRPC1−/− mice showed inhibited expression of M1-associated signature molecules. Furthermore, in human patients with systemic inflammatory response syndrome, the level of TRPC1 expression in circulating macrophages directly correlated with M1 inflammatory mediators. Overall, TRPC1-mediated Ca2+ influx is essential for the induction/shaping of macrophage polarization to M1 inflammatory phenotype., Graphical Abstract, Highlights • TRPC1 mediates sterile or infection-induced Ca2+ influx and M1 phenotype in macrophages • ORAI1 mediates the basal Ca2+ influx in macrophages • In patients with SIRS, the TRPC1 level correlates with M1 inflammatory mediators in macrophages, Biological Sciences; Immune Response; Immunology

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of N- vs O-linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for

Tamoxifen induces autophagy-dependent NET formation in CGD patient neutrophils and improves their antimicrobial activity. Given the genetic defect in their ROS-generation machinery, this study highlights repurposing of tamoxifen to potentiate host defense in CGD patients.

Complete cleaning and shaping is the key to successful endodontic treatment. A thorough understanding of the internal anatomy and morphology of the root canal system is an important consideration when performing cleaning and shaping procedures. Mandibular premolars are one of the most difficult teeth to treat endodontically because of aberrant root canal anatomy. This article describes case series of mandibular premolars with variations in root canal anatomy treated successfully by conventional endodontic treatment.

In continuance of our search for anticancer agents, we report herein the synthesis and anticancer activity of some novel oxadiazole analogues. The compounds were screened for anticancer activity as per National Cancer Institute (NCI US) protocol on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers cell lines.N-(2,4-Dimethylphenyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine (4s) showed maximum activity with mean growth percent (GP) of 62.61 and was found to be the most sensitive on MDA-MB-435 (melanoma), K-562 (leukemia), T-47D (breast cancer), and HCT-15 (colon cancer) cell lines with GP of 15.43, 18.22, 34.27, and 39.77, respectively. Maximum GP was observed on MDA-MB-435 (melanoma) cell line (GP=6.82) by compoundN-(2,4-dimethylphenyl)-5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-amine (4u).

Lower respiratory tract infection with bacteria can lead to sepsis development, which is a complex immune disorder characterized by a systemic hyperinflammation. There are currently no effective therapies for sepsis, which results in 750 000 hospitalizations annually in the United States and has a mortality rate of 20%–50% [1, 2]. Nosocomial infections with the opportunistic pathogen Klebsiella pneumoniae account for 5%–20% of cases of gram-negative bacterial sepsis [3–5]. Additionally, emergence of multidrug-resistant isolates of K. pneumoniae in clinical settings is a serious health concern. Because innate mucosal immunity plays a direct role in bacterial killing and immunomodulation in this acute infection [6–10], an understanding of functioning of host innate immune components might provide targets for modulation of the host immune system in a beneficial manner. Mincle is a C-type lectin receptor (CLR) belonging to the dectin-2 subfamily of innate immune receptors that can function as an activating receptor for host-associated molecular patterns (alarmins) and pathogen-associated molecular patterns (PAMPs) [11, 12]. It is an inducible receptor, expressed mainly by myeloid cells such as macrophages, neutrophils, myeloid dendritic cells, and some B-cell subsets [11, 13, 14]. Functional analysis of this receptor in macrophages has received the most attention, where its association with FcRγ activates downstream signaling cascades involving Syk kinases, resulting in induction of protective inflammatory response [15–17]. While the function of Mincle in chronic bacterial infections such as tuberculosis and fungal infections was examined in these studies, its role in acute pneumonic infections leading to sepsis development has not been explored. Furthermore, its functions other than as an inflammatory pattern-recognition receptor (PRR) have received little, if any, attention. Neutrophil-mediated responses are essential for combating pneumonic bacterial infection, and their protective role in sepsis and K. pneumoniae infection in particular has been described [18, 19]. The professional antimicrobial program of neutrophils mainly constitutes phagocytosis of infectious agents, followed by production of noxious agents, such as reactive oxygen species, that kill the internalized microbes. Another recently established mechanism of microbial killing by neutrophils is by their formation of extracellular traps (NETs), which are DNA fibrils expelled by neutrophils and are decorated with granular contents, such as various proteases, that can ensnare and kill microbes without phagocytosis [20–22]. Mincle has been shown to be expressed by neutrophils, and although it plays a role in neutrophil-mediated protective responses against Candida species and mycobacteria [23, 24], its direct role in bacterial phagocytosis and NET formation is not known. Because neutrophils are a key cell type in controlling K. pneumoniae infection, Mincle signaling in neutrophils may be a key event in control of K. pneumoniae infection and sepsis. In this study, we examined the role of Mincle in acute K. pneumoniae infection causing pneumonic sepsis. Our results suggest a novel protective function of Mincle as a nonopsonic phagocytic receptor for bacteria and in regulation of NET formation, indicating the importance of this CLR in neutrophil-specific bacterial clearance mechanisms in pneumonic infections.

Pneumonia is frequently associated with sepsis, characterized by a nonresolving hyperinflammation. However, specific host components of the pulmonary milieu that regulate the perpetuation of inflammation and tissue destruction observed in this immune disorder are not clearly understood. We examined the function of Clec4d, an orphan mammalian CLR, in Gram negative pneumonic sepsis caused by KPn. Whereas the WT mice infected with a sublethal dose of bacteria could resolve the infection, the Clec4d−/− mice were highly susceptible with a progressive increase in bacterial burden, hyperinflammatory response typical of sepsis, and severe lung pathology. This correlated with a massive accumulation of neutrophils in lungs of infected Clec4d−/− mice, which was in contrast with their WT counterparts, where neutrophils transiently infiltrated the lungs. Interestingly, the Clec4d−/− neutrophils did not exhibit any defect in bacterial clearance. These results suggest that Clec4d plays an important role in resolution of inflammation, possibly by facilitating neutrophil turnover in lungs. This is the first report depicting the physiological function of Clec4d in a pathological condition. The results can have implications not only in sepsis but also in other inflammatory diseases, where nonresolving inflammation is the root cause of disease development.

Background: Codon usage bias is the unequal use of synonymous codons; some codons are more preferred than others. However codon usage pattern is different among genes and among organisms. The bias is found more often in highly expressed genes. The greatest diversity of insects among the animal kingdom makes insects appropriate for co- don bias study at variable evolutionary time scales. Result and Discussion: we analyzed pattern of codon usage in two insect species of Lepidopteran order. The ENC values range from 51 to 60 with a mean value of 57.75 in B. mandarina but from 52 to 60 with a mean value of 57.17 in O. furnacalis, respectively. In B. mandarina, a significant positive correlation was observed between A% and A3% (r = 0.608**, p 0.05), T % and C3 % ( r = - 0.871**p

The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent Francisella novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant. EF-Tu was immunodominant in mice vaccinated and protected from virulent F. novicida. Moreover, recombinant EF-Tu induced macrophages to produce inflammatory cytokines in a TLR4 dependent manner. This study shows immune stimulatory properties of a cytoplasmic protein EF-Tu expressed on the membrane of virulent Francisella strains.

The macrophage is a versatile cell type that can sense and respond to a particular need based on the conditions of the microenvironment. Some studies have recently suggested that pathogens can directly influence the polarization of macrophages. As Francisella infections are characterized by intense necrotic infiltrates in the lung as well as in distal sites of infection, we sought to investigate whether pulmonary Francisella infections could cause the polarization of alternatively activated macrophages (M2/aaMs). Our results indicate that Francisella infections can cause the polarization of M2/aaM in vivo and that macrophages can be polarized toward an M2/aaM phenotype more potently if dead cell debris is used for stimulation in the presence and absence of Francisella infections. Finally, we also demonstrate that efferocytosis is inhibited in macrophages infected with Francisella, thus providing a potential explanation for the lack of clearance and eventual accumulation of dead cell debris associated with this disease.

Neutrophil extracellular trap (NET) formation represents an important antimicrobial function of neutrophils, which partly depends on reactive oxygen species (ROS) and autophagy activation. However, the factors governing these processes remain unknown. In this study, we demonstrate that nonselective calcium permeable cation channel Transient receptor potential melastatin 2 (TRPM2), senses ROS and contributes to the NET formation. The NET forming function of TRPM2 in response to ROS stimulation requires autophagy activation through ADP ribose, AMPK phosphorylation and p38 MAPK activation. TRPM2 deficient neutrophils fail to activate this pathway with a block of NET release and a concomitant impairment of antimicrobial capacity. Trpm2−/− mice were found to be highly susceptible to pneumonic infection with Klebsiella pneumoniae owing to an impaired NET formation and high bacterial burden despite increased neutrophil infiltration in their lungs. Taken together our data identifies a key role of TRPM2 in NET formation by ROS sensing and links it to autophagy activation via AMPK/p38 pathway. Our study also provides evidence for a protective anti-microbial role of TRPM2 in pneumonic bacterial infection thus positing TRPM2-mediated NET formation as a valuable target for modulating NETs and related pathologies.

Respiratory Francisella infections induce a delayed innate immune response followed by a severe sepsis like condition. In this study, mice infected intranasally with Francisella novicida showed a depletion of alphabeta T cells in lungs while exhibiting large accumulations of other leukocytes correlating with disease severity. The depleted T cells were predominantly CD4(+). The alphabeta T cells in infected mice showed significantly higher levels of Annexin V binding than those in mock control mice suggesting increased apoptosis of T cells. These results suggest that lack of transition from an innate to adaptive host response is associated with lethality of respiratory tularemia.

In neurocysticercosis, parasite-induced immune suppressive effects are thought to play an important role in enabling site-specific inhibition of inflammatory responses to infections. It is axiomatic that microglia-mediated (M1 proinflammatory) response causes central nervous system inflammation; however, the mechanisms by which helminth parasites modulate microglia activation remain poorly understood. Here, we show that microglia display a diminished expression of M1-inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide synthase 2 (NOS2) in murine neurocysticercosis. Microglia also exhibited a lack of myeloid cell maturation marker major histocompatibility complex (MHC)-II in these parasite-infected brains. Treatment of microglia with helminth soluble/secreted factors (HSFs) in vitro did not induce expression of M1-inflammatory signature molecule NOS2 as well as MHC-II in primary microglia. However, HSF treatment completely inhibited lipopolysaccharide-induced increase in expression of MHC-II, NOS2 and nitric oxide production in these cells. As epigenetic modulation of chromatin states that regulates recruitment of RNA polymerase II (Pol-II) is a key regulatory step in determining gene expression and functional outcome, we next evaluated whether HSF induced modulation of these phenomenon in microglia in vitro. Indeed, HSF downregulated Pol-II recruitment to the promoter region of TNF-α, IL-6, NOS2, MHC-II, and transcription factor CIITA (a regulator of MHC-II expression), by itself. Moreover, HSF suppressed the lipopolysaccharide-induced increase in Pol-II recruitment as well. In addition, HSF exposure reduced the positive histone marks H3K4Me3 and H3K9/14Ac at the promoter of TNF-α, IL-6, NOS2, MHC-II, and CIITA. These studies provide a novel mechanistic insight into helminth-mediated immune suppression in microglia via modulation of epigenetic processes.

Sepsis is a complex immune disorder that is characterized by systemic hyperinflammation. Alarmins, which are multifunctional endogenous factors, have been implicated in exacerbation of inflammation in many immune disorders including sepsis. Here we show that Galectin-9, a host endogenous β-galactoside binding lectin, functions as an alarmin capable of mediating inflammatory response during sepsis resulting from pulmonary infection with Francisella novicida, a Gram negative bacterial pathogen. Our results show that this galectin is upregulated and is likely released during tissue damage in the lungs of F. novicida infected septic mice. In vitro, purified recombinant galectin-9 exacerbated F. novicida-induced production of the inflammatory mediators by macrophages and neutrophils. Concomitantly, Galectin-9 deficient (Gal-9-/-) mice exhibited improved lung pathology, reduced cell death and reduced leukocyte infiltration, particularly neutrophils, in their lungs. This positively correlated with overall improved survival of F. novicida infected Gal-9-/- mice as compared to their wild-type counterparts. Collectively, these findings suggest that galectin-9 functions as a novel alarmin by augmenting the inflammatory response in sepsis development during pulmonary F. novicida infection.

Using antibodies raised with chlamydial fusion proteins, we have localized a protein encoded by hypothetical open reading frame CT813 in the inclusion membrane of Chlamydia trachomatis . The detection of the C. trachomatis inclusion membrane by an anti-CT813 antibody was blocked by the CT813 protein but not unrelated fusion proteins. The CT813 protein was detected as early as 12 h after chlamydial infection and was present in the inclusion membrane during the entire growth cycle. All tested serovars from C. trachomatis but not other chlamydial species expressed the CT813 protein. Exogenously expressed CT813 protein in HeLa cells displayed a cytoskeleton-like structure similar to but not overlapping with host cell intermediate filaments, suggesting that the CT813 protein is able to either polymerize or associate with host cell cytoskeletal structures. Finally, women with C. trachomatis urogenital infection developed high titers of antibodies to the CT813 protein, demonstrating that the CT813 protein is not only expressed but also immunogenic during chlamydial infection in humans. In all, the CT813 protein is an inclusion membrane protein unique to C. trachomatis species and has the potential to interact with host cells and induce host immune responses during natural infection. Thus, the CT813 protein may represent an important candidate for understanding C. trachomatis pathogenesis and developing intervention and prevention strategies for controlling C. trachomatis infection.

The available chlamydial genome sequences have made it possible to comprehensively analyze host responses to all chlamydial proteins, which is essential for further understanding of chlamydial pathogenesis and development of effective chlamydial vaccines. Microplates arrayed with 156Chlamydia trachomatisfusion proteins were used to evaluate antibody responses in women urogenitally infected withC. trachomatis. Based on both the antibody recognition frequency and titer, seven chlamydial antigens encoded by open reading frames (ORFs) CT089, CT147, CT226, CT681, CT694, CT795, and CT858, respectively, were identified as relatively immunodominant; six of these are encoded by hypothetical ORFs. Antibody binding to these chlamydial fusion proteins was blocked byC. trachomatis-infected but not by normal HeLa cell lysates or irrelevant bacterial lysates. These results have revealed novel immune-reactive chlamydial antigens, not only indicating that the hypothetical ORF-encoded proteins are expressed during chlamydial infection in humans but also providing the proof of principle that the fusion protein-based approach can be used to profile human immune responses to chlamydial infection at the whole-genome scale.

A typical endodontic procedure includes access preparation, root canal preparation and root canal filling. This treatment process is time-consuming and prone to human error. The outcome relies on the clinician's master skills that can be gained only through years of training and practice. In order to improve the quality and reliability of endodontic therapy, an endodontic micro robot is proposed to be built. The objective is to modernize the traditional treatment, moving from a “manual art” to science-based automation. This computer controlled micro machine is mounted on the teeth within patient's mouth. With on-line monitoring and positioning control, the multipurpose micro robot performs automatic treatment procedures, including probing, drilling, filing, cleaning and filling. This article gives an insight to design and applications of the intelligent robotic micro-machine applicable to root canal preparation with on-line monitoring capabilities that will increase the accuracy and efficiency of the treatment.

We have previously identified a chlamydia-secreted protein (designated chlamydial proteasome/protease-like activity factor, or CPAF) in the cytosol of chlamydia-infected cells. Although CPAF is known to degrade host transcription factors required for major histocompatibility complex antigen expression in cultured cells, it is not clear whether CPAF is produced and maintains similar functions in humans infected with chlamydial organisms. We now report that CPAF does not preexist in chlamydial organisms and that CPAF synthesis requires live organism replication in cultured cells. Mice inoculated with live, but not mice inoculated with dead, chlamydial organisms produced a strong antibody response to CPAF, correlating CPAF-specific antibody production with CPAF synthesis in animals. Sera from women diagnosed withChlamydia trachomatiscervicitis displayed higher levels of antibodies to CPAF than to either chlamydial major outer membrane protein or heat shock protein 60, suggesting that CPAF is both produced and immunogenic during human chlamydial infection.

We have previously shown that infection with Chlamydia pneumoniae can significantly exacerbate atherosclerotic lesions in LDLR-/- mice concurrently fed a high cholesterol diet in 6 or 9 months. We now report that a period of 4 month was sufficient for demonstrating the C. pneumoniae atherogenicity. However, heat inactivation of C. pneumoniae organisms completely abolished the ability of C. pneumoniae to exacerbate the atherosclerotic lesions, suggesting that viable organism infection may be required for the C. pneumoniae atherogenicity.

Chlamydia trachomatis is one of the most common bacterial pathogens and is the etiological agent of debilitating sexually transmitted and ocular diseases in humans. The organism is an obligate intracellular prokaryote characterized by a highly specialized biphasic developmental cycle. We have performed genomic transcriptional analysis of the chlamydial developmental cycle. This approach has led to the identification of a small subset of genes that control the primary (immediate-early genes) and secondary (late genes) differentiation stages of the cycle. Immediate-early gene products initiate bacterial metabolism and potentially modify the bacterial phagosome to escape fusion with lysosomes. One immediate early gene (CT147) is a homolog of the human early endosomal antigen-1 that is localized to the chlamydial phagosome; suggesting a functional role for CT147 in establishing the parasitophorous vacuole in a nonfusogenic pathway. Late gene products terminate bacterial cell division and constitute structural components and remodeling activities involved in the formation of the highly disulfide cross-linked outer-membrane complex that functions in attachment and invasion of new host cells. Many of the genes expressed during the immediate-early and late differentiation stages are Chlamydia -specific and have evolutionary origins in eukaryotic lineages.

Alternatively activated macrophages (M2 MΦ) function to dampen host inflammatory responses and promote wound healing and tissue repair. As the blood brain barrier (BBB) restricts the flow of cells into the brain, understanding how M2 MΦ extravagate into the central nervous system (CNS) is of great interest. In a mouse model of neurocysticercosis (NCC) we previously demonstrated that M2 MΦ are essential in containing neuropathology in helminth infected CNS. Analysis of helminth infected mice revealed an abundant expression of galectin-7 that recognize galactose moieties in brain endothelial cells. The Galectin-7−/− NCC mice displayed a reduced accumulation of M2 MΦ in parasite infected brain. This reduction in M2 MΦ numbers in the CNS was correlated with an increased neuropathology and shortened survival. The use of adoptive transfer of fluorescent labeled cells demonstrated a decreased influx of M2 MΦ into the CNS of helminth infected NCC brains. The use of in-vitro transmigration assay conclusively demonstrated a key function of Galectin-7 expressed on endothelial cells in centrally regulating M2 MΦ entry into the CNS.

Calcium influx mediated by plasma membrane calcium permeable cation channels play essential role in immune homeostasis. Transient receptor potential melastatin 2 (Trpm2) is a trp superfamily member cationic channel which is reported to shape several effector functions of myeloid cells. Because of its role in neutrophil chemotaxis, we examined a yet undetermined function of Trpm2 in pathogenesis of Klebsiella pneumoniae (KPn), a causative agent of pneumonic sepsis with a significant neutrophil component. Here we show that Trpm2−/− mice exhibit significantly increased susceptibility to pulmonary KPn infection corresponding with an increased bacterial burden and exacerbated tissue pathology. This correlated with a modulation of local and systemic inflammatory response in-vivo in KPn infected Trpm2−/− mice as well as altered calcium influx, ROS generation and MAP kinase activity in Trpm2 deficient neutrophils compared with their WT counterparts. Our data suggest that Trpm2 regulates KPn pathogenesis by controlling neutrophil mediated inflammation and oxidative stress.