RIPK3 Activates MLKL-mediated Necroptosis and Inflammasome Signaling during Streptococcus Infection.

Community-acquired pneumonia is the most common type of pneumonia and remains a leading cause of morbidity and mortality worldwide. Although many different pathogens can contribute to pneumonia, Streptococcus pneumoniae is one of the common bacterial pathogens that underlie community-acquired pneumonia. RIPK3 (receptor-interacting protein kinase 3) is widely recognized as a key modulator of inflammation and cell death. To elucidate a potential role of RIPK3 in pneumonia, we examined plasma from healthy control subjects and patients positive for streptococcal pneumonia. In human studies, RIPK3 protein concentrations were significantly elevated and were identified as a potential plasma marker of pneumococcal pneumonia. To expand these findings, we used an in vivo murine model of pneumococcal pneumonia to demonstrate that RIPK3 deficiency leads to reduced bacterial clearance, severe pathological damage, and high mortality. Our results illustrated that RIPK3 forms a complex with RIPK1, MLKL (mixed-lineage kinase domain-like protein), and MCU (mitochondrial calcium uniporter) to induce mitochondrial calcium uptake and mitochondrial reactive oxygen species(mROS) production during S. pneumoniae infection. In macrophages, RIPK3 initiated necroptosis via the mROS-mediated mitochondrial permeability transition pore opening and NLRP3 inflammasome activation via the mROS-AKT pathway to protect against S. pneumoniae. In conclusion, our study demonstrated a mechanism by which RIPK3-initiated necroptosis is essential for host defense against S. pneumoniae.

LL37 Inhibits Aspergillus fumigatus Infection via Directly Binding to the Fungus and Preventing Excessive Inflammation.

The incidence of Aspergillus fumigatus infection and the rate of resistance to antifungal drugs have sharply increased in recent years. LL37 has been reported as a host defense peptide with broad-spectrum antibacterial activities. However, the role of LL37 during A. fumigatus infection remains unclear. Here, we examined the interaction between LL37 and A. fumigatus and found that synthetic LL37 could directly bind to the surface of A. fumigatus, disrupting the integrity of the cell wall in vitro. LL37 inhibited mycelial growth in a concentration-dependent manner, rather than fungicidal effect even at high concentration (e.g., 20 µM). Interestingly, low concentrations of LL37 (e.g., 4 µM) significantly attenuated mycelial adhesion and prevented the invasion and destruction of epithelial cells. Following LL37 treatment, the levels of proinflammatory cytokines released by A. fumigatus-stimulated macrophages decreased significantly, accompanied by downregulation of M1 type markers. In a mouse model of pulmonary A. fumigatus infection, LL37-treated mice showed lower amounts of fungi load, moderate pathological damage, and reduced proinflammatory cytokines. Further, LL37 transgenic mice (LL37+/+) were examined to investigate the effects of endogenous LL37 in an A. fumigatus infection model and showed lower susceptibility to A. fumigatus infection in comparison with wild-type mice. In addition, LL37 also played a protective role in an immunosuppressed mouse model of A. fumigatus infection. Thus, LL37 inhibits A. fumigatus infection via directly binding to mycelia and reducing excessive inflammation. LL37 or its analogs may therefore constitute potential drug components for A. fumigatus infection.

Mitochondrial Dysfunction in Aged Macrophages and Lung during Primary Streptococcus pneumoniae Infection is Improved with Pirfenidone.

Pneumococcal infections remain a leading cause of death in older adults, with the most serious cases occurring in persons ≥65 years of age. There is an urgent need to investigate molecular pathways underlying these impairments and devise new therapeutics to modulate innate immunity. The goal of our current study is to understand the impact of chronological aging on mitochondrial function in response to Streptococcus pneumoniae, a causative agent of bacterial pneumonia. Using chronologically aged murine models, our findings demonstrate that decreased ATP production is associated with dysregulated mitochondrial complex expression, enhanced oxidative stress, diminished antioxidant responses, and decreased numbers of healthy mitochondria in aged adult macrophages and lung in response to S. pneumoniae. Pre-treatment of aged macrophages with pirfenidone, an anti-fibrotic drug with antioxidant and anti-inflammatory properties, improved mitochondrial function and decreased cellular oxidative stress responses. In vivo administration of pirfenidone decreased superoxide formation, increased healthy mitochondria number, improved ATP production, and decreased inflammatory cell recruitment and pulmonary oedema in aged mouse lung during infection. Taken together, our data shed light on the susceptibility of older persons to S. pneumoniae and provide a possible therapeutic to improve mitochondrial responses in this population.

Dectin-3 Recognizes Glucuronoxylomannan of Cryptococcus neoformans Serotype AD and Cryptococcus gattii Serotype B to Initiate Host Defense Against Cryptococcosis.

Cryptococcus neoformans and Cryptococcus gattii cause life-threatening meningoencephalitis or lung diseases in immunocompetent individuals or immunocompromised ones. C. neoformans and C. gattii are subdivided into five serotypes based on their capsular glucuronoxylomannan (GXM). C. neoformans consists of serotypes A, D, and AD hybrid, and C. gattii consists of serotypes B and C. Given structural differences of GXM between C. neoformans and C. gattii, it remains unclear that how innate immune system recognizes GXM. Here, we report that C-type lectin receptor Dectin-3 (MCL encoded by Clec4d) is a direct receptor for GXMs from C. neoformans serotype AD (C.n-AD) and C. gattii serotype B (C.g-B). GXMs from C.n-AD and C.g-B activated NF-κB and ERK pathways to induce pro-inflammatory cytokine production, whereas it was completely abolished due to deficiency of Dectin-3 or caspase recruitment domain family member 9 (CARD9). Upon pulmonary C.n-AD and C.g-B infection, Dectin-3- and CARD9-deficient mice were highly susceptible and showed augmented lung injury due to impairment of alveolar macrophage accumulation and killing activities. Our study provides the first biological and genetic evidence demonstrating that Dectin-3 recognizes GXM of C.n-AD and C.g-B to initiate host defense against cryptococcosis.

Mechanistic Study of Inhibitory Effects of Metformin and Atorvastatin in Combination on Prostate Cancer Cells in Vitro and in Vivo.

Metformin is a commonly used drug for the treatment of type II diabetes and atorvastatin is the most prescribed cholesterol-lowering statin. The present study investigated the effects and mechanisms of metformin and atorvastatin in combination on human prostate cancer cells cultured in vitro and grown as xenograft tumor in vivo. Metformin in combination with atorvastatin had stronger effects on growth inhibition and apoptosis in PC-3 cells than either drug alone. The combination also potently inhibited cell migration and the formation of tumorspheres. Metformin and atorvastatin in combination had a potent inhibitory effect on nuclear factor-kappaB (NF-κB) activity and caused strong decreases in the expression of its downstream anti-apoptotic gene Survivin. Moreover, strong decreases in the levels of phospho-Akt and phosphor-extracellular signal-regulated kinase (Erk)1/2 were found in the cells treated with the combination. The in vivo study showed that treatment of severe combined immunodeficient (SCID) mice with metformin or atorvastatin alone resulted in moderate inhibition of tumor growth while the combination strongly inhibited the growth of the tumors. Results of the present study indicate the combination of metformin and atorvastatin may be an effective strategy for inhibiting the growth of prostate cancer and should be evaluated clinically.

Evaluation of a new cryptococcal antigen lateral flow immunoassay in serum, cerebrospinal fluid and urine for the diagnosis of cryptococcosis: a meta-analysis and systematic review.

BACKGROUND: A new lateral flow immunoassay (LFA) for the detection of cryptococcal antigen was developed. OBJECTIVE: We aimed to systematically review all relevant studies to evaluate the diagnostic accuracy of the cryptococcal antigen LFA on serum, CSF and urine specimens. METHODS: We searched public databases including PubMed, Web of Science, Elsevier Science Direct and Cochrane Library for the English-language literature published up to September 2014. We conducted meta-analyses of sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratios (DOR) and SROC of LFA in serum and CSF, respectively. The sensitivity of LFA in urine was also analyzed. Subgroup analyses were carried out to analyze the potential heterogeneity. RESULTS: 12 studies were included in this study. The pooled sensitivity and specificity values of LFA in serum were 97.6% (95% CI, 95.6% to 98.9%) and 98.1% (95% CI, 97.4% to 98.6%), respectively. The average PLR of LFA in serum was 43.787 (95% CI, 22.60-84.81) and the NLR was 0.03 (95% CI, 0.01-0.09). The pooled DOR was 2180.30 (95% CI, 868.92-5471.00) and the AUC was 0.9968. The pooled sensitivity and specificity values of LFA in CSF were 98.9% (95% CI, 97.9% to 99.5%) and 98.9% (95% CI, 98.0% to 99.5%), respectively. The average PLR of LFA in serum was 48.83 (95% CI, 21.59-110.40) and the NLR was 0.02 (95% CI, 0.01-0.04). The pooled DOR was 2931.10 (95% CI, 1149.20-7475.90) and the AUC was 0.9974. The pooled sensitivity value of LFA in urine was 85.0% (95% CI, 78.7% to 90.1%). CONCLUSIONS: The study demonstrates a very high accuracy of LFA in serum and CSF for the diagnosis of cryptococcosis in patients at risk. LFA in urine can be a promising sample screening tool for early diagnosis of cryptococcosis.

Differential gene expression profiles of peripheral blood mononuclear cells in childhood asthma.

OBJECTIVE: Asthma is a common childhood disease with strong genetic components. This study compared whole-genome expression differences between asthmatic young children and healthy controls to identify gene signatures of childhood asthma. METHODS: Total RNA extracted from peripheral blood mononuclear cells (PBMC) was subjected to microarray analysis. QRT-PCR was performed to verify the microarray results. Classification and functional characterization of differential genes were illustrated by hierarchical clustering and gene ontology analysis. Multiple logistic regression (MLR) analysis, receiver operating characteristic (ROC) curve analysis, and discriminate power were used to scan asthma-specific diagnostic markers. RESULTS: For fold-change>2 and p < 0.05, there were 758 named differential genes. The results of QRT-PCR confirmed successfully the array data. Hierarchical clustering divided 29 highly possible genes into seven categories and the genes in the same cluster were likely to possess similar expression patterns or functions. Gene ontology analysis presented that differential genes primarily enriched in immune response, response to stress or stimulus, and regulation of apoptosis in biological process. MLR and ROC curve analysis revealed that the combination of ADAM33, Smad7, and LIGHT possessed excellent discriminating power. CONCLUSIONS: The combination of ADAM33, Smad7, and LIGHT would be a reliable and useful childhood asthma model for prediction and diagnosis.

Effects of azathioprine and infliximab on mesenchymal stem cells derived from the bone marrow of rats in vitro.

Mesenchymal stem cell (MSC) transplantation has been demonstrated to be promising in the treatment of inflammatory bowel disease (IBD). Azathioprine (AZA) is widely used in IBD patients. Infliximab, as a representative of biological therapy for IBD, is important in the treatment regimen. In the present study we investigated the effects of AZA and infliximab on the cell proliferation, cell cycle and apoptosis of the MSCs derived from the bone marrow of Sprague­Dawley (SD) rats in vitro in order to provide preliminary data for optimizing the treatment of IBD. MSCs derived from the bone marrow of rats were either cultured in various concentrations of AZA­ or infliximab­supplemented medium for 24, 48 and 72 h, respectively. The growth curves of MSCs were obtained. The apoptosis and the cell cycle of the MSCs were analyzed by flow cytometry. AZA decreased the proliferation of MSCs by 66% and increased apoptosis at 0.20 mg/ml for 72 h (P<0.05). The percentage of necrotic cells increased markedly in MSCs treated with 0.30 mg/ml AZA for 72 h (P<0.05). As the exposure time increased, the percentage of MSCs in phase G0­G1 increased and that in phase S decreased in AZA groups exceeding 0.20 mg/ml (P<0.05). However, infliximab had a minimal impact on the cell proliferation, apoptosis and cell cycle of the MSCs. AZA was able to inhibit cell proliferation and induce apoptosis of the MSCs in vitro. Infliximab did not affect the cell proliferation, apoptosis and cell cycle of the MSCs derived from rats.

1,4-Bis[4-(meth-oxy-carbon-yl)benz-yl]-1H-1,2,4-triazol-4-ium bromide.

In the title salt, C(20)H(20)N(3)O(4) (+)·Br(-), the dihedral angle between the benzene rings is 8.69 (16)°, and those between the benzene rings and the triazole ring are 69.98 (18) and 72.17 (18)°. In the crystal, C-H⋯Br hydrogen bonds link the cations and anions into chains along the c axis.

The association between IFN-γ and IL-4 genetic polymorphisms and childhood susceptibility to bronchial asthma.

The present study aims to investigate the association between the genetic polymorphisms of interferon (IFN)-γ and interleukin (IL)-4 with childhood susceptibility to asthma and the levels of IFN-γ, IL-4, and immunoglobulin (Ig) E among asthmatic children. A total of 100 asthmatic children and 122 control children were enrolled in the present study. The genotypes of the IFN-γ gene at the -179G/T locus and the IL-4 gene at the -33C/T and -589C/T loci were detected using polymerase chain reaction with restriction fragment length polymorphism. The IFN-γ gene at the +874A/T locus and the IFN-γ CA repeats were tested using allele-specific and capillary electrophoresis, respectively, whereas the IFN-γ, IL-4, and total IgE levels were measured using enzyme-linked immunosorbent assays. The 100 asthmatic children and the 122 control children were all GG homozygous in the -179 locus of the IFN-γ gene, which shows that the IFN-γ gene is not mutated at the -179 locus. No significant differences were found in terms of genotypic and allelic frequency distribution in the IFN-γ gene or the CA repeat at the +874A/T locus between the asthmatic children and the control (P>0.05). An association was found between the polymorphism of the IFN-γ gene at +874A/T and IFN-γ levels. IFN-γ expression was lower among patients with the AA genotype than those with the AT genotype (P<0.05); the genotypic and allelic frequency distributions of the IL-4 gene at -33C/T and -589C/T were significantly different between the asthmatic children and the control (P<0.05). The levels of IL-4 and IgE among children with TT genotype at the -33 and -589 loci were higher than those with the CT genotype, but only the polymorphism at -33C/T was associated with IL-4 levels (P<0.05). The polymorphisms of the IFN-γ gene at +874A/T or the CA repeats are not correlated with susceptibility to asthma. Thus, the polymorphism at +874A/T is correlated with IFN-γ level. The TT genotypes of the IL-4 gene at the -33 and -589 loci are associated with asthma susceptibility in children, and polymorphism at the -33 locus may be associated with IL-4 level.

Histone deacetylase inhibitor promotes differentiation of embryonic stem cells into neural cells in adherent monoculture.

BACKGROUND: Embryonic stem (ES) cells poss unlimited self-renewal capacity and the ability to differentiate into cell of all three germ layers in vitro. Induced differentiation of ES cells to neural lineage cells has great potential in basic study of neurogenesis and regeneration therapy of neurodegenerative diseases. Histone deacetylase (HDAC) inhibitors enhance histone acetylation so that globularly activate gene expression and may initiate multilineage differentiation. In this study, we aimed to develop a method to induce the differentiation of ES cells to neural cells combining HDAC inhibition and neural cell selection. METHODS: In this study, we used HDAC inhibitor sodium butyrate (NaB) to induce the differentiation of mouse embryonic stem cells to neural cells through monolayer culture. After differentiation initiation by histone deacetylase inhibitor sodium butyrate, neural cells were induced and selected with a serum free culture system. RESULTS: Homogeneous neurons without glial cells demonstrated by molecular marker expression were differentiated with the method. The resultant neurons were excitable. CONCLUSION: The method combined differentiation induction effect of HDAC inhibitors and selective culture system to derive neural cells from ES cells, and implied the involvement of epigenetic regulation in neural differentiation.

Methyl 4-bromo-3-hy-droxy-benzoate.

In the title compound, C(8)H(7)BrO(3), the meth-oxy-carbonyl group is twisted at a dihedral angle of 8.06 (4)° with respect to the benzene ring. In the crystal, mol-ecules are connected by O-H⋯O hydrogen bonds into helical chains running along the b axis.

(3E,5E)-3,5-Bis(4-hy-droxy-benzyl-idene)oxan-4-one.

In the title compound, C(19)H(16)O(4), there are two 4-hy-droxy-benzyl substituents on the oxan-4-one (tetra-hydro-pyran-4-one) ring, which exhibits an envelope conformation. The dihedral angles between pyran-one ring and the two benzene rings are 26.69 (9) and 36.01 (9)° while the benzene rings make a dihedral angle of 20.88 (10)°. In the crystal, mol-ecules are linked by inter-molecular O-H⋯O hydrogen bonds into a supra-molecular three-dimensional twofold inter-penetrating hydrogen-bonded network.

1,3-Bis[4-(meth-oxy-carbon-yl)benz-yl]benzimidazolium bromide monohydrate.

In the title compound, C(25)H(23)N(2)O(4) (+)·Br(-)·H(2)O, the dihedral angles between the benzimidazole ring system and the two benzene rings are 87.77 (11) and 63.05 (11)°; the dihedral angle between the two benzene rings is 66.25 (13)°. The crystal structure exhibits C-H⋯O and O-H⋯Br inter-actions; it is also stabilized by π-π stacking inter-actions, with a face-to-face separation of 3.456 Šbetween parallel benzimidazole ring systems.

(3E,5E)-3,5-Bis(4-hy-droxy-3,5-di-methoxy-benzyl-idene)oxan-4-one monohydrate.

In the title compound, C(23)H(24)O(8)·H(2)O, the six-membered ring of the oxan-4-one (tetra-hydro-pyran-4-one) ring displays an envelope conformation with the heterocyclic O atom at the flap position. The dihedral angles between the terminal benzene rings is 37.23 (10)°. Classical intermolecular O-H⋯O and weak C-H⋯O hydrogen bonds are present in the crystal structure.

[Effect of dexamethasone on the expression of aquaporin-5 in the lungs of mice with acute allergic asthma].

OBJECTIVE: To detect the expression of lung aquaporin 5 (AQP5) in mice with acute allergic asthma and the effect of dexamethasone (DEX) treatment on AQP5 expression, and investigate the role of AOP5 in asthma pathogenesis. METHODS: Mouse models of acute allergic asthma were randomly divided into acute asthma group, normal control group and DEX treatment group. The total number of white blood cells, the subpopulations, and the levels of IL-5 and IFN-gamma were detected in the bronchoalveolar larvage fluid (BALF). The lung tissue AQP5 mRNA expression was detected by RT-PCR, and AQP5 distribution by immunohistochemical method. RESULTS: In asthma group, the total white blood cells, eosinophils and IL-5 levels were all significantly higher (P<0.01) and IFN-gamma levels lower than those of the control group (P<0.01). After DEX treatment, the levels underwent a significant reverse change (P<0.05, P<0.01, P<0.01, and P<0.01, respectively). AQP5 mRNA expression in the asthma group was significantly higher than that in the control group (P<0.01), and was significantly lowered with DEX treatment (P<0.01). Extensive inflammatory changes, mucus hypersecrection, several edema and inflammatory cell infitration around the blood vessels were observed in the lung tissue of the mice in the asthma group. The morphological changes of the treatment group were significantly ameliorated. AQP5 protein was detected in the type I alveolar epithelial cells, the airway columnar epithelial cells and the apical membranes of the submucosal gland acinar cells in the control group. Stronger AQP5 protein expression was found in the asthma group. CONCLUSION: AQP5 is over-expressed in mice with acute asthma which is possibly associated with mucus hypersecrection. DEX can inhibit AQP5 expression and ameliorate allergic airway inflammation, edema and mucus hypersecrection.

3-[(R)-3,3-Dichloro-2-hydroxy-prop-yl]-8-hydr-oxy-6-meth-oxy-1H-isochromen-1-one.

The title compound, C(13)H(12)Cl(2)O(5), is an isocoumarin compound which has been isolated from the ethyl acetate extract of the fermentation broth of actinomycete Streptomyces sp. (V(4)) from the South China Sea. There are intra- and inter-molecular hydrogen bonds and halogen bonds [Cl⋯Cl = 3.434 (2) Å; C-Cl⋯Cl = 121.6°]. The intermolecular O-H⋯O hydrogen bonds link mol-ecules into chains along the b axis.

5-Hydr-oxy-1-methyl-3,4-dihydro-2H-pyrrolium hydrogensulfate.

The title compound, C(5)H(10)NO(+)·HSO(4) (-), has been synthesized by reaction of 1-methyl-pyrrolidin-2-one with H(2)SO(4) in a 1:1 molar ratio. The substituted pyrrolium ring adopts an envelope conformation. The hydrogensulfate anions form infinite helical chains parallel to the a axis via strong O-H⋯O hydrogen bonds. The pyrrolium cations are pendant from the chains. These cations are the hydrogen donors in the strong O-H⋯O hydrogen bonds to the hydrogensulfates. In addition, there are weak C-H⋯O hydrogen bonds in the structure.

(1)H and (13)C NMR assignments for five anthraquinones from the mangrove endophytic fungus Halorosellinia sp. (No. 1403).

We report the unambiguous assignments of the (1)H and (13)C NMR spectra of two new natural products, namely, 1,4,5,6,7,9-hexahydroxy-2-methoxy-7-methyl-5beta,9beta,8abeta, 6alpha,10aalpha-hexahydroanthracen-10 (10aH)-one (1) and 1,4,6-trihydroxy-2-methoxy-7-methylanthracene-9, 10-dione (2), together with three known anthraquinones. These compounds were all isolated from the marine endophytic fungus No. 1403 collected from the South China Sea. Compounds 3 and 4 were isolated from the marine fungus for the first time. The structures were elucidated by the spectroscopic methods 1D and 2D NMR including COSY, HMQC, HMBC and NOE, and HREIMS. In our cytotoxicity assays, compound 5 showed cytotoxicity toward KB and KBv-200 cells with IC(50) of 1.40 and 2.58 microg/ml, respectively. In addition, the plausible biogenic relationship of compounds 1, 2, 3 and 4 is discussed.

[Expression of transcription factors T-bet/GATA-3 mRNA and its effect on Tc1/Tc2 balance in asthmatic children].

OBJECTIVE: In contrast to CD(4)(+) helper T-lymphocytes (T(H)), little is known about the transcriptional regulation of CD(8)(+) cytotoxic T-lymphocytes (Tc) and its role in the pathogenesis of asthma is unclear. This study was conducted to investigate the effect of T-bet and GATA-3 mRNA expression on profiles of type 1 and type 2 cytotoxic T lymphocytes in asthmatic children. METHOD: Totally 38 asthmatic children, including acute attack group composed of 20 cases (age 3 - 13 years, mean 6.2 +/- 2.9), remission group with 18 cases (age 3 - 12 years, mean 6.1 +/- 2.5) and 20 healthy control children (age 3 - 12, 6.9 +/- 2.7) were recruited in this study from Sep. 2005 to Mar. 2006. The mRNA expression of T-bet and GATA-3 in the peripheral blood mononuclear cells were detected by using semi-quantitative PCR and Tc1, Tc2 cell numbers by flow cytometry analysis system. RESULT: T-bet mRNA in asthmatic children was lower than that in control group and lower in attack stage than in remission stage (0.14 +/- 0.04, 0.21 +/- 0.03, 0.28 +/- 0.03, P < 0.05). In contrast, GATA-3 mRNA was higher in asthmatic children than in control group and higher in attack stage than in remission stage (0.49 +/- 0.09, 0.44 +/- 0.08, 0.37 +/- 0.04, P < 0.05). It was shown that Tc1 percentage was lower in asthmatic children than those of control group and lower in attack stage than those of remission stage (6.6 +/- 2.4, 14.2 +/- 4.3, 31.2 +/- 3.8, P < 0.05). Tc2 percentage in asthmatic children was higher than that of control group and higher in attack stage than that of remission stage (10.0 +/- 4.2, 5.4 +/- 2.2, 3.5 +/- 1.1, P < 0.05). Spearman correlation analysis revealed that T-bet mRNA was positively correlated with Tc1 percentage (r = 0.704) and negatively correlated with Tc2 percentage (r = -0.629). GATA3 mRNA was negatively correlated with Tc1 percentage (r = -0.612) and positively correlated with Tc2 percentage (r = 0.673). The T-bet/GATA-3 mRNA ratio was positively correlated with Tc1 percentage (r = 0.731) and Tc1/Tc2 (r = 0.773), while negatively correlated with Tc2 percentage (r = -0.642). CONCLUSION: The imbalance of T-bet/GATA-3 mRNA expression is closely correlated with skewed Tc2 dominance in asthmatic children.