Tumor necrosis factor-alpha activation of the c-Jun N-terminal kinase pathway in human neutrophils. Integrin involvement in a pathway leading from cytoplasmic tyrosine kinases apoptosis.

The intensity and duration of an inflammatory response depends on the balance of factors that favor perpetuation versus resolution. At sites of inflammation, neutrophils adherent to other cells or matrix components are exposed to tumor necrosis factor-alpha (TNFalpha). Although TNFalpha has been implicated in induction of pro-inflammatory responses, it may also inhibit the intensity of neutrophilic inflammation by promoting apoptosis. Since TNFalpha is not only an important activator of the stress-induced pathways leading to p38 MAPk and c-Jun N-terminal kinase (JNK) but also a potent effector of apoptosis, we investigated the effects of TNFalpha on the JNK pathway in adherent human neutrophils and the potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNFalpha was found to result in beta2 integrin-mediated activation of the cytoplasmic tyrosine kinases Pyk2 and Syk, and activation of a three-part MAPk module composed of MEKK1, MKK7, and/or MKK4 and JNK1. JNK activation was attenuated by blocking antibodies to beta2 integrins, the tyrosine kinase inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor, and piceatannol, a Syk-specific inhibitor. Exposure of adherent neutrophils to TNFalpha led to the rapid onset of apoptosis that was demonstrated by augmented annexin V binding and caspase-3 cleavage. TNFalpha-induced increases in annexin V binding to neutrophils were attenuated by blocking antibodies to beta2 integrins, and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence, exposure of adherent neutrophils to TNFalpha leads to utilization of the JNK-signaling pathways that may contribute to diverse functional responses including induction of apoptosis and subsequent resolution of the inflammatory response.

Depletion of extracellular RANTES during human cytomegalovirus infection of endothelial cells.

Human cytomegalovirus (CMV) infection results in pneumonitis in bone-marrow and lung-transplant recipients. The source of CMV infection contributing to the onset of pneumonitis is unclear, but may involve infection of the lung endothelium in the presence of infiltrating mononuclear cells. Viral infection stimulates the host cell to express chemokines as signals to recruit specific immune cells to the site of injury. CMV encodes a chemokine receptor that may function to reduce host cell expression of chemokines. In the study reported here we found that extracellular concentrations of the chemokine regulated on activation, normal T cell expressed and secreted (RANTES) are depleted during productive infection of primary endothelial cells with CMV strain 4010, an endothelial-adapted strain of CMV. Utilizing adenovirus-transformed human kidney epithelial cells (type 293 cells) that stably express the CMV-encoded chemokine receptor US28, we found that depletion of extracellular RANTES during infection is attributable to US28, which binds and internalizes extracellular RANTES.

Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils.

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.

Intracellular signaling by the chemokine receptor US28 during human cytomegalovirus infection.

In patients with impaired cell-mediated immune responses (e.g., lung transplant recipients and AIDS patients), cytomegalovirus (CMV) infection causes severe disease such as pneumonitis. However, although immunocompetency in the host can protect from CMV disease, the virus persists by evading the host immune defenses. A model of CMV infection of the endothelium has been developed in which inflammatory stimuli, such as the CC chemokine RANTES, bind to the endothelial cell surface, stimulating calcium flux during late times of CMV infection. At 96 h postinfection, CMV-infected cells express mRNA of the CMV-encoded CC chemokine receptor US28 but do not express mRNA of other CC chemokine receptors that bind RANTES (CCR1, CCR4, CCR5). Cloning and stable expression of the receptor CMV US28 in human kidney epithelial cells (293 cells) with and without the heterotrimeric G protein alpha16 indicated that CMV US28 couples to both Galphai and Galpha16 proteins to activate calcium flux in response to the chemokines RANTES and MCP-3. Furthermore, cells that coexpress US28 and Galpha16 responded to RANTES stimulation with activation of extracellular signal-regulated kinase, which could be attributed, in part, to specific Galpha16 coupling. Thus, through expression of the CC chemokine receptor US28, CMV may utilize resident G proteins of the infected cell to manipulate cellular responses stimulated by chemokines.

Antiviral efficacy of disinfectant solution MRI-1.

Disinfectant MRI-1 was prepared by dissolution of non-ionic and ionic detergent in ethanol. The disinfectant inactivated extracellular and intracellular enveloped and non-enveloped viruses including herpes viruses, influenza A and human immunodeficiency disease virus in suspension or on surfaces by pre-exposure or post-exposure to the disinfectant; in addition, cells were disabled as potential hosts for viral infection using concentrations of MRI-1 which were 50-fold less than the operative concentration for disinfection. There was no evidence of in vitro mutagenicity using Salmonella typhimurium or sensitization or other adverse effect in a guinea pig model or in human subjects.

A system for isolation, transport and storage of herpes simplex viruses.

A major difficulty with diagnostic virus isolation concerns the relative thermolability of certain viruses, e.g. herpes simplex virus type 2, which may, therefore, lose infectivity during transport to the laboratory. This study describes a system of virus isolation and transport, which depends on direct inoculation at the bedside or clinic, to a monolayer or suspension of susceptible cells with subsequent incubation for 10 h at approximately 32 degrees C, whereupon the newly synthesised virus becomes very stable if the cells are subsequently maintained at room temperature. This system was found to increase the sensitivity of isolation of herpes simplex virus, particularly under conditions of asymptomatic virus excretion or if there was significant delay in transportation of clinical samples to the virus laboratory. It is envisaged that this system will allow clinical self-sampling by the patient with application to epidemiological surveys in both the developed and underdeveloped world.

Postoligomerization folding of human cytomegalovirus glycoprotein B: identification of folding intermediates and importance of disulfide bonding.

Human cytomegalovirus glycoprotein B (gB or UL55) has been demonstrated to be a disulfide-linked homodimer within the envelope of mature virions. Previously, it has been shown that gB undergoes a rapid dimerization nearly coincident with its synthesis. Following dimerization, the molecule slowly folds into a form which can be transported from the endoplasmic reticulum. In this study we have examined the prolonged folding of gB by using a set of defined gB-reactive murine monoclonal antibodies and gB expressed as a recombinant protein in the absence of other human cytomegalovirus proteins. Our results have documented a folding pathway consistent with the relatively rapid dimerization of the translation product followed by delayed conversion into a fully folded molecule. Assembly of the dominant antigenic domain of gB, AD-1, preceded dimerization and folding of the molecule. The fully folded dimer was heat stable, but its conformation was altered by treatment with 2% sodium dodecyl sulfate (SDS), whereas an oligomeric folding intermediate was both heat and SDS stable. Postoligomerization disulfide bond formation could be demonstrated during folding of gB, suggesting that the formation of these covalent bonds could contribute to the prolonged folding of this glycoprotein.

Development of subunit preparations of cytomegalovirus antigen by aqueous extraction for immunization against cytomegalovirus infection.

This study has examined different methods of preparation of a subunit vaccine from cytomegalovirus (CMV)-infected MRC cells in terms of protein and DNA content, antigenicity and immunogenicity. Two preparations have been developed where CMV proteins were obtained by extraction of infected cells with water. Virus particles were removed from the preparations by ultracentrifugation and residual virus was inactivated by formaldehyde in the WUF preparations or by chloroform in the WUCh preparations. The preparations contained CMV proteins which may play a role in protective immune responses, and the preparations were antigenic as determined by immunodiffusion, ELISA and immunoblotting. Immunogenicity as evaluated in rabbits indicated that the preparations stimulated neutralizing and immunoprecipitating antibody. These results suggest that the gentle method of water extraction of viral antigens may be a useful protocol for vaccine preparation.

In vivo model for evaluation of species-specific virus vaccines.

It is difficult to evaluate the protective efficacy of species-specific viruses of humans and expensive companion animals where there is no non-human animal model. This study describes an in vivo model system which allows simultaneous operation of humoral, cell-mediated, interferon-like or other unidentified immunological defence mechanisms. There was evidence of in vivo inactivation of both enveloped and unenveloped DNA and RNA viruses including retrovirus mouse sarcoma virus/mouse leukaemia virus as evaluated by assay of the enzyme reverse transcriptase. This model will allow examination of vaccine efficacy in immunocompetent host animals while avoiding morbidity and/or mortality from virus infection in these animals.

Immune inhibition of virus release from human and nonhuman cells by antibody to viral and host cell determinants.

Immune inhibition of release of the DNA viruses, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and surprisingly two herpes viruses, bovine mamillitis and equine abortion, were not inhibited by either anti-viral or anti-host sera. Using the herpes simplex virus model, inhibition of virus release was detected in different cells of human and nonhuman origin with cross-inhibition between cell lines of different origin; thus, this form of immunotherapy may not require antibody to be tissue or organ specific. Evidence of inhibition of virus release from neoplastic and leukemic cell lines suggests possible application of this approach to control of virus-mediated leukoproliferative pathology (e.g. Burkitt's lymphoma or adult T cell leukemia).

Inhibition of rate of tumour growth in rodent species by inoculation of herpesviruses and encephalomyocarditis virus.

Inoculation of herpesviruses and encephalomyocarditis virus into subcutaneous tumours in hamsters and mice reduced the rate of tumour growth compared to untreated tumours or secondary tumours which had arisen following surgical excision of the primary tumour; in addition, survival times were increased in animals whose tumours were inoculated with virus. It is suggested that the role of virus in the modification of tumour growth merits further exploration.

Reduced tumorigenicity of rodent tumour cells and tumour explants following infection with wild type and mutant herpes simplex virus, bovine mammillitis virus and encephalomyocarditis virus.

The tumorigenicity of neoplastic hamster and mouse cell lines and tumour explants was reduced by infection with herpes simplex virus (HSV-1), a thymidine-kinaseless mutant of herpes simplex virus, namely 'MDK', encephalomyocarditis virus (EMC) and bovine mammillitis virus (BMV). There was an approximate relationship between duration of virus infection in vitro and reduction in incidence and/or rate of tumour development. The rate of tumour development was also reduced by 'site inoculation' of virus (HSV-1) at various time intervals following inoculation of tumorigenic BHK 21 cells indicating that virus was capable of reducing the rate of tumour development in a situation where the neoplastic cells were already transplanted into the susceptible host species. It is suggested that the therapeutic role of wild type, mutant or recombinant viruses merits further exploration towards prevention and treatment of human cancer.