Ex vivo tumor necrosis factor-alpha response of blood leukocytes in Danish Holstein-Friesian cows stimulated by Gram-positive and Gram-negative bacteria isolated from mastitic milk.

A whole blood stimulation assay was used to investigate the effects of parity, number of weeks after calving and Gram-positive and Gram-negative bacteria on the ex vivo TNF-α responsiveness of Danish Holstein-Friesian cows of first to third lactation (n = 28). Blood samples were collected in weeks 2, 3, 5 and 8 after parturition and stimulated with Escherichia coli LPS (10 µg/mL), Staphylococcus aureus peptidoglycan (PGN, 10 µg/mL) and dead Escherichia coli, Streptococcus uberis, Staphylococcus aureus, and Streptococcus dysgalactiae at a concentration of 2.5 × 106/mL. The antibiotic polymyxin-B (100 µg/mL) was added to the Gram-positive bacteria to avoid the influence of environmental endotoxin by ELISA test. Overall, parity had no effect, whereas number of weeks after calving altered the TNF-α responsiveness of the majority of the stimulants. Ex vivo, Gram-positive bacteria always resulted in a higher TNF-α response than Gram-negative bacteria with large differences within the individual cows. High correlations were found within the Gram-negative stimulants panel (r = 0.83) and within the Gram-positive (r = 0.81 to 0.86) stimulants panel except PGN. The higher TNF-α responsiveness by Gram-positive bacteria is in agreement with in vitro studies in human but in contrast to the in vivo TNF-α responsiveness in bovine udder.

A novel electrochemical biosensor based on peptidoglycan and platinum-nickel-copper nano-cube for rapid detection of Gram-positive bacteria.

A novel non-enzyme electrochemical biosensor for the rapid detection of Gram-positive bacteria has been constructed that relys on a stable and efficient combination between the peptidoglycan layer and platinum-nickel-copper nanocubes (Pt-Ni-Cu NCs). Briefly, bacteria were first captured by a specific antibody. Then, the electrochemical signal materials (Pt-Ni-Cu NCs) were bound to the bacteria peptidoglycan layer using specific structural and surface features. The rapid and sensitive bacterial detection was then achieved using intrinsic electrochemical characteristics and superoxidase-like activity of the Pt-Ni-Cu NCs. Moreover, the nature of peptidoglycan covering the whole bacteria provided the premise for signal amplification. Under optimal conditions, the electrochemical signal variation was proportional to the concentration of bacteria ranging from 1.5 × 102 to 1.5 × 108 CFU/mL with a detection limit of 42 CFU/mL using a working potential of - 0.4 V. This electrochemical biosensor has been successfully applied to detect bacteria concentrations in urine samples, and the recoveries range from 90.4 to 107%. The proposed biosensor could be applied for broad-spectrum detection of Gram-positive bacteria since most Gram-positive bacteria possess a thick peptidoglycan layer. The developed electrochemical biosensing strategy might be used as a potential tool for clinical pathogenic bacteria detection and point-of-care testing (POCT).

Effectiveness of Two Types of Photostimulable Phosphor Plate Plastic Barrier Envelopes for Prevention of Microbiological Contamination

Abstract Objective: To compare the effectiveness of two types of commercially available photostimulable phosphor plate (PSP) protective barrier envelopes to prevent microbiological contamination. Material and Methods: In this cross-sectional study, 80 barrier envelopes were tested in 40 volunteers. The PSP plates were placed individually in Asia Teb and Soredex protective barrier envelopes and were placed in the mouth for two minutes, similar to periapical films. The protective barrier envelopes were then removed under sterile conditions, and the sensors were placed on different culture media. The number of colonies on each plate was counted. Data were analyzed using SPSS via McNemar and Wilcoxon tests. Results: Bacterial growth was noted in 17.5% of PSPs with Soredex, and 32.5% of PSPs with Asia Teb barrier envelopes. Gram-positive bacilli were the most commonly isolated bacteria. The difference between the Asia Teb and Soredex barrier envelopes for the protection of microbiological contamination was not significant (p>0.05). Conclusion: The use of different types of protective barrier envelopes was not sufficient for prevention of microbiological contamination of PSP plates, and some adjunct modalities were required to decrease microbiological contamination of PSP plates.

IFN-mediated negative feedback supports bacteria class-specific macrophage inflammatory responses.

Despite existing evidence for tuning of innate immunity to different classes of bacteria, the molecular mechanisms used by macrophages to tailor inflammatory responses to specific pathogens remain incompletely defined. By stimulating mouse macrophages with a titration matrix of TLR ligand pairs, we identified distinct stimulus requirements for activating and inhibitory events that evoked diverse cytokine production dynamics. These regulatory events were linked to patterns of inflammatory responses that distinguished between Gram-positive and Gram-negative bacteria, both in vitro and after in vivo lung infection. Stimulation beyond a TLR4 threshold and Gram-negative bacteria-induced responses were characterized by a rapid type I IFN-dependent decline in inflammatory cytokine production, independent of IL-10, whereas inflammatory responses to Gram-positive species were more sustained due to the absence of this IFN-dependent regulation. Thus, disparate triggering of a cytokine negative feedback loop promotes tuning of macrophage responses in a bacteria class-specific manner and provides context-dependent regulation of inflammation dynamics.

Bactericidal/Permeability-Increasing Protein Is an Enhancer of Bacterial Lipoprotein Recognition.

Adequate perception of immunologically important pathogen-associated molecular patterns like lipopolysaccharide and bacterial lipoproteins is essential for efficient innate and adaptive immune responses. In the context of Gram-negative infection, bactericidal/permeability-increasing protein (BPI) neutralizes endotoxic activity of lipopolysaccharides, and thus prohibits hyperactivation. So far, no immunological function of BPI has been described in Gram-positive infections. Here, we show a significant elevation of BPI in Gram-positive meningitis and, surprisingly, a positive correlation between BPI and pro-inflammatory markers like TNFα. To clarify the underlying mechanisms, we identify BPI ligands of Gram-positive origin, specifically bacterial lipopeptides and lipoteichoic acids, and determine essential structural motifs for this interaction. Importantly, the interaction of BPI with these newly defined ligands significantly enhances the immune response in peripheral blood mononuclear cells (PBMCs) mediated by Gram-positive bacteria, and thereby ensures their sensitive perception. In conclusion, we define BPI as an immune enhancing pattern recognition molecule in Gram-positive infections.

Kunitz-type serine protease inhibitor is a novel participator in anti-bacterial and anti-inflammatory responses in Japanese flounder (Paralichthys olivaceus).

Kunitz-type serine protease inhibitor (KSPI) interacts with serine protease (SP) to regulate cascade reactions in vivo and plays essential roles in innate immunity. Theoretical considerations support various functions of kspi, but further studies are required for full characterization of these functions. In this study, a KSPI molecule was identified from Japanese flounder (Paralichthys olivaceus), and was named Pokspi. The full-length cDNA sequence of Pokspi was 2810 nt, containing an open reading frame of 1527 nt, which encoded a polypeptide of 509 amino acid residues. PoKspi protein contained five conversed domains, namely, MANEC, PKD, LDLa and two Kunitz domains. Homology analysis revealed that Pokspi shared the highest similarity (83%) with its homolog in Cynoglossus semilaevis. Phylogenetic analysis indicated that Pokspi clustered with the homologs in other fishes. The mRNA transcripts of Pokspi were detected in all tested tissues, with the highest expression level in gill, followed by kidney and intestine. Its elevated expression in response to the application of Edwardsiella tarda (in vivo) and pathogen-associated molecular pattern (in vitro) suggested the involvement of Pokspi in the essential immune defense against various pathogens. Recombinant PoKspi (rPoKspi) purified from Escherichia coli exhibited not only serine protease inhibitor activities but also a broad spectrum of anti-microbial effect in a manner that was independent of any host factors. In addition, the recombinant PoKspi protein could cause the down-regulation of pro-inflammatory factors TNF-α and IL-1ß. In conclusion, Pokspi is a biologically active serine protease inhibitor endowed with anti-bacterial and anti-inflammatory property. This study provides strong evidences for understanding the innate immune defense in Japanese flounder.

Chicken-type lysozyme functions in the antibacterial immunity in red swamp crayfish, Procambarus clarkii.

Lysozymes possess antibacterial activities, making them crucial defense proteins in innate immunity. In this study, a chicken-type (c-type) lysozyme (designated PcLyzc) was cloned and characterized from red swamp crayfish Procambarus clarkii. The full-length cDNA had an open reading frame of 435 base pairs encoding a polypeptide of 144 amino acid residues. Multiple alignments and phylogenetic analysis revealed that PcLyzc shared high similarity to the other known invertebrate c-type lysozymes. PcLyzc transcripts were steadily expressed in a wide range of tissues in healthy crayfish, and were prominently up-regulated in the hepatopancreas and gills after Vibrio anguillarum or Aeromonas hydrophila challenge. Recombinant PcLyzc showed inhibitory activity in vitro against both Gram-positive bacteria, including Staphylococcus aureus, Micrococcus luteus and Bacillus thuringiensis, and Gram-negative bacteria, including A. hydrophila, V. anguillarum and Escherichia coli. By overexpressing PcLyzc through introducing exogenous recombinant protein, or silencing PcLyzc expression through injecting double strand RNA, it was found that PcLyzc could help eliminate the invading bacteria in crayfish hemolymph and could protect crayfish from death, possibly by promoting the hemocytic phagocytosis. These results indicated that PcLyzc played a role in the antibacterial immunity of crustaceans, and laid a foundation of developing new therapeutic agents in aquaculture.

Subversion of Macrophage Functions by Bacterial Protein Toxins and Effectors.

Macrophages represent one of the first lines of host immune defenses against the invasion of pathogenic bacteria. Many receptors, immune signaling pathways and cellular processes in macrophages, including Toll-like receptors, Nod-like receptors, phagocytosis, autophagy and programmed cell death, are involved in combating the infection of bacterial pathogens. For efficient colonization in the host, bacterial pathogens have evolved diverse mechanisms to interfere with macrophage functions to evade host defenses. The major weapons utilized by bacterial pathogens are protein toxins and effectors secreted via specific bacterial secretion systems, including type I-VII secretion apparatuses. In recent years, great advances have been achieved in understanding how bacterial toxins and effectors subvert immune signaling and cellular processes of macrophages. In this review, we focus on the toxins and effectors that modulate the phagocytosis, intracellular immune signaling pathways, autophagy and programmed cell death processes of macrophages from the bacterium Legionella pneumophila, Shigella flexneri, Listeria monocytogenes, Salmonella spp., Yersinia spp., enteropathogenic E. coli and Mycobacterium tuberculosis.

Isolation of bacterial DNA followed by sequencing and differing cytokine response in peritoneal dialysis effluent help in identifying bacteria in culture negative peritonitis.

AIM: The treatment of peritoneal dialysis related culture negative peritonitis is empirical which increases the cost of therapy and moreover antibiotic resistance. We aimed the study to isolate bacterial DNA from PD effluent and indentify bacteria causing peritonitis in culture negative situations. We have also studied the cytokine response with different bacteria causing peritonitis. METHODS: We have isolated bacterial DNA from PD effluent of culture negative and culture positive peritonitis patients. Bacterial DNA was subjected to polymerase chain reaction using universal bacteria specific primers and subsequently to Gram type specific primers for the differentiation of the etiologic agents into Gram-positive and Gram-negative. The amplified products were sequenced and subjected to blast search to identify agent at genus/ species level. RESULTS: Of the 30 molecular method positive samples, 16 (53.33%) samples were positive for Gram-negative bacteria and 4 (13.33%) for Gram-positive, while the remaining10 (33.33%) were positive for both Gram-positive and Gram-negative bacteria. We have found organisms that usually do not grow on normal culture methods. TNF-α was significantly associated with Gram-positive peritonitis and regulatory cytokine IL-10 with Gram-negative peritonitis. CONCLUSIONS: The molecular techniques are helpful in detecting and identifying organisms from culture negative PD effluent.

Identification of neuroglobin as a novel player in anti-bacterial responses in amphioxus.

Theoretical considerations support various functions of neuroglobin (Ngb), but further studies are required for full characterization of these functions. In this study, we identified the presence of a single Ngb gene, BjNgb, in the amphioxus Branchiostoma japonicum. BjNgb was expressed in various tissues including the notochord, gonads (ovary and testis) and gill, and up-regulated significantly in response to the challenge with LPS and LTA, suggesting involvement in immune response of amphioxus against bacterial infection. In accord, we demonstrated for the first time that recombinant BjNgb (rBjNgb) not only interacted with the Gram-positive and negative bacteria as well as their conserved surface components LPS and LTA, but also enhanced the phagocytosis of bacteria by macrophages. Collectively, these data suggest that BjNgb is a novel player in amphioxus, via functioning as a pattern recognition molecule and an opsonin.

Scavengers for bacteria: Rainbow trout have two functional variants of MARCO that bind to gram-negative and -positive bacteria.

Class A scavenger receptors (SR-As) are a family of surface-expressed receptors who bind a wide range of polyanionic ligands including bacterial components and nucleic acids and play a role in innate immunity. Macrophage receptor with collagenous structure (MARCO) is a SR-A family member that has been studied in mammals largely for its role in binding bacteria. To date there is little information about SR-As in general and MARCO specifically in fish, particularly what ligands individual SR-A family members bind remains largely unknown. In the present study two novel rainbow trout MARCO transcript variants have been identified and their sequence and putative protein domains have been analyzed. When overexpressed in CHSE-214, a cell line that appears to lack functional scavenger receptors, GFP-tagged rtMARCO-1 and rtMARCO-2 were able to bind gram-positive, and gram-negative bacteria of both mammalian and aquatic sources. rtMARCO appears to bind bacteria via its scavenger receptor cysteine-rich (SRCR) domain, because SRCR deleted rtMARCO-1 and -2 were unable to bind bacteria. rtMARCO did not show any binding to the yeast cell wall component zymosan or to double-stranded (ds)RNA. This is the first time rainbow trout MARCO sequences have been identified and the first in-depth study exploring their ligand binding profile. This study provides novel insight into the role of rainbow trout MARCO in bacterial innate immunity.

Characterization and function of a tachylectin 5-like immune molecule in Penaeus monodon.

Tachylectin5A and its homolog, tachylectin5B both contain a fibrinogen-related domain (FReD) and have been studied in horseshoe crabs, Tachypleus tridentatus and Carcinoscorpius rotundicauda and shown to be involved in host defense. Here, we demonstrate the presence of tachylectin5-like genes in shrimp, Penaeus monodon, designated as Penlectin5-1 (PL5-1) and Penlectin5-2 (PL5-2), which both contain a signal peptide and a single FReD with an acetyl group and a calcium binding sites and they are both structurally similar to horseshoe crab tachylectin/carcinolectin5. The PL5-1and PL5-2 transcript were expressed in various shrimp tissues in normal shrimp, and their expression was upregulated in tissues such as hemocytes and hindgut following challenge with pathogenic Vibrio harveyi. The PL5-2 protein was detected in various tissues as well as in cell-free hemolymph. The biological function of the PL5-2 protein is to recognize some Gram-positive and Gram-negative bacteria regardless whether they are non-pathogenic or pathogenic. They have hemagglutination activity on human erythrocyte and bacterial agglutination activity to both Gram negative and Gram positive bacteria. Possible binding sites of PL5-2 to bacteria could be at the N-acetyl moiety of the GlcNAc-MurNAc cell wall of the peptidoglycan since the binding could be inhibited by GlcNAc or GalNAC. The presence of PL5-2 protein in both circulating hemolymph and intestine, where host and microbes are usually interacting, may suggest that the physiological function of shrimp tachylectin-like proteins is to recognize and bind to invading bacteria to immobilize and entrap these microbes and subsequently clear them from circulation and the host body, and probably to control and maintain the normal flora in the intestine.

A chymotrypsin-like serine protease from Portunus trituberculatus involved in pathogen recognition and AMP synthesis but not required for prophenoloxidase activation.

Clip domain serine proteases (clip-SPs) play critical roles in various immune responses in arthropods, such as hemolymph coagulation, antimicrobial peptide (AMP) synthesis, cell adhesion and melanization. In the present study, we report the molecular and functional characterization of a clip domain serine protease (PtcSP2) from the swimming crab Portunus trituberculatus. The N-terminal clip domain and the C-terminal SP-like domain of PtcSP2 were expressed in Escherichia coli system, and assayed for their activities. Sequence similarity and phylogenetic analysis revealed that PtcSP2 may belong to the chymotrypsin family, which was confirmed by protease activity assay of the recombinant SP-like domain. The clip domain of PtcSP2 exhibited strong antibacterial activity and microbial-binding activity, suggesting the potential role in immune defense and recognition. Knockdown of PtcSP2 by RNA interference could significantly reduce PtcSP2 transcript levels, but neither decrease the total phenoloxidase (PO) activity in crab nor significantly alter the expression levels of serine protease inhibitors PtPLC and PtSerpin. These results indicate that PtcSP2 is not involved in the proPO system. However, suppression of PtcSP2 led to a significant change in the expression of AMP genes PtALFs and PtCrustin but not PtALF5. All these findings suggest that PtcSP2 is a multifunctional chymotrypsin-like serine protease and may participate in crab innate immunity by its antibacterial activity, immune recognition or regulation of AMP expression.

Human α-Defensin 6: A Small Peptide That Self-Assembles and Protects the Host by Entangling Microbes.

Human α-defensin 6 (HD6) is a 32-residue cysteine-rich peptide that contributes to innate immunity by protecting the host at mucosal sites. This peptide is produced in small intestinal Paneth cells, stored as an 81-residue precursor peptide named proHD6 in granules, and released into the lumen. One unusual feature of HD6 is that it lacks the broad-spectrum antimicrobial activity observed for other human α-defensins, including the Paneth cell peptide human α-defensin 5 (HD5). HD6 exhibits unprecedented self-assembly properties, which confer an unusual host-defense function. HD6 monomers self-assemble into higher-order oligomers termed "nanonets", which entrap microbes and prevent invasive gastrointestinal pathogens such as Salmonella enterica serovar Typhimurium and Listeria monocytogenes from entering host cells. One possible advantage of this host-defense mechanism is that HD6 helps to keep microbes in the lumen such that they can be excreted or attacked by other components of the immune system, such as recruited neutrophils. In this Account, we report our current understanding of HD6 and focus on work published since 2012 when Bevins and co-workers described the discovery of HD6 nanonets in the literature. First, we present studies that address the biosynthesis, storage, and maturation of HD6, which demonstrate that nature uses a propeptide strategy to spatially and temporally control the formation of HD6 nanonets in the small intestine. The propeptide is stored in Paneth cell granules, and proteolysis occurs during or following release into the lumen, which affords the 32-residue mature peptide that self-assembles. We subsequently highlight structure-function studies that provide a foundation for understanding the molecular basis for why HD6 exhibits unusual self-assembly properties compared with other characterized defensins. The disposition of hydrophobic residues in the HD6 primary structure differs from that of other human α-defensins and is an important structural determinant for oligomerization. Lastly, we consider functional studies that illuminate how HD6 contributes to mucosal immunity. We recently discovered that in addition to blocking bacterial invasion into host epithelial cells by Gram-negative and Gram-positive gastrointestinal pathogens, HD6 suppresses virulence traits displayed by the opportunistic human fungal pathogen Candida albicans. In particular, we found that C. albicans biofilm formation, which causes complications in the treatment of candidiasis, is inhibited by HD6. This observation suggests that HD6 may contribute to intestinal homeostasis by helping to keep C. albicans in its commensal state. We intend for this Account to inspire further biochemical, biophysical, and biological investigations that will advance our understanding of HD6 in mucosal immunity and the host-microbe interaction.

Genomic organization, expression and antimicrobial activity of a hepcidin from taimen (Hucho taimen, Pallas).

Hepcidin, an antimicrobial peptide, plays a crucial role in innate immune system of teleost fish. As a cysteine-rich peptide, hepcidin possesses a dual function including iron regulation and innate immunity. In the present study, a full-length hepcidin cDNA (HtHep) was cloned and characterized by RT-PCR and RACE techniques from taimen (Hucho taimen, Pallas), which is a type of rare, precious and cold-water fish species in China. The cDNA contains an open reading frame (ORF) of 267 bp encoding 88 amino acid (aa), with 170 bp located in the 5(') untranslated region (UTR) and 151 bp in the 3' UTR. The genomic sequences analysis showed that the HtHep gene consisted of three exons and two introns (with the length 94 and 251 bp, respectively). With a predicted molecular mass of 2881.4 Da and a theoretical pI of 8.53, the deduced amino acid encodes a signal peptide of 24 aa, prodomain of 39 aa and mature peptide of 25 aa. The signal peptidase (SA-VP) and the motif RX (K/R)R of propeptide convertase suggested the cleavage site of signal and mature peptide. Eight conserved cysteine residues were also identified and formed four disulfide bonds. Pair-wise alignments showed that HtHep clustered together with two fish species of Salmonidae family (Salmo salar and Oncorhynchus mykiss) in HAMP1 branch. Quantitative RT-PCR analysis indicated that the mRNA levels of HtHep were detected in a wide range of tissues and the highest level was detected in the liver. Its expression was also detected early during embryonic stage and could be up-regulated in the liver when challenged with pathogenic bacteria (Yersinia ruckeri). The recombinant HtHep (rHtHep) had antimicrobial activity against both gram-positive (Micrococcus lysodeikticus and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). Our results suggested that HtHep might be involved in the innate immune defense against bacterial pathogens in taimen.

Novel Innate Immune Genes Regulating the Macrophage Response to Gram Positive Bacteria.

Host variation in Toll-like receptors and other innate immune signaling molecules alters infection susceptibility. However, only a portion of the variability observed in the innate immune response is accounted for by known genes in these pathways. Thus, the identification of additional genes that regulate the response to Gram positive bacteria is warranted. Bone marrow-derived macrophages (BMMs) from 43 inbred mouse strains were stimulated with lipotechoic acid (LTA), a major component of the Gram positive bacterial cell wall. Concentrations of the proinflammatory cytokines IL-6, IL-12, and TNF-α were measured. In silico whole genome association (WGA) mapping was performed using cytokine responses followed by network analysis to prioritize candidate genes. To determine which candidate genes could be responsible for regulating the LTA response, candidate genes were inhibited using RNA interference (RNAi) and were overexpressed in RAW264.7 macrophages. BMMs from Bdkrb1-deficient mice were used to assess the effect of Bdkrb1 gene deletion on the response to LTA, heat-killed Streptococcus pneumoniae, and heat-killed Staphylococcus aureus WGA mapping identified 117 loci: IL-6 analysis yielded 20 loci (average locus size = 0.133 Mb; 18 genes), IL-12 analysis produced 5 loci (0.201 Mb average; 7 genes), and TNF-α analysis yielded 92 loci (0.464 Mb average; 186 genes of which 46 were prioritized by network analysis). The follow-up small interfering RNA screen of 71 target genes identified four genes (Bdkrb1, Blnk, Fbxo17, and Nkx6-1) whose inhibition resulted in significantly reduced cytokine production following LTA stimulation. Overexpression of these four genes resulted in significantly increased cytokine production in response to LTA. Bdkrb1-deficient macrophages were less responsive to LTA and heat-killed S. aureus, validating the genetic and RNAi approach to identify novel regulators of the response to LTA. We have identified four innate immune response genes that may contribute to Gram positive bacterial susceptibility.

Differential effects of gram-positive and gram-negative bacterial products on morphine induced inhibition of phagocytosis.

Opioid drug abusers have a greater susceptibility to gram positive (Gram (+)) bacterial infections. However, the mechanism underlying opioid modulation of Gram (+) versus Gram (-) bacterial clearance has not been investigated. In this study, we show that opioid treatment resulted in reduced phagocytosis of Gram (+), when compared to Gram (-) bacteria. We further established that LPS priming of chronic morphine treated macrophages leads to potentiated phagocytosis and killing of both Gram (+) and Gram (-) bacteria in a P-38 MAP kinase dependent signaling pathway. In contrast, LTA priming lead to inhibition of both phagocytosis and bacterial killing. This study demonstrates for the first time the differential effects of TLR4 and TLR2 agonists on morphine induced inhibition of phagocytosis. Our results suggest that the incidence and severity of secondary infections with Gram (+) bacteria would be higher in opioid abusers.

Gut microbiome and innate immune response patterns in IgE-associated eczema.

BACKGROUND: Gut microbiome patterns have been associated with predisposition to eczema potentially through modulation of innate immune signalling. OBJECTIVE: We examined gut microbiome development in the first year of life in relation to innate immune responses and onset of IgE-associated eczema over the first 2.5 years in predisposed children due to maternal atopy [www.anzctr.org.au, trial ID ACTRN12606000280505]. METHODS: Microbial composition and diversity were analysed with barcoded 16S rRNA 454 pyrosequencing in stool samples in pregnancy and at ages 1 week, 1 month and 12 months in infants (n = 10) who developed IgE-associated eczema and infants who remained free of any allergic symptoms at 2.5 years of age (n = 10). Microbiome data at 1 week and 1 month were analysed in relation to previously assessed immune responses to TLR 2 and 4 ligands at 6 months of age. RESULTS: The relative abundance of Gram-positive Ruminococcaceae was lower at 1 week of age in infants developing IgE-associated eczema, compared with controls (P = 0.0047). At that age, the relative abundance of Ruminococcus was inversely associated with TLR2 induced IL-6 (-0.567, P = 0.042) and TNF-α (-0.597, P = 0.032); there was also an inverse association between the abundance of Proteobacteria (comprising Gram-negative taxa) and TLR4-induced TNF-α (rs = -0.629, P = 0.024). This relationship persisted at 1 month, with inverse associations between the relative abundance of Enterobacteriaceae (within the Proteobacteria phylum) and TLR4-induced TNF-α (rs = -0.697, P = 0.038) and Enterobacteriaceae and IL-6 (rs = -0.709, P = 0.035). Mothers whose infants developed IgE-associated eczema had lower α-diversity of Bacteroidetes (P = 0.04) although this was not seen later in their infants. At 1 year, α-diversity of Actinobacteria was lower in infants with IgE-associated eczema compared with controls (P = 0.002). CONCLUSION AND CLINICAL RELEVANCE: Our findings suggest that reduced relative abundance of potentially immunomodulatory gut bacteria is associated with exaggerated inflammatory cytokine responses to TLR-ligands and subsequent development of IgE-associated eczema.

La pubertad en niños chilenos muestra un adelantamiento en el inicio del crecimiento testicular / Age of onset of puberty in Chilean boys according to testicular volume and Tanner stage

Background: A secular trend towards a younger age of puberty onset has been reported in Chilean girls. Aim: To evaluate the age of onset of puberty and prevalence of early puberty in Chilean boys. Material and Methods: A pediatric endocrinologist examined 319 children attending schools in central Santiago. Pubertal development was assessed by testicular volume (TV) and genital inspection (GI) using Tanner graduation. Precocious and early puberty development was diagnosed if TV ≥ 4 ml or GI > stage 2 occurred in boys younger than 9 years and at 9-10 years of age, respectively. Results: Pubertal onset occurred at 10.2 ± 1.5 years according to TV and at 11.1 ± 1.6 years according to GI (p < 0.01). Before the age of nine, 15.2% of children had a VT ≥ 4 ml, 3% had genital changes in GI and only 3% had both changes simultaneously. Early puberty was observed in 23.8% of children according to TV and 9.5% according to GI. However, no child of less than 11 years old had a TV ≥ 4 ml, genital changes and pubic hair simultaneously. Late pubertal stages occurred at the same age according to both criteria used. Body mass index z score was not associated with the age of pubertal onset. Conclusions: Testicular enlargement occurs one year earlier than changes in genitalia according to inspection. Testicular growth, but not late stages of puberty, are occurring one year earlier than previously reported in Chile 10 years ago.

Commensal Gram-positive bacteria initiates colitis by inducing monocyte/macrophage mobilization.

Breakdown of the intestinal epithelial layer's barrier function results in the inflow of commensal flora and improper immune responses against the commensal flora, leading to inflammatory bowel disease (IBD) development. Using a mouse dextran sodium sulfate (DSS)-induced colitis model, we show here that commensal Gram-positive bacteria trigger the mobilization of inflammatory monocytes and macrophages into the colon. Monocytes/macrophages are major producers of tumor necrosis factor-α (TNF-α), a representative cytokine that aggravates colitis. Notably, pretreating mice with vancomycin, which eliminated Gram-positive bacteria, particularly the Lachnospiraceae family, significantly reduced the severity of the colitis by selectively blocking the recruitment of monocytes/macrophages, but not of other cells. Importantly, vancomycin treatment specifically downregulated the colonic epithelial cell (cEC) expression of C-C chemokine receptor type-2 (CCR2) ligands, which are critical chemokines for monocyte/macrophage mobilization into the inflamed colon. Collectively, these results provide previously undiscovered evidence that Gram-positive commensal bacteria induce colitis by recruiting colitogenic monocytes and macrophages. Our findings may lead to new avenues of treatment for IBD.