Nutritional ecology of a prototypical generalist predator, the red fox (Vulpes vulpes).

Generalist species, which exploit a wide range of food resources, are expected to be able to combine available resources as to attain their specific macronutrient ratio (percentage of caloric intake of protein, lipids and carbohydrates). Among mammalian predators, the red fox Vulpes vulpes is a widespread, opportunistic forager: its diet has been largely studied, outlining wide variation according to geographic and climatic factors. We aimed to check if, throughout the species' European range, diets vary widely in macronutrient composition or foxes can combine complementary foods to gain the same nutrient intake. First, we assessed fox's intake target in the framework of nutritional geometry. Secondly, we aimed to highlight the effects of unbalanced diets on fox density, which was assumed as a proxy for Darwinian fitness, as assessed in five areas of the western Italian Alps. Unexpectedly, the target macronutrient ratio of the fox (52.4% protein-, 38.7% lipid- and 8.9% carbohydrate energy) was consistent with that of hypercarnivores, such as wolves and felids, except for carbohydrate intakes in urban and rural habitats. The inverse relation between density and the deviation of observed macronutrient ratios from the intake target suggests that fox capability of surviving in a wide range of habitats may not be exempt from fitness costs and that nutrient availability should be regarded among the biotic factors affecting animal abundance and distribution.

Staphylococcus aureus adlb gene is associated with high prevalence of intramammary infection in dairy herds of northern Italy: A cross-sectional study.

Staphylococcus aureus is a major mastitis pathogen in dairy cattle worldwide, responsible for substantial economic losses. Environmental factors, milking routine, and good maintenance of milking equipment have been described as important factors to prevent intramammary infections (IMI). Staphylococcus aureus IMI can be widespread within the farm or the infection can be limited to few animals. Several studies have reported that Staph. aureus genotypes differ in their ability to spread within a herd. In particular, Staph. aureus belonging to ribosomal spacer PCR genotype B (GTB)/clonal complex 8 (CC8) is associated with high within-herd prevalence of IMI, whereas other genotypes are generally associated with individual cow disease. The adlb gene seems to be strictly related to Staph. aureus GTB/CC8, and is a potential marker of contagiousness. We investigated Staph. aureus IMI prevalence in 60 herds in northern Italy. In the same farms, we assessed specific indicators linked to milking management (e.g., teat condition score and udder hygiene score) and additional milking risk factors for IMI spread. Ribosomal spacer-PCR and adlb-targeted PCR were performed on 262 Staph. aureus isolates, of which 77 underwent multilocus sequence typing. In most of the herds (90%), a predominant genotype was identified, especially Staph. aureus CC8 (30%). In 19 of 60 herds, the predominant circulating Staph. aureus was adlb-positive and the observed IMI prevalence was relevant. Moreover, the adlb gene was detected only in genotypes of CC8 and CC97. Statistical analysis showed a strong association between the prevalence of Staph. aureus IMI, the specific CCs, and carriage of adlb, with the predominant circulating CC and presence of the gene alone explaining the total variation. Interestingly, the difference in the odds ratio obtained in the models for CC8 and CC97 suggests that it is carriage of the adlb gene, rather than the circulation of these CCs per se, that leads to higher within-herd prevalence of Staph. aureus. In addition, the model showed that environmental and milking management factors had no or minimal effect on Staph. aureus IMI prevalence. In conclusion, the circulation of adlb-positive Staph. aureus strains within a herd has a strong effect on the prevalence of IMI. Thus, adlb can be proposed as a genetic marker of contagiousness for Staph. aureus IMI in cattle. However, further analyses using whole-genome sequencing are required to understand the role of genes other than adlb that may be involved in the mechanisms of contagiousness of Staph. aureus strains associated with high prevalence of IMI.

Short communication: Characterization of Staphylococcus aureus from bulk tank milk of dairy cattle in Lombardy (northern Italy).

Staphylococcus aureus is one of the most important pathogens causing mastitis in cattle, and it is responsible for economic losses in dairy herds worldwide. The PCR amplification of the 16S-23S rRNA intergenic spacer (ribosomal spacer PCR, RS-PCR) allows a rapid classification of the strains in genotypes and genotypic clusters (CL), which are characterized by different epidemiological and clinical properties. Both RS-PCR and multi-locus sequence typing (MLST) were performed on strains isolated from bovine bulk tank milk (BTM) collected from dairy herds located in the Lombardy region (northern Italy), to outline the distribution of Staph. aureus genotypes in this geographical area. Out of 844 examined samples, 398 were positive for Staph. aureus, with a variable count (cfu/mL) Up to 8 colonies from each sample were genotyped. A total of 1,101 Staph. aureus strains were analyzed with RS-PCR, and only a selection of them (n = 86), in relation to their frequency and geographical origin, underwent MLST. This study revealed 8 major genotypic clusters (CLB, CLC, CLR, CLS, CLI, CLF, CLAO, and CLZ), of which Staph. aureus CLB (29.3%) was the most common. Samples of BTM positive for CLB had a Staph. aureus cfu/mL count significantly higher than the non-CLB positive ones. Our MLST analysis showed genotypes already known as bovine-associated in literature, such as clonal complexes CC8, CC97, and CC151. The same selection of 86 strains was also analyzed for the presence of the adlb gene, which was recently proposed as a possible marker of contagiousness. Most Staph. aureus belonging to CLB or CC8 carried the adlb gene (85%), whereas this gene was detected in only 9% of non-CLB strains (CLAA, CLBI, CLBJ, CLS). In conclusion, the present study confirms that Staph. aureus CLB, which is recognized as a contagious genotype, is a particularly relevant agent of intramammary infection in dairy cows in Lombardy, and indirectly supports the idea that adlb can be a possible marker of contagiousness of isolates.

Ficoll-hypaque separation vs whole blood lysis: Comparison of efficiency and impact on minimal residual disease analysis.

INTRODUCTION: The high-throughput era remarkably changed molecular laboratory practice. Actually, the increasing number of processed samples requires to reduce the risk of operator biases, by automating or simplifying as much as possible both the analytical and the pre-analytical phases. Minimal residual disease (MRD) studies in hematology often require a simultaneous processing of many bone marrow and peripheral blood samples from patients enrolled in prospective, multicenter, clinical trials, monitored at several planned time points. METHODS: In this study, we demonstrate that red blood cell lysis (RBL) pre-analytical procedure can replace the time-consuming Ficoll stratification as cell recovering step. Here, we show a MRD comparison study using both total white blood cells and mononuclear cells recovered by the 2 procedures from 46 follicular lymphoma (FL), 15 multiple myeloma (MM), and 11 mantle cell lymphoma (MCL) patients enrolled in prospective clinical trials. RESULTS: The experiments were performed in the 4 laboratories of the Fondazione Italiana Linfomi (FIL) MRD Network and showed superimposable results, in terms of good correlation (R = 0.87) of the MRD data obtained by recovering blood cells by the 2 approaches. CONCLUSION: Based on these results, the FIL MRD Network suggests to optimize the pre-analytical phases introducing RBL approach for cell recovery in the clinical trials including MRD analysis.

Impact of viral hepatitis aetiology on survival outcomes in hepatocellular carcinoma: A large multicentre cohort study.

While HBV and HCV are risk factors for HCC, uncertainty exists as to whether these viral infections have prognostic significance in HCC. Thus, we compared the overall survival of patients with HBV, HCV and nonviral HCC, and evaluated whether the presence of HBV and HCV predicts patient outcomes. We conducted a multicentre study of HCC cases diagnosed at six Melbourne tertiary hospitals between Jan 2000-Dec 2014. Patient demographics, liver disease and tumour characteristics and patient outcomes were obtained from hospital databases, computer records and the Victorian Death Registry. Survival outcomes were compared between HBV, HCV and nonviral hepatitis cases and predictors of survival determined using Cox proportional hazards regression. There were 1436 new HCC cases identified including 776 due to viral hepatitis (HBV 235, HCV 511, HBV-HCV 30) and 660 from nonviral causes. The median survival of HBV, HCV and nonviral HCC patients was 59.1, 28.4 and 20.9 months, respectively (P<.0001). On multivariate analysis, independent risk factors for survival included HCC aetiology, gender, BCLC stage, serum AFP, total number and size of lesions, and serum creatinine and albumin. After adjusting for these and method of detection, HBV remained an independent predictor of improved overall survival when compared to both nonviral (HR 0.60%, 95% CI 0.35-0.98; P=.03) and HCV-related HCC (HR 0.51%, 95% CI 0.30-0.85; P=.01). In this large multicentre study, HBV is independently associated with improved overall survival compared with HCV and nonviral-related HCC. Further studies are needed to determine the underlying factor(s) responsible.

Pathogenetic and diagnostic significance of microRNA deregulation in peripheral T-cell lymphoma not otherwise specified.

Peripheral T-cell lymphomas not otherwise specified (PTCLs/NOS) are rare and aggressive tumours whose molecular pathogenesis and diagnosis are still challenging. The microRNA (miRNA) profile of 23 PTCLs/NOS was generated and compared with that of normal T-lymphocytes (CD4+, CD8+, naive, activated). The differentially expressed miRNA signature was compared with the gene expression profile (GEP) of the same neoplasms. The obtained gene patterns were tested in an independent cohort of PTCLs/NOS. The miRNA profile of PTCLs/NOS then was compared with that of 10 angioimmunoblastic T-cell lymphomas (AITLs), 6 anaplastic large-cell lymphomas (ALCLs)/ALK+ and 6 ALCLs/ALK-. Differentially expressed miRNAs were validated in an independent set of 20 PTCLs/NOS, 20 AITLs, 19 ALCLs/ALK- and 15 ALCLs/ALK+. Two hundred and thirty-six miRNAs were found to differentiate PTCLs/NOS from activated T-lymphocytes. To assess which miRNAs impacted on GEP, a multistep analysis was performed, which identified all miRNAs inversely correlated to different potential target genes. One of the most discriminant miRNAs was selected and its expression was found to affect the global GEP of the tumours. Moreover, two sets of miRNAs were identified distinguishing PTCL/NOS from AITL and ALCL/ALK-, respectively. The diagnostic accuracy of this tool was very high (83.54%) and its prognostic value validated.

Platelet-derived growth factor alpha mediates the proliferation of peripheral T-cell lymphoma cells via an autocrine regulatory pathway.

Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) are very aggressive tumors characterized by consistent aberrant expression of platelet-derived growth factor receptor alpha (PDGFRA). In this study, we aimed to identify the determinants of PDGFRA activity in PTCL/NOS and to elucidate the biological consequences of its activation. We observed overexpression of the PDGFRA gene by gene expression profiling in most of the tested PTCLs and confirmed the expression of PDGFRA and phospho-PDGFRA using immunohistochemistry. The integrity of the PDFGRA locus was demonstrated using several different approaches, including massive parallel sequencing and Sanger sequencing. PDGF-AA was found to be expressed and secreted by PTCL/NOS cells and to be necessary and sufficient for PDGFRA phosphorylation ex vivo by sustaining an autocrine stimulation. We documented consistently high PDGF-A expression in primary biopsies and patients' plasma and tracked PDGFRA signaling in primary tumors, achieving evidence of its activation. Indeed, we found that STAT1 and STAT5 are implicated in PDGFRA signaling transduction. Finally, we demonstrated that PDGFRA activation supported tumor cell proliferation and provided the first evidence of the anti-lymphoma activity of PDGRA inhibition in a PTCL/NOS patient. Altogether, our data demonstrated that PDGFRA activity fosters PTCL/NOS proliferation via an autocrine loop.

Molecular profiling of blastic plasmacytoid dendritic cell neoplasm reveals a unique pattern and suggests selective sensitivity to NF-kB pathway inhibition.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease of controversial origin recently recognized as a neoplasm deriving from plasmacytoid dendritic cells (pDCs). Nevertheless, it remains an orphan tumor with obscure biology and dismal prognosis. To better understand the pathobiology of BPDCN and discover new targets for effective therapies, the gene expression profile (GEP) of 25 BPDCN samples was analyzed and compared with that of pDCs, their postulated normal counterpart. Validation was performed by immunohistochemistry (IHC), whereas functional experiments were carried out ex vivo. For the first time at the molecular level, we definitely recognized the cellular derivation of BPDCN that proved to originate from the myeloid lineage and in particular, from resting pDCs. Furthermore, thanks to an integrated bioinformatic approach we discovered aberrant activation of the NF-kB pathway and suggested it as a novel therapeutic target. We tested the efficacy of anti-NF-kB-treatment on the BPDCN cell line CAL-1, and successfully demonstrated by GEP and IHC the molecular shutoff of the NF-kB pathway. In conclusion, we identified a molecular signature representative of the transcriptional abnormalities of BPDCN and developed a cellular model proposing a novel therapeutic approach in the setting of this otherwise incurable disease.

Partial nodal involvement by marginal zone lymphoma. Use of IGK gene rearrangement analysis in diagnostic work-up.

Nodal marginal zone lymphoma (NMZL) is an indolent B-cell lymphoma that originates from the marginal zone of B-cell follicles. The tumour is rather uncommon, and shares some morphologic and immunophenotypic similarities with the extranodal form of marginal zone lymphomas. However, diagnosis of NMZL implies the exclusion of lymphoplasmacytic lymphoma, follicular lymphoma, and lymph node involvement by extra nodal or splenic marginal zone B-cell lymphoma In addition, its distinction from reactive conditions, including T-zone hyperplasia, are sometimes problematic based on morphologic grounds. We describe a patient who presented with cervical and inguinal lymphadenopathies and high inflammation indexes. Bone marrow and lymph node biopsies were performed for definitive diagnosis. Bone marrow histological and immunophenotypic examinations were normal and excluded haematological disease. In contrast, lymph node evaluation showed some features compatible with a possible lymphoproliferative disorder, even though no definite diagnosis could be made based on morphologic and immunohistochemical investigation. In particular, the problem of a differential diagnosis between NMZL and a florid hyperplasia of monocytoid B-elements was posed. Thus, in order to assess the nature (neoplastic vs. reactive) of the lesion, molecular analysis of the immunoglobulin genes was performed by PCR. Notably, although no clonal rearrangements were revealed by IGHV@ analysis, further evaluation of the immunoglobulin light chain (IGKV@) confirmed the presence of a clonal B-cell population. Accordingly, a final diagnosis of NMZL was made. In conclusion, this case is a good example of the crucial role of complete molecular analysis in the diagnostic work up of lymphoproliferative disorders.

Polymorphism of glutathione S-transferases M1 and T1: susceptibility to solar keratoses in an Italian population.

BACKGROUND: Polymorphisms of glutathione S-transferases (GSTs) are linked to skin cancer, but data on their association with solar keratosis (SK) are few and conflicting. AIM: To verify the possible association between the development of SK and the 'null' GSTM1 and/or T1 genotype. METHODS: Analysis of the GSTM1 and T1 genotype of 33 subjects with ≥3 solar keratoses and of 150 controls, before and after stratification based on smoking habits, sun exposure and immunosuppression. RESULTS: The GST T1 null allele is significantly (P < 0.03) associated with increased prevalence of SK in our population. CONCLUSIONS: Our study, the first on a Mediterranean population, shows the existence of a correlation between SK and the GST T1 null genotype. This result points out significant differences between subjects of different ethnic and geographical origin and warrants further investigation on a larger population, and ethnically different populations.

Peripheral T cell lymphoma, not otherwise specified: the stuff of genes, dreams and therapies.

Peripheral T cell lymphomas (PTCL) account for about 12% of lymphoid tumours worldwide. Almost half show such morphological and molecular variability as to hamper any further classification, and to justify their inclusion in a waste-basket category termed "not otherwise specified (NOS)". The latter term is used for neoplasms with aggressive presentation, poor response to therapy and dismal prognosis. In contrast to B cell lymphomas, PTCL have been the subject of only a limited number of studies to elucidate their pathobiology and identify novel pharmacological approaches. Herewith, the authors revise the most recent contributions on the subject based on the experience they have gained in the extensive application of microarray technologies. PTCL/NOS are characterised by erratic expression of T cell associated antigens, including CD4 and CD52, which have recently been proposed as targets for ad hoc immunotherapies. PTCL/NOS also show variable Ki-67 marking, with rates >80% heralding a worse prognosis. Gene expression profiling studies have revealed that PTCL/NOS derive from activated T lymphocytes, more often of the CD4+ type, and bear a signature composed of 155 genes and related products that play a pivotal role in cell signalling transduction, proliferation, apoptosis and matrix remodelling. This observation seems to pave the way for the use of innovative drugs such as tyrosine kinase and histone deacetylase inhibitors whose efficacy has been proven in PTCL primary cell cultures. Gene expression profiling also allows better distinction of PTCL/NOS from angioimmunoblastic T cell lymphoma, the latter being characterised by follicular T helper lymphocyte derivation and CXCL13, PD1 and vascular endothelial growth factor expression.

Calcium and phosphorus balance in very low birth weight babies on total parenteral nutrition.

A balance study of Ca and P has been performed in 12 Very Low Birth Wt babies receiving prolonged Total Parenteral Nutrition. The mean intake of both minerals was 54.4 mg/kg/day (range 40-70). In order to avoid the formation and precipitation of CaP crystals in the solution, fructose-1,6-diphosphate was used as a source of P. 30 balance studies were performed between the seven and 63 day of life: they were always positive with a mean retention of 47.4 mg/kg/day of Ca and 48.1 mg/kg/day of P. For both minerals, 88% of the amount infused was retained: the correlation between intake and retention was linear and statistically significative (Ca:r = 0.9, p < 0.0001; P:r = 0.68, p < 0.0001). The post-natal and post-conceptional ages of the babies had no influence on Ca and P balance. The blood levels of Ca and P were poorly correlated to both intake and excretion, and were not as indicative of the mineral balance as the retention rates calculated on the basis of the 24 h urinary excretion of the minerals. A very useful test for clinical monitoring of Ca and P balance proved to be the Ca/creatinine and P/creatinine ratios measured on simple urine samples, which were strongly correlated to 24 h excretion. All infants developed radiological signs of mild osteopenia, but there was no case of acute metabolic derangement or rickets. Our data demonstrated that even in sick VLBW infants on TPN it is possible to achieve good retention rates of Ca and P, which are not different from those observed in well VLBW babies fed a 'standard' premature formula.

Repeated rescue of a Broviac catheter: fixation of exposed cuff by a skin flap.

A cutaneous infection exposed the cuff of a Broviac catheter employed for home-TPN in a 3-month-old child with ultra-short bowel syndrome. In order to avoid removal of the catheter, sepsis was abated by antibiotic administration through its lumen, then the exposed cuff was covered and fixed by a skin flap. The advantages proceeding from this sort of "emergency rescue" of the Broviac catheter have been: (1) to avoid a new cutdown in a child already submitted to several attempts of cannulation with sacrifice of major vessels; (2) to resume home total parenteral nutrition (TPN) in a short time, being the patient strictly dependent upon his parenteral intake and to spare a well-functioning catheter. Ten months after the last cuff covering by skin flap, the catheter is safely fixed in place and currently employed for home TPN.