Antibiotic susceptibility in prostate-derived Propionibacterium acnes isolates

The aim of this study was to determine antibiotic susceptibility of Propionibacterium acnes isolates from prostate. Prostate‐derived P. acnes isolates (n = 24, Umeå & Örebro, Sweden, 2007–2010) and a panel of control strains (n = 25, Sweden) collected from skin and deep infections were assessed for resistance to penicillin G, piperacillin–tazobactam, imipenem, gentamicin, azithromycin, erythromycin, vancomycin, ciprofloxacin, moxifloxacin, tetracycline, tigecycline, fusidic acid, clindamycin, rifampicin, linezolid, daptomycin, trimethoprim–sulfamethoxazole, and metronidazole. In addition, the isolates were tested for inducible clindamycin resistance. All prostate derived P. acnes isolates displayed wild‐type distribution of MIC‐values, without evidence of acquired resistance. In the reference panel, 5 of 25 isolates had acquired macrolide resistance with cross‐resistance to azithromycin, clindamycin, and erythromycin. In addition, one of these isolates was resistant to tetracycline.     

H‐ficolin binds Aspergillus fumigatus leading to activation of the lectin complement pathway and modulation of lung epithelial immune responses

Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H‐Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H‐ficolin in fungal defence. Additionally, we investigated the presence of H‐ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H‐Ficolin exhibited demonstrable binding to A. fumigatus conidia via l ‐fucose, d ‐mannose and N‐acetylglucosamine residues in a calcium‐ and pH‐dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H‐ficolin opsonization manifested an increase in interleukin‐8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen‐activated protein kinase MAPK kinase 1/2, p38 MAPK and c‐Jun N‐terminal kinase. Finally, H‐ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H‐ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H‐ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus–host interactions and modulated immune responses.     

No associations established between single nucleotide polymorphisms in human Toll‐like receptor 2 and Toll‐interacting protein and Staphylococcus aureus bloodstream infections

Staphylococcus aureus bloodstream infections (SABSI) are associated with high morbidity and mortality. The Toll‐like receptor 2 (TLR2) and Toll‐interacting protein (TOLLIP) are important in recognition and regulation of human innate immunity response to S. aureus. Single nucleotide polymorphisms (SNPs) in the TLR2 and TOLLIP encoding genes have been associated with disease, including BSI. The aim of this study was to examine potential associations between a selection of SNPs in the genes encoding TLR2 and TOLLIP, and predisposition, severity, and outcome of SABSI. All patients ≥18 years of age with at least one S. aureus positive blood culture collected from March 2011 through February 2014 at Akershus University Hospital, Lørenskog, Norway, were considered for inclusion. Patients attending elective orthopaedic surgery (total hip and knee replacements, lumbar surgery) served as a control group. The TLR2 Arg753Gln, TLR2 Pro631His, TOLLIP rs5743942, and rs5743867 polymorphisms were analysed using TaqMan SNP Genotyping Assays. A total of 209 SABSI patients and 295 controls were included. The TLR2 Arg753Gln and TLR2 Pro631His polymorphisms were infrequent with no homozygotes and <10% heterozygotes. The included TLR2 and TOLLIP polymorphisms were not associated with susceptibility to SABSI, severity, 30‐day all‐cause mortality, or SABSI caused by the clonal complex 30 (CC30) genotype.     

Interferon‐γ enhances both the anti‐bacterial and the pro‐inflammatory response of human mast cells to Staphylococcus aureus

Human mast cells (huMCs) are involved in both innate and adaptive immune responses where they release mediators including amines, reactive oxygen species (ROS), eicosanoids and cytokines. We have reported that interferon‐γ (IFN‐γ) enhances FcγR‐dependent ROS production. The aim of this study was to extend these observations by investigating the effect of IFN‐γ on the biological responses of huMCs to Staphylococcus aureus. We found that exposure of huMCs to S. aureus generated intracellular and extracellular ROS, which were enhanced in the presence of IFN‐γ. IFN‐γ also promoted bacteria killing, β‐hexosaminidase release and eicosanoid production. Interferon‐γ similarly increased expression of mRNAs encoding CCL1 to CCL4, granulocyte–macrophage colony‐stimulating factor (GM‐CSF), tumour necrosis factor‐α and CXCL8 in S. aureus‐stimulated huMCs. The ability of IFN‐γ to increase CXCL8 and GM‐CSF protein levels was confirmed by ELISA. Fibronectin or a β₁ integrin blocking antibody completely abrogated IFN‐γ‐dependent S. aureus binding and reduced S. aureus‐dependent CXCL8 secretion. These data demonstrate that IFN‐γ primes huMCs for enhanced anti‐bacterial and pro‐inflammatory responses to S. aureus, partially mediated by β₁ integrin.     

Interleukin‐17A participates in podocyte injury by inducing IL‐1β secretion through ROS‐NLRP3 inflammasome‐caspase‐1 pathway

Studies show that the Th17/IL ‐17A axis plays an important role in the pathogenesis of kidney diseases. Previously, we also showed that IL ‐17A may play a role in the pathogenesis of primary nephrotic syndrome; however, the underlying mechanism(s) is unclear. The aim of this study was to explore the molecular mechanism of IL ‐17A‐inducing podocyte injury in vitro. In this study, the NLRP 3 inflammasome activation and the morphology of podocytes were detected by Western blot and immunofluorescence. The results showed that podocytes persistently expressed IL ‐17A receptor and that NLRP 3 inflammasome in these cells was activated upon exposure to IL ‐17A. Also, activity of caspase‐1 and secretion of IL ‐1β increased in the presence of IL ‐17A. In addition, IL ‐17A disrupted podocyte morphology by decreasing expression of podocin and increasing expression of desmin. Blockade of intracellular ROS or inhibition of caspase‐1 prevented activation of the NLRP 3 inflammasome, thereby restoring podocyte morphology. Taken together, the results suggest that IL ‐17A induces podocyte injury by activating the NLRP 3 inflammasome and IL ‐1β secretion and contributes to disruption of the kidney's filtration system.     

Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2‐binding domain

Filamentous hemagglutinin (FHA) is a major adhesion and virulence factor of Bordetella pertussis and also a main component of acellular pertussis vaccines. Interaction of FHA with different receptors on human epithelial and immune cells facilitates entrance and colonization of bacteria as well as immunomodulation of the host immune response. Three overlapping segments of the FHA gene were cloned in a prokaryotic expression vector and the recombinant proteins were purified. These recombinant fragments along with the native FHA protein were employed to assess their potential Toll‐like receptor (TLR) stimulatory effects and to localize the TLR binding region. TLR stimulation was monitored by applying HEK293‐Blue cell lines cotransfected with TLR2, 4, or 5 and a NF‐κB reporter gene. Culture supernatants were checked for secretion of the reporter gene product and IL‐8 as indicators of TLR stimulation. Native FHA was found to strongly stimulate TLR2, but not TLR4 or TLR5 transfected cells. Among recombinant FHA fragments only the fragment spanning amino acid residues 1544–1917 was able to exhibit the TLR2 stimulating property of FHA. Interaction of FHA with TLR2 suggests its involvement in induction of the innate immune system against Bordetella pertussis. The TLR2‐binding domain of FHA may contribute to immunoprotection against pertussis infection.     

Interrelationships of interleukin-8 with interleukin-1beta and neutrophils in vaginal fluid of healthy and bacterial vaginosis positive women

Vaginal innate immunity in response to microbial perturbation is still poorly understood and could be crucial for protection from adverse outcomes. We investigated the relationship between interleukin (IL)-8, IL-1beta and neutrophils in vaginal fluid obtained from 60 healthy women and 51 women who were bacterial vaginosis (BV) positive. Concentrations of IL-8 and IL-1beta were highly correlated with counts of neutrophils in vaginal fluid of the entire population examined (111 subjects). Vaginal IL-1beta concentrations were significantly higher (P < 0.001) in BV positive women. There was no significant difference in IL-8 levels or number of neutrophils between healthy controls and BV positive women. None of the healthy controls with high neutrophil counts (> or =75th percentile, 14 average count per field) had high concentrations of IL-1beta (> or =75th percentile, 220 pg/ml), whereas 84% of BV positive women with high neutrophil counts had high IL-1beta concentrations (P < 0.001). On the contrary, no difference in the percentage of subjects with elevated concentrations of IL-8 (> or =75th percentile, 2842 pg/ml) was found between healthy and BV positive women with high numbers of neutrophils (55.5% of healthy versus 53% of BV positive women). Our findings show that BV causes a large increase in IL-1beta concentrations which is not paralleled by an increase in IL-8 concentrations in vaginal fluid, suggesting that BV-associated factors more specifically dampen IL-8 rather than IL-1beta. The lack of an increase in IL-8 may explain the absence of an increase in neutrophil numbers in most women exposed to abnormal vaginal colonization (BV).     

Innate,antigen‐independent role for T cells in the activation of the immune system by Propionibacterium acnes

Propionibacterium acnes is a human commensal but also an opportunistic pathogen. In mice, P. acnes exerts strong immunomodulatory activities, including formation of intrahepatic granulomas and induction of LPS hypersensitivity. These activities are dependent on P. acnes recognition via TLR9 and subsequent IL‐12‐mediated IFN‐γ production. We show that P. acnes elicits IL‐12p40 and p35 mRNA expression in macrophages, and IFN‐γ mRNA in liver CD4⁺ T cells and NK cells. After priming with P. acnes, CD4⁺ T cells serve as the major IFN‐γ mRNA source. In the absence of CD4⁺ T cells, CD8⁺ T cells (regardless of antigenic specificity) or NK cells can produce sufficient IFN‐γ to induce the P. acnes‐driven immune effects. Moreover, in the absence of αβT cells, γδT cells also enable the development of strongly enhanced TNF‐α and IFN‐γ responses to LPS and intrahepatic granuloma formation. Thus, under microbial pressure, different T‐cell types, independent of their antigen specificity, exert NK‐cell‐like functions, which contribute decisively to the activation of the innate immune system.     

Influence of NKG2C gene deletion and CCR5Δ32 in Pre‐eclampsia—Approaching the effect of innate immune gene variants in pregnancy

Pre‐eclampsia (PE) is a hypertensive disorder that affects an important number of pregnant women worldwide. The exact causes of PE remain poorly understood. However, inflammation and deregulation of innate immune cells, such as natural killer (NK) cells, contribute to PE pathogenesis. Besides, the mother's genetic background also impacts on PE susceptibility. Thus, genetic variants that potentially modify the behaviour of inflammatory cells may help us to understand the causes of PE. Variants of genes encoding NKG2C (expressed in NK cells) and C–C chemokine receptor type 5 (CCR5) (expressed mainly in leucocytes) are important targets in the study of gestational disorders. In this context, we evaluated the impact of both NKGC2 gene deletion and CCR5Δ32 gene variant on PE susceptibility in a population sample from central‐southeast Brazil composed by 369 women (156 with PE and 213 healthy pregnant women). No statistically significant association between the NKG2C gene deletion and susceptibility to PE was observed. However, taking into consideration the important role of NK cells in pregnancy, the influence of NKG2C gene deletion on PE pathogenesis should not be ruled out and deserves further studies in populations with different genetic/ethnic backgrounds. In addition, our results regarding CCR5Δ32 corroborate previous data from our group approaching a distinct cohort and reinforce CCR5Δ32 as a protective factor against PE development (p < 0.05).     

Microbiology of folliculitis: a histological study of 39 cases

Folliculitis is a common inflammatory skin syndrome. Several microbial organisms have been put forward as causative agents, but few studies visualized microbes directly in inflamed hair follicles. This retrospective study investigated bacterial and fungal colonization of inflamed hair follicles in patients with clinically diagnosed non‐infectious folliculitis. Skin biopsies from 39 folliculitis patients and 27 controls were screened by fluorescence in situ hybridization (FISH) using broad‐range bacterial and fungal probes and by immunofluorescence microscopy using a monoclonal antibody towards Gram‐positive bacteria. Specific monoclonal and polyclonal antibodies towards Staphylococcus spp. and Propionibacterium acnes were applied for further species identification. Inflamed follicles were associated with bacterial colonization in 10 samples (26%) and fungal colonization in three samples (8%). Staphylococcus spp. were observed in inflamed follicles in seven samples (18%). Two samples were positive for P. acnes, which were identified as either type II or type IB/type III. Both Staphylococcus spp. and P. acnes were seen in macrocolonies/biofilm structures. In conclusion, one‐third of patients with clinically diagnosed, non‐infectious folliculitis exhibited microbial colonization with predominance of Staphylococcus spp.     

High expression of CD26 accurately identifies human bacteria‐reactive MR1‐restricted MAIT cells

Mucosa‐associated invariant T (MAIT) cells express the semi‐invariant T‐cell receptor TRAV1–2 and detect a range of bacteria and fungi through the MHC‐like molecule MR1. However, knowledge of the function and phenotype of bacteria‐reactive MR1‐restricted TRAV1–2⁺ MAIT cells from human blood is limited. We broadly characterized the function of MR1‐restricted MAIT cells in response to bacteria‐infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria‐reactive MR1‐restricted T cells shared effector functions of cytolytic effector CD8⁺ T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8⁺ subsets we demonstrated that high expression of CD26 on CD8⁺ TRAV1–2⁺ cells identified with high specificity and sensitivity, bacteria‐reactive MR1‐restricted T cells from human blood. CD161^hi was also specific for but lacked sensitivity in identifying all bacteria‐reactive MR1‐restricted T cells, some of which were CD161^dim. Using cell surface expression of CD8, TRAV1–2, and CD26^hi in the absence of stimulation we confirm that bacteria‐reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis.     

Molecular alterations of Ras‐Raf‐mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase‐Akt signaling pathways in colorectal cancers from a tertiary hospital at Kuala Lumpur,Malaysia

Molecular alterations in KRAS, BRAF, PIK3CA, and PTEN have been implicated in designing targeted therapy for colorectal cancer (CRC). The present study aimed to determine the status of these molecular alterations in Malaysian CRCs as such data are not available in the literature. We investigated the mutations of KRAS, BRAF, and PTEN, the gene amplification of PIK3CA, and the protein expression of PTEN and phosphatidylinositol 3‐kinase (PI3K) catalytic subunit (p110α) by direct DNA sequencing, quantitative real‐time PCR, and immunohistochemistry, respectively, in 49 CRC samples. The frequency of KRAS (codons 12, 13, and 61), BRAF (V600E), and PTEN mutations, and PIK3CA amplification was 25.0% (11/44), 2.3% (1/43), 0.0% (0/43), and 76.7% (33/43), respectively. Immunohistochemical staining demonstrated loss of PTEN protein in 54.5% (24/44) of CRCs and no significant difference in PI3K p110α expression between CRCs and the adjacent normal colonic mucosa (p = 0.380). PIK3CA amplification was not associated with PI3K p110α expression level, but associated with male cases (100% of male cases vs 56% of female cases harbored amplified PIK3CA, p = 0.002). PI3K p110α expression was significantly higher (p = 0.041) in poorly/moderately differentiated carcinoma compared with well‐differentiated carcinoma. KRAS mutation, PIK3CA amplification, PTEN loss, and PI3K p110α expression did not correlate with Akt phosphorylation or Ki‐67 expression. KRAS mutation, PIK3CA amplification, and PTEN loss were not mutually exclusive. This is the first report on CRC in Malaysia showing comparable frequency of KRAS mutation and PTEN loss, lower BRAF mutation rate, higher PIK3CA amplification frequency, and rare PTEN mutation, as compared with published reports.     

Immunology in clinic review series; focus on autoinflammatory diseases: role of inflammasomes in autoinflammatory syndromes

OTHER THEMES PUBLISHED IN THIS IMMUNOLOGY IN THE CLINIC REVIEW SERIES Allergy, Host Responses, Cancer, Type 1 diabetes and viruses, Metabolic diseases. SUMMARY: Autoinflammatory syndromes are disorders characterized by the hyperactivation of the innate immune system in the absence of microbial infection or autoantibody production. Some autoinflammatory syndromes are associated with recurrent episodes of fever and systemic inflammation that are caused by dysregulated activation of inflammasomes, molecular platforms responsible for the activation of caspase-1 and the production of interleukin (IL)-1β. In this review we will discuss the role of IL-1β and the inflammasomes in host defence and how mutations of two genes, NLRP3 and PYRIN, leads to the autoinflammatory syndromes, cryopyrin-associated periodic syndromes (CAPS) and familial Mediterranean fever (FMF). Both CAPS and FMF are characterized by increased inflammasome activity and overproduction of IL-1β which is ultimately responsible for disease manifestations. Importantly, understanding the molecular mechanisms of these syndromes has led to effective treatment for these rare diseases with biological drugs that target IL-1β-mediated signalling.     

Amomum tsao‐ko fruit extract suppresses lipopolysaccharide‐induced inducible nitric oxide synthase by inducing heme oxygenase‐1 in macrophages and in septic mice

Amomum tsao‐ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti‐inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao‐ko (AOM) in lipopolysaccharide (LPS)‐induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS‐induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase‐1 (HO‐1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO‐1) and silencing HO‐1 using siRNA prevented the AOM‐mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF‐E2‐related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N‐acetyl‐l ‐cysteine (NAC; a ROS scavenger) diminished the AOM‐induced phosphorylation of ERK and JNK and AOM‐induced HO‐1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM‐induced signalling of HO‐1 expression. In LPS‐induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO‐1 expression and survival rates. These results indicate that AOM strongly inhibits LPS‐induced NO production by activating the ROS/MAPKs/Nrf2‐mediated HO‐1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.