Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo

Notch receptors and their ligands contribute to many developmental systems, but it is not apparent how they function after birth, as their null mutants develop severe defects during embryogenesis. Here we used the Cre-loxP system to delete the Delta-like 1 gene (Dll1) after birth and demonstrated the complete disappearance of splenic marginal zone B cells in Dll1-null mice. In contrast, T cell development was unaffected. These results demonstrated that Dll1 was dispensable as a ligand for Notch1 at the branch point of T cell-B cell development but was essential for the generation of marginal zone B cells. Thus, Notch signaling is essential for lymphocyte development in vivo, but there is a redundancy of Notch-Notch ligand signaling that can drive T cell development within the thymus.     

Meningitis due to mixed infection with penicillin-resistant and penicillin-susceptible strains of Streptococcus pneumoniae

Streptococcus pneumoniae is the major cause of bacterial meningitis. We report a case of meningitis due to a mixed infection with two distinct strains of S. pneumoniae: one penicillin-resistant strain of serotype 9V and one penicillin-susceptible strain of serotype 7. The two strains exhibited different pulsed-field gel electrophoresis profiles.     

Development of DNA vaccines against hemolytic-uremic syndrome in a murine model

Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2 Delta A). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.     

HLA-DRB1*1602 allele is positively associated with HPV cervical infection in Bolivian Andean women

Incidence of cervical cancer is high among Bolivian Andean women. Human papillomavirus (HPV) infection is known as the major risk factor of cervical cancer. The host immune system plays an important role in the outcome of HPV infection and associated malignancies. In order to study the immunogenetic background of Bolivian Andean women with regard to HPV infection status, we compared HLA class I and class II allele frequencies between 37 HPV positive and 68 HPV negative Bolivian women. Demographic variables, including distribution of Andean ethnicities, were similar in both groups. Comparison of HLA class I allele frequencies between both groups indicated no significant difference. In contrast, HLA class II DRB1*1602 allele, an Amerindian allele, was significantly higher in the HPV positive women compared with HPV negative controls (chi(2) = 5.2, p < 0.05, odds ratio = 3.17; 95% confidence interval = 1.4-8.8). HPV types present in the HPV positive group were HPV-18, -16, -31, -33, and -58. These results suggest that HLA class II DRB1*1602 may confer susceptibility to infection with genetically related HPV types. This is the first report of an HLA class II association with HPV infection in an Andean population.     

Missed diagnosis in hematological patients—an autopsy study

Autopsy findings of missed diagnoses that would probably have changed management or prognosis occur in up to 29% of cases in general hospitals. Such proportions may be higher in subsets of patients with complex diseases. We reviewed 2908 consecutive autopsies performed over a period of 29 months in a large-volume hospital, analyzing 118 autopsies of patients with hematological malignancies or severe aplastic anemia. A review of macroscopic reports as well as microscopic examination of tissue samples was performed. Medical records were reviewed for clinical diagnoses. Discordances between clinical and autopsy diagnoses were classified using Goldmans criteria. Additionally, we searched for clinical parameters correlated with occurrence of class-I discrepancy using a multivariate method. Median age was 46.5 years, and 25.4% had received a hematopoietic stem-cell transplant. Overall, 11.9% (6.6–19.1%) of patients died before conclusion of the hematological diagnosis and 33% (24.6–42.3%) died with no active hematological disease. We found class-I discrepancy in 31.3% (23.1–40.5 %) of cases. The most common among these diagnoses were hematological disease, pneumonia and gastrointestinal bleeding. In a univariate analysis, being elderly (P=0.04) was positively correlated with the finding of class-I discrepancies; while, having received previous specific hematological treatment (P=0.0005) or hematopoietic stem-cell transplants (P=0.013), or being admitted to a specialized hematology unit (P=0.0006) were negatively correlated to the occurrence of such discrepancies. Multivariate analysis showed that care in a specialized hematology unit (OR 0.34, 0.12–0.93) was independently associated with lower occurrence of discrepancies. We concluded that critical diagnoses are often missed in highly complex hematological patients especially in the absence of admission to specialized hematology units.     

Multilocus sequence typing of methicillin-resistant Staphylococcus aureus from an area of low endemicity by real-time PCR

A protocol for multilocus sequence typing (MLST) of methicillin-resistant Staphylococcus aureus (MRSA) was adapted to real-time LightCycler System PCR for efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products. The method was evaluated on a representative and well-characterized collection of clinical MRSA isolates (n = 57) obtained from an area of low endemicity. Twenty sequence types (STs) and nine clonal complexes were identified. Combining STs and the staphylococcal cassette chromosome mec (SCCmec) type identified 27 different genotypes, and type IV SCCmec was present in 11 different STs. The presence of the Panton Valentine leukocidin (PVL) genes was found in isolates of four different STs. Eleven different STs were found among the community-acquired as well as among the hospital-acquired MRSA. The genetic heterogeneity was also denoted by pulsed-field gel electrophoresis analysis that showed 24 different pulsotypes among the 57 MRSA isolates. The presence of more than one different type of SCCmec in the same ST indicates that the MRSA clones have arisen at several occasions in the same genetic background by independent acquisition of SCCmec into methicillin-sensitive strains. This circumstance shows the importance of combining MLST data with SCCmec-typing results when investigating the origins of MRSA.     

Presence of the cfxA gene in Bacteroides distasonis

In this study we investigated the presence of the cfxA gene (encoding a class A beta-lactamase) in 73 strains of the Bacteroides fragilis group belonging to the species B. distasonis (34), B. vulgatus (14), B. thetaiotaomicron (8), B. merdae (6), B. caccae (9) and B. ovatus (2) isolated from human intestinal microflora of healthy children and adults. Employing specific primers to the cfxA gene, a 312-bp amplified fragment was obtained in 2 strains of B. vulgatus and 9 strains, the majority from children, of B. distasonis. The expression of this enzyme was analysed by determining the MICs to cefoxitin and cefotaxime and values varied from 2 to >256 microg/ml of both cefoxitin and cefotaxime. Sequence analysis of the amplicons corresponding to the cfxA gene from B. distasonis and B. vulgatus revealed identical sequences between these isolates and high similarity with other beta-lactamase genes of anaerobes such as cfxA of B. vulgatus (99%) and cfxA2 of Prevotella intermedia (99%), both sequences of which deposited in Genbank under accession numbers U38243 and AF118110, respectively. However, a fragment obtained from a B. distasonis strain (EC17-4) showed a unique RFLP profile and 87% nucleotide similarity with cfxA and cfxA2 genes. These results seem to suggest a dissemination of these resistance determinants among Bacteroides species.     

Changes in peripheral energy metabolism during audiogenic seizures in rats

Plasma glucose and lactate, hepatic glycogen and epididymal adipose tissue lipogenesis and lipolysis were studied in Wistar audiogenic rats (WARs), a genetic model of epilepsy, under three experimental conditions, i.e., before, 3 min after and 10 min after seizures induced by intense sound exposure. Plasma glucose increased 3 min after the seizure onset and rose to a peak after 10 min. Hepatic glycogen decreased significantly in susceptible audiogenic rats compared to nonepileptic controls, even before sound stimulation. A marked ( approximately 10-fold) rise was observed in plasma lactate levels 3 and 10 min after the seizures compared to the response of the control group. Lipogenic activity showed a marked decrease even after stimulation with 25 ng/ml insulin. Based on these results, WARs showed reduced isoproterenol-stimulated lipolysis compared to control animals, whereas basal levels only differed significantly at 10 min after seizure activity. In conclusion, it can be inferred from these results that (a) the increase in plasma glucose after stimulation might result from sequential interaction of autonomic activation at seizure onset; (b) excessive muscular activity was at least partially responsible for the steady rise in plasma lactate concentrations; (c) audiogenic seizures, which increase adrenergic activity, induce desensitization of the beta-adrenergic lipolytic pathway in epididymal adipose tissue; (d) genetic selection for audiogenic seizure susceptibility results in pronounced alterations at multiple levels of metabolic regulation.     

Characterization and identification of an iron-oxidizing,Leptospirillum-like bacterium,present in the high sulfate leaching solution of a commercial bioleaching plant

Most copper bioleaching plants operate with a high concentration of sulfate salts, caused by the continuous addition of sulfuric acid and the recycling of the leaching solution. Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population present in the high-sulfate (150 gl(-1)) leaching solution, employed in a copper production plant, was investigated. The iron-oxidizing bacteria able to grow in the leaching solution were enriched by several batch cultivations and, after serial dilution, an abundant bacterial strain was isolated. This strain, called LA, exhibited a relatively constant rate of iron-oxidation in media containing sulfate ions at concentrations ranging from 10 to 150 gl(-1). Culture collection strains of Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans showed limited abilities to grow at sulfate ion concentrations higher than 70 gl(-1). In spite of its tolerance to high sulfate concentrations, strain LA was as sensitive to NaCl as A. ferrooxidans. Comparative sequence analysis of the 16S rRNA gene of strain LA indicated that it is phylogenetically related to strains described as Leptospirillum ferrooxidans. Bacterial community DNA restriction patterns of 16S rRNA genes suggested that strain LA was a minor component of the bacterial population present in leaching solution, but is abundant in ore leached with this solution.