Immunization with a novel recombinant protein (YidR) reduced the risk of clinical mastitis caused by Klebsiella spp. and decreased milk losses and culling risk after Escherichia coli infections.

The primary objective of this study was to evaluate the protective efficacy of a novel recombinant subunit vaccine containing the protein YidR (rYidR) against clinical mastitis (CM) caused by Klebsiella spp. and Escherichia coli. Given that E. coli infection is known to cause metritis, we also evaluated the effect of rYidR vaccination on the incidence of metritis and conception at the first artificial insemination. Retained placenta and abortion incidence, milk production and composition, and serological responses to specific antigens were also evaluated. In total, 3,107 cows were blocked by parity and randomly allocated into 1 of 3 treatment groups: experimental recombinant subunit vaccine containing the YidR protein (rYidR); commercial vaccine composed of Klebsiella pneumoniae siderophore receptors and porin protein (Kleb-SRP; KlebVax, Epitopix, Willmar, MN); and sterile water adjuvanted with aluminum hydroxide (20%; placebo). Vaccinations were performed at the dry-off for cows, and at 223 ± 3 d of pregnancy for pre-fresh heifers. A second administration was given at 21 ± 3 d after the first injection. Vaccination with rYidR significantly reduced the incidence of CM caused by Klebsiella spp. (3.2%) when compared with the placebo (5.1%) group. No difference was observed on risk of Klebsiella CM between Kleb-SRP (5.9%) and placebo groups. Cows in the rYidR group that experienced E. coli CM had a lower risk of death or culling (12.5%) compared with the Kleb-SRP (27.6%) and placebo groups (27.8%). Furthermore, among cows that developed E. coli CM, rYidR-immunized cows produced more milk than did cows in the placebo and Kleb-SRP groups. Regardless of CM occurrence, rYidR-immunized cows tended to have higher milk production up to the eighth month of lactation than cows in the other groups. No significant effect of treatment was observed on the overall incidence of abortion and metritis; however, the risk of retained placenta tended to be lower for the rYidR group (4.7%) compared with the placebo group (6.7%). In addition, primiparous cows in the rYidR group had the highest conception risk at the first artificial insemination (48.3%) compared with the placebo (39.5%) group, and no significant difference was observed when the Kleb-SRP (40.1%) group was compared with the placebo group. Generally, higher antibody serum titers (IgM and IgG) were observed for the immunized groups compared with the placebo. In conclusion, the rYidR vaccine reduced the risk of CM caused by Klebsiella spp. and the mortality or culling of cows with E. coli infections. Other benefits of the novel vaccine include maintenance of milk production after CM caused by E. coli, and higher conception risk at the first service in primiparous cows compared with cows in the placebo and Kleb-SRP groups.

Development and validation of I-DNA1: a 15-Loci multiplex system for identity testing.

This paper presents a system for the multiplex amplification of 15 loci, known as I-DNA1, which combines mini and midiSTR technology, with amplicon sizes ranging from 49 to 297 bp. I-DNA1 analyses all the STR loci included in the CODIS and the Interpol Standard Set of loci, nine of the ten European core loci and seven of the eight German core loci, making it suitable for use in identifying humans. Moreover, its high sensitivity and the small size of its amplicons mean that I-DNA1 is potentially highly useful for analysing highly degraded and/or very small DNA samples.

SNPSTR rs59186128_D7S820 polymorphism distribution in European Caucasoid, Hispanic, and Afro-American populations.

Single nucleotide polymorphisms (SNPs) in the flanking regions of microsatellite loci (SNPSTRs) help to increase the power of discrimination of short tandem repeat (STR) loci. SNPs are positions in the genome that have been well-conserved over the course of evolution, so analysing them can help distinguish between STR alleles in which the number of repetitions matches due to descent from those which match by chance. This provides support for the determination of biological paternity and other kinship analyses in which mutation needs to be ruled out as grounds for exclusion. Locus D7S820 shows a variable position, SNP rs59186128, in the 5' flanking region. This study is set out (1) to determine the frequencies of SNP rs59186128 in populations with various geographical origins and (2) to estimate the possible contribution of rs59186128 to the allele discrimination of locus D7S820. To that end, individuals from European Caucasoid, Hispanic, and Afro-American populations are studied using denaturing high-performance liquid chromatography, which enables locus rs59186128 to be quickly and highly cost-effectively screened. Moreover, a method is established for determining the haplotypes of SNPSTR rs59186128_D7820. The results show that SNP rs59186128 has a T allele frequency of more than 0.15 in one of the Afro-American populations studied, and the haplotype analysis shows that there is no preferential association between the alleles of SNPSTR rs59186128_D7S820, which supports the idea that they could be useful in forensic applications.

Cytological damage of nasal epithelium associated with decreased glutathione peroxidase in residents from a heavily polluted city.

OBJECTIVE: To evaluate the cytological damage and glutathione peroxidase (GPX) content in the nasal epithelium of residents of Southwest Metropolitan Mexico City (SWMMC) along 1 year of ozone and PM(10) exposure. METHOD: Four nasal scrapings were obtained in 20 volunteers from a control low polluted city and SWMMC permanent residents (n = 20) during 1 year. The scrapings were obtained in September and December 2004, and February and May 2005. One part of the scraping was stained by hematoxylin-eosin technique for cytological evaluation and a second part was stained by immunocytochemistry method to evaluate GPX concentration by morphometry. RESULTS: Control subjects: in total, 30% had no cytological alterations and 70% showed only mild or moderate inflammation in four nasal scrapings. All SWMMC residents showed moderate to severe inflammatory processes in some scrapings. Additionally, dysplasia was found once (in 2 cases) or more than on scraping in five cases (25%). GPX concentration in the control group remained highest in median values throughout the study. SWMMC residents with the highest median values of GPX content were found in the May and September scrapings, and the lowest median values were found in December and February when Ozone and PM(10) levels are increased (P < or = 0.05). A lower GPX content was found as the cytological damage increased (P < or = 0.001). CONCLUSION: Cytological evaluation of nasal epithelium and GPX immunodetection are satisfactory methods to evaluate the earliest damage produced by atmospheric pollution in heavily contaminated cities.

Phenotypic and genotypic characterization of Staphylococcus spp. isolated from mastitis milk and cheese processing: Study of adherence and biofilm formation.

The aim of this study was to identify the phenotypic and genotypic profiles of Staphylococcus spp. isolated from mastitis milk and cheese processing plant.To evaluate the biofilm production of wild-type strains on contact surfaces by testing different factors through adhered cells and biofilm quantifications, finally, these biofilms were observed by Scanning Electron Microscopy (SEM). Congo red agar (CRA) plate method was used to identify slime production by strains. Screening of genes encoding adhesion factors and biofilm formation was carried out using PCR. After strains selection, adhesion and biofilm assays were designed testing different times (12, 48, 96 h), strains (n = 13), contact surfaces (stainless steel and polypropylene), and temperatures (5 °C and 25 °C); and then, bacterial count and crystal violet staining were conducted. Relative frequencies of positive on CRA and genes presence were determined, and Friedman test was applied for bacterial counts and OD values. Additionally, significant factors (P ≤ .05) were subjected to multiple comparisons using the Nemenyi test. The slime production in CRA was observed by visual inspection in 38.7% of strains. A large distribution of genes was described among strains, implying a high variability of genotypic profiles. Moreover, relative frequencies of CRA positive and gene presence were described. The developed assay showed that the strain, temperature, contact surface, were significant for both variables. The SEM corroborated the findings, showing greater biofilm formation on stainless steel at 25 °C. Thus, it is essential to highlight the importance of temperature control and material with low superficial energy to avoid biofilm formation by staphylococci.

Retinoblastoma and optic nerve enhancement in a brain magnetic resonance scan: is it always a metastasis? / Retinoblastoma y realce del nervio óptico en la resonancia magnética cerebral: ¿es siempre una metástasis?

CASE REPORT: The case is presented on a girl with a unilateral retinoblastoma that required treatment with intra-arterial chemotherapy. In the nuclear magnetic resonance imaging of the brain performed 1 month after intra-arterial chemotherapy treatment, post-laminar optic nerve (ON) enhancement was observed, leading to the suspicion of an ON tumour infiltration. Additional examinations were requested by which a probable optic neuropathy was diagnosed. DISCUSSION: The ON enhancement in magnetic resonance imaging of the brain in retinoblastoma generally corresponds to tumour invasion of the ON. However, other diagnostic alternatives associated with the use of new treatments, such as intra-arterial chemotherapy, should be considered.

Simvastatin interferes with cancer 'stem-cell' plasticity reducing metastasis in ovarian cancer.

Cell plasticity of 'stem-like' cancer-initiating cells (CICs) is a hallmark of cancer, allowing metastasis and cancer progression. Here, we studied whether simvastatin, a lipophilic statin, could impair the metastatic potential of CICs in high-grade serous ovarian cancer (HGS-ovC), the most lethal among the gynecologic malignancies. qPCR, immunoblotting and immunohistochemistry were used to assess simvastatin effects on proteins involved in stemness and epithelial-mesenchymal cell plasticity (EMT). Its effects on tumor growth and metastasis were evaluated using different models (e.g., spheroid formation and migration assays, matrigel invasion assays, 3D-mesomimetic models and cancer xenografts). We explored also the clinical benefit of statins by comparing survival outcomes among statin users vs non-users. Herein, we demonstrated that simvastatin modifies the stemness and EMT marker expression patterns (both in mRNA and protein levels) and severely impairs the spheroid assembly of CICs. Consequently, CICs become less metastatic in 3D-mesomimetic models and show fewer ascites/tumor burden in HGS-ovC xenografts. The principal mechanism behind statin-mediated effects involves the inactivation of the Hippo/YAP/RhoA pathway in a mevalonate synthesis-dependent manner. From a clinical perspective, statin users seem to experience better survival and quality of life when compared with non-users. Considering the high cost and the low response rates obtained with many of the current therapies, the use of orally or intraperitoneally administered simvastatin offers a cost/effective and safe alternative to treat and potentially prevent recurrent HGS-ovCs.

A GHEP-ISFG collaborative study on the genetic variation of 38 autosomal indels for human identification in different continental populations.

A collaborative effort was carried out by the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) to promote knowledge exchange between associate laboratories interested in the implementation of indel-based methodologies and build allele frequency databases of 38 indels for forensic applications. These databases include populations from different countries that are relevant for identification and kinship investigations undertaken by the participating laboratories. Before compiling population data, participants were asked to type the 38 indels in blind samples from annual GHEP-ISFG proficiency tests, using an amplification protocol previously described. Only laboratories that reported correct results contributed with population data to this study. A total of 5839 samples were genotyped from 45 different populations from Africa, America, East Asia, Europe and Middle East. Population differentiation analysis showed significant differences between most populations studied from Africa and America, as well as between two Asian populations from China and East Timor. Low FST values were detected among most European populations. Overall diversities and parameters of forensic efficiency were high in populations from all continents.

Mutation rates at Y chromosome specific microsatellites.

A collaborative work was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) to estimate Y-STR mutation rates. Seventeen Y chromosome STR loci (DYS19, DYS385, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460, DYS461, DYS635 [GATA C4], GATA H4, and GATA A10) were analyzed in a sample of 3,026 father/son pairs. Among 27,029 allele transfers, 54 mutations were observed, with an overall mutation rate across the 17 loci of 1.998 x 10(-3) (95% CI, 1.501 x 10(-3) to 2.606 x 10(-3)). With just one exception, all of the mutations were single-step, and they were observed only once per gametogenesis. Repeat gains were more frequent than losses, longer alleles were found to be more mutable, and the mutation rate seemed to increase with the father's age. Hum Mutat 26(6), 520-528, 2005. (c) 2005 Wiley-Liss, Inc.

A new oral direct thrombin inhibitor, dabigatran etexilate, compared with enoxaparin for prevention of thromboembolic events following total hip or knee replacement: the BISTRO II randomized trial.

BACKGROUND: Dabigatran etexilate is an oral direct thrombin inhibitor undergoing evaluation for the prevention of venous thromboembolism (VTE) following orthopedic surgery. METHODS: In a multicenter, parallel-group, double-blind study, 1973 patients undergoing total hip or knee replacement were randomized to 6-10 days of oral dabigatran etexilate (50, 150 mg twice daily, 300 mg once daily, 225 mg twice daily), starting 1-4 h after surgery, or subcutaneous enoxaparin (40 mg once daily) starting 12 h prior to surgery. The primary efficacy outcome was the incidence of VTE (detected by bilateral venography or symptomatic events) during treatment. RESULTS: Of the 1949 treated patients, 1464 (75%) patients were evaluable for the efficacy analysis. VTE occurred in 28.5%, 17.4%, 16.6%, 13.1% and 24% of patients assigned to dabigatran etexilate 50, 150 mg twice daily, 300 mg once daily, 225 mg twice daily and enoxaparin, respectively. A significant dose-dependent decrease in VTE occurred with increasing doses of dabigatran etexilate (P < 0.0001). Compared with enoxaparin, VTE was significantly lower in patients receiving 150 mg twice daily [odds ratio (OR) 0.65, P = 0.04], 300 mg once daily (OR 0.61, P = 0.02) and 225 mg twice daily (OR 0.47, P = 0.0007). Compared with enoxaparin, major bleeding was significantly lower with 50 mg twice daily (0.3% vs. 2.0%, P = 0.047) but elevated with higher doses, nearly reaching statistical significance with the 300 mg once-daily dose (4.7%, P = 0.051). CONCLUSIONS: Oral administration of dabigatran etexilate, commenced early in the postoperative period, was effective and safe across a range of doses. Further optimization of the efficacy/safety balance will be addressed in future studies.

Development of a simple embedding procedure allowing immunocytochemical localization at the ultrastructural level.

Immunobed solution A is a water-soluble acrylic compound recently developed for immunocytochemical localization at the light microscopic level. In this study, we combined it with methyl methacrylate (MMA) to achieve sufficient hardness to obtain ultra-thin sections. Samples of platelets were dehydrated and embedded in the water-soluble acrylic mixture (WSAM). The embedding process was carried out at 4 degrees C and final polymerization was induced with either chemical (benzoyl peroxide) or physical (UV light) catalysts. Tubulin was localized at the ultrastructural level in sections embedded according to these two methods. Results were compared with those obtained in platelets processed in Lowicryl. Dehydration and embedding with the WSAM yielded a preservation of antigenicity similar to that obtained in Lowicryl. The new procedure benefits from the low temperature achieved during polymerization, providing good ultrastructural morphology and immunolocalization of protein antigens with the simplicity of a routine embedding procedure for light microscopy.

Increased prevalence of cysticercosis and taeniasis among professional fried pork vendors and the general population of a village in the Peruvian highlands. Cysticercosis Working Group in Peru.

Two different populations in Saylla, a Peruvian village near Cusco, known for chicharrones, a local pork dish, were surveyed by serology and stool examination to determine the prevalence and epidemiologic characteristics of Taenia solium infection. Group I (n=43), the chicharroneros, were members of families professionally devoted to the making and selling of chicharrones, and Group II (n=102) was a sample of the general population of the same village. Unlike people in Group I, general villagers only occasionally prepare or sell this food product, and then only to their neighbors or relatives. The prevalence of taeniasis was extremely high (8.6%) for the chicharroneros and 3% for the general villagers. Seroprevalence for cysticercosis by immunoblot was similarly high in both groups (23.3% and 23.8%, respectively). Being female, older than 30, and having daily contact with pork were factors strongly associated with a positive serologic result for cysticercosis in the chicharroneros, whereas males were more frequently seropositive in the general villagers group. Antibody reaction to more antigen bands in immunoblots and neurologic symptoms were more common among the chicharroneros. Also, in the general villagers group, seroprevalence increased with each exposure factor, ranging from 9.4% in individuals who did not raise pigs to 50% in the small subgroup that raised pigs, butchered their own animals, sold pork, and sold chicharrones, suggesting that these activities are related to increased risk for tapeworm or larval infection.

Gastrotoxic activity and inhibitory effects on gastric mucosal PGE2 production with different non-steroidal anti-inflammatory drugs: modifications induced by pretreatment with zinc acexamate.

Gastrotoxic activities of different non-steroidal anti-inflammatory drugs (NSAIDs) (diclofenac, indomethacin, ketoprofen, naproxen and piroxicam) administered per os were compared with their ability to inhibit gastric prostaglandin E2 (PGE2) synthesis in the rat. In a parallel study, effects of pretreatment with zinc acexamate (ZAC) were also assessed. NSAIDs invariably caused gastric mucosal damage and a decrease of PGE2 levels. A good correlation between the decrease of PGE2 levels and the index of gastric lesion (r = 0.41; p < 0.021) was observed when results obtained with the different NSAIDs were pooled. ZAC pretreatment significantly decreased the overall severity of lesions induced by NSAIDs. However, no correlation between gastric lesion index and depletion of PGE2 gastric levels was observed after treatment with ZAC (r = 0.012; p < 0.948). These data corroborate the hypothesis that preservation of the capability to synthesize endogenous PGs is of critical importance in the maintenance of gastric mucosal integrity. The gastroprotective action observed with ZAC involves alternative mechanisms other than modification of PGE2 levels.

Cytoprotective activity in the gastric mucosa of rats exposed to carbon tetrachloride-induced liver injury.

The present study was undertaken to evaluate the cytoprotective activity in the gastric mucosa of rats subjected to CCl4-induced liver injury. Response of gastric mucosa to absolute ethanol insult or acid (pylorus ligation) after CCl4 challenge was analyzed. Intraperitoneal administration of CCl4 increased plasma AST and ALT, but liver protein and glycogen levels were decreased; in addition, gastric acid secretion was significantly increased with respect to control animals (1541 +/- 266 vs. 629 +/- 25 mu eq H+; p < 0.001). Microscopical gastric erosions were observed in 3/10 animals after CCl4 challenge. Pylorus-ligated as well as CCl4-challenged rats developed increased susceptibility to gastric lesions, compared to control (lesion indices: 4.6 +/- 0.20 vs 2.8 +/- 0.13; p < 0.05), while showing increased resistance to absolute ethanol-induced gastric damage (30.4 +/- 11.2 vs 89.7 +/- 9.7 mm, p < 0.01). PGE2 levels in the gastric mucosa were not influenced by exposure to CCl4. Ultrastructural studies revealed the presence of continuous ethanol-resistant and apparently more adherent layer of mucus in CCl4-challenged animals. Morphological evaluation revealed an increase in Alcian blue-stained mucus. A dual condition of enhanced sensitivity to HCl with increased tolerance to ethanol was observed in gastric mucosa of CCl4-treated animals. These observations could be explained by the amount and/or composition of protective mucus layer in the gastric mucosa.

Evaluation of cutaneous absorption and safety of idoxuridine after topical administration.

The absorption of a preparation of the antiviral drug 5-iodo-2'-deoxyuridine (idoxuridine or IDU] has been assessed in rats. A 40% w/v solution of 3H-IDU in dimethyl sulfoxide (DMSO) was topically applied to rats; cutaneous absorption was determined by morphometric evaluation of autoradiographic preparations of the skin. Passage of the drug to the bloodstream was followed using radioisotopic techniques. In addition, the potential irritative action of the same preparation of IDU was investigated in a more suitable animal model. A predictive guinea pig sensitization study was performed according to the Magnusson and Kligman method. 3H-IDU was detected in the epidermis of the rat 60 min after local application. However, it was not possible to determine noticeable levels of the radiolabelled drug in the circulating blood. Experiments on guinea pigs showed slight transient erythematous responses in three animals of the test group following first challenge, and in one animal of the test group and one of the control group, following the second challenge. The overall results of the sensitization test were negative. These results give experimental support for the safety of the IDU preparation studied.

[Multiple sclerosis: epidemiological-genetic studies in the population of Antioquia, Colombia. Disequilibrium of HLA DQ alpha]. / Esclerosis múltiple: aproximación epidemiológico-genética en habitantes de Antioquia, Colombia. Desequilibrio de ligamiento al HLA DQ alpha.

INTRODUCTION AND OBJECTIVE: Discrimination and quantification of the environmental and genetic components involved in developing multiple sclerosis (MS) have not been made. In order to discriminate these components we have ascertained affected individuals by MS belonging to the Paisa community from Antioquia, Colombia, a state localized in the tropical area of South America, to detect eventual linkage disequilibrium to HLA, locus DQ alpha, which could demonstrate the relevance of the genetic component. DEVELOPMENT: A contingence analysis among case-control HLA DQ alpha genotype distributions, by using Monte Carlo resampling method to solve small number sample, showed that there are significant differences between the two groups. We observe that HLA DQ alpha 1.1, 1.2 allele frequencies were higher in the cases than in the controls. Also, there was significant HLA DQ alpha 3 allele lower frequency (p < 0.05) in the cases than in the controls. CONCLUSIONS: Similar results have been described in other Caucasian populations living in non tropical areas. Before results could indicate that the Caucasoid populations genetic component implied in the susceptibility to MS have remained in Paisa community, whether the environmental component, being meaningful to develop MS.