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Introduction: Typhoid incidence in children is higher in urban areas than in rural areas of Bangladesh. This study examined whether healthy urban children harboured higher levels of Salmonella genes than healthy rural children. Methodology: Stool samples from 140 children were studied: 70 from rural areas and 70 from urban metropolitan areas. Results: The stool samples of urban children contained more Salmonella genes (median 4, IQR 3-4) than those of rural children (median 3, IQR 3-4). This suggests that urban Bangladeshi children have more Salmonella genes in their guts than rural children. Especially, in those under 12 months of age, the Salmonella gene prevalence in urban children was unique. They had more Salmonella genes (median 4, IQR 4-5) than rural children in the same age group (median 3, IQR 2.5-4). We also found more Salmonella genes in urban children who drank tap water (median 4, IQR 3-5) than in rural children whose water source was tube well water (median 3, IQR 2-4) and boiled pond water (median 3, IQR 3-3.5). However, there was no significant difference of Salmonella genes between urban children who drank tap-water and children whose water source was a tube well (median 4, IQR 3-4). Conclusions: These data suggest that the urban environment, including the drinking water supply system, increases the likelihood of healthy children in urban areas harbouring more potentially pathogenic Salmonella organisms in their gut than found in rural healthy children.

Inhibition of immune checkpoint proteins like programmed death 1 (PD-1) is a promising therapeutic approach for several cancers, including non-small cell lung cancer (NSCLC). Although PD-1 ligand (PD-L1) expression is used to predict anti-PD-1 therapy responses in NSCLC, its accuracy is relatively less. Therefore, we sought to identify a more accurate predictive blood biomarker for evaluating anti-PD-1 response. We evaluated the frequencies of T cells, B cells, natural killer (NK) cells, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), mononuclear myeloid-derived suppressor cells (M-MDSCs), and Lox-1+ PMN-MDSCs in peripheral blood samples of 62 NSCLC patients before and after nivolumab treatment. Correlation of immune-cell population frequencies with treatment response, progression-free survival, and overall survival was also determined. After the first treatment, the median NK cell percentage was significantly higher in responders than in non-responders, while the median Lox-1+ PMN-MDSC percentage showed the opposite trend. NK cell frequencies significantly increased in responders but not in non-responders. NK cell frequency inversely correlated with that of Lox-1+ PMN-MDSCs after the first treatment cycle. The NK cell-to-Lox-1+ PMN-MDSC ratio (NMR) was significantly higher in responders than in non-responders. Patients with NMRs ≥ 5.75 after the first cycle had significantly higher objective response rates and longer progression-free and overall survival than those with NMRs

Activation of the nuclear factor B (NF-κB) signaling pathway by pattern recognition receptors (PRRs) is regarded as a crucial mechanism of neuroinflammation and brain injury after acute ischemic stroke. The stimulation of alpha-kinase 1 (ALPK1), a newly identified PRR, triggers NF-κB activation and an inflammatory response. Longitudinal population-based genetic epidemiological studies suggest that the ALPK1 gene is a susceptible site to ischemic stroke. However, the function of ALPK1 in the central nervous system remains unclear. The present study explored the role of ALPK1 in acute ischemic stroke.BV2 microglial cells were stimulated with conditioned medium (CM) that was collected from oxygen and glucose deprivation (OGD)-treated HT22 neurons, and a murine brain ischemia model was established to detect the changes of ALPK1 expression. We used lentivirus to knockdown ALPK1 to explore the effects of ALPK1 in cerebral ischemia models in vitro and in vivo.We observed a significant increase of ALPK1 expression in BV2 cells that were stimulated with OGD CM. The knockdown of ALPK1 inhibited the phosphorylation of tumor necrosis factor receptor associated factor-interacting protein with a forkhead-associated domain (TIFA), the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), the activation of NF-κB, and the levels of proinflammatory factors in the BV2 cells. We also verified a neuroprotective effect of ALPK1 knockdown against ischemic brain injury through inhibition of the TIFA/TRAF6/NF-κB pathway and neuroinflammation in mice.This study demonstrates that ALPK1 is implicated in sterile inflammatory injury after acute brain ischemia, which provides first evidence for the therapeutic potential of ALPK1 inhibition in ischemic stroke.

Calcium (Ca2+) is an important element for many physiological functions of the uterus, including embryo implantation. Here, we investigated the possible involvement of altered intracellular Ca2+ levels in decidualization in human endometrial stromal cells (hEMSCs). hEMSCs showed high levels of mesenchymal stem cell marker expression (CD73, CD90, and CD105) and did not express markers of hematopoietic progenitor cells (CD31, CD34, CD45, and HLA-DR). Decidualization is a process of ovarian steroid-induced endometrial stromal cell proliferation and differentiation. Several types of ion channels, which are regulated by the ovarian hormones progesterone and estradiol, as well as growth factors, are important for endometrial receptivity and embryo implantation. The combined application of progesterone (1 μM medroxyprogesterone acetate) and cyclic AMP (0.5 mM) for 6 days not only elevated inositol 1,4,5-triphosphate receptor (IP3R)-mediated Ca2+ release and IP3R expression, it also promoted ORAI and STIM expression as well as cyclopiazonic acid-induced Ca2+ release. Finally, intracellular Ca2+ levels and ion channel gene expression influenced hEMSC proliferation. These results suggest that cytosolic Ca2+ dynamics, mediated by specific ion channels, serve as an important step in the decidualization of hEMSCs.

Alzheimer’s disease (AD) is the most common cause of dementia due to inflammatory neurodegeneration in the brain. Neuroinflammation caused by damage associated molecular pattern (DAMP: Aβ, ATP) leads to neuronal apoptosis. From our study HY209, a NCAIDs and a TGR5 agonist, inhibits neuroinflammation and neurodegeneration. In vitro, HY209 suppressed NLRP3-ASC oligomerization and production of mature IL-1β/IL-18 in microglia cells by Aβ ± ATP. HY209 inhibited Ca++ influx incurred by ATP or BzATP (P2X7 agonists). Ca++ influx of microglia from TGR5 KO was like that of P2X7 KO compared to wild type mice in response to BzATP. Suggesting that TGR5 is essential in P2X7-mediated Ca++ signaling and HY209 might play a role as an inverse agonist for TGR5 colocalized with P2X7. HY209 activates adenylate cyclase and cAMP. cAMP inhibits priming phase of inflammasomal activation, suppresses pro-inflammatory cytokine (TNF-α) production and oligomerization of NLRP3-ASC complex. Indicating that HY209 suppresses both priming and activation phase of NLRP3 inflammasome. In the 5x Familial Alzheimer’s disease (5xFAD) mice model, HY209 (1 mpk, i.p. or 30 mpk, p.o.) improves memory function, decreases Aβ plaque and neuronal apoptosis in the brain. Structure activity relationships were studied to screen out more potent HY209 derivatives (next generation NCAIDs) with increased bioavailability and higher BBB permeability. Considering efficacies of NSAIDs, PPARγ agonists, specific inhibitors (for TNF-α, ROS, RAGE, HMG-CoA, p38 MAPK, P2X7 and NLRP3) targeting limited number of proinflammatory pathways, NCAIDs could ameliorate broad spectrum of neuroinflammation that is crucial in preventing neurodegeneration and improving cognition and memory in AD.

Amyloid β (Aβ) and/or ATP activates NLRP3 inflammasome (N3I) by P2 × 7R ion channel of microglia, which is crucial in neuroinflammation shown in Alzheimer’s disease (AD). Due to polymorphisms, subtypes, and ubiquitous expression of P2 × 7R, inhibition of P2 × 7R has not been effective for AD. We first report that GPCR19 is a prerequisite for P2 × 7R-mediated N3I activation and Taurodeoxycholate (TDCA), a GPCR19 ligand, inhibited the priming phase of N3I activation, suppressed P2 × 7R expression and P2 × 7R-mediated Ca++ mobilization, and N3I oligomerization which is essential for production of IL-1β/IL-18. Further, TDCA increased expression of scavenger receptor (SR) A, enhanced phagocytosis of Aβ, and decreased Aβ plaque numbers in the brain of 5x Familial Alzheimer’s disease (5xFAD) mice. TDCA also reduced microgliosis, prevented neuronal loss, and improved memory function of 5xFAD mice. The pleiotropic roles of GPCR19 in P2 × 7-mediated N3I activation suggest that targeting GPCR19 might resolve neuroinflammation in AD patients.

Mitochondrial defects and antimitochondrial cardiolipin (CL) antibodies are frequently detected in autoimmune disease patients. CL from dysregulated mitochondria activates various pattern recognition receptors, such as NLRP3. However, the mechanism by which mitochondrial CL activates APCs as a damage-associated molecular pattern to prime antigen-specific naive T cells, which is crucial for T-cell-dependent anticardiolipin IgG antibody production in autoimmune diseases is unelucidated. Here, we show that CL increases the expression of costimulatory molecules in CD11c+ APCs both in vitro and in vivo. CL activates CD11c+ APCs via TLR2-PI3K-PKN1-AKT/p38MAPK-NF-κB signaling. CD11c+ APCs that have been activated by CL are sufficient to prime H-Y peptide-specific naive CD4+ T cells and OVA-specific naive CD8+ T cells. TLR2 is necessary for anti-CL IgG antibody responses in vivo. Intraperitoneal injection of CL does not activate CD11c+ APCs in CD14 KO mice to the same extent as in wild-type mice. CL binds to CD14 (Kd = 7 × 10-7 M). CD14, but not MD2, plays a role in NF-kB activation by CL, suggesting that CD14+ macrophages contribute to recognizing CL. In summary, CL activates signaling pathways in CD11c+ APCs through a mechanism similar to gram (+) bacteria and plays a crucial role in priming antigen-specific naive T cells.

Type 2 diabetic nephropathy (T2DN) progresses with an increasingly inflammatory milieu, wherein various immune cells are relevant. Herein, we investigated the levels of myeloid-derived suppressor cells (MDSCs) and their clinical implication in patients with T2DN. A total of 91 subjects (T2DN, n=80; healthy, n=11) were recruited and their PBMCs were used for flow cytometric analysis of polymorphonuclear (PMN-) and monocytic (M-) MDSCs, in addition to other immune cell subsets. The risk of renal progression was evaluated according to the quartiles of MDSC levels using the Cox model. The proportion of MDSCs in T2DN patients was higher than in healthy individuals (median, 6.7% vs. 2.5%). PMN-MDSCs accounted for 96% of MDSCs, and 78% of PMN-MDSCs expressed Lox-1. The expansion of PMN-MDSCs was not related to the stage of T2DN or other kidney disease parameters such as glomerular filtration rate and proteinuria. The production of ROS in PMN-MDSCs of patients was higher than in neutrophils of patients or in immune cells of healthy individuals, and this production was augmented under hyperglycemic conditions. The 4th quartile group of PMN-MDSCs had a higher risk of renal progression than the 1st quartile group, irrespective of adjusting for multiple clinical and laboratory variables. In conclusion, PMN-MDSCs are expanded in patients with T2DN, and may represent as an immunological biomarker of renal progression.

Sodium taurodeoxycholate (TDCA) has been investigated for various inflammatory disorders such as sepsis. We recently evaluated nonclinical safety profile of TDCA using rats infused intravenously. As a series of preclinical safety investigations, we further conducted toxicity studies with TDCA delivered to dogs

The anti-inflammatory effects of HY209 have been identified in sepsis. However, the anti-inflammatory potential of HY209 in colitis has not yet been investigated. In here, we evaluated the therapy efficacy of HY209 in an acute colitis mice model which induced by using dextran sulfate sodium (DSS). Compare to vehicle mice, HY209 treated mice showed significantly less body weight loss, lower disease activity index, longer colon length, as well as a lower histology disease scores. Meanwhile, the expression of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) were reduced in colon tissue after HY209 treatment. Furthermore, HY209 treated mice significantly reduced inflammatory macrophage, but increased anti-inflammatory macrophage in lamina propria. In vitro, HY209 showed a strong inhibition to LPS and ATP/BzATP activated NLRP3 inflammasome formation in the bone marrow derived macrophages (BMDMs). In summary, our findings demonstrated that HY209 alleviates inflammation in colitis mice through NLRP3 inflammasome inhibition, discovery the potential of HY209 as a therapeutic option for colitis.

Alzheimer’s disease (AD) is an inflammatory neurodegenerative condition characterized by deposition of amyloid beta (Abeta) in the brain and origins dementia. Neuronal apoptosis following neuroinflammation by Abeta impairs cognition and memory. In this study we focused on Taurodeoxyxcholate (TDCA) is an active bile acid derivative with potent anti-inflammatory function. In 5× Familial Alzheimer’s disease (5xFAD) mice model, chronic administration of TDCA in 5xFAD mice improved learning and memory in different memory test compared with PBS group. TDCA treatment significantly decreased neuronal apoptosis, load of Abeta plaques, the number of activated GFAP+ astrocytes and Iba-1+ microglia in different brain region of 5xFAD mice. CD11bhiGr1intF4/80+ myeloid cells in brain and spleen increased significantly in TDCA-treated group compared to PBS-treated group when brain single cell were immune-stained. In vitro, TDCA increased phagocytosis of Abeta. TDCA suppressed NLRP3-ASC oligomerization and production of mature IL-1β/IL-18 in Microglia by Abeta. TDCA inhibited Ca++ influx incurred by ATP or BzATP (P2X7 agonists). Compared with wild type, Ca++ influx of macrophages from TGR5 KO mice was like that of P2X7 KO mice in response to BzATP, suggesting that TDCA might play role as an inverse agonist for TGR5 complexed with P2X7. TDCA activates adenylate cyclase and cAMP inhibit priming phase of inflammasomal activation and oligomerization of NLRP3-ASC complex, suggesting that TDCA suppresses both priming and activation phase of NLRP3 inflammasome. TDCA and its novel chemical derivatives could be better choice in ameliorating broad spectrum of neuroinflammation to improve cognition and memory in AD patients.

HY209 has been reported to be a ligand for the membrane G protein–coupled receptor, TGR5. HY209 could inhibit inflammatory responses in sepsis by increasing number of immunosuppressive myeloid-derived suppressor cells (MDSC), suggesting the potent role of HY209 in the treatment of atopic dermatitis (AD). Here HY209 was evaluated whether it controls inflammatory immune responses in AD. We employed mouse AD model using a topical treatment of dinitrochlorobenzene (DNCB) on the dorsal skin of Balb/c mice. HY209-treated mice showed significantly reduction in Th2 immune responses, shown as lower IgE serum, and inhibition of T cells mast cells, eosinophilia and neutrophil recruitment accompanied with reduced in epidermal and dermal thickness in skin of HY209 treated mice. Interestingly, regulatory T cells noticeably increased upon to HY209 treatment along with inhibition of type 2 cytokines production. Proteogenomic analyses of mouse skin demonstrated upregulated expression of molecules responsible for tissue regeneration and downregulated expression of proinflammatory molecules. Consistent with these findings, in vitro data indicated that HY209 decreased production of thymic stromal lymphopoietin (TSLP) and CCL17 (TARC) through inhibition of NF-KB and MAPK (P38) pathway. Taken together, HY209 ameliorates AD in mice by normalizing Th1/Th2 imbalance immune responses and inhibiting various pro-inflammatory responses via TGR5-cAMP-PKA axis, Suggesting HY209 could be a promising therapeutic candidate for AD.

Taurodeoxycholate (TDCA) inhibits various inflammatory responses suggesting potential clinical application. However, the toxicity of TDCA has not been evaluated in detail in vivo. We investigated the acute toxicity and 4-week repeated-dose toxicity of TDCA following intravenous infusion under Good Laboratory Practice regulations. In the sighting study of acute toxicity, one of two rats (one male and one female) treated with 300 mg/kg TDCA died with hepatotoxicity, suggesting that the approximate 50% lethal dose of TDCA is 300 mg/kg. Edema and discoloration were observed at the injection sites of tails when rats were infused with 150 mg/kg or higher amount of TDCA once. In 4-week repeated-dose toxicity study, no treatment-related mortality or systemic changes in hematology and serum biochemistry, organ weights, gross pathology, or histopathology were observed. However, the tail injection site showed redness, discharge, hardening, and crust formation along with histopathological changes such as ulceration, edema, fibrosis, and thrombosis when rats were infused with 20 mg/kg TDCA. Taken together, TDCA induced no systemic toxicity or macroscopic lesions at the injection site at a dose of 10 mg/kg/day, which is 33 times higher than the median effective dose observed in a mouse sepsis model. These findings suggest that TDCA might have a favorable therapeutic index in clinical applications.

Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disease. The precise pathogenic mechanisms of the disease remain uncertain, and as of yet, there is no effective cure. Human adipose stem cells (hASC) can be easily obtained during operative procedures. hASC have a clinically feasible potential to treat neurodegenerative disorders, since cytosolic extract of hASC contain a number of essential neurotrophic factors. In this study, we investigated effects of hASC extract on the SOD1 G93A mouse model of ALS and in vitro test. Administration of hASC extract improved motor function and prolonged the time until symptom onset, rotarod failure, and death in ALS mice. In the hASC extracts group, choline acetyltransferase immunostaining in the ventral horn of the lumbar spinal cord showed a large number of motor neurons, suggesting normal morphology. The neuroprotective effect of hASC extract in ALS mice was also suggested by western blot analysis of spinal cord extract from ALS mice and in vitro test. hASC extract treatment significantly increased expression of p-Akt, p-CREB, and PGC-1α in SOD1 G93A mouse model and in vitro test. Our results indicated that hASC extract reduced apoptotic cell death and recovered mutant SOD1-induced mitochondrial dysfunction. Moreover, hASC extract reduced mitochondrial membrane potential. In conclusion, we have demonstrated, for the first time, that hASC extract exert a potential therapeutic action in the SOD1 G93A mouse model of ALS and in vitro test. These findings suggest that hASC hold promise as a novel therapeutic strategy for treating ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by selective degeneration of motor neurons. Mutant superoxide dismutase 1 (SOD1) is often found as aggregates in the cytoplasm in motor neurons of various mouse models and familial ALS patients. The interplay between motor neurons and astrocytes is crucial for disease outcome, but the mechanisms underlying this phenomenon remain unknown. In this study, we investigated whether transient transfection with wild-type and mutant-type SOD1 may lead to amplification of mutant SOD1-mediated toxicity in cortical neurons and astrocytes derived from wild-type and mutant-type (human G93A-SOD1) mice. In transgenic mice expressing either wild- or mutant-type SOD1, we found that green fluorescent protein (GFP)-wtSOD1 was present in the cytoplasm and nuclei of wild-type cortical neurons and astrocytes, whereas GFP-mutant SOD1 was mainly cytoplasmic in wild- and mutant-type cortical neurons and astrocytes. These findings indicate that intracellular propagation of misfolding of GFP-wt or mtSOD1 are possible mediators of toxic processes involved in initiating mislocalization and aggregation. Here, we provide evidence that cytoplasmic aggregates induce apoptosis in G93A-SOD1 mouse cortical neurons and astrocytes and that the toxicity of mutant SOD1 in astrocytes is similar to the pathological effects of ALS on neurons in vitro. Collectively, our results indicate that mtSOD1 probably interacts with wtSOD1 via an unknown mechanism to produce augmented toxicity and may influence aggregate formation and apoptosis.

Aim of the study To identify proteins of which depletion are associated with the poor 6-month neurological outcome of out-of-hospital cardiac arrest survivors. Methods Seven healthy volunteers and 34 out-of-hospital cardiac arrest survivors admitted to the intensive care unit (ICU) and underwent targeted-temperature management were enrolled. According to the 6-month cerebral performance category (CPC) scale, patients were divided into the good (CPC 1–2) and poor (CPC 3–5) outcome groups. Blood samples were obtained at 0, 24, and 72 h after admission to the ICU. Results With proteomic approaches, we found 23 proteins that showed group-differences between the sera pooled from 7 study groups: healthy volunteers, the good outcome groups (0, 24, and 72 h), and the poor outcome groups (0, 24, and 72 h). We selected 7 candidate proteins of which intensities were different between the good and poor outcome groups (>2-fold change) and excluded 5 proteins related to haemolysis or remaining high abundant proteins. To confirm the 2 identified proteins: retinal dehydrogenase 1 and Kallistatin, we performed enzyme-linked immunosorbent assay with individual serum. Finally, old age (odds ratio = 1.055; 95% confidence interval, 1.002–1.112; p = 0.043) and low serum kallistatin level at 0 h (odds ratio = 0.784; 95% confidence interval, 0.618–0.995; p = 0.046) were independently associated with the poor 6-month neurological outcome. Conclusion The depletion of serum kallistatin at admission to the ICU was associated with the poor neurological outcome of out-of-hospital cardiac arrest survivors.

Introduction: Split hand is considered to be a spe- cific feature of amyotrophic lateral sclerosis (ALS). Methods: We evaluated the pattern difference of intrinsic hand muscles of upper limb-onset ALS (UL-ALS), upper limb-onset progressive muscular atrophy (UL-PMA), brachial amyotrophic diplegia (BAD), and Hirayama disease (HD) by measuring objective electrophysiological markers. Results: The abductor digiti minimi (ADM)/abductor pollicis brevis (APB) compound muscle action potential (CMAP) amplitude ratio was significantly higher in UL- ALS than other variants, but a considerable proportion of UL- ALS cases had an amplitude ratio in the range of other var- iants. Absent APB CMAP and abnormally high ADM/APB CMAP amplitude ratio (4) occurred only with UL-ALS. Con- versely, an absent ADM CMAP was identified only in UL-PMA and BAD. Conclusions: The absolute ADM/APB CMAP ampli- tude ratio was not specific for ALS; however, several findings from simple electrophysiological measurements may help pre- dict prognosis in patients with motor neuron diseases and may be early diagnostic markers for ALS. Muscle Nerve 51: 333-337, 2015

Adaptation of tumor cells to the host is a major cause of cancer progression, failure of therapy, and ultimately death. Immune selection drives this adaptation in human cancer by enriching tumor cells with a cancer stem cell–like (CSC-like) phenotype that makes them resistant to CTL-mediated apoptosis; however, the mechanisms that mediate CSC maintenance and proliferation are largely unknown. Here, we report that CTL-mediated immune selection drives the evolution of tumor cells toward a CSC-like phenotype and that the CSC-like phenotype arises through the Akt signaling pathway via transcriptional induction of Tcl1a by Nanog. Furthermore, we found that hyperactivation of the Nanog/Tcl1a/Akt signaling axis was conserved across multiple types of human cancer. Inhibition of Nanog in a murine model of colon cancer rendered tumor cells susceptible to immune-mediated clearance and led to successful, long-term control of the disease. Our findings establish a firm link among immune selection, disease progression, and the development of a stem-like tumor phenotype in human cancer and implicate the Nanog/Tcl1a/Akt pathway as a central molecular target in this process.

DNA vaccines have emerged as an attractive approach to generate antigen-specific T-cell immune response. Nevertheless, the potency of DNA vaccines still needs to be improved for cancer immunotherapy. In this study, we explored whether functional linkage of a Th1-polarizing chemokine, IP-10, to a model tumor antigen, human papillomavirus type 16 (HPV-16) E7, enhanced DNA vaccine potency. IP-10 linkage changed the location of E7 from the nucleus to the endoplasmic reticulum and led to the secretion of functionally chemoattractive chimeric IP-10/E7 protein. In addition, this linkage drastically enhanced the endogenous processing of E7 antigen through MHC class I. More importantly, we found that C57BL/6 mice intradermally vaccinated with IP-10/E7 DNA exhibited a dramatic increase in the number of E7-specific CD4(+) Th1 T-cells and CD8(+) T-cells and, consequently, were strongly resistant over the long term to E7-expressing tumors compared to mice vaccinated with wild-type E7 DNA. Thus, because of the increase in tumor antigen-specific T-cell immune responses obtained through both enhanced antigen presentation and chemoattraction, vaccination with DNA encoding IP-10 linked to a tumor antigen holds great promise for treating tumors.

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular pathogen. Previously, we reported that the 56-kDa type-specific antigen (TSA56), a major outer membrane protein of O. tsutsugamushi , binds to fibronectin and facilitates bacterial entry into the host cell, potentially via an interaction with integrins. Here, we demonstrated that O. tsutsugamushi colocalizes with integrin α5β1 and activates integrin signaling effectors, including focal adhesion kinase, Src kinase, and RhoA GTPase, and also recruits signaling adaptors, such as talin and paxillin, to the site of infection. Inhibition of protein tyrosine kinases or RhoA reduced intracellular invasion. We also observed substantial actin reorganization and membrane protrusions at the sites of infection of nonphagocytic host cells. Finally, we identified a region in the extracellular domain of TSA56 that binds to fibronectin. A peptide containing this region was able to significantly reduce bacterial invasion. Taken together, these results clearly indicate that O. tsutsugamushi exploits integrin-mediated signaling and the actin cytoskeleton for invasion of eukaryotic host cells.

Both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) activate antigen-presenting cells, often through the same pattern recognition receptors (PRR), such as Toll-like receptors (TLR). The TLR4-CD14-MD2 and TLR2-CD14 complexes have been shown to play a role in the recognition of lipopolysaccharide (LPS) and peptidoglycan (PG), respectively. Since many DAMPs have also been known to activate TLR2 or TLR4 pathways, we dissected the role of each molecule in the receptor complexes (TLR2-D14-MD2) responding to DAMP (necrotic cells) or PAMP (LPS and PG). CD14 played a significant role in the activation of NF-kappaB in response to necrotic cells in the presence or absence of TLR2. However, MD2 did not play a significant role in NF-kappaB activation by necrotic cells. Intriguingly, MD2 did play a significant role in activating NF-kappaB by PG in the presence of TLR2-CD14. Compared with CD14(pos) B6 mice, CD14(neg) B6 mice showed delayed production of IL12p40 in response to necrotic cells in vivo. Microarray analysis showed that various pro-inflammatory genes of peritoneal cells were regulated in response to necrotic cells, in a CD14-dependent manner. The CD14 appears to recognize necrotic cells in addition to LPS, PG, apoptotic cells, and lipids, suggesting that CD14 might be a universal adaptor for DAMP and PAMP. On the contrary, MD2 recognizes only exogenous PAMP, when complexed with TLR2-CD14 or TLR4-CD14. Taken together, MD2 appears to discriminate between DAMP and PAMP.

SummaryWe suggested earlier that the hydrophobic portions (Hyppos) of mole-cules, which are normally embedded in the membranes of cells or the coreof molecular structures so as to be separated from the aqueous environ-ment, might serve as evolutionarily ancient alarm signals of injury orstress to initiate innate immune responses when they are exposed on thesurface. Under normal physiological conditions, the Hyppos released fromendogenous or exogenous sources might be handled by ‘Hyppo-quench-ers’ in vivo to maintain the tissue homeostasis and immune modulation.To test this idea, we selected apolipoproteins, which have been known totransport blood lipids and play a role in a number of pathological inflam-matory conditions. We examined their role as Hyppo-quenchers in earlyimmune responses and found that apolipoproteins showed significantinhibition of the nuclear factor-jB-dependent gene expression in recombi-nant Chinese hamster ovary (CHO) cells and dendritic cells stimulated bynecrotic cells or bacterial endotoxin. In addition, our results indicate thatapolipoproteins could dramatically abrogate complement fixation on thesurface of necrotic cells. These findings suggest that apolipoproteins,besides having known functions in lipid metabolism, also have a role inpreventing the initiation of innate immunity, potentially through neutral-izing Hyppos from injured cells or exogenous endotoxin.Keywords: apolipoproteins; complement; endotoxin; innate immunity;necrotic cell

SummaryMucin antigen 1 (MUC1) is overexpressed on various human adenocarcinomas and haematological malignancies and has long been used as a target antigen for cancer immunotherapy. Most of the preclinical and clinical studies using MUC1 have used the tandem repeat region of MUC1, which could be presented by only a limited set of major histocompatibility complex haplotypes. Here, we evaluated N-terminal region (2–147 amino acids) of MUC1 (MUC1-N) for dendritic cell (DC)-based cancer immunotherapy. We used Esherichia coli-derived MUC1-N that was fused to the protein transduction domain of human immunodeficiency virus Tat protein for three reasons. First, mature DCs do not phagocytose soluble protein antigens. Secondly, tumour cells express underglycosylated MUC1, which can generate epitopes repertoire that differs from normal cells, which express hyperglycosylated MUC1. Finally, aberrantly glycosylated MUC1 has been known to impair DC function. In our study, Tat-MUC1-N-loaded DCs induced type 1 T cell responses as well as cytotoxic T lymphocytes efficiently. Furthermore, they could break tolerance in the transgenic breast tumour mouse model, where MUC1-positive breast cancers grow spontaneously. Compared with DCs pulsed with unconjugated MUC1-N, DCs loaded with Tat-conjugated MUC1-N could delay tumour growth more effectively in the transgenic tumour model as well as in the tumour injection model. These results suggest that the recombinant N-terminal part of MUC1, which may provide a diverse epitope repertoire, could be utilized as an effective tumour antigen for DC-based cancer immunotherapy.

Part of pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) activate antigen-presenting cells through Toll-like receptors (TLRs) to initiate immune responses. However, controversy remains if TLR4 mediates DAMP signaling due to the confounding effects of potential LPS contamination. To test if TLR4 functions as a true receptor for DAMP, we compared TLR4(pos)- and TLR4(neg)-responders in vitro and in vivo after stimulation with whole necrotic cell (NC) lysates. Using CHO reporter cells transfected with anti-TLR4-siRNAs, TLR4 was found to partially mediate NF-kappaB activation in response to NC lysates. TLR4(neg) DCs exhibited less I-A(b) expression and nitric oxide secretion than TLR4(pos) DCs upon NC stimulation and this defect was well correlated with diminished presentation of H-Y antigen by TLR4(neg) DCs to I-A(b)-restricted CD4(pos) Marilyn T cells in vitro. Similarly, TLR4(neg) DCs showed significantly less expression of I-A(b), CD80, CD86, and CD40 than TLR4(pos) DCs when NC lysates were injected into peritoneal cavity. Finally, delayed type hypersensitivity response to OVA was significantly decreased in TLR4(neg) mice when NCs were used as an adjuvant. Taken together, our data support the idea that part of the endogenous ligands presented by NCs could activate APCs thru TLR4 and contribute to the development of antigen-specific adaptive immunity. Therefore, endogenous DAMP ligands themselves, not contaminated LPS, activate TLR4 signaling leading to activation of professional antigen-presenting cells.

Summary Carcinoembryonic antigen (CEA) is over-expressed on various human cancer cells and has been the target of immunotherapies using dendritic cells (DCs) pulsed with CEA-specific RNA or peptides, or transduced by CEA-expressing adenovirus or vaccinia virus. Because activated DCs do not phagocytose soluble protein antigens efficiently and pure immature DCs are not obtained easily ex vivo, an efficacious whole CEA protein-loaded DC vaccine has not been reported. To improve the antigen delivery into DCs, we utilized CEA conjugated to a protein-transduction domain, human immunodeficiency virus transactivating Tat. Furthermore, we purified the truncated non-glycosylated CEA from Escherichia coli to overcome the safety concerns and immunosuppressive functions associated with the native CEA protein. Using confocal microscopy and fluorescence activating cell sorter analysis, we demonstrated that the Tat-CEA protein entered the cytoplasm of DCs efficiently within 10 min of co-culture, compared with the negligible amount of CEA into DCs 30 min later. CEA-specific T cell proliferation and cytotoxic T cell responses were enhanced significantly in mice immunized with Tat-CEA-pulsed DCs [DC (Tat-CEA)] compared with those immunized with CEA-pulsed DCs [DC (CEA)]. T helper type 1 responses were more prominent in the DC (Tat-CEA) immunized mice whose splenocytes secreted more interferon-γ and less interleukin-4 than those from DC (CEA) immunized mice. In vivo, the DC (Tat-CEA) vaccine delayed tumour growth significantly and prolonged survival of tumour-bearing mice. These results suggest that protective epitopes are well preserved on bacteria-derived recombinant Tat-CEA. This strategy may provide a basic platform for DC-based anti-CEA vaccines that could be utilized in combination with advanced immune-enhancing therapeutics.

Most amyotrophic lateral sclerosis (ALS) patients show focal onset of upper and lower motor neuron signs and spread of symptoms to other regions or the other side clinically. Progression patterns of sporadic ALS are unclear. The aim of this study was to evaluate the pattern of respiratory deterioration in sporadic ALS according to the onset site by using respiratory function tests. Study participants included 63 (42 cervical-onset [C-ALS] and 21 lumbosacral-onset [L-ALS]) ALS patients and 31 healthy controls. We compared respiratory function test parameters among the 3 groups. Age was 57.4±9.6 (mean±SD), 60.8±9, and 60.5±7 years, and there were 28, 15, and 20 male participants, in the C-ALS, L-ALS, and control groups, respectively. Disease duration did not differ between C-ALS and L-ALS patients. Sniff nasal inspiratory pressure (SNIP) was significantly low in C-ALS patients compared with controls. Maximal expiratory pressure (MEP) and forced vital capacity percent predicted (FVC% predicted) were significantly low in C-ALS and L-ALS patients compared with controls. Maximal inspiratory pressure to maximal expiratory pressure (MIP:MEP) ratio did not differ among the 3 groups. Eighteen C-ALS and 5 L-ALS patients were followed up. ΔMIP, ΔMEP, ΔSNIP, ΔPEF, and ΔFVC% predicted were higher in C-ALS than L-ALS patients without statistical significance. Fourteen C-ALS (77.8%) and 3 L-ALS (60%) patients showed a constant MIP:MEP ratio above or below 1 from the first to the last evaluation. Our results suggest that vulnerability of motor neurons in sporadic ALS might follow a topographic gradient.

To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production in susceptible (C3H/HeN) and resistant (BALB/c) mice after infection with O. tsutsugamushi Gilliam. C3H/HeN mice produced high levels of chemokines macrophage inflammatory proteins 1 alpha (MIP-1 alpha ), MIP-2, monocyte chemoattractant protein 1 (MCP-1), and cytokines gamma-interferon (IFN-gamma ), interleukin-12 (IL-12), IL-10, and tumor necrosis factor alpha (TNF-alpha ) in response to O. tsutsugamushi infection, compared to BALB/c mice. Chemokine profiles in infected mice correlated well with the kinetics of inflammatory cell infiltration. Hyperproduction of chemokines and cytokines was observed in another susceptible-infection model (BALB/c-Karp). These results suggest that hyperproduction of chemokines and cytokines are associated with susceptibility during O. tsutsugamushi infection.

Orientia tsutsugamushi , an obligate intracellular bacterium, is the causative agent of scrub typhus which is histopathologically characterized by inflammatory manifestations, indicating that rickettsiae induce mechanisms that amplify the inflammatory response. To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production after infection with O. tsutsugamushi in mice. The mRNAs that were upregulated included lymphotactin, RANTES ( r egulated upon a ctivation, n ormal T -cell e xpressed and s ecreted), macrophage inflammatory proteins 1α/β (MIP-1α/β), MIP-2, monocyte chemoattractant protein 1, lymphotoxin β, tumor necrosis factor α, interleukin-6, γ-interferon, transforming growth factor β1, and migration inhibition factor. Peak expression of these chemokines and cytokines was observed between 4 and 8 days after infection. Gene induction was followed by the secretion of chemokine and cytokine proteins. Chemokine profile in infected mice was well correlated with kinetics of inflammatory cell infiltration. Thus, O. tsutsugamushi appears to be a strong inducer of chemokines and cytokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation observed in scrub typhus.

Human macrophage chemoattractant protein 1 (MCP-1) is a potent mediator of macrophage migration and therefore plays an essential role in early events of inflammation. In endothelial cells, at least three independent pathways regulate MCP-1 expression by NF-κB and AP-1.Orientia tsutsugamushicauses vasculitis in humans by replicating inside macrophages and endothelial cells. In the present study, we investigated thecis-acting andtrans-acting elements involved inO. tsutsugamushi-induced MCP-1 gene expression in human umbilical vein endothelial cells (HUVEC). Although NF-κB activation was observed in HUVEC infected withO. tsutsugamushi, inhibition of NF-κB activation did not affect the MCP-1 expression. However, treatment of HUVEC with extracellular signal-regulated kinase (ERK) kinase inhibitor or p38 mitogen-activated protein kinase (MAPK) inhibitor suppressed expression of MCP-1 mRNA concomitant with downregulation of activator protein 1 (AP-1) activation. Deletion of triphorbol acetate response elements (TRE) at position −69 to −63 of MCP-1 gene abolished inducible promoter activity. Deletion of TRE at position −69 to −63−96 to −90 or deletion of NF-κB-binding site at position −69 to −63−88 to −79 did not affect the inducibility of promoter. Site-directed mutagenesis of the NF-κB binding sites at positions −2640 to −2632, −2612 to −2603 in the enhancer region, or the AP-1 biding site at position −2276 to −2270 decreased the inducible activity of the promoter. Taken together, AP-1 activation by both the ERK pathway and the p38 MAPK pathway as well as their binding to TRE at position −69 to −63 in proximal promoter and TRE at position −2276 to −2270 in enhancer region is altogether essential in induction of MCP-1 mRNA in HUVEC infected withO. tsutsugamushi. Although NF-κB activation is not essential per se, the κB site in the enhancer region is important in MCP-1 induction of HUVEC. This discrepancy in the involvement of the NF-κB may be due to the function of chromatin structures and other transcription cofactors in the regulation of MCP-1 gene expression in response toO. tsutsugamushiinfectioin.

Orientia tsutsugamushishows both pro- and antiapoptotic activities in infected vertebrate cells. Apoptosis of THP-1 cells induced by beauvericin was inhibited byO. tsutsugamushiinfection. Beauvericin-induced calcium redistribution was significantly reduced and retarded in cells infected withO. tsutsugamushi. Antiapoptotic activities ofO. tsutsugamushiin infected cells are most probably due to inhibition of the increase in the cytosolic calcium concentration.

Optimizing conditions for the microarraying of protein antigens onto glass slides were studied. Various vendors, surface functional groups, buffers, and fixatives were evaluated to enhance protein binding. A total of 125 pg of human immunoglobulin was detectable with this assay system, suggesting that protein microarray can be applied for routine immunodiagnosis.

Reactive oxygen species (ROS) play an important role in cellular signaling as second messengers. However, studying the role of ROS in physiological redox signaling has been hampered by technical difficulties in controlling their generation within cells. Here, we utilize two inert components, a photosensitizer and light, to finely manipulate the generation of intracellular ROS and examine their specific role in activating dendritic cells (DCs). Photoswitchable generation of intracellular ROS rapidly induced cytosolic mobilization of Ca(2+), differential activation of mitogen-activated protein kinases, and nuclear translocation of NF-κB. Moreover, a transient intracellular ROS surge could activate immature DCs to mature and potently enhance migration in vitro and in vivo. Finally, we observed that intracellular ROS-stimulated DCs enhanced antigen specific T-cell responses in vitro and in vivo, which led to delayed tumor growth and prolonged survival of tumor-bearing mice when immunized with a specific tumor antigen. Therefore, a transient intracellular ROS surge alone, if properly manipulated, can cause immature DCs to differentiate into a motile state and mature forms that are sufficient to initiate adaptive T cell responses in vivo.

Hepatitis C virus (HCV) infection in hepatocytes stimulates innate antiviral responses including the production of type III interferons (IFN-λ), including IL-28A, IL-28B, and IL-29 (Figure ​(Figure1).1). However, the molecular mechanism(s) regulating the expression of IFN-λ genes in HCV-infected hepatocytes remains undefined. In this study, we examined regulatory elements involved in the induction of IFN-λ genes following HCV infection in hepatocytes and further determined the binding of specific transcription factor(s) to promoter regions of IFN-λ genes. Our studies reveal that the regulatory portion for IL-28A, IL-28B, andIL-29 genes is localized to a 1-kb region in their respective promoters (Figure ​(Figure2,2, ​,4).4). Notably, interferon regulatory factor (IRF)-3 and -7 are the key transcriptional factors for the induction of IL-28A and IL-28Bgenes (Figure ​(Figure5,5, ​,6),6), whereas NF-κB is an additional requirement for the induction of the IL-29 gene (Figure ​(Figure3).3). Ligation of Toll-like receptors (TLR) 3, 7, 8, and 9, which also activate IRFs and NF-κB, resulted in more robust production of IFN-λ than that observed with HCV infection, verifying the importance of TLR pathways in IFN-λ production (Figure ​(Figure8).8). Furthermore, the addition of IFN-λ to HCV-infected hepatocytes decreased viral replication and produced a concurrent reduction in microRNA-122 (miR-122). The decrease in viral replication was enhanced by the co-administration of IFN-λ and miR-122 inhibitor (miRIDIAN) (Figure ​(Figure7),7), suggesting that such combinatorial therapies may be beneficial for the treatment of chronic HCV infection. Figure 1 Hepatic induction of IFN-λ genes during HCV infection. A, B. Total RNA was extracted from liver tissues of healthy and chronic HCV patients for IL-28A/B (A) and IL-29 (B) mRNA quantification by real-time PCR. C-F. Human primary hepatocytes were ... Figure 2 Transcriptional activity at the IL-28A, IL-28B, and IL-29 promoter by HCV infection. PH5CH8 cells were transfected with a mixture of the luciferase constructs containing various 5' deletions of the human IL-28A (A), IL-28B (B), or IL-29 (C) promoters ... Figure 3 IRF-7, and NF-κB differentially determine expression of IL-28A/B and IL-29. Nucleotide sequence from 1.0 kb upstream of the transcription start site of human IL-28A, IL-28B, and IL-29. The positions of the IRF, NF-κB, AP-1, and GATA-1 ... Figure 4 Overexpression of IRFs and NF-κB confirms their role in expression of IFN-λ genes. PH5CH8 cells were transiently transfected with plasmids encoding different promoter regions of human IL-28A (pGL3-IL-28A 1kb) (A-C), IL-28B (pGL3-IL-28B ... Figure 5 IRFs are core transcription factors of IFN-λ genes. IL-28B (B), or IL-29 (C) in combination with siRNA specific for IRF-3, IRF-7, RELA(NF-κB p65), or control. At 36 h post-transfection, cells were stimulated with poly(I:C) for 6 hours ... Figure 6 IRFs and NF-κB directly bind the promoters of IFN-λ genes. PH5CH8 cells were stimulated with poly(I:C) (A and C) or infected with JFH-1 (B and D) for the indicated lengths of time. Chromatin was immunoprecipitated with anti-IRF-3, anti-IRF-7, ... Figure 7 miR-122-dependent replication of HCV is suppressed by IFN-λ stimulation. Huh 7.5.1 cells infected with JFH-1 for the indicated number of days and HCV RNA or miR-122 levels were quantified by real time PCR. The mean value of day 1 post-infection ... Figure 8 TLR3, 7, 8, and 9 induce human IFN-λ gene expression in human primary hepatocytes. Human primary hepatocytes were incubated with JFH-1, Pam3CSK4 (1 μg/ml), HKLM (108 cells/ml), Poly (I-C) (5 μg/ml), LPS (10 μg/ml), Flagellin ...