Susan B. Dillon

Role of p38 Mitogen-Activated Protein Kinase in Rhinovirus-Induced Cytokine Production by Bronchial Epithelial Cells

The stress-activated protein kinase p38 plays a central role in the regulation of cytokine biosynthesis by various cell types in response to a wide range of stimuli. Because the local inflammatory response and the infiltration of neutrophils is thought to contribute to the symptoms and sequelae of rhinovirus infection, we investigated the role of p38 kinase in cytokine and chemokine elaboration in airway epithelial cells infected with human rhinovirus. Rhinovirus-39 infection of BEAS-2B cells resulted in synthesis of cytokines (IL-1, IL-6, G-CSF, and GM-CSF) and CXC chemokines (IL-8, epithelial neutrophil-activating protein-78, and growth-related oncogene-α), evident 24–72 h postinfection. Rhinovirus infection induced a time- and dose-dependent increase in tyrosine phosphorylation of p38 kinase, which peaked 30 min postinfection and remained elevated for 1 h. Treatment of infected cells with SB 239063, a potent pyridinyl imidazole inhibitor of p38 kinase, resulted in up to 100% inhibition of mediator production and partially reduced levels of IL-8 mRNA as determined by quantitative RT-PCR. Treatment with SB 239063 had no effect on virus replication and was not cytotoxic at concentrations ≤ 70 μM. These studies provide the first evidence that early activation of p38 kinase by rhinovirus infection is a key event in regulation of virus-induced cytokine transcription, and may provide a new target for inhibition of symptoms and airway inflammation associated with rhinovirus infection.

A Direct Comparison of the Activities of Two Humanized Respiratory Syncytial Virus Monoclonal Antibodies: MEDI-493 and RSHZl9

Two humanized monoclonal antibodies, MEDI-493 and RSHZ19, were developed independently as potential improvements over RSV-IGIV for prevention of respiratory syncytial virus (RSV) infection. RSV-IGIV is a polyclonal human antibody preparation for intravenous infusion enriched for RSV neutralizing activity. A phase III clinical trial showed that MEDI-493 significantly reduced hospitalizations due to RSV infection. In a separate trial, RSHZ19 failed to show significant efficacy. In new studies, the in vitro and in vivo activities of MEDI-493 and RSHZ19 were compared to determine whether the different clinical results are related to differences in biologic activity. MEDI-493 was consistently 4- to 5-fold more potent than RSHZ19 in antigen binding, RSV neutralization, and fusion inhibition assays. Although both MEDI-493 and RSHZ19 were effective against A and B subtypes of RSV in the cotton rat model of RSV infection, 2- to 4-fold higher doses of RSHZ19 were required for similar protection. The enhanced activity of MEDI-493 compared with RSHZ19 may, in part, explain its better clinical effect.

Differences in pathogenicity of herpes simplex virus serotypes 1 and 2 may be observed by histopathology and high-resolution magnetic resonance imaging in a murine encephalitis model.

The mouse model for herpes simplex-induced encephalitis (HSE) is an established preclinical tool for evaluating the efficacy of new therapeutic interventions. We evaluated the utility of high-resolution in vivo MRI in observing the progression of experimental HSE during the first week postinfection. Female BALB/c mice were inoculated intracerebrally with HSV-1 or HSV-2 by microinjection. Each animal was evaluated daily by high-resolution (4.7 Tesla) T2 weighted MRI and clinical disease scoring (neurological and behavioral). Lesions induced by a high dose of HSV-1 (1000 PFU) were detectable by MRI without administration of contrast agent whereas for low dose HSV-1 (100 PFU), administration of contrast agent was necessary to visualize the lesions in the brain. The correlation between the MRI and histologic results was excellent. No HSV-2 induced lesions were observed by MRI. Although both HSV serotypes caused similar clinical disease, significant type differences were found by histologic and MRI examinations. HSV-1 caused necrotizing meningoencephalitis, whereas HSV-2 induced mostly meningitis. The data indicate that in vivo high-resolution MRI may be useful to longitudinally evaluate HSV-1-related pathology in a mouse model of HSE and potentially could be used for monitoring the efficacy of anti-infective therapeutic approaches.

Identification of upregulated early response genes in g-csf treated bone marrow

Abstract G-CSF acts on progenitor cells in the bone marrow, causing differentiation to the myeloid lineage for maturation as neutrophils in peripheral blood. Suppressive subtractive hybridization was used to identify early genes induced in bone marrow specifically in response to treatment with G-CSF. Bone marrow was treated with G-CSF (100 ng/ml) and cells were harvested at 1 and 12 hr. A cDNA library was prepared from difference products obtained from subtractions between 0 and 1 hr treated, and 0 and 12 hr treated samples. The 3′ ends of thirty differentially expressed clones were sequenced. Of these, fifteen sequences were identified by BLAST searches as known genes including, among others, human neutrophil peptide (HNP), DAXX, and a NF-E2 like protein. While these genes have not been previously reported to be early response genes induced by G-CSF, their presence could be anticipated. Quantitative PCR analysis was used to determine the differential expression of several of these newly identified genes in three additional G-CSF treated bone marrows and in marrow treated with EPO, TPO, SCF, G-CSF or IL-3. An upregulation of HNP was demonstrated by Taqman in marrow 12 hours following treatment with G-CSF. The identification of genes induced by G-CSF by methods such as SSH furthers our understanding of the mechanisms involved in neutrophil differentiation.