Maternal treatment with oral intestinal alkaline phosphatase mitigates high fat diet-induced cognitive disorders in offspring mice.

Intestinal alkaline phosphatase (IAP) is an endogenous enzyme that promotes gastrointestinal homeostasis by detoxifying inflammatory mediators, tightening the gut barrier and promoting a healthy microbiome. Oral IAP administration was efficacious in ameliorating diabetes in a high fat diet (HFD)-induced murine model. In humans, maternal obesity and diabetes during pregnancy have been associated with an increased risk of autism spectrum disorders (ASD). In mice, HFD-induced maternal obesity leads to offspring with cognitive deficiency. Here we investigated whether IAP administration to obese dams could ameliorate autism-like disorders in mice. Using a HFD murine model, we recapitulated that maternal obesity leads to male offspring with social deficits as shown by the three chamber test and reciprocal social interaction analyses. Notably, oral delivery of IAP to dams improved those deficiencies. In addition, a jumping behavior was noted in pups from obese dams, which was rescued by maternal IAP treatment. Our findings suggest that maternal treatment with IAP can relieve some ASD-like symptoms in offspring mice.

Current Concepts for the IND-Directed Development of Microbicide Products to Prevent the Sexual Transmission of HIV.

In the absence of an approved and effective vaccine, topical microbicides have become the strategy of choice to provide women with the ability to prevent the sexual transmission of HIV. Topical microbicides are chemical and physical agents specifically developed and formulated for use in either the vaginal or rectal environment to prevent the sexual transmission of infectious organisms. Although a microbicide product will have many of the same properties as other anti-infective therapeutic agents, the microbicide development pathway has significant differences which reflect the complex biological environment in which the products must act. These challenges to the development of an effective microbicide are reflected in the recently released FDA Guidance document which defines the microbicide development algorithm and includes the evaluation of preclinical efficacy and toxicity, and safety and toxicology, and indicates the necessity of testing of the active pharmaceutical product as well as an optimal formulation for delivery of the microbicide product. The microbicide development algorithm requires evaluation of the potential microbicidal agent and final formulated product in assays which mimic the microenvironment of the vagina and rectum during the sexual transmission of HIV, including the evaluation of activity and cytotoxicity in the appropriate biological matrices, toxicity testing against normal vaginal flora and at standard vaginal pH, testing in ectocervical and colorectal explant tissue, and irritation studies to vaginal, rectal and penile tissue. Herein, we discuss currently accepted practices required for the development of a successful microbicide product which will prevent virus transmission in the vaginal and rectal vaults.

Caspase-1-mediated pathway promotes generation of thromboinflammatory microparticles.

Extracellular ATP is a signal of tissue damage and induces macrophage responses that amplify inflammation and coagulation. Here we demonstrate that ATP signaling through macrophage P2X7 receptors uncouples the thioredoxin (TRX)/TRX reductase (TRXR) system and activates the inflammasome through endosome-generated ROS. TRXR and inflammasome activity promoted filopodia formation, cellular release of reduced TRX, and generation of extracellular thiol pathway-dependent, procoagulant microparticles (MPs). Additionally, inflammasome-induced activation of an intracellular caspase-1/calpain cysteine protease cascade degraded filamin, thereby severing bonds between the cytoskeleton and tissue factor (TF), the cell surface receptor responsible for coagulation activation. This cascade enabled TF trafficking from rafts to filopodia and ultimately onto phosphatidylserine-positive, highly procoagulant MPs. Furthermore, caspase-1 specifically facilitated cell surface actin exposure, which was required for the final release of highly procoagulant MPs from filopodia. Together, the results of this study delineate a thromboinflammatory pathway and suggest that components of this pathway have potential as pharmacological targets to simultaneously attenuate inflammation and innate immune cell-induced thrombosis.

Organ-specific protection against lipopolysaccharide-induced vascular leak is dependent on the endothelial protein C receptor.

OBJECTIVE: To study the role of the endothelial protein C receptor (EPCR) in the modulation of susceptibility to inflammation-induced vascular leak in vivo. APPROACH AND RESULTS: Genetically modified mice with low, <10% EPCR expression (EPCR(low)) and control mice were challenged with lipopolysaccharides in a mouse model of endotoxemia. Infrared fluorescence and quantification of albumin-bound Evans Blue in tissues and intravascular plasma volumes were used to assess plasma extravasation. Pair-wise analysis of EPCR(low) and control mice matched for sex, age, and weight allowed determination of EPCR-dependent vascular leak. Kidney, lung, and brain were the organs with highest discriminative increased Evans Blue accumulation in EPCR(low) versus control mice in response to lipopolysaccharides. Histology of kidney and lung confirmed the EPCR-specific pathology. In addition to severe kidney injury in response to lipopolysaccharides, EPCR(low) and anti-EPCR-treated wild-type mice suffered from enhanced albuminuria and profound renal hemorrhage versus controls. Intravascular volume loss at the same extent of weight loss in EPCR(low) mice compared with control mice provided proof that plasma leak was the predominant cause of Evans Blue tissue accumulation. CONCLUSIONS: This study demonstrates an important protective role for EPCR in vivo against vascular leakage during inflammation and suggests that EPCR-dependent vascular protection is organ-specific.

Infrared fluorescence for vascular barrier breach in vivo--a novel method for quantitation of albumin efflux.

Vascular hyperpermeability contributes to morbidity in inflammation. Current methodologies for in vivo assessment of permeability based on extravasation of Evans Blue (EB)-bound albumin are cumbersome and often lack sensitivity. We developed a novel infrared fluorescence (IRF) methodology for measurement of EB-albumin extravasation to quantify vascular permeability in murine models. Vascular permeability induced by endotoxaemia was examined for all solid organs, brain, skin and peritoneum by IRF and the traditional absorbance-based measurement of EB in tissue extracts. Organ IRF increased linearly with increasing concentrations of intravenous EB (2.5-25 mg/kg). Tissue IRF was more sensitive for EB accumulation compared to the absorbance-based method. Accordingly, differences in vascular permeability and organ EB accumulation between lipopolysaccharide-treated and saline-treated mice were often significant when analysed by IRF-based detection but not by absorbance-based detection. EB was detected in all 353 organs analysed with IRF but only in 67% (239/353) of organs analysed by absorbance-based methodology, demonstrating improved sensitivity of EB detection in organs with IRF. In contrast, EB in plasma after EB administration was readily measured by both methods with high correlation between the two methods (n=116, r2=0.86). Quantitation of organ-specific EB-IRF differences due to endotoxin was optimal when IRF was compared between mice matched for weight, gender, and age, and with appropriate corrections for organ weight and EB plasma concentrations. Notably, EB-IRF methodology leaves organs intact for subsequent histopathology. In summary, EB-IRF is a novel, highly sensitive, rapid, and convenient method for the relative quantification of EB in intact organs of treatment versus control mice.

Tissue factor-protease-activated receptor 2 signaling promotes diet-induced obesity and adipose inflammation.

Tissue factor, the initiator of the coagulation cascade, mediates coagulation factor VIIa-dependent activation of protease-activated receptor 2 (PAR2). Here we delineate a role for this signaling pathway in obesity and its complications. Mice lacking PAR2 (F2rl1) or the cytoplasmic domain of tissue factor were protected from weight gain and insulin resistance induced by a high-fat diet. In hematopoietic cells, genetic ablation of tissue factor-PAR2 signaling reduced adipose tissue macrophage inflammation, and specific pharmacological inhibition of macrophage tissue factor signaling rapidly ameliorated insulin resistance. In contrast, nonhematopoietic cell tissue factor-VIIa-PAR2 signaling specifically promoted obesity. Mechanistically, adipocyte tissue factor cytoplasmic domain-dependent VIIa signaling suppressed Akt phosphorylation with concordant adverse transcriptional changes of key regulators of obesity and metabolism. Pharmacological blockade of adipocyte tissue factor in vivo reversed these effects of tissue factor-VIIa signaling and rapidly increased energy expenditure. Thus, inhibition of tissue factor signaling is a potential therapeutic avenue for improving impaired metabolism and insulin resistance in obesity.

P2X7 receptor signaling contributes to tissue factor-dependent thrombosis in mice.

Thrombosis is initiated by tissue factor (TF), a coagulation cofactor/receptor expressed in the vessel wall, on myeloid cells, and on microparticles (MPs) with variable procoagulant activity. However, the molecular pathways that generate prothrombotic TF in vivo are poorly defined. The oxidoreductase protein disulfide isomerase (PDI) is thought to be involved in the activation of TF. Here, we found that in mouse myeloid cells, ATP-triggered signaling through purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7 receptor; encoded by P2rx7) induced activation (decryption) of TF procoagulant activity and promoted release of TF+ MPs from macrophages and SMCs. The generation of prothrombotic MPs required P2X7 receptor-dependent production of ROS leading to increased availability of solvent-accessible extracellular thiols. An antibody to PDI with antithrombotic activity in vivo attenuated the release of procoagulant MPs. In addition, P2rx7-/- mice were protected from TF-dependent FeCl3-induced carotid artery thrombosis. BM chimeras revealed that P2X7 receptor prothrombotic function was present in both hematopoietic and vessel wall compartments. In contrast, an alternative anti-PDI antibody showed activities consistent with cellular activation typically induced by P2X7 receptor signaling. This anti-PDI antibody restored TF-dependent thrombosis in P2rx7-/- mice. These data suggest that PDI regulates a critical P2X7 receptor-dependent signaling pathway that generates prothrombotic TF, defining a link between inflammation and thrombosis with potential implications for antithrombotic therapy.

Endogenous EPCR/aPC-PAR1 signaling prevents inflammation-induced vascular leakage and lethality.

Protease activated receptor 1 (PAR1) signaling can play opposing roles in sepsis, either promoting dendritic cell (DC)-dependent coagulation and inflammation or reducing sepsis lethality due to activated protein C (aPC) therapy. To further define this PAR1 paradox, we focused on the vascular effects of PAR1 signaling. Pharmacological perturbations of the intravascular coagulant balance were combined with genetic mouse models to dissect the roles of endogenously generated thrombin and aPC during escalating systemic inflammation. Acute blockade of the aPC pathway with a potent inhibitory antibody revealed that thrombin-PAR1 signaling increases inflammation-induced vascular hyperpermeability. Conversely, aPC-PAR1 signaling and the endothelial cell PC receptor (EPCR) prevented vascular leakage, and pharmacologic or genetic blockade of this pathway sensitized mice to LPS-induced lethality. Signaling-selective aPC variants rescued mice with defective PC activation from vascular leakage and lethality. Defects in the aPC pathway were fully compensated by sphingosine 1 phosphate receptor 3 (S1P3) deficiency or by selective agonists of the S1P receptor 1 (S1P1), indicating that PAR1 signaling contributes to setting the tone for the vascular S1P1/S1P3 balance. Thus, the activating proteases and selectivity in coupling to S1P receptor subtypes determine vascular PAR1 signaling specificity in systemic inflammatory response syndromes in vivo.

Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation.

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.

Emerging role of regulatory T cells in gene transfer.

Induction and maintenance of immune tolerance to therapeutic transgene products are key requirements for successful gene replacement therapies. Gene transfer may also be used to specifically induce immune tolerance and thereby augment other types of therapies. Similarly, gene therapies for treatment of autoimmune diseases are being developed in order to restore tolerance to self-antigens. Regulatory T cells have emerged as key players in many aspects of immune tolerance, and a rapidly increasing body of work documents induction and/or activation of regulatory T cells by gene transfer. Regulatory T cells may suppress antibody formation and cytotoxic T cell responses and may be critical for immune tolerance to therapeutic proteins. In this regard, CD4(+)CD25(+) regulatory T cells have been identified as important components of tolerance in several gene transfer protocols, including hepatic in vivo gene transfer. Augmentation of regulatory T cell responses should be a promising new tool to achieve tolerance and avoid immune-mediated rejection of gene therapy. During the past decade, it has become obvious that immune regulation is an important and integral component of tolerance to self-antigens and of many forms of induced tolerance. Gene therapy can only be successful if the immune system does not reject the therapeutic transgene product. Recent studies provide a rapidly growing body of evidence that regulatory T cells (T(reg)) are involved and often play a crucial role in tolerance to proteins expressed by means of gene transfer. This review seeks to provide an overview of these data and their implications for gene therapy.

Characterization of mild coagulation factor VII deficiency: activity and clearance of the Arg315Trp and Arg315Lys variants in the Cys310-Cys329 loop (c170s).

BACKGROUND AND OBJECTIVES: Arginine 315 in factor VII (FVII) belongs to a solvent-exposed loop involved in direct interaction with the co-factor (tissue factor, TF), in transmission of TF-induced effects and potentially in FVIIa inactivation. Natural FVII variants at position 315 provide peculiar models for structure-function studies. DESIGN AND METHODS: We characterized a mild coagulation FVII deficiency associated with reduced FVII activity (26%) and antigen (67%). Mutations were searched by FVII gene sequencing. FVII variants were created by mutagenesis of FVII cDNA and characterized through expression in HEK293 cells followed by functional studies. FVII antigen in media was estimated by immunoassay while FVII activity was assessed by prothrombin-time based and FXa generation assays. FVII variants were injected into mice to investigate their recovery and half-life. One-way ANOVA was used to test statistical significance. RESULTS: The patient was double heterozygous for a novel R315W mutation and for the R304Q substitution (FVII Padua) previously demonstrated to impair TF binding. The recombinant 315W-FVII was normally expressed in medium but showed a markedly reduced coagulant function (52%) and activity towards factor X (FX) in plasma (34%). Moreover, the 315W-FVII showed significantly decreased recovery of the protein (20%) and a slightly shorter half-life (8.6 min) as compared to wt-FVII (50% and 10.7 min). We also studied the conservative R315K change that was responsible for low recovery (20%) and a decreased half-life (7 min) of a FVII variant with virtually normal FVII antigen and activity levels. INTERPRETATION AND CONCLUSIONS: These findings suggest a dual role of R315 for FVII function and clearance, and indicate that substitutions at this position have appreciable effects on human FVII biology, compatible with residual FVII function and thus with mild FVII deficiency.