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.

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.

Non-specific antiviral activity of antisense molecules targeted to the E1 region of human papillomavirus.

Antisense phosphorothioate oligonucleotides (ODN1 0x5 OMe) directed against the E1 start region of human papillomavirus 11 (HPV11) can inhibit papillomavirus induced growth of implanted human foreskin in a mouse xenograft model. Administration of a mismatch control oligonucleotide (ODN9 0x5 OMe), in which guanine was replaced with adenine in the same model, had no effect on papilloma induced growth. However, the apparent antiviral activity of ODN1 0x5 OMe was also shown in a lethal mouse cytomegalovirus (CMV) model, in which the oligonucleotides are not expected to have antisense activity. To understand the mechanisms of action of these oligonucleotides, a mismatch oligonucleotide (ODN61 0x5 OMe) was prepared which retained the CpG motifs of ODN1 0x5 OMe. This was tested in the mouse xenograft model and shown to have moderate inhibitory activity. As a definitive experiment, a comparison was made between the efficacy of the active oligonucleotide ODN1 0x5 OMe against two papilloma viruses HPV11 and HPV40. Both these viruses cause benign genital warts, but differ by four bases in their E1 sequence that was the target for ODN1 0x5 OMe. Papillomavirus induced growth in the mouse xenograft model was inhibited by ODN1 0x5 OMe in both cases, suggesting that oligonucleotide molecules have a non-specific antiviral activity that is not directly related to their antisense sequence.

The basics of heart failure management: are they being ignored?

BACKGROUND: Advances have been made in the medical management of congestive heart failure. However, there is concern that these changes may not be transmitted to the heart failure population in the community. Other impediments to improved prognosis, such as failure to apply non-pharmacological strategies and poor patient comprehension may also be prevalent in the community. AIMS: The purpose of this study was to assess physician practice and patient knowledge in a heart failure population admitted to a University Hospital in Ireland. METHODS: Patients admitted with a primary diagnosis of heart failure were studied. Estimation of ejection fraction was used to subdivide the population into heart failure with impaired and normal systolic function. Patients' course in hospital was noted with reference to management by cardiology or internal medicine, use of angiotensin-converting enzyme inhibition therapy and digoxin and application of dietary and rehabilitative services. Patient knowledge was assessed by questionnaire. RESULTS: Eighty patients were included in this study. Two-thirds of the population had impaired systolic function. The majority of patients were managed by internal medicine physicians, and this population was older and more likely to have normal systolic function. Prescription of converting enzyme inhibitor therapy was more frequently used in cardiology-managed patients (96 vs. 70%, P<0.05). Neither group applied dietary or rehabilitative advice to a significant level. Patient comprehension was poor, especially with regard to understanding of medicine and the value of weight measurement. CONCLUSION: The above data demonstrate a lack of use of rehabilitative and dietary services and poor patient knowledge. These deficiencies may play a role in determining outlook and may impede the expected improvement in prognosis that has been witnessed in large randomised studies.

The bisindolylmaleimide protein kinase C inhibitor, Ro 32-2241, reverses multidrug resistance in KB tumour cells.

Ro 32-2241 is a bisindolylmaleimide that selectively inhibits protein kinase C (PKC) as compared with other protein kinases. Experiments were carried out to examine its potential as a multidrug resistance-reversing agent. Ro 32-2241 inhibited efflux, and increased accumulation, of [3H]-daunomycin in multidrug-resistant (MDR) KB-8-5 and KB-8-5-11 cells and had no effect on drug-sensitive KB-3-1 cells. Ro 32-2241 completely reversed the doxorubicin resistance of KB-8-5 and KB-8-5-11 cells, showing no effect on the sensitivity of drug-sensitive KB-3-1 cells. The potency of Ro 32-2241 was comparable with that of cyclosporin A and better than that of verapamil, known modulators of multidrug resistance. Ro 32-2241 also completely reversed the taxol resistance of KB-8-5 cells and partially reversed the resistance of KB-8-5-11 cells. Vinblastine resistance was also partially reversed. Mechanistic experiments were carried out to determine whether Ro 32-2241 interacted with P-glycoprotein (Pgp) directly. Increased efflux of [14C]-Ro 32-2241 was seen with the more resistant KB-8-5-11 cells (although the percentage effluxed was very low as compared with [3H]-daunomycin), suggesting that Ro 32-2241 can act as a substrate for Pgp. Direct interaction of Ro 32-2241 with Pgp was confirmed by demonstration that it inhibited binding of [3H]-azidopine to Pgp in KB-8-5-11 membranes. In conclusion, Ro 32-2241, acting directly on Pgp (rather than, or in addition to, an effect on PKC), is effective in reducing or reversing resistance to doxorubicin, taxol and vinblastine in human tumour cells with a clinically relevant degree of MDR. However, results of in vivo experiments conducted to investigate the effects of Ro 32-2241 on resistance to doxorubicin suggest that it may not be possible to achieve sufficiently high levels of Ro 32-2241 in vivo to modulate MDR.

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.

Novel, potent and selective inhibitors of protein kinase C show oral anti-inflammatory activity.

Clarification of the precise role of protein kinase C (PKC) in cellular functional responses has been hampered by a lack of potent, selective inhibitors. The structural lead provided by staurosporine, a potent but non-selective protein kinase (PK) inhibitor, was used to derive a series of bis(indolyl)maleimides of which the most potent, Ro 31-8425 (I50: PKC = 8 nM) showed 350-fold selectivity for PKC over cAMP-dependent protein kinase. Ro 31-8425 antagonised cellular processes triggered by phorbol esters (potent, specific PKC activators) and inhibited the allogeneic mixed lymphocyte reaction, suggesting a role for PKC in T-cell activation. Methylation of the primary amine in Ro 31-8425 produced an analogue. Ro 31-8830 which, when administered orally, produced a dose-dependent inhibition of a phorbol ester-induced paw oedema in mice (minimum effective dose = 15 mg/kg). Ro 31-8830 also selectively inhibited the secondary inflammation in a developing adjuvant arthritis model in the rat. The results presented here suggest that these selective inhibitors of PKC may have therapeutic value in the treatment of T-cell-mediated autoimmune diseases.

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 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.

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.