The role of endogenous interleukin-12 in resistance to murine cytomegalovirus (MCMV) infection and a novel action for endogenous IL-12 p40.

Biologically active interleukin-12 (IL-12), comprising a 40 kDa subunit (p40) covalently linked to a 35 kDa subunit (p35), is produced in response to a range of infectious stimuli. Here, we demonstrate that mice deficient in either IL-12 p40 (p40-/-) or IL-12 p35 (p35-/-) are susceptible to murine cytomegalovirus (MCMV) infection in terms of survival (Balb/c p35-/-) and viral clearance (Balb/c p35-/- and Balb/c p40-/-), and this susceptibility may be correlated to a deficiency in serum interferon-gamma (IFN-gamma) levels. These data support a role for endogenous IL-12 in controlling MCMV infection. The IL-12 p40 subunit is produced in excess of IL-12 p35, and to date the function of the excess endogenous p40 has been assumed to be one of IL-12 antagonism, as demonstrated by experiments with exogenous p40 both in vivo and in vitro. We show that Balb/c p35-/- alone are significantly compromised in survival of a sublethal infection and in clearance of virus from the spleen. These mice produce a very early IFN-gamma spike (8 h after infection) and an aberrant tumor necrosis factor-alpha (TNF-alpha) spike (day 2 after infection). MCMV infection has revealed an altered Balb/c p35-/- phenotype compared with Balb/c p40-/-, and this indicates that endogenous p40 may have an activity independent of and additional to IL-12 antagonism in vivo.

Inhibition of virus-encoded thymidine kinase suppresses herpes simplex virus replication in vitro and in vivo.

Both herpes simplex virus type 1 (HSV-1) and HSV-2 encode a thymidine kinase enzyme which differs from cellular thymidine kinase in substrate specificity. Viral thymidine kinase enables the virus to replicate in cells that lack cellular thymidine kinase, namely those of the sensory neurons where the virus establishes, and periodically reactivates from, a latent state. Thymidine kinase-dependent HSV replication following viral reactivation at the site of latency is thought to precede the emergence of virus at mucosal surfaces. The ability to inhibit such an essential viral enzyme would potentially prevent HSV from replicating within neuronal tissue, and thus stop the recurrent disease cycle. Ro 32-2313 was designed as a selective and competitive inhibitor of HSV thymidine kinase and in vitro studies have confirmed this mechanism of action. In vivo evaluation of a soluble prodrug of Ro 32-2313, Ro 32-4397, was undertaken in murine models where pathogenesis was dependent upon viral replication in neuronal tissue. It was shown that in vivo administration of Ro 32-4397 (i) significantly reduced the viral titre detected in isolated dorsal root ganglia; (ii) prevented HSV-2-induced lethality in a systemic infection model; and (iii) reduced zosteriform lesion development in a model of dermal infection. Administration of Ro 32-4397 produced dose-related changes in viral pathogenicity towards those of the phenotype of a thymidine kinase-deficient virus. Overall, the study confirmed that thymidine kinase inhibitors can suppress the replication of HSV in vivo, and suggest that such inhibitors may reduce reactivation of the virus from latency if used prophylactically in recurrent HSV infection.

Interleukin-12 exhibits potent antiviral activity in experimental herpesvirus infections.

The prophylactic and therapeutic efficacy of interleukin-12 was studied by using murine models of herpes simplex virus infection. Prophylactic administration consisted of two intraperitoneal doses of interleukin-12 given 48 and 24 h prior to infection. Therapeutic intraperitoneal administration of interleukin-12 commenced 6 h after the mice were infected with herpes simplex virus and was continued daily for a total of 5 days. Interleukin-12 therapy improved the survival rates of mice with systemic herpes simplex virus infection compared with those of placebo-treated infected mice. Subcutaneous administration of interleukin-12 also improved the rate of survival of mice after systemic herpes simplex virus infection, although higher doses were required to give comparable effects. Combined prophylactic and therapeutic administration of interleukin-12 produced the greatest effect on survival after an otherwise lethal systemic infection. Intraperitoneal administration of interleukin-12 for 2 days before and 3 days after systemic infection with herpes simplex virus resulted in survival of 80% of the mice. These surviving mice were resistant to subsequent reinfection with herpes simplex virus. Such resistance was apparently specific for herpes simplex virus infection, since a second group of survivors succumbed to a lethal infection with murine cytomegalovirus. Infectious virus was recovered from lumbar ganglia explants dissected from survivors of prophylactic interleukin-12 therapy and cultured for 5 days in vitro, suggesting that interleukin-12 treatment did not prevent the establishment of latent herpes simplex virus infection. One action of interleukin-12 may be to enhance natural killer cell-mediated clearance of the virus. However, interleukin-12 therapy was also effective in mice carrying the beige mutation, which reduces natural killer cell lytic activity, suggesting that interleukin-12 has additional activities in vivo.

Interleukin-12 and infectious diseases: a potential novel therapy.

Interleukin-12 (IL-12) is emerging as a central component of both innate and acquired immunity. The multiplicity of biological activities associated with this cytokine, particularly the stimulation of cell-mediated immunity, suggests that it may be crucial in the control of extracellular and intracellular infections. In in vitro studies, IL-12 production is initiated rapidly after infection with a variety of viral, parasitic, fungal and bacterial agents. This induction correlates well with the reported resistance or susceptibility of animals to infection with these agents. Other factors may, however, influence responses in vivo, including host genetic make-up, microbial load and the induction of antagonistic cytokine pathways, notably IL-4 and IL-10. In some situations, IL-12 may direct immune responses to inappropriate pathways, and worsen disease, so that careful consideration of the type of required immune response is needed before IL-12 therapy is initiated. IL-12 treatment may also be useful in promoting protective immune responses to vaccines, allowing systemic immunisation with lower doses, or even normally non-immunogenic preparations, of antigen. Finally, IL-12 has been demonstrated to act in concert with standard antimicrobial chemotherapy in viral, parasitic, fungal and bacterial infections, allowing a reduction in the dose of the agent used and providing hope that such combination therapy may more effectively control drug-resistant strains of infectious agents.

Interleukin-12: potential clinical applications in the treatment and prevention of infectious diseases.

Interleukin-12 (IL-12) is a cytokine that promotes cell-mediated immunity by facilitating type 1 helper T-lymphocyte responses, enhancing the lytic activity of natural killer cells, augmenting specific cytolytic T-lymphocyte responses, and inducing the secretion of interferon-gamma. It can also boost the production of some subclasses of IgG antibodies. IL-12 has demonstrated activity in several mouse models of infectious diseases caused by viruses, protozoans, fungi, and mycobacteria. It has the potential for use either as a single immunotherapeutic agent, in combination with chemotherapeutic agents, or as a vaccine adjuvant. Endogenous IL-12 appears to play an important role in the host defence against intracellular pathogens; poor IL-12 production observed in mononuclear cells from patients infected with the human immunodeficiency virus may be involved in the susceptibility of this patient group to opportunistic infections. Clinical trials are being initiated to evaluate the possible therapeutic uses of IL-12.

Oral, anti-inflammatory activity of a potent, selective, protein kinase C inhibitor.

The protein kinase C family of enzymes is thought to be important in mediating signal transduction. Ro 31-8830 is a novel, potent inhibitor of protein kinase C, derived from the non-selective protein kinase inhibitor staurosporine. In this paper we demonstrate the selectivity of Ro 31-8830 for protein kinase C over other protein kinases and its ability to inhibit protein kinase-C-mediated events in platelets and lymphocytes. In addition, we describe a novel system for the in vivo evaluation of inhibitors of protein kinase C, and we demonstrate the oral anti-inflammatory activity of Ro 31-8830. This finding has implications for the treatment of inflammatory disorders in the clinic.