Fabio Gentilini

Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs

Endotoxic shock, one of the most prominent causes of mortality in intensive care units, is characterized by pulmonary hypertension, systemic hypotension, heart failure, widespread endothelial activation/injury, and clotting culminating in disseminated intravascular coagulation and multi‐organ system failure. In the last few years, studies in rodents have shown that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. In this study, we have administered CO (one our pretreatment at 250 ppm) in a clinically relevant, well‐characterized model of LPS‐induced acute lung injury in pigs. Pretreatment only with inhaled CO significantly ameliorated several of the acute pathological changes induced by endotoxic shock. In terms of lung physiology, CO pretreatment corrected the LPS‐induced changes in resistance and compliance and improved the derangement in pulmonary gas exchange. In terms of coagulation and inflammation, CO reduced the development of disseminated intravascular coagulation and completely suppressed serum levels of the proinflammatory IL‐1β in response to LPS, while augmenting the anti‐inflammatory cytokine IL‐10. Moreover, the effects of CO blunted the deterioration of kidney and liver function, suggesting a beneficial effect in terms of end organ damage associated with endotoxic shock. Lastly, CO pretreatment prevents LPS‐induced ICAM expression on lung endothelium and inhibits leukocyte marginalization on lung parenchyma.

The Effects of Hydrocortisone on Systemic Arterial Blood Pressure and Urinary Protein Excretion in Dogs

BACKGROUND Hypertension and proteinuria are commonly recognized in dogs with spontaneous hypercortisolism. There is, however, little information regarding the effect of exogenous glucocorticoids on blood pressure (BP) and proteinuria and whether these changes are reversible. HYPOTHESIS Hydrocortisone administration increases systemic BP and urinary protein excretion, and these effects are reversible after hydrocortisone withdrawal. ANIMALS Six control dogs and 6 dogs treated with hydrocortisone. METHODS BP, urine protein : creatinine ratio (UPC), microalbuminuria (MALB), urine albumin : creatinine ratio (UAC), and urine gel electrophoresis were evaluated before, during, and after administration of hydrocortisone (8 mg/kg PO q12h for 12 weeks) or placebo. RESULTS BP and UPC increased substantially during hydrocortisone administration from 123 mmHg (range 114-136 mmHg) and 0.17 (0.15-0.28) to a maximum of 143 mmHg (128-148 mmHg) and 0.38 (0.18-1.78), respectively, on day 28. MALB developed in 4 dogs and UAC significantly increased in all dogs during hydrocortisone administration with the maximum on day 84. Both increases in BP and proteinuria were reversible and completely resolved within 1 month after stopping hydrocortisone administration. SDS-AGE revealed the proteinuria to be primarily albuminuria with a pronounced increase during hydrocortisone treatment. Furthermore, a protein of 25-30 kDa was found in male dogs, identified by mass spectrometry to be arginine esterase, the major secretory prostatic protein. CONCLUSIONS AND CLINICAL IMPORTANCE Long-term hydrocortisone treatment results in significant but only mild increases in systemic BP and urinary protein excretion, which are both reversible within 1 month after discontinuation of hydrocortisone.

Validation of a Human Immunoturbidimetric Assay to Measure Canine Albumin in Urine and Cerebrospinal Fluid

The aim of this study was to validate an automated immunoturbidimetric assay used to quantify human albumin in urine and to accurately measure canine albumin concentrations in both urine and cerebrospinal fluid. The partial homology existing between human and canine albumin limited the accuracy of the human assays in measuring canine albumin without method modifications. Thus, the assay was modified by calibrating the analyzer with calibrators made in the laboratory containing known concentrations of canine albumin. To prepare the set of calibrators, the albumin concentration of pooled sera of healthy dogs was assessed in 5 replicates using the BromocresolGreen assay. Pooled samples were aliquoted and serially diluted to obtain the expected concentrations of albumin (0.5, 1, 5, 13, and 30 mg/dl) for establishing the canine calibration curve. Thereafter, the performance was assessed by analyzing canine urine and CSF. The modified assay accurately quantified canine albumin in both specimens, as indicated by the following. Intra- and interassay variability was 0.92% and 2.74%, respectively; recovery was 99.66% and 99.07% in urine and 105.02% in CSF. No interference was detected when hemolysate and glucose were added to urine. The test was linear within the verified range (0–225 mg/dl). These results demonstrate that the modified human albumin immunoturbidimetric assay can be a useful tool in the veterinary diagnostic laboratory. It is accurate and tends itself to automatization on chemistry analyzers.

Hematologic and biochemical profiles in Standardbred mares during peripartum

The purposes of this study were to determine physiological changes occurring in hematologic and biochemical parameters in mares between the last month of gestation and the first week after parturition. If a significant change was observed with respect to the reference interval of an adult horse, a further aim of the study was to establish different reference intervals. Blood samples were collected from 62 healthy pregnant Standardbred mares. Seventeen nonpregnant and nonlactating mares were used as a control group. In pregnant mares, blood sampling was conducted every three days from 1 month before the expected foaling date (335 days after the last mating), at parturition, and 7 days after foaling. The barren mares in the control group were sampled once. Results from samples collected 20 and 10 days before parturition, at parturition, and 7 days after were considered in the statistical analysis. A parametric method for all the parameters studied was used to establish reference intervals. Results were compared by repeated measures ANOVA. When significant differences were observed in relation to sampling time, a post hoc analysis was performed (Tukey test). The one-way ANOVA test followed by Dunnetts test was performed to evaluate the presence of a significant difference between each sampling time and the control group. Any significant difference in the blood count parameters at different sampling times was observed by repeated measure ANOVA. Hemoglobin (P < 0.01) and hematocrit (P < 0.01) 7 days after parturition and white blood cell count (P < 0.01) at parturition were significantly different from the control group. Erythrocyte indices and platelet count were within the normal reference intervals as established in the control group. In the biochemical panel, gamma-glutamyltransferase, creatinine, glucose, biliar acids, total protein, albumin-to-globulin ratio, and calcium were significantly different at different sampling times. Moreover, serum concentration of creatine kinase, aspartate aminotransferase, creatinine, blood urea nitrogen, glucose, lactate, total protein, albumin, albumin-to-globulin ratio, calcium, magnesium, sodium, chloride, potassium, and total, direct, and indirect bilirubin was different from that of the control group. Remarkable changes were not observed in alkaline phosphatase, triglyceride, and fibrinogen concentrations. Temporal changes in the hematologic and biochemical parameters observed in the present study in the peripartum and the differences with reference intervals made up for nonpregnant and nonlactating mares could be used to better evaluate the conditions of periparturient mares.

Intermittent Gastroesophageal Intussusception in a Dog: Clinical Features, Radiographic and Endoscopic Findings, and Surgical Management

M. Pietra1*, F. Gentilini1, S. Pinna2, F. Fracassi1, A. Venturini2 and M. Cipone1 Department of Veterinary Clinical Science (1Section of Internal Medicine; 2Section of Surgery), Faculty of Veterinary Medicine, University of Bologna, 40064 Ozzano dell’Emilia (BO), Italy *Correspondence: Dipartimento Clinico Veterinario, Universita degli Studi di Bologna, V ia T olara di Sopra 50, 40064, Ozzano dell’Emilia (BO), Italy E-mail: mpietra@vet.unibo.it

Prognostic significance of Kit receptor tyrosine kinase dysregulations in feline cutaneous mast cell tumors.

Feline cutaneous mast cell tumors (FeCMCTs) are characterized by variable biological behavior. Development of multiple nodules and potential visceral involvement, along with inconsistency of conventional prognostic aids, justify uncertainty in differentiating benign from malignant forms. c-Kit proto-oncogene activating mutations have been reported in feline mast cell tumors (MCTs), but their prognostic relevance was not investigated. This study was performed on FeCMCTs with variable clinical outcome to assess whether Kit cytoplasmic immunohistochemical labeling can be regarded as indicative of c-Kit mutations and to evaluate the relationship between Kit dysregulation and survival. Twenty-four cats diagnosed with a primary cutaneous MCT were enrolled. Kit immunohistochemical pattern and c-Kit (exons 8, 9, 11) mutational status were assessed in 34 tumor samples. Risk factors affecting survival were a number of mitoses greater than 5 per 10 HPFs (P = .017) and cytoplasmic Kit labeling (P = .045). Increased mitotic activity was associated with Kit cytoplasmic expression (P = .01). c-Kit encoding mutations were present in 19 (56%) tumors (exon 8, 19%; exon 9, 71%; exon 11, 10%), however, they were not significantly related to protein expression and they had no influence on prognosis. Additionally, in 6 of 9 (67%) cats, multiple nodules from the same cat had different mutational statuses. Mutations in the fifth immunoglobulin-like domain of Kit occur frequently in FeCMCT, but they are variably associated with aberrant protein expression and do not appear to be strictly correlated with biological behavior. These findings need to be confirmed in larger series, and exploration of further genomic regions of c-Kit is warranted.

Raphanus sativus cv. Sango Sprout Juice Decreases Diet-Induced Obesity in Sprague Dawley Rats and Ameliorates Related Disorders.

Background Obesity is recognized as a leading global health problem, correlated with an increased risk for several chronic diseases. One strategy for weight control management includes the use of vegetables rich in bioactive compounds to counteract weight gain, improve the antioxidant status and stimulate lipid catabolism. Aim of the Study The aim of this study was to investigate the role of Raphanus sativus Sango sprout juice (SSJ), a Brassica extraordinarily rich in anthocyanins (AC) and isothiocyanates (ITCs), in a non-genetic model of obesity (high fat diet-HFD induced). Methods Control groups were fed with HFD or regular diet (RD). After a 10-week period, animals were assigned to experimental units and treated by gavage for 28 days as follows: HFD and RD control groups (rats fed HFD or RD and treated with vehicle only) and HFD-treated groups (rats fed HFD and treated with 15, 75 or 150 mg/kg b.w. of SSJ). Body weight and food consumption were recorded and serum lipid profile was measured (total cholesterol, triglycerides, and non-esterified fatty acids). Hepatic phase-I, phase-II as well as antioxidant enzymatic activities were assessed. Results SSJ lowered total cholesterol level, food intake and liver weight compared with HFD rodents. SSJ at medium dose proved effective in reducing body-weight (~19 g reduction). SSJ was effective in up-regulating the antioxidant enzymes catalase, NAD(P)H:quinone reductase, oxidised glutathione reductase and superoxide dismutase, which reached or exceeded RD levels, as well as the phase II metabolic enzyme UDP-glucuronosyl transferase (up to about 43%). HFD up-regulated almost every cytochrome P450 isoform tested, and a mild down-regulation to baseline was observed after SSJ intervention. Conclusion This work reveals, for the first time, the antioxidant, hypolipidemic and antiobesity potential of SSJ, suggesting its use as an efficient new functional food/nutraceutical product.

Detection of Wolbachia DNA in Blood for Diagnosing Filaria-Associated Syndromes in Cats

ABSTRACT A fundamental role for the endosymbiotic bacteria Wolbachia pipientis in the pathogenesis of Dirofilaria immitis infections has emerged in recent years. Diagnostic opportunities arising from this breakthrough have not yet been fully exploited. This study was aimed at developing conventional and real-time PCR assays to carry out a molecular survey in a convenience sample of cats living in an area where D. immitis is endemic and to evaluate the detection of bacterial DNA in blood as a surrogate assay for diagnosing filaria-associated syndromes in cats. COI and FtsZ loci were used as targets for D. immitis and Wolbachia PCR assays, respectively, and real-time TaqMan PCR assays were used only for Wolbachia. A convenience sample of 307 disease-affected or healthy cats examined at a University facility were PCR tested, and their medical records were investigated. Conventional nested PCR for Wolbachia amplified the endosymbionts of both D. immitis and D. repens, while real-time PCR was highly specific only for the former. Observed prevalences of 0.3 and 10.4% were found using conventional nested PCR assays for D. immitis and real-time PCR for Wolbachia, respectively. Similar prevalences were established using the Wolbachia nested PCR (98% concordance with real-time PCR). The group of Wolbachia-positive samples had a significantly higher proportion of subjects with respiratory signs (29.0% versus 9.7%; P = 0.002). The findings of this study indicate that a highly sensitive PCR assay can be used to detect the Wolbachia organism in the peripheral blood of cats with respiratory signs.

Recruited leukocytes and local synthesis account for increased matrix metalloproteinase-9 activity in cerebrospinal fluid of dogs with central nervous system neoplasm

Matrix metalloproteinases (MMP) 2 and 9 are enzymes known to degrade several protein components of the extracellular matrix. In humans, increased concentrations of these enzymes have been demonstrated in the cerebrospinal fluid (CSF) of subjects affected by many neurological conditions including brain tumours; nevertheless comparative data in dogs are completely lacking. Aim of this study was to investigate these molecules in CSF of dogs diagnosed with CNS neurological diseases. Higher activity of MMP 2 and 9 was revealed in dogs with space occupying lesions of likely neoplastic origin in comparison to dogs with idiopathic epilepsy. Statistical modelling reveals that increased MMP 9 activity is mainly due to leukocytes recruitment and local synthesis.

Serum amyloid A, haptoglobin, and ferritin in horses with colic: Association with common clinicopathological variables and short-term outcome

Equine colic may be associated with an acute phase response (APR). Measurement of acute phase proteins (APPs) allows the detection of an APR and may help clinicians in monitoring the disease; however, the role of APPs in colic is unclear. This study aimed to evaluate the clinical usefulness of serum amyloid A (SAA), haptoglobin and ferritin in combination with an extended clinicopathological profile in equine colic. The medical records of 54 horses were retrospectively selected. Horses were grouped based on outcome (survivors vs. non-survivors), diagnosis (ischaemic/strangulating vs. non-ischaemic/non-strangulating), and treatment (medical treatment vs. surgery). Laboratory data were compared, and a logistic regression analysis was performed for outcome prediction upon admission. A high percentage of horses had abnormal SAA (29/54), haptoglobin (20/54), and ferritin (31/54) concentrations. In particular, haptoglobin was below the reference interval in 13/54 horses. Non-survivors had significantly decreased haptoglobin and increased ferritin concentrations compared with survivors. The ischaemic/strangulating group had significantly increased creatinine and ferritin and decreased haptoglobin concentrations compared with the non-ischaemic/non-strangulating group. Creatinine was the only significant predictor of mortality in the regression analysis. In conclusion, APPs including SAA, haptoglobin, and ferritin combined with clinicopathological variables may help clinicians to understand the pathogenesis of APR and underline potential complications of equine colic. The reduction in haptoglobin concentration may suggest haemolysis or muscle fibre damage; ferritin may indicate alteration in iron metabolism and tissue damage. Further prospective studies are needed to assess diagnostic and prognostic values of APPs in colic horses.