WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in SCLC.

Small cell lung cancers (SCLCs) have high mutational burden but are relatively unresponsive to immune checkpoint blockade (ICB). Using SCLC models, we demonstrate that inhibition of WEE1, a G2/M checkpoint regulator induced by DNA damage, activates the STING-TBK1-IRF3 pathway, which increases type I interferons (IFN-α and IFN-ß) and pro-inflammatory chemokines (CXCL10 and CCL5), facilitating an immune response via CD8+ cytotoxic T cell infiltration. We further show that WEE1 inhibition concomitantly activates the STAT1 pathway, increasing IFN-γ and PD-L1 expression. Consistent with these findings, combined WEE1 inhibition (AZD1775) and PD-L1 blockade causes remarkable tumor regression, activation of type I and II interferon pathways, and infiltration of cytotoxic T cells in multiple immunocompetent SCLC genetically engineered mouse models, including an aggressive model with stabilized MYC. Our study demonstrates cell-autonomous and immune-stimulating activity of WEE1 inhibition in SCLC models. Combined inhibition of WEE1 plus PD-L1 blockade represents a promising immunotherapeutic approach in SCLC.

Cytotoxic granzyme C-expressing ILC1s contribute to antitumor immunity and neonatal autoimmunity.

Innate lymphocytes are integral components of the cellular immune system that can coordinate host defense against a multitude of challenges and trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we showed that the cytolytic molecule granzyme C was expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C-expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s. These granzyme C-expressing ILC1s required the transcription factors T-bet and, to a lesser extent, Eomes and support from transforming growth factor-ß (TGF-ß) signaling for their maintenance in the salivary gland. In a transgenic mouse breast cancer model, depleting ILC1s caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Constitutive activation of STAT5, a transcription factor regulated by IL-15, in granzyme C-expressing ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond "helper-like" lymphocytes.

A Urinary Drug-Disposing Approach as an Alternative to Intravesical Chemotherapy for Treating Nonmuscle Invasive Bladder Cancer.

The standard treatment of nonmuscle invasive bladder cancer (NMIBC) is transurethral resection of the tumors, followed by intravesical therapy (IT), which comprises a direct instillation of a solution of Bacillus Calmette-Guérin vaccine or chemotherapy into the bladder. However, the recurrence rate in this disease remains unacceptably high. IT is a local treatment that fails to reach tumors developed in the upper urinary tract (ureter and renal pelvis). The catheterization procedure required for IT is invasive, painful, and poses an increased infection risk, resulting in poor patient quality of life and compliance. There is an unmet need for a potent, comprehensive, and noninvasive option. Without chemical modifications, peptides are rapidly removed by renal clearance. This "shortcoming" can be advantageous when used as a drug carrier for directing therapy to NMIBC. Here we develop a urinary drug-disposing (UDD) approach to improve NMIBC treatment. A 12-amino acid bio-inert peptide (Bdd) that can be exclusively eliminated via renal filtration was generated for delivering the microtubule inhibitor DM1 to NMIBC with minimal nonspecific accumulation in other organs. The UDD approach prolonged survival of mice bearing human bladder tumors. Unlike IT, the treatment was given noninvasively (intravenously). Furthermore, it was more effective at suppressing tumor growth than clinically used IT (mitomycin) and safer than free DM1. The application of this UDD approach to treat kidney tumors and deliver other drugs such as doxorubicin was also demonstrated. Overall, the rapid renal clearance of peptides can be exploited to direct cancer therapies to the urinary system. SIGNIFICANCE: A noninvasive drug delivery approach that targets the urinary system overcomes the current barriers facing effective treatment of bladder cancer.

Chaphamaparvovirus antigen and nucleic acids are not detected in kidney tissues from cats with chronic renal disease or immunocompromised cats.

Chronic kidney disease (CKD) is a common cause of morbidity and mortality in domestic cats, but the cause is still largely elusive. While some viruses have been associated with this disease, none have been definitively implicated as causative. Recently, Rodent chaphamaparvovirus 1 was recognized as the cause of murine inclusion body nephropathy, a disease reported for over 40 years in laboratory mice. A novel virus belonging to the same genus, Carnivore chaphamaparvovirus 2, was recently identified in the feces of cats with diarrhea. The goal of this study was to investigate the possible role of chaphamaparvoviruses including members of Rodent chaphamaparvovirus 1 and Carnivore chaphamaparvovirus 2 in the development of feline CKD. The presence of these viruses was retrospectively investigated in formalin-fixed paraffin-embedded feline kidney samples using polymerase chain reaction, in situ hybridization, and immunohistochemistry. Cats were divided into 3 groups: normal (N = 24), CKD (N = 26), and immunocompromised (N = 25). None of the kidney tissues from any of the 75 cats revealed the presence of chaphamaparvovirus DNA, RNA, or antigen. We conclude that viruses belonging to the chaphamaparvovirus genus are unlikely to contribute to the occurrence of feline CKD.

Isoform specific anti-TGFß therapy enhances antitumor efficacy in mouse models of cancer.

TGFß is a potential target in cancer treatment due to its dual role in tumorigenesis and homeostasis. However, the expression of TGFß and its inhibition within the tumor microenvironment has mainly been investigated in stroma-heavy tumors. Using B16 mouse melanoma and CT26 colon carcinoma as models of stroma-poor tumors, we demonstrate that myeloid/dendritic cells are the main sources of TGFß1 and TGFß3. Depending on local expression of TGFß isoforms, isoform specific inhibition of either TGFß1 or TGFß3 may be effective. The TGFß signature of CT26 colon carcinoma is defined by TGFß1 and TGFß1 inhibition results in tumor delay; B16 melanoma has equal expression of both isoforms and inhibition of either TGFß1 or TGFß3 controls tumor growth. Using T cell functional assays, we show that the mechanism of tumor delay is through and dependent on enhanced CD8+ T cell function. To overcome the local immunosuppressive environment, we found that combining TGFß inhibition with immune checkpoint blockade results in improved tumor control. Our data suggest that TGFß inhibition in stroma poor tumors shifts the local immune environment to favor tumor suppression.

Biology and Cellular Tropism of a Unique Astrovirus Strain: Murine Astrovirus 2.

Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains-a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD- Prkdcem26Cd52Il2rgem26Cd22 /NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

ERK Inhibition Improves Anti-PD-L1 Immune Checkpoint Blockade in Preclinical Pancreatic Ductal Adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma (PDAC) do not benefit from immune checkpoint blockade (ICB) along the PD-1/PD-L1 axis. Variable PD-L1 expression in PDAC indicates a potential access issue of PD-L1-targeted therapy. To monitor target engagement of PD-L1-targeted therapy, we generated a PD-L1-targeted PET tracer labeled with zirconium-89 (89Zr). As the MAPK signaling pathway (MEK and ERK) is known to modulate PD-L1 expression in other tumor types, we used [89Zr]Zr-DFO-anti-PD-L1 as a tool to noninvasively assess whether manipulation of the MAPK signaling cascade could be leveraged to modulate PD-L1 expression and thereby immunotherapeutic outcomes in PDAC. In this study, we observed that the inhibition of MEK or ERK is sufficient to increase PD-L1 expression, which we hypothesized could be leveraged for anti-PD-L1 immune checkpoint therapy. We found that the combination of ERK inhibition and anti-PD-L1 therapy corresponded with a significant improvement of overall survival in a syngeneic mouse model of PDAC. Furthermore, IHC analysis indicates that the survival benefit may be CD8+ T-cell mediated. The therapeutic and molecular imaging tool kit developed could be exploited to better structure clinical trials and address the therapeutic gaps in challenging malignancies such as PDAC.

Transarterial Embolization of Liver Cancer in a Transgenic Pig Model.

PURPOSE: To develop and characterize a porcine model of liver cancer that could be used to test new locoregional therapies. MATERIALS AND METHODS: Liver tumors were induced in 18 Oncopigs (transgenic pigs with Cre-inducible TP53R167H and KRASG12D mutations) by using an adenoviral vector encoding the Cre-recombinase gene. The resulting 60 tumors were characterized on multiphase contrast-enhanced CT, angiography, perfusion, micro-CT, and necropsy. Transarterial embolization was performed using 40-120 µm (4 pigs) or 100-300 µm (4 pigs) Embosphere microspheres. Response to embolization was evaluated on imaging. Complications were determined based on daily clinical evaluation, laboratory results, imaging, and necropsy. RESULTS: Liver tumors developed at 60/70 (86%) inoculated sites. Mean tumor size was 2.1 cm (range, 0.3-4 cm) at 1 week. Microscopically, all animals developed poorly differentiated to undifferentiated carcinomas accompanied by a major inflammatory component, which resembled undifferentiated carcinomas of the human pancreatobiliary tract. Cytokeratin and vimentin expression confirmed epithelioid and mesenchymal differentiation, respectively. Lymph node, lung, and peritoneal metastases were seen in some cases. On multiphase CT, all tumors had a hypovascular center, and 17/60 (28%) had a hypervascular rim. After transarterial embolization, noncontrast CT showed retained contrast medium in the tumors. Follow-up contrast-enhanced scan showed reduced size of tumors after embolization using either 40-120 µm or 100-300 µm Embosphere microspheres, while untreated tumors showed continued growth. CONCLUSIONS: Liver tumors can be induced in a transgenic pig and can be successfully treated using bland embolization.

Phaeohyphomycosis due to Veronaea botryosa in cultured white sturgeon (Acipenser transmontanus Richardson) from California USA during 2006 to 2015.

Infection with Veronaea botryosa can result in rare cutaneous or disseminated, granulomatous to pyogranulomatous phaeohyphomycosis in humans, although disease due to the fungus has also been reported in non-mammalian vertebrates. This report documents disease due to V. botryosa in captive, juvenile to subadult or young adult white sturgeon (Acipenser transmontanus Richardson) from California USA and complements a previous report of the disease in captive Siberian sturgeon (Acipenser baerii) from Florida USA. Pathological examinations revealed granulomatous to pyogranulomatous inflammation of multiple organs. Isolates of the fungal agent were phenotypically consistent with V. botryosa, and molecular analyses of the D1/D2 region of the fungal 28S rRNA gene and the internal transcribed spacer (ITS) region located between the fungal 18S and 28S rRNA genes confirmed the aetiologic agent as V. botryosa. The disease in captive sturgeon results in a considerable economic encumbrance to the producer due to the loss of the cumulative financial resources invested in the production of older subadult to young adult sturgeon.

A Self-Assembling and Disassembling (SADA) Bispecific Antibody (BsAb) Platform for Curative Two-step Pretargeted Radioimmunotherapy.

PURPOSE: Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index. EXPERIMENTAL DESIGN: The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA-BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses. RESULTS: SADA-BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA-BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver. CONCLUSIONS: The SADA-BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA-BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose.See related commentary by Capala and Kunos, p. 377.

Ethmoidal meningoencephalocele in a C57BL/6J mouse.

An otherwise healthy two-month-old female C57BL/6J mouse presented with a left-sided head tilt. Differential diagnoses included idiopathic necrotizing arteritis, bacterial otitis media/interna (Pasteurella pneumotropica, Pseudomonas aeruginosa, Streptococcus sp., Mycoplasma pulmonis and Burkholderia gladioli), encephalitis, an abscess, neoplasia, a congenital malformation and an accidental or iatrogenic head trauma. Magnetic resonance imaging (MRI) revealed a large space-occupying right olfactory lobe intra-axial lesion with severe secondary left-sided subfalcine herniation. Following imaging, the animal was euthanized due to poor prognosis. Histopathologic examination revealed a unilateral, full-thickness bone defect at the base of the cribriform plate and nasal conchae dysplasia, resulting in the herniation of the olfactory bulb into the nasal cavity. There was also a left midline-shift of the frontal cortex and moderate catarrhal sinusitis in the left mandibular sinus. The MRI and histopathologic changes are consistent with a congenital malformation of the nasal cavity and frontal aspect of the skull known as an ethmoidal meningoencephalocele. Encephaloceles are rare abnormalities caused by herniation of contents of the brain through a defect in the skull which occur due to disruption of the neural tube closure at the level anterior neuropore or secondary to trauma, surgical complications, cleft palate or increased intracranial pressure. The etiology is incompletely understood but hypotheses include genetics, vitamin deficiency, teratogens, infectious agents and environmental factors. Ethmoidal encephaloceles have been reported in multiple species including humans but have not been reported previously in mice. There are multiple models for spontaneous and induced craniofacial malformation in mice, but none described for ethmoidal encephaloceles.

Alpha radioimmunotherapy using 225Ac-proteus-DOTA for solid tumors - safety at curative doses.

This is the initial report of an α-based pre-targeted radioimmunotherapy (PRIT) using 225Ac and its theranostic pair, 111In. We call our novel tumor-targeting DOTA-hapten PRIT system "proteus-DOTA" or "Pr." Herein we report the first results of radiochemistry development, radiopharmacology, and stoichiometry of tumor antigen binding, including the role of specific activity, anti-tumor efficacy, and normal tissue toxicity with the Pr-PRIT approach (as α-DOTA-PRIT). A series of α-DOTA-PRIT therapy studies were performed in three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2), including evaluation of chronic toxicity at ~20 weeks of select survivors. Methods: Preliminary biodistribution experiments in SW1222 tumor-bearing mice revealed that 225Ac could not be efficiently pretargeted with current DOTA-Bn hapten utilized for 177Lu or 90Y, leading to poor tumor uptake in vivo. Therefore, we synthesized Pr consisting of an empty DOTA-chelate for 225Ac, tethered via a short polyethylene glycol linker to a lutetium-complexed DOTA for picomolar anti-DOTA chelate single-chain variable fragment (scFv) binding. Pr was radiolabeled with 225Ac and its imaging surrogate, 111In. In vitro studies verified anti-DOTA scFv recognition of [225Ac]Pr, and in vivo biodistribution and clearance studies were performed to evaluate hapten suitability and in vivo targeting efficiency. Results: Intravenously (i.v.) administered 225Ac- or 111In-radiolabeled Pr in mice showed rapid renal clearance and minimal normal tissue retention. In vivo pretargeting studies show high tumor accumulation of Pr (16.71 ± 5.11 %IA/g or 13.19 ± 3.88 %IA/g at 24 h p.i. for [225Ac]Pr and [111In]Pr, respectively) and relatively low uptake in normal tissues (all average ≤ 1.4 %IA/g at 24 h p.i.). Maximum tolerated dose (MTD) was not reached for either [225Ac]Pr alone or pretargeted [225Ac]Pr at administered activities up to 296 kBq/mouse. Single-cycle treatment consisting of α-DOTA-PRIT with either huA33-C825 bispecific anti-tumor/anti-DOTA-hapten antibody (BsAb), anti-HER2-C825 BsAb, or hu3F8-C825 BsAb for targeting GPA33, HER2, or GD2, respectively, was highly effective. In the GPA33 model, no complete responses (CRs) were observed but prolonged overall survival of treated animals was 42 d for α-DOTA-PRIT vs. 25 d for [225Ac]Pr only (P < 0.0001); for GD2, CRs (7/7, 100%) and histologic cures (4/7, 57%); and for HER2, CRs (7/19, 37%) and histologic cures (10/19, 56%) with no acute or chronic toxicity. Conclusions: [225Ac]Pr and its imaging biomarker [111In]Pr demonstrate optimal radiopharmacologic behavior for theranostic applications of α-DOTA-PRIT. For this initial evaluation of efficacy and toxicity, single-cycle treatment regimens were performed in all three systems. Histologic toxicity was not observed, so MTD was not observed. Prolonged overall survival, CRs, and histologic cures were observed in treated animals. In comparison to RIT with anti-tumor IgG antibodies, [225Ac]Pr has a much improved safety profile. Ultimately, these data will be used to guide clinical development of toxicity and efficacy studies of [225Ac]Pr, with the goal of delivering massive lethal doses of radiation to achieve a high probability of cure without toxicity.

Induction and characterization of pancreatic cancer in a transgenic pig model.

BACKGROUND: Preclinical testing of new locoregional therapies for pancreatic cancer has been challenging, due to the lack of a suitable large animal model. PURPOSE: To develop and characterize a porcine model of pancreatic cancer. Unlike small animals, pigs have similar physiology, drug dosing, and immune response to humans. Locoregional therapy in pigs can be performed using the same size catheters and devices as in humans. METHODS: The Oncopig is a transgenic pig with Cre-inducible TP53R167H and KRASG12D mutations. In 12 Oncopigs, CT-guided core biopsy of the pancreas was performed. The core biopsy was incubated with an adenoviral vector carrying the Cre recombinase gene. The transformed core biopsy was injected back into the pancreas (head, tail, or both). The resulting tumors (n = 19) were characterized on multi-phase contrast-enhanced CT, and on pathology, including immunohistochemistry. Angiographic characterization of the tumors was performed in 3 pigs. RESULTS: Pancreatic tumors developed at 19 out of 22 sites (86%) that were inoculated. Average tumor size was 3.0 cm at 1 week (range: 0.5-5.1 cm). H&E and immunohistochemical stains revealed undifferentiated carcinomas, similar to those of the pancreatobiliary system in humans. Neoplastic cells were accompanied by a major inflammatory component. 1 of 12 pigs only had inflammatory nodules without evidence of neoplasia. On multiphase CT, tumors were hypovascular compared to the normal pancreas. There was no pancreatic duct dilation. In 3 pigs, angiography was performed, and in all 3 cases, the artery supplying the pancreatic tumor could be catheterized using a 2.4 F microcatheter. Selective angiography showed the pancreatic tumor, without extra-pancreatic perfusion. CONCLUSION: Pancreatic cancer can be induced in a transgenic pig. Intra-arterial procedures using catheters designed for human interventions were technically feasible in this large animal model.

A combined approach of convection-enhanced delivery of peptide nanofiber reservoir to prolong local DM1 retention for diffuse intrinsic pontine glioma treatment.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a highly lethal malignancy that occurs predominantly in children. DIPG is inoperable and post-diagnosis survival is less than 1 year, as conventional chemotherapy is ineffective. The intact blood-brain barrier (BBB) blocks drugs from entering the brain. Convection-enhanced delivery (CED) is a direct infusion technique delivering drugs to the brain, but it suffers from rapid drug clearance. Our goal is to overcome the delivery barrier via CED and maintain a therapeutic concentration at the glioma site with a payload-adjustable peptide nanofiber precursor (NFP) that displays a prolonged retention property as a drug carrier. METHODS: The post-CED retention of 89Zr-NFP was determined in real time using PET/CT imaging. Emtansine (DM1), a microtubule inhibitor, was conjugated to NFP. The cytotoxicity of the resulting DM1-NFP was tested against patient-derived DIPG cell lines. The therapeutic efficacy was evaluated in animals bearing orthotopic DIPG, according to glioma growth (measured using bioluminescence imaging) and the long-term survival. RESULTS: DM1-NFP demonstrated potency against multiple glioma cell lines. The half-maximal inhibitory concentration values were in the nanomolar range. NFP remained at the infusion site (pons) for weeks, with a clearance half-life of 60 days. DM1-NFP inhibited glioma progression in animals, and offered a survival benefit (median survival of 62 days) compared with the untreated controls (28 days) and DM1-treated animal group (26 days). CONCLUSIONS: CED, in combination with DM1-NFP, complementarily functions to bypass the BBB, prolong drug retention at the fusion site, and maintain an effective therapeutic effect against DIPG to improve treatment outcome.

Clostridium perfringens epsilon toxin induces blood brain barrier permeability via caveolae-dependent transcytosis and requires expression of MAL.

Clostridium perfringens epsilon toxin (ETX) is responsible for causing the economically devastating disease, enterotoxaemia, in livestock. It is well accepted that ETX causes blood brain barrier (BBB) permeability, however the mechanisms involved in this process are not well understood. Using in vivo and in vitro methods, we determined that ETX causes BBB permeability in mice by increasing caveolae-dependent transcytosis in brain endothelial cells. When mice are intravenously injected with ETX, robust ETX binding is observed in the microvasculature of the central nervous system (CNS) with limited to no binding observed in the vasculature of peripheral organs, indicating that ETX specifically targets CNS endothelial cells. ETX binding to CNS microvasculature is dependent on MAL expression, as ETX binding to CNS microvasculature of MAL-deficient mice was not detected. ETX treatment also induces extravasation of molecular tracers including 376Da fluorescein salt, 60kDA serum albumin, 70kDa dextran, and 155kDA IgG. Importantly, ETX-induced BBB permeability requires expression of both MAL and caveolin-1, as mice deficient in MAL or caveolin-1 did not exhibit ETX-induced BBB permeability. Examination of primary murine brain endothelial cells revealed an increase in caveolae in ETX-treated cells, resulting in dynamin and lipid raft-dependent vacuolation without cell death. ETX-treatment also results in a rapid loss of EEA1 positive early endosomes and accumulation of large, RAB7-positive late endosomes and multivesicular bodies. Based on these results, we hypothesize that ETX binds to MAL on the apical surface of brain endothelial cells, causing recruitment of caveolin-1, triggering caveolae formation and internalization. Internalized caveolae fuse with early endosomes which traffic to late endosomes and multivesicular bodies. We believe that these multivesicular bodies fuse basally, releasing their contents into the brain parenchyma.

Ceftolozane-Tazobactam in the Treatment of Experimental Pseudomonas aeruginosa Pneumonia in Persistently Neutropenic Rabbits: Impact on Strains with Genetically Defined Mechanisms of Resistance.

Ceftolozane-tazobactam (C/T) is a novel cephalosporin with in vitro activity against Pseudomonas aeruginosa that is resistant to extended-spectrum penicillins and antipseudomonal cephalosporins. In order to assess the antimicrobial effect of C/T in treatment of Pseudomonas pneumonia, we investigated the pharmacokinetics and efficacy of C/T in persistently neutropenic rabbits. Pseudomonas pneumonia was established by direct endotracheal inoculation. Treatment groups consisted of C/T, ceftazidime (CAZ), piperacillin-tazobactam (TZP), and untreated controls (UC). Rabbits received a dosage of C/T of 80 mg/kg every 4 h (q4h) intravenously (i.v.) (53 mg/kg ceftolozane/26 mg/kg tazobactam) to match the free drug time above the MIC as well as a comparable plasma area under the concentration-time curve (AUC) (humanized doses of ceftolozane-tazobactam of 3 g [2 g/1 g]) q8h, due to the more rapid elimination of ceftolozane in rabbits (0.75 h) than in humans (2.5 h). Four molecularly characterized clinical P. aeruginosa isolates from patients with pneumonia were studied, including one isolate from each classification group: pan-susceptible (PS), outer membrane porin D (OPRD) porin loss (OPRDPL), efflux pump expression (EPE), and AmpC hyperexpression (ACHE). Treatment was continued for 12 days. Treatment with ceftolozane-tazobactam resulted in a ≥105 reduction in residual pulmonary and bronchoalveolar lavage (BAL) fluid bacterial burdens caused by all 4 strains (P ≤ 0.01). This antibacterial activity coincided with reduction of lung weight (an organism-mediated pulmonary injury marker) (P < 0.05). CAZ was less active in ACHE-infected rabbits, and TZP had less activity against EPE, ACHE, and OPRDPL strains. Survival was prolonged in the C/T and CAZ treatment groups in comparison to the TZP and UC groups (P < 0.001). Ceftolozane-tazobactam is highly active in treatment of experimental P. aeruginosa pneumonia in persistently neutropenic rabbits, including infections caused by strains with the most common resistance mechanisms.

Equine idiopathic hemorrhagic cystitis: Clinical features and comparison with bladder neoplasia.

BACKGROUND: A new syndrome of hematuria in horses has been documented. HYPOTHESIS/OBJECTIVES: Hemorrhagic cystitis is a novel cause of stranguria and hematuria in horses. This syndrome may be difficult to differentiate from bladder neoplasia because they share several clinical features. ANIMALS: Eleven horses with idiopathic hemorrhagic cystitis and 7 horses with bladder neoplasia. METHODS: Retrospective cohort study. RESULTS: Hemorrhagic cystitis was detected on cystoscopy of affected horses, with hemorrhagic and thickened apical bladder mucosa. Clinical signs and endoscopic appearance of the bladder resolved within 3-8 weeks. Histopathology of bladder mucosal biopsy specimens featured neutrophilic and hemorrhagic cystitis. Histopathology was suggestive of dysplasia or neoplasia in 3 horses with hemorrhagic cystitis, yet the horses experienced complete resolution, suggesting that small biopsy specimens obtained by endoscopy can be difficult to interpret. Horses with bladder neoplasia had lower hematocrits, were older, more likely to be female, and more likely to have a mass detected on ultrasonographic examination of the bladder than horses with hemorrhagic cystitis syndrome. CONCLUSIONS AND CLINICAL IMPORTANCE: Hemorrhagic cystitis represents a novel differential diagnosis for horses with hematuria, and is associated with a favorable prognosis. Although histopathology may suggest a neoplastic process, affected horses should be monitored cystoscopically, because complete resolution of hemorrhagic cystitis occurs. The cause of this disease is unknown, and warrants investigation.

Bushmeat Hunting and Zoonotic Transmission of Simian T-Lymphotropic Virus 1 in Tropical West and Central Africa.

Simian T-lymphotropic virus 1 (STLV-1) enters human populations through contact with nonhuman primate (NHP) bushmeat. We tested whether differences in the extent of contact with STLV-1-infected NHP bushmeat foster regional differences in prevalence of human T-lymphotropic virus 1 (HTLV-1). Using serological and PCR assays, we screened humans and NHPs at two Sub-Saharan African sites where subsistence hunting was expected to be less (Taï region, Côte d'Ivoire [CIV]) or more (Bandundu region, Democratic Republic of the Congo [DRC]) developed. Only 0.7% of human participants were infected with HTLV-1 in CIV (n = 574), and 1.3% of humans were infected in DRC (n = 302). Two of the Ivorian human virus sequences were closely related to simian counterparts, indicating ongoing zoonotic transmission. Multivariate analysis of human demographic parameters and behavior confirmed that participants from CIV were less often exposed to NHPs than participants from DRC through direct contact, e.g., butchering. At the same time, numbers of STLV-1-infected NHPs were higher in CIV (39%; n = 111) than in DRC (23%; n = 39). We conclude that similar ultimate risks of zoonotic STLV-1 transmission-defined as the product of prevalence in local NHP and human rates of contact to fresh NHP carcasses-contribute to the observed comparable rates of HTLV-1 infection in humans in CIV and DRC. We found that young adult men and mature women are most likely exposed to NHPs at both sites. In view of the continued difficulties in controlling zoonotic disease outbreaks, the identification of such groups at high risk of NHP exposure may guide future prevention efforts.IMPORTANCE Multiple studies report a high risk for zoonotic transmission of blood-borne pathogens like retroviruses through contact with NHPs, and this risk seems to be particularly high in tropical Africa. Here, we reveal high levels of exposure to NHP bushmeat in two regions of Western and Central tropical Africa. We provide evidence for continued zoonotic origin of HTLV-1 in humans at CIV, and we found that young men and mature women represent risk groups for zoonotic transmission of pathogens from NHPs. Identifying such risk groups can contribute to mitigation of not only zoonotic STLV-1 transmission but also transmission of any blood-borne pathogen onto humans in Sub-Saharan Africa.

Acquired tricuspid valve stenosis due to intentionally redundant transvenous lead placement for VDD pacing in two small dogs.

Placement of an endocardial VDD pacing lead in small dogs (<12 kg) may necessitate a redundant lead remaining looped in the right atrium for appropriate sensing and pacing. This report documented acquired tricuspid valve stenosis in two small dogs between 8 months and 4 years after VDD pacemaker placement for third-degree atrioventricular block. Echocardiography and Doppler echocardiography identified elevated transtricuspid flow velocities, prolonged pressure half-times, decreased valve leaflet excursions, and tricuspid regurgitation in both cases. Both cases were euthanized secondary to this pacing complication. Necropsy was performed in one case and confirmed adherence between the redundant lead loop, atrial and valve tissue. While VDD pacing in dogs has proven hemodynamic benefits, these benefits have not been demonstrated in terms of survival benefit or clinical signs. The requirement of redundant lead placement in small dogs for appropriate VDD lead function creates potential deleterious effects that should be weighed against the possible clinical value of VDD pacing in these patients.

Babesia spp. in European wild ruminant species: parasite diversity and risk factors for infection.

Babesia are tick-borne parasites that are increasingly considered as a threat to animal and public health. We aimed to assess the role of European free-ranging wild ruminants as maintenance mammalian hosts for Babesia species and to determine risk factors for infection. EDTA blood was collected from 222 roe deer (Capreolus c. capreolus), 231 red deer (Cervus e. elaphus), 267 Alpine chamois (Rupicapra r. rupicapra) and 264 Alpine ibex (Capra i. ibex) from all over Switzerland and analysed by PCR with pan-Babesia primers targeting the 18S rRNA gene, primers specific for B. capreoli and Babesia sp. EU1, and by sequencing. Babesia species, including B. divergens, B. capreoli, Babesia sp. EU1, Babesia sp. CH1 and B. motasi, were detected in 10.7% of all samples. Five individuals were co-infected with two Babesia species. Infection with specific Babesia varied widely between host species. Cervidae were significantly more infected with Babesia spp. than Caprinae. Babesia capreoli and Babesia sp. EU1 were mostly found in roe deer (prevalences 17.1% and 7.7%, respectively) and B. divergens and Babesia sp. CH1 only in red deer. Factors significantly associated with infection were low altitude and young age. Identification of Babesia sp. CH1 in red deer, co-infection with multiple Babesia species and infection of wild Caprinae with B. motasi and Babesia sp. EU1 are novel findings. We propose wild Caprinae as spillover or accidental hosts for Babesia species but wild Cervidae as mammalian reservoir hosts for B. capreoli, possibly Babesia sp. EU1 and Babesia sp. CH1, whereas their role regarding B. divergens is more elusive.