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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.

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 vivohas greater relevance for in vitrostudies. 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 StatementMast cells (MCs) are key effector and immunoregulatory cells in immune disorders; however, their role is not fully understood. Few studies have investigated human ex vivoMCs 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.

Influenza A virus (IAV) claims ∼250,000–500,000 lives annually worldwide. Currently, there are a few in vitromodels 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 vivolower 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.

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 vivotreatment 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.

Bovine coronavirus (BoCV; Betacoronavirus 1) infections are associated with varied clinical presentations including neonatal diarrhea, winter dysentery in dairy cattle, and respiratory disease in various ages of cattle. The current report presents information on BoCV infections associated with enteric disease of postweaned beef cattle in Oklahoma. In 3 separate accessions from a single herd, 1 in 2012 and 2 in 2013, calves were observed with bloody diarrhea. One calf in 2012 died and was necropsied, and 2 calves from this herd died in 2013 and were necropsied. A third calf from another herd died and was necropsied. The gross and histologic diagnosis was acute, hemorrhagic colitis in all 4 cattle. Colonic tissues from all 4 animals were positive by fluorescent antibody testing and/or immunohistochemical staining for BoCV antigen. Bovine coronavirus was isolated in human rectal tumor cells from swabs of colon surfaces of all animals. The genomic information from a region of the S envelope region revealed BoCV clade 2. Detection of BoCV clade 2 in beef cattle in Oklahoma is consistent with recovery of BoCV clade 2 from the respiratory tract of postweaned beef calves that had respiratory disease signs or were healthy. Further investigations on the ecology of BoCV in cattle are important, as BoCV may be an emerging disease beyond the initial descriptions. Challenge studies are needed to determine pathogenicity of these strains, and to determine if current BoCV vaccines are efficacious against the BoCV clade 2 strains.

We demonstrate the feasibility of diffuse optical tomography (DOT) of the proximal pancreas by using optical applicator channels deployed longitudinally along the exterior surface of a duodenoscope. As the duodenum that nearly encircles the proximal pancreas forms a natural “C-loop” that is approximately three-quarters of a circle of 5–6 cm in diameter, a multichannel optical applicator attached to a duodenoscope has the potential to perform transduodenal DOT sampling of the bulk proximal pancreas wherein most cancers and many cystic lesions occur. The feasibility of transduodenal DOT is demonstrated on normal porcine pancreas tissues containing an introduced gelatinous inclusion of approximately 3 cm in diameter, by using nine source channels and six detector channels attached to a duodenoscope. Concurrent ultrasonography of the gelatinous inclusion in the porcine pancreas parenchyma provided a coarse, albeit indispensable, anatomic prior to transduodenal DOT in reconstructing a contrast of optical properties in the pancreas.

A 12-year-old intact male Miniature Schnauzer dog with chronic diarrhea that was unresponsive to empirical treatment was presented to a referring veterinarian. A laparotomy was performed, and formalin-fixed biopsies of duodenum, jejunum, and colon were sent to Oklahoma Animal Disease Diagnostic Laboratory for evaluation. Histologic examination revealed a severe, diffuse, granulomatous enteritis and colitis with intralesional yeast and hyphal forms. Grocott methenamine silver stains revealed short, aseptate hyphae co-mingled with 2–8 µm, oval to round yeast organisms consistent with Histoplasma capsulatum. The atypical presentation of both yeast and hyphal forms prompted identification of the organism. Direct sequencing of a polymerase chain reaction product from paraffin-embedded intestinal samples confirmed the presence of Ajellomyces capsulatuswith a homology over 99% to several sequences in GenBank. Ajellomyces capsulatusis the holomorphic name for H. capsulatum. Therefore, the mycelial form of a dimorphic fungus such as H. capsulatumcan coexist with yeast cells within lesions of histoplasmosis. Following diagnosis, the dog was treated with itraconazole for 6 months and has improved.

Extracellular ATP and adenosine have immunoregulatory roles during inflammation. Elevated extracellular ATP is known to exacerbate GVHD, and the pharmacologic activation of the adenosine A2A receptor is protective. However, the role of endogenous adenosine is unknown. We used gene-targeted mice and a pharmacologic inhibitor to test the role of adenosine generated by CD73/ecto-5′-nucleotidase in GVHD. In allogeneic transplants, both donor and recipient CD73 were protective, with recipient CD73 playing the dominant role. CD73 deficiency led to enhanced T-cell expansion and IFN-γ and IL-6 production, and the migratory capacity of Cd73−/− T cells in vitro was increased. However, the number of regulatory T cells and expression of costimulatory molecules on antigen-presenting cells were unchanged. A2A receptor deficiency led to increased numbers of allogeneic T cells, suggesting that signaling through the A2A receptor via CD73-generated adenosine is a significant part of the mechanism by which CD73 limits the severity of GVHD. Pharmacologic blockade of CD73 also enhanced graft-versus-tumor activity. These data have clinical implications, as both the severity of GVHD and the strength of an alloimmune antitumor response could be manipulated by enhancing or blocking CD73 activity or adenosine receptor signaling depending on the clinical indication.

Extracellular ATP and adenosine have immunoregulatory roles during inflammation. Elevated extracellular ATP is known to exacerbate GVHD, and the pharmacologic activation of the adenosine A2Areceptor is protective. However, the role of endogenous adenosine is unknown. We used gene-targeted mice and a pharmacologic inhibitor to test the role of adenosine generated by CD73/ecto-5′-nucleotidase in GVHD. In allogeneic transplants, both donor and recipient CD73 were protective, with recipient CD73 playing the dominant role. CD73 deficiency led to enhanced T-cell expansion and IFN-γ and IL-6 production, and the migratory capacity of Cd73−/−T cells in vitro was increased. However, the number of regulatory T cells and expression of costimulatory molecules on antigen-presenting cells were unchanged. A2Areceptor deficiency led to increased numbers of allogeneic T cells, suggesting that signaling through the A2Areceptor via CD73-generated adenosine is a significant part of the mechanism by which CD73 limits the severity of GVHD. Pharmacologic blockade of CD73 also enhanced graft-versus-tumor activity. These data have clinical implications, as both the severity of GVHD and the strength of an alloimmune antitumor response could be manipulated by enhancing or blocking CD73 activity or adenosine receptor signaling depending on the clinical indication.

Objective:To evaluate the effects of low level laser therapy (LLLT) on healing of full thickness symmetrical skin wounds in horses. LLLT is a therapeutic modality using the application of light, usually a low power laser or light emitting diode in the power range of 1 mW to 12 W that, in practical terms, promotes tissue regeneration as well as reducing inflammation and pain.

A 7-year-old, intact male standard Poodle dog with hypothyroidism and atypical hyperadrenocorticism developed acute signs of lethargy, weakness, inappetence, vomiting, and diarrhea. Clinical signs progressed to hind limb proprioceptive deficits, aggressive behavior with obtundation, and an equivocal seizure. Necropsy revealed a mass in the sellar region that histologically consisted of multinucleated giant cells, macrophages, and hemosiderin-laden macrophages with fewer lymphocytes and plasma cells admixed with large regions of cholesterol cleft deposition, fibrin, and prominent Rosenthal fibers. Pituitary tissue was not identified on gross or histologic examination, but the mass was partially bordered by epithelial cells. The histologic characteristics are similar to changes described in the human medical literature as xanthogranuloma of the sellar region and xanthogranulomatous hypophysitis.

Needle-based core-biopsy to locate prostate cancer relies heavily upon trans-rectal ultrasound (TRUS) imaging guidance. Ultrasonographic findings of classic hypoechoic peripheral zone lesions have a low specificity of ~28%, a low positive predictive value of ~29%, and an overall accuracy of ~43%, in prostate cancer diagnosis. The prevalence of isoechoic or nearly invisible prostate cancers on ultrasonography ranges from 25 to 42%. As a result, TRUS is useful and convenient to direct the needle trajectory following a systematic biopsy sampling template rather than to target only the potentially malignant lesion for focal-biopsy. To address this deficiency in the first-line of prostate cancer imaging, a trans-rectal ultrasound-coupled spectral tomography (TRUST) approach is being developed to non-invasively resolve the likely optical signatures of prostate malignancy. The approach has evolved from using one NIR wavelength to two NIR bands, and recently to three bands of NIR spectrum information. The concept has been evaluated on one normal canine prostate and three dogs with implanted prostate tumor developed as a model. The initial results implementing TRUST on the canine prostate tumor model includes: (1) quantifying substantially increased total hemoglobin concentration over the time-course of imaging in a rapidly growing prostate tumor; (2) confirming hypoxia in a prostatic cystic lesion; and (3) imaging hypoxic changes of a necrotic prostate tumor. Despite these interesting results, intensive technologic development is necessary for translating the approach to benefiting clinical practice, wherein the ultimate utility is not possibly to eliminate needle-biopsy but to perform focal-biopsy that is only necessary to confirm the cancer, as well as to monitor and predict treatment responses.

Different optical spectral characteristics were observed in a necrotic transmissible venereal tumor (TVT) and a cystic lesion in the same canine prostate by triple-wavelength trans-rectal optical tomography under trans-rectal ultrasound (TRUS) guidance. The NIR imager acquiring at 705nm, 785nm and 808nm was used to quantify both the total hemoglobin concentration (HbT) and oxygen saturation (StO2) in the prostate. The TVT tumor in the canine prostate as a model of prostate cancer was induced in a 7-year old, 27 kg dog. A 2 mL suspension of 2.5x106cells/mL of homogenized TVT cells recovered from an in vivo subcutaneously propagated TVT tumor in an NOD/SCID mouse were injected in the cranial aspect of the right lobe of the canine prostate. The left lobe of the prostate had a cystic lesion present before TVT inoculation. After the TVT homogenate injection, the prostate was monitored weekly over a 9-week period, using trans-rectal NIR and TRUS in grey-scale and Doppler. A TVT mass within the right lobe developed a necrotic center during the later stages of this study, as the mass presented with substantially increased [HbT] in the periphery, with an area of reduced StO2 less than the area of the mass itself shown on ultrasonography. Conversely, the cystic lesion presented with slightly increased [HbT] in the periphery of the lesion shown on ultrasound with oxygen-reduction inside and in the periphery of the lesion. There was no detectable change of blood flow on Doppler US in the periphery of the cystic lesion. The slightly increased [HbT] in the periphery of the cystic lesion was correlated with intra-lesional hemorrhage upon histopathologic examination.

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 prioremployed 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.

Conceptus-uterine communication is established during trophoblastic elongation when the conceptus synthesizes and releases estrogen, the maternal recognition signal in the pig. Interleukin-1β (IL-1β) is a differentially expressed gene during rapid trophoblastic elongation in the pig. The current investigation determined conceptus and endometrial changes in gene expression for IL-1β, IL-1 receptor antagonist (IL-1Rant), IL-1 receptor type 1 (IL-1RT1), and IL-1 receptor accessory protein (IL-1RAP) in developing peri- and postimplantation conceptuses as well as uterine endometrium collected from cyclic and pregnant gilts. Conceptus IL-1β gene expression was enhanced during the period of rapid trophoblastic elongation compared with earlier spherical conceptuses, followed by a dramatic decrease in elongated Day 15 conceptuses. IL-1RT1 and IL-1RAP gene expression was greater in Day 12 and 15 filamentous conceptuses compared with earlier morphologies while IL-1Rant gene expression was unchanged by conceptus development. The uterine lumenal content of IL-1β increased during the process of trophoblastic elongation on Day 12. Uterine IL-1β content declined on Day 15, reaching a nadir by Day 18 of pregnancy. IL-1β gene expression in porcine conceptuses was temporally associated with an increase in endometrial IL-1RT1 and IL-1RAP gene expression in pregnant gilts. Endometrial IL-1β and IL-1Rant gene expression were lowest during Days 10–15 of the estrous cycle and pregnancy. The temporal expression of IL-1β during conceptus development and the initiation of conceptus-uterine communication suggests conceptus IL-1β synthesis plays an important role in porcine conceptus elongation and the establishment of pregnancy in the pig.

Hairy vetch poisoning (vetch-associated disease) of cattle is a generalized disease characterized pathologically by infiltration of skin and many internal organs by monocytes, lymphocytes, plasma cells, and often eosinophils and multinucleated giant cells and clinically by dermatitis, pruritis, often diarrhea, wasting, and high mortality. The disease was experimentally reproduced in an adult Angus female that had recovered from the natural disease 1 year earlier. She developed dermatitis on the 11th day of vetch feeding, and despite withdrawal from the vetch diet on the 12th day, death occurred 24 days after first day of vetch feeding. The cow developed lymphocytosis and hyperproteinemia. The results of other hematologic evaluations, blood chemical profiles, urinalysis, and cutaneous hypersensitivity tests using vetch lectin were normal. Lymphocyte blastogenesis studies with vetch lectin were not interpretable. Necropsy revealed gross lesions characteristic of the disease in the skin, heart, kidney, adrenal, and lymphoid tissues. Microscopically there was typical cellular infiltration in those organs and in the thyroid, liver, pancreas, salivary and mammary glands, urinary bladder, corpus luteum, and cerebral meninges. Cutaneous apocrine gland necrosis was present. The inflammatory reaction has qualities of a type-IV hypersensitivity reaction. Hypersensitivity may occur when constituents of the ingested plant are absorbed and act as antigens that sensitize lymphocytes and evoke the multisystemic granulomatous inflammatory response that characterizes the disease. Alternatively, vetch lectin may directly activate T lymphocytes to initiate the cellular response. Vetch-like diseases have been associated with a variety of diets that did not contain hairy vetch. The gross and histopathologic lesions of the vetch-associated and the vetch-like diseases are not mimicked in quality and distribution by any other disease.

Renal papillary necrosis was diagnosed during postmortem examination of a juvenile white-tailed deer (Odocoileus virginianus) from Oklahoma. The deer was surgically treated for a Salter Harris type II fracture of the proximal tibia of the left hind limb. The animal was administered multiple nonsteroidal anti-inflammatory drugs (NSAIDs), including meloxicam, flunixin meglumine, and ketoprofen for pain management. At postmortem examination, gross lesions included a proximal tibial Salter Harris type II fracture with an associated fibrinonecrotizing myositis and bilateral renal papillary necrosis. Histologically, the kidneys exhibited coagulation necrosis of the renal medulla and renal papilla, thrombosis of renal blood vessels, and interstitial medullary edema. The gross and microscopic lesion coupled with the clinical history of multiple NSAID administration suggests NSAID-induced renal papillary necrosis.

Similar to human immunodeficiency virus, feline immunodeficiency virus (FIV) induces immunodeficiency and enhanced susceptibility to secondary pathogens. To explore cytokine alterations in lentivirus immunodeficiency, constitutive mRNA expression was measured in lymph nodes of healthy and FIV-infected cats before and after challenge with Toxoplasma gondii. Cytokine mRNA expression was similar in control and FIV-infected cats during the first 10 weeks after infection. At 16 weeks, interferon (IFN)-γ, tumor necrosis factor-α, and interleukin (IL)-10 mRNA were increased in FIV-infected cats. Challenge with T. gondii induced an increase in IL-2, IFN-γ, and IL-12 in the lymph nodes of control cats, whereas IFN-γ and IL-10 but not IL-2 or IL-12 increased in the lymph nodes of FIV-T. gondii coinfected cats. These results indicate that FIV immunodeficiency may derive from a failure to generate an IL-12-dependent type 1 response and that an elevated level of IL-10 mRNA expression is a predictor of lentivirus immunodeficiency.