Phosphoinositide 3-kinase γ mediates microglial phagocytosis via lipid kinase-independent control of cAMP.

Microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. Here we investigated the regulatory function of phosphoinositide 3-kinase γ (PI3Kγ) in phagocytosis of bacteria and Zymosan particles by mouse brain microglia in vitro and in vivo. Using genetic and pharmacological approaches our data revealed PI3Kγ as an essential mediator of microglial phagocytosis. Unexpectedly, microglia expressing lipid kinase deficient mutant PI3Kγ exhibited similar phagocytosis as wild-type cells. These data suggest kinase-independent stimulation of cAMP phosphodiesterase activity by PI3Kγ as a crucial mediator of phagocytosis. In sum our findings indicate PI3Kγ-dependent suppression of cAMP signaling as a critical regulatory element of microglial phagocytosis.

Improved strategies for HIV diagnosis among men who have sex with men (MSM) in Buenos Aires, Argentina, a population with a high prevalence and incidence of HIV infection.

BACKGROUND: In Argentina, HIV diagnosis in adults is made using one or two enzyme immunoassay tests and a confirmatory test. These strategies may fail to identify infected individuals during early primary infection, which represents an important public health problem among groups with a high HIV incidence, such as men who have sex with men (MSM) (6.3% persons/year). The general objective of this study was to contribute to reducing HIV transmission among MSM through the identification of antibody-negative, nucleic acid-positive individuals. FINDINGS: A total of 1549 MSM were recruited for an HIV seroprevalence study. A total of 161 (10.4%) MSM were HIV-positive and 14 (0.9%) were indeterminate. Among the 1374 negative individuals, 16 (1.2%) exhibited reactive results in the screening assay. Indeterminate Western blot (WB) samples and negative WB samples (with discordant results in the screening) were analysed to detect HIV nucleic acid by viral load testing. Up to 23.1% of HIV-indeterminate WB samples and 7.1% of HIV-negative WB samples with discordant results in the screening assays had detectable nucleic acid. Overall, 14.8% of the samples with discordant or indeterminate results were identified as HIV-positive using direct diagnosis. With the identification of four new cases using the nucleic acid detection test, the HIV prevalence in MSM increased by 0.3% (from 10.4 to 10.7%). CONCLUSIONS: The results of this study suggest the importance of including nucleic acid detection in the HIV algorithm for MSM with HIV-indeterminate WB results and those with HIV-negative WB results and discordant results in screening assays, in order to decrease HIV transmission among this population with a high HIV prevalence and incidence.

Incidence of HIV type 1 infection, antiretroviral drug resistance, and molecular characterization in newly diagnosed individuals in Argentina: A Global Fund Project.

An HIV incidence estimation was performed among men who have sex with men (MSM), drug users (DUs), sex workers (SWs), and pregnant women (PW) from Argentina. Volunteers older than 18 years old without a previous HIV-positive diagnosis were included. HIV-positive samples were analyzed by the Serological Testing Algorithm for Recent HIV Seroconversion (STARHS) to estimate incidence. By partial RT-PCR and sequencing of the HIV pol gene, an HIV subtype and resistance profile were determined. A total of 12,192 volunteers were recruited from October 2006 to September 2008. A higher HIV prevalence was detected among trans SWs (33.9%, 38/112), male SWs (10.8%, 12/111), and MSM 10.4% (161/1549). HIV incidence estimates by STARHS was also higher on trans SWs (11.31 per 100 person-years), male SWs (6.06 per 100 person-years), and MSM (6.36 per 100 person-years). Antiretroviral primary resistant mutations were detected in 8.4% of the study group, with a higher frequency in female DUs (33.3%). Phylogenetic analysis showed that 124 (57.9%) samples were subtype B, 84 (39.3%) intersubtype BF recombinants, 5 (2.3%) subtype C, and 1 (0.5%) subtype F in the pol region. Subtype B was most commonly found in MSM and male SWs whereas the intersubtype BF recombinant was more prevalent in female DUs, female SWs, and PW. Given the high HIV prevalence and incidence found in most of these groups, monitoring the continuing spread of the HIV epidemic is essential for determining public health priorities, assessing the impact of interventions, and estimating current and future health care needs.

The impact of environmental risk factors on HIV-associated cognitive decline in children.

Both the human immunodeficiency virus (HIV) and environmental stress have been independently associated with decreased cognitive functioning in children. Given that they are also known to have a strong relationship with each other, the present study sought to test the hypothesis that children in conditions of high environmental risk would be at greater risk for the cognitive complications related to immunosuppression. A retrospective review was conducted to examine the records of 141 children treated at a large pediatric AIDS clinic from 1993 to 2000. CD4+ lymphocyte levels were recorded from laboratory results and IQ scores were recorded from routine psychological evaluations. Key indicators of environmental risk were collected and combined into one measure of overall environmental risk. Pearson product moment correlations were conducted to examine the relationship between environmental risk, age-adjusted CD4 and IQ. Results indicated a significant correlation between CD4 and IQ, with higher levels of immunocompetence predicting higher IQ scores. When subjects were dichotomized based on their environmental risk score, there was no relationship between CD4 count and IQ in the low environmental risk group. In contrast, CD4 was positively associated with IQ in the high environmental risk group. It is proposed that this may be due to gp120 levels in immunocompromised children being particularly toxic to the hippocampus and cortex under conditions of high stress but not so under conditions of low stress.

Comparative analysis of two controlled proliferation strategies regarding product quality, influence on tetracycline-regulated gene expression, and productivity.

Overexpression of the cyclin-dependent kinase inhibitor p27 and exposure to low temperature (30 degrees C) represent two strategies to establish controlled proliferation processes for production of therapeutic proteins using Chinese hamster ovary (CHO) cells. Here we analyze the effect of growth inhibition on the quality of the human model glycoprotein SEAP (secreted alkaline phosphatase) for both strategies in monoclonal CHO-derived cell lines. Separation of purified SEAP samples using two-dimensional gel electrophoresis showed that production by proliferation-controlled CHO cultures did not alter the overall integrity of the product. Further, oligosaccharide profiles were compared using HPEC-PAD analysis. No differences were detectable between SEAP profiles obtained from p27 growth-arrested and proliferating cultures. However, production at 30 degrees C led to a significant increase in the degree of sialylation, an effect that is generally considered beneficial for the in vivo efficacy of protein therapeutics. In the production context presented here, SEAP expression is controlled by the tetracycline- (tet) repressible gene regulation system. Here we show low temperature-induced upregulation of the tetracycline-dependent transactivator (tTA). This induction has been shown by Northern blot analysis to occur at the mRNA level and is independent of the promoters driving the transactivator. We also describe a novel bottleneck in productivity at low temperature found in p27 growth-arrested CHO cells cultivated at 30 degrees C.

Characterization of an N-terminally truncated cyclin A isoform in mammalian cells.

Cyclin A is essential for regulating key transitions in the eukaryotic cell cycle including initiation of DNA replication and mitosis. This paper describes the characterization of a truncated cyclin A isoform (cyclin A(t)) in vitro in cultured mammalian cells and in mouse tissues. The presence of cyclin A(t) in specific cell types correlates with the ability of cell extracts to cleave in vitro translated cyclin A. In CHO-K1 cells, cyclin A processing to cyclin A(t) occurs at the N terminus; it does not involve the 26 S proteasome, nor could it be induced by conditional overexpression of the cyclin-dependent kinase inhibitor p27(Kip1). However, high cell densities lead to increased cyclin A(t) levels. Unlike full-length cyclin A, cyclin A(t) localizes to the cytoplasm, where it binds Cdk2. The data suggest that cyclin A processing occurs in vivo to yield an N-terminally truncated isoform by an unknown mechanism that is regulated by cell density. Differential subcellular localization may provide the first insights into the physiological role of cyclin A(t).

The MEDLINE experience at Yale: 1986-1996.

The Cushing/Whitney Medical Library began providing end-user access to MEDLINE in 1986 and switched to the OVID system in 1993. MEDLINE is a core service of the library, and the choice of delivery systems has had a significant impact throughout the Yale-New Haven Medical Center. This paper describes the user response to MEDLINE, discusses the effects of MEDLINE on other library services, and suggests ways in which technology, policy, and funding have influenced use. Yale's experience suggests that removing barriers in all three areas can dramatically expand the points of access, the number of users, and the amount of use with manageable effects on other library services.

Bronchiectasis in children with lymphocytic interstitial pneumonia and acquired immune deficiency syndrome. Plain film and CT observations.

In a review of 77 HIV positive children seen between 1981 and 1990, 32 were diagnosed as having lymphocytic interstitial pneumonitis). Four of the LIP group developed bronchiectasis, a finding not previously reported. The precise factors leading to the bronchiectasis are unclear. All patients had chronically consolidated lung with volume loss. A history of recurrent bacterial superinfection was not noted in any of the cases. With more cases of HIV positive children living longer, bronchiectasis, long known to occur in primary immunologic disorders, will probably be more frequently noted.

Nature of the repair of methyl methanesulfonate-induced damage in Bacillus subtilis.

A nuclease present in extracts of Bacillus subtilis inserts breaks in deoxyribonucleic acid (DNA) treated with the monofunctional alkylating agent, methyl methanesulfonate (MMS), but the nature of the sites within the alkylated macromolecule at which these breaks occur is not known. DNA extracted from B. subtilis cells that have recovered from MMS damage has lost its susceptibility to enzyme action. The recovery process is accompanied by some DNA breakdown and by the incorporation of thymidine. Some recovery from ultraviolet irradiation (UV) and MMS occurred in organisms starved for thymine or adenine, but UV recovery was stimulated by their addition. It is possible that MMS recovery proceeds by a process of excision and repair similar to, but not identical with, UV repair.