Human platelet concentrates treated with microbicidal 405 nm light retain hemostasis activity.

Chemical and UV light-based pathogen reduction technologies are currently in use for human platelet concentrates (PCs) to enhance safety from transfusion-transmitted infections. Relative to UV light, 405 nm violet-blue light in the visible spectrum is known to be less harmful. Hence, in this report for the first time, we have assessed the global hemostasis activity of PCs stored in plasma and the activities of six plasma coagulation factors (CFs) as a measure of in vitro hemostatic activity following exposure to the microbicidal 405 nm light. Apheresis PC samples collected from each screened human donor (n = 22) were used for testing of PCs and platelet poor plasma (PPP). Both PCs and PPPs were treated for 5 h with 405 nm light to achieve a previously established microbicidal light dose of 270 J/cm2. Activated partial thromboplastin time and prothrombin time-based potency assays using a coagulation analyzer and hemostatic capacity via Thromboelastography were analyzed. Thromboelastography analysis of the light-treated PCs and plasma present in the PCs showed little difference between the treated and untreated samples. Further, plasma present in the PCs during the light treatment demonstrated a better stability in potency assays for several coagulation factors compared to the plasma alone prepared from PCs first and subjected to the light treatment separately. Overall, PCs stored in plasma treated with 405 nm violet-blue light retain activity for hemostasis.

Preclinical evaluation of Tc-99m p5+14 peptide for SPECT detection of cardiac amyloidosis.

INTRODUCTION: Amyloid deposition is a cause of restrictive cardiomyopathy. Patients who present with cardiac disease can be evaluated for transthyretin (TTR)-associated cardiac amyloidosis using nuclear imaging with 99mTc-labeled pyrophosphate (PYP); however, light chain-associated (AL) cardiac amyloid is generally not detected using this tracer. As an alternative, the amyloid-binding peptide p5+14 radiolabeled with iodine-124 has been shown to be an effective pan-amyloid radiotracer for PET/CT imaging. Here, a 99mTc-labeled form of p5+14 peptide has been prepared to facilitate SPECT/CT imaging of cardiac amyloidosis. METHOD: A synthesis method suitable for clinical applications has been used to prepare 99mTc-labeled p5+14 and tested for peptide purity, product bioactivity, radiochemical purity and stability. The product was compared with99mTc-PYP for cardiac SPECT/CT imaging in a mouse model of AA amyloidosis and for reactivity with human tissue sections from AL and TTR patients. RESULTS: The 99mTc p5+14 tracer was produced with >95% yields in radiopurity and bioactivity with no purification steps required and retained over 95% peptide purity and >90% bioactivity for >3 h. In mice, the tracer detected hepatosplenic AA amyloid as well as heart deposits with uptake ~5 fold higher than 99mTc-PYP. 99mTc p5+14 effectively bound human amyloid deposits in the liver, kidney and both AL- and ATTR cardiac amyloid in tissue sections in which 99mTc-PYP binding was not detectable. CONCLUSION: 99mTc-p5+14 was prepared in minutes in >20 mCi doses with good performance in preclinical studies making it suitable for clinical SPECT/CT imaging of cardiac amyloidosis.

Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm.

BACKGROUND: Fluorogenic thrombin generation (TG) is a global hemostasis assay that provides an overall representation of hemostasis potential. However, the accurate detection of thrombin activity in plasma may be affected by artifacts inherent to the assay-associated fluorogenic substrate. The significance of the fluorogenic artifacts or their corrections has not been studied in hemophilia treatment applications. METHODS: We sought to investigate TG in hemophilia plasma samples under typical and worst-case fluorogenic artifact conditions and assess the performance of artifact correction algorithms. Severe hemophilic plasma with or without added Factor VIII (FVIII) was evaluated using commercially available and in-house TG reagents, instruments, and software packages. The inner filter effect (IFE) was induced by spiking elevated amounts of fluorophore 7-amino-4-methylcoumarin (AMC) into plasma prior to the TG experiment. Substrate consumption was modeled by adding decreasing amounts of Z-Gly-Gly-Arg-AMC (ZGGR-AMC) to plasma or performing TG in antithrombin deficient plasma. RESULTS: All algorithms corrected the AMC-induced IFE and antithrombin-deficiency induced substrate consumption up to a certain level of either artifact (edge of failure) upon which TG results were not returned or overestimated. TG values in FVIII deficient (FVIII-DP) or supplemented plasma were affected similarly. Normalization of FVIII-DP resulted in a more accurate correction of substrate artifacts than algorithmic methods. CONCLUSIONS: Correction algorithms may be effective in situations of moderate fluorogenic substrate artifacts inherent to highly procoagulant samples, but correction may not be required under typical conditions for hemophilia treatment studies if TG parameters can be normalized to a reference plasma sample.

Development and characterization of a prototypic pan-amyloid clearing agent - a novel murine peptide-immunoglobulin fusion.

Introduction: Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues. Amyloid accumulation causes organ dysfunction and is not generally cleared by the immune system. Current treatment focuses on reducing amyloid precursor protein synthesis and slowing amyloid deposition. However, curative interventions will likely also require removal of preexisting amyloid deposits to restore organ function. Here we describe a prototypic pan-amyloid binding peptide-antibody fusion molecule (mIgp5) that enhances macrophage uptake of amyloid. Methods: The murine IgG1-IgG2a hybrid immunoglobulin with a pan amyloid-reactive peptide, p5, fused genetically to the N-terminal of the immunoglobulin light chain was synthesized in HEK293T/17 cells. The binding of the p5 peptide moiety was assayed using synthetic amyloid-like fibrils, human amyloid extracts and amyloid-laden tissues as substrates. Binding of radioiodinated mIgp5 with amyloid deposits in vivo was evaluated in a murine model of AA amyloidosis using small animal imaging and microautoradiography. The bioactivity of mIgp5 was assessed in complement fixation and in vitro phagocytosis assays in the presence of patient-derived amyloid extracts and synthetic amyloid fibrils as substrates and in the presence or absence of human serum. Results: Murine Igp5 exhibited highly potent binding to AL and ATTR amyloid extracts and diverse types of amyloid in formalin-fixed tissue sections. In the murine model of systemic AA amyloidosis, 125I-mIgp5 bound rapidly and specifically to amyloid deposits in all organs, including the heart, with no evidence of non-specific uptake in healthy tissues. The bioactivity of the immunoglobulin Fc domain was uncompromised in the context of mIgp5 and served as an effective opsonin. Macrophage-mediated uptake of amyloid extract and purified amyloid fibrils was enhanced by the addition of mIgp5. This effect was exaggerated in the presence of human serum coincident with deposition of complement C5b9. Conclusion: Immunostimulatory, amyloid-clearing therapeutics can be developed by incorporating pan-amyloid-reactive peptides, such as p5, as a targeting moiety. The immunologic functionality of the IgG remains intact in the context of the fusion protein. These data highlight the potential use of peptide-antibody fusions as therapeutics for all types of systemic amyloidosis.

Collagen inhibits phagocytosis of amyloid in vitro and in vivo and may act as a 'don't eat me' signal.

BACKGROUND: Systemic amyloidosis refers to a group of protein misfolding disorders characterized by the extracellular deposition of amyloid fibrils in organs and tissues. For reasons heretofore unknown, amyloid deposits are not recognized by the immune system, and progressive deposition leads to organ dysfunction. METHODS: In vitro and in vivo phagocytosis assays were performed to elucidate the impact of collagen and other amyloid associated proteins (eg serum amyloid p component and apolipoprotein E) had on amyloid phagocytosis. Immunohistochemical and histopathological staining regimens were employed to analyze collagen-amyloid interactions and immune responses. RESULTS: Histological analysis of amyloid-laden tissue indicated that collagen is intimately associated with amyloid deposits. We report that collagen inhibits phagocytosis of amyloid fibrils by macrophages. Treatment of 15 patient-derived amyloid extracts with collagenase significantly enhanced amyloid phagocytosis. Preclinical mouse studies indicated that collagenase treatment of amyloid extracts significantly enhanced clearance as compared to controls, coincident with increased immune cell infiltration of the subcutaneous amyloid lesion. CONCLUSIONS: These data suggest that amyloid-associated collagen serves as a 'don't eat me' signal, thereby hindering clearance of amyloid. Targeted degradation of amyloid-associated collagen could result in innate immune cell recognition and clearance of pathologic amyloid deposits.

Treatment of glioblastoma with current oHSV variants reveals differences in efficacy and immune cell recruitment.

Oncolytic herpes simplex viruses (oHSVs) have demonstrated efficient lytic replication in human glioblastoma tumors using immunodeficient mouse models, but early-phase clinical trials have reported few complete responses. Potential reasons for the lack of efficacy are limited vector potency and the suppressive glioma tumor microenvironment (TME). Here we compare the oncolytic activity of two HSV-1 vectors, a KOS-strain derivative KG4:T124 and an F-strain derivative rQNestin34.5v.1, in the CT2A and GL261N4 murine syngeneic glioma models. rQNestin34.5v1 generally demonstrated a greater in vivo viral burden compared to KG4:T124. However, both vectors were rapidly cleared from CT2A tumors, while virus remained ensconced in GL261N4 tumors. Immunological evaluation revealed that the two vectors induced similar changes in immune cell recruitment to either tumor type at 2 days after infection. However, at 7 days after infection, the CT2A microenvironment displayed the phenotype of an untreated tumor, while GL261N4 tumors exhibited macrophage and CD4+/CD8+ T cell accumulation. Furthermore, the CT2A model was completely resistant to virus therapy, while in the GL261N4 model rQNestin34.5v1 treatment resulted in enhanced macrophage recruitment, impaired tumor progression, and long-term survival of a few animals. We conclude that prolonged intratumoral viral presence correlates with immune cell recruitment, and both are needed to enhance anti-tumor immunity.

Considerations on activity assay discrepancies in factor VIII and factor IX products.

New modified coagulation factor VIII (FVIII) and factor IX (FIX) products have been designed to improve the treatment of individuals with hemophilia A and B by increasing the interval between dosing. Although these FVIII and FIX molecules have been structurally modified to improve the circulation time, the changes have also influenced their behavior in functional assays in comparison with traditional plasma-derived or recombinant coagulation factors. The assignment of potencies for these products can be problematic because discordance in factor activity values between the commonly used one-stage clotting and chromogenic substrate assays is often observed. Discrepancies in potency assay values also exist when different assay kits and reagents are used in the same assay type. Ideally, all FVIII and FIX products should be calibrated against the World Health Organization (WHO) International Standards (IS) because the assignment of potencies in international units (IU) helps maintain treatment tradition and meaningful references for manufacturers, patients, and clinicians. The discrepant measurements, attributed to the modified structural and functional properties of these products, are manifested in their lack of commutability with the WHO IS for FVIII or FIX. Herein, we discuss the considerations upon which an assay is chosen for potency assignment and postadministration monitoring of a new factor product, which include the validity of the assay calibrated with the IS, the meaning of the potency values in IU, standards of care for patients, clinical relevance between the assigned potency value and recovery value from clinical laboratories, and patient safety.

Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples.

INTRODUCTION: The thrombin generation (TG) test is a global hemostasis assay sensitive to procoagulant conditions. However, some TG assays may underestimate elevated TG when the thrombin fluorogenic substrate is depleted or fluorescence is attenuated by the inner filter effect (IFE). OBJECTIVES: We sought to elucidate the extent to which procoagulant conditions require correcting for fluorogenic substrate depletion and/or IFE. METHODS: We analyzed corrections for substrate depletion and IFE and their effect on TG parameters in plasma samples with elevated blood coagulation factors in the presence or absence of thrombomodulin via commercial calibrated automated thrombogram (CAT) platform and in-house software capable of internal thrombin calibration with or without CAT-like artifact correction. RESULTS: Elevated thrombin peak height (TPH) and endogenous thrombin potential (ETP) were detected with 2× and 4× increases in blood coagulation factors I, V, VIII, IX, X, and XI, or prothrombin in the presence or absence of artifact correction. The effect of the CAT algorithm was evident in TG curves from both low procoagulant (thrombomodulin-supplemented) and procoagulant (factor-supplemented) plasma samples. However, in all samples, with the exception of elevated prothrombin, CAT's correction was small (<10%) and did not affect detection of procoagulant samples versus normal plasma. For elevated prothrombin samples, uncorrected TPH or ETP values were underestimated, and CAT correction produced drastically elevated TG curves. CONCLUSIONS: Our data suggest that correction for substrate consumption and IFE, as offered by the CAT algorithm, is critical for detecting a subset of extremely procoagulant samples, such as elevated prothrombin, but is not necessary for all other conditions, including elevated factors XI and VIII.

Detecting factor XIa in immune globulin products: Commutability of international reference materials for traditional and global hemostasis assays.

BACKGROUND: Activated coagulation factor XIa (FXIa) is an impurity and primary source of procoagulant activity in thrombosis-implicated immune globulin (IG) products. Several assays, of varying quality and precision are used to assess FXIa-like procoagulant activity in units relevant to their respective principles. OBJECTIVES: To advance unified reporting, we sought to employ the World Health Organization reference reagents (RRs) to present the results of differing methodologies in units of FXIa activity and rank the sensitivity and robustness of these methodologies. METHODS: RR 11/236 served as a calibrator in several FXIa-sensitive blood coagulation tests: two commercial chromogenic FXIa assays (CAs); a nonactivated partial thromboplastin time (NaPTT); an in-house fibrin generation (FG) assay; an in-house thrombin generation (TG) assay; and an assay for FXIa- and kallikrein-like proteolytic activities based on cleavage of substrate SN13a. Some assays were tested in either normal or FXI-deficient plasma. RESULTS: Each method demonstrated a sigmoidal dose-response to RRs. NaPTT was the least sensitive to FXIa and the least precise; our in-house TG was the most sensitive; and the two CAs were the most precise. All methods, except for SN13a, which is less specific for thrombotic impurities, gave comparable (within 20% difference) FXIa activity assignments for IG lots. CONCLUSIONS: Purified FXIa reference standards support quantitation of FXIa levels in IG products in all tested assay methodologies. This should help to standardize the measurement of thrombotic potentials in IG products and prevent products exhibiting high procoagulant activity from distribution for patient use. Further research is needed to address the effect of IG product-specific matrixes on assay performance.

Oncolytic HSV Vectors and Anti-Tumor Immunity.

The therapeutic promise of oncolytic viruses (OVs) rests on their ability to both selectively kill tumor cells and induce anti-tumor immunity. The potential of tumors to be recognized and eliminated by an effective anti-tumor immune response has been spurred on by the discovery that immune checkpoint inhibition can overcome tumor-specific cytotoxic T cell (CTL) exhaustion and provide durable responses in multiple tumor indications. OV-mediated tumor destruction is now recognized as a powerful means to assist in the development of anti-tumor immunity for two important reasons: (i) OVs, through the elicitation of an anti-viral response and the production of type I interferon, are potent stimulators of inflammation and can be armed with transgenes to further enhance anti-tumor immune responses; and (ii) lytic activity can promote the release of tumor-associated antigens (TAAs) and tumor neoantigens that function as in situ tumor-specific vaccines to elicit adaptive immunity. Oncolytic herpes simplex viruses (oHSVs) are among the most widely studied OVs for the treatment of solid malignancies, and Amgen's oHSV Imlygic® for the treatment of melanoma is the only OV approved in major markets. Here we describe important biological features of HSV that make it an attractive OV, clinical experience with HSV-based vectors, and strategies to increase applicability to cancer treatment.

Pancreatic, but not myeloid-cell, expression of interleukin-1alpha is required for maintenance of insulin secretion and whole body glucose homeostasis.

OBJECTIVE: The expression of the interleukin-1 receptor type I (IL-1R) is enriched in pancreatic islet ß-cells, signifying that ligands activating this pathway are important for the health and function of the insulin-secreting cell. Using isolated mouse, rat, and human islets, we identified the cytokine IL-1α as a highly inducible gene in response to IL-1R activation. In addition, IL-1α is elevated in mouse and rat models of obesity and Type 2 diabetes. Since less is known about the biology of IL-1α relative to IL-1ß in pancreatic tissue, our objective was to investigate the contribution of IL-1α to pancreatic ß-cell function and overall glucose homeostasis in vivo. METHODS: We generated a novel mouse line with conditional IL-1α alleles and subsequently produced mice with either pancreatic- or myeloid lineage-specific deletion of IL-1α. RESULTS: Using this in vivo approach, we discovered that pancreatic (IL-1αPdx1-/-), but not myeloid-cell, expression of IL-1α (IL-1αLysM-/-) was required for the maintenance of whole body glucose homeostasis in both male and female mice. Moreover, pancreatic deletion of IL-1α led to impaired glucose tolerance with no change in insulin sensitivity. This observation was consistent with our finding that glucose-stimulated insulin secretion was reduced in islets isolated from IL-1αPdx1-/- mice. Alternatively, IL-1αLysM-/- mice (male and female) did not have any detectable changes in glucose tolerance, respiratory quotient, physical activity, or food intake when compared with littermate controls. CONCLUSIONS: Taken together, we conclude that there is an important physiological role for pancreatic IL-1α to promote glucose homeostasis by supporting glucose-stimulated insulin secretion and islet ß-cell mass in vivo.

Comparative Analysis of Thrombin Calibration Algorithms and Correction for Thrombin-α2macroglobulin Activity.

BACKGROUND: The thrombin generation (TG) test is useful for characterizing global hemostasis potential, but fluorescence substrate artifacts, such as thrombin-α2macroglobulin (T-α2MG) signal, inner filter effect (IFE), substrate consumption, and calibration algorithms have been suggested as sources of intra- and inter-laboratory variance, which may limit its clinical utility. METHODS: Effects of internal vs. external normalization, IFE and T-α2MG on TG curves in normal plasma supplemented with coagulation factors, thrombomodulin, and tissue factor were studied using the Calibrated Automated Thrombinography (CAT; Diagnostica Stago, Parsippany, NJ, USA) and in-house software. RESULTS: The various calibration methods demonstrated no significant difference in producing TG curves, nor increased the robustness of the TG assay. Several TG parameters, including thrombin peak height (TPH), produced from internal linear calibration did not differ significantly from uncalibrated TG parameters. Further, TPH values from internal linear and nonlinear calibration with or without T-α2MG correction correlated well with TPH from external calibration. Higher coefficients of variation (CVs) for TPH values were observed in both platelet-free and platelet-rich plasma with added thrombomodulin. CONCLUSIONS: Our work suggests minimal differences between distinct computational approaches toward calibrating and correcting fluorescence signals into TG levels, with most samples returning similar or equivalent TPH results.

Novel Mechanism of Microvesicle Regulation by the Antiviral Protein Tetherin During HIV Infection.

Background Microvesicles are cell membrane-derived vesicles that have been shown to augment inflammation. Specifically, monocyte-derived microvesicles (MDMVs), which can express the coagulation protein tissue factor, contribute to thrombus formation and cardiovascular disease. People living with HIV experience higher prevalence of cardiovascular disease and also exhibit increased levels of plasma microvesicles. The process of microvesicle release has striking similarity to budding of enveloped viruses. The surface protein tetherin inhibits viral budding by physically tethering budding virus particles to cells. Hence, we investigated the role of tetherin in regulating the release of MDMVs during HIV infection. Methods and Results The plasma of aviremic HIV-infected individuals had increased levels of tissue factor + MDMVs, as measured by flow cytometry, and correlated to reduced tetherin expression on monocytes. Superresolution confocal and electron microscopy showed that tetherin localized at the site of budding MDMVs. Mechanistic studies revealed that the exposure of monocytes to HIV-encoded Tat triggered tetherin loss and subsequent rise in MDMV production. Overexpression of tetherin in monocytes led to morphologic changes in the pseudopodia directly underneath the MDMVs. Further, tetherin knockout mice demonstrated a higher number of circulating MDMVs and less time to bleeding cessation. Conclusions Our studies define a novel regulatory mechanism of MDMV release through tetherin and explore its contribution to the procoagulatory state that is frequently observed in people with HIV. Such insights could lead to improved therapies for individuals infected with HIV and also for those with cardiovascular disease.

Induction of neutrophil extracellular traps by Campylobacter jejuni.

Campylobacter jejuni is the leading cause of bacterial-derived gastroenteritis worldwide and can lead to several post-infectious inflammatory disorders. Despite the prevalence and health impacts of the bacterium, interactions between the host innate immune system and C. jejuni remain poorly understood. To expand on earlier work demonstrating that neutrophils traffic to the site of infection in an animal model of campylobacteriosis, we identified significant increases in several predominantly neutrophil-derived proteins in the faeces of C. jejuni-infected patients, including lipocalin-2, myeloperoxidase and neutrophil elastase. In addition to demonstrating that these proteins significantly inhibited C. jejuni growth, we determined they are released during formation of C. jejuni-induced neutrophil extracellular traps (NETs). Using quantitative and qualitative methods, we found that purified human neutrophils are activated by C. jejuni and exhibit signatures of NET generation, including presence of protein arginine deiminase-4, histone citrullination, myeloperoxidase, neutrophil elastase release and DNA extrusion. Production of NETs correlated with C. jejuni phagocytosis/endocytosis and invasion of neutrophils suggesting that host- and bacterial-mediated activities are responsible for NET induction. Further, NET-like structures were observed within intestinal tissue of C. jejuni-infected ferrets. Finally, induction of NETs significantly increased human colonocyte cytotoxicity, indicating that NET formation during C. jejuni infection may contribute to observed tissue pathology. These findings provide further understanding of C. jejuni-neutrophil interactions and inflammatory responses during campylobacteriosis.

Pharmacologic dissection of the overlapping impact of heat shock protein family members on platelet function.

BACKGROUND: Platelets play a pivotal role in hemostasis, wound healing, and inflammation, and are thus implicated in a variety of diseases, including cancer. Platelet function is associated with release of granule content, cellular shape change, and upregulation of receptors that promote establishment of a thrombus and maintenance of hemostasis. OBJECTIVES: The role of heat shock proteins (Hsps) in modulating platelet function has been studied for a number of years, but comparative roles of individual Hsps have not been thoroughly examined. METHODS: We utilized a panel of specific inhibitors of Hsp40, Hsp70, Hsp90, and Grp94 (the endoplasmic reticulum homolog of Hsp90) to assess their impact on several aspects of platelet function. RESULTS: Inhibition of each of the aforementioned Hsps reduced alpha granule release. In contrast, there was some selectivity in impacts on dense granule release. Thromboxane synthesis was impaired after exposure to inhibitors of Hsp40, Hsp90, and Grp94, but not after inhibition of Hsp70. Both expression of active glycoprotein IIb/IIIa (GPIIb/IIIa) and fibrinogen-induced platelet shape change were diminished by our inhibitors. In contrast, aggregation was selectively abrogated after inhibition of Hsp40 or Hsp90. Lastly, activated platelet-cancer cell interactions were reduced by inhibition of both Hsp70 and Grp94. CONCLUSIONS: These data suggest the importance of Hsp networks in regulating platelet activity.

Pathways That Synthesize Phosphatidylethanolamine Impact Candida albicans Hyphal Length and Cell Wall Composition through Transcriptional and Posttranscriptional Mechanisms.

Candida albicans is a leading cause of systemic bloodstream infections, and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce PE by the Kennedy pathway, exhibits decreased kidney fungal burden in systemically infected mice. Conversely, overexpression of EPT1 results in a hypervirulent phenotype in this model. Thus, mutations that increase PE synthesis increase virulence, and mutations that decrease PE synthesis decrease virulence. However, the mechanism by which virulence is regulated by PE synthesis is only partially understood. RNA sequencing was performed on strains with deficient or excessive PE biosynthesis to elucidate the mechanism. Decreased PE synthesis from loss of EPT1 or PSD1 and PSD2 leads to downregulation of genes that impact mitochondrial function. Losses of PSD1 and PSD2, but not EPT1, cause significant increases in transcription of glycosylation genes, which may reflect the substantial cell wall defects in the psd1Δ/Δ psd2Δ/Δ mutant. These accumulated defects could contribute to the decreased virulence observed for mutants with deficient PE synthesis. In contrast to mutants with decreased PE synthesis, there were no transcriptional differences between the EPT1 overexpression strain and the wild type, indicating that the hypervirulent phenotype is a consequence of posttranscriptional changes. It was found that overexpression of EPT1 causes increased chitin content and increased hyphal length. These phenotypes may help to explain the previously observed hypervirulence in the EPT1 overexpressor.

One week of continuous corticosterone exposure impairs hepatic metabolic flexibility, promotes islet ß-cell proliferation, and reduces physical activity in male C57BL/6 J mice.

Clinical glucocorticoid use, and diseases that produce elevated circulating glucocorticoids, promote drastic changes in body composition and reduction in whole body insulin sensitivity. Because steroid-induced diabetes is the most common form of drug-induced hyperglycemia, we investigated mechanisms underlying the recognized phenotypes associated with glucocorticoid excess. Male C57BL/6 J mice were exposed to either 100ug/mL corticosterone (cort) or vehicle in their drinking water. Body composition measurements revealed an increase in fat mass with drastically reduced lean mass during the first week (i.e., seven days) of cort exposure. Relative to the vehicle control group, mice receiving cort had a significant reduction in insulin sensitivity (measured by insulin tolerance test) five days after drug intervention. The increase in insulin resistance significantly correlated with an increase in the number of Ki-67 positive ß-cells. Moreover, the ability to switch between fuel sources in liver tissue homogenate substrate oxidation assays revealed reduced metabolic flexibility. Furthermore, metabolomics analyses revealed a decrease in liver glycolytic metabolites, suggesting reduced glucose utilization, a finding consistent with onset of systemic insulin resistance. Physical activity was reduced, while respiratory quotient was increased, in mice receiving corticosterone. The majority of metabolic changes were reversed upon cessation of the drug regimen. Collectively, we conclude that changes in body composition and tissue level substrate metabolism are key components influencing the reductions in whole body insulin sensitivity observed during glucocorticoid administration.

The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo.

Human cytomegalovirus (HCMV) is a betaherpesvirus that is a significant pathogen within newborn and immunocompromised populations. Morbidity associated with HCMV infection is the consequence of viral dissemination. HCMV has evolved to manipulate the host immune system to enhance viral dissemination and ensure long-term survival within the host. The immunomodulatory protein vCXCL-1, a viral chemokine functioning primarily through the CXCR2 chemokine receptor, is hypothesized to attract CXCR2+ neutrophils to infection sites, aiding viral dissemination. Neutrophils harbor HCMV in vivo; however, the interaction between vCXCL-1 and the neutrophil has not been evaluated in vivo Using the mouse model and mouse cytomegalovirus (MCMV) infection, we show that murine neutrophils harbor and transfer infectious MCMV and that virus replication initiates within this cell type. Utilizing recombinant MCMVs expressing vCXCL-1 from the HCMV strain (Toledo), we demonstrated that vCXCL-1 significantly enhances MCMV dissemination kinetics. Through cellular depletion experiments, we observe that neutrophils impact dissemination but that overall dissemination is largely neutrophil independent. This work adds neutrophils to the list of innate cells (i.e., dendritic and macrophages/monocytes) that contribute to MCMV dissemination but refutes the hypothesis that neutrophils are the primary cell responding to vCXCL-1.IMPORTANCE An adequate in vivo analysis of HCMV's viral chemokine vCXCL-1 has been lacking. Here we generate recombinant MCMVs expressing vCXCL-1 to study vCXCL-1 function in vivo using MCMV as a surrogate. We demonstrate that vCXCL-1 increases MCMV dissemination kinetics for both primary and secondary dissemination. Additionally, we provide evidence, that the murine neutrophil is largely a bystander in the mouse's response to vCXCL-1. We confirm the hypothesis that vCXCL-1 is a HCMV virulence factor. Infection of severely immunocompromised mice with MCMVs expressing vCXCL-1 was lethal in more than 50% of infected animals, while all animals infected with parental virus survived during a 12-day period. This work provides needed insights into vCXCL-1 function in vivo.

Anticytomegalovirus Peptides Point to New Insights for CMV Entry Mechanisms and the Limitations of In Vitro Screenings.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that can cause severe disease following in utero exposure, during primary infection, or latent virus reactivation in immunocompromised populations. These complications lead to a 1- to 2-billion-dollar economic burden, making vaccine development and/or alternative treatments a high priority. Current treatments for HCMV include nucleoside analogues such as ganciclovir (GCV), foscarnet, and cidofovir. Recently, letermovir, a terminase complex inhibitor, was approved for prophylaxis after stem cell transplantation. These treatments have unwanted side effects, and HCMV is becoming resistant to them. Therefore, we sought to develop an alternative treatment that targets a different stage in viral infection. Currently, small antiviral peptides are being investigated as anti-influenza and anti-HIV treatments. We have developed heparan sulfate-binding peptides as tools for preventing CMV infections. These peptides are highly effective at stopping infection of fibroblasts with in vitro-derived HCMV and murine cytomegalovirus (MCMV). However, they do not prevent MCMV infection in vivo Interestingly, these peptides inhibit infectivity of in vivo-derived CMVs, albeit not as well as tissue culture-grown CMVs. We further demonstrate that this class of heparan sulfate-binding peptides is incapable of inhibiting MCMV cell-to-cell spread, which is independent of heparan sulfate usage. These data indicate that inhibition of CMV infection can be achieved using synthetic polybasic peptides, but cell-to-cell spread and in vivo-grown CMVs require further investigation to design appropriate anti-CMV peptides.IMPORTANCE In the absence of an effective vaccine to prevent HCMV infections, alternative interventions must be developed. Prevention of viral entry into susceptible cells is an attractive alternative strategy. Here we report that heparan sulfate-binding peptides effectively inhibit entry into fibroblasts of in vitro-derived CMVs and partially inhibit in vivo-derived CMVs. This includes the inhibition of urine-derived HCMV (uCMV), which is highly resistant to antibody neutralization. While these antiviral peptides are highly effective at inhibiting cell-free virus, they do not inhibit MCMV cell-to-cell spread. This underscores the need to understand the mechanism of cell-to-cell spread and differences between in vivo-derived versus in vitro-derived CMV entry to effectively prevent CMV's spread.