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 cleared >90% 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.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract 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 ( S taphylococcus aureus ) bacteria, fungi ( C andida 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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 15–50% 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia 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.
We 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.
Organ-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.
1 Obesity Research Center, 2 Department of Medicine and Department of Genetics and Genomics, Boston University, Boston, Massachusetts; 3 Department of Surgery, Creighton University, Omaha, Nebraska; ...4 Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston Massachusetts; and 5 Mayo Clinic Foundation, Rochester, Minnesota
Submitted 26 April 2006
; accepted in final form 6 September 2006
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.
visceral fat; homeotic genes; telomerase; metabolic syndrome
Address for reprint requests and other correspondence: J. L. Kirkland, Boston University Medical Center, 88 East Newton St., Boston, MA 02118
Commensal bacteria within the gut microbiome contribute to development of host tolerance to infection, however, identifying specific microbes responsible for this response is difficult. Here we ...describe methods for developing microfluidic organ-on-a-chip models of small and large intestine lined with epithelial cells isolated from duodenal, jejunal, ileal, or colon organoids derived from wild type or transgenic mice. To focus on host-microbiome interactions, we carried out studies with the mouse Colon Chip and demonstrated that it can support co-culture with living gut microbiome and enable assessment of effects on epithelial adhesion, tight junctions, barrier function, mucus production, and cytokine release. Moreover, infection of the Colon Chips with the pathogenic bacterium,
, resulted in epithelial detachment, decreased tight junction staining, and increased release of chemokines (CXCL1, CXCL2, and CCL20) that closely mimicked changes previously seen in mice. Symbiosis between microbiome bacteria and the intestinal epithelium was also recapitulated by populating Colon Chips with complex living mouse or human microbiome. By taking advantage of differences in the composition between complex microbiome samples cultured on each chip using 16s sequencing, we were able to identify
as a positive contributor to host tolerance, confirming past findings obtained in mouse experiments. Thus, mouse Intestine Chips may represent new experimental
platforms for identifying particular bacterial strains that modulate host response to pathogens, as well as for investigating the cellular and molecular basis of host-microbe interactions.
Aging, Depot Origin, and Preadipocyte Gene Expression Cartwright, Mark J.; Schlauch, Karen; Lenburg, Marc E. ...
The journals of gerontology. Series A, Biological sciences and medical sciences,
03/2010, Volume:
65A, Issue:
3
Journal Article
Peer reviewed
Open access
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.
Current therapeutic strategies against bacterial infections focus on reduction of pathogen load using antibiotics; however, stimulation of host tolerance to infection in the presence of pathogens ...might offer an alternative approach. Computational transcriptomics and Xenopus laevis embryos are used to discover infection response pathways, identify potential tolerance inducer drugs, and validate their ability to induce broad tolerance. Xenopus exhibits natural tolerance to Acinetobacter baumanii, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pneumoniae bacteria, whereas Aeromonas hydrophila and Pseudomonas aeruginosa produce lethal infections. Transcriptional profiling leads to definition of a 20‐gene signature that discriminates between tolerant and susceptible states, as well as identification of a more active tolerance response to gram negative compared to gram positive bacteria. Gene pathways associated with active tolerance in Xenopus, including some involved in metal ion binding and hypoxia, are found to be conserved across species, including mammals, and administration of a metal chelator (deferoxamine) or a HIF‐1α agonist (1,4‐DPCA) in embryos infected with lethal A. hydrophila increased survival despite high pathogen load. These data demonstrate the value of combining the Xenopus embryo infection model with computational multiomics analyses for mechanistic discovery and drug repurposing to induce host tolerance to bacterial infections.
An integrated experimental‐bioinformatics screening approach in Xenopus embryos is described and used to discover broad‐spectrum tolerance‐inducing drugs. Drawing inspiration from natural infection tolerance observed in Xenopus, mice, and primates, tolerance inducing pathways are identified and it is shown that metal ion chelators and a prolyl‐4 hydroxylase inhibitor can artificially promote tolerance to bacterial infection.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Complement alternative pathway (AP) dysregulation has been implicated in geographic atrophy, an advanced form of age-related macular degeneration. Danicopan is an investigational, first-in-class ...inhibitor of factor D, an essential AP activation enzyme. We assessed danicopan distribution to the posterior segment of the eye after oral dosing.
Tissue distribution of drug-derived radioactivity was evaluated using whole-body autoradiography following oral administration of 14C-danicopan to pigmented and albino rats. Pharmacokinetics and ocular tissue distribution were studied in pigmented and albino rabbits following single and multiple oral dosing of danicopan. The melanin binding property was characterized in vitro.
Radioactivity was distributed widely in rats and became nonquantifiable in most tissues 24 hours postdose except in the pigmented rat uvea (quantifiable 672 hours postdose). Danicopan binding to melanin was established in vitro. After single dosing, the maximum concentration (Cmax) and area under the curve (AUC) in neural retina and plasma were similar in both rabbit types. After multiple dosing, AUC in neural retina was 3.4-fold higher versus plasma in pigmented rabbits. Drug levels in choroid/Bruch's membrane (BrM)/retinal pigment epithelium (RPE) were similar to plasma in albino rabbits but higher in pigmented rabbits: Cmax and AUC were 2.9- and 23.8-fold higher versus plasma after single dosing and 5.8- and 62.7-fold higher after multiple dosing. In pigmented rabbits, ocular tissue exposures slowly declined over time but remained quantifiable 240 hours postdose.
The results demonstrate that danicopan crosses the blood-retina barrier and binds melanin reversibly, leading to a higher and more sustained exposure in melanin-containing ocular tissues (choroid/BrM/RPE) and in the neural retina as compared to in plasma after repeated oral dosing in pigmented animals.
These findings suggest that oral danicopan possesses potential for treating geographic atrophy because AP dysregulation in the posterior segment of the eye is reported to be involved in the disease pathogenesis.
Background: Fc-mannose-binding lectin (FcMBL), an engineered version of the blood opsonin MBL that contains the carbohydrate recognition domain (CRD) and flexible neck regions of MBL fused to the Fc ...portion of human IgG1, has been shown to bind various microbes and pathogen-associated molecular patterns (PAMPs). FcMBL has also been used to create an enzyme-linked lectin sorbent assay (ELLecSA) for use as a rapid (<1 h) diagnostic of bloodstream infections.
Methods: Here we extended this work by using the ELLecSA to test FcMBL's ability to bind to more than 190 different isolates from over 95 different pathogen species.
Results: FcMBL bound to 85% of the isolates and 97 of the 112 (87%) different pathogen species tested, including bacteria, fungi, viral antigens and parasites. FcMBL also bound to PAMPs including, lipopolysaccharide endotoxin (LPS) and lipoteichoic acid (LTA) from Gram-negative and Gram-positive bacteria, as well as lipoarabinomannan (LAM) and phosphatidylinositol mannoside 6 (PIM
6) from
Mycobacterium tuberculosis.
Conclusions: The efficiency of pathogen detection and variation between binding of different strains of the same species could be improved by treating the bacteria with antibiotics, or mechanical disruption using a bead mill, prior to FcMBL capture to reveal previously concealed binding sites within the bacterial cell wall. As FcMBL can bind to pathogens and PAMPs in urine as well as blood, its broad-binding capability could be leveraged to develop a variety of clinically relevant technologies, including infectious disease diagnostics, therapeutics, and vaccines.
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