Characterization of a novel leucine-rich repeat protein antigen from group B streptococci that elicits protective immunity

Group B streptococci (GBS) usually behave as commensal organisms that asymptomatically colonize the gastrointestinal and urogenital tracts of adults. However, GBS are also pathogens and the leading bacterial cause of life-threatening invasive disease in neonates. While the events leading to transmission and disease in neonates remain unclear, GBS carriage and level of colonization in the mother have been shown to be significant risk factors associated with invasive infection. Surface antigens represent ideal vaccine targets for eliciting antibodies that can act as opsonins and/or inhibit colonization and invasion. Using a genetic screen for exported proteins in GBS, we identified a gene, designated lrrG, that encodes a novel LPXTG anchored surface antigen containing leucine-rich repeat (LRR) motifs found in bacterial invasins and other members of the LRR protein family. Southern blotting showed that lrrG was present in all GBS strains tested, representing the nine serotypes, and revealed the presence of an lrrG homologue in Streptococcus pyogenes. Recombinant LrrG protein was shown in vitro to adhere to epithelial cells in a dose-dependent manner, suggesting that it may function as an adhesion factor in GBS. More importantly, immunization with recombinant LrrG elicited a strong immunoglobulin G response in CBA/ca mice and protected against lethal challenge with virulent GBS. The data presented in this report suggest that this conserved protein is a highly promising candidate antigen for use in a GBS vaccine.     

The Complete Set of Predicted Genes from Saccharomyces cerevisiae in a Readily Usable Form

Nearly all of the open reading frames (ORFs) of the yeast Saccharomyces cerevisiae have been synthesized by PCR using a set of ~6000 primer pairs. Each of the forward primers has a common 22-base sequence at its 5′ end, and each of the back primers has a common 20-base sequence at its 5′ end. These common termini allow reamplification of the entire set of original PCR products using a single pair of longer primers—in our case, 70 bases. The resulting 70-base elements that flank each ORF can be used for rapid and efficient cloning into a linearized yeast vector that contains these same elements at its termini. This cloning by genetic recombination obviates the need for ligations or bacterial manipulations and should permit convenient global approaches to gene function that require the assay of each putative yeast gene.     

Mnemonic coding system for clinical data entry into laboratory computers: its effect on quality and efficiency.

A simplified coding method for entering the clinical details found on pathology request cards was developed. The method uses a basic four letter code, derived from the initial character of the first four words in a clinical detail, being expanded to four characters with letters from the final word if the number of words is less than four. Rules were devised to cope with common medical terminology. In excess of 90% of clinical details on request cards are readily input by clerical staff using our coding system, and 8% of clinical details are used intelligently by the computer in scheduling further tests or automatically commenting on results. A carefully designed coding system such as the one outlined above could greatly facilitate input of clinical detail without the penalty of reduced throughput.     

Improving the teaching skills of residents as tutors/facilitators and addressing the shortage of faculty facilitators for PBL modules

Background  

Residents play an important role in teaching of medical undergraduate students. Despite their importance in teaching undergraduates they are not involved in any formal training in teaching and leadership skills. We aimed to compare the teaching skills of residents with faculty in facilitating small group Problem Based Learning (PBL) sessions.     

Anti-inflammatory activity of salmeterol: down-regulation of cytokine production.

Elevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a beta 2-agonist which activates adenylate cyclase, its ability to inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol, did not inhibit IL-1 beta production in vitro, both drugs did inhibit tumour necrosis factor-alpha (TNF-alpha) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar IC50s of approximately 0.1 microM. This inhibition was effectively reversed by the beta 2-antagonist oxprenolol, indicating that the inhibition was mediated through the beta 2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human T cells, as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-alpha production, and was not reversed by a beta 2-antagonist, indicating that a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-alpha inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with ED50s of approximately 0.1 mg/kg. This inhibition could be abrogated by dosing orally with the beta-blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3 h, while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-alpha production. Though salmeterol inhibited serum TNF-alpha levels by up to 94% in this assay, it protected less than 50% of the animals from the lethal effects of the LPS/galactosamine mixture. This observation suggests that functional levels of TNF-alpha localized in tissues may not be accurately reflected by serum levels.     

Results of a high-resolution genome screen of 437 Alzheimer's disease families

Alzheimer's disease (AD) is a devastating neurodegenerative disorder of late life with complex inheritance. Mutations in three known genes lead to the rare early-onset autosomal dominant form of AD, while a common polymorphism (epsilon 4) in the gene encoding apolipoprotein E (APOE ) is a risk factor for more typical late-onset (>60 years) AD. A recent study concluded that there are up to four additional genes with an equal or greater contribution to the disease. We performed a 9 cM genome screen of 437 families with AD, the full National Institute of Mental Health (NIMH) sample, which has been carefully ascertained, evaluated and followed by our group over the last decade. Performing standard parametric and non-parametric linkage analyses, we observed a 'highly significant' linkage peak by Lander and Kruglyak criteria on chromosome 19q13, which probably represents APOE. Twelve additional locations-on 1q23, 3p26, 4q32, 5p14, 6p21, 6q27, 9q22, 10q24, 11q25, 14q22, 15q26 and 21q22-met criteria for 'suggestive' linkage [i.e. two-point lod score (TLS) >/=1.9 and/or multipoint lod score (MLS) >/=2.2] in at least one of our analyses. Although some of these will surely prove to be false positives, these linkage signals should provide a valuable framework for future studies aimed at identifying additional susceptibility genes for late-onset AD.     

Fine-needle aspiration of a primary mediastinal large B-cell lymphoma: a case report with cytologic, histologic, and flow cytometric considerations

Fine-needle aspiration (FNA) cytology and immunophenotyping by flow cytometry (FCM) are increasingly being used for diagnosing and subclassifying lymphoma in the REAL/WHO classification. Herein, we report a case of primary mediastinal large B-cell lymphoma (PMBL), a subtype of diffuse large B-cell lymphoma in the WHO classification, diagnosed by FNA cytology in conjunction with FCM. This, to our knowledge, has not previously been reported. A 57-yr-old woman presented with bilateral axillary lymphadenopathy and intermittent shortness of breath. CT scan revealed a 5-cm anterior mediastinal mass and mediastinal lymphadenopathy. Endoscopic ultrasound-guided FNA of a 4.5-cm subcarinal lymph node showed medium to large atypical lymphocytes with scant to moderate finely vacuolated cytoplasm. Nuclei were enlarged, cleaved, noncleaved, lobulated, and hyperchromatic. The background showed lymphoglandular bodies. Malignant large cell lymphoma was cytologically diagnosed. FCM, performed on a portion of the FNA specimen, demonstrated large B cells devoid of surface immunoglobulin expression, the characteristic immunophenotype of PMBL. The histologic diagnosis was PMBL. Touch-imprint cytology of the histologic specimen showed large cells with a narrow rim of clear cytoplasm and prominent outer cell border. Nuclear features were similar to the FNA specimen. In the presence of a mediastinal mass, FNA cytology in conjunction with FCM can effectively diagnose PMBL in the appropriate clinical setting.