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Rapid clearance of thrombolytics from blood following intravenous injection is a major clinical challenge in cardiovascular medicine. To overcome this barrier, nanoparticle (NP) based drug delivery systems have been reported. Although superior than conventional therapy, a large proportion of the injected NP is still cleared by the reticuloendothelial system. Previously, we and others showed that ex vivo attachment of bioscavengers, thrombolytics, and nanoparticles (NPs) to glycophorin A receptors on red blood cells (RBCs) improved the blood half-life. This is promising, but ex-vivo approaches are cumbersome and challenging to translate clinically. Here, we developed a novel Ter119-polymeric NP containing tissue plasminogen activator for on-demand targeting of GPA receptors in vivo. Upon intravenous injection, the Ter119-NPs achieved remarkable RBC labeling efficiencies (>95%), resulting in marked enhancement of blood residence time of tPA from minutes to several days without any morphological, hematological, and histological complications. Our approach of RBC labeling with the NPs also prevented reticuloendothelial detections and the activations of innate and adaptive immune system. Data suggest that real-time targeting of therapeutics to RBC with NPs can potentially improve outcomes and reduce complications against a variety chronic disease.

Retinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation., Lingyan Wang et al. report that the autophagy-associated protein FIP200 interacts with the RNA sensor RIG-I to trigger activation of the type I interferon pathway.

The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. This study validates a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with severe COVID-19 in humans. Intra-tracheal inoculation of concentrated SARS-CoV-2 caused infected cats to develop clinical disease consistent with that observed in the early exudative phase of COVID-19. A novel clinical scoring system for feline respiratory disease was developed and utilized, documenting a significant degree of lethargy, fever, dyspnea, and dry cough in infected cats. In addition, histopathologic pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were observed due to SARS-CoV-2 infection, imitating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation exists between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were quantified in nasal turbinates, distal trachea, lung, and various other organs. Natural ACE2 expression, paired with clinicopathologic correlates between this feline model and human COVID-19, encourage use of this model for future translational studies.Author SummaryIdentifying an ideal animal model to study COVID-19 has been difficult, and current models come with challenges that restrict their potential in translational studies. Few lab animals naturally express the receptors necessary for viral infection (ACE2), and many fail to manifest clinical signs or pathology similar to that seen in humans. Other models (non-human primates, mink) are ideal for disease and transmission studies, but are restricted by cost, husbandry challenges, and scarce availability. Alternatively, cats naturally express ACE2 receptors, are naturally infected with SARS-CoV-2 and can transmit virus from cat-to-cat. Prior to this study, cats infected by oral/nasal routes have not displayed significant clinical disease or lung pathology. However, we demonstrate that direct inoculation of concentrated SARS-CoV-2 virus in the trachea of cats induces analogous clinical and pathologic features to hospitalized patients with acute COVID-19. Our results show that infected cats exhibit significant clinical signs during experimental infection (coughing, increased respiratory effort, lethargy, and fever) and exhibit extensive lung lesions that mimic severe COVID-19 pathology such as diffuse alveolar damage and hyaline membrane formation – highlighting the immeasurable potential for this feline model to address translational approaches for COVID-19 and to better understand the role of cats in transmission and disease.

The global outbreak and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an urgent need for large-scale testing of populations. There is a demand for high-throughput testing protocols that can be used for efficient and rapid testing of clinical specimens. We evaluated a pooled PCR protocol for testing nasopharyngeal (NP) swabs using known positive/negative and untested clinical samples that were assigned to pools of 5 or 10., The global outbreak and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have created an urgent need for large-scale testing of populations. There is a demand for high-throughput testing protocols that can be used for efficient and rapid testing of clinical specimens. We evaluated a pooled PCR protocol for testing nasopharyngeal (NP) swabs using known positive/negative and untested clinical samples that were assigned to pools of 5 or 10. In total, 630 samples were used in this study. Individual positive samples with cycle threshold (CT) values as high as 33 could be consistently detected when pooled with 4 negative samples (pool of 5), and individual positive samples with CT values up to 31 could be consistently detected when pooled with 9 negative samples (pool of 10). Pooling of up to 5 samples can be employed in laboratories for the diagnosis of COVID-19 for efficient utilization of resources, rapid screening of a greater number of people, and faster reporting of test results.

The onset of type 2 diabetes in obesity is associated with gut dysbiosis and a failure to confine commensal bacteria and toxins to the gut lumen while prebiotics may prevent these effects. This study evaluated the effects of pinto beans (PB) supplementation on cecal bacteria, short-chain fatty acids (SCFAs), distal ileal antigen presentation marker (major histocompatibility complex [MHC] II) and antimicrobial peptide genes during short-term high-fat, high sucrose (HFS) feeding. Six-week-old, male C57BL/6J mice were randomly assigned to four groups (n=12/group), and fed a control (C) or HFS diet with or without cooked PB (10%, wt/wt) for 30 days. Supplemental PB in both the C and HFS diets decreased the abundance of Tenericutes and the sulfate-reducing bacteria Bilophila. In contrast, PB raised the abundance of taxa within the SCFAs-producing family, Lachnospiraceae, compared to groups without PB. Consequently, fecal butyric acid was significantly higher in PB-supplemented groups compared to C and HFS groups. PB reversed the HFS-induced ablation of the distal ileal STAT3 phosphorylation, and up-regulated antimicrobial peptide genes (Reg3γ and Reg3β). Furthermore, the expression of MHC II protein was elevated in the PB supplemented groups compared to C and HFS. Tenericutes and Bilophilia negatively correlated with activated STAT3 and MHC II proteins. Finally, supplemental PB improved fasting blood glucose, glucose tolerance and suppressed TNFα and inducible nitric oxide synthase mRNA in the visceral adipose tissue. Put together, the beneficial impact of PB supplementation on the gut may be central to its potential to protect against diet-induced inflammation and impaired glucose tolerance.

An 8-year-old, intact female, mixed-breed dog presented to the Oklahoma State University Boren Veterinary Medical Teaching Hospital for evaluation of progressive lameness and joint effusion of multiple joints. Physical examination revealed joint effusion of the elbow, hock, and stifle joints bilaterally, enlarged left axillary and right popliteal lymph nodes, a subcutaneous mass over the left elbow, and a subcutaneous mass involving the left second and third mammary glands. Cytologic examination of the mammary mass, enlarged lymph nodes, and joint fluid from most affected joints revealed a monomorphic population of loosely cohesive neoplastic epithelial cells. The patient was humanely euthanized, and subsequent necropsy with histopathologic examination revealed a complex mammary carcinoma with metastases to enlarged lymph nodes, subcutaneous tissue over the left elbow, and the synovium of multiple joints. Immunohistochemical stains were performed and showed diffusely positive pan cytokeratin, CK8/18, and CK19 staining in the neoplastic luminal epithelial cells of the mammary carcinoma, synovium, and lymph nodes, and showed diffusely positive vimentin staining of the myoepithelial cells. Myoepithelial calponin positivity was diffuse in the mammary mass and lymph nodes but minimal in the synovium. Only the mammary mass showed p63 positivity. Metastatic mammary neoplasia is relatively common in dogs; however, metastasis to the synovium has only been reported once previously in the literature. This is the first case utilizing immunohistochemistry for confirmation and characterization of metastases.

Diabetes has been identified as major risk factor for atrial fibrillation (AF). Although glucose and insulin disturbances during diabetes may affect atrial function, little is known about the potential pathogenic role of glucose metabolism during AF. Glucose transport into the cell via glucose transporters (GLUTs) is the rate-limiting step of glucose utilization. Although GLUT4 is the major isoform, GLUT8 has emerged as a novel insulin-sensitive cardiac isoform. We hypothesized that atrial glucose homeostasis will be impaired during insulin resistance-induced AF. AF was induced by transesophageal atrial pacing in healthy mice and following a long-term high-fat-diet-induced insulin resistance. Active cell surface GLUT content was measured using the biotinylated photolabeling assay in the intact perfused heart. Atrial fibrosis, advanced glycation end products (AGEs) and glycogen were measured in the atria using histological analyses. Animals fed a high-fat-diet were obese and mildly hyperglycemic, and developed insulin resistance compared to controls. Insulin-resistant (IR) animals demonstrated an increased vulnerability to induced AF, as well as spontaneous AF. Insulin-stimulated translocation of GLUT4 and GLUT8 was down-regulated in the atria of IR animals, as well as their total protein expression. We also reported the absence of fibrosis, glycogen and AGE accumulation in the atria of IR animals. In the absence of structural remodeling and atrial fibrosis, these data suggest that insulin signaling dysregulation, resulting in impaired glucose transport in the atria, could provide a metabolic arrhythmogenic substrate and be a novel early pathogenic factor of AF.

Diabetes has been identified as an independent risk factor for atrial fibrillation (AF), the most common chronic cardiac arrhythmia. Whether or not glucose and insulin disturbances observed during diabetes enhance arrhythmogenicity of the atria, potentially leading to AF, is not well-known. We hypothesized that insulin deficiency and impaired glucose transport provide a metabolic substrate for the development and maintenance of AF during diabetes. Transesophageal atrial pacing was used to induce AF in healthy, streptozotocin-induced insulin-deficient type 1 diabetic, and insulin-treated diabetic mice. Translocation of insulin-sensitive glucose transporters (GLUTs) to the atrial cell surface was measured using a biotinylated photolabeling assay in the perfused heart. Fibrosis and glycogen accumulation in the atrium were measured using histological analysis. Diabetic mice displayed mild hyperglycemia, increased duration and frequency of AF episodes vs. age-matched controls (e.g., AF duration: 19.7 ± 6.8 s vs. 1.8 ± 1.1 s, respectively, p = 0.032), whereas insulin-treated diabetic animals did not. The translocation of insulin-sensitive GLUT-4 and -8 to the atrial cell surface was significantly downregulated in the diabetic mice (by 67 and 79%, respectively; p ≤ 0.001), and rescued by insulin treatment. We did not observe fibrosis or glycogen accumulation in the atria of diabetic mice. Therefore, these data suggest that insulin and glucose disturbances were sufficient to induce AF susceptibility during mild diabetes.

Exaggerated host innate immune response has been implicated in severe influenza pneumonia. We have previously demonstrated that excessive neutrophils recruited during influenza infection drive pulmonary pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. Chemokine receptors (CRs) are essential in the recruitment and activation of leukocytes. Although neutrophils have been implicated in influenza pathogenesis, little is known about their phenotypic changes, including expression of CRs occurring in the infected-lung microenvironment. Here, we examined expression of CC and CXC CRs expressions in circulating as well as lung-recruited neutrophils during influenza infection in mice using flow cytometry analyses. Our studies revealed that lung-recruited neutrophils displayed induction of CRs including CCR1, CCR2, CCR3, CCR5, CXCR1, CXCR3 and CXCR4, all of which were marginally induced in circulating neutrophils. CXCR2 is most predominant CR observed in both circulating and lung-infiltrated neutrophils after infection. The stimulation of these induced CRs modulated neutrophil phagocytic activity, ligand-specific neutrophil migration, bacterial killing and NETs induction ex vivo. These findings indicate that neutrophils induce a novel CR repertoire in the infectious lung microenvironment, which alters their functionality during influenza pneumonia.

Mast cells (MCs) play critical roles in the pathogenesis of IgE- and non-IgE-mediated immune responses, as well as host defense against parasites, bacteria, and viruses. Due to the effect of extracellular matrix components on tissue morphogenesis and cell behavior, utilizing a tissue model that mimics MC microenvironmental conditions in vivo has greater relevance for in vitro studies. For this work, MCs were developed within a connective tissue-equivalent model and cell function was examined in response to an allergen. MCs are located in proximity to fibroblasts and endothelial cells (ECs) that play a role in MC development and maturity. Accordingly, MC progenitors isolated from human peripheral blood were co-cultured with human primary fibroblasts in a 3D collagen matrix to represent the connective tissue. The matrix was coated with type IV collagen and fibronectin before seeding with primary human ECs, representing the capillary wall. The stem cell-derived cells demonstrated MC characteristics, including typical MC morphology, and the expression of cytoplasmic granules and phenotypic markers. Also, the generated cells released histamine in IgE-mediated reactions, showing typical MC functional phenotype in an immediate-type allergenic response. The created tissue model is applicable to a variety of research studies and allergy testing. Impact Statement Mast cells (MCs) are key effector and immunoregulatory cells in immune disorders; however, their role is not fully understood. Few studies have investigated human ex vivo MCs in culture, due to the difficulties in isolating large numbers. Our study demonstrates, for the first time, the generation of cells exhibiting MC phenotypic and functional characteristics from hematopoietic stem cells within a connective tissue-equivalent model with ancillary cells. Utilizing the 3D matrix-embedded cells can advance our understanding of MC biological profile and immunoregulatory roles. The tissue model can also be used for studying the mechanism of allergic diseases and other inflammatory disorders.

Condensins play a key role in global chromosome packing. Pseudomonas aeruginosa encodes two condensins, SMC-ScpAB and MksBEF. We report here that the two proteins are involved in the differentiation of the bacterium and impose opposite physiological states. The inactivation of SMC induced a state characterized by increased adhesion to surfaces as well as defects in competitive growth and colony formation. In contrast, MksB-deficient cells were impaired in biofilm formation with no obvious defects during planktonic growth. The phenotype of the double mutant was dominated by the absence of MksB, indicating that the observed growth defects are regulatory in their nature rather than structural. ATPase mutations recapitulated many of the phenotypes of the condensins, indicating their requirement for a functional protein. Additionally, inactivation of condensins dramatically reduced the virulence of the bacterium in a murine model of lung infection. These data demonstrate that condensins are involved in the differentiation of P. aeruginosa and reveal their importance for pathogenicity. IMPORTANCE Adaptation and differentiation play key roles in bacterial pathogenicity. In Pseudomonas aeruginosa , an opportunistic human pathogen, these processes are mediated by the activity of an intricate regulatory network. We describe here novel members of this network, condensins. We show that the two P. aeruginosa condensins specialize in the establishment of the sessile and planktonic states of the bacterium. Whereas condensins have well-established roles in global chromosome organization, their roles in regulating bacterial physiology have remained unknown. Our data indicate that the two programs may be linked. We further show that condensins are essential for the pathogenicity of P. aeruginosa .

Radiation-induced pulmonary fibrosis is a severe side effect of thoracic irradiation, but its pathogenesis remains poorly understood and no effective treatment is available. In this study, we investigated the role of the extracellular adenosine as generated by the ecto-5′-nucleotidase CD73 in fibrosis development after thoracic irradiation. Exposure of wild-type C57BL/6 mice to a single dose (15 Gray) of whole thorax irradiation triggered a progressive increase in CD73 activity in the lung between 3 and 30 weeks postirradiation. In parallel, adenosine levels in bronchoalveolar lavage fluid (BALF) were increased by approximately 3-fold. Histologic evidence of lung fibrosis was observed by 25 weeks after irradiation. Conversely, CD73-deficient mice failed to accumulate adenosine in BALF and exhibited significantly less radiation-induced lung fibrosis (P < 0.010). Furthermore, treatment of wild-type mice with pegylated adenosine deaminase or CD73 antibodies also significantly reduced radiation-induced lung fibrosis. Taken together, our findings demonstrate that CD73 potentiates radiation-induced lung fibrosis, suggesting that existing pharmacologic strategies for modulating adenosine may be effective in limiting lung toxicities associated with the treatment of thoracic malignancies. Cancer Res; 76(10); 3045–56. ©2016 AACR.

The success of melanoma immunotherapy is dependent on the presence of activated and functional T-cells in tumors. The objective of this study was to investigate the impact of local-focused ultrasound (FUS) heating (∼42-45 °C) and in-situ anti-CD-40 agonistic antibody in enhancing T-cell function for melanoma immunotherapy. We compared the following groups of mice with bilateral flank B16 F10 melanoma: (1) Control, (2) FUS, (3) CD-40, and (4) CD-40 + FUS (FUS40). FUS heating was applied for ∼15 min in right flank tumor, and intratumoral injections of CD-40 were performed sequentially within 4 h. A total of 3 FUS and 4 anti-CD-40 treatments were administered unilaterally 3 days apart. Mice were sacrificed 30 days post-inoculation, and the treated tumor and spleen tissues were profiled for T-cell function and macrophage polarization. Compared to all other groups, histology and flow cytometry showed that FUS40 increased the population of tumor-specific CD-4+ and CD-8+ T cells rich in Granzyme B+, interleukin-2 (IL-2) and IFN-γ production and poor in PD-1 expression. In addition, FUS40 promoted the infiltration of tumor-suppressing M1 phenotype macrophages in the treated mice. The resultant immune-enhancing effects of FUS40 suppressed B16 melanoma growth at the treated site by 2-3-folds compared to control, FUS, and CD-40, and also achieved significant abscopal effects in untreated tumors relative to CD40 alone. Additionally, the local FUS40 prevented adverse liver toxicities in the treated mice. Our study suggests that combined FUS and CD-40 can enhance T-cell and macrophage functions to aid effective melanoma immunotherapy.

BACKGROUND A link between high-fat diet consumption and obesity-related diseases is the disruption of the gut bacterial population, which promotes local and systemic inflammation. Wheat germ (WG) is rich in bioactive components with antioxidant and anti-inflammatory properties. OBJECTIVE The aim of this study was to investigate the effects of WG supplementation in modulating the gut bacterial population and local and systemic inflammatory markers of mice fed a high-fat, high-sucrose (HFS) diet. METHODS Six-week-old male C57BL/6 mice were randomly assigned to 4 groups (n = 12/group) and fed a control (C; 10% kcal fat, 10% kcal sucrose) or HFS (60% kcal fat, 20% kcal sucrose) diet with or without 10% WG (wt:wt) for 12 wk. Cecal bacteria was assessed via 16S rDNA sequencing, fecal short-chain fatty acids by GC, small intestinal CD4+ lymphocytes using flow cytometry, and gut antimicrobial peptide genes and inflammatory markers by quantitative polymerase chain reaction. Statistical analyses included Kruskal-Wallis/Dunn's test and 2-factor ANOVA using HFS and WG as factors. RESULTS There was a 4-fold increase (P = 0.007) in the beneficial bacterial family, Lactobacillaceae, in the HFS + WG compared with the HFS group. Fecal propionic and n-butyric acids were elevated at least 2-fold in C + WG compared with the other groups (P

Diabetes has been identified as a major independent risk factor for atrial fibrillation (AF), the most common sustained cardiac arrhythmia. However, it is unknown whether glucose and insulin disturbances observed during diabetes directly affect the function of the atria, leading to AF. Thus, we hypothesized that insulin deficiency and impaired glucose transport provide a metabolic substrate for the development and maintenance of AF during diabetes. Transesophageal atrial pacing was used to induce AF in healthy, streptozotocin-induced type 1 diabetic and insulin-treated (via a subcutaneous pump) diabetic mice (n=4-11/group). Translocation of insulin-sensitive glucose transporters (GLUTs) was measured using a photolabeled biotinylation assay in the perfused heart. Atrial fibrosis and glycogen accumulation were measured using histological analyses. As expected, insulin-deficient diabetic mice displayed hyperglycemia (434 ± 50.9 mg/dl) while insulin-treated diabetic mice remained euglycemic (118.8 ± 3.3 mg/dl, p Disclosure Z. Maria: None. A. Campolo: None. B.J. Scherlag: None. J.W. Ritchey: None. V. Lacombe: None.

Influenza A virus (IAV) claims ∼250,000-500,000 lives annually worldwide. Currently, there are a few in vitro models available to study IAV immunopathology. Monolayer cultures of cell lines and primary lung cells (two-dimensional [2D] cell culture) is the most commonly used tool, however, this system does not have the in vivo-like structure of the lung and immune responses to IAV as it lacks the three-dimensional (3D) tissue structure. To recapitulate the lung physiology in vitro, a system that contains multiple cell types within a 3D environment that allows cell movement and interaction would provide a critical tool. In this study, as a first step in designing a 3D-Human Tissue-Engineered Lung Model (3D-HTLM), we describe the 3D culture of primary human small airway epithelial cells (HSAEpCs) and determined the immunophenotype of this system in response to IAV infections. We constructed a 3D chitosan-collagen scaffold and cultured HSAEpCs on these scaffolds at air-liquid interface (ALI). These 3D cultures were compared with 2D-cultured HSAEpCs for viability, morphology, marker protein expression, and cell differentiation. Results showed that the 3D-cultured HSAEpCs at ALI yielded maximum viable cells and morphologically resembled the in vivo lower airway epithelium. There were also significant increases in aquaporin-5 and cytokeratin-14 expression for HSAEpCs cultured in 3D compared to 2D. The 3D culture system was used to study the infection of HSAEpCs with two major IAV strains, H1N1 and H3N2. The HSAEpCs showed distinct changes in marker protein expression, both at mRNA and protein levels, and the release of proinflammatory cytokines. This study is the first step in the development of the 3D-HTLM, which will have wide applicability in studying pulmonary pathophysiology and therapeutics development.

Objective To assess the effects of a crosslinked, modified hyaluronic acid (xCMHA-S) gel on equine tendon healing using an in vivo surgical model. Study Design In vivo experimental study. Animals Adult horses (n = 5). Methods Full thickness bilateral forelimb window tenectomies were surgically created in both forelimb superficial digital flexor tendons and xCMHA-S gel was implanted intraoperatively into the right forelimb lesion of each horse whereas the left forelimb served as the untreated control. Healing was monitored by serial ultrasound examinations every 14 days over the course of the 84 day study. In addition, gross pathology, scanning electron microscopy for fiber diameter, and histological scoring were completed on tendon samples harvested after euthanasia at 84 days. Results Ultrasound assessment demonstrated a significant decrease in mean lesion size of treated (0%) compared to control (30%) tendons at 84 days. Mean (±SD) cumulative histologic tendon scores for control tendons (17.7 ± 2.7) were significantly higher than treated tendons (13.6 ± 1.9), indicating less advanced healing in the control group. Tendon cell density was increased and neovascularization, intensity of inflammation, and uniformity of fiber diameter were increased in control compared to treated tendons. There were no differences in fibroblast shape, levels of intralesional hemorrhage, linearity of collagen fibers, or collagen fiber diameter or distribution between treated and control tendons. Conclusion Tendons treated with xCMHA-S gel at the time of model induction had superior histologic healing scores and sonographically smaller lesions compared to controls, suggesting that xCMHA-S gel may aid the natural healing process.

Geropathology is the study of aging and age-related lesions and diseases in the form of whole necropsies/autopsies, surgical biopsies, histology, and molecular biomarkers. It encompasses multiple subspecialties of geriatrics, anatomic pathology, molecular pathology, clinical pathology, and gerontology. In order to increase the consistency and scope of communication in the histologic and molecular pathology assessment of tissues from preclinical and clinical aging studies, a Geropathology Research Network has been established consisting of pathologists and scientists with expertise in the comparative pathology of aging, the design of aging research studies, biostatistical methods for analysis of aging data, and bioinformatics for compiling and annotating large sets of data generated from aging studies. The network provides an environment to promote learning and exchange of scientific information and ideas for the aging research community through a series of symposia, the development of uniform ways of integrating pathology into aging studies, and the statistical analysis of pathology data. The efforts of the network are ultimately expected to lead to a refined set of sentinel biomarkers of molecular and anatomic pathology that could be incorporated into preclinical and clinical aging intervention studies to increase the relevance and productivity of these types of investigations.

Background Glioblastoma is a malignant World Health Organization (WHO) grade IV glioma with a poor prognosis in humans. New therapeutics are desperately required. The nitrone OKN-007 (2,4-disulfophenyl-PBN) has demonstrated effective anti-glioma properties in several rodent models and is currently being used as a clinical investigational drug for recurrent gliomas. We assessed the regional effects of OKN-007 in the tumor necrotic core and non-necrotic tumor parenchyma. Methods An F98 rat glioma model was evaluated using proton magnetic resonance spectroscopy (1H-MRS), diffusion-weighted imaging (DWI), morphological T2-weighted imaging (T2W) at 7 Tesla (30 cm-bore MRI), as well as immunohistochemistry and microarray assessments, at maximum tumor volumes (15–23 days following cell implantation in untreated (UT) tumors, and 18–35 days in OKN-007-treated tumors). Results 1H-MRS data indicates that Lip0.9/Cho, Lip0.9/Cr, Lip1.3/Cho, and Lip1.3/Cr ratios are significantly decreased (all P

OBJECTIVE To determine whether prophylactic administration of valacyclovir hydrochloride versus initiation of treatment at the onset of fever would differentially protect horses from viral replication and clinical disease attributable to equine herpesvirus type-1 (EHV-1) infection. ANIMALS 18 aged mares. PROCEDURES Horses were randomly assigned to receive an oral placebo (control), treatment at detection of fever, or prophylactic treatment (initiated 1 day prior to viral challenge) and then inoculated intranasally with a neuropathogenic strain of EHV-1. Placebo or valacyclovir was administered orally for 7 or 14 days after EHV-1 inoculation or detection of fever (3 horses/group). Effects of treatment on viral replication and clinical disease were evaluated. Plasma acyclovir concentrations and viremia were assessed to determine inhibitory concentrations of valacyclovir. RESULTS Valacyclovir administration decreased shedding of virus and viremia, compared with findings for control horses. Rectal temperatures and clinical disease scores in horses that received valacyclovir prophylactically for 2 weeks were lower than those in control horses. The severity of but not the risk for ataxia was decreased by valacyclovir administration. Viremia was decreased when steady-state trough plasma acyclovir concentrations were > 0.8 μg/mL, supporting the time-dependent activity of acyclovir. CONCLUSIONS AND CLINICAL RELEVANCE Valacyclovir treatment significantly decreased viral replication and signs of disease in EHV-1–infected horses; effects were greatest when treatment was initiated before viral inoculation, but treatment was also effective when initiated as late as 2 days after inoculation. During an outbreak of equine herpesvirus myeloencephalopathy, antiviral treatment may be initiated in horses at various stages of infection, including horses that have not yet developed signs of viral disease.

Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro , in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza–challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia.

Monkey B virus (Macacine herpesvirus 1; BV) is noted for its extreme neurovirulence in humans. Since the vhs protein encoded by the UL41 gene has been shown to be a neurovirulence factor in the related human herpes simplex viruses, the role of the UL41 gene in BV neurovirulence was investigated. BV mutants were constructed that lacked the entire UL41 ORF (Δ41) or had the RNase active site mutated (Δ41A). Neither mutant shut off host protein synthesis, degraded β-actin mRNA, or prevented an IFN-β response, indicating that the vhs protein and its RNase activity are both necessary for these activities. Replication of both mutants in primary mouse cells was impaired and they exhibited a prolonged disease course in mice. Whereas Δ41 infected mice were euthanized for symptoms related to central nervous system (CNS) infection, Δ41A infected mice were euthanized primarily for symptoms of autonomic nervous system dysfunction. While neuroinvasiveness was not affected, lesions in the CNS were more limited in size, anatomical distribution, and severity than for wild-type virus. These results indicate that the vhs protein affects the general replicative efficiency of BV in vivo rather than being a specific neurovirulence factor critical for invasion of or preferential replication in the CNS.

Summary Alaskan racing sled dogs are a well-established model of exercise-induced gastric disease. The aim of this study was to define the temporal development of microscopical gastric lesions during long distance racing. Two groups of dogs were examined: group I comprised conditioned dogs that were exercising and group II were conditioned dogs not exercising. The gastric mucosa was examined endoscopically and sampled for routine histopathology and microscopical scoring, immunohistochemistry (IHC) and detection of apoptotic epithelial cells. Overall, group I dogs exhibited more significant epithelial lesions, including ulcers, compared with dogs in group II. Group II dogs exhibited the most severe mucosal inflammatory infiltrates. Although the intensity of inflammation differed, the nature of the inflammation was similar between groups, consisting of diffuse lymphocytic infiltration and a unique interface-type infiltrate that obscured the basement membrane zone and was accompanied by intraepithelial infiltration of lymphocytes. IHC confirmed the presence of CD3 + T and CD79 + B lymphocytes within the mucosal infiltrates; however, most of the intraepithelial and interface infiltrates were CD3 + T cells. Spiral-shaped bacterial organisms were seen in the gastric tissues; however, their presence did not correlate with either the severity of epithelial lesions, inflammation or the pattern of interface inflammation. The number of apoptotic epithelial cells was widely variable and not significantly different between groups. These findings confirm previous observations that gastric ulcers develop in conditioned dogs under racing stress. The unique nature of the interface-type gastric inflammation is similar to that of human lymphocytic gastritis and may suggest an immune-mediated mechanism for the changes seen in Alaskan racing sled dogs.

Leukotoxin (LKT) is a virulence factor for Mannheimia haemolytica. In this study, bovine alveolar macrophages (BAMs) were challenged with wild type (wt) and LKT deficient (lkt(-)) M. haemolytica at a concentration of 1 bacterium/BAM and the cytokine response was quantified by ELISA and real-time reverse transcriptase-PCR. Significant increases in protein concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-10 were observed in supernatants obtained from BAMs challenged with the lkt(-) strain of M. haemolytica compared with wt challenged BAMs. There were no significant differences in mRNA expression of TNFα, IL-1ß, IL-6, IL-8 or IL-10 between BAMs challenged with the lkt(-) strain of M. haemolytica compared with wt challenged BAMs. BAMs challenged with the wt strain exhibited, on average, 43% more cytotoxicity than lkt(-) challenged BAMs (P0.01).

Mannheimia haemolytica serotype S1 is considered the predominant cause of bovine pneumonic pasteurellosis, or shipping fever. Various virulence factors allow M haemolytica to colonize the lungs and establish infection. These virulence factors include leukotoxin (LKT), lipopolysaccharide, adhesins, capsule, outer membrane proteins, and various proteases. The effects of LKT are species specific for ruminants, which stem from its unique interaction with the bovine β2 integrin receptor present on leukocytes. At low concentration, LKT can activate bovine leukocytes to undergo respiratory burst and degranulation and stimulate cytokine release from macrophages and histamine release from mast cells. At higher concentration, LKT induces formation of transmembrane pores and subsequent oncotic cell necrosis. The interaction of LKT with leukocytes is followed by activation of these leukocytes to undergo oxidative burst and release proinflammatory cytokines such as interleukins 1, 6, and 8 and tumor necrosis factor α. Tumor necrosis factor α and other proinflammatory cytokines contribute to the accumulation of leukocytes in the lung. Formation of transmembrane pores and subsequent cytolysis of activated leukocytes possibly cause leakage of products of respiratory burst and other inflammatory mediators into the surrounding pulmonary parenchyma and so give rise to fibrinous and necrotizing lobar pneumonia. The effects of LKT are enhanced by lipopolysaccharide, which is associated with the release of proinflammatory cytokines from the leukocytes, activation of complement and coagulation cascade, and cell cytolysis. Similarly, adhesins, capsule, outer membrane proteins, and proteases assist in pulmonary colonization, evasion of immune response, and establishment of the infection. This review focuses on the roles of these virulence factors in the pathogenesis of shipping fever.

Fowlicidin-1 is a newly identified alpha-helical cathelicidin host defense peptide. We have shown that fowlicidin-1 possesses potent antibacterial activity, but also displays considerable toxicity toward mammalian cells. To further identify fowlicidin-1 analog(s) with enhanced therapeutic potential, a series of amino-terminal truncation analogs were synthesized and functionally evaluated. Relative to the full-length peptide, fowl-1(6-26), an analog with omission of five amino-terminal amino acid residues, maintained the antibacterial potency against a range of Gram-negative and Gram-positive bacteria including antibiotic-resistant strains. Fowl-1(6-26)-NH(2), a carboxyl-terminal amidated form of fowl-1(6-26), retained the antibacterial activity for a minimum of 2h in the presence of 100% serum. In addition, an intraperitoneal administration of 10mg/kg of fowl-1(6-26)-NH(2) led to a 50% increase in the survival of neutropenic mice over a 7-day period from a lethal dose of methicillin-resistant Staphylococcus aureus (MRSA), concomitant with a reduction in the bacterial titer in both peritoneal fluids and spleens of mice 24h post-infection. Fowl-1(6-26)-NH(2) at 20 microM was further found to suppress lipopolysaccharide-mediated production of TNF-alpha and nitric oxide in macrophages by 77% and 96%, respectively. Therefore, with potent endotoxin-neutralizing and bactericidal activities, fowlicidin-1(6-26)-NH(2), may have strong therapeutic potential for drug-resistant infections and sepsis.

This paper presents a transrectal dual-modality near-infrared (NIR) diffuse optical tomography technique coupled with ultrasonography that provides an integrated method for detecting prostate cancer (PCa). The study that provides an alternative transrectal prostate imaging system stems from the perceived inadequacy of conventional transrectal ultrasound (TRUS) in PCa imaging. The transrectally applied diffuse optical tomography aims to characterize the spatially resolved optical properties of the intact prostate that are known to have intraorgan and intersubject heterogeneities. A canine model of PCa using canine transmissible venereal tumor was used to demonstrate the utility of this technology in detecting PCa. Tumors in the pelvic canal, including tumors in the prostate, were found to be detectable at 2-week postinjection based upon the NIR absorption contrast, which was detected a few weeks earlier than using NIR-reduced scattering and effective attenuation contrasts, as well as TRUS. Transrectal optical tomography detection of cancerous tissues in vivo in intact canine prostate based upon NIR contrasts may prove useful for diagnostic imaging of PCa and potentially aid in pretreatment planning for phototherapy applications.

This is the first tomography-presentation of the optical properties of a normal canine prostate, in vivo, in its native intact environment in the pelvic canal. The imaging was performed by trans-rectal near-infrared (NIR) optical tomography in steady-state measurement at 840 nm on three sagittal planes across the right lobe, middle-line, and left lobe, respectively, of the prostate gland. The NIR imaging planes were position-correlated with concurrently applied trans-rectal ultrasound, albeit there was no spatial prior employed in the NIR tomography reconstruction. The reconstructed peak absorption coefficients of the prostate on the three planes were 0.014, 0.012, and 0.014 mm-1. The peak reduced scattering coefficients were 5.28, 5.56, and 6.53 mm-1. The peak effective attenuation coefficients were 0.45, 0.43, and 0.50 mm-1. The absorption and effective attenuation coefficients were within the ranges predictable at 840 nm by literature values which clustered sparsely from 355 nm to 1064 nm, none of which were performed on a canine prostate with similar conditions. The effective attenuation coefficients of the gland were shown to be generally higher in the internal aspects than in the peripheral aspects, which is consistent with the previous findings that the urethral regions were statistically more attenuating than the capsular regions.

Objective – To describe the clinical and pathological findings in 2 adult horses with documented increases in intra-abdominal pressure (IAP), and to describe the direct puncture technique used to measure this pressure. Series Summary – Two adult horses developed increases in IAP secondary to large-volume abdominal effusion. A 9-year-old Quarter Horse cross gelding was presented for evaluation of urinary and neurologic signs. Abdominal ultrasonographic examination showed a hepatic abscess along with abdominal effusion. A 4-year-old Quarter Horse gelding was presented for evaluation of castration complications. A castration site infection extended into surrounding tissues, resulting in peritonitis, abdominal effusion, and severe cellulitis of the limbs. IAP measured in both horses was increased relative to reported equine reference values. Changes in hemodynamic parameters in both horses, notably increased central venous pressure, were consistent with those seen in other species in which intra-abdominal hypertension (IAH) and its sequellae have been documented. New or Unique Information Provided – Extensive research and clinical literature guides management of humans with IAH and abdominal compartment syndrome. Knowledge of these conditions in companion animal and large domestic species is less well developed. Recent research has established reference values for standing, sedated and recumbent, anesthetized horses. Detailed reports of equine clinical cases of IAH have not been reported in the literature. This report provides information on the clinical, hemodynamic, and pathologic characteristics of 2 horses with measured increases in IAP, and describes the direct puncture technique used to perform these measurements.

Isolates of baboon alpha-herpesvirus Papiine herpesvirus 2 (HVP2) exhibit one of two distinct phenotypes in mice: extremely neurovirulent or apathogenic. Previous studies implicated the type I interferon (IFN) response as being a major factor in controlling infection by apathogenic isolates. To further investigate the possibility that the host IFN-beta response underlies the pathogenicity of the two HVP2 subtypes, the susceptibility of mice lacking the IFN-beta receptor (IFNAR(-/-)) to infection was examined. Apathogenic isolates of HVP2 (HVP2ap) replicated in IFNAR(-/-) primary mouse dermal fibroblast (PMDF) cultures as well as neurovirulent (HVP2nv) isolates. IFNAR(-/-) mice were also susceptible to lethal infection by HVP2ap isolates. Unlike Balb/c or parental 129 mice, LD(50) and ID(50) values for HVP2ap were the same in IFNAR(-/-) mice indicating that in these mice infection always progressed to death. HVP2ap replicated in the skin at the site of inoculation and invaded dorsal root ganglia as efficiently as HVP2nv in IFNAR(-/-) mice. Since the virion host shutoff (vhs) protein encoded by the UL41 gene of herpes simplex virus has been implicated in circumventing the host IFN-beta response and the phenotype of UL41 deletion mutants of HSV is very similar to that of HVP2ap isolates, the UL41 gene was deleted from HVP2nv (Delta 41) and replaced with the UL41 ORF from HVP2ap (Delta 41C). Like the parental HVP2nv virus, the Delta 41C recombinant replicated efficiently in Balb/c PMDFs and did not induce a strong IFN-beta response. The neuropathogenicity of the Delta 41C recombinant was also the same as the parental HVP2nv virus in Balb/c mice, indicating that the vhs protein does not underlie the different neuropathogenic phenotype of HVP2ap and HVP2nv. In contrast, the Delta 41 deletion virus induced a strong IFN-beta response but was still able to undergo multiple rounds of replication in PMDF cultures, albeit at a slower pace than the parental HVP2nv. This was reflected in vivo as the Delta 41 mutant had an LD(50) equivalent to that of the parental HVP2nv virus although the time to death was longer. These results indicate that while the vhs protein is involved in preventing and/or suppressing an IFN-beta response, it is not responsible for the ability of HVP2nv to overcome IFN-beta induced resistance of uninfected cells and does not underlie the divergent pathogenicity of the two HVP2 subtypes in mice.

A 2-year-old Hereford cow was presented to the teaching hospital for increased respiratory noise and bilateral bloody nasal discharge. A nodular rhinitis was detected clinically, and the nasal biopsy revealed a granulomatous and eosinophilic rhinitis with intralesional fungi. The cow was euthanized due to financial constraints. Grossly multifocal-to-coalescing, raised, ulcerated firm nodules were present in both nares. Histologically, the lamina propria was expanded by intense infiltrates of eosinophils, epithelioid macrophages, multinucleate giant cells, lymphocytes, and plasma cells. Associated with the inflammatory cells were variably sized, septate hyphae, 5–8 μm in diameter, admixed with numerous, terminal conidia, 6–30 μm in diameter, with a discrete outer wall identified by culture as Pseudallescheria boydii species complex (anamorph Scedosporium apiospermum). The infection was localized to the nasal cavity with no gross or microscopic evidence of a systemic dissemination.

This study explores percutaneous single-fiber spectroscopy (SfS) of rat livers undergoing fatty infiltration. Eight test rats were fed a methionine-choline-deficient (MCD) diet, and four control rats were fed a normal diet. Two test rats and one control rat were euthanized on days 12, 28, 49, and 77 following initiation of the diet, after percutaneous SfS of the liver under transabdominal ultrasound guidance. Histology of each set of the two euthanized test rats showed mild and mild hepatic lipid accumulations on day 12, moderate and severe on day 28, severe and mild on day 49, and moderate and mild on day 77. Livers with moderate or higher lipid accumulation generally presented higher spectral reflectance intensity when compared to lean livers. Livers of the eight test rats on day 12, two of which had mild lipid accumulation, revealed an average scattering power of 0.37±0.14 in comparison to 0.07±0.14 for the four control rats (p

Cercopithecine herpesvirus 1 (monkey B virus; BV) produces extremely severe and usually fatal infections when transmitted from macaque monkeys to humans. Cercopithecine herpesvirus 16 (herpesvirus papio 2; HVP2) is very closely related to BV, yet cases of human HVP2 infection are unknown. However, following intramuscular inoculation of mice, HVP2 rapidly invades the peripheral nervous system and ascends the central nervous system (CNS) resulting in death, very much like human BV infections. In this study, the neurovirulence of HVP2 in mice was further evaluated as a potential model system for human BV infections. HVP2 was consistently neurovirulent when administered by epidermal scarification, intracranial inoculation and an eye splash. Quantitative real-time PCR, histopathology and immunohistochemistry were used to follow the temporal spread of virus following skin scarification and to compare the pathogenesis of neurovirulent and apathogenic isolates of HVP2. Apathogenic isolates were found to be capable of reaching the CNS but were extremely inefficient at replicating within the CNS. It is concluded that neurovirulent strains of HVP2 exhibit a pathogenesis in mice that parallels that observed in human BV infections and that this model system may prove useful in dissecting the viral determinants underlying the extreme severity of zoonotic BV infections.

1,3,5-Trinitrobenzene (TNB) is a munitions chemical that causes gliovascular lesions in the brain stem of rats similar to those produced by thiamine deficiency and nitroaromatic compounds, including m-dinitrobenzene. To identify neuropathic indices of toxicity, the effects of varying concentrations (0 to 2 mM) of TNB on cytotoxicity and cellular metabolic activity were examined using cultured astrocytes from Fischer-344 rats. The cytotoxicity was assessed by lactate dehydrogenase (LDH) leakage into the culture medium. Astrocyte metabolic activity was assessed by measuring the conversion of a tetrazolium salt to a formazan product. Additionally, the effects of oxidative stress on cellular metabolic activity - were determined by varying oxygen tension via alteration of culture media depth. In vitro, the toxic concentration 50% (TC50) of TNB, which induced cell death, was 16 μM following a 24-h exposure. The concentration of TNB that reduced cellular metabolic activity by 50% was 29 μM following a 24-h exposure. Varying the depth of the culture media did not influence the cellular metabolic activity in control or TNB-treated astrocytes. These results support the hypothesis that TNB induced neurotoxicity could partially be mediated via injury to astrocytes, a major component of the blood-brain barrier.

Objective— To confirm in vivo optimal laser energy settings previously determined in vitro for lithotripsy, to develop a technique for Ho:YAG laser lithotripsy, and to evaluate acute and chronic tissue effects of laser lithotripsy. Study design— Prospective, randomized preclinical trial. Animals or Sample Population— Nineteen intact, adult male dogs. Methods— Via cystotomy, a single urolith was inserted into the urethra to the level of the base of the os penis to simulate obstruction. Uroliths (calcium oxalate, urate, or magnesium ammonium phosphate) were fragmented by Ho:YAG laser, in contact mode through a 320 μ optic fiber, passed through the operating channel of a 2.8 mm flexible endoscope. The time and total energy to fragmentation were recorded. Dogs were euthanatized immediately after lithotripsy (3 dogs) or at 3 days (7 dogs), and urethral lesions and any stone remnants were evaluated. Urethral integrity was also evaluated in 9 other dogs by endoscopy on day 10; these were also monitored clinically for 30 days. Results— The mean time for adequate fragmentation was 166.7 seconds (range, 47–494.5 seconds). The mean±SD energy used was 1418±851.2 J. In part 1, 2 dogs were obstructed with urolith fragments at necropsy. Eight dogs had minimal (

In comparisons of the pathogenicity of simian alphaherpesviruses in mice, two isolates of the baboon virus HVP2 were nearly as lethal as monkey B virus, a biological safety level 4 agent (J. W. Ritchey, K. A. Ealey, M. Payton, and R. Eberle, J. Comp. Pathol. 127: 150-161, 2002). To confirm these results, mice were inoculated intramuscularly with 10 5 PFU of HVP2 isolates obtained from different baboon subspecies and primate centers. Some of the HVP2 isolates (6 of 13) caused paralysis and death in the mice, while 7 of 13 HVP2 isolates produced no clinical signs of disease. The apathogenic HVP2 isolates (HVP2 ap ) induced only low levels of serum antiviral immunoglobulin G relative to levels observed in sera from mice infected with the neurovirulent isolates of HVP2 (HVP2 nv ). Histological examination of tissues from mice inoculated with HVP2 nv isolates showed extensive neural tissue destruction, while mice infected with HVP2 ap isolates showed no lesions. Tissue samples collected at 48-h intervals postinfection suggested that HVP2 ap isolates failed to replicate at the site of inoculation. There was no significant difference in the in vitro replication, plaque size, or cytopathic effect morphology of HVP2 ap versus HVP2 nv isolates. While HVP2 isolates replicated better in Vero monkey kidney cells than in murine L cells, plaquing efficiency of individual isolates did not correlate with the dichotomous pathogenic properties seen in mice. Phylogenetic analyses of both coding and intergenic regions (US4-6) of the HVP2 genome separated isolates into two distinct clades that correlated with the two in vivo virulence phenotypes. Taken together, these results demonstrate that two subtypes of HVP2 exist that are very closely related but differ dramatically in their ability to cause disease in a murine model.