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P J Murray

Publications and source records attributed to P J Murray.

At least 19 recordsLinked to original sources
PubMed2001

Apoptosis-inducing factor mediates microglial and neuronal apoptosis caused by pneumococcus.

Streptococcus pneumoniae is the major cause of bacterial meningitis and it damages the hippocampus by inducing neuronal apoptosis. The blocking of caspases provides only partial protection in experimental meningitis, which suggests that there is an additional apoptotic pathway. A trigger of this pathway is the bacterium itself, as exposure of microglia or neurons to live pneumococci induces rapid apoptosis. In this study, apoptosis was not associated with the activation of caspases-1-10 and was not inhibited by z-VAD-fmk, a broad-spectrum caspase inhibitor. Rather, apoptosis was attributed to damage to mitochondria, which was followed by the release of apoptosis-inducing factor (AIF) from the mitochondria, large-scale DNA fragmentation, and hypodiploidy. Furthermore, intracytoplasmatic microinjection of AIF-specific antiserum markedly impaired pneumococcus-induced apoptosis. These findings indicate that AIF may play a central role in brain cell apoptosis and bacterial pathogenesis.

Animals↗
PubMed2001

Yeast artificial chromosome targeting technology: an approach for the deletion of genes in the C57BL/6 mouse.

An approach is described to modify yeast artificial chromosomes (YACs) with cassettes that can be easily excised for embryonic stem (ES) cell gene targeting experiments. YAC targeting technology (YTT) uses the WIBR/MIT-820 C57BL/6-mapped YAC library derived from the C57BL/6 mouse as the starting point for Internet- or PCR-based clone isolation, although in principle any YAC system can be used. Homologous recombination is initially performed in yeast using cassettes that function in Saccharomyces cerevisiae, Escherichia coli, and ES cells, followed by cloning or conversion of the targeted locus into a plasmid. The completed targeting vector can be transfected into C57BL/6 ES cells and clones selected with G418 followed by injection into Balb/c blastocysts. YTT increases the speed of targeting vector construction and obviates the need for extensive backcrossing to the C57BL/6 background.

Animals↗
PubMed2001

TNF-alpha controls intracellular mycobacterial growth by both inducible nitric oxide synthase-dependent and inducible nitric oxide synthase-independent pathways.

The role of TNF-alpha in the control of mycobacterial growth in murine macrophages was studied in vitro. Infection of macrophages from TNF-alpha gene disrupted (TNF-knockout (KO)) mice with recombinant Mycobacterium bovis bacillus Calmette Guérin (BCG) expressing the vector only (BCG-vector) resulted in logarithmic growth of the intracellular bacilli. Infection with BCG-secreting murine TNF-alpha (BCG-TNF) led to bacillary killing. Killing of BCG-TNF was associated with rapid accumulation of inducible NO synthase (iNOS) protein and the production of nitrite. The uncontrolled growth of BCG-vector was associated with low iNOS expression but no nitrite production. Thus, iNOS expression appears to be TNF-alpha independent but iNOS generation of NO requires TNF-alpha. In cultures of TNF-KO macrophages infected with BCG-TNF, inhibition of iNOS by aminoguanidine (AMG) abolished the killing of the bacilli. However, the growth of the organisms was still inhibited, suggesting an iNOS-independent TNF-alpha-mediated growth inhibition. To confirm this, macrophages from iNOS-KO mice were infected with either BCG-vector or BCG-TNF. As expected, no nitrite was detected in the culture medium. TNF-alpha was detected only when the cells were infected with BCG-TNF. In the iNOS-KO macrophages, the growth of BCG was inhibited only in the BCG-TNF infection. These results suggest that in the absence of iNOS activity, TNF-alpha stimulates macrophages to control the growth of intracellular BCG. Thus, there appears to be both a TNF-alpha-dependent-iNOS-dependent killing pathway as well as a TNF-alpha-dependent-iNOS-independent growth inhibitory pathway for the control of intracellular mycobacteria in murine macrophages.

Animals↗
PubMed2001

Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production.

The cytokines IL-4 and IL-13 inhibit the production of NO from activated macrophages through an unresolved molecular mechanism. We show here that IL-4 and IL-13 regulate NO production through depletion of arginine, the substrate of inducible NO synthase (iNOS). Inhibition of NO production from murine macrophages stimulated with LPS and IFN-gamma by IL-4 or IL-13 was dependent on Stat6, cell density in the cultures, and pretreatment for at least 6 h. IL-4/IL-13 did not interfere with the expression or activity of iNOS but up-regulated arginase I (the liver isoform of arginase) in a Stat6-dependent manner. Addition of exogenous arginine completely restored NO production in IL-4-treated macrophages. Furthermore, impaired killing of the intracellular pathogen Toxoplasma gondii in IL-4-treated macrophages was overcome by supplementing L-arginine. The simple system of regulated substrate competition between arginase and iNOS has implications for understanding the physiological regulation of NO production.

Animals↗
PubMed2001

Screening for chronic hepatitis B and C virus infections in an urban sexually transmitted disease clinic: rationale for integrating services.

BACKGROUND AND OBJECTIVES: Clients attending sexually transmitted disease (STD) clinics are at risk for multiple infections (e.g., STDs, HIV, and infectious viral hepatitis). Risk assessment and serosurveys can document the need for hepatitis screening and vaccination services. GOAL: To determine hepatitis C and B virus seroprevalence, identify predictive risk factors, and provide a rationale for integrating hepatitis services in an STD clinic. METHODS: During various periods in 1998, consecutive clients completed a self-administered risk assessment and were offered screening for markers of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection (HBV core antibody and anti-HCV [enzyme-linked immunosorbent assay 3.0, confirmed by recombinant immunoblot assay 2.0]). RESULTS: Sixteen percent of 300 clients tested for an anti-HBV core were positive, with injecting-drug users (IDUs) and men who have sex with men (MSM) having higher prevalences (50% and 37%, respectively). Of 615 clients tested for anti-HCV, 21 (3.4%) were positive. Injecting-drug users (n = 34) had a 38% anti-HCV prevalence compared with 1.1% for non-IDUs. Of 66 non-IDU MSM tested, none was HCV infected. IDUs had a high prevalence of past STDs (> 50%) and unsafe sexual behavior. CONCLUSIONS: Injecting drug users and MSM are at high risk for STDs, HIV, and hepatitis infections and could benefit from a "one-stop" STD clinic that included hepatitis prevention services.

Adult↗
PubMed2000

Aerosol infection of mice with recombinant BCG secreting murine IFN-gamma partially reconstitutes local protective immunity.

To better understand the contribution of interferon-gamma (IFN-gamma) to the immune response during the first 60 days of mycobacterial infection in the lungs, IFN-gamma gene disrupted (IFN-gamma-/-) mice were infected via aerosol with recombinant Mycobacterium bovis Bacillus Calmette-Guerin (BCG) secreting murine IFN-gamma (BCG-IFN-gamma) and compared to mice infected with recombinant BCG containing the vector only (BCG-vector). When IFN-gamma-/- mice were infected with BCG-vector, increasing bacillary loads and large undifferentiated granulomas that did not express inducible nitric oxide synthase (iNOS) were observed in the lungs. In contrast, infection with BCG-IFN-gamma resulted in reduced bacillary load and better differentiated granulomas containing epithelioid macrophages expressing iNOS as well as reduced levels of interleukin 10 (IL-10) mRNA. However, local production of IFN-gamma by the recombinant BCG did not protect IFN-gamma-/- mice from subsequent challenge with M. tuberculosis. Infection of IFN-gamma-/- peritoneal macrophages in vitro with BCG-IFN-gamma led to induction of iNOS expression and lower IL-10 mRNA levels. Nevertheless, the growth of the intracellular BCG was unaffected. Since IFN-gamma induced-iNOS protein and reduced IL-10 production were insufficient to control mycobacterial growth in vitro, the results suggest that additional mediator(s) present in vivo are required for control of mycobacterial growth.

Aerosols↗
PubMed2000

Phospholipase Cgamma2 is essential in the functions of B cell and several Fc receptors.

Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent antibody production is absent. The similarity to Btk- or Blnk-deficient mice demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function. The results define a signal transduction pathway broadly utilized by immunoglobulin superfamily receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Ubiquitous expression and embryonic requirement for RNA polymerase II coactivator subunit Srb7 in mice.

Mammalian RNA polymerase II complexes and coactivators containing homologs of yeast Srb/Med proteins have been isolated recently from tissue culture cells. The yeast Srb/Med complex is involved in global gene expression and is essential, but it is not yet known if its mammalian counterparts are broadly expressed in tissues or if they are essential. We have isolated the murine gene encoding Srb7, an Srb/Med complex protein whose sequence and function is highly conserved between yeast and humans. The mouse Srb7 gene is single copy, and Northern analysis showed that it is expressed in all tissues examined. Disruption of the gene in embryonic stem cells revealed that it is essential for cell viability and murine embryonic development. These results, together with evidence that murine Srb7 is associated exclusively with high molecular weight forms of RNA polymerase II in extracts, suggest that Srb7-containing polymerase complexes occur in most tissues and have essential roles in expression of protein coding genes.

Amino Acid Sequence↗
PubMed1999

Defining the requirements for immunological control of mycobacterial infections.

Tuberculosis remains a major public health problem throughout the world. Research into the immunological basis of the host-pathogen relationship has recently benefited from the fascinating convergence of genetic data from mouse models and from humans. Latency - the seemingly quiescent phase of bacterial persistence - is the central problem in controlling tuberculosis and will be the next frontier of research.

Animals↗
PubMed1999

Efficacy of methylphenidate among preschool children with developmental disabilities and ADHD.

OBJECTIVE: This was a double-blind, placebo-controlled, crossover design study of the safety and efficacy of methylphenidate (MPH) in 11 preschool children (aged 4.0-5.11 years) with developmental disabilities and attention-deficit hyperactivity disorder (ADHD). METHOD: MPH doses of 0.3 and 0.6 mg/kg per dose and a placebo were given. Drug response was evaluated via teacher-completed behavior checklists and clinic-based observations of activity level, attention, and compliance to adult requests. A side effects checklist was also completed by teachers and parents. RESULTS: Significant improvement on teacher ratings of hyperactivity and inattention as well as clinic-based observations of activity level and compliance were associated with MPH. Eight of 11 preschool children were medication responders (based on a minimum 40% decrease between placebo and one drug condition on either the teacher-rated Conners Hyperactivity Index or the Hyperactive-Distractible subscale of the Preschool Behavior Questionnaire). Five children exhibited significant adverse drug side effects such as severe social withdrawal, increased crying, and irritability, especially at the higher dose (0.6 mg/kg). CONCLUSIONS: Results suggest that preschool children with developmental disabilities and ADHD respond to MPH at rates similar to those of school-age children with mental retardation and ADHD. However, this population appears to be especially susceptible to adverse drug side effects.

Analysis of Variance↗
PubMed1999

Increased antimycobacterial immunity in interleukin-10-deficient mice.

Macrophage effector functions are essential for clearing mycobacterial infections. Interleukin 10 (IL-10) negatively regulates macrophages and could be a factor inhibiting effective antimycobacterial immunity. We previously showed that transgenic mice which produce excess IL-10 from T cells are susceptible to infection, even though these mice continue to produce gamma interferon (IFN-gamma) at levels similar to those in controls. Here, we extend our genetic analysis of the functions of IL-10 in antimycobacterial immunity by testing the hypothesis that IL-10-deficient (IL-10(-/-)) mice should be more resistant to mycobacteria than control mice. Mycobacterium bovis bacillus Calmette-Guérin-infected IL-10(-/-) mice had significantly lower bacterial burdens than control mice early in the infection. Contrary to expectations, however, IL-10(-/-) mice did not have increased levels of IFN-gamma, either from T cells or in the plasma, suggesting that other mechanisms are responsible for the increased resistance. However, macrophages from IL-10(-/-) mice produced increased levels of inflammatory cytokines, including IFN-gamma, as well as nitric oxide and prostaglandins, which could account for increased antimycobacterial immunity. Our genetic analysis revealed that IL-10 is an inhibitor of early mycobacterial clearance. The data also suggest that IL-10 negatively regulates numerous macrophage functions as well as playing a role in down-regulating the general inflammatory response, especially in conditions where an infection must be controlled through macrophage activity.

Animals↗
PubMed1999

Pneumolysin, a protein toxin of Streptococcus pneumoniae, induces nitric oxide production from macrophages.

Nitric oxide (NO) production by inducible NO synthase (iNOS) during inflammation is an essential element of antimicrobial immunity but can also contribute to host-induced tissue damage. Under conditions of bacterial sepsis, large amounts of NO are produced, causing hypotension, a critical pathological feature of septic shock. In sepsis caused by gram-positive organisms, the bacterial factors contributing to host NO production are poorly characterized. We show that a soluble toxin of Streptococcus pneumoniae, pneumolysin (Pln), is a key component initiating NO production from macrophages. In contrast to wild-type bacteria, a mutant of S. pneumoniae lacking Pln failed to elicit NO production from murine macrophages. Purified recombinant Pln induced NO production at low concentrations and independently of exogenous gamma interferon (IFN-gamma) priming of RAW 264.7 macrophages. However, IFN-gamma was essential for Pln-induced NO production, since primary macrophages from mice lacking the IFN-gamma receptor or interferon regulatory factor 1, a transcription factor essential for iNOS expression, failed to produce NO when stimulated with Pln. In addition, Pln acts as an agonist of tumor necrosis factor alpha and interleukin 6 production in macrophages. The properties of Pln, previously identified as a pore-forming hemolysin, also include a role as a general inflammatory agonist.

Animals↗
PubMed1998

Hematopoietic remodeling in interferon-gamma-deficient mice infected with mycobacteria.

Control of intracellular bacterial infections requires interferon-gamma (IFN-gamma) both for establishing a Th1 T-cell response and for activating macrophages to kill the bacteria. Exposure of mice deficient in IFN-gamma to mycobacterial infection produces an immune response characterized by a Th2 T-cell phenotype, florid bacterial growth, and death. We report here that IFN-gamma-deficient mice infected with mycobacteria also undergo a dramatic remodeling of the hematopoietic system. Myeloid cell proliferation proceeds unchecked throughout the course of mycobacterial infection, resulting in a transition to extramedullary hematopoiesis. The splenic architecture of infected IFN-gamma-deficient mice is completely effaced by expansion of macrophages, granulocytes, and extramedullary hematopoietic tissue. These features coincide with splenomegaly, an increase in splenic myeloid colony-forming activity, and marked granulocytosis in the peripheral blood. Systemic levels of cytokines are elevated, particularly interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF). These results suggest that in addition to its central role in cellular immunity, IFN-gamma may be a key cytokine in coordinate regulation of immune effector cells and myelopoiesis. This model should be valuable for deciphering the cross-talk between the immune response and hematopoiesis during bacterial infection and for improving our understanding of the mechanisms that control chronic infections.

Animals↗