Indicators of surgical stress and influence of clinical experience, simulation models, and cadaveric laboratory on the stress response of third-year veterinary students performing first elective surgery.

OBJECTIVE: To evaluate the role of simulation models and previous surgical experience on subjective and objective stress levels of students performing their 1st elective surgery within the veterinary curriculum. SAMPLE: 141 third-year veterinary students. METHODS: Using a pre-post experimental design, salivary alpha-amylase, and cortisol were evaluated as markers of physiologic stress response before students' first elective surgery. Student self-reported State-Trait Anxiety Inventory (STAI) scores and quantitative measures of experience were correlated to biomarker results. RESULTS: No association was found for change in salivary biomarkers of stress, alpha-amylase, and cortisol, between baseline and presurgical samples accounting for gender, age, type of elective surgery performed, previous surgical experience, or simulation model use. Salivary cortisol levels were markedly elevated falling between the 66th and 99th percentile compared to an age and gender-matched population. Salivary alpha-amylase levels were also 2 to 3 times higher than those recorded by other health professionals. Veterinary student STAI scores were high falling between the 65th and 73rd percentile compared to working adults in the general population. CLINICAL RELEVANCE: Veterinary students' salivary cortisol, alpha-amylase, and STAI scores fell into the upper 2/3rds of the general population, demonstrating a high level of stress. Simulation models and previous surgical experience were not associated with decreased stress. Further evaluation of the implementation of high-fidelity simulation models and the role of stress on performance is indicated.

Optical coherence tomography for surgical margin evaluation of excised canine cutaneous and subcutaneous tumours.

Currently, intraoperative tumour margin imaging is not routinely utilized in veterinary medicine. Optical coherence tomography (OCT) allows for real-time assessment of tissue morphology of 1-2 mm depth. The aims of this study were (1) to compare the histologic and OCT features of excised canine skin and subcutaneous specimens, and (2) to determine the diagnostic accuracy of OCT for surgical margin evaluation. The authors hypothesized that OCT imaging would correlate well with histopathology and that OCT would be sensitive for detection of incomplete margins. Eighty dogs were prospectively enrolled. Tumours were excised, and the surgical margins were imaged using a spectral domain OCT system. The tumour type and completeness of excision were determined by histopathology. Nine blinded observers received training in OCT image interpretation and were then given a set of OCT images and videos. The observers assigned each image/video a grade from 1 (no tumour) to 4 (tumour) and the results were compared to histopathology. The overall median sensitivity and specificity of OCT imaging for detection of incomplete margins were 86.7% and 84.6%, respectively. A potential limitation is that observers had varied experience with OCT image interpretation, ranging from no prior experience to participating in a previous OCT project. OCT is sensitive for detection of incomplete margins and could be a promising real-time surgical margin imaging modality. Further study is needed to evaluate intraoperative applications of OCT and its impact on tumour recurrence and long-term outcome.

Renal Senescence, Telomere Shortening and Nitrosative Stress in Feline Chronic Kidney Disease.

Kidney tissues from cats with naturally occurring chronic kidney disease (CKD) and adult and senior cats without CKD were assessed to determine whether telomere shortening and nitrosative stress are associated with senescence in feline CKD. The histopathologic assessment of percent global glomerulosclerosis, inflammatory infiltrate, and fibrosis was performed. Senescence and nitrosative stress were evaluated utilizing p16 and iNOS immunohistochemistry, respectively. Renal telomere length was evaluated using telomere fluorescent in situ hybridization combined with immunohistochemistry. CKD cats were found to have significantly increased p16 staining in both the renal cortex and corticomedullary junction compared to adult and senior cats. Senior cats had significantly increased p16 staining in the corticomedullary junction compared to adult cats. p16 staining in both the renal cortex and corticomedullary junction were found to be significantly correlated with percent global glomerulosclerosis, cortical inflammatory infiltrate, and fibrosis scores. p16 staining also correlated with age in non-CKD cats. Average telomere length was significantly decreased in CKD cats compared to adult and senior cats. CKD cats had significantly increased iNOS staining compared to adult cats. Our results demonstrate increased renal senescence, telomere shortening, and nitrosative stress in feline CKD, identifying these patients as potential candidates for senolytic therapy with translational potential.

Incidence, survival time, and surgical treatment of parathyroid carcinomas in dogs: 100 cases (2010-2019).

OBJECTIVE: To evaluate outcomes of dogs with parathyroid carcinoma (PTC) treated by surgical excision and to describe the incidence of postoperative hypocalcemia, degree of hypocalcemia, duration of hospitalization, duration of calcium supplementation, and survival time. ANIMALS: 100 client-owned dogs with PTC admitted to academic, referral veterinary institutions. PROCEDURES: In a retrospective multi-institutional study, medical records of dogs undergoing surgical excision of PTC between 2010 to 2019 were reviewed. Signalment, relevant medical history, clinical signs, clinicopathologic testing, imaging, surgical findings, intraoperative complications, histologic examination, and survival time were recorded. RESULTS: 100 dogs with PTC were included, and 96 dogs had clinical or incidental hypercalcemia. Common clinical signs included polyuria (44%), polydipsia (43%), hind limb paresis (22%), lethargy (21%), and hyporexia (20%). Cervical ultrasonography detected a parathyroid nodule in 91 of 91 dogs, with a single nodule in 70.3% (64/91), 2 nodules in 25.3% (23/91), and ≥ 3 nodules in 4 (4/91)% of dogs. Hypercalcemia resolved in 89 of 96 dogs within 7 days after surgery. Thirty-four percent of dogs developed hypocalcemia, on the basis of individual analyzer ranges, within 1 week after surgery. One dog had metastatic PTC to the prescapular lymph node, and 3 dogs were euthanized for refractory postoperative hypocalcemia. Estimated 1-, 2-, and 3-year survival rates were 84%, 65%, and 51% respectively, with a median survival time of 2 years. CONCLUSIONS AND CLINICAL RELEVANCE: Excision of PTC results in resolution of hypercalcemia and excellent long-term tumor control. Surgical excision of PTC is recommended because of resolution of hypercalcemia and a good long-term prognosis. Future prospective studies and long-term follow-up are needed to further assess primary tumor recurrence, metastasis, and incidence of postoperative hypocalcemia.

In vitro holding strength of the laparoscopic Miller's knot compared with open Miller's knot, open surgeon's throw, and laparoscopic surgeon's throw in a vascular pedicle model.

OBJECTIVE: To compare in vitro knot holding strength of the laparoscopic Miller's knot (LMK), open Miller's knot (MK), open surgeon's throw (Sx), and laparoscopic surgeon's throw (LSx) in a vascular pedicle model when used as the first throw for vascular ligation. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Ten constructs each of the Miller's knot and surgeon's throw performed openly and laparoscopically with 2-0 polyglyconate suture. METHODS: Knot holding strengths of the LMK, MK, LSx, and Sx knots were evaluated on balloon dilation catheters used as vascular pedicle models. Laparoscopic knots were tied in a laparoscopic box trainer. Knot constructs were pressure tested to failure. Results were compared by Kruskal-Wallis and Steel-Dwass comparisons. RESULTS: Both MK and LMK had mean leakage pressures above 300 mm Hg. The MK leaked at higher pressure than all other knots, including the LMK (P < .001). The LMK leaked at greater pressures compared with the Sx and the LSx (P < .001). No difference was detected between leaking pressures of the Sx and the LSx (P = .226), with both leaking at pressures below 40 mm Hg. CONCLUSION: The LMK created a more secure first throw compared with the Sx and leaked at supraphysiologic pressures. CLINICAL SIGNIFICANCE: The LMK has excellent knot holding strength on a vascular pedicle model and may be further evaluated for clinical application.

The Antibiotic Neomycin Enhances Coxsackievirus Plaque Formation.

Coxsackievirus typically infects humans via the gastrointestinal tract, which has a large number of microorganisms collectively referred to as the microbiota. To study how the intestinal microbiota influences enteric virus infection, several groups have used an antibiotic regimen in mice to deplete bacteria. These studies have shown that bacteria promote infection with several enteric viruses. However, very little is known about whether antibiotics influence viruses in a microbiota-independent manner. In this study, we sought to determine the effects of antibiotics on coxsackievirus B3 (CVB3) using an in vitro cell culture model in the absence of bacteria. We determined that an aminoglycoside antibiotic, neomycin, enhanced the plaque size of CVB3 strain Nancy. Neomycin treatment did not alter viral attachment, translation, or replication. However, we found that the positive charge of neomycin and other positively charged compounds enhanced viral diffusion by overcoming the negative inhibitory effect of sulfated polysaccharides present in agar overlays. Neomycin and the positively charged compound protamine also enhanced plaque formation of reovirus. Overall, these data provide further evidence that antibiotics can play noncanonical roles in viral infections and that this should be considered when studying enteric virus-microbiota interactions.IMPORTANCE Coxsackieviruses primarily infect the gastrointestinal tract of humans, but they can disseminate systemically and cause severe disease. Using antibiotic treatment regimens to deplete intestinal microbes in mice, several groups have shown the bacteria promote infection with a variety of enteric viruses. However, it is possible that antibiotics have microbiota-independent effects on viruses. Here we show that an aminoglycoside antibiotic, neomycin, can influence quantification of coxsackievirus in cultured cells in the absence of bacteria.

Bacteria Facilitate Enteric Virus Co-infection of Mammalian Cells and Promote Genetic Recombination.

RNA viruses exist in genetically diverse populations due to high levels of mutations, many of which reduce viral fitness. Interestingly, intestinal bacteria can promote infection of several mammalian enteric RNA viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact infection of poliovirus, a model enteric virus. Most bacterial strains, including those extracted from cecal contents of mice, bound poliovirus, with each bacterium binding multiple virions. Certain bacterial strains increased viral co-infection of mammalian cells even at a low virus-to-host cell ratio. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated genetic recombination between two different viruses, thereby removing deleterious mutations and restoring viral fitness. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections.

Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies.

The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, the number of plaques is expected to correlate linearly with the virus dilution plated, and each plaque should be formed by a single founder virus. Here, we examined whether more than one virus can contribute to plaque formation. By using genetic and phenotypic assays with genetically marked polioviruses, we found that multiple parental viruses are present in 5 to 7% of plaques, even at an extremely low multiplicity of infection. We demonstrated through visual and biophysical assays that, like many viral stocks, our viral stocks contain both single particles and aggregates. These data suggest that aggregated virions are capable of inducing coinfection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased coinfection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher coinfection frequencies due to processes restoring fitness, such as complementation and recombination. Indeed, we found that coinfection frequency correlated with mutation load, with 17% chimeric plaque formation for heavily mutagenized viruses. Importantly, the frequency of chimeric plaques may be underestimated by up to threefold, since coinfection with the same parental virus cannot be scored in our assay. This work indicates that more than one virus can contribute to plaque formation and that coinfection may assist plaque formation in situations where the amount of genome damage is high.IMPORTANCE One of the most common methods to quantify viruses is the plaque assay, where it is generally presumed that each plaque represents a single infectious virus. Using genetically marked polioviruses, we demonstrate that a plaque can contain more than one parental virus, likely due to aggregates within virus stocks that induce coinfection of a cell. A relatively small number of plaques are the products of coinfection for our standard virus stocks. However, mutagenized virus stocks with increased genome damage give rise to a higher amount of plaques that are chimeric. These results suggest that coinfection may aid plaque formation of viruses with genome damage, possibly due to processes such as complementation and recombination. Overall, our results suggest that the relationship between viral dilution and plaque number may not be linear, particularly for mutagenized viral populations.

Comparison of three neurotropic viruses reveals differences in viral dissemination to the central nervous system.

Neurotropic viruses initiate infection in peripheral tissues prior to entry into the central nervous system (CNS). However, mechanisms of dissemination are not completely understood. We used genetically marked viruses to compare dissemination of poliovirus, yellow fever virus 17D (YFV-17D), and reovirus type 3 Dearing in mice from a hind limb intramuscular inoculation site to the sciatic nerve, spinal cord, and brain. While YFV-17D likely entered the CNS via blood, poliovirus and reovirus likely entered the CNS by transport through the sciatic nerve to the spinal cord. We found that dissemination was inefficient in adult immune-competent mice for all three viruses, particularly reovirus. Dissemination of all viruses was more efficient in immune-deficient mice. Although poliovirus and reovirus both accessed the CNS by transit through the sciatic nerve, stimulation of neuronal transport by muscle damage enhanced dissemination only of poliovirus. Our results suggest that these viruses access the CNS using different pathways.

Dairy consumption and insulin resistance: the role of body fat, physical activity, and energy intake.

The relationship between dairy consumption and insulin resistance was ascertained in 272 middle-aged, nondiabetic women using a cross-sectional design. Participants kept 7-day, weighed food records to report their diets, including dairy intake. Insulin resistance was assessed using the homeostatic model assessment (HOMA). The Bod Pod was used to measure body fat percentage, and accelerometry for 7 days was used to objectively index physical activity. Regression analysis was used to determine the extent to which mean HOMA levels differed across low, moderate, and high dairy intake categories. Results showed that women in the highest quartile of dairy consumption had significantly greater log-transformed HOMA values (0.41 ± 0.53) than those in the middle-two quartiles (0.22 ± 0.55) or the lowest quartile (0.19 ± 0.58) (F = 6.90, P = 0.0091). The association remained significant after controlling for each potential confounder individually and all covariates simultaneously. Adjusting for differences in energy intake weakened the relationship most, but the association remained significant. Of the 11 potential confounders, only protein intake differed significantly across the dairy categories, with those consuming high dairy also consuming more total protein than their counterparts. Apparently, high dairy intake is a significant predictor of insulin resistance in middle-aged, nondiabetic women.

Spectrum of disease outcomes in mice infected with YFV-17D.

The host and viral factors that influence disease outcome during flavivirus infections are not fully understood. Using the live attenuated yellow fever virus (YFV) vaccine strain 17D as a model system we evaluated how viral dose, inoculation route and immunopathogenesis contributed to disease outcome in mice deficient in the type I IFN response. We found that YFV-17D infection of IFN-α/ß receptor knockout mice resulted in three distinct disease outcomes: no clinical signs of disease, fatal viscerotropic disease or fatal neurotropic disease. Interestingly, viral load at disease onset did not correlate with disease outcome. However, we found increased immune infiltrates in the brain tissues of mice that developed neurotropic disease. Additionally, mice that developed viscerotropic disease, as characterized by liver and spleen pathology and/or intestinal haemorrhage, had significantly elevated levels of alanine aminotransferase, monocyte chemotactic protein and IFN-inducible protein (IP)-10 as compared with mice with no clinical signs of disease or neurotropic disease. Furthermore, mice treated with recombinant IP-10 throughout YFV-17D infection showed increased mortality and an increased percentage of mice with viscerotropic disease. Our results demonstrated that viral load did not correlate with pathogenesis, and the host immune response played a pivotal role in disease outcome and contributed to YFV-17D pathogenesis in mice.

Dynamic viral dissemination in mice infected with yellow fever virus strain 17D.

Arboviruses such as yellow fever virus (YFV) are transmitted between arthropod vectors and vertebrate hosts. While barriers limiting arbovirus population diversity have been observed in mosquitoes, whether barriers exist in vertebrate hosts is unclear. To investigate whether arboviruses encounter bottlenecks during dissemination in the vertebrate host, we infected immunocompetent mice and immune-deficient mice lacking alpha/beta interferon (IFN-α/ß) receptors (IFNAR⁻/⁻ mice) with a pool of genetically marked viruses to evaluate dissemination and host barriers. We used the live attenuated vaccine strain YFV-17D, which contains many mutations compared with virulent YFV. We found that intramuscularly injected immunocompetent mice did not develop disease and that viral dissemination was restricted. Conversely, 32% of intramuscularly injected IFNAR⁻/⁻ mice developed disease. By following the genetically marked viruses over time, we found broad dissemination in IFNAR⁻/⁻ mice followed by clearance. The patterns of viral dissemination were similar in mice that developed disease and mice that did not develop disease. Unlike our previous results with poliovirus, these results suggest that YFV-17D encounters no major barriers during dissemination within a vertebrate host in the absence of the type I IFN response.

Antiviral potency analysis and functional comparison of consensus interferon, interferon-alpha2a and pegylated interferon-alpha2b against hepatitis C virus infection.

BACKGROUND: Current treatments for chronic hepatitis C virus (HCV) employing pegylated interferon (PEG-IFN) plus ribavirin are successful in approximately 50% of patients. Consensus IFN (CIFN) is a recombinant type I IFN that has demonstrated efficacy where conventional therapy has failed. We evaluated the host cell antiviral response and anti-HCV actions induced by IFN-alpha2a, PEG-IFN-alpha2b or CIFN on cultured immortalized human hepatocytes, Huh7 human hepatoma cells and Huh7 cells that harboured genetically distinct HCV RNA replicons or were infected with HCV 2a. METHODS: Cultured cells were treated with each IFN at relevant dosing based upon the pharmacological attainable in vivo serum maximum IFN concentrations. Gene expression and antiviral properties were measured using protein, RNA and virus quantification assays. RESULTS: CIFN treatment maximally triggered Janus kinase signal transducer and activator of transcription signalling in association with enhanced IFN-stimulated gene (ISG) expression. Increased antiviral potency of CIFN was associated with enhancement of IFN-induced blockade upon viral protein synthesis, protection of the cellular IFN promoter stimulator-1 (IPS-1) protein from HCV proteolysis and reduced replication of an IFN-resistant HCV replicon variant. Microarray analyses revealed that CIFN treatment induced a distinct pattern of ISG expression in cultured hepatocytes compared with other IFNs. CONCLUSIONS: CIFN exhibits increased anti-HCV potency over IFN-alpha2a and PEG-IFN through maximal and distinct induction of ISG expression and enhanced intracellular innate antiviral response, while protecting IPS-1 from HCV proteolysis. CIFN might offer a treatment regimen imparting translational control programmes and restoration of the retinoic acid-inducible gene-1/IPS-1 pathway and could be considered for previous treatment failures.

Innate immune evasion by hepatitis C virus and West Nile virus.

Antiviral immunity in mammals involves several levels of surveillance and effector actions by host factors to detect viral pathogens, trigger alpha/beta interferon production, and to mediate innate defenses within infected cells. Our studies have focused on understanding how these processes are regulated during infection by hepatitis C virus (HCV) and West Nile virus (WNV). Both viruses are members of the Flaviviridae and are human pathogens, but they each mediate a very different disease and course of infection. Our results demonstrate common and unique innate immune interactions of each virus that govern antiviral immunity and demonstrate the central role of alpha/beta interferon immune defenses in controlling the outcome of infection.

Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C.

Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from fibrocystin located in the cell membrane to the nucleus.

Viral and therapeutic control of IFN-beta promoter stimulator 1 during hepatitis C virus infection.

Viral signaling through retinoic acid-inducible gene-I (RIG-I) and its adaptor protein, IFN promoter-stimulator 1 (IPS-1), activates IFN regulatory factor-3 (IRF-3) and the host IFN-alpha/beta response that limits virus infection. The hepatitis C virus (HCV) NS3/4A protease cleaves IPS-1 to block RIG-I signaling, but how this regulation controls the host response to HCV is not known. Moreover, endogenous IPS-1 cleavage has not been demonstrated in the context of HCV infection in vitro or in vivo. Here, we show that HCV infection transiently induces RIG-I- and IPS-1-dependent IRF-3 activation. This host response limits HCV production and constrains cellular permissiveness to infection. However, HCV disrupts this response early in infection by NS3/4A cleavage of IPS-1 at C508, releasing IPS-1 from the mitochondrial membrane. Cleavage results in subcellular redistribution of IPS-1 and loss of interaction with RIG-I, thereby preventing downstream activation of IRF-3 and IFN-beta induction. Liver tissues from chronically infected patients similarly demonstrate subcellular redistribution of IPS-1 in infected hepatocytes and IPS-1 cleavage associated with a lack of ISG15 expression and conjugation of target proteins in vivo. Importantly, small-molecule inhibitors of NS3/4A prevent cleavage and restore RIG-I signaling of IFN-beta induction. Our results suggest a dynamic model in which early activation of IRF-3 and induction of antiviral genes are reversed by IPS-1 proteolysis and abrogation of RIG-I signaling as NS3/4A accumulates in newly infected cells. HCV protease inhibitors effectively prevent IPS-1 proteolysis, suggesting they may be capable of restoring this innate host response in clinical practice.