Evaluating the ability of non-tuberculous mycobacteria to induce non-specific reactions in bovine tuberculosis diagnostic tests in guinea pigs and cattle.

Limitations in diagnostic test performance are one of the major challenges hampering the eradication of bovine tuberculosis (bTB). Non-tuberculous mycobacteria (NTM) are considered one of the main causes of non-specific reactions in the intradermal tuberculin test, the most widely used bTB diagnostic test. To determine the role of NTMs in bTB misdiagnosis in Spain, an experimental study including the NTM species most commonly found in bTB-positive animals from bTB-free farms in the country (M. avium subsp. avium (Maa), "Mycobacterium avium subsp. hominissuis" (Mah), M. bourgelatii, M. intermedium, M. kansasii and M. nonchromogenicum) was carried out on guinea pigs and cattle. First, guinea pigs were sensitized with the selected NTMs, and six weeks post-sensitization four antigen mixtures (bovine-PPD, avian-PPD, P22 and ESAT6-CFP10) were inoculated intradermally and their effect was measured 24- and 48-h post-inoculation. Larger erythematous reactions were observed in guinea pigs sensitized with Mah, M. kansasii, and Maa, with significant differences in the reactions measured at the bovine-PPD inoculation site for the two first bacteria compared with other NTMs. The sensitization process was repeated in cattle, and five months post-sensitization the same antigen mixtures were inoculated in the cervical region and responses were measured at 48- and 72-h post-inoculation. A significantly higher increase in the skinfold thickness measured at the bovine-PPD inoculation site was observed in calves sensitized with Mah, Maa, M. intermedium and M. kansasii. These results demonstrate that certain NTM species may play a more significant role in bTB diagnostic interferences and show that results obtained in guinea pig and bovine models do not always coincide.

Prospective, randomized, clinical trial on the effects of laparoscopic insufflation pressures on portal pressures in dogs.

OBJECTIVE: The adverse effects of intra-abdominal pressure from capnoperitoneum on cardiovascular and pulmonary systems have been well documented, but the effects on portal pressures in dogs with various insufflation pressures is poorly defined. The aim of the present study was to measure the effect of a range of insufflation pressures on the portal pressure, using direct pressure measurements in patients undergoing laparoscopy. STUDY DESIGN: Clinical randomized prospective study. ANIMALS: Nine client-owned dogs undergoing routine laparoscopy. METHODS: Two rounds of direct portal pressure assessments were performed, at insufflation pressures of 0, 6, 10, and 14 mmHg in a predetermined randomized sequence. The data were analyzed for effects of insufflation pressure, hemodynamic alterations, and round. A best-fit exponential model of the relationship between portal pressure and insufflation pressure was created. RESULTS: Portal pressure increased by 38% at 6 mmHg, 95% at 10 mmHg, and 175% at 14 mmHg compared to baseline. Portal pressure increased at an average rate of 7.45% per mmHg of insufflation pressure. Effects of weight, weight/insufflation pressure interaction, and round of insufflation were not statistically significant. No systemic hemodynamic adverse events were observed. CONCLUSION: Portal pressure increased as insufflation pressure increased. There was no clinically significant difference in baseline portal pressure between rounds of insufflation. CLINICAL SIGNIFICANCE: This exponential model of portal pressure supports the use of the minimum insufflation pressure to allow visualization during laparoscopy. The return of portal pressure to baseline following desufflation supports the comparison of portal pressure measurements before and after laparoscopic shunt attenuation.

Clustering analysis of lipoprotein profiles to identify subtypes of hypertriglyceridemia in Miniature Schnauzers.

BACKGROUND: Hypertriglyceridemia (HTG) is prevalent in Miniature Schnauzers, predisposing them to life-threatening diseases. Varied responses to management strategies suggest the possibility of multiple subtypes. HYPOTHESIS/OBJECTIVE: To identify and characterize HTG subtypes in Miniature Schnauzers through cluster analysis of lipoprotein profiles. We hypothesize that multiple phenotypes of primary HTG exist in this breed. ANIMALS: Twenty Miniature Schnauzers with normal serum triglyceride concentration (NTG), 25 with primary HTG, and 5 with secondary HTG. METHODS: Cross-sectional study using archived samples. Lipoprotein profiles, generated using continuous lipoprotein density profiling, were clustered with hierarchical cluster analysis. Clinical data (age, sex, body condition score, and dietary fat content) was compared between clusters. RESULTS: Six clusters were identified. Dogs with primary HTG were dispersed among 4 clusters. One cluster showed the highest intensities for triglyceride-rich lipoprotein (TRL) and low-density lipoprotein (LDL) fractions and also included 4 dogs with secondary HTG. Two clusters had moderately high TRL fraction intensities and low-to-intermediate LDL intensities. The fourth cluster had high LDL but variable TRL fraction intensities with equal numbers of NTG and mild HTG dogs. The final 2 clusters comprised only NTG dogs with low TRL intensities and low-to-intermediate LDL intensities. The clusters did not appear to be driven by differences in the clinical data. CONCLUSIONS AND CLINICAL IMPORTANCE: The results of this study support a spectrum of lipoprotein phenotypes within Miniature Schnauzers that cannot be predicted by triglyceride concentration alone. Lipoprotein profiling might be useful to determine if subtypes have different origins, clinical consequences, and response to treatment.

A behavior screening questionnaire improves problem identification in veterinary primary care with implications for patient health.

OBJECTIVE: To compare how often owners identified concerns about their pets' behavior during routine examinations in 2 test conditions (voluntarily or when completing a questionnaire), and to test the efficacy of a simple behavior screening questionnaire to be used by practicing veterinarians. ANIMALS: Privately owned dogs and cats presenting for routine examinations to 1 of 2 primary care clinics. METHODS: The study was conducted in 2 sequential phases. In Phase 1, veterinary staff recorded whether pet owners or veterinarians inquired about behavior during routine examinations. In Phase 2, a different set of pet owners completed a short behavioral screening questionnaire as they waited for the veterinarian. We compared the 2 phases for how often behavior concerns were identified, the types of concerns, the pets' ages, and the owners' levels of satisfaction and desire for help. RESULTS: Dog and cat owners were more likely to identify behavior concerns when prompted than they were to volunteer this information, especially for older patients in which behavior changes may be the first sign of disease. Most owners were satisfied with their pets' behavior, though owners were more likely to be unsatisfied with certain identified concerns. Owners who were dissatisfied were much more likely to want help. CLINICAL RELEVANCE: A behavior screening questionnaire enhances detection of behavior problems that may have medical implications and impact the security of the pet in its home. A behavior screening questionnaire can elevate standard of care by enabling veterinarians to quickly assess behavior during every examination.

Evaluation of the efficacy of the Lentulo spiral filler operated at four different speeds and with two different techniques in cadaveric canine teeth of dogs.

This study evaluated the effect of filling technique and rotational speed on the efficacy of sealer cement placement using the Lentulo spiral filler. Cadaveric maxillary and mandibular canine teeth (n = 74) from age and breed matched fresh-frozen adult canine cadaver heads were utilized. Following routine mechano-chemical preparation, teeth were randomly divided into 8 treatment groups with varying rotational speeds [250-2,000 revolutions per minute (RPM)] and fill techniques (backfill technique versus pumping technique). The quality of the sealer cement fill was evaluated radiographically before and after master gutta percha cone placement. Percentage of voids present in the apical, midbody, and coronal portions of the tooth were subjectively scored as: no voids present, voids present in less than 25% of the area, voids present in 25-50% of the area, voids present in greater than 50% of the area. The apex was also scored as underfilled, adequately filled, or overfilled. Operating the Lentulo spiral filler at 2,000 RPM resulted in the best quality fill regardless of filling technique. The pumping technique with placement of the master gutta percha cone had a higher probability of success as compared to the backfill technique, but this finding lacked significance. Regardless of speed or technique, the apical region had the highest quality of fill. The addition of the master gutta percha cone improved the quality of fill. Therefore, we recommend using the Lentulo spiral filler at higher speeds with a pumping technique (followed by addition of a master gutta percha cone) to improve the quality of epoxy resin-based sealer cement placement.

Cell Count and Cell Density Decrease as Age Increases in Cadaveric Pediatric Medial Menisci.

Purpose: To examine the histologic changes in terms of cellularity, cell density, and nuclear shape in medial meniscal cellularity during skeletal development using pediatric cadaver specimens. Methods: Medial menisci from 26 pediatric cadavers, 11 female and 15 male (total 36 menisci), were obtained from tissue bank. Mean age of female donors was 34 months (1-108 months) and of male donors was 52 months (1-132 months). Menisci were processed and embedded in paraffin blocks. Each tissue block containing 6 representative areas of meniscus (anterior root, anterior horn, body [n = 2], posterior horn, and posterior root) was sectioned at 4 microns and stained with hematoxylin and eosin for evaluation of chondrocyte nuclei. Each of the 6 representative areas was imaged at 10×; one image on peripheral one-third of section, the second image on central two-thirds of the section. FIJI imaging software was used to measure cell count, cell density, and nuclear morphology (1 = perfect circle). Data analysis included linear mixed models, Type II analysis of variance tests, and pairwise tests with the Tukey correction to assess statistical significance. Results: Peripheral meniscus was more cellular than central meniscus. The cell count was found to decrease by 14% per year of age. Peripheral cell count decreased at a rate similar to the cell count in the central meniscus. Meniscal cell density was 2× higher peripherally than centrally. Overall average cell density in all locations in the menisci decreased by an average of 14% per year of age. Conclusions: The results of this study reveal decreases in cell count, cell density, and circularity as age increases in cadaveric pediatric medial menisci. Clinical Relevance: To better understand the development of pediatric menisci at a cellular level and use this knowledge in the future on how to maintain the menisci in a younger, healthier state.

Clinical success of guided tissue regeneration for treating vertical bone and furcation defects in dogs.

This study evaluated the clinical success rate of guided tissue regeneration (GTR) for treating advanced periodontal disease in a large canine cohort. A total of 112 GTR procedures performed from 2003-2021 were retrospectively evaluated, including pre- and post-treatment (3-12 months) periodontal probing depths of 104 treated teeth, dental radiographs of 73 treated teeth, and both diagnostic modalities in 64 treated teeth. Probing depth, radiographically apparent bone height, bone graft material, barrier membrane material, and tooth extraction adjacent to the GTR site were investigated as factors affecting success. Vertical bone defects were evaluated separately from furcation defects. GTR was clinically successful, defined as objective improvement in probing depth, objective decrease in radiographic vertical bone defect, and subjective radiographic gain in bone height in 90.3% of vertical bone defects. Success was significantly associated with the magnitude of initial probing depth and the type of barrier membrane used. GTR was clinically successful, defined as objective improvement in furcation probing and subjective radiographic improvement of the bone in the furcation in 22.2% of furcation defects. When F3 lesions were excluded, GTR was successful in 64.3% of furcation defects. GTR is an appropriate treatment to maintain teeth in the oral cavity of dogs with proper client counseling and patient selection, but it is most likely to be successful in vertical defects.

Retrospective evaluation of the incidence of presumed feline urethral obstruction during a prepandemic year compared to a pandemic year.

OBJECTIVE: To investigate the influence of the coronavirus disease 2019 (COVID-19) pandemic on the incidence of urethral obstruction (UO) in male cats. DESIGN: Retrospective study. SETTING: One veterinary university teaching hospital and 6 private practice veterinary specialty and emergency centers. ANIMALS: A total of 24,937 total feline cases presenting to the emergency room (ER) between March 2019 and March 2021. MEASUREMENTS AND MAIN RESULTS: Out of 24,937 total cases, 1793 male cats met the inclusion criteria for diagnosis of UO. Of those, 327 cases were identified in which an additional diagnosis of either urolithiasis or neoplasia was made and were therefore excluded. The remaining 1466 UO cases were presumed to be idiopathic urethral obstruction (iUO) caused by feline interstitial cystitis (FIC) alone. Of those, 637 cats presented during the prepandemic year and 829 cats presented during the pandemic year. KEY FINDINGS: Incidence of presumptive iUO increased by 30% during the COVID-19 pandemic. Total feline emergency caseload increased by 38%. iUO accounted for 6.08% and 5.73% of total feline emergency cases during the prepandemic and pandemic years, respectively. CLINICAL SIGNIFICANCE: The reported increase in incidence of feline UO is likely due to the increase in overall emergency feline caseload.

Evaluation of an HMGA2 variant contribution to height and basal insulin concentrations in ponies.

BACKGROUND: The HMGA2:c.83G>A variant was identified in Welsh ponies having pleiotropic effects on height and insulin concentration. OBJECTIVE: Determine whether the HMGA2:c.83G>A variant is associated with decreased height and higher basal insulin concentrations across pony breeds. ANIMALS: Two hundred thirty-six ponies across 6 breeds. METHODS: Cross-sectional study. Ponies were genotyped for the HMGA2:c.83G>A variant and phenotyped for height and basal insulin concentrations. Stepwise regression was performed for model analysis using a linear regression model for height and mixed linear model for insulin with farm as a random effect. Coefficient of determination, pairwise comparison of the estimated marginal means and partial correlation coefficients (parcor) were calculated to assess the relationship between HMGA2 genotype and height or insulin. RESULTS: Breed and genotype accounted for 90.5% of the variation in height across breeds, and genotype explained 21% to 44% of the variation within breeds. Breed, genotype, cresty neck score, sex, age, and farm accounted for 45.5% of the variation in insulin, with genotype accounting for 7.1%. The HMGA2 A allele frequency was 62% and correlated with both height (parcor = -0.39; P < .001) and insulin (parcor = 0.22; P = .02). Pairwise comparisons found A/A ponies were >10 cm shorter than other genotypes. Compared with G/G individuals, A/A and G/A individuals had 4.3 µIU/mL (95% confidence interval [CI]: 1.8-10.5) and 2.7 µIU/mL (95% CI: 1.4-5.3) higher basal insulin concentrations, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: These data demonstrate the pleiotropic effects of the HMGA2:c.83G>A variant and its role in identifying ponies at increased risk for insulin dysregulation.

Expression and Prognostic Evaluation of the Receptor Tyrosine Kinase MET in Canine Malignant Melanoma.

The overexpression and activation of the MET receptor tyrosine kinase has been identified in many human malignancies, but its role in canine cancer has only been minimally investigated. In this study we evaluated the expression of MET in two canine malignant melanoma (CMM) cell lines as well as in 30 CMM tissue samples that were collected from the clinical service at our institution. We were able to confirm the expression of the MET protein in both melanoma cell lines, and we demonstrated MET activation by its ligand, HGF, through phosphorylation, in Western blot analysis. We were also able to demonstrate, by immunohistochemistry, the expression of MET in 63% of the tumor tissue samples analyzed, with the majority demonstrating a relatively low expression profile. We then evaluated the association of MET expression scores with histologic parameters, metastasis, and survival. While statistically significant associations were not found across these parameters, an inverse relationship between MET expression levels and time to lymph node versus distant metastasis was suggested in our cohort. These findings may require assessment in a larger group of specimens to further evaluate the role of MET expression in the homing of metastasis in lymph nodes versus that in distant organs.

A retrospective evaluation of hops ingestion in 177 dogs (2005-2018).

OBJECTIVE: To describe the clinical signs and outcomes observed after Humulus lupulus (hops) ingestion in dogs. A secondary objective was to note any trends in the number of hops-related phone calls made to an animal poison control center over a 13-year period. DESIGN: Retrospective study (2005-2018). SETTING: An animal poison control center. ANIMALS: One hundred and seventy-seven dogs with known or suspected hops ingestion. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 177 calls were made to Pet Poison Helpline between 2005 and 2018 involving hops ingestion in dogs. Outcomes were determined in 83 cases; 79 of 83 (95.2%) survived. Clinical signs associated with hops ingestion were observed in 74.0% (131/177). Commonly observed clinical signs were tachypnea (98/131), hyperthermia (65/131), and vomiting (44/131). Severe hyperthermia (>41.4°C, [>106°F]) developed in 8 dogs and 3 of those dogs did not survive. A fourth nonsurvivor was found deceased at home. The majority of symptomatic dogs developed clinical signs between 2 and 8 hours postingestion. Resolution of clinical signs occurred in less than 24 hours in all survivors except in one. Cases consulted with Pet Poison Helpline related to hops ingestion increased from 2005 to 2018 relative to the total amount of cases managed overall. CONCLUSIONS: The most common clinical signs associated with hops ingestion include tachypnea, hyperthermia, and vomiting; however, not all dogs develop clinical signs. While prognosis is good with 95.2% of dogs in this population surviving, some dogs can develop a severe and fatal hyperthermia.

Effect of Holstein genotype on ex-vivo interleukin-1ß response to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and heat-killed Gram-negative and Gram-positive bacteria.

Effects of Holstein genotype on interleukin-1ß response were assessed by ex-vivo stimulation of whole blood with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or sonicated, heat-killed Gram-negative or Gram-positive bacteria. Holstein genotypes were unselected Holsteins (UH, n = 14) not subjected to selection pressures since the mid-1960s and contemporary Holsteins (CH, n = 13). Milk yield of UH and CH cows differ by more than 4500 kg/lactation. Whole blood was mixed with 0.01 µg LPS, 10 µg LTA or 2.5 × 106 CFU of sonicated, heat-killed E. coli, K. pneumoniae, S. marcescens, S. aureus, S. dysgalactiae, or S. uberis per mL of blood and incubated (4 h, 37 °C). Plasma IL-1ß was quantified by ELISA and log10-transformed concentrations analyzed with a multivariate linear mixed effects model. Responses to bacteria were greater than responses to LPS or LTA. Responses to LPS, LTA and the Gram-negative stimulants were greater in UH than in CH cows while responses to the Gram-positive bacteria did not differ between Holstein genotypes. In both genotypes, strong correlations were detected among IL-1ß responses to the Gram-negative stimulants and to LTA. There were strong correlations among IL-1ß responses to the Gram-positive bacteria in CH cows but only between S. aureus and S. dysgalactiae in UH cows. The IL-1ß response to S. uberis was highly correlated with responses to all of the Gram-negative stimulants in CH cows but only with E. coli in the UH cows. The reduced immune response could make contemporary cows more susceptible to infection by Gram-negative bacteria. Results confirm selection practices since the mid-1960s have altered immune response in the Holstein, at least to Gram-negative bacteria, and validate the need for additional studies to further evaluate the impacts of these selection practices on immune function in contemporary Holsteins.

Assessment of anti-CD20 antibody pre-treatment for augmentation of CAR-T cell therapy in SIV-infected rhesus macaques.

During chronic HIV and SIV infections, the majority of viral replication occurs within lymphoid follicles. In a pilot study, infusion of SIV-specific CD4-MBL-CAR-T cells expressing the follicular homing receptor, CXCR5, led to follicular localization of the cells and a reduction in SIV viral loads in rhesus macaques. However, the CAR-T cells failed to persist. We hypothesized that temporary disruption of follicles would create space for CAR-T cell engraftment and lead to increased abundance and persistence of CAR-T cells. In this study we treated SIV-infected rhesus macaques with CAR-T cells and preconditioned one set with anti-CD20 antibody to disrupt the follicles. We evaluated CAR-T cell abundance and persistence in four groups of SIVmac239-infected and ART-suppressed animals: untreated, CAR-T cell treated, CD20 depleted, and CD20 depleted/CAR-T cell treated. In the depletion study, anti-CD20 was infused one week prior to CAR-T infusion and cessation of ART. Anti-CD20 antibody treatment led to temporary depletion of CD20+ cells in blood and partial depletion in lymph nodes. In this dose escalation study, there was no impact of CAR-T cell infusion on SIV viral load. However, in both the depleted and non-depleted animals, CAR-T cells accumulated in and around lymphoid follicles and were Ki67+. CAR-T cells increased in number in follicles from 2 to 6 days post-treatment, with a median 15.2-fold increase in follicular CAR-T cell numbers in depleted/CAR-T treated animals compared to an 8.1-fold increase in non-depleted CAR-T treated animals. The increase in CAR T cells in depleted animals was associated with a prolonged elevation of serum IL-6 levels and a rapid loss of detectable CAR-T cells. Taken together, these data suggest that CAR-T cells likely expanded to a greater extent in depleted/CAR-T cell treated animals. Further studies are needed to elucidate mechanisms mediating the rapid loss of CAR-T cells and to evaluate strategies to improve engraftment and persistence of HIV-specific CAR-T cells. The potential for an inflammatory cytokine response appears to be enhanced with anti-CD20 antibody treatment and future studies may require CRS control strategies. These studies provide important insights into cellular immunotherapy and suggest future studies for improved outcomes.

Nine-axis inertial measurement unit output discriminates activities of varying intensity in the dog.

OBJECTIVE: To explore relationships between 9-axis inertial measurement unit (IMU) output and activities of varying intensity in dogs of various sizes. ANIMALS: 20 healthy, agility course-trained dogs of various ages and sizes. PROCEDURES: Height, weight, body condition score, age, length from IMU to the ischium, and height of IMU to the floor were recorded. Dogs performed a series of activities (rest, walk, trot, and agility course) while wearing the IMU device. IMU and video output were reviewed by independent investigators. Correlations and multiple regression models were used to explore relationships between independent variables and IMU output. RESULTS: Calibration demonstrated excellent correlation and concordance between IMUs (intraclass correlation > 0.9) and that the IMUs reliably measured a known acceleration (gravity at rest). Resultant vector magnitude {sqrt[(x^2) + (y^2) + (z^2)]} normalized to body size was calculated from the data. IMU output clearly discriminates between activities of varying intensity in the dog. CLINICAL RELEVANCE: The inability to accurately measure chronic pain is a barrier to the development of new, or critical evaluation of, therapeutics. Activity monitors (AM) may be the ideal diagnostic target since they are small and provide objective data that can be collected while the pet remains in its natural environment. These results demonstrate the concurrent and predictive validity of the IMU tested. Our long-range goal is to validate an open-source algorithm for the IMU so activity in a pet's natural environment can be used as an outcome measure in future studies.

Retrospective study of 20 cats surgically treated for insulinoma.

OBJECTIVE: To report the clinical signs, histopathology results, and prognostic factors for outcomes following excision for feline insulinoma (INS). STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Twenty client-owned cats. METHODS: Medical records from 2006 to 2020 were reviewed by Veterinary Society of Surgical Oncology members for cats with hypoglycemia resulting from INS, with surgical excision and follow up. Clinical signs and histopathology results were summarized. Factors potentially related to disease-free interval (DFI), disease-related death (DRD), and overall survival time (OST) were analyzed with a Cox proportional hazards regression analysis. RESULTS: All cats were hypoglycemic on presentation with neurologic signs in 18 out of 20 and inappropriate insulin levels in 12/13. Excision of insulinomas resulted in immediate euglycemia or hyperglycemia in 18 cats. Eighteen cats survived to hospital discharge. The median time to death or last postoperative follow up was 664 days (range: 2-1205 days). Prognostic factors included age at presentation (for DFI); time to postoperative euglycemia (for DRD); preoperative and postoperative serum blood glucose concentrations; metastasis at the time of surgery (DFI and DRD), and histopathologic tumor invasion (for OST). The median OST for all cats was 863 days. The 1-, 2- and 3-year survival rates were 75%, 51%, and 10%, respectively. CONCLUSION: Excision of insulinoma resulted in euglycemia or hyperglycemia in most cats. Negative prognostic factors included young age, low serum glucose concentrations, metastasis at time of surgery, tumor invasion, and shorter time to euglycemia. CLINICAL SIGNIFICANCE: Surgical excision resulted in survival times comparable to those of canine INS.

Development of an anti-CAR antibody response in SIV-infected rhesus macaques treated with CD4-MBL CAR/CXCR5 T cells.

T cells expressing a simian immunodeficiency (SIV)-specific chimeric antigen receptor (CAR) and the follicular homing molecule, CXCR5, were infused into antiretroviral therapy (ART) suppressed, SIV-infected rhesus macaques to assess their ability to localize to the lymphoid follicle and control the virus upon ART interruption. While the cells showed evidence of functionality, they failed to persist in the animals beyond 28 days. Development of anti-CAR antibodies could be responsible for the lack of persistence. Potential antigenic sites on the anti-SIV CAR used in these studies included domains 1 and 2 of CD4, the carbohydrate recognition domain (CRD) of mannose-binding lectin (MBL), and an extracellular domain of the costimulatory molecule, CD28, along with short linker sequences. Using a flow cytometry based assay and target cells expressing the CAR/CXCR5 construct, we examined the serum of the CD4-MBL CAR/CXCR5-T cell treated animals to determine that the animals had developed an anti-CAR antibody response after infusion. Binding sites for the anti-CAR antibodies were identified by using alternative CARs transduced into target cells and by preincubation of the target cells with a CD4 blocking antibody. All of the treated animals developed antibodies in their serum that bound to CD4-MBL CAR/CXCR5 T cells and the majority were capable of inducing an ADCC response. The CD4 antibody-blocking assay suggests that the dominant immunogenic components of this CAR are the CD4 domains with a possible additional site of the CD28 domain with its linker. This study shows that an anti-drug antibody (ADA) response can occur even when using self-proteins, likely due to novel epitopes created by abridged self-proteins and/or the self-domain of the CAR connection to a small non-self linker. While in our study, there was no statistically significant correlation between the ADA response and the persistence of the CD4-MBL CAR/CXCR5-T cells in rhesus macaques, these findings suggest that the development of an ADA response could impact the long-term persistence of self-based CAR immunotherapies.

HIV-Specific CAR T Cells with CD28 or 4-1BB Signaling Domains Are Phenotypically and Functionally Distinct and Effective at Suppressing HIV and Simian Immunodeficiency Virus.

Despite mounting a robust antiviral CD8 T cell response to HIV infection, most infected individuals are unable to control HIV viral load without antiretroviral therapy (ART). Chimeric Ag receptor (CAR) T cell treatment is under intensive investigation as an alternative therapy for ART-free remission of chronic HIV infection. However, achieving durable remission of HIV will require a successful balance between CAR T cell effector function and persistence. CAR T cells with CD28 costimulatory domains have robust effector function but limited persistence in vivo, whereas CAR T cells with 4-1BB costimulatory domains present a more undifferentiated phenotype and greater in vivo persistence. We compared the in vitro phenotype and function of rhesus macaque and human CAR T cells that contained either the CD28 or 4-1BB costimulatory domain; both constructs also included CARs that are bispecific for gp120 of HIV or SIV and the CXCR5 moiety to promote in vivo homing of CAR/CXCR5 T cells to B cell follicles. Cells were transduced using a gammaretroviral vector and evaluated using flow cytometry. 4-1BB-CAR/CXCR5 T cells were phenotypically distinct from CD28-CAR/CXCR5 T cells and showed increased expression of CAR and CD95. Importantly, both CD28- and 4-1BB-CAR/CXCR5 T cells retained equal capacity to recognize and suppress SIV in vitro. These studies provide new insights into rhesus macaque and human 4-1BB- and CD28-bearing CAR T cells.

Evaluation of dam parity and internal biosecurity practices in influenza infections in piglets prior to weaning.

Influenza is an important respiratory disease of pigs and humans. Controlling influenza in pigs is challenging due to the substantial genetic diversity of influenza A virus (IAV). In this study, we assessed the impact of internal biosecurity practices directed at limiting exposure of piglets to IAV before weaning; evaluated the association of sow parity with IAV prevalence in piglets and the levels of maternally derived antibodies (MDA), and documented the frequency of detection of IAV on farmworkers' hands and the instruments used when handling pigs. The control group included litters in rooms where no specific changes were made to standard farm procedures. The treatment group included litters in rooms where no cross-fostering or nurse sows use was allowed, and where farmworkers were required to change gloves between litters when handling pigs. Both, younger (≤ Parity 3) and older parity sows (>Parity 3) were represented in all rooms included in the study. Overall, litters in the treatment group had lower IAV prevalence (29.9 %) than litters in the control group (44.2 %) (p < 0.001), and at day 8 of age the litters from the control group had 7.5 times higher IAV prevalence than the litters from the treatment group. However, at weaning differences were not found (77.2 % vs. 81 % for treatment vs. control, respectively, p = 0.41). There were no differences in IAV detection between parity groups at any of the sampling points (p = 0.86) and incidence of detection in sows from farrowing to weaning was 29 %. Piglets that tested ELISA negative were 1.3 times more likely to test IAV positive than piglets that were ELISA positive for IAV antibody test, suggesting that effective colostrum intake may reduce the likelihood of infection. IAV was detected on 46 % of the instruments used when handling piglets and on 58 % of farmworkers' hands, indicating the potential risk for mechanical transmission of IAV in pigs. Overall, we showed that the implementation of internal biosecurity practices that limit IAV exposure to newborn piglets helped delay IAV infections but were not sufficient to reduce the prevalence of IAV infection in litters at weaning.

The anatomical relationship between the mandibular first molar roots and the mandibular canal based on breed size and skull type.

The first molar is the largest tooth in the dog mandible with roots often extending to the level of the mandibular canal (MC). The anatomical relationship between the roots and MC is variable and the normal relationship between those structures in a diverse population of dogs has not been established. The lingual location of the roots relative to the MC poses a risk for iatrogenic trauma during dentoalveolar surgery, and it is unknown if certain skull conformations are predisposed to this relationship. This study aimed to identify associations between patient weight and skull type with molar tooth root location. CT scans performed for reasons unrelated to the study were retrospectively analyzed. Subjects were sorted into one of 12 groups (n=16 per group) based on skull type (brachycephalic, mesaticephalic, and dolichocephalic) and weight (extra small: ≤ 6.8 kg, small: >6.8 to ≤ 13.6 kg, medium: >13.6 to ≤ 25 kg, and large >25 to ≤ 38.6 kg). The mandibular first molar roots were categorized as lingual, buccal, or dorsal relative to the MC. Lingual root location was diagnosed in 50.0% of all roots evaluated, and 64.2% of all dogs assessed had at least one root in the lingual position. The size was shown to be protective, with lingual root location being significantly less likely as size increased. The exception to this was in large brachycephalic patients, which had rates of lingual roots similar to smaller dogs. Buccal roots were rarest, diagnosed in only 9.7% of all roots, and were most common in brachycephalic patients, which had 83.8% of all buccal roots. Additional caution should be employed when removing alveolar bone during surgical extraction in dogs ≤ 13.6 kg and in large brachycephalic patients (boxers) to avoid iatrogenic trauma to the neurovascular bundle.

Evaluation of coated platelets, a subset of highly procoagulant platelets, in healthy dogs and dogs with neoplasia.

OBJECTIVE: To determine if dogs with neoplasia produce more coated platelets, a subpopulation of activated platelets generated by dual stimulation with thrombin and convulxin, a glycoprotein VI agonist, than healthy control dogs. ANIMALS: Client-owned dogs diagnosed with lymphoma (n = 19) or solid tumors (14) and healthy control dogs (14). PROCEDURES: Platelets were stimulated ex vivo with thrombin and convulxin. Flow cytometry was used to quantify the percentage of coated platelets based on high levels of surface fibrinogen. To compare the percentage of coated platelets between the three groups, an ANOVA was performed followed by pairwise 95% confidence intervals (CI) adjusted for multiple comparisons using Tukey's method. RESULTS: We observed a greater mean percentage of coated platelets in dogs with solid tumors, compared with healthy control dogs, by 10.9 percentage points (95% CI: -1.0, 22.8), and a mean percentage of coated platelets in dogs with lymphoma that was less than healthy control dogs by 0.3 percentage points (95% CI: -11.4, 10.8). CLINICAL RELEVANCE: This study provides the first data-based evidence that dogs with solid tumors may have a greater mean coated platelet percentage when compared with healthy control dogs, although there is overlap between groups. Further studies are needed investigating coated platelets in specific subsets of neoplasia and investigating additional mechanisms of hypercoagulability in dogs with neoplasia.