Reovirus triggers cell type-specific proinflammatory responses dependent on the autocrine action of IFN-beta.

Resident cells of the respiratory and gastrointestinal tracts, including epithelial and fibroblast cells, are the initial sites of entry for many viral pathogens. We investigated the role that these cells play in the inflammatory process in response to infection with reovirus 1/L. In A549 human bronchial or HT-29 human colonic epithelial cells, interferon (IFN)-beta, regulated on activation T cell expressed and secreted (RANTES), IFN-gamma-inducible protein (IP)-10, and interleukin-8 were upregulated regardless of whether cells were infected with replication-competent or replication-deficient reovirus 1/L. However, in CCD-34Lu human lung fibroblast cells, IFN-beta, IP-10, and RANTES were expressed only after infection with replication-competent reovirus 1/L. Expression of interleukin-8 in CCD-34Lu fibroblast cells was viral replication independent. This differential expression of IFN-beta, RANTES, and IP-10 was shown to be due to the lack of induction of IFN regulatory factor-1 and -2 in CCD-34Lu fibroblast cells treated with replication-deficient reovirus 1/L. We have shown that cytokine and/or chemokine expression may not be dependent on viral replication. Therefore, treatment of viral infections with inhibitors of replication may not effectively alleviate inflammatory mediators because most viral infections result in the generation of replication-competent and replication-deficient virions in vivo.

Polychlorinated biphenyl mixtures (Aroclors) inhibit LPS-induced murine splenocyte proliferation in vitro.

The immune system is believed to be a sensitive indicator for adverse polychlorinated biphenyl (PCB)-induced health effects. Four commercial PCB mixtures (Aroclors) or six individual PCB congeners were evaluated for their effect on splenocyte viability and lipopolysaccharide (LPS)-induced splenocyte proliferation in vitro in two strains of mice, C57B1/6 (high affinity aromatic hydrocarbon receptor (AhR) complex) and DBA/J (low affinity AhR complex). All four Aroclors, the selected individual noncoplanar congeners, or two tertiary mixtures containing one congener from each class significantly decreased the in vitro LPS-induced proliferation of murine splenocytes in either strain of mice without inducing a significant decrease in viability. In contrast, selected individual coplanar or mono-ortho-coplanar congeners did not inhibit splenocyte proliferation or viability at any concentration. These results suggest that mixtures of PCBs and/or congener class (specifically, noncoplanar congeners) may be more highly immunotoxic than individual planar and mono-ortho-coplanar congeners alone. Thus, this in vitro assay has revealed a more complex pattern of immunotoxicity of Aroclors versus individual congeners than has previously been reported or anticipated based on both in vivo derived immunotoxic data and standard comparisons to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These results have important practical significance since mixtures of PCB congeners were used industrially and now contaminate the environment.

Prolonged induction of IL-8 gene expression in a human fibroblast cell line infected with reovirus serotype 1 strain Lang.

Viruses which infect mucosal surfaces commonly infect these particular anatomical sites based on both the virion structure and the interaction of the virus with a particular microenvironment. We infected a human lung epithelial cell line, a human gut epithelial cell line, and a human lung fibroblast cell line with reovirus 1/L to explore how this natural isolate of both the lung and the gut may interact with mucosal surfaces. While reovirus infection of the gut and lung epithelial cell lines was lytic, a chronic infection was established in the human lung fibroblast cell line. All three cell lines also produced interleukin-8 (IL-8) after infection with reovirus 1/L, and IL-8 production was not dependent upon viral replication. A prolonged production of IL-8 was observed in the chronically infected lung fibroblast cell line, suggesting that this mucosal population may be involved in the generation of inflammatory responses after the resolution of the initial lytic infection of the epithelium. These studies provide an in vitro model system for analyzing the interaction of reovirus 1/L with resident mucosal cell populations.

Clonidine challenge in childhood anxiety disorder.

OBJECTIVE: To evaluate the neurohormonal and subjective mood response of children with anxiety disorder to clonidine challenge METHOD: Children with DSM-IV diagnoses of anxiety disorder (ANX) (n = 24) and normal controls (n = 15) were given a challenge of intravenous clonidine (1.3 micrograms/kg) and provided neurohormonal and mood self-report assessment over a 180-minute period. RESULTS: The ANX group differed from normal controls in Hamilton Anxiety Rating, Revised Children's Manifest Anxiety Scale score, and maximum change from baseline (delta max) in growth hormone (GH). Clonidine-stimulated GH concentration of the ANX group was significantly elevated compared with that of controls but returned to baseline within 2 hours. A subgroup with obsessive-compulsive disorder (OCD) (n = 9) had significantly higher delta max GH (17.5 +/- 10.1 ng/mL) than the group with other anxiety disorders (ANX-OCD) (9.1 +/- 5.8 ng/mL) and controls (5.7 +/- 4.1 ng/mL). CONCLUSION: GH response to clonidine challenge is not blunted in ANX subjects. This finding is in contrast to adult disorder and suggests that adrenergic postsynaptic receptor down-regulation is not a feature of childhood anxiety. These findings suggest enhanced central adrenergic sensitivity in ANX which is most pronounced in OCD and argue against a neurobiological continuum from childhood to adult anxiety disorder.

Expression of beta-galactosidase in mouse brain: utilization of a novel nonreplicative Sindbis virus vector as a neuronal gene delivery system.

Sindbis virus expression has been used for in vitro investigations of antigen processing, presentation and epitope mapping. The recent development of a replication-deficient recombinant Sindbis virus expression vector has made in vivo expression possible with minimal pathogenic risk. Advantages of Sindbis virus over other available viral systems include a comparatively smaller genome size making it possible to clone larger inserts, the ability to infect a wide range of host cell types with reduced pathogenicity for humans. These features suggest the possible utility of Sindbis virus for the in vivo delivery of genes to neural cells. We used the recombinant Sindbis viral expression system to target delivery of the lacZ gene to neuronal cells of mice by stereotactic surgery. Sindbis viral mRNA obtained by in vitro transcription was used to transfect baby hamster kidney (BHK) cells. As shown by histochemistry, beta-galactosidase (beta-gal) was expressed in approximately 50% of transfected cells. Cells were then cotransfected with DH-BB helper sequences that enabled the recombinant Sindbis virus RNA packaging. Nonreplicative Sindbis viral stock was collected 24 h after transfection. BHK cells were then infected with viral stock and histochemistry analysis was performed. Again, approximately 50% of the cells expressed beta-gal. The same viral stock was infused into the nucleus caudatus/putamen and nucleus accumbens septi and histochemical analysis of frozen sections from the relevant brain areas confirmed that beta-gal was expressed in neurons in a time-dependent manner. beta-Gal was detected at 24 h after inoculation and was present for at least 14 days, with maximum expression at 48 h. These results suggest that a nonreplicative Sindbis virus expression system may be useful for delivery of foreign genes into the central nervous system (CNS).

The effects of phosphorylation on the functional regulation of an expressed recombinant human dopamine transporter.

Metabolic labeling experiments were performed using eukaryotic cells transfected with the human dopamine (DA) transporter cDNA. Autophosphorylation in the presence and absence of the transporter substrate DA, was analyzed. Dopamine transporter (DAT) was phosphorylated in the absence of DA and dephosphorylated in the presence of the substrate. The functional significance of this phenomenon was checked by incubating cells with phosphorylation promoting agents, all of which reduced substrate uptake and ligand binding significantly. It is shown that at least one site of phosphorylation on DAT is a serine residue. These experiments suggest that the state of phosphorylation of the DAT may play an important role in its biological function.