Reference ranges for haematology, biochemical profile and electrophoresis in a single herd of Ragusana donkeys from Sicily (Italy)

Donkeys, an endangered species, have recently gained a new application with the use of their milk to feed humans with allergic processes. The Ragusana donkey breed from Sicily is used to produce milk for humans with allergic diseases. In order to evaluate the hygienic, nutritional and management measures on a farm of Ragusana donkeys, complete blood counts, extended biochemical profiles and serum protein electrophoresis, as part of metabolic profile test (MPT), were performed in Ragusana donkeys. Fifty-four donkeys were studied and grouped according to their age, (1) 29 females and a single stallion (n=30), (2) young females, 1 – 3 years old (n=10) and (3) young of both sexes under 1 year old (n=14). The RBC count, RDW value, Lymp, and Mono counts, and PDW values were statistically greater in donkeys under one year old than in adult donkeys, while the Seg Neu count was lower. The CPK, ALP, iPhos, and HCO₃, values were statistically higher in the group of donkeys under 1 year of age than adult donkeys while Cl and LDH values were statistically lower in donkeys under 1 year than adult donkeys. Additionally, statistically significant increased values for CPK, ALP, Alb, Chol, iPhos, HCO₃, and UIBC in young donkeys under 1 year when compared with young donkeys, 1 – 3 years were observed. A statistically significant decreased value for Urea and an increased value for Crea in young donkeys, 1 – 3 years old were found as compared to adults. The serum protein fractions recognised by electrophoresis were: albumin, alpha globulin (subdivided into alpha-1 and alpha-2-globulins), beta globulin, and gamma globulin. In the alpha-1-globulin region three small peaks were constantly noticed, and alpha-2-globulins were statistically different between the three groups being greater in young donkeys under 1 year of age. The results obtained were used both to establish reference ranges and a data bank for the farm of Ragusana donkeys for future needs in assessing the metabolic status and health of the animals.     

Association between atopy and recurrent vaginal candidiasis

To determine whether there is an association between atopy and recurrent vaginal candidiasis (RVC) and to evaluate the type-2 immune response in patients with RVC. Evaluation of immediate hypersensitivity skin tests to aeroallergens, measurement of total IgE and Candida albicans specific IgE and levels of IL-5 in 44 women with RVC and 26 with sporadic vaginal candidiasis (SVC). Statistical analyses were performed by Mann-Whitney test and chi(2) test with Yates correction. History of atopy (68%) and positive skin test (42%) were higher (P < 0.05) in RVC than in patients with SVC. No significant difference was found in total IgE, C. albicans specific IgE and IL-5 levels. There was a strong association between atopy and RVC, but type-2 immune response to C. albicans antigen was absent or similar in the two groups of patients.     

Intracellular calcium mobilization by muscarinic receptors is regulated by micromolar concentrations of external Ca2+

Carbachol-induced contractions of rat stomach fundus strips, obtained in a nutrient solution containing 1.8 mM Ca2+, were resistant to Ca2+ withdrawal, even after 1 h of bathing the tissues in a nominal 0 Ca2+ solution. This was not observed when K+ was used to evoke contractions, which were rapidly inhibited after Ca2+ removal (t1/2=2 min). The effect of carbachol in 0 Ca2+ solution was reduced by using drugs that reduce intracellular pools of Ca2+, such as caffeine (1-3 mM), ryanodine (30 microM) or thapsigargin (1 microM), corroborating the involvement of intracellular Ca2+ stores. On the other hand, when the 0 Ca2+ solution contained EGTA, a complete decline of carbachol effects was observed within about 8 min, indicating the involvement of extracellular Ca2+. Atomic absorption spectrometry showed that our 0 Ca2+ solution still contained 45 microM Ca2+, which was drastically reduced to 5.9 nM in the presence of EGTA. Taken together, our results indicate that the effects of carbachol are due to the mobilization of caffeine-, ryanodine- and thapsigargin-sensitive intracellular Ca2+ stores, and that these stores are not inactivated or depleted if micromolar concentrations (45 microM), but not nanomolar concentrations (5.9 nM) of Ca2+ are maintained in the extracellular milieu.     

Comparison of C(18)-carboxypropylbetaine and standard N-acetyl-L-cysteine-NaOH processing of respiratory specimens for increasing tuberculosis smear sensitivity in Brazil

Techniques to improve the sensitivity of smear microscopy would facilitate early tuberculosis (TB) diagnosis and disease control, especially in low-income countries where the positive predictive value is high. C(18)-carboxypropylbetaine (CB-18) is a zwitterionic detergent that helps to compensate for the innate buoyancy of mycobacteria, potentially enhancing recovery by centrifugation. Previous data suggest that CB-18 may increase the sensitivity of smear, culture, and molecular amplification diagnostic testing. The goal of the present study was to evaluate if the sensitivity of the smear technique using light microscopy could be improved by treating respiratory samples with CB-18. In the first phase, respiratory specimens were collected consecutively from patients with suspected pulmonary tuberculosis in a tertiary-care hospital in Rio de Janeiro, Brazil (236 specimens were analyzed). After protocol modifications, another 120 respiratory specimens were evaluated. The standard technique was N-acetyl-L-cysteine with sodium hydroxide (NALC-NaOH) treatment, smear concentration with centrifugation, and Ziehl-Neelsen staining. Culture on L?wenstein-Jensen slants was performed on all specimens for use as the "gold standard." No specimens from patients undergoing active TB treatment were included. The initial protocol for CB-18 processing resulted in a sensitivity of 59.6% and specificity of 96.8% compared to standard processing with a sensitivity of 66.0% and specificity of 96.8%. Using the modified protocol, the sensitivity of CB-18 increased to 71.4% with a specificity of 97.0% versus standard processing with a sensitivity of 61.9% and a specificity of 99.0%. The diagnostic yield of acid-fast bacillus smear with CB-18 in the absence of fluorescence microscopy and PCR compared to standard processing with NALC-NaOH was not significantly different, although the power to detect a difference by the modified assay was low.     

Extracellular HIV-1 Tat protein activates phosphatidylinositol 3- and Akt/PKB kinases in CD4+ T lymphoblastoid Jurkat cells

The biological basis for the pleiotropic activity of extracellular human immunodeficiency virus (HIV)-1 Tat protein on lymphoid T cell survival is not well understood. We have here demonstrated that the addition in culture of 0.1–10 nM Tat protein to 36-h serum-starved lymphoblastoid Jurkat T cells rapidly stimulates the catalytic activity of phosphatidylinositol 3-kinase (PI 3-K). The peak of activation was observed 30 min after Tat addition. Extracellular Tat also stimulated the catalytic activity of the Akt/PKB kinase, a major target of PI 3-K lipid products. Pretreatment of serum-starved Jurkat cells with 100 nM wortmannin (WT) or 10 μM LY294002, two unrelated pharmacological inhibitors of PI 3-K, markedly suppressed the catalytic activity of both PI 3-K and Akt/PKB in Jurkat cells. Moreover, at low concentrations (0.1–1 nM), extracellular Tat showed a small but reproducible protection of Jurkat cells from apoptosis induced by serum deprivation (p < 0.05), while the combination of Tat plus 100 nM WT significantly (p < 0.05) increased the percentage of apoptosis with respect to cells left untreated or treated with Tat alone. Taken together, these data suggest that the anti-apoptotic activity of low concentrations of Tat protein on Jurkat cells is mediated by a PI 3-kinase/Akt pathway.     

Multiclonal Leishmania braziliensis population structure and its clinical implication in a region of endemicity for American tegumentary leishmaniasis

In Corte de Pedra (CP), northeastern Brazil, Leishmania braziliensis causes three distinct forms of American tegumentary leishmaniasis (ATL). To test the hypothesis that strain polymorphism may be involved in this disease spectrum and accurately characterize the parasite population structure in CP, we compared one L. major, two non-CP L. braziliensis, one CP L. amazonensis, and 45 CP L. braziliensis isolates, obtained over a 10-year period from localized cutaneous, mucosal, and disseminated leishmaniasis patients, with randomly amplified polymorphic DNA (RAPD). Electrophoretic profiles were mostly unique across species. All typing protocols revealed polymorphism among the 45 CP L. braziliensis isolates, which displayed eight different RAPD patterns and greater than 80% overall fingerprint identity, attesting to the adequacy of the tools to assess strain variability in CP's geographically limited population of parasites. The dendrogram based on the sum of RAPD profiles of each isolate unveiled nine discrete typing units clustered into five clades. Global positioning showed extensive overlap of these clades in CP, precluding geographic sequestration as the mechanism of the observed structuralization. Finally, all forms of ATL presented a statistically significant difference in their frequencies among the clades, suggesting that L. braziliensis genotypes may be accompanied by specific disease manifestation after infection.     

Histology of the kidney and urinary bladder of Siphonops annulatus (Amphibia-Gymnophiona)

The histology of the kidney and urinary bladder of Siphonops annulatus was studied by light microscopy in semithin sections of tissue embedded in hydrophilic resin. The kidney's nephron comprises the renal corpuscle, neck segment, proximal tubule, intermediate segment, distal tubule and collecting tubule. Nephrostomes are present. This structure, the neck segment, and intermediate tubules present long cilia, and probably play important roles in the propulsion of the peritoneal fluid and glomerular filtrate. The proximal tubule cells possess loosely packed microvilli and contain abundant polymorphic granules and vesicles that assume the aspect of lysosomes in different stages of intracellular digestion. The distal tubules are characterized by large, vertically disposed mitochondria assuming the aspect of ions transporting cells. The urinary bladder is lined with a transitional epithelium, whose aspect varies according to the quantity of urine.     

Antibody against the carboxyl terminus of intimin alpha reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization

The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin alpha.     

Molecular epidemiologic evaluation of endocarditis due to Oerskovia turbata and CDC group A-3 associated with contaminated homograft valves

Oerskovia turbata is an unusual bacterial cause of endocarditis and septicemia in immunocompromised patients. In this study, we compared 12 isolates from a 1975 medical center cluster, 11 originally identified as O. turbata (four from the blood of a homograft aortic valve-associated endocarditis patient and seven from contaminated homograft valves) and one CDC group A-3 strain from the blood of a second endocarditis patient with fatal outcome, with eight control strains from unrelated locations. The control strains included type and reference strains of O. turbata, Cellulomonas hominis, and CDC group A-3. The four blood isolates from the first patient and six of the valve isolates shared identical biochemical, antimicrobial susceptibility, and BglI ribotype patterns that differed from the second patient's isolate and control strains. The blood isolate from the second patient and the remaining valve isolate shared a phenotypic and genotypic profile and were phenotypically identical to, but epidemiologically different from, the CDC group A-3 reference strain with the strain-specific enzyme. Also, these isolates differed from the type strain and the other reference strains of C. hominis and O. turbata. Our results indicate that the four blood isolates from the first patient and six of the homograft valve isolates represent a single clone of O. turbata associated with endocarditis. Additionally, our results indicate that the blood isolate from the second patient and one of the homograft valve isolates differ from O. turbata and C. hominis and represent a unique clone of CDC group A-3 associated with fatal endocarditis.     

In Situ Detection of Apoptosis at Sites of Chronic Bacterially Induced Inflammation in Human Gingiva

Apoptosis is a key phenomenon in the regulation of the life span of terminally differentiated leukocytes. Human gingiva represents an established model to study immune responses to bacterial infection. In this investigation, we used the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) technique to evaluate presence and topographic location of apoptosis-associated DNA damage in human gingival biopsies along with the expression of the p53 and Bcl-2 apoptosis-regulating proteins. Qualitative data analysis showed high densities of cells expressing DNA damage and p53 both within the epithelial attachment to the tooth and in the perivascular infiltrate (infiltrated connective tissue [ICT]) immediately underlying the site of chronic bacterial aggression. Topographic consistency between DNA damage- and p53-positive cells was consistently observed. Quantitative analysis of the ICT showed mean densities of DNA damage- and p53-positive cells of 345 ± 278 and 403 ± 182 cells/mm², respectively. Numerical consistency was confirmed by multivariate regression analysis: densities of DNA damage-positive cells were significantly predicted by densities of p53-positive cells (P = 0.001, r² = 0.84). In the ICT, cells displaying biotinylated DNA nicks were 3.8% ± 2.7% of total cellularity, while p53- and Bcl-2-positive cells represented 4.4% ± 1.7% and 15.4% ± 6.7% of total cells, respectively. It is suggested that p53 expression associated with DNA damage is a prevalent phenomenon in chronically inflamed human gingiva, and that apoptosis may be a relevant process for the maintenance of local immune homeostasis at sites of chronic bacterial challenge in vivo.