Detection and biochemical characterization of circulating proteasomes in dog plasma.

A growing body of evidence convincingly indicates that proteasomes are not located exclusively within cells but also in different extracellular compartments. In humans, in fact, this large multimeric protease has been identified in many body fluids and secretions such as blood, urine, tears, sweat, saliva, milk, and cerebrospinal and pericardial fluid. Intriguingly, the exact origins of these extracellular proteasomes as well as the specific biological functions they perform are largely unknown. As no data on this important subject is yet available in domestic animals, the present study was undertaken to investigate the presence of extracellular proteasomes in canine blood. As a result, for the first time, circulating proteasomes could be clearly detected in the plasma of a cohort of 20 healthy dogs. Furthermore, all three main proteasomal peptidase activities were measured and characterized using fluorogenic peptides and highly specific inhibitors. Finally, the effect of ATP and PA28 family activators on this circulating proteasome was investigated. Collectively, our data indicate that at least a part of the proteasome present in dog plasma consists of a particle that in vitro displays the enzymatic properties of the 20S proteasome.

Prevalence of Proteinuria in Owned Dogs from Italy: A Multicentric Study.

Even though proteinuria is related to different causes, when it is persistent and associated with inactive urinary sediment, it is primarily due to kidney disease. Early detection of proteinuria allows us to identify several pathological conditions. The aim of the study was screening a canine population not known as being proteinuric, by the urinary dipstick. The study was carried out in seven Italian veterinary clinics during a period of six weeks. Dogs were enrolled with no restriction of sex or age. Females in estrus, dogs with signs of genitourinary diseases, or those previously diagnosed with proteinuric nephropathy were excluded. Dogs were considered "nonproteinuric" (NP) in case of negative dipstick test or "suspected proteinuric" (SP), if positive at the dipstick. When possible, proteinuria was confirmed by UPC ratio. A total of 1156 dogs were evaluated: 414 were from northern Italy and 742 from southern Italy. Based on dipstick test, 655 (56.6%) dogs were NP, while 501 (43.3%) were SP. Among the NP dogs 225 out of 414 (54.3%) were in northern Italy and 430 of 742 (57.9%) in southern Italy. One hundred eighty-nine of 414 (45.7%) SP dogs were identified in northern Italy and 312 of 742 (42.1%) in southern Italy. No statistical difference was found between the North and the South of Italy. UPC was available in 412 out of 501 SP samples: proteinuria was confirmed in 263 (63.86%) samples. Results from our study showed a high percentage of suspected proteinuric dogs, apparently not affected by renal diseases, together with the absence of statistically significant differences based on geographical area.

Mendelian obesity, molecular pathways and pharmacological therapies: a review.

OBJECTIVE: In this qualitative review we analyze the major pathways and mechanisms involved in the onset of genetically-determined obesity (Mendelian obesity), identifying possible pharmacological treatments and trials. MATERIALS AND METHODS: We searched PubMed with the keywords (obesity[Title/Abstract]) AND mutation[Title/Abstract], and OMIM with the keyword "obesity". In both cases, we selected non-syndromic Mendelian obesity. We then searched ClinicalTrials.gov with the following criteria: "recruitment status: active, not recruiting and completed"; "study type: interventional (clinical trial)"; "study results: with results"; type of intervention: "drug or dietary supplement". RESULTS: From the PubMed and OMIM searches we obtained a total of 15 genes associated with monogenic Mendelian obesity. From ClinicalTrials.gov we retrieved 46 completed or active trials of pharmacological treatments. CONCLUSIONS: We summarized the molecular bases of Mendelian obesity and searched for any clinical trials completed or underway for the treatment of severe forms of obesity. Most Mendelian obesities are linked to dysfunctions in the leptin/melanocortin signaling pathway, and most of the possible drugs target this pathway in order to improve energy expenditure and reduce food intake.

Flow Cytometric Characterization of S-phase Fraction and Ploidy in Lymph Node Aspirates from Dogs with Lymphoma.

Canine lymphoma is a multifaceted disease encompassing numerous entities with different prognosis. Objective assessment of the proliferation rate is of importance from the pathological and clinical perspectives. Different methods have been described in the literature to assess proliferation rate, including evaluation of Ki67 expression in fresh lymph node (LN) aspirates measured by flow cytometry (FC). This test has a high accuracy in discriminating between low- and high-grade lymphomas, and provides prognostic information among high-grade B-cell lymphomas. DNA content analysis is less expensive and suitable for well-preserved samples. We describe DNA-content analysis using LN aspirates from 112 dogs with lymphoma. S-phase fraction (SPF) accurately discriminated between low- and high-grade lymphomas, with 3.15% being the best discriminating cut-off value. SPF values strongly correlated with Ki67 expression as assessed by FC. Survival analyses were restricted to 33 dogs with high-grade B-cell lymphoma receiving standardized multi-agent chemotherapy, but no significant result was obtained for SPF. We also describe a subset of aneuploid cases and their respective follow-up. We conclude that DNA content analysis may be combined with morphological examination of LN aspirates to improve the objectivity in lymphoma subtype classification in dogs. Further studies are needed to assess the possible prognostic role of SPF and ploidy status within specific lymphoma subtypes in dogs.

Accuracy of Ultrasonographic Measurements of Adrenal Glands in Dogs: Comparison with Necroscopic Findings.

Ultrasonographic evaluation of the adrenal glands was performed in 85 dogs, followed by macroscopic and histopathological examination either post-mortem or after adrenalectomy. This retrospective cross-sectional study evaluated the difference between gross and ultrasonographic measurements to determine the diagnostic accuracy of ultrasonography in the evaluation of canine adrenal gland size. The differences were assessed for gland length, thickness at cranial, middle and caudal regions, and surface area. In our sample, ultrasound error accuracy ranged between 0% in measurement of the right adrenal gland surface area and 25.21% for left cranial pole thickness. The parameters with minor errors were caudal pole thickness (3.64% right side and 3.49% left side) and length (5.75% right side and 2.19% left side). The ultrasonographic measurements generally underestimated the actual size of the adrenal glands. No statistically significant differences were observed for measurement errors between normal and pathological adrenal glands. This study confirmed that the caudal pole of both glands is the best parameter for ultrasonographic evaluation of normal and pathological adrenal glands size in dog. Furthermore, the surface area could be considered as a dimensional parameter for better assessment of the complex shape and the global aspect of the adrenal glands, while standardize ultrasonographic projections are needed to measure the cranial pole of both adrenal glands.

Identification of a suitable internal control for fluorescence analysis on canine peripheral blood samples.

Reliable detection of fluorescence intensity (FI) by flow cytometry (FC) is fundamental. FI depends on instrument settings and sample processing procedures: thus, measurements should be done using internal controls with known FI. Commercially available beads-based standards are expensive, thus reducing their usability in the veterinary practice. Cell subsets with stable mean FI (MFI) within the population have been proposed as acceptable surrogates in human medicine. In veterinary medicine, no data exist about stability of antigen expression among different subjects or upon sample storage. The aim of the present study was to evaluate MFI variability of main lymphocytes antigens among the lymphoid cells within each subject, among different subjects, and upon 24-h storage, in order to identify the antigen most suitable as stable internal control in MFI analyses. Peripheral blood samples from 18 healthy dogs were analysed by FC within 3h from sampling to assess the expression of CD3, CD5, CD4, CD8, CD21 and cyCD79b using conjugated monoclonal antibodies. Analyses were restricted to the lymphoid population. Fluorescent microbeads were added to each tube, and antigen MFI was calculated as Relative Fluorescence Intensity RFI (CD/beads). Fluorescence histogram CV (fhCV) for each CD was regarded as an index of the variability of expression among lymphocytes within each subject (cell-to-cell variability); whereas the CV of RFI was regarded as an index of inter-subjects variability (dog-to-dog variability). In 11 cases, FC analyses were repeated after 24h storage at 4°C and RFI and CVs of fresh and stored samples were compared to assess variability linked to storage. CD4 was identified as the best antigen to be used as an internal control for MFI analyses in canine peripheral blood samples because of low cell-to-cell and dog-to-dog variability, and optimal stability upon 24-h storage. Blood samples from a second group of 21 healthy dogs were labelled only with CD4, in order to assess the influence of breed, sex and age on the expression of CD4 in a larger case series. Based on univariate GLMs, none of these variables influenced CD4 RFI. Normalizing fluorescence data using lymphoid CD4 MFI as a reference would improve the comparison of results obtained by different laboratories, patients or times in diagnostic and research analyses of FI. Further studies are needed to confirm our results with different FC approaches.

Effect of rMnSOD on survival signaling in pediatric high risk T-cell acute lymphoblastic leukaemia.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that defends against oxidative damage due to reactive oxygen species (ROS). A new isoform of MnSOD with cytotoxic activity was recently discovered in liposarcoma cells. Here, we tested the effectiveness of a recombinant form of this isoform (rMnSOD) on leukemic T cells, Jurkat cells, and lymphocytes. Our results confirm that leukemic T cells can internalize rMnSOD and that rMnSOD causes apoptosis of 99% of leukemic cells without showing toxic effects on healthy cells. Using light and electron microscopy, we determined that an rMnSOD concentration of 0.067 µM most effective on apoptosis induction. Western blot analysis showed that treatment with 0.067 µM rMnSOD resulted in high expression of the pro-apoptotic protein Bax and low expression of the anti-apoptotic protein Bcl-2 in leukemia cells. Concerning signal transduction pathway no influence was observed after treatment except for Jurkat cells showing a slightly decreased expression of ERK phosphorylation. These results suggest that rMnSOD may be an effective and non-toxic treatment option for T-cell leukemia.

ST segment elevations: always a marker of acute myocardial infarction?

Chest pain is one of the chief presenting complaints among patients attending Emergency department. The diagnosis of acute myocardial infarction may be a challenge. Various tools such as anamnesis, blood sample (with evaluation of markers of myocardial necrosis), ultrasound techniques and coronary computed tomography could be useful. However, the interpretation of electrocardiograms of these patients may be a real concern. The earliest manifestations of myocardial ischemia typically interest T waves and ST segment. Despite the high sensitivity, ST segment deviation has however poor specificity since it may be observed in many other cardiac and non-cardiac conditions. Therefore, when ST-T abnormalities are detected the physicians should take into account many other parameters (such as risk factors, symptoms and anamnesis) and all the other differential diagnoses. The aim of our review is to overview of the main conditions that may mimic a ST segment Elevation Myocardial Infarction (STEMI).

Sepsis and bacterial suppurative meningitis-meningoencephalitis in critically ill neonatal Piedmontese calves: clinical approach and laboratory findings.

Sepsis (S) and bacterial suppurative meningitis-meningoencephalitis (M-ME) are common causes of death in bovine neonates. The aim of this prospective study was to evaluate the prevalence of S and M-ME in critically ill neonatal Piedmontese calves. Critically ill animals up to 15 days old referred by practitioners were registered according to their status and subsequently assigned to clinical standardized score. Calves with a clinical score > = 5 were further assessed under a clinical and clinical-pathological protocol to strengthen the suspicion of S and M-ME. Critically ill neonatal calves sent for necropsy were included in the study as well. Fifty-nine calves were investigated, 26 of which referred alive and 33 dead. Ten out of the 26 clinically evaluated calves were classified as suspicious of S on the basis of the clinical and clinical-pathological protocols. S was confirmed by positive bacteriologic culture in 7 cases and in 3 cases on the basis of necroptic lesions. Concomitant suppurative M-ME suspected in 6 of these 10 calves was subsequently confirmed by CSF analysis or histological findings. Of the 33 calves examined only post-mortem, 20 showed pathognomonic findings of S and 14 signs of M-ME. The prevalence of S and M-ME was 46 and 36 %, respectively. Clinical signs of S were confirmed to be vague and overlapping with other diseases. The developed protocol was highly accurate in predicting S in these neonatal calves.

Is it worthwhile using a transcutaneous bilirubinometer in the nursery?

Jaundice is a potential threat to neonatal health and/or life. The advantages and limitations of transcutaneous determination of bilirubin concentration and current devices are briefly discussed in this paper.

[A single effective way to reduce post-thyroidectomy discomfort: a clinical trial]. / Un metodo semplice ed efficace per ridure il discomfort dopo tiroidectomia: un trial clinico.

AIM: Nausea, with or without vomiting (postoperative nausea and vomiting, PONV), occurs up to 60-76% after thyroidectomy and other head and neck surgeries. Due to the fact that patients typically have only mild-to-moderate pain after thyroid or parathyroid surgery, PONV might be the main source of discomfort, and it may be perceived as the most unpleasant aspect of postoperative recovery. This study aims to assess the effects of a preoperative single dose of 8 mg dexamethasone on the nausea, vomiting, pain, and subjective vocal function after thyroidectomy in patients undergoing surgery for benign disease. METHODS: Seventy patients operated on for thyroidectomy were randomized in two groups: Group A, 8 mg/2 mL of dexamethasone administered in 100 mL of physiologic saline given intravenously (i.v.) 20 minutes before the induction of anesthesia; group B, 2 mL NaCl 0.9% in 100 mL of physiologic saline. Postoperative therapy has been standardized. PONV have been evaluated with a scale of 4, degrees (0-3), pain by a Visual Analog Scale (0-100) and subjective vocal function by a Visual Analog Scale (0-100) at 8, 24, 32 and 48 hours after surgery. RESULTS: The severity of nausea was less in patients of group A (P=0.0001); Dexamethasone patients reported significantly less pain (P=0.008); no differences were noted about the subjective voice analysis (P=0.693). No steroid-related complications occurred. CONCLUSION: Dexamethasone 8 mg i.v. is a safe and effective method to reduce PONV and pain after thyroid resection and we advise its routine use.

Occlusal alterations in unilateral coronal craniosynostosis.

There are few studies on maxillomandibular skeletal alterations. Twenty-one patients with unilateral coronal craniosynostosis were analysed and compared with controls. Landmarks analysed were: sella-nasion-point A and B angles, point A-nasion-point B angle, interincisal angle, angle of superior incisor axis on the sella-nasion plane, lower incisor to mandibular plane angle, Frankfort mandibular plane angle, zygomatic-frontal suture (Z), point on the most concave part of pyramidal apophysis of the upper maxilla (Mx), antegonial incisure (AG), upper (UMT) and lower (LMT) molar teeth. Differences were significant for class II dentoskeletal occlusion (p<0.0001), mandibular hyperdivergence (p<0.0001), lingualization of superior incisor (p<0.005), deviation of inferior interincisal contralateral line to the synostosis (p<0.0001) in the plagiocephalic population. Compared with contralateral counterpoints, Z (p<0.05), Mx (p<0.005) and UMT (p<0.0005) on the affected side were closer to the midline; AG (p<0.0005) and LMT (p<0.05) were further from it. On the frontal plane, Z, Mx, UMT, LMT and AG on the affected side were higher. Vertical and transversal contraction of the jaw of the synostotic side and laterodeviation of the mandibular interincisal line of the contralateral synostotic were clear. The altered position of the glenoid cavity, anteriorized in unilateral coronal craniosynostosis, could be the cause of mandibular dentoskeletal asymmetry.

[Feasibility and effectiveness of laparoscopic right colectomy with extracorporeal anastomosis]. / Emicolectomia destra laparoscopia con anastomosi intracorporea: fattibilità ed efficacia.

AIM: Despite the laparoscopic right hemicolectomy has been validated by many randomized prospective trials, clear evidences on the validity of the totally mini-invasive technique, namely, through intracorporeal anastomosis, are still lacking. The aim of this study was the assessment of short-term outcome within three months from laparoscopic right colectomy with intra- or extra-corporeal anastomosis. METHODS: With no exclusion, all patients undergoing laparoscopic right hemicolectomy at our institution have been enrolled in this study. Group A included patients undergoing laparoscopic right hemicolectomy with extracorporeal anastomosis (LAC) and Group B, included patients undergoing laparoscopic right hemicolectomy with intracorporeal anastomosis (TLC). Patients' data, surgery details, results of postoperative period and histological tests have been prospectively recorded in a database and analysed. RESULTS: Between December 2006 and December 2008, 45 patients underwent right hemicolectomy, 21 with extracorporeal anastomosis and 24 had intracorporeal ones. As to patients' characteristics and histopathological results there are no difference between the groups. Anastomotic dehiscence occurred one in group A and one in group B (P>0.05). Both patients underwent reoperation. We recorded 6 postoperative ileus with vomiting in the LAC group and only 1 in the TLC group (P<0.05). The incidence of Non-Surgical Site Complications (NSSC) was of 4.54% in LAC group and 8.33% in TLC group (P>0.05). Hospitalization was of 5 days for both groups. CONCLUSION: In conclusion, we believe that this technique is feasible in terms of safety; it doesn't significatively affect the length of surgical procedure and guarantees maintenance of oncological radicality standards of reference. Besides it significatively improves quality of the post-operative period.

Changes in the gene expression profile of gastric cancer cells in response to ibuprofen: a gene pathway analysis.

Nonsteroidal anti-inflammatory drugs possess antiproliferative activities that can affect cancer cells. The aim of this study was to examine the antiproliferative effects of ibuprofen on the MKN-45 cell line. Cells were treated with ibuprofen for 24, 48 or 72 h, and cell proliferation was evaluated by cell counting and [(3)H]-thymidine incorporation. Using microarray technology, we studied changes in the gene expression profiles over time after ibuprofen treatment. Ibuprofen induced a dose- and time-dependent reduction in cell number without altering cell viability. Genes involved in the 'biological oxidation' and 'G(1)/S checkpoint' pathways were the most significantly represented at 24 h, whereas genes involved in the 'cell cycle' and 'DNA replication' pathways were represented at 48 and 72 h. Genes associated with the 'apoptosis' pathway were also significantly represented at 72 h. Modulation of the expression of p53 and p53-induced genes (CDKN1A/p21 and GADD45), which are involved in the G(1)/S transition, suggested an effect of ibuprofen on cell-cycle progression. Using flow cytometry, we observed an early block in the G(1) phase of the cell cycle after ibuprofen treatment. In addition, P450 family transcripts were upregulated and intracellular reactive oxygen species (ROS) was increased following 12 h of ibuprofen treatment. Ibuprofen induced ROS, which resulted in cellular alterations that promoted a p53-dependent G(1) blockade. These findings suggest that ibuprofen exerts its antiproliferative actions through cell-cycle control and the induction of apoptosis. Both of these mechanisms appear to be independent of ibuprofen's anti-inflammatory effects.

[A new effective method to reduce pain after laparoscopic cholecystectomy]. / Un nuovo efficace sistema per ridurre il dolore dopo videolaparocolecistectomia.

BACKGROUND: The primary aim of this randomized controlled prospective study (www.clinicaltrials.gov NCT00599144) was assessing effectiveness on pain control after videolaparoscopic cholecystectomy (VLC) of 0.5% Bupivacaine-soaked sheet of regenerated oxidized cellulose positioned in the gallbladder bed. PATIENTS AND METHODS: We randomized in three equal groups 45 patients that underwent elective videolaparoscopic cholecystectomy: group A - Bupivacaine-soaked sheet of regenerated oxidized cellulose positioned in the gallbladder bed; group B - Bupivacaine infiltrated in the muscular fasciae of the trocars' seat; group C - control group, not using local anesthetic. Six and 24 hours after the intervention we recorded the type of the pain and its intensity, using a visual analog scale (VAS). RESULTS: We observed no statistically significant difference in pain intensity. Considering the type of pain, 55,56% of patients referred visceral pain, 62,22% parietal pain and 44,44% shoulder pain. Group A shows, at both endpoints, a statistically significant reduction (p < 0.05) of visceral and shoulder pain versus other groups. Moreover, the use of a local anesthetic significantly reduces the use of post-operative pain drugs, both for group A and B. CONCLUSION: A Bupivacaine-soaked sheet of regenerated oxidized cellulose positioned in the gallbladder bed post-VLC is safe and not economically demanding, and can give advantages in increasing post-operative comfort.

[The development of a fast track surgery program after laparoscopic colonic surgery in a General Surgery Unit]. / Lo sviluppo di un Protocollo di Fast Track Surgery dopo colectomia laparoscopica in una Unità di Chirurgia Generale.

AIM: The aim of the "Fast-Track Surgery" program is to decrease the perioperative stress response to surgical trauma and thus to decrease the complication rates after elective surgery. Critics of the "Fast-Track"(FT) rehabilitation may argue that all reports of successful programs came from major specialised hospital units and that implementation in smaller or less specialised units may be difficult, if not impossible. METHODS: We retrospectively studied 101 patients that, from November 2004 to October 2007, underwent laparoscopic colorectal surgery in our institute. A detailed "Fast-Track Surgery" protocol was prepared and given to patients, physicians and nurses, with the aim to create a standard treatment. Data about demographics, ASA score, preoperative complicating diseases, diagnosis, type of surgery and postoperative clinical data were analyzed. Univariate analysis of the relationship among all factors (patient characteristics, intervention characteristics, protocol compliance and presence of complications) described here and length of hospital stay was performed. RESULTS: We compared our results to those of major trials previously published and observed no substantial differences in morbidity, mortality and length of postoperative hospital stay between the two. Univariate analysis showed that compliance to the elements of the FT protocol influences the length of postoperative period more significantly than the patient characteristics or surgical procedure. CONCLUSION: Based on six comparative single-centre studies, the FT program was found to reduce length of hospital stay and was deemed safe for major abdominal surgeries. The present study shows that enhanced recovery or FT program can also be implemented safely in a General Surgery Unit.

Late onset and rapid progression of cerebellar abiotrophy in a domestic shorthair cat.

A two-year-old male domestic shorthair cat was seen because of sudden onset of incoordination and tremors that had rapidly worsened over five days. Neurological examination revealed severe cerebellar ataxia, intention tremors and bilaterally decreased menace response. Blood work evaluation included a complete cell blood count (CBC), serum biochemistry profile, urinalysis, faecal flotation, cerebrospinal fluid (CSF) analysis and retroviral testing. Except for testing positive for feline immunodeficiency virus (FIV) antibodies, all other results were within the normal range. The patient was euthanased two days later because of progression of clinical signs, and a necropsy was performed. Histologically, lesions were limited to the cerebellum and consistent with cerebellar abiotrophy. No secondary diseases that could explain the rapid development of clinical signs were found. It was considered unlikely that cerebellar degeneration was related to FIV positivity, as virus invasion of the central nervous system (CNS) is mainly limited to the cerebral cortex. This case report is the first to describe late onset and rapid progression cerebellar abiotrophy in a cat.

Cachexia secondary to intracranial anaplastic (malignant) ependymoma in a boxer dog.

An eight-year-old female boxer was referred because of weight loss of several months' duration. On physical examination, cachexia was the only reported abnormality. Neurological symptoms were shown only in the last days preceding death and consisted of altered mental status, compulsive behaviour and left rear proprioception deficit. At post-mortem examination, a voluminous, soft, haemorrhagic mass was found invading the floor of the brain. Based on the morphological features of the tumour, marked parenchymal invasion, extensive necrosis and cellular atypia, the mass was classified as an anaplastic ependymoma. This case report shows similarities to the diencephalic syndrome reported in human paediatric medicine in which the main clinical sign is a profound emaciation in spite of normal or slightly diminished caloric intake. Weight loss and cachexia are clinically relevant problems in small animals and these clinical signs should raise a suspicion, among the other differentials, of a brain tumour, even in absence of neurologic signs.

Analysis of cerebrospinal fluid from 20 calves after storage for 24 hours.

Samples of CSF collected from 20 normal healthy calves were analysed either immediately or after having been stored for 24 hours at 4 degrees C in the presence of 11 per cent autologous serum. There were no significant differences between the total and differential cells counts of the fresh and stored samples, but there was a positive linear correlation between them. There were some morphological changes to the nuclei of the mononuclear cells in the stored samples.

[Endometrial ablation versus hysterectomy in women treated with tamoxifen]. / Ablazione endometriale versus isterectomia in pazienti in trattamento con tamoxifene.

AIM: The aim of our study is the assessment of the importance of the endometrial ablation versus hysterectomy in patients treated with tamoxifen for previous breast cancer. METHODS: Fifty-eight outpatients in therapy with tamoxifen for 1 year were controlled in the Department of Gynaecology of the University of Naples. We have selected these patients in two groups: group A, with 28 women with abnormal uterine bleeding and endometrial thickness >8 mm and group B, with 30 normal endometrium asymptomatic women. All patient of group A and 18 of group B were treated with endometrial ablation. RESULTS: Next follow-up showed normal hysteroscopy figures in 89% of cases and 5% of cases needed a hysterectomy for new abnormal uterine bleeding and cytology. CONCLUSION: Our results show the utility of endometrial ablation especially in selected cases in therapy with tamoxifen for previous breast cancer.