Plasmid Acquisition Alters Vancomycin Susceptibility in Clostridioides difficile.

The increasing incidence of primary and recurring Clostridioides difficile infections (CDI), which evade current treatment strategies, reflects the changing biology of C difficile. Here, we describe a putative plasmid-mediated mechanism potentially driving decreased sensitivity of C difficile to vancomycin treatment. We identified a broad host range transferable plasmid in a C difficile strain associated with lack of adequate response to vancomycin treatment. The transfer of this plasmid to a vancomycin-susceptible C difficile isolate decreased its susceptibility to vancomycin in vitro and resulted in more severe disease in a humanized mouse model. Our findings suggest plasmid acquisition in the gastrointestinal tract to be a possible mechanism underlying vancomycin treatment failure in patients with CDI, but further work is needed to characterize the mechanism by which plasmid genes determine vancomycin susceptibility in C difficile.

Viral Clearance and Neuroinflammation in Acute TMEV Infection Vary by Host Genetic Background.

A wide range of viruses cause neurological manifestations in their hosts. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the brain, depending in part on host genetic background. The interaction between host genetic background, neurological response to viral infection, and subsequent clinical manifestations remains poorly understood. In the present study, we used the genetically diverse Collaborative Cross (CC) mouse resource to better understand how differences in genetic background drive clinical signs and neuropathological manifestations of acute Theiler's murine encephalomyelitis virus (TMEV) infection. For the first time, we characterized variations of TMEV viral tropism and load based on host genetic background, and correlated viral load with microglial/macrophage activation. For five CC strains (CC002, CC023, CC027, CC057, and CC078) infected with TMEV, we compared clinical signs, lesion distribution, microglial/macrophage response, expression, and distribution of TMEV mRNA, and identified genetic loci relevant to the early acute (4 days post-infection [dpi]) and late acute (14 dpi) timepoints. We examined brain pathology to determine possible causes of strain-specific differences in clinical signs, and found that fields CA1 and CA2 of the hippocampal formation were especially targeted by TMEV across all strains. Using Iba-1 immunolabeling, we identified and characterized strain- and timepoint-specific variation in microglial/macrophage reactivity in the hippocampal formation. Because viral clearance can influence disease outcome, we used RNA in situ hybridization to quantify viral load and TMEV mRNA distribution at both timepoints. TMEV mRNA expression was broadly distributed in the hippocampal formation at 4 dpi in all strains but varied between radiating and clustered distribution depending on the CC strain. We found a positive correlation between microglial/macrophage reactivity and TMEV mRNA expression at 4 dpi. At 14 dpi, we observed a dramatic reduction in TMEV mRNA expression, and localization to the medial portion of field CA1 and field CA2. To better understand how host genetic background can influence pathological outcomes, we identified quantitative trait loci associated with frequency of lesions in a particular brain region and with microglial/macrophage reactivity. These QTL were located near several loci of interest: lysosomal trafficking regulator (Lyst) and nidogen 1 (Nid1), and transmembrane protein 106 B (Tmem106b). Together, these results provide a novel understanding about the influences of genetic variation on the acute neuropathological and immunopathological environment and viral load, which collectively lead to variable disease outcomes. Our findings reveal possible avenues for future investigation which may lead to more effective intervention strategies and treatment regimens.

Implementing Digital Pathology into Veterinary Academics and Research.

The advantages of digital pathology (DP) have been recognized as early as 1963, but only within the last decade or so have the advancements of slide scanners and viewing software made the use and implementation of DP feasible in the classroom and in research. Several factors must be considered prior to undertaking the project of implementing the DP workflow in any setting, but particularly in an academic environment. Sustained and open dialogue with information technology (IT) is critical to the success of this enterprise. In addition to IT, there is a multitude of criteria to consider when determining the best hardware and software to purchase to support the project. The goals and limitations of the laboratory and the requirements of its users (students, instructors, and researchers) will ultimately direct these decisions. The objectives of this article are to provide an overview of the opportunities and challenges associated with the integration of DP in education and research, to highlight some important IT considerations, and to discuss some of the requirements and functionalities of some hardware and software options.

Computed tomographic features of confirmed gallbladder pathology in 34 dogs.

Gallbladder pathology is common in dogs, but published studies describing the computed tomographic (CT) appearance of many gallbladder pathologies are currently lacking. This retrospective, multicenter, cases series, descriptive study evaluated the CT features of confirmed gallbladder pathology in 34 dogs. In this subset of dogs, the most common pathologies included cystic mucosal hyperplasia (15/34, 44.1%), gallbladder wall edema (9/34, 26.5%), gallbladder mucocele (8/34, 23.5%), bactibilia (7/34, 20.6%), cholecystitis (6/34, 17.6%), white bile (6/34, 17.6%), and cholelithiasis (4/34, 11.8%). The presence of intraluminal nodules, gallbladder wall thickening, hyperattenuating material (35-100 HU), and mineral attenuating material (>100 HU) were the most common abnormalities detected. However, overlap of each of these findings with a variety of gallbladder pathologies showed that none of the findings were pathognomonic for any of these pathologies. The presence of any of these CT abnormalities should increase the suspicion of gallbladder pathology and prompt further evaluation of the gallbladder for definitive diagnosis.

Concurrent bullous emphysema, bronchointerstitial pneumonia with necrosis, and tension pneumothorax in an 8-week-old puppy.

An 8-week-old, male, mixed breed puppy was presented because of progressive respiratory distress. Thoracic radiographs revealed innumerable coalescing pulmonary bullae throughout the lungs with increased soft tissue opacity of the remaining pulmonary parenchyma and tension pneumothorax. Necropsy and histopathological findings were compatible with bullous emphysema and marked neutrophilic to histiocytic bronchointerstitial pneumonia with necrosis, bronchiectasis, fibrosis, dystrophic mineralization, and vasculitis with thrombosis. Tests for agents of infectious disease did not reveal an underlying etiological agent. This case highlights the radiographic, gross necropsy, and histopathologic features of a puppy with bullous emphysema and severe bronchointerstitial pneumonia with necrosis.

Un chiot mâle croisé âgé de 8 semaines fut présenté à cause de détresse respiratoire progressive. Des radiographies thoraciques ont révélé un nombre incalculable de bulles pulmonaires coalescentes à travers les poumons avec augmentation de l'opacité des tissus mous du parenchyme pulmonaire restant et un pneumothorax de tension. La nécropsie et les trouvailles histopathologiques étaient compatibles avec de l'emphysème bulleux et une pneumonie broncho-interstitielle neutrophilique et histiocytaire marquée avec nécrose, de la bronchiectasie, de la fibrose, une minéralisation dystrophique et une vasculite avec thrombose. Des tests pour agents infectieux de maladie n'ont pas permis de mettre en évidence un agent étiologique sous-jacent. Ce cas met en évidence les trouvailles radiographiques, de nécropsie et histopathologiques chez un chiot avec emphysème bulleux et pneumonie broncho-interstitielle avec nécrose.(Traduit par Dr Serge Messier).

Induction of acute graft vs. host disease in lymphopenic mice.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially life-saving treatment for refractory/relapsing hematological malignancies, blood disorders or autoimmune diseases. However, approximately 40-50% of patients undergoing allogeneic HSCT will develop a multi-organ, inflammatory disorder called acute graft vs. host disease (aGVHD). Experimental and clinical studies suggest that intestinal injury due to toxic, pre-transplant conditioning protocols (e.g. lethal irradiation and/or chemotherapy) may play a major role in the development of aGVHD. However, recent studies from our laboratory suggest that this may not be the case. The objective of this study was to quantify and compare the onset and severity of aGVHD induced by the adoptive transfer of allogeneic T cells into untreated lymphopenic mice. Four million allogeneic or syngeneic CD4+CD62L+CD25- T cells were transferred (i.p.) into NK cell-depleted RAG1-/- mice or RAG2-/-IL2rγ-/-double knock-out (DKO) mice and assessed daily for signs of aGVHD. We found that adoptive transfer of allogeneic but not syngeneic T cells into NK cell-depleted RAG1-/- or DKO mice induced many of the clinical and histological features of aGVHD including weight loss, inflammatory cytokine production and tissue inflammation. In addition, adoptive transfer of allogeneic T cells into each recipient induced severe anemia as well as dramatic reductions in bone marrow and spleen cellularity. Taken together, we conclude that allogeneic CD4+ T cells are both necessary and sufficient to induce aGVHD in lymphopenic recipients in the absence of toxic, pre-transplant conditioning.

Age, sex, and TNF associated differences in the gut microbiota of mice and their impact on acute TNBS colitis.

Mouse models are often used to determine the interactions between the microbiota and inflammatory processes and overcome the confounding effect of the naturally high inter-individual variation of the gut microbiota in humans. However, the microbiomes of mice are also variable and data detailing the degree to which factors like mouse sex and age contribute to mouse gut microbiota variation is limited. Our objective was to determine the impact sex and age have on the mouse gut microbiota and the severity of acute 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) induced colitis. We used Illumina sequencing of 16S rRNA genes to characterize the fecal microbiota of B6.129S wild-type (WT) mice and mice lacking tumor necrosis factor (Tnf-/-) before and after acute TNBS colitis. There were differences between the fecal microbiota of male and female WT mice as well as Tnf-/- mice, both pre-and post-colitis. Male WT mice had more severe colitis than female WT mice and Tnf-/- mice of both sexes. We also identified microbial taxa differences between 4-5 and 6-7-week old WT and Tnf-/- mice both pre-and post-colitis. Here we provide evidence that the mouse fecal microbiome is shaped, in part, by sex, age and TNF production and that these effects correlate with the degree of animals' colitis.

Immunopathological characterization of selected mouse models of inflammatory bowel disease: Comparison to human disease.

Inflammatory bowel diseases (IBD) are chronic, relapsing conditions of multifactorial etiology. The two primary diseases of IBD are Crohn's disease (CD) and ulcerative colitis (UC). Both entities are hypothesized to occur in genetically susceptible individuals due to microbial alterations and environmental contributions. The exact etiopathogenesis, however, is not known for either disease. A variety of mouse models of CD and UC have been developed to investigate the pathogenesis of these diseases and evaluate treatment modalities. Broadly speaking, the mouse models can be divided into 4 categories: genetically engineered, immune manipulated, spontaneous and erosive/chemically induced. No one mouse model completely recapitulates the immunopathology of CD or UC, however each model possesses particular similarities to human IBD and offers advantageous for specific details of IBD pathogenesis. Here we discuss the more commonly used models in each category and critically evaluate how the immunopathology induced compares to CD or UC, as well as the advantages and disadvantages associated with each model.

Disruption of Circadian Clock Induces Abnormal Mammary Morphology and Aggressive Basal Tumorigenesis by Enhancing LILRB4 Signaling.

Epidemiological studies have shown that circadian rhythm disruption (CRD) is associated with the risk of breast cancer. However, the role of CRD in mammary gland morphology and aggressive basal mammary tumorigenesis and the molecular mechanisms underlying CRD and cancer risk remain unknown. To investigate the effect of CRD on aggressive tumorigenesis, a genetically engineered mouse model that recapitulates the human basal type of breast cancer was used for this study. The effect of CRD on mammary gland morphology was investigated using wild-type mice model. The impact of CRD on the tumor microenvironment was investigated using the tumors from LD12:12 and CRD mice via scRNA seq. ScRNA seq was substantiated by multiplexing immunostaining, flow cytometry, and realtime PCR. The effect of LILRB4 immunotherapy on CRD-induced tumorigenesis was also investigated. Here we identified the impact of CRD on basal tumorigenesis and mammary gland morphology and identified the role of LILRB4 on CRD-induced lung metastasis. We found that chronic CRD disrupted mouse mammary gland morphology and increased tumor burden, and lung metastasis and induced an immunosuppressive tumor microenvironment by enhancing LILRB4a expression. Moreover, CRD increased the M2-macrophage and regulatory T-cell populations but decreased the M1-macrophage populations. Furthermore, targeted immunotherapy against LILRB4 reduced CRD-induced immunosuppressive microenvironment and lung metastasis. These findings identify and implicate LILRB4a as a link between CRD and aggressive mammary tumorigenesis. This study also establishes the potential role of the targeted LILRB4a immunotherapy as an inhibitor of CRD-induced lung metastasis.

Characterization of synergistic anti-cancer effects of docosahexaenoic acid and curcumin on DMBA-induced mammary tumorigenesis in mice.

BACKGROUND: The major obstacles to the successful use of individual nutritional compounds as preventive or therapeutic agents are their efficacy and bioavailability. One approach to overcoming this problem is to use combinations of nutrients to induce synergistic effects. The objective of this research was to investigate the synergistic effects of two dietary components: docosahexaenoic acid (DHA), an omega-3 fatty acid present in cold-water fish, and curcumin (CCM), an herbal nutrient present in turmeric, in an in vivo model of DMBA-induced mammary tumorigenesis in mice. METHODS: We used the carcinogen DMBA to induce breast tumors in SENCAR mice on control, CCM, DHA, or DHA + CCM diets. Appearance and tumor progression were monitored daily. The tumors were harvested 15 days following their first appearance for morphological and immunohistological analysis. Western analysis was performed to determine expression of maspin and survivin in the tumor tissues. Characterization of tumor growth was analyzed using appropriate statistical methods. Otherwise all other results are reported as mean ± SD and analyzed with one-way ANOVA and Tukey's post hoc procedure. RESULTS: Analysis of gene microarray data indicates that combined treatment with DHA + CCM altered the profile of "PAM50" genes in the SK-BR-3 cell line from an ER⁻/Her-2⁺ to that resembling a "normal-like" phenotype. The in vivo studies demonstrated that DHA + CCM treatment reduced the incidence of breast tumors, delayed tumor initiation, and reduced progression of tumor growth. Dietary treatment had no effect on breast size development, but tumors from mice on a control diet (untreated) were less differentiated than tumors from mice fed CCM or DHA + CCM diets. The synergistic effects also led to increased expression of the pro-apoptotic protein, maspin, but reduced expression of the anti-apoptotic protein, survivin. CONCLUSIONS: The SK-BR-3 cells and DMBA-induced tumors, both with an ER⁻ and Her-2⁺ phenotype, were affected by the synergistic interaction of DHA and CCM. This suggests that the specific breast cancer phenotype is an important factor for predicting efficacy of these nutraceuticals. The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon the molecular phenotype of the cancer.

Micro-CT Features of Lung Consolidation, Collagen Deposition and Inflammation in Experimental RSV Infection Are Aggravated in the Absence of Nrf2.

Severe respiratory syncytial virus (RSV) infections in early life have been linked to the development of chronic airway disease. RSV triggers the production of reactive oxygen species (ROS), which contributes to inflammation and enhanced clinical disease. NF-E2-related factor 2 (Nrf2) is an important redox-responsive protein that helps to protect cells and whole organisms from oxidative stress and injury. The role of Nrf2 in the context of viral-mediated chronic lung injury is not known. Herein, we show that RSV experimental infection of adult Nrf2-deficient BALB/c mice (Nrf2-/-; Nrf2 KO) is characterized by enhanced disease, increased inflammatory cell recruitment to the bronchoalveolar compartment and a more robust upregulation of innate and inflammatory genes and proteins, compared to wild-type Nrf2+/+ competent mice (WT). These events that occur at very early time points lead to increased peak RSV replication in Nrf2 KO compared to WT mice (day 5). To evaluate longitudinal changes in the lung architecture, mice were scanned weekly via high-resolution micro-computed tomography (micro-CT) imaging up to 28 days after initial viral inoculation. Based on micro-CT qualitative 2D imaging and quantitative reconstructed histogram-based analysis of lung volume and density, we found that RSV-infected Nrf2 KO mice developed significantly greater and prolonged fibrosis compared to WT mice. The results of this study underscore the critical role of Nrf2-mediated protection from oxidative injury, not only in the acute pathogenesis of RSV infection but also in the long-term consequences of chronic airway injury.

Combining gamma-tocopherol and aspirin synergistically suppresses colitis-associated colon tumorigenesis and modulates the gut microbiota in mice, and inhibits the growth of human colon cancer cells.

Despite being shown to be effective for chemoprevention of colorectal cancer, aspirin has limitations including adverse effects and inability to block colitis-associated colon cancer (CAC). γ-Tocopherol (γT), a vitamin E form, has been reported to mitigate experimental colitis and CAC, prolong the anti-inflammatory activity of aspirin and alleviate aspirin-induced adverse effect. We therefore hypothesize that combining γT and aspirin is better than either compound singly for suppressing CAC. This hypothesis was tested in the murine azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC model and with human HCT116 colon cancer cells. Compared to the control, combining aspirin (250 ppm) and γT (500 ppm) but not either compound alone significantly reduced AOM/DSS-induced tumor area and multiplicity of large-size tumors by 60% and 50%, respectively. Meanwhile, γT mitigated aspirin-promoted inflammation and stomach lesions in mice. Moreover, the combination appeared to cause favorable changes of gut microbiota compared to the control and synergistically suppressed the growth of HCT116 cells. Our study demonstrates that combining aspirin and γT improves anticancer effects and counteracts side effects compared to aspirin and may therefore be a novel combinatory chemopreventive agent against CAC.

Hypoxia-inducible-factors differentially contribute to clinical disease and the control of viral replication during RSV infection.

Hypoxia-inducible-factors (HIF) are transcription factors that regulate cellular adaptation to hypoxic conditions, enabling cells to survive in low-oxygen environments. Viruses have evolved to stabilize this pathway to promote successful viral infection, therefore modulation of HIFs could represent a novel antiviral strategy. In previous in vitro studies, we found that respiratory syncytial virus (RSV), a leading cause of respiratory illness, stabilizes HIFs under normoxic conditions, with inhibition of HIF-1α resulting in reduced viral replication. Despite several HIF modulating compounds being tested/approved for use in other non-infectious models, little is known about their efficacy against respiratory viruses using relevant animal models. This study aimed to characterize the disease modulating properties and antiviral potential of anti-HIF-1α (PX478) and anti-HIF-2α (PT2385) in RSV-infected BALB/c mice. We found that inhibition of HIF-1α worsen clinical disease parameters, while simultaneously improving airway function. Additionally, anti-HIF-1α results in significantly reduced viral titer at early and peak time points of RSV replication, followed by a loss in viral clearance when given every day, but not every-other-day. In contrast, inhibition of HIF-2α was associated with improved clinical parameters, with no changes in airway function, and amelioration of interstitial pneumonia. Furthermore, anti-HIF-2α reduced early and peak lung viral replication, with no impairment of viral clearance. Analysis of lung cells found significant modification in the T cell compartment that correlated with changes in lung pathology and viral titers in response to each HIF inhibitor administration. These data underscore the complex role of HIFs in RSV infection and highlight the need for careful therapeutic consideration.

Influence of Housing Temperature and Genetic Diversity on Allogeneic T Cell-Induced Tissue Damage in Mice.

The objective of this study was to determine how housing temperature and genetic diversity affect the onset and severity of allogeneic T cell-induced tissue damage in mice subjected to reduced intensity conditioning (RIC). We found that adoptive transfer of allogeneic CD4+ T cells from inbred donors into sub-lethally irradiated inbred recipients (I→I) housed at standard housing temperatures (ST; 22-24 °C) induced extensive BM and spleen damage in the absence of injury to any other tissue. Although engraftment of T cells in RIC-treated mice housed at their thermo-neutral temperature (TNT; 30-32 °C) also developed similar BM and spleen damage, their survival was markedly and significantly increased when compared to their ST counterparts. In contrast, the adoptive transfer of allogeneic T cells into RIC-treated outbred CD1 recipients failed to induce disease in any tissue at ST or TNT. The lack of tissue damage was not due to defects in donor T cell trafficking to BM or spleen but was associated with the presence of large numbers of B cells and myeloid cells within these tissues that are known to contain immunosuppressive regulatory B cells and myeloid-derived suppressor cells. These data demonstrate, for the first time, that housing temperature affects the survival of RIC-treated I→I mice and that RIC-conditioned outbred mice are resistant to allogeneic T cell-induced BM and spleen damage.

SARS-CoV-2 Inhibits NRF2-Mediated Antioxidant Responses in Airway Epithelial Cells and in the Lung of a Murine Model of Infection.

Several viruses have been shown to modulate the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of redox homeostasis. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, also seems to disrupt the balance between oxidants and antioxidants, which likely contributes to lung damage. Using in vitro and in vivo models of infection, we investigated how SARS-CoV-2 modulates the transcription factor NRF2 and its dependent genes, as well as the role of NRF2 during SARS-CoV-2 infection. We found that SARS-CoV-2 infection downregulates NRF2 protein levels and NRF2-dependent gene expression in human airway epithelial cells and in lungs of BALB/c mice. Reductions in cellular levels of NRF2 seem to be independent of proteasomal degradation and the interferon/promyelocytic leukemia (IFN/PML) pathway. Furthermore, lack of the Nrf2 gene in SARS-CoV-2-infected mice exacerbates clinical disease, increases lung inflammation, and is associated with a trend toward increased lung viral titers, indicating that NRF2 has a protective role during this viral infection. In summary, our results suggest that SARS-CoV-2 infection alters the cellular redox balance by downregulating NRF2 and its dependent genes, which exacerbates lung inflammation and disease, therefore, suggesting that the activation of NRF2 could be explored as therapeutic approach during SARS-CoV-2 infection. IMPORTANCE The antioxidant defense system plays a major function in protecting the organism against oxidative damage caused by free radicals. COVID-19 patients often present with biochemical characteristics of uncontrolled pro-oxidative responses in the respiratory tract. We show herein that SARS-CoV-2 variants, including Omicron, are potent inhibitors of cellular and lung nuclear factor erythroid 2-related factor 2 (NRF2), the master transcription factor that controls the expression of antioxidant and cytoprotective enzymes. Moreover, we show that mice lacking the Nrf2 gene show increased clinical signs of disease and lung pathology when infected with a mouse-adapted strain of SARS-CoV-2. Overall, this study provides a mechanistic explanation for the observed unbalanced pro-oxidative response in SARS-CoV-2 infections and suggests that therapeutic strategies for COVID-19 may consider the use of pharmacologic agents that are known to boost the expression levels of cellular NRF2.

Ixodes scapularis nymph saliva protein blocks host inflammation and complement-mediated killing of Lyme disease agent, Borrelia burgdorferi.

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent.

Supplementation of polyphenol-rich grapes attenuates colitis, colitis-associated colon cancer, and disease-associated dysbiosis in mice, but fails to mitigate colitis in antibiotic-treated mice.

Polyphenols are known to interact with gut microbes that play key roles in maintaining gut health, but the role of gut microbiota modulation by polyphenols in mitigating colonic diseases is not fully established. We hypothesize that the interaction of polyphenols with the gut microbiota contributes to the attenuation of colitis and colitis-associated colon cancer (CAC). To test this hypothesis, we examined the effects of dietary supplementation of polyphenol-rich grape powder (GP) on azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced colitis, CAC, and the gut microbiota in mice (study 1), and further compared anti-colitis effects of GP in regular and antibiotic-treated mice (study 2). Compared to the control diet that has matched non-polyphenol contents, 10% GP, but not 3% GP, attenuated AOM-DSS-induced colitis and tumor multiplicity by 29% (P<.05). Ten percent GP increased gut bacterial evenness and counteracted CAC-induced decrease of bacterial evenness and changes in microbial composition. Remarkably, the estimated gut bacterial functional profiles of healthy mice and diseased mice fed 10% GP were similar, and both were significantly different from those of diseased mice fed the control diet. Furthermore, 10% GP increased the relative abundance of butyrate-producing bacteria in the Lachnospiraceae family and enhanced the concentrations of fecal butyrate. Additionally, 10% GP mitigated DSS-induced colitis in conventional mice, but not the antibiotic-treated, gut microbe-depleted mice. Collectively, our studies demonstrate that grape polyphenols alleviate colonic diseases and prevent disease-associated dysbiosis, and their interaction with the gut microbiota may play a causative role in the protection of gut health.

Nested PCR Detection of Pythium sp. from Formalin-Fixed, Paraffin-Embedded Canine Tissue Sections.

Pythium insidiosum is an infectious oomycete affecting dogs that develop the cutaneous or gastrointestinal form of pythiosis with a poor prognosis. If left untreated, pythiosis may be fatal. This organism is not a true fungus because its cell wall and cell membrane lack chitin and ergosterol, respectively, requiring specific treatment. Identifying the organism is challenging, as a hematoxylin and eosin (H&E) stain poorly stain the P. insidiosum hyphae and cannot be differentiated conclusively from other fungal or fungal-like organisms (such as Lagenidium sp.) morphologically. Our study aimed to develop a nested PCR to detect P. insidiosum and compare it with the traditional histopathologic detection of hyphae. Formalin-fixed, paraffin-embedded (FFPE) tissue scrolls from 26 dogs with lesions suggesting the P. insidiosum infection were assessed histologically, and DNA was extracted from the FFPE tissue sections for nested PCR. Agreement between the histologic stains, (H&E), periodic acid-Schiff (PAS), and/or Grocott methenamine silver (GMS) and the nested PCR occurred in 18/26 cases. Hyphae consistent with Pythium sp. were identified via histopathology in 57.7% of the samples, whereas the nested PCR detected P. insidiosum in 76.9% of samples, aiding in the sensitivity of the diagnosis of pythiosis in dogs. Using this combination of techniques, we report 20 canine cases of pythiosis over 18 years in Indiana and Kentucky, an unexpectedly high incidence for temperate climatic regions. Using a combination of histopathology evaluation and nested PCR is recommended to aid in the accurate diagnosis of pythiosis.

An engineered dual function peptide to repair fractured bones.

Targeted drug delivery, often referred to as "smart" drug delivery, is a process whereby a therapeutic drug is delivered to specific parts of the body in a manner that increases its concentration at the desired sites relative to others. This approach is poised to revolutionize medicine as exemplified by the recent FDA approval of Cytalux (FDA approves pioneering drug for ovarian cancer surgery - Purdue University News) which is a folate-receptor targeted intraoperative near infrared (NIR) imaging agent that was developed in our laboratories. Fracture-associated morbidities and mortality affect a significant portion of world population. United states, Canada and Europe alone spent $48 billion in treating osteoporosis related fractures although this number doesn't count the economic burden due to loss in productivity. It is estimated that by 2050 ca 21 million hip fractures would occur globally which will be leading cause of premature death and disability. Despite the need for improvement in the treatment for fracture repair, methods for treating fractures have changed little in recent decades. Systemic delivery of fracture-homing bone anabolics holds great promise as a therapeutic strategy in this regard. Here we report the design of a fracture-targeted peptide comprised of a payload that binds and activates the parathyroid hormone receptor (PTHR1) and is linked to a targeting ligand comprised of 20 D-glutamic acids (D-Glu20) that directs accumulation of the payload specifically at fracture sites. This targeted delivery results in reduction of fracture healing times to <1/2 while creating repaired bones that are >2-fold stronger than saline-treated controls in mice. Moreover, this hydroxyapatite-targeted peptide can be administered without detectable toxicity to healthy tissues or modification of healthy bones in dogs. Additionally, since similar results are obtained upon treatment of osteoporotic and diabetic fractures in mice, and pain resolution is simultaneously accelerated by this approach, we conclude that this fracture-targeted anabolic peptide displays significant potential to revolutionize the treatment of bone fractures.

Vitamin E alpha- and gamma-tocopherol mitigate colitis, protect intestinal barrier function and modulate the gut microbiota in mice.

Inflammatory bowel diseases (IBDs) including colitis are intestinal disorders characterized by chronic inflammation, barrier dysfunction and dysbiosis. Specific forms of vitamin E have been shown to attenuate colitis, but the mechanisms are not fully understood. The objective of this study is to examine the impact of α-tocopherol (αT) and γ-tocopherol-rich tocopherols (γTmT) on gut inflammation, barrier integrity and microbiota in dextran sulfate sodium (DSS)-induced colitis in mice. We observe that αT and γTmT mitigated DSS-caused fecal bleeding, diarrhea and elevation of IL-6. These vitamin E forms inhibited colitis-induced loss of the tight junction protein occludin, and attenuated colitis-caused elevation of LPS-binding protein in the plasma, a surrogate marker of intestinal barrier dysfunction, suggesting protection of gut barrier integrity. Consistently, αT and γT mitigated TNF-α/IFN-γ-induced impairment of trans-epithelial electrical resistance in human intestinal epithelial Caco-2 cell monolayer. Using 16S rRNA gene sequencing of fecal DNA, we observe that DSS reduced gut microbial evenness and separated microbial composition from healthy controls. In colitis-induced mice, γTmT but not αT separated gut microbial composition from controls, and attenuated DSS-caused depletion of Roseburia, which contains butyrate producing bacteria and is decreased in IBD patients. Canonical correspondence analysis also supports that γTmT favorably altered gut microbial community. In contrast, neither αT nor γTmT affected gut microbes in healthy animals. These results provide evidence supporting protective effects of αT and γT on intestinal barrier function and that γTmT caused favorable changes of the gut microbiota in colitis-induced mice.