Tumor Suppression by Anti-Fibroblast Activation Protein Near-Infrared Photoimmunotherapy Targeting Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) constitute a prominent cellular component of the tumor stroma, with various pro-tumorigenic roles. Numerous attempts to target fibroblast activation protein (FAP), a highly expressed marker in immunosuppressive CAFs, have failed to demonstrate anti-tumor efficacy in human clinical trials. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor therapy that utilizes an antibody-photo-absorbing conjugate activated by near-infrared light. In this study, we examined the therapeutic efficacy of CAF depletion by NIR-PIT in two mouse tumor models. Using CAF-rich syngeneic lung and spontaneous mammary tumors, NIR-PIT against FAP or podoplanin was performed. Anti-FAP NIR-PIT effectively depleted FAP+ CAFs, as well as FAP+ myeloid cells, and suppressed tumor growth, whereas anti-podoplanin NIR-PIT was ineffective. Interferon-gamma production by CD8 T and natural killer cells was induced within hours after anti-FAP NIR-PIT. Additionally, lung metastases were reduced in the treated spontaneous mammary cancer model. Depletion of FAP+ stromal as well as FAP+ myeloid cells effectively suppressed tumor growth in bone marrow chimeras, suggesting that the depletion of both cell types in one treatment is an effective therapeutic approach. These findings highlight a promising therapy for selectively eliminating immunosuppressive FAP+ cells within the tumor microenvironment.

The effect of aerosolized bacterial lysate on experimentally induced Mannheimia haemolytica pneumonia in calves.

Bovine respiratory disease (BRD) often occurs during specific periods of increased susceptibility when stress, viral infection, or reduced air quality are thought to suppress respiratory defences. The innate immune system is rapidly responsive and broadly protective and could be a target for preventing BRD during these periods of increased susceptibility. This study tested the hypothesis that stimulation of pulmonary innate immune responses by aerosol delivery of a lysate of killed Escherichia coli and Staphylococcus aureus bacteria would protect calves against Mannheimia haemolytica pneumonia. Ten clean-catch colostrum-deprived Holstein calves were randomly assigned to receive either aerosolized bacterial lysate or saline 24 hours before M. haemolytica challenge. Effects of this treatment on clinical, hematologic, microbiologic, and pathologic outcomes were assessed. Compared to controls, lysate-treated calves had lower serum haptoglobin and blood leukocyte and neutrophil concentrations following M. haemolytica challenge. There were no differences in temperature, heart and respiratory rates, clinical scores, ultrasound lesions, or number of M. haemolytica in the nasal cavity or lung. Thus, treatment with bacterial lysate prior to M. haemolytica challenge appeared to ameliorate early measures of inflammation but did not provide sufficient protection to substantially alter the course of disease.

La maladie respiratoire bovine (BRD) survient souvent pendant des périodes spécifiques de sensibilité accrue lorsque le stress, une infection virale ou une qualité de l'air réduite sont censés supprimer les défenses respiratoires. Le système immunitaire inné est rapidement réactif et largement protecteur et pourrait être une cible pour prévenir la BRD pendant ces périodes de sensibilité accrue. Cette étude a testé l'hypothèse selon laquelle la stimulation des réponses immunitaires innées pulmonaires par la délivrance d'aérosols d'un lysat de bactéries Escherichia coli et Staphylococcus aureus tuées protégerait les veaux contre la pneumonie à Mannheimia haemolytica. Dix veaux Holstein dont on a limité la contamination bactérienne et privés de colostrum ont été répartis au hasard pour recevoir soit un lysat bactérien en aérosol, soit une solution saline 24 heures avant une infection défi par M. haemolytica. Les effets de ce traitement sur les résultats cliniques, hématologiques, microbiologiques et pathologiques ont été évalués. Comparativement aux témoins, les veaux traités au lysat présentaient des concentrations sériques d'haptoglobine et de leucocytes et de neutrophiles sanguins plus faibles après la provocation par M. haemolytica. Il n'y avait aucune différence dans la température, les fréquences cardiaques et respiratoires, les scores cliniques, les lésions échographiques ou le nombre de M. haemolytica dans la cavité nasale ou les poumons. Ainsi, le traitement avec un lysat bactérien avant la provocation par M. haemolytica a semblé améliorer les réactions précoces de l'inflammation mais n'a pas fourni une protection suffisante pour modifier substantiellement l'évolution de la maladie.(Traduit par Docteur Serge Messier).

Novel Humanized Mesothelin-Expressing Genetically Engineered Mouse Models Underscore Challenges in Delivery of Complex Therapeutics to Pancreatic Cancers.

Antibody-based therapies designed for human use frequently fail to cross-react with the murine isoform of their target. Because of this problem, preclinical studies of antibody-based mesothelin (Msl)-targeted therapeutics in immunocompetent systems have been limited by the lack of suitable mouse models. Here, we describe two immunocompetent humanized mesothelin transgenic mouse lines that can act as tolerant hosts for C57Bl/6-syngeneic cell lines expressing the human isoform of mesothelin. Thyroid peroxidase (TPO) mice have thyroid-restricted human mesothelin expression. Mesothelin (Msl) mice express human mesothelin in the typical serosal membrane distribution and can additionally be utilized to assess on-target, off-tumor toxicity of human mesothelin-targeted therapeutics. Both transgenic strains shed human mesothelin into the serum like human mesothelioma and patients with ovarian cancer, and serum human mesothelin can be used as a blood-based surrogate of tumor burden. Using these models, we examined the on-target toxicity and antitumor activity of human mesothelin-targeted recombinant immunotoxins. We found that immunotoxin treatment causes acute and chronic histologic changes to serosal membranes in Msl mice, while human mesothelin-expressing thyroid follicular cells in TPO mice are resistant to immunotoxin despite excellent drug delivery. Furthermore, poor delivery of immunotoxin to syngeneic orthotopic human mesothelin-expressing pancreatic adenocarcinoma limits antitumor activity both alone and in combination with immune checkpoint inhibition. In summary, we have developed two high-fidelity, immunocompetent murine models for human cancer that allow for rigorous preclinical evaluation of human mesothelin-targeted therapeutics.

Effect of aerosolized bacterial lysate on development of naturally occurring respiratory disease in beef calves.

BACKGROUND: Bovine respiratory disease (BRD) is a major problem affecting beef cattle after arrival to feedlots. Alternatives to antibiotics are needed for prevention. HYPOTHESIS: Stimulation of pulmonary innate immune responses at the time of arrival to a feedlot reduces the occurrence and severity of BRD. ANIMALS: Sixty beef steers at high risk of BRD. METHODS: Randomized, double-blinded, placebo-controlled study. Calves received saline or a lysate of Staphylococcus aureus and Escherichia coli by aerosol, at 16 hours after feedlot arrival. Calves were monitored for 28 days for disease outcomes and levels of Mycoplasma bovis and Mannheimia haemolytica in nasal swabs. RESULTS: Death from M bovis pneumonia was significantly greater in lysate-treated animals (6/29, 24%) compared to controls (1/29, 3%; odds ratio = 10.2; 95% confidence interval [CI] = 1.1-96.0; P = .04). By 28 days after arrival, 29/29 lysate-treated calves had ultrasonographic pulmonary consolidation compared to 24/29 control calves (P = .05). Lysate-treated calves had lower weight gain compared to control calves (-8.8 kg, 95% CI = -17.1 to -0.5; P = .04), and higher body temperatures on days 4, 7, and 21 (0.19°C; 95% CI = 0.01-0.37; P = .04). Nasal M bovis numbers increased over time and were higher in lysate-treated calves (0.76 log CFU, 95% CI = 0.3-1.2; P = .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Aerosol administration of a bacterial lysate exacerbated BRD in healthy high-risk beef calves, suggesting that respiratory tract inflammation adversely affects how calves respond to subsequent natural infection with M bovis and other respiratory pathogens.

Naturally Acquired Mouse Kidney Parvovirus Infection Produces a Persistent Interstitial Nephritis in Immunocompetent Laboratory Mice.

Mouse kidney parvovirus (MKPV), also known as murine chapparvovirus (MuCPV), is an emerging, highly infectious agent that has been isolated from laboratory and wild mouse populations. In immunocompromised mice, MKPV produces severe chronic interstitial nephropathy and renal failure within 4 to 5 months of infection. However, the course of disease, severity of histologic lesions, and viral shedding are uncertain for immunocompetent mice. We evaluated MKPV infections in CD-1 and Swiss Webster mice, 2 immunocompetent stocks of mice. MKPV-positive CD-1 mice (n = 30) were identified at approximately 8 weeks of age by fecal PCR (polymerase chain reaction) and were subsequently housed individually for clinical observation and diagnostic sampling. Cage swabs, fecal pellets, urine, and blood were evaluated by PCR at 100 and 128 days following the initial positive test, which identified that 28 of 30 were persistently infected and 24 of these were viremic at 100 days. Histologic lesions associated with MKPV in CD-1 (n = 31) and Swiss mice (n = 11) included lymphoplasmacytic tubulointerstitial nephritis with tubular degeneration. Inclusion bodies were rare; however, intralesional MKPV mRNA was consistently detected via in situ hybridization within tubular epithelial cells of the renal cortex and within collecting duct lumina. In immunocompetent CD-1 mice, MKPV infection resulted in persistent shedding of virus for up to 10 months and a mild tubulointerstitial nephritis, raising concerns that this virus could produce study variations in immunocompetent models. Intranuclear inclusions were not a consistent feature of MKPV infection in immunocompetent mice.

Pulmonary and systemic responses to aerosolized lysate of Staphylococcus aureus and Escherichia coli in calves.

BACKGROUND: Constitutive and inducible defenses protect the respiratory tract from bacterial infection. The objective of this study was to characterize the response to an aerosolized lysate of killed bacteria, as a basis for studying the regulation and in vivo effects of these inducible innate immune responses. RESULTS: Bacterial lysate consisting of heat-killed and sonicated Staphylococcus aureus and Escherichia coli was aerosolized to 6 calves and systemic and pulmonary innate immune and inflammatory responses were measured in the first 24 h relative to baseline. Evaluated parameters included clinical parameters (body temperature and heart and respiratory rates), blood acute phase proteins and leukocyte counts, and leukocytes and proteins in bronchoalveolar lavage fluid. Mild clinical signs with increased heart rates and rectal temperatures developed following administration of the lysate, with resolution by 24 h. Serum haptoglobin and plasma fibrinogen concentrations were elevated at 24 h relative to baseline. Bronchoalveolar lavage fluid (BALF) had increased cellularity and increased proportion of neutrophils, as well as higher concentrations of interleukin (IL)-8, IL-10 and total protein at 24 h relative to baseline. Mass spectrometry identified 965 unique proteins in BALF: 19 proteins were increased and 26 proteins were decreased relative to baseline. The upregulated proteins included those involved in innate immunity including activation of complement, neutrophils and platelets. At postmortem examination, calves receiving higher doses of lysate had areas of lobular consolidation and interlobular edema. Histologically, neutrophils were present within bronchioles and to a lesser extent within alveoli. Calves receiving highest doses of lysate had patchy areas of neutrophils, hemorrhage and hyaline membranes within alveoli. CONCLUSIONS: Aerosolization of bacterial lysate stimulated an innate immune response in lungs and airways, with alveolar damage observed at higher doses. Such a stimulus could be of value for investigating the effects of inducible innate immune responses on occurrence of disease, or for evaluating how stress, drugs or genetics affect these dynamic responses of the respiratory tract.

Host Tolerance to Infection with the Bacteria that Cause Bovine Respiratory Disease.

Calves vary considerably in their pathologic and clinical responses to infection of the lung with bacteria. The reasons may include resistance to infection because of pre-existing immunity, development of effective immune responses, or infection with a minimally virulent bacterial strain. However, studies of natural disease and of experimental infections indicate that some calves develop only mild lung lesions and minimal clinical signs despite substantial numbers of pathogenic bacteria in the lung. This may represent "tolerance" to pulmonary infection because these calves are able to control their inflammatory responses or protect the lung from damage, without necessarily eliminating bacterial infection. Conversely, risk factors might predispose to bovine respiratory disease by triggering a loss of tolerance that results in a harmful inflammatory and tissue-damaging response to infection.

Effect of tracheal antimicrobial peptide on the development of Mannheimia haemolytica pneumonia in cattle.

Bacterial pneumonia causes significant economic loss to the beef industry and occurs at times of stress and viral infection. Administering antibiotics to at-risk calves is often used to prevent the disease, but alternatives to mass treatment with antibiotics are needed. Tracheal antimicrobial peptide (TAP), a ß-defensin naturally produced by bovine airways, has bactericidal activity against the pathogens that cause pneumonia in cattle. However, TAP expression is suppressed by glucocorticoid (stress) and viral infection. We hypothesized that delivering TAP to the respiratory tract would prevent development of pneumonia in calves infected with Mannheimia haemolytica. Clean-catch calves (i.e. obtained prior to contact with the dam) were challenged by aerosol with M. haemolytica, and TAP or water was delivered to the respiratory tract at 0.3, 2 and 6 hours post-infection. TAP treatment did not protect against development of disease. Calves treated with TAP had similar bacterial loads in the nasal cavity and lung compared to calves treated with water. Similarly, TAP treatment did not affect the development of clinical signs, elevated rectal temperatures, or increased levels of blood neutrophils, haptoglobin and fibrinogen that occurred after bacterial challenge. Postmortem gross and histologic lung lesions were also similar in the two groups. To determine why there was a lack of protective effect, we tested the effect of substances in respiratory lining fluid on the bactericidal activity of TAP. Physiologic concentrations of sodium chloride inhibited TAP bactericidal activity in vitro, as did serum at concentrations of 0.62 to 2.5%, but concentrated bronchoalveolar lavage fluid had no consistent effect. These findings suggest that TAP does not have in vivo bactericidal activity against M. haemolytica because of interference by physiological sodium chloride levels and by serum. Thus, administration of TAP may not be effective for prevention of M. haemolytica pneumonia.

Development of an aerosolized Mannheimia haemolytica experimental pneumonia model in clean-catch colostrum-deprived calves.

Mannheimia haemolytica is an important cause of bovine respiratory disease (BRD). BRD is usually a multifactorial disease with host factors and viral infections influencing pathogenesis. Previous studies that have attempted to experimentally induce pneumonia using aerosolized M. haemolytica alone have produced inconsistent results, yet an aerosol model would be useful to study the details of early infection and to investigate the role of innate defences in pathogenesis. The objective of these studies was to develop and characterize an aerosolized M. haemolytica disease model. In an initial study, conventionally raised calves with higher levels of antibody against M. haemolytica leukotoxin developed acute respiratory distress and diffuse alveolar damage, but did not develop bronchopneumonia, following challenge with M. haemolytica serotype 1. Clean-catch colostrum-deprived calves challenged with 1 × 1010 colony forming units of M. haemolytica serotype 1 consistently developed bronchopneumonia, with elevations in rectal temperature, serum haptoglobin, plasma fibrinogen, and blood neutrophils. Mannheimia haemolytica serotype 1 was consistently isolated from the nasal cavities and lungs of challenged calves. Despite distribution of aerosol and isolation of M. haemolytica in all lung lobes, gross lesions were mainly observed in the cranioventral area of lung. Gross and histologic lesions included neutrophilic bronchopneumonia and fibrinous pleuritis, with oat cells (necrotic neutrophils with streaming nuclei), and areas of coagulative necrosis, which are similar to lesions in naturally occurring BRD. Thus, challenge with M. haemolytica serotype 1 and use of clean-catch colostrum-deprived calves with low or absent antibody titres allowed development of an effective aerosol challenge model that induced typical clinical disease and lesions.

Observational Study Design in Veterinary Pathology, Part 2: Methodology.

Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research-histopathologic scoring, immunohistochemistry, and polymerase chain reaction-to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.

Observational Study Design in Veterinary Pathology, Part 1: Study Design.

Observational studies are the basis for much of our knowledge of veterinary pathology and are highly relevant to the daily practice of pathology. However, recommendations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offer advice on planning and conducting an observational study with examples from the veterinary pathology literature. Investigators should recognize the importance of creativity, insight, and innovation in devising studies that solve problems and fill important gaps in knowledge. Studies should focus on specific and testable hypotheses, questions, or objectives. The methodology is developed to support these goals. We consider the merits and limitations of different types of analytic and descriptive studies, as well as of prospective vs retrospective enrollment. Investigators should define clear inclusion and exclusion criteria and select adequate numbers of study subjects, including careful selection of the most appropriate controls. Studies of causality must consider the temporal relationships between variables and the advantages of measuring incident cases rather than prevalent cases. Investigators must consider unique aspects of studies based on archived laboratory case material and take particular care to consider and mitigate the potential for selection bias and information bias. We close by discussing approaches to adding value and impact to observational studies. Part 2 of the series focuses on methodology and validation of methods.

Bovine neutrophils in health and disease.

Bovine neutrophils have similarities to those of other species with respect to mechanisms of their activation and migration into tissue, modulation of immune responses and the balance between microbial killing and host tissue damage. However, bovine neutrophils have biochemical and functional differences from those of other species, which may yield insights about the comparative biology of neutrophils. Neutrophils play protective and harmful roles in the infectious diseases of cattle that occur at times of transition: respiratory disease in beef calves recently arrived to feedlots and mastitis and other diseases of postparturient dairy cows. An important research focus is the mechanisms by which risk factors for these diseases affect neutrophil function and thereby lead to disease and the prospect of genetic or pharmacologic improvement of disease resistance. Further, in keeping with the One Health paradigm, cattle can be considered a model for studying the role of neutrophils in naturally occurring diseases caused by host-adapted pathogens and are thus an intermediary between studies of mouse models and investigations of human disease. Finally, the study of bovine neutrophils is important for agriculture, to understand the pathogenesis of these production-limiting diseases and to develop novel methods of disease prevention that improve animal health and reduce the reliance on antimicrobial use.

Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats.

Bordetella bronchiseptica frequently causes nonfatal tracheobronchitis, but its role in fatal pneumonia is less recognized. Our study evaluated histologic identification of cilia-associated bacteria as a method for diagnosis of B. bronchiseptica pneumonia. Cases of fatal bronchopneumonia were studied retrospectively, excluding neonates and cases of aspiration pneumonia, minor lung lesions, or autolysis. The study population comprised 36 canine and 31 feline cases of bronchopneumonia. B. bronchiseptica was identified in 8 of 36 canine and 14 of 31 feline cases based on immunohistochemistry (IHC) using serum from a rabbit hyperimmunized with pertactin, PCR testing (Fla2/Fla12), and/or bacterial culture data when available. Of these, IHC was positive in 4 canine and 7 feline cases, PCR was positive in 8 canine and 14 feline cases, and B. bronchiseptica was isolated in 2 of 5 canine and 3 of 9 feline cases tested. Examination of histologic sections stained with hematoxylin and eosin revealed bronchial cilia-associated bacteria in 4 of 36 canine and 5 of 31 feline cases; these were all positive by IHC and PCR. The presence of cilia-associated bacteria had been noted in the pathology report for only 2 of these 9 cases. Thus, the presence of cilia-associated bacteria seems frequently overlooked by pathologists, but is a diagnostically significant feature of B. bronchiseptica pneumonia. A specific diagnosis of B. bronchiseptica pneumonia is important because it suggests primary or opportunistic bacterial pneumonia rather than aspiration pneumonia, and because of the risk of animal-to-animal transmission of B. bronchiseptica, the availability of vaccines for disease prevention, and the potential zoonotic risk to immunocompromised pet owners.

Regulation of tracheal antimicrobial peptide gene expression in airway epithelial cells of cattle.

ß-defensins are an important element of the mucosal innate immune response against bacterial pathogens. Tracheal antimicrobial peptide (TAP) has microbicidal activity against the bacteria that cause bovine respiratory disease, and its expression in tracheal epithelial cells is upregulated by bacterial products including lipopolysaccharide (LPS, a TLR4 agonist), Pam3CSK4 (an agonist of Toll-like receptor 2/1), and interleukin (IL)-17A. The objectives of this study were to identify the signalling pathway by which LPS, Pam3CSK4 and IL-17A induce TAP gene expression, and to determine the effect of glucocorticoid as a model of stress on this epithelial innate immune response. In primary cultures of bovine tracheal epithelial cells (bTEC), LPS, Pam3CSK4 and IL-17A each stimulated TAP gene expression. This effect was abrogated by caffeic acid phenylester (CAPE), an inhibitor of NF-κB. Similarly, western analysis showed that LPS, Pam3CSK4 and IL-17A each induced translocation of NF-κB p65 from the cytoplasm to the nucleus, but pre-treatment with CAPE inhibited this response. Finally, pre-treatment of bTEC with the glucocorticoid dexamethasone abolished the stimulatory effect of LPS, Pam3CSK4 and IL-17A on upregulation of TAP gene expression. These findings indicate that NF-κB activation is necessary for induction of TAP gene expression by LPS (a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist), or IL-17A. Furthermore, this stimulatory response is inhibited by glucocorticoid, suggesting this as one mechanism by which stress increases the risk of bacterial pneumonia. These findings have implications for understanding the pathogenesis of stress-associated bacterial pneumonia, and for developing methods to stimulate innate immune responses in the respiratory tract of cattle.

Diagnosis and Management of Polycythemia Vera in a Ferret (Mustela putorius furo).

A 5-y-old female ferret (Mustela putorius furo) was evaluated for diarrhea, anorexia, and lethargy for 1 wk. Only mild dehydration was detected on physical examination. CBC analysis revealed marked erythrocytosis with an unremarkable plasma biochemistry panel; follow-up CBC analyses revealed a consistent primary erythrocytosis. Whole-body radiographs and abdominal ultrasonography were unremarkable except for a small nephrolith in the right kidney and a small cyst in the left kidney. The plasma erythropoietin level was 17.0 mIU/mL and considered normal. In light of the diagnostic work-up and consistent erythrocytosis, a diagnosis of polycythemia vera (primary erythrocytosis) was made. The initial presentation of diarrhea resolved after treatment with oral metronidazole (20 mg/kg PO BID for 7 d). Treatment for the polycythemia consisted of a phlebotomy initially followed by chemotherapy with hydroxyurea (10 mg/kg PO BID). During the subsequent 12 mo, the hydroxyurea dose adjusted according to follow-up CBC results, and finding an optimal dosage regimen proved to be challenging. One year after the initial diagnosis, the ferret presented to an emergency clinic for acute and severe hemorrhagic diarrhea and died shortly thereafter. The postmortem diagnosis was acute venous infarction of the small and large intestine. To our knowledge, this report is the first to describe the diagnosis and long-term management of polycythemia vera in a ferret and the use of hydroxyurea for this purpose.