Sub-acute exposure of male guppies (Poecilia reticulata) to environmentally relevant concentrations of PFOA and GenX induces significant changes in the testis transcriptome and reproductive traits.

Poly- and perfluoroalkyl substances (PFAS) are frequently detected in the environment and are linked to adverse reproductive health outcomes in humans. Although legacy PFAS have been phased out due to their toxicity, alternative PFAS are increasingly used despite the fact that information on their toxic effects on reproductive traits is particularly scarce. Here, we exposed male guppies (Poecilia reticulata) for a short period (21 days) to an environmentally realistic concentration (1 ppb) of PFOA, a legacy PFAS, and its replacement compound, GenX, to assess their impact on reproductive traits and gene expression. Exposure to PFAS did not impair survival but instead caused sublethal effects. Overall, PFAS exposure caused changes in male sexual behaviour and had detrimental effects on sperm motility. Sublethal variations were also seen at the transcriptional level, with the modulation of genes involved in immune regulation, spermatogenesis, and oxidative stress. We also observed bioaccumulation of PFAS, which was higher for PFOA than for GenX. Our results offer a comprehensive comparison of these two PFAS and shed light on the toxicity of a newly emerging alternative to legacy PFAS. It is therefore evident that even at low concentrations and with short exposure, PFAS can have subtle yet significant effects on behaviour, fertility, and immunity. These findings underscore the potential ramifications of pollution under natural conditions and their impact on fish populations.

Expression of Ki67, BCL-2, and COX-2 in canine cutaneous mast cell tumors: association with grading and prognosis.

The expression of Ki67, BCL-2, and COX-2 was investigated in 53 canine cutaneous mast cell tumors (MCTs) by immunohistochemistry and quantitative real time polymerase chain reaction (qPCR) to evaluate their prognostic significance and the association with the histologic grading and the mitotic index (MI). MCTs were graded according to the Patnaik grading system and the novel 2-tier grading system proposed by Kiupel. The numbers of mitotic figures/10 high-power fields (MI) were counted. Both grading systems were significantly associated with prognosis. The Patnaik grading was of limited prognostic value for grade 2 MCTs, with 23% being associated with mortality. The concordance among pathologists was strongly improved by the application of the 2-tier grading system, and 71% of high-grade MCTs were associated with a high mortality rate. MI and Ki67 protein expression were significantly associated with grading and survival. No significant association between BCL-2 protein expression and either grading system or health status was observed. BCL-2 mRNA expression was significantly higher in grade 2 than in grade 1 MCTs, while no statistically significant differences were detected between low- and high-grade MCTs. The increased BCL-2 mRNA level was significantly associated with increased mortality rate. The COX-2 protein expression was detected in 78% of the MCTs investigated. However, neither association with the tumor grade nor with the health status was observed. COX-2 mRNA was significantly up-regulated in MCTs compared to surgical margins and control skin tissue, but it was neither associated with tumor grade nor with survival.

Tissue distribution and phenobarbital induction of target SLC- and ABC- transporters in cattle.

In veterinary pharmaco-toxicological sciences, few data about uptake and efflux drug transporters (DTs) expression and regulation phenomena have been published. In this study, the tissue distribution and transcriptional modulation of solute carrier (SLC) and ATP-binding cassette (ABC) DTs were investigated in cattle orally administered with phenobarbital (PB) by using a quantitative real-time RT-PCR approach. The criterion for target gene selection was the PB-responsiveness in human and rodent model species. All target DTs were expressed in the liver. Only two of the seven PB-responsive target DTs (SLCO1B3 and SLC10A1) were not constitutively expressed in cattle extra-hepatic tissues. The greatest number of DTs (SLCO2B1, ABCB1, ABCC2, ABCG2) were expressed in intestine and testis, followed by, adrenal gland (SLCO2B1, ABCB1, ABCG2), lung (ABCB1, ABCG2), kidney, and skeletal muscle (ABCG2). PB administration never altered DTs mRNA levels, except for an increase in hepatic ABCC2 mRNA and a down-regulation of renal ABCG2. Altogether, these results confirm only to some extent data obtained in humans and laboratory species; clearly, they should be considered a preliminary step for further molecular investigations about species-differences in DT gene expression and regulation as well as in DT expression and function.

Short communication: CSN1S1-CSN3 (α(S1)-κ-casein) composite genotypes affect detailed milk protein composition of Mediterranean water buffalo.

The aim of the study was to investigate the effect of composite CSN1S1-CSN3 [α(S1)-κ-casein (CN)] genotype on milk protein composition in Mediterranean water buffalo. Content of α(S1)-CN, α(S2)-CN, ß-CN, γ-CN, κ-CN, glycosylated and unglycosylated κ-CN, α-lactalbumin, and ß-lactoglobulin was measured by reversed-phase HPLC using 621 individual milk samples. Genotypes at CSN1S1 and CSN3 were also obtained by reversed-phase HPLC. Two alleles were detected at CSN1S1 (corresponding to the A and B variants, O62823: p.Leu193Ser,) and at CSN3 (corresponding to the X1 and X2 variants, CAP12622.1: p.Ile156Thr). Increased proportions of α(S1)-CN in total casein (TCN) were associated with genotypes carrying CSN1S1 A. Genotypes associated with a marked decrease of the proportion of α(S1)-CN in TCN (composite genotypes AB-X1X1 and BB-X1X2) were associated with marked increases in the proportion of α(S2)-CN. In addition, composite genotypes carrying the X1 allele at CSN3 were associated with a greater proportion of α(S2)-CN in TCN relative to those carrying CSN3 X2. Composite genotypes greatly affected also the variability of ratios of κ-CN to TCN, with genotypes carrying the X1 allele at CSN3 being associated with decreased ratios. The decreased content of glycosylated κ-CN associated with CSN3 X1 was responsible for the overall lower content of total κ-CN in milk of X1-carrying animals. Increasing the frequency of specific genotypes might be an effective way to alter milk protein composition, namely the proportion of α(S1)-CN, α(S2)-CN, and κ-CN in TCN, and the degree of glycosylation of κ-CN.

Effect of CSN1S1-CSN3 (α(S1)-κ-casein) composite genotype on milk production traits and milk coagulation properties in Mediterranean water buffalo.

The aim of this study was to estimate effects of CSN1S1-CSN3 (α(S1)-κ-casein) composite genotypes on milk production traits and milk coagulation properties (MCP) in Mediterranean water buffalo. Genotypes at CSN1S1 and CSN3 and coagulation properties [rennet clotting time (RCT), curd firming time (K20), and curd firmness (A30)] were assessed by reversed-phase HPLC and computerized renneting meter analysis, respectively, using single test-day milk samples of 536 animals. Alternative protein variants of α(S1)-CN and κ-CN were detected by HPLC, and identification of the corresponding genetic variants was carried out by DNA analysis. Two genetic variants were detected at CSN1S1 (A and B variants) and 2 at CSN3 (X1 and X2 variants). Statistical inference was based on a linear model including the CSN1S1-CSN3 composite genotype effect (7 genotypes), the effects of herd-test-day (8 levels), and a combined days in milk (DIM)-parity class. Composite genotype AB-X2X2 was associated with decreased test-day milk yield [-0.21 standard deviation (SD) units of the trait] relative to genotype BB-X2X2. Genotypes did not affect milk protein content, but genotype AB-X1X1 was associated with increased fat content compared with genotype BB-X2X2 (+0.28 SD units of the trait) and AB-X1X1 (+0.43 SD units of the trait). For RCT, the largest difference (+1.91 min; i.e., 0.61 SD units of the trait) was observed between genotype AA-X1X2 and AB-X1X1. Direction of genotype effects on K(20) was consistent with that for RCT. The maximum variation in K20 due to genotype effects (between AA-X1X2 and AB-X1X1 genotypes) was almost 0.9 SD units of the trait. Magnitude of genotype effects was smaller for A30 than for RCT and K20, with a maximum difference of 0.5 SD units of the trait between genotype AA-X1X2 and AA-X1X1. The B allele at CSN1S1 was associated with increased RCT and K20 and with weaker curds compared with allele A. Allele X2 at CSN3 exerted opposite effects on MCP relative to CSN1S1 B. Because of linkage disequilibrium, allele B at CSN1S1 and allele X2 at CSN3 tend to be associated and this likely makes their effects cancel each other. This study indicates a role for casein genes in variation of MCP of buffalo milk. Further studies are necessary to estimate the effects of casein genetic variants on variation of cheese yield.

Effect of the dietary supplementation with extracts of chestnut wood and grape pomace on performance and jejunum response in female and male broiler chickens at different ages.

BACKGROUND: Recently, interest in the use of herbs and phytogenic compounds has grown because of their potential role in the production and health of livestock animals. Among these compounds, several tannins have been tested in poultry, but those from chestnut wood and grape-industry byproducts have attracted remarkable interest. Thus, the present study aimed to gain further insights into the mechanisms involved in the response to the dietary supplementation with extracts of chestnut wood or grape pomace. To this purpose, 864 broiler chickens were fed a control diet (C) or the same diet supplemented 0.2% chestnut wood (CN) extract or 0.2% grape pomace (GP) extract from hatching until commercial slaughtering (at 45 days of age) to assess their effects on performance, meat quality, jejunum immune response and whole-transcriptome profiling in both sexes at different ages (15 and 35 d). RESULTS: Final live weight and daily weight gain significantly increased (P < 0.01) in chickens fed GP diets compared to CN and C diets. The villi height was lower in chickens fed the CN diet than in those fed the C diet (P < 0.001); moreover, a lower density of CD45+ cells was observed in chickens fed the CN diet (P < 0.05) compared to those fed the C and GP diets. Genes involved in either pro- or anti-inflammatory response pathways, and antimicrobial and antioxidant responses were affected by GP and CN diets. There was no effect of the dietary treatment on meat quality. Regarding sex, in addition to a lower growth performance, females showed a lower occurrence of wooden breast (16.7% vs. 55.6%; P < 0.001) and a higher occurrence of spaghetti meat (48.6% vs. 4.17%; P < 0.001) in pectoralis major muscles after slaughtering than those in males. Based on the results of whole-transcriptome profiling, a significant activation of some molecular pathways related to immunity was observed in males compared with those of females. CONCLUSIONS: The GP supplementation improved chicken performance and promoted immune responses in the intestinal mucosa; moreover, age and sex were associated with the most relevant transcriptional changes.

Testosterone hydroxylation in bovine liver: enzyme kinetic and inhibition study.

In order to sort out the involvement of cytochrome P450 (CYP) 3A and possibly CYP2B in testosterone hydroxylation in cattle, enzyme kinetic and inhibition studies were performed. Most relevant kinetic constants (Km and Vmax) for 6beta-, 16beta- and 2beta-testosterone hydroxylase (OHT) activities were determined and accounted for 93.4 +/- 13.8, 36.4 +/- 6.1 and 110.8 +/- 15.2 muM, respectively, for Km and 0.558 +/- 0.03, 0.280 +/- 0.013, and 0.338 +/- 0.017 nmol min-1 mg-1 protein, respectively, for Vmax. Eadie-Hofstee plot analysis pointed out how these enzymatic activities in cattle follow a monophasic kinetic pattern. Preliminary inhibition studies conducted with the CYP3A inhibitor ketoconazole and the CYP2B inhibitors orphenadrine and 9-ethynylphenanthrene seemed to suggest the major involvement of CYP3A in testosterone hydroxylation in cattle. Immuno-inhibition studies with an anti-peptide antibody against bovine CYP3A4 confirmed the predominant role of CYP3A in testosterone hydroxylation in bovine liver, proving the usefulness of anti-peptide antibodies in defining the contribution of specific P450 isoforms in drug metabolism in veterinary species.

Proposed new nomenclature for Bos taurus cytochromes P450 involved in xenobiotic drug metabolism.

The cytochrome P450 (CYP) superfamily of drug metabolizing enzymes (DMEs) plays a central role in the oxidative metabolism of xenobiotics to which living organisms are exposed. In Bos taurus (cattle), a definitive nomenclature for CYP proteins is still lacking, and to unambiguously settle cattle nomenclature a phylogenetic analysis of proteins belonging to CYP 1-4 families was performed. Sequences collected from GenBank and Dr Nelson's P450 homepage databases were analyzed according to the maximum likelihood method. Phylogenetic outputs showed that CYPs sharing the same name and collected from different species did not form, in several instances, monophyletic groups. Some cattle CYPs did not group with their supposed human orthologous counterparts, thus requiring a new nomenclature. Name changes mostly mirrored the orthologous counterparts established for other species, and new names were created when no clear orthologous sequences were identified. The new nomenclature will allow a more appropriate investigation of biochemical and molecular mechanisms involved in the expression and regulation of these DMEs.

Whole-transcriptome profiling of sheep fed with a high iodine-supplemented diet.

Iodine (I) is a micronutrient that mammals need for proper functionality of thyroid gland since it is the main component of thyroid hormones. Besides studies that have investigated the role of I in livestock nutrition, it is also important to know the transcriptomics changes in small ruminants following I supplementation. Therefore, the aim of this study was to investigate the effects of I on the whole blood transcriptome in sheep. Fifteen lactating cross-bred ewes (3 to 4-year-old, 55 to 65 kg BW) at their late lactation period were enrolled in this study. At the beginning, all the animals had a 2-week acclimation period where they were fed with a basal diet which includes an adequate level of I (2 mg I/animal per day) in the form of calcium iodate (CaI2O6). Then, the ewes were randomly divided into two groups and fed in individual troughs: the control group (n = 5) was maintained on basal diet and the experimental group (I, n = 10) was fed for 40 days with a diet containing a high I supplementation (equivalent to 30 mg I/animal per day), in the form of potassium iodide. Whole blood and milk were collected individually at the beginning (T0) and after the 40 days of supplementation (T40). Iodine quantification was assessed in serum and milk sample. Microarray gene expression analysis was performed on whole blood and, filtering data using a fold change >2 with an adjusted P < 0.05, we identified 250 differentially expressed genes (DEGs) in the I group (T40 v. T0). Looking for biological processes associated with our DEGs, we found significant association with cell growth regulation. Thus, our study unveils the role of I supplementation on gene expression in sheep improving the knowledge about micronutrients in animal nutrition.

Effect of dietary supplementation with yeast cell wall extracts on performance and gut response in broiler chickens.

BACKGROUND: The dietary supplementation of yeast cell wall extracts (YCW) has been found to reduce pathogenic bacteria load, promote immunoglobulin production, prevent diseases by pro-inflammatory responses, and alter gut microbiota composition. This study evaluated growth and slaughter results, health, gut morphology, immune status and gut transcriptome of 576 male chickens fed two diets, i.e. C (control) or Y (with 250-500 g/t of YCW fractions according to the growth period). At 21 and 42 d the jejunum of 12 chickens per diet were sampled and stained with hematoxylin/eosin for morphometric evaluation, with Alcian-PAS for goblet cells, and antibodies against CD3+ intraepithelial T-cells and CD45+ intraepithelial leukocytes. The jejunum sampled at 42 d were also used for whole-transcriptome profiling. RESULTS: Dietary YCW supplementation did not affect final live weight, whereas it decreased feed intake (114 to 111 g/d; P ≤ 0.10) and improved feed conversion (1.74 to 1.70; P ≤ 0.01). Regarding the gut, YCW supplementation tended to increase villi height (P = 0.07); it also increased the number of goblet cells and reduced the density of CD45+ cells compared to diet C (P < 0.001). In the gut transcriptome, four genes were expressed more in broilers fed diet Y compared to diet C, i.e. cytochrome P450, family 2, subfamily C, polypeptide 23b (CYP2C23B), tetratricopeptide repeat domain 9 (TTC9), basic helix-loop-helix family member e41 (BHLHE41), and the metalloreductase STEAP4. Only one gene set (HES_PATHWAY) was significantly enriched among the transcripts more expressed in broilers fed diet Y. However, a total of 41 gene sets were significantly over-represented among genes up-regulated in control broilers. Notably, several enriched gene sets are implicated in immune functions and related to NF-κB signaling, apoptosis, and interferon signals. CONCLUSIONS: The dietary YCW supplementation improved broiler growth performance, increased gut glycoconjugate secretion and reduced the inflammatory status together with differences in the gut transcriptome, which can be considered useful to improve animal welfare and health under the challenging conditions of intensive rearing systems in broiler chickens.

Effects of nonselective and selective cyclooxygenase inhibitors on small intestinal motility in the horse.

We investigated the effects of nonselective cyclooxygenase (COX) inhibitors (indomethacin and flunixin meglumine) and selective COX-1 (SC-560) or COX-2 (celecoxib, DUP-398 and NS-697) inhibitors on horse small bowel motility in vitro. At this purpose, samples of equine ileum were put in isolated organ baths for the motility experiments. Nonselective COX inhibitors were devoid of major effects on motility, except for an inhibition of tonic contraction shown by flunixin meglumine. SC-560, selective COX-1 inhibitor, was devoid of significant effects on ileal motility. Selective COX-2 inhibitors reduced both tonic contraction and spontaneous phasic contractions, while prostaglandin (PG) receptor antagonists were uneffective. Some of the intestinal samples were submitted to histological investigation or reverse transcription-polymerase chain reaction (RT-PCR), which revealed the presence of an inflammation reaction and the presence of both COX isoforms mRNAs. Present data support the hypothesis that the effects of COX inhibitors on horse small intestinal motility are not linked to PG depletion.

Effects of fenbendazole and triclabendazole on the expression of cytochrome P450 1A and flavin-monooxygenase isozymes in bovine precision-cut liver slices.

Combinations of the anthelmintics fenbendazole (FBZ) and triclabendazole (TCBZ) have shown enhanced efficacy against the liver fluke Fasciola hepatica. This study aimed to measuring the constitutive expression of CYP1A1, CYP1A2, FMO1 and FMO3, thought to be involved in the metabolism of those compounds, by using an absolute quantitative real time (RT)-PCR approach in bovine precision-cut liver slices (PCLS). It also aimed to characterize the effects of FBZ and TCBZ (alone and in combination) on the expression and activity of the aforementioned isozymes. Both FMO1 and FMO3 were equally represented in control PCLS, whereas CYP1A2 was expressed more than CYP1A1 (P<0.05). PCLS cultured in the presence of beta naphthoflavone (ß-NF; CYP1A inducer) had higher mRNA levels of CYP1A1, CYP1A2, FMO1 and FMO3 (P<0.05). No clear-cut evidence of transcriptional effects of the anthelmintics were recorded. After incubation of PCLS with FBZ, there was a significant increase (P<0.05) vs. controls and TBCZ was observed for CYP1A1. PCLS treated with FBZ showed a higher (P<0.05) expression of CYP1A2 compared to controls, TCBZ alone, and the combination FBZ+TCBZ. The gene expression profiles of FMO1 and FMO3 were not affected by the presence of the anthelmintics; the only exception was an upregulation of FMO3 by TCBZ alone. The observed transcriptional effects of the xenobiotics were not mirrored by increased enzyme activities using prototypical substrates of the isozymes under study. Although further confirmatory studies are needed, these results suggest that PCLS represent an alternative in vitro tool for studies on the expression, regulation and function of relevant xenobiotic-metabolizing enzymes in cattle.

Features and prognostic impact of distant metastases in 45 dogs with de novo stage IV cutaneous mast cell tumours: A prospective study.

BACKGROUND: Distant metastases in dogs with cutaneous mast cell tumors (cMCT) are rare and incurable. The aims of this prospective study were to clarify the clinico-pathological features of stage IV cMCTs and to identify possible prognostic factors for progression-free interval (PFI) and survival time (ST). MATERIAL AND METHODS: Dogs were eligible for recruitment if they had a previously untreated, histologically confirmed cMCT and if they underwent complete staging demonstrating stage IV disease. Dogs were uniformly followed-up, whereas treatment was not standardized and included no therapy, surgery, radiation therapy, chemotherapy, tyrosine-kinase inhibitors or a combination of these. RESULTS: 45 dogs with stage IV cMCT were enrolled. All dogs had distant metastatic disease, and 41 (91.1%) dogs had also metastasis in the regional lymph node. Histopathological grade and mutational status greatly varied among dogs. Median ST was 110 days. Notably, PFI and ST were independent of well-known prognostic factors, including anatomic site, histological grade, and mutational status. Conversely, tumor diameter >3 cm, more than 2 metastatic sites, bone marrow infiltration, and lack of tumor control at the primary site were confirmed to be negative prognostic factors by multivariate analysis. CONCLUSION: Currently, there is no satisfactory treatment for stage IV cMCT. Asymptomatic dogs with tumor diameter <3 cm and a low tumor burden, without bone marrow infiltration may be candidates for multimodal treatment. Stage IV dogs without lymph node metastasis may enjoy a surprisingly prolonged survival. The achievement of local tumor control seems to predict a better outcome in dogs with stage IV cMCT.

Kit receptor tyrosine kinase dysregulations in feline splenic mast cell tumours.

This study investigated Kit receptor dysregulations (cytoplasmic immunohistochemical expression and/or c-KIT mutations) in cats affected with splenic mast cell tumours. Twenty-two cats were included. Median survival time was 780 days (range: 1-1219). An exclusive splenic involvement was significantly (P = 0.042) associated with longer survival (807 versus 120 days). Eighteen tumours (85.7%) showed Kit cytoplasmic expression (Kit pattern 2, 3). Mutation analysis was successful in 20 cases. Fourteen missense mutations were detected in 13 out of 20 tumours (65%). Eleven (78.6%) were located in exon 8, and three (21.6%) in exon 9. No mutations were detected in exons 11 and 17. Seven mutations corresponded to the same internal tandem duplication in exon 8 (c.1245_1256dup). Although the association between Kit cytoplasmic expression and mutations was significant, immunohistochemistry cannot be considered a surrogate marker for mutation analysis. No correlation was observed between c-Kit mutations and tumour differentiation, mitotic activity or survival.

Conformity and controversies in the diagnosis, staging and follow-up evaluation of canine nodal lymphoma: a systematic review of the last 15 years of published literature.

Diagnostic methods used in the initial and post-treatment evaluation of canine lymphoma are heterogeneous and can vary within countries and institutions. Accurate reporting of clinical stage and response assessment is crucial in determining the treatment efficacy and predicting prognosis. This study comprises a systematic review of all available canine multicentric lymphoma studies published over 15 years. Data concerning diagnosis, clinical stage evaluation and response assessment procedures were extracted and compared. Sixty-three studies met the eligibility criteria. Fifty-five (87.3%) studies were non-randomized prospective or retrospective studies. The survey results also expose variations in diagnostic criteria and treatment response assessment in canine multicentric lymphoma. Variations in staging procedures performed and recorded led to an unquantifiable heterogeneity among patients in and between studies, making it difficult to compare treatment efficacies. Awareness of this inconsistency of procedure and reporting may help in the design of future clinical trials.

Transcriptomic signature of high dietary organic selenium supplementation in sheep: A nutrigenomic insight using a custom microarray platform and gene set enrichment analysis.

The objective of this study was to investigate the effect of a high dietary Se supplementation on the whole transcriptome of sheep. A custom sheep whole-transcriptome microarray, with more than 23,000 unique transcripts, was designed and then used to profile the global gene expression of sheep after feeding a high dietary supplementation of organic Se. Lactating crossbred ewes ( = 10; 3 to 4 yr of age and 55 to 65 kg BW) at late lactation (100 ± 8 d in milk) were acclimated to indoor individual pen feeding of a basal control diet (0.40 mg Se/d, sodium selenite) for 4 wk. Sheep were then kept on a diet with an extra (high) supplementation of organic Se (1.45 mg Se/d as Sel-Plex; Alltech Biotechnology Pty Ltd, Dandenong, Victoria, Australia) for 40 d. Whole blood was collected at 2 time points (last day of the acclimatization period [T0] and after 40 d of the organic Se supplementation [T40]), and then total RNA was isolated and labeled for the subsequent microarray analysis. Significance Analysis of Microarrays, using the -statistic, of the microarray data (T40 versus T0) evidenced the up- and downregulation of 942 and 244 transcripts (false discovery rate < 0.05), respectively. Seven genes showed the same trend of expression (up- or downregulation) when tested by quantitative real-time PCR (qPCR) in a cross-validation step. The microarray showed significant upregulation of the following selenoproteins at T40: selenium binding protein 1 (SELENBP1), selenoprotein W1 (SEPW1), glutathione peroxidase 3 (GPX3), and septin 8 (SEPT8). And the expression trends for SEPW1 and SEPT8 were validated using qPCR. Functional annotation of the differentially expressed genes showed the enrichment of several immune system-related biological processes (lymphocyte activation, cytokine binding, leukocyte activation, T cell differentiation, and B cell activation) and pathways (cytokine and interleukin signaling). Moreover, Gene Set Enrichment Analysis evidenced the enrichment of B and T cell receptors signaling pathways, with an enrichment score of 0.63 and 0.59, respectively. Overall, from a global gene expression (whole-transcriptome) point of view, short-term supplementation of a high dietary organic Se to Se-nondeficient sheep results in a transcriptomic signature that mainly reflects an induced immune system and a modulation of transcription effect. Also, the present study provides a custom whole-transcriptome microarray platform that can be used in further global gene expression studies in the ovine species.

Efficacy and side effects of radiation therapy in comparison with radiation therapy and temozolomide in the treatment of measurable canine malignant melanoma.

Prognosis for unresectable canine malignant melanoma (MM) is typically poor, and therapeutic approaches remain largely palliative. A bi-institutional trial was conducted to compare efficacy and safety of radiation therapy (RT) and RT with post-radiation temozolomide in dogs with chemotherapy-naïve, measurable MM. RT consisted of 5 × 6 Gy fractions over 2.5 weeks. Dogs whose owners wished to pursue chemotherapy received adjuvant oral temozolomide (60 mg m-2 for 5 days every 28 days). Fifteen dogs were treated with RT only (Group 1) and 12 dogs subsequently received temozolomide (Group 2). Overall response rate was similar between Group 1 (86.7%) and Group 2 (81.1%). Median time to progression (TTP) was significantly longer in Group 2 (205 days) compared to Group 1 (110 days; p = 0.046). Survival time was not significantly different between groups. Both treatments were well tolerated. Post-radiation temozolomide has a good safety profile, and may improve TTP in MM when compared to coarse fractionated RT.

An open-label phase 1 dose-escalation clinical trial of a single intravenous administration of gemcitabine in dogs with advanced solid tumors.

BACKGROUND: A broad range of gemcitabine dosages have been used in dogs. HYPOTHESIS/OBJECTIVES: To determine maximally tolerated dose (MTD), dose-limiting toxicity (DLT), and preliminary antitumor activity of intravenous administration of gemcitabine in dogs with advanced solid tumors. ANIMALS: Twenty-two client-owned dogs. METHODS: Dogs with advanced cancer were prospectively enrolled in an open-label Phase 1 study of gemcitabine. Gemcitabine was administered as a 30-minute intravenous bolus starting at 800 mg/m(2), using escalation of 50 mg/m(2) increments with 3 dogs per dose level. MTD was established based on the number of dogs experiencing DLT assessed after 1 cycle. Treatment continued until disease progression or unacceptable toxicosis. Additional dogs were enrolled at MTD to better characterize tolerability, and to assess the extent and duration of gemcitabine excretion. RESULTS: Twenty-two dogs were treated at 4 dose levels, ranging from 800 to 950 mg/m(2). Neutropenia was identified as DLT. MTD was 900 mg/m(2). DLT consisting of grade 4 febrile neutropenia was observed at 950 mg/m(2) in 2 dogs. There were no nonhematologic DLTs. Twenty dogs received multiple doses, and none had evidence of severe toxicosis from any of their subsequent treatments. At 900 mg/m(2), 2 complete and 5 partial responses were observed in dogs with measurable tumors. The amount of gemcitabine excreted in urine decreased over time, and was undetectable after the first 24 hours. CONCLUSIONS AND CLINICAL IMPORTANCE: The recommended dose of gemcitabine for future Phase 2 studies is weekly 900 mg/m(2). In chemotherapy-naïve dogs with advanced solid tumor this dose level merits further evaluation.

Comparative effects of cytokines on constitutive and inducible expression of the gene encoding for the cytochrome P450 3A6 isoenzyme in cultured rabbit hepatocytes: consequences on progesterone 6beta-hydroxylation.

Cultured rabbit hepatocytes were used to compare the relative activities of cytokines to inhibit the constitutive or rifampicin (RIF)-induced expression of the cytochrome P450 3A6 gene (CYP3A6). Human recombinant cytokines tested were interleukin-1beta (IL-1beta) (2 U/mL), interleukin-2 (IL-2) (5,000 U/mL) and interferon-gamma (IFN-gamma) (50 U/mL). Hepatocytes were cultured in the presence or absence of 25 microM RIF for 24 hr, with or without cytokines alone or in combination. All these cytokines inhibited RIF-induced P4503A6 expression without apparent cellular toxicity. By contrast, only IFN-gamma treatment provided a significant decrease (41%) in the constitutive P4503A6 protein level. Moreover, cytokines differed in their ability to repress RIF-dependent transcriptional induction of CYP3A6: IL-1beta and IL-2 were approximately equipotent, causing an almost 40-50% suppression of CYP3A6 mRNA and protein levels, whereas IFN-gamma exerted repressive effects only on P4503A6-related erythromycin N-demethylase activity and inducible protein expression. In fact, although strongly reducing P4503A6 protein content (an approximate 70% decrease), IFN-gamma did not exhibit any influence on CYP3A6 mRNAs with the exception of its association with interleukins. All these results suggest that IL-1beta and IL-2 mainly promote a transcriptional repression mechanism, given the absence of effect of these cytokines on the basal P4503A6 level, whereas IFN-gamma exerts a post-transcriptional suppressive action on both induced and constitutive P4503A6 expression. Consequently, P4503A6-dependent progesterone 6beta-hydroxylase activity also presented a cytokine-specific pattern of inhibition, with a much greater sensitivity than P4503A6 immunoreactive protein to IL-1beta and IL-2 + IFN-gamma treatments. Thus, this study underlines the significant impact of inflammation on steroid metabolism.

Triphenyltin acetate-induced cytotoxicity and CD4(+) and CD8(+) depletion in mouse thymocyte primary cultures.

Organotin compounds (OTs) find application worldwide as catalysts, stabilizers and biocides. Triphenyltin derivatives (TPs), including the fungicide triphenyltin acetate (TPTA), are OTs mostly used in our country. Some OTs were proved to be immunotoxic and in this paper the cytotoxicity, the possible selective activity upon definite lymphocyte subsets as well as the antiproliferative effect of TPTA was investigated in vitro by using primary cultures of mouse thymocytes. TPTA (5, 10 and 25 microM) was cytotoxic to these cells, as demonstrated by the significant (P<0.05) reduction of the cell viability percentage (trypan blue dye exclusion test), the neutral red uptake and the reduction of tetrazolium salts to formazan products (MTT assay). These overt effects were already noticed after 4 h of exposure to TPTA. The fungicide otherwise significantly reduced, after 24 h of incubation, the percentage of mature single positive thymocytes, particularly the CD4(+)/CD8(-) one. Finally, a significative dose-dependent inhibition of the T-cell mitogen-induced cell proliferation was observed in thymocytes exposed to 1 and 8 microM TPTA. These results are indicative of the TPTA immunotoxic properties, according to previous published reports concerning the in vitro and in vivo toxicity of some di- and triorganotin compounds.