Gammaherpesvirus-induced lung pathology is altered in the absence of macrophages.

The purpose of this study was to examine the lung pathogenesis of murine gammaherpesvirus (MHV-68) infection in mice that lack CC chemokine receptor CCR2, an important receptor for macrophage recruitment to sites of inflammation. BALB/c and CCR2(-/-) mice were inoculated intranasally (i.n.) with MHV-68 and samples were collected during acute infection (6 dpi) and following viral clearance (12 dpi). Immunohistochemistry was used to determine which cells types responded to MHV-68 infection in the lungs. Lung pathology in infected BALB/c mice was characterized by a mixed inflammatory cell infiltrate, necrosis, and increased alveolar macrophages by 12 dpi. Immunohistochemistry showed intense positive staining for macrophages. CCR2(-/-) mice showed greater inflammation in the lungs at 12 dpi than did BALB/c mice, with more necrosis and diffuse neutrophil infiltrates in the alveoli. Immunohistochemistry demonstrated much less macrophage infiltration in the CCR2(-/-) mice than in the BALB/c mice. These studies show that CCR2 is involved in macrophage recruitment in response to MHV-68 infection and illustrates how impairments in macrophage function affect the normal inflammatory response to this viral infection.

Activation of the Epstein-Barr virus lytic cycle by the latex of the plant Euphorbia tirucalli.

Exposure to the plant Euphorbia tirucalli has been proposed to be a cofactor in the genesis of endemic Burkitt's lymphoma (eBL). The purpose of this study was to examine the effects of unpurified E. tirucalli latex on Epstein-Barr virus (EBV) gene expression. A Burkitt lymphoma cell line was treated with varying dilutions of the latex and the effects on EBV gene expression were measured. We observed that the latex was capable of reactivating the EBV lytic cycle in a dose-dependent manner and at dilutions as low as 10(-6). Simultaneous treatment of cells with E. tirucalli latex and the protein kinase C inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride blocked lytic cycle activation. These data suggest that environmental exposure to the latex of E. tirucalli could directly activate the EBV lytic cycle and provide further evidence of a role for E. tirucalli in the aetiology of eBL.

Kinetics of murine gammaherpesvirus 68 gene expression following infection of murine cells in culture and in mice.

A model system to study the pathogenesis of gammaherpesvirus infections is the infection of mice with murine gammaherpesvirus 68 (MHV-68). To define the kinetics of infection, we developed an RNase protection assay to quantitate gene expression from lytic (K3, Rta, M8, DNA polymerase [DNA pol], and gB) and candidate latency (M2, M3, M9, M11, ORF73, and ORF74) genes. All candidate latency genes were expressed during lytic infection of 3T3 cells. Four kinetic classes of transcripts were observed following infection of 3T3 cells: immediate-early (K3, Rta, M8, and ORF73), early (DNA pol), early-late (M3, M11, and ORF74), and late (M2, M9, and gB). To assess the kinetics of viral gene expression in vivo, lungs, spleens, and mediastinal lymph nodes (MLN) were harvested from MHV-68-infected mice. All transcripts were expressed between 3 and 6 days postinfection (dpi) in the lungs. In the spleen, K3, M3, M8, and M9 transcripts were expressed between 10 and 16 dpi when latency is established. The K3, M3, M8, M9, and M11 transcripts were detected in the MLN from 2 through 16 dpi. This is the first demonstration of MHV-68 gene expression in the MLN. Importantly, our data showed that MHV-68 has different kinetics of gene expression at different sites of infection. Furthermore, we demonstrated that K3, a gene recently shown to encode a protein that downregulates major histocompatibility complex class I on the surface of cells, is expressed during latency, which argues for a role of K3 in immune evasion during latent infection.

Reliability of the Fagerstrom Tolerance Questionnaire and the Fagerstrom Test for Nicotine Dependence.

The purpose of this study was to assess the test-retest stability of the Fagerstrom Tolerance Questionnaire (FTQ) in two samples: (a) paid subjects in an American laboratory; data were collected via telephone screen and subsequently via questions embedded in a written history; and (b) smokers hospitalized for depression in Paris; data were collected via a written questionnaire upon admission and again after 3 weeks of treatment for depression. Reliability data are also presented for a recently revised version of the FTQ, the Fagerstrom Test for Nicotine Dependence (FTND), and compared with FTQ data collected in a subsample of subjects in the American database who received both versions of the questionnaire. Both the FTQ (in both samples) and the FTND proved to be highly reliable. The validity of the scales, using cotinine, number of years smoked, and the "addictive" factor on the Classification of Smoking by Motives questionnaire as criterion variables, was also supported. No relationship between FTQ score and severity of depression was detected in either sample. Internal consistency was somewhat higher for the FTND than for the FTQ, replicating previous findings in the literature.

Purification of PKC-I, an endogenous protein kinase C inhibitor, and types II and III protein kinase C isoenzymes from human neutrophils.

Human neutrophil protein kinase C (PKC) activity is inhibited by an endogenous protein found primarily in the pellet fraction from homogenized specific granules, which was both heat- and proteinase-sensitive [Balazovich, Smolen & Boxer (1986) J. Immunol. 137, 1665-1673]. We now report that two PKC isoenzymes and the endogenous PKC inhibitor, which we named PKC-I, were purified from human neutrophils. A neutrophil soluble fraction that was subjected to DEAE-Sephacel chromatography yielded highly enriched PKC because, by definition, enzymic activity was strictly dependent on Ca2+ and phosphatidylserine. Hydroxyapatite chromatography resolved two peaks of PKC activity. Type II and Type III PKC isoenzymes were each identified on Western blots by using isoenzyme-specific monoclonal antibodies. Unlike rat brain, from which PKC isoenzymes were also purified, Type I PKC was not detected in human neutrophils. Western blots indicated that both Type II and Type III PKC isoenzymes had molecular masses near 80 kDa. In agreement with other reports, PKC was autophosphorylated in vitro. PKC-I, an endogenous neutrophil inhibitor of PKC, was purified to apparent homogeneity by DEAE-Sephacel and S-400 Sephacel chromatography. PKC-I had a molecular mass of 41 kDa. PKC-I inhibited purified PKC activity stimulated by 1,2-diacylglycerols in a concentration-dependent manner, and inhibited PKC-dependent phosphorylation of proteins present in neutrophil cytosol.