Altered splenic B cell subset development in mice lacking phosphoinositide 3-kinase p85alpha

The signaling enzyme phosphoinositide 3-kinase (PI3K) is activated following B cell receptor (BCR) engagement and by many other receptors on B lymphocytes. Mice lacking p85alpha, the predominant PI3K regulatory isoform, exhibit defects in B cell development and activation that are grossly similar to those found in mice lacking Bruton's tyrosine kinase (Btk) and other critical signaling molecules. However, a detailed analysis of splenic B cell subsets in p85alpha-deficient mice has not been reported. Here we show that these mice are deficient in four major B cell subsets: transitional-1, transitional-2, follicular and marginal zone. These defects are distinct from those observed in Xid mice that express a mutant Btk unable to interact with PI3K lipid products. Moreover, mice with both genetic lesions exhibit even greater impairment in B cell development. Finally, we show that transgenic expression of the anti-apoptotic protein Bcl-2 in p85alpha-deficient mice restores the transitional B cell subsets but not the marginal zone subset, and produces a follicular population with an aberrant phenotype. These findings establish a role for PI3K-p85alpha in differentiation of both follicular and marginal zone B cells, and suggest that these functions are required not solely for the propagation of anti-apoptotic signals.     

Quantitative evaluation of recall and precision of CAT Crawler,a search engine specialized on retrieval of Critically Appraised Topics

Background

Critically Appraised Topics (CATs) are a useful tool that helps physicians to make clinical decisions as the healthcare moves towards the practice of Evidence-Based Medicine (EBM). The fast growing World Wide Web has provided a place for physicians to share their appraised topics online, but an increasing amount of time is needed to find a particular topic within such a rich repository.

Methods

A web-based application, namely the CAT Crawler, was developed by Singapore's Bioinformatics Institute to allow physicians to adequately access available appraised topics on the Internet. A meta-search engine, as the core component of the application, finds relevant topics following keyword input. The primary objective of the work presented here is to evaluate the quantity and quality of search results obtained from the meta-search engine of the CAT Crawler by comparing them with those obtained from two individual CAT search engines. From the CAT libraries at these two sites, all possible keywords were extracted using a keyword extractor. Of those common to both libraries, ten were randomly chosen for evaluation. All ten were submitted to the two search engines individually, and through the meta-search engine of the CAT Crawler. Search results were evaluated for relevance both by medical amateurs and professionals, and the respective recall and precision were calculated.

Results

While achieving an identical recall, the meta-search engine showed a precision of 77.26% (±14.45) compared to the individual search engines' 52.65% (±12.0) (p < 0.001).

Conclusion

The results demonstrate the validity of the CAT Crawler meta-search engine approach. The improved precision due to inherent filters underlines the practical usefulness of this tool for clinicians.

     

A screening assay for detecting CD8+ cell non-cytotoxic anti-HIV responses

The rate of HIV-1 disease progression is influenced by several factors that include pathogen and host genetic variations and the quality of antiviral immune responses. The CD8+ cell non-cytotoxic antiviral response (CNAR) substantially suppresses HIV replication in CD4+ cells and is positively associated with an asymptomatic clinical state. Traditionally, the measurement of CNAR has required several culture procedures and costly reagents. Here we report the development and validation of a screening assay for detection of CNAR that accurately identifies individuals benefiting from this response. Use of the CNAR screening assay should facilitate the evaluation of this important immune parameter in studies of HIV pathogenesis, resistance to infection, and vaccine development.     

Genetic imbalances in pleomorphic xanthoastrocytoma detected by comparative genomic hybridization and literature review.

Pleomorphic xanthoastrocytoma (PXA) is a rare, low-grade astrocytic tumor found in the central nervous system. Histologically, the tumor is characterized by markedly pleomorphic and lipidized cells. Although most of the patients have a favorable prognosis, a small number of cases undergoing recurrence or progression to anaplastic astrocytoma were reported. Very few genetic studies have been performed on PXA because of its rarity and the pathogenesis of this neoplasm is largely unknown. In order to provide an overview of genetic alterations in PXA, we performed comparative genomic hybridization to identify chromosomal imbalances (DNA gains and losses) in three cases of PXA. Genetic imbalance was detected on at least one chromosome for each case. One case, which revealed multiple genetic alterations, showed a poor prognosis. DNA gain on chromosome 7 and loss on 8p were demonstrated in two of three cases, suggesting that the candidate gene(s) located on these regions may play a role in the development of PXA. Further studies are needed to identify the residing candidate genes that are involved in the tumorigenesis of PXA. In addition, the histopathological features and previous genetic studies on PXA are reviewed.     

Tyrosine phosphatase-like protein (IA-2) and glutamic acid decarboxylase (GAD65) autoantibodies: a study of Chinese patients with diabetes mellitus

Type 1 diabetes in most Asian populations may not have a salient autoimmune basis when assessed with single determinations of the major markers, islet cell antibodies (ICAs) and glutamic acid decarboxylase antibodies (GAD65ab). With the inclusion of antibodies to tyrosine phosphatase-like protein IA-2 (IA-2ab) as an additional major marker, we re-examined autoimmune diabetes in a group of Chinese patients. We studied 272 subjects at various stages of disease with blood samples procured for biochemical analysis. ICAs were measured by immunofluorescence, GAD65ab and IA-2ab by radioimmunoassay. Sixty-seven patients fulfilled clinical diagnosis of type 1 diabetes and the remaining 205 patients were type 2. Prevalence of single autoantibody type in recent-onset type 1 diabetes ( < 1 year duration; n = 47) showed 10.6% with ICAs, 44.7% GAD65ab and 36.2% IA-2ab. GAD65ab account for more than two-thirds of the markers found in type 1 diabetes. Combined analysis further showed that 51.1% had at least one antibody type, 31.9% with two or more antibodies and 8.5% with all three antibodies. Islet autoimmunity presence in childhood-onset type 1 diabetes improved with the addition of IA-2ab, though less impact was seen in the adult-onset. Similarly, combined analysis for type 2 patients with recent diabetes showed a modest increase to 13% with islet autoimmunity compared to 8% when assessed by GAD65ab alone. Combining IA-2ab and GAD65ab assays results detected slightly more immune-mediated diabetes, compared to using a single GAD65ab determination. Non-autoimmune causes need to be considered in the pathogenesis of type 1 diabetes in Chinese, particularly in adults.     

Spinocerebellar ataxia type 7 (SCA7): a neurodegenerative disorder with neuronal intranuclear inclusions

Autosomal dominant cerebellar ataxia with progressive macular degeneration is caused by a CAG/glutamine repeat expansion in the SCA7 gene/protein. Neuronal intranuclear inclusions were detected in the brain of an early onset SCA7 case with the 1C2 antibody directed against an expanded polyglutamine domain. Nuclear inclusions were most frequent in the inferior olivary complex, a site of severe neuronal loss in SCA7. They were also observed in other brain regions, including the cerebral cortex, not considered to be affected in the disease. Using confocal microscopy we showed that some inclusions were ubiquitinated, but to varying degrees, ranging from <1% in the cerebral cortex to 60% in the inferior olive. In addition, we also observed cytoplasmic staining using the 1C2 antibody, particularly in the supramarginal gyrus, the hippocampus, the thalamus, the lateral geniculate body and the pontine nuclei. These data confirm that the presence of intranuclear inclusions in neurons is a common characteristic of disorders caused by CAG/polyglutamine expansions, but unlike what has been reported for Huntington's disease, SCA1 and SCA3/MJD, in SCA7 the inclusions were not restricted to the sites of severe neuronal loss.      

Human micro-insemination by injection of single or multiple sperm: ultrastructure

The process of micro-insemination by single or muhiple spermtransfer into the perivitelline space (PVS) or by direct sperminjection into oocytes was examined by transmission electronmicroscopy. Spermatozoa from normal and oligozoospermic menwere injected into oocytes, obtained from consenting IVF patients,mostly by zona-puncture using micromanipulators. Spermatozoawere washed by the Percoll or Ficoll methods and capacitatedusing Whittingham's T₆ or modified Tyrode's medium or incubatedin strontium medium before injection. The women were stimulatedby three IVF methods and oocytes were recovered by laparoscopyor ultrasonography. Sixty-one oocytes were cultured in T₆ orHam's F-10 media (3–24 h) and were subjected to micromanipulation.Four oocytes were also studied after zona-drilling. Normal 2-pronuclearova were developed after single-sperm transfer satisfying allmorphological criteria of fertilization. Both monospermic andpolyspermic fertilization resulted after multiple sperm transfer,indicating that a vitelline block to polyspermy may exist inhumans. The majority of oocytes examined were unfertilized.Spermatozoa with intact or reacted acrosomes and those undergoingthe acrosome reaction were found in the PVS and in the ooplasm.Abnormal spermatozoa were also seen in these locations. Quantitatlonof acrosomal status in 16 oocytes after multiple-sperm transfer,revealed that 24% of spermatozoa were acrosome reacted or reactingin the PVS following Ficoll entrapment, while 76% of spermatozoawere intact (33% of these abnormal). Sperm transfer seemed tobe the least invasive, while direct sperm injection was comparativelydestructive to oocytes. Drilling with acid made larger breachesin the zona when compared with mechanical perforation and spermatozoaoccasionally escaped through breaches. Three 2-pronuclear ovaobtained after multiple sperm transfer have resulted in twopregnancies, in cases of severe oligozoo spermia, during thecourse of this study.     

Presence of pro-opiomelanocortin peptides and corticotropin-releasing factor in human placenta

Immunoreactive adrenocorticotropin (ACTH), beta-endorphin (BEP) and corticotropin-releasing factor (CRF) were detected in human term placenta obtained from elective Caesarian surgery. The concentrations of ACTH, BEP and CRF in placenta detected by radioimmunoassay (RIA) were 2.83 +/- 0.36, 0.52 +/- 0.05 and 0.56 +/- 0.15 ng/g wet weight of tissue respectively. Pro-opiomelanocortin (POMC) peptides were also detected in the amnion and chorion membranes and in the decidua. The concentrations of ACTH were 1.72 +/- 0.20, 4.43 +/- 0.39 and 5.80 +/- 0.17 ng/g and the levels of BEP were 0.42 +/- 0.18, 0.65 +/- 0.20 and 3.66 +/- 1.10 ng/g in the amnion, chorion and decidua respectively. In contrast to placenta, immunoreactive CRF was not detected in the amnion, chorion and decidua. Immunoreactive N-acetylated BEP was also not detected in all the placental subfractions. Comparison of the amounts of both ACTH and BEP in the various placental components indicated the following distribution: decidua > chorion > placenta > amnion. In decidua, POMC peptides were present in an equi-molar ratio but in the other three placental fractions, ACTH levels were three to five-fold higher than BEP. In immunohistochemical studies, only a positive staining for ACTH was obtained for decidua. Our results confirm the presence of POMC peptides and CRF in placenta and their physiological roles in pregnancy and parturition.