Enrico Bollo

Biofilm development by clinical isolates of Malassezia pachydermatis.

Malassezia pachydermatis fungemia has been reported in patients receiving parenteral nutrition. Biofilm formation on catheters may be related to the pathogenesis of this mycosis. We investigated the biofilm-forming ability of 12 M. pachydermatis strains using a metabolic activity plate-based model and electronic microscopic evaluation of catheter surfaces. All M. pachydermatis strains developed biofilms but biofilm formation showed variability among the different strains unrelated to their clinical origin. This study demonstrates the ability of M. pachydermatis to adhere to and form biofilms on the surfaces of different materials, such as polystyrene and polyurethane.

Effects of dexamethasone, administered for growth promoting purposes, upon the hepatic cytochrome P450 3A expression in the veal calf.

Dexamethasone (DEX) exerts its known anti-inflammatory and immunosuppressant activities through the interaction with the glucocorticoid receptor (GR). In human liver, DEX is metabolized by cytochrome P450 3A (CYP3A); moreover, it is among those xenobiotics which induce CYP3A itself. The transcriptional regulation of CYP3A involves GR and nuclear receptors (NRs). In cattle, DEX is used at low dosages as a growth promoter; besides, CYP3A is expressed in the liver. In the present study, the effects of two illicit DEX protocols upon liver CYP3A were investigated in the veal calf. Dexamethasone, administered per os (DOS) or injected intramuscularly (DIM) at growth promoting purposes, increased GR mRNA (+25.62% and +73.02% of CTRL for DOS and DIM, respectively), while tyrosine aminotransferase (TAT) and NRs gene expression profiles were unaffected; decreased CYP3A mRNA (-20.64% and -16.07% with Q RT-PCR; -30.55% and -34.31% with Northern blotting); at the post-translational level, decreased TAT activity (-19.84% and 44.34%), CYP3A apoprotein (-27.65% and -42.85%) and CYP3A-dependent enzyme activities (erythromycin N-demethylase, -78.89% and -23.87%; ethylmorphine N-demethylase, -44.26% and -28.37%; testosterone 6beta-hydroxylase, -44.60% and -18.07%; testosterone 2beta-hydroxylase, -43.95% and -11.69%); by contrast, an increase (about 2-fold) of the urinary 6beta-hydroxycortisol:cortisol ratio was observed in vivo. In summary, DEX modulates cattle liver CYP3A at pre- and post-translational level. Species-differences in GR-NRs-CYP3A regulation and in their response to differing DEX dosages might justify present results. Furthermore, the urinary 6beta-hydroxycortisol:cortisol ratio is not useful to monitor in vivo CYP3A activity in DEX-treated individuals.

Effects of anabolic and therapeutic doses of dexamethasone on thymus morphology and apoptosis in veal calves

Three groups of 10 veal calves were treated, respectively, with 5 mg of dexamethasone-21-isonicotinate administered intramuscularly on days 0 and 7 (group A); 0·4 mg/day of dexamethasone-21-phosphate administered orally for 20 days (group B); or left untreated as controls (group C). Two animals from each group were slaughtered on day 3, 7, 14, 32 and 52. The size and weight of the thymus decreased progressively in both treated groups until day 32. On day 14, in comparison with the controls, there was a mean reduction of 76 per cent in the thymus weight of group A and 35 per cent in group B. On day 32, the reductions were 13 per cent in group A and 50 per cent in group B, but the thymus weight of both groups had recovered completely by day 52. Dexamethasone-induced changes in thymus weight associated with lymphoid depletion and fat replacement, and there were clear correlations between these changes and apoptosis of thymocytes.

Antioxidant capacity as a reliable marker of stress in dairy calves transported by road

TRANSPORTATION of cattle is a routine management practice. However, mixing groups of unfamiliar animals and loading, unloading and driving the cattle are associated with psychological stress, physical damage and injury. These stresses have adverse effects on animal welfare and lead to reduced meat quality and economic losses to the farmer (Knowles 1999, Eicher 2001). Biochemical parameters in the blood (for example, levels of cortisol, adrenaline, non-esteridied fatty acids, iron, urea and glucose) are useful in the assessment of transport stress (Grasso and others 1989, Agnes and others 1990, Warriss and others 1995, Steinhardt and others 1997). The most commonly measured indicator of shortterm stress is cortisol, but its levels are highly variable and comparisons should not be made between different studies (Grandin 1997). The evaluation of antioxidant capacity (that is, the ability of a compound to reduce pro-oxidants) is commonly used in human medicine, in cystic fibrosis (Lands and others 2000), diabetes (Ceriello and others 1997) and HIV infection (De Martino and others 2001). This short communication describes a study to determine whether antioxidant capacity is a reliable marker in the evaluation of transport stress in veal calves transported by road. Fifty calves aged between one and two months were purchased in Rumilly, France, and transported by road to Centallo, northern Italy, over a distance of approximately 330 km, a journey which took five hours. Transportation took place at the end of January 2000, at a mean atmospheric temperature of 5°C. There were 20 male and 30 female cattle, all Garonnaise crosses, from different facilities, weighing 60 to 65 kg. They were all transported in one two-floor trailer at a stocking density of 0·65 m2 per calf, and they were not given food or water during the journey; this has been shown to lead to weight loss and dehydration, especially in young calves (Knowles 1995). Blood samples were collected immediately after the journey by jugular venipuncture, and the sera obtained were immediately frozen. The calves were boxed singly and fed a milk replacer. They were in good health and two months after their arrival, when a second blood sampling was performed, they weighed approximately 110 to 120 kg. Each animal acted as its own control in the study; as suggested by Palme and others (2000), this reduces the influence of variation between individuals. The sera were tested by means of the PAO kit (MED.DIA) in order to determine their antioxidant capacity, according to the manufacturer’s instructions. This test is based on the evaluation of the concentration of Cu+ ion obtained from the reduction of a known amount of Cu2+ ion by antioxidant substances in the sample. The antioxidant capacity of the sample can be quantified by comparing the obtained value with a standard curve. Briefly, serum samples were diluted in the buffer supplied with the kit and placed in 96-well plates, and their absorbance at 490 nm was determined in a microplate reader (Bio-Rad) to obtain blank values. After the addition of the Cu+-containing solution and incubation for three minutes at room temperature, stopping solution was added and a second absorbance value at 490 nm was determined. The antioxidant capacity value for each sample, expressed as reducing equivalents in μmol/litre, was obtained by subtracting the first measure from the second one, comparing the result with the standard curve and multiplying by a correction factor. The tests were performed in duplicate. The results were analysed using Student’s t test for paired values and InStat software (GraphPad Software). The antioxidant capacity values were normalised by applying the formula:

Genital Lesions following Long-term Administration of Clenbuterol in Female Pigs

Pathologic findings, lectin histochemistry, and nuclear estrogen receptors were studied in the reproductive organs of gilts treated with clenbuterol. A ration containing 1 ppm of clenbuterol was fed for 40 days to four Landrace x Large white, 9-month-old gilts, weighing 134 to 172 kg at slaughter (gilt Nos. 5–8). Four gilts (Nos. 1–4) served as controls. Treated animals had macroscopic lesions characterized by microcystic ovaries and uterine atrophy. Histopathologic lesions included atretic degeneration of many ovarian follicles, complete absence of functional corpora lutea, a reduction in the number of endometrial glands, and a decrease in cytoplasmic volume of endometrial and glandular epithelial cells. In ovaries, uterus, and vagina lectin histochemistry, performed with thirteen different biotinylated lectins, revealed a different staining distribution between control and treated gilts. The binding pattern of Ricinus communis agglutinin-1 (RCA-I) and -II (RCA-II) in the ovaries of control gilts, displayed labeling of cytoplasm in theca interna cells of Graafian follicles. There was no labeling of the same cells in treated gilts. Labeling patterns with Griffonia simplicifolia agglutinin-I (GS-I), Phaseolus vulgaris agglutinin (PHA), RCA-I and RCA-II documented a difference in the vascularis of the theca interna between Graafian follicles of control and treated gilts. The GS-1 and Ulex europaeus agglutinin-I (UEA-I) binding patterns in uterus and vagina of treated gilts when compared to control gilts suggested that there was a block of the cycling activity in the proliferative stage. Immunohistochemical staining for estrogen receptors in the endometrium was positive in all but one treated gilts, and negative to weakly positive in control gilts. Serum progesterone concentrations were decreased in treated animals when compared to control: estradiol concentrations were similar in both group of gilts. Cystic ovaries, uterine atrophy, and reduction in progesterone concentrations suggested that clenbuterol changed ovarian hormonal activity in treated animals.

Actividad enzimática extracelular en Cryptococcus neoformans en diferentes países

Three hundred and ten Cryptococcus neoformans strains isolated from AIDS patients in five different countries (151 from Brazil, 23 from Italy, 28 from Spain, 104 from Thailand and four from Turkey) were tested by the API-ZYM kit to detect their extracellular enzymatic activity. The enzymes esterase (C4) (no 3), esterase lipase (C8) (no 4), leucine arylamidase (no 6) and acid phosphatase (no 11) were commonly positive in most of the strains (more than 95%). These enzymes could be considered a useful tool not only for C. neoformans identification, but in particular for their possible relationship to new C. neoformans virulence factors and also for epidemiological research. Interestingly, it is also the high positive percentage of alpha-glucosidase and beta-glucosidase detected in all isolates. The serotype A was the most predominant serotype in all countries, except for Italy where the serotype D was predominant. Further studies are needed to draw a clear correlation between the API-ZYM profile and serotype.Three hundred and ten Cryptococcus neoformans strains isolated from AIDS patients in five different countries (151 from Brazil, 23 from Italy, 28 from Spain, 104 from Thailand and four from Turkey) were tested by the API-ZYM kit to detect their extracellular enzymatic activity. The enzymes esterase (C4) (no 3), esterase lipase (C8) (no 4), leucine arylamidase (no 6) and acid phosphatase (no 11) were commonly positive in most of the strains (more than 95%). These enzymes could be considered a useful tool not only for C. neoformans identification, but in particular for their possible relationship to new C. neoformans virulence factors and also for epidemiological research. Interestingly, it is also the high positive percentage of alpha-glucosidase and beta-glucosidase detected in all isolates. The serotype A was the most predominant serotype in all countries, except for Italy where the serotype D was predominant. Further studies are needed to draw a clear correlation between the API-ZYM profile and serotype.

Thymus atrophy and regeneration following dexamethasone administration to beef cattle

Thymus atrophy and regeneration were studied in 13- to 22-month-old beef calves treated with dexamethasone (DMT), using anabolic dosages and implementing different withdrawal times. Two trials were conducted. In trial 1, group A (n=6) received 0.7 mg/day DMT orally for 40 days, group B (n=6) received 1.4 mg/day orally for 40 days and group C (n=6) was the control. In trial 2, group D (n=6) received 0.7 mg/day DMT orally for 40 days, group E (n=6) received 1.4 mg/day orally for 40 days and group K (n=6) was the control. DMT withdrawal times before slaughter were six days (groups A and B) and 26 days (groups D and E). At slaughter, thymus atrophy was severe and progressive in animals from groups A and B. In contrast, thymus weight and volume of the animals from groups D and E were almost normal. Slight atrophy was also detected in the calves in these groups. Histological changes and Ki67 immunostaining revealed a large number of positive lymphoid cells, mostly in the cortical area, associated with higher expression of apoptosis in the medulla compared with controls. This demonstrated that the thymus of beef cattle is still able to regenerate following DMT administration.

Triphenyltin acetate toxicity : a biochemical and ultrastructural study on mouse thymocytes

1 Triphenyltin acetate (TPTA) has been shown to exert in vivo a selective toxic effect on the immune system. To assess in vitro possible alterations induced by TPTA exposure, primary cultures of mouse thymocytes were incubated up to 24 h with graded amounts (1-12 μM) ofthe organotin. 2 The cytotoxic activity has been evaluated with the MTT colorimetric assay, the neutral red (NR) assay and the lactic dehydrogenase (LDH) cellular release. Cell pellets were fixed with 2.5% glutaraldehyde, resin-embedded and ultrathin sections were observed through transmission electron microscopy. 3 After 2 h of incubation, dose-dependent increases of cytotoxicity were observed in thymocytes submitted to MTT and NR tests (up to 41.43% and 18.9%, respectively), while 22 h later this overt effect on cell viability was noticed merely in cells exposed to 12 μM TPTA. Dose- dependent increases of LDH leakage in the culture medium were observed all throughout the study. 4 Morphological investigations revealed features (chro matin condensation, cell membranes fragmentation and formation of membrane bound apoptotic bodies) sugges tive of apoptosis. 5 This study indicates that TPTA is cytotoxic to mouse thymocytes: morphologically, the rising of apoptosis is likely to be recognized, as previously reported in different in vitro studies with other immunosuppressive agents as dioxin and corticosteroids.

17β-oestradiol-induced gene expression in cattle prostate: biomarkers to detect illegal use of growth promoters

The effects of 17β-oestradiol (E2) on gene expression in cultures of bovine primary prostate stromal cells (BPSCs) and prostate gland tissue were studied. In the first part of the study, BPSCs were grown in the presence of E2 from the first passage to the end of the experiment; a second group was treated in the same way but the treatment was suspended for 48 hours before the end of the experiment; a third group of BPSCs served as a control. In the second part of the study, five male veal calves, aged 130 days, were treated four times intramuscularly with 10 mg of E2 at intervals of two weeks and then euthanased two weeks after the last treatment. Quantitative PCR and immunohistochemistry were used to evaluate the expression of fibroblast growth factor (FGF) receptors (FGFRs), FGFs, progesterone receptor, androgen receptor and oestrogen receptor in BPSCs and prostate tissue. E2 induced a significant over-expression of progesterone receptor in both BPSCs and prostate tissue. There was also a marked up-regulation of FGFR types 1, 2 and 3 genes observed in the BPSCs.

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.