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BackgroundSpecies-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic E. coli (EHEC) infection whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities.ResultsWe utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites — 4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid — that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity.ConclusionOrgan on chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection.

Background Blood cultures, and molecular diagnostic tests that directly detect pathogen DNA in blood, fail to detect bloodstream infections in most infected patients. Thus, there is a need for a rapid test that can diagnose the presence of infection to triage patients, guide therapy, and decrease the incidence of sepsis. Methods An Enzyme-Linked Lectin-Sorbent Assay (ELLecSA) that uses magnetic microbeads coated with an engineered version of the human opsonin, Mannose Binding Lectin, containing the Fc immunoglobulin domain linked to its carbohydrate recognition domain (FcMBL) was developed to quantify pathogen-associated molecular patterns (PAMPs) in whole blood. This assay was tested in rats and pigs to explore whether it can detect infections and monitor disease progression, and in prospectively enrolled, emergency room patients with suspected sepsis. These results were also compared with data obtained from non-infected patients with or without traumatic injuries. Results The FcMBL ELLecSA was able to detect PAMPS present on, or released by, 85% of clinical isolates representing 47 of 55 different pathogen species, including the most common causes of sepsis. The PAMP assay rapidly ( 81%), specificity (> 89%), and diagnostic accuracy of 0·87. It also distinguished infection from trauma-related inflammation in the same patient cohorts with a higher specificity than the clinical sepsis biomarker, C-reactive Protein. Conclusion The FcMBL ELLecSA-based PAMP assay offers a rapid, simple, sensitive and specific method for diagnosing infections, even when blood cultures are negative and antibiotic therapy has been initiated. It may help to triage patients with suspected systemic infections, and serve as a companion diagnostic to guide administration of emerging dialysis-like sepsis therapies., Highlights • The FcMBL ELLecSA-based PAMP assay offers a rapid, simple, sensitive and specific method for diagnosing infections. • The FcMBL ELLecSA distinguished infection from trauma-related inflammation. • It can detect infection even when blood cultures are negative and antibiotic therapy has been initiated. Current diagnostics of sepsis using blood cultures and molecular diagnostic tests fail to detect bloodstream infections in most infected patients, whereas the inflammatory biomarkers of infection that have a higher sensitivity of detection, lack specificity in distinguishing infection from trauma-related inflammation. Therefore we have leveraged a broad-spectrum pathogen binding opsonin and developed a rapid test to directly diagnose the presence of infection in the blood to triage patients and guide antibiotic therapy.

We describe the discovery of MK-6169, a potent and pan-genotype hepatitis C virus NS5A inhibitor with optimized activity against common resistance-associated substitutions. SAR studies around the combination of changes to both the valine and aminal carbon region of elbasvir led to the discovery of a series of compounds with substantially improved potency against common resistance-associated substitutions in the major genotypes, as well as good pharmacokinetics in both rat and dog. Through further optimization of key leads from this effort, MK-6169 (21) was discovered as a preclinical candidate for further development.

Fat distribution changes with aging. Inherent changes in fat cell progenitors may contribute because fat cells turn over throughout life. To define mechanisms, gene expression was profiled in preadipocytes cultured from epididymal and perirenal depots of young and old rats. 8.4% of probe sets differed significantly between depots, particularly developmental genes. Only 0.02% differed with aging, despite using less stringent criteria than for comparing depots. Twenty-five genes selected based on fold change with aging were analyzed in preadipocytes from additional young, middle-aged, and old animals by polymerase chain reaction. Thirteen changed significantly with aging, 13 among depots, and 9 with both. Genes involved in inflammation, stress, and differentiation changed with aging, as occurs in fat tissue. Age-related changes were greater in perirenal than epididymal preadipocytes, consistent with larger declines in replication and adipogenesis in perirenal preadipocytes. Thus, age-related changes in preadipocyte gene expression differ among depots, potentially contributing to fat redistribution and dysfunction.

Fat mass and tissue distribution change dramatically throughout life. Fat depot sizes reach a peak by middle or early old age, followed by a substantial decline, together with fat tissue dysfunction and redistribution in advanced old age. These changes are associated with health complications, including type 2 diabetes, atherosclerosis, dyslipidemia, thermal dysregulation, and skin ulcers, particularly in advanced old age. Fat tissue growth occurs through increases in size and number of fat cells. Fat cells turn over throughout the lifespan, with new fat cells developing from preadipocytes, which are of mesenchymal origin. The pool of preadipocytes comprises 15 to 50% of the cells in fat tissue. Since fat tissue turns over throughout life, characteristics of these cells very likely have a significant impact on fat tissue growth, plasticity, function, and distribution. The aims of this review are to highlight recent findings regarding changes in preadipocyte cell dynamics and function with aging, and to consider how inherent characteristics of these cells potentially contribute to age- and depot-dependent changes in fat tissue development and function.

Here we describe development of an extracorporeal hemoadsorption device for sepsis therapy that employs commercially available polysulfone or polyethersulfone hollow fiber filters similar to those used clinically for hemodialysis, covalently coated with a genetically engineered form of the human opsonin Mannose Binding Lectin linked to an Fc domain (FcMBL) that can cleanse a broad range of pathogens and endotoxin from flowing blood without having to first determine their identity. When tested with human whole blood in vitro, the FcMBL hemoadsorption filter (FcMBL-HF) produced efficient (90-99%) removal of Gram negative (Escherichia coli) and positive (Staphylococcus aureus) bacteria, fungi (Candida albicans) and lipopolysaccharide (LPS)-endotoxin. When tested in rats, extracorporeal therapy with the FcMBL-HF device reduced circulating pathogen and endotoxin levels by more than 99%, and prevented pathogen engraftment and inflammatory cell recruitment in the spleen, lung, liver and kidney when compared to controls. Studies in rats revealed that treatment with bacteriocidal antibiotics resulted in a major increase in the release of microbial fragments or 'pathogen-associated molecular patterns' (PAMPs) in vivo, and that these PAMPs were efficiently removed from blood within 2 h using the FcMBL-HF; in contrast, they remained at high levels in animals treated with antibiotics alone. Importantly, cleansing of PAMPs from the blood of antibiotic-treated animals with the FcMBL-hemoadsorbent device resulted in reduced organ pathogen and endotoxin loads, suppressed inflammatory responses, and resulted in more stable vital signs compared to treatment with antibiotics alone. As PAMPs trigger the cytokine cascades that lead to development of systemic inflammatory response syndrome and contribute to septic shock and death, co-administration of FcMBL-hemoadsorption with antibiotics could offer a more effective approach to sepsis therapy.

Here we describe a blood-cleansing device for sepsis therapy inspired by the spleen, which can continuously remove pathogens and toxins from blood without first identifying the infectious agent. Blood flowing from an infected individual is mixed with magnetic nanobeads coated with an engineered human opsonin--mannose-binding lectin (MBL)--that captures a broad range of pathogens and toxins without activating complement factors or coagulation. Magnets pull the opsonin-bound pathogens and toxins from the blood; the cleansed blood is then returned back to the individual. The biospleen efficiently removes multiple Gram-negative and Gram-positive bacteria, fungi and endotoxins from whole human blood flowing through a single biospleen unit at up to 1.25 liters per h in vitro. In rats infected with Staphylococcus aureus or Escherichia coli, the biospleen cleared90% of bacteria from blood, reduced pathogen and immune cell infiltration in multiple organs and decreased inflammatory cytokine levels. In a model of endotoxemic shock, the biospleen increased survival rates after a 5-h treatment.

Here we describe how Staphylococcus aureus bacteria can be rapidly isolated from clinical samples of articular fluid and synovial tissue using magnetic beads coated with the engineered chimeric human opsonin protein, Fc-mannose-binding lectin (FcMBL). The FcMBL-beads were used to capture and magnetically remove bacteria from purified cultures of 12 S. aureus strains, and from 8 articular fluid samples and 4 synovial tissue samples collected from patients with osteoarthritis or periprosthetic infections previously documented by positive S. aureus cultures. While the capture efficiency was high (85%) with purified S. aureus strains grown in vitro, direct FcMBL-bead capture from the clinical samples was initially disappointing (< 5% efficiency). Further analysis revealed that inhibition of FcMBL binding was due to coating of the bacteria by immunoglobulins and immune cells that masked FcMBL binding sites, and to the high viscosity of these complex biological samples. Importantly, capture of pathogens using the FcMBL-beads was increased to 76% efficiency by pretreating clinical specimens with hypotonic washes, hyaluronidase and a protease cocktail. Using this approach, S. aureus bacteria could be isolated from infected osteoarthritic tissues within 2 hours after sample collection. This FcMBL-enabled magnetic method for rapid capture and concentration of pathogens from clinical samples could be integrated upstream of current processes used in clinical microbiology laboratories to identify pathogens and perform antibiotic sensitivity testing when bacterial culture is not possible or before colonies can be detected.

Fat depot sizes peak in middle age but decrease by advanced old age. This phenomenon is associated with ectopic fat deposition, decreased adipocyte size, impaired differentiation of preadipocytes into fat cells, decreased adipogenic transcription factor expression, and increased fat tissue inflammatory cytokine generation. To define the mechanisms contributing to impaired adipogenesis with aging, we examined the release of TNFalpha, which inhibits adipogenesis, and the expression of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), which blocks activity of adipogenic C/EBP family members, in preadipocytes cultured from young, middle-aged, and old rats. Medium conditioned by fat tissue, as well as preadipocytes, from old rats impeded lipid accumulation by preadipocytes from young animals. More TNFalpha was released by preadipocytes from old than young rats. Differences in TNFalpha-converting enzyme, TNFalpha degradation, or the presence of macrophages in cultures were not responsible. TNFalpha induced rat preadipocyte CHOP expression. CHOP was higher in undifferentiated preadipocytes from old than younger animals. Overexpression of CHOP in young rat preadipocytes inhibited lipid accumulation. TNFalpha short interference RNA reduced CHOP and partially restored lipid accumulation in old rat preadipocytes. CHOP normally increases during late differentiation, potentially modulating the process. This late increase in CHOP was not affected substantially by aging: CHOP was similar in differentiating preadipocytes and fat tissue from old and young animals. Hypoglycemia, which normally causes an adaptive increase in CHOP, was less effective in inducing CHOP in preadipocytes from old than younger animals. Thus increased TNFalpha release by undifferentiated preadipocytes with elevated basal CHOP contributes to impaired adipogenesis with aging.

Anatomically separate fat depots differ in size, function, and contribution to pathological states, such as the metabolic syndrome. We isolated preadipocytes from different human fat depots to determine whether the basis for this variation is partly attributable to differences in inherent properties of fat cell progenitors. We found that genome-wide expression profiles of primary preadipocytes cultured in parallel from abdominal subcutaneous, mesenteric, and omental fat depots were distinct. Interestingly, visceral fat was not homogeneous. Preadipocytes from one of the two main visceral depots, mesenteric fat, had an expression profile closer to that of subcutaneous than omental preadipocytes, the other main visceral depot. Expression of genes that regulate early development, including homeotic genes, differed extensively among undifferentiated preadipocytes isolated from different fat depots. These profiles were confirmed by real-time PCR analysis of preadipocytes from additional lean and obese male and female subjects. We made preadipocyte strains from single abdominal subcutaneous and omental preadipocytes by expressing telomerase. Depot-specific developmental gene expression profiles persisted for 40 population doublings in these strains. Thus, human fat cell progenitors from different regions are effectively distinct, consistent with different fat depots being separate mini-organs.

Functional alpha1- and alpha2-adrenoreceptor subtype pharmacology was characterized in an in vitro human nasal mucosa contractile bioassay.Nasal mucosa was obtained from 49 donor patients and mucosal strips were placed in chambers filled with Krebs-Ringer solution and attached to isometric force transducers.Nonselective a-adrenoreceptor agonists epinephrine, norepinephrine, and oxymetazoline produced concentration-dependent contractions of isolated human nasal mucosa (pD2 = 5.2, 4.9, and 6.5, respectively). The alpha2-adrenoreceptor agonist BHT-920 (10 microM)-induced contractions were blocked by yohimbine (0.01-1 microM) and prazosin (0.01-1 microM) inhibited the contractile response to the alpha1-adrenoreceptor agonist phenylephrine (10 microM). Histological analysis showed that phenylephrine and BHT-920 differentially contracted the arteries and veins of human nasal mucosa, respectively.Our results indicate that functional alpha1- and alpha2-adrenoceptors are present and functional in human nasal mucosa. The alpha2-adrenoceptors display a predominant role in contracting the veins and the alpha1-adrenoceptors appear to preferentially constrict the human nasal arteries.

We produce here a model to explain the control of testosterone secretion. In this model the hypothalamic secretion of the hormone LHRH (luteinizing hormone releasing hormone) is controlled by a combination of local testosterone concentration and of the local concentration of the pituitary hormone LH (luteinizing hormone). Since LHRH stimulates the release of LH, and LH in turn stimulates the release of testosterone, the three hormones constitute a three-component "feedback" network. We show how this model is able to account for the pulsatility of the release of these three hormones. Furthermore, the model is consistent with results obtained from a wide range of experimental manipulations of the system. For example, it accounts for the changes observed in hormone release patterns after castration. In particular, it follows that no "neural clock", or "neural pulse-generator", is required to force the system into pulsatile behaviour.