Malaria: host-pathogen interactions, immunopathological complications and therapy.

Malaria is a global tropical disease causing more than 1 million deaths and 300 million clinical cases every year. It is caused by parasites from the genus Plasmodium and is transmitted by Anopheles mosquitoes. Approximately 3 billion people live in malaria-endemic regions and a majority of them are infected. In this review, we discuss the life cycle of the parasite, the complex interactions with the human host and the ensuing immune reactions and complications. The immune system plays a dual role in malaria, by providing life-saving immunity against the parasite, but also by causing often lethal complications in a number of patients. Cytokines, chemokines and proteases are key players in the immunopathological complications, and we propose immunomodulation with dexamethasone as a promising strategy for the therapy of malaria-associated acute respiratory distress syndrome.

Virulent Toxoplasma gondii strain RH promotes T-cell-independent overproduction of proinflammatory cytokines IL12 and gamma-interferon.

The aim of this study was the analysis of the cytokine response in BALB/c mice infected with the highly virulent RH or the weakly virulent Beverley strains of Toxoplasma gondii. Analysis of cytokine messages showed increased expression of IL12, IFN-gamma and TNF-alpha, but not IL4 mRNAs in spleen cells after infection with the T. gondii strains RH and Beverley. High levels of circulating IL12 and IFN-gamma were detected in the serum of mice infected with strain RH, although TNF-alpha levels remained low. In contrast, the same cytokines were detected at only low levels in the serum of mice infected with the Beverley strain. Administration of antibody against IL12 or IFN-gamma significantly delayed time to death of mice infected with strain RH compared to controls. T-Cell-deficient as well as normal mice were equally infected by strain RH, suggesting that T lymphocytes do not contribute to the response. Depletion of natural killer cells from the splenocyte population abolished the in vitro production of IFN-gamma. Together, our data suggest that the virulent strain RH induces in BALB/c mice a type 1 cytokine pattern with T-cell-independent overproduction of IL12 and IFN-gamma that may be involved in the pathogenesis of this micro-organism.

Role of interleukin-12 in the induction of mucosal inflammation and abrogation of regulatory T cell function in chronic experimental colitis.

IL-12 promotes Th1 cell differentiation and cell-mediated immunity. In the present study, the potential role of IL-12 was analyzed in an experimental colitis model in scid mice reconstituted with syngeneic CD45RBhighCD4+ T cells. Real-time reverse transcription-PCR studies demonstrated that IL-12 p40 mRNA in inflamed colon is induced shortly after T cell transfer and maintained at a stable level after week 4, at the time when wasting disease starts. Administration of anti-IL-12 on days 0,14, and 28 (early treatment) or on days 28, 42, and 56 (delayed treatment) after T cell transfer, effectively prevented or, respectively cured wasting disease and colitis in scid recipients. Anti-IL-12 treatment abrogated mucosal inflammation with significantly diminished leukocyte infiltration (CD4 cells, macrophages) and CD54 expression, and down-regulated proinflammatory cytokines IFN-gamma and IL-2. Of note, although splenic CD4+ T cells are unable to induce disease as a result of the presence of regulatory CD45RBlow cells, splenic CD4+ T cells, preactivated by IL-12 and anti-CD3 in vitro, were highly pathogenic in inducing severe mucosal inflammation, suggesting that IL-12 and anti-CD3 abrogated regulatory T cell function. These findings indicate that IL-12 is important for the induction of experimental colitis through effects on proinflammatory cytokine production and on regulatory T cell function.

B7 interactions with CD28 and CTLA-4 control tolerance or induction of mucosal inflammation in chronic experimental colitis.

CD28-B7 interaction plays a critical costimulatory role in inducing T cell activation, while CTLA-4-B7 interaction provides a negative signal that is essential in immune homeostasis. Transfer of CD45RB(high)CD4(+) T cells from syngeneic mice induces transmural colon inflammation in SCID recipients. This adoptive transfer model was used to investigate the contribution of B7-CD28/CTLA-4 interactions to the control of intestinal inflammation. CD45RB(high)CD4(+) cells from CD28(-/-) mice failed to induce mucosal inflammation in SCID recipients. Administration of anti-B7.1 (but not anti-B7.2) after transfer of wild-type CD45RB(high)CD4(+) cells also prevented wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of proinflammatory cytokines IL-2 and IFN-gamma by lamina propria CD4(+) cells. In contrast, anti-CTLA-4 treatment led to deterioration of disease, to more severe inflammation, and to enhanced production of proinflammatory cytokines. Of note, CD25(+)CD4(+) cells from CD28(-/-) mice similar to those from the wild-type mice were efficient to prevent intestinal mucosal inflammation induced by the wild-type CD45RB(high) cells. The inhibitory functions of these regulatory T cells were effectively blocked by anti-CTLA-4. These data show that the B7-CD28 costimulatory pathway is required for induction of effector T cells and for intestinal mucosal inflammation, while the regulatory T cells function in a CD28-independent way. CTLA-4 signaling plays a key role in maintaining mucosal lymphocyte tolerance, most likely by activating the regulatory T cells.

The protective effect of IFN-gamma in experimental autoimmune diseases: a central role of mycobacterial adjuvant-induced myelopoiesis.

The study of animal models for organ-specific autoimmune disease contributes to our understanding of human diseases such as multiple sclerosis and rheumatoid arthritis. Although experimental autoimmune diseases develop spontaneously in certain strains of mice, others need to be induced by administration of organ-specific autoantigen, often together with complete Freund's adjuvant (CFA), containing heat-killed mycobacteria. In the two types of models, the role of endogenous interferon-gamma (IFN-gamma) has extensively been investigated by using neutralizing anti-IFN-gamma antibodies and by employing mice genetically deficient in IFN-gamma or its receptor. In these studies disease-promoting as well as disease-protective roles of endogenous IFN-gamma have been described. Remarkably, in most models that rely on the use of CFA, there is abundant evidence for a protective role. Here, we review evidence that this role derives from an inhibitory effect of IFN-gamma on myelopoiesis elicited by the killed mycobacteria. These findings explain the bimodal role of IFN-gamma in different models of autoimmune disease and raise questions regarding the clinical relevance of these models.

In vivo neutrophil recruitment by granulocyte chemotactic protein-2 is assisted by gelatinase B/MMP-9 in the mouse.

Granulocyte chemotactic protein-2 (GCP-2) of the mouse is a potent neutrophil chemotactic and activating factor in vitro and in vivo. Gelatinase B/matrix metalloproteinase-9 is released from neutrophils within 1 h after stimulation with GCP-2. In vitro neutrophil chemotaxis by GCP-2 was not impaired by specific inhibitory monoclonal antibodies (mAb) against gelatinase B, indicating that gelatinase B is not involved in chemotaxis of neutrophils through polycarbonate filters. To investigate if gelatinase B degranulation is involved in in vivo cell migration toward GCP-2, experiments were performed with gelatinase B knockout mice. When mouse GCP-2 was injected intradermally in mice, a dose-dependent neutrophil chemotactic response was observed, and this cell migration was significantly impaired in young mice by genetic gelatinase B knockout. In adult vs. young gelatinase B-deficient mice, such compensatory mechanisms as higher basal neutrophil counts and less impairment of chemotaxis toward local GCP-2 injection were observed. These experiments prove the concept that gelatinase B release under pressure of GCP-2 is a relevant, but not exclusive, effector mechanism of neutrophil chemotaxis in vivo and that known mechanisms, other than the release of gelatinase B, allow for a full-blown chemotactic response and compensate for gelatinase B deficiency in adult life in the mouse.

Prevention of experimental colitis in SCID mice reconstituted with CD45RBhigh CD4+ T cells by blocking the CD40-CD154 interactions.

Increased expression of CD40 and CD40 ligand (CD40L or CD154) has been found in inflamed mucosa of human inflammatory bowel disease (IBD), and interactions between these molecules seem to be involved in local cytokine production by macrophages. However, the precise role of CD40 signaling in the pathogenesis of IBD is still poorly understood. The aim of the present study was to investigate the in vivo relevance of CD40 signaling in experimental colitis in SCID mice reconstituted with syngeneic CD45RBhighCD4+ T cells. The results demonstrated that CD40+ and CD40L+ cells as well as their mRNA levels were significantly increased in inflamed mucosa. Administration of anti-CD40L neutralizing mAb over an 8-wk period starting immediately after CD45RBhighCD4+ T cell reconstitution completely prevented symptoms of wasting disease. Intestinal mucosal inflammation was effectively prevented, as revealed by abrogated leukocyte infiltration and decreased CD54 expression and strongly diminished mRNA levels of the proinflammatory cytokines IFN-gamma, TNF, and IL-12. When colitic SCID mice were treated with anti-CD40L starting at 5 wk after T cell transfer up to 8 wk, this delayed treatment still led to significant clinical and histological improvement and down-regulated proinflammatory cytokine secretion. These data suggest that the CD40-CD40L interactions are essential for the Th1 inflammatory responses in the bowel in this experimental model of colitis. Blockade of CD40 signaling may be beneficial to human IBD.

IFN-gamma, IL-4, IL-10 and IL-12 gene expression in BCG-Leishmania vaccination of Trypanosoma cruzi-infected mice.

We have previously shown that vaccination of BALB/c mice with a combination of BCG plus killed Leishmania promastigotes, applied by the i.p. route 10 and 3 days before Trypanosoma cruzi inoculation, prolonged their survival and decreased their parasitaemia. In the present study we show that the BCG-Leishmania vaccine induced higher levels of circulating IFN-gamma in acute and chronic infection of mice [on day 25 and 40 post-infection (p.i.) respectively], in comparison to unvaccinated animals (PBS-treated). Though the IFN-gamma mRNA content of spleen cells of vaccinated and infected mice (on day 25 p.i.) was similar to that of unvaccinated animals, the BCG-Leishmania vaccine enhanced significantly the production of IFN-gamma by spleen cells stimulated with T. cruzi antigens. This effect was observed to a lower extent in BCG- and Leishmania-treated mice. The BCG-Leishmania vaccine reduced the expression of the IL-10 mRNA of splenocytes as soon as day 12 p.i., before the peak parasitaemia. Such this effect was not observed in BCG- or Leishmania-treated animals. On day 25 p.i., the BCG plus Leishmania- or BCG-treatment of mice abolished the capacity of spleen cells to produce IL-10 in response to T. cruzi antigens. The levels of mIL-4 RNA and protein production were not modified in any group of mice. T. cruzi infection in BCG-Leishmania-vaccined mice stimulated an early and high production of IL-12 transcripts in spleen cells during the acute phase of the infection, that was prolonged during the chronic phase of infection. This effect was weaker or absent in BCG- and Leishmania-treated animals, respectively. These results indicate that the BCG-Leishmania vaccine stimulates the production of IL-12 and IFN-gamma, but inhibits that of IL-10 and is without effect on IL-4 when mice are infected with T. cruzi. This highlights the key role of endogenously produced IFN-gamma, IL-10 and IL-12 in the control of T. cruzi acute and chronic infection in mice and the favorable modulation of their balance by a vaccination combining BCG and Leishmania.

Role of interferon-gamma and nitric oxide in pulmonary edema and death induced by lipopolysaccharide.

Mice given lipopolysaccharide (LPS) intravenously developed lung edema, which was maximum after 6 h. Tumor necrosis factor, interleukin 12 (IL-12), IL-6, and interferon-gamma (IFN-gamma) appeared in the serum, and levels of nitrogen oxide (NO) derivatives were increased in serum and bronchoalveolar fluid. Mice pretreated with neutralizing anti-IFN-gamma antibodies had lower serum levels of IFN-gamma, and fewer died. However, levels of other cytokines and NO derivatives as well as lung edema were unchanged. If IFN-gamma and LPS were given together, pulmonary edema was less, but levels of cytokines and NO derivatives in serum were raised, and the mortality was greater. IFN-gamma receptor knockout mice had more edema after LPS, but were less sensitive to the lethal effects. Treatment with anti-IL-12 antibody inhibited IFN-gamma induction and reduced mortality, but had no effect on the lung edema; exogenous IL-12 also failed to affect edema, but boosted serum cytokine levels and increased the mortality. Aminoguanidine, an inhibitor of NO synthase, protected against pulmonary edema, but did not modify the lethal effects of LPS. Clearly, in this model, early pulmonary edema and lethality are not directly related, and induced IFN-gamma has no role in causing early lung edema, but augments other events that result in death.

Bimodal role of endogenous interleukin-6 in concanavalin A-induced hepatitis in mice.

Acute concanavalin A (Con A)-induced hepatitis in mice is an animal model for hepatic injury induced by activated T cells. The evolution of hepatic involvement can be followed from hour to hour by measuring serum transaminase levels. We investigated the possible role of endogenous interleukin-6 (IL-6) in this model. We found serum IL-6 levels and splenic IL-6 mRNA during Con A-induced hepatitis to be significantly lower in interferon-gamma (IFN-gamma)-deficient mice, which are resistant against the Con A-induced syndrome, than in wild-type ones, suggesting that systemic IL-6 production favors development of hepatic injury. However, IL-6-deficient mice proved to be more susceptible to the disease than wild-type mice, indicating that endogenous IL-6 plays a predominantly hepatoprotective role. Experiments in which wild-type mice were treated with anti-IL-6 antibodies, before or after Con A challenge, allowed us to reconcile these contrasting observations. The antibody injections resulted in a biphasic alteration of serum IL-6 levels, initial neutralization being followed by rebound increased levels due to accumulation of IL-6 in the form of antigen-antibody complexes. The effect of antibody on disease severity differed depending on the time of injection. Antibody injection at 2.5 h post Con A resulted in delayed disease manifestation, whereas treatment initiated before Con A resulted in accelerated disease. We conclude that endogenous IL-6 plays a bimodal role. IL-6 present before Con A challenge as well as that induced in the very early phase after Con A injection triggers hepatoprotective pathways. Continuation of IL-6 production beyond this early phase, by some other pathway, seems to be harmful to hepatocytes.

Resistance of young gelatinase B-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions.

Regulated expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) plays a role in various physiological processes. To determine in vivo how unbalanced expression of these factors can promote or affect the course of pathologies, we knocked out the mouse gelatinase B gene by replacing the catalytic and zinc-binding domains with an antisense-oriented neomycin resistance gene. Adult gelatinase B-deficient mice and wild-type controls could be induced to develop experimental autoimmune encephalomyelitis (EAE) with similar scores for neurologic disease, blood-brain barrier permeability, and central nervous system histopathology. However, whereas diseased control animals showed necrotizing tail lesions with hyperplasia of osteocartilaginous tissue, adult gelatinase B-deficient mice were resistant to this tail pathology. Gelatinase B-deficient mice younger than 4 weeks of age were significantly less susceptible to the development of EAE than were age matched controls and, even as they aged, they remained resistant to tail lesions. These data illustrate that gelatinase B expression plays a role in the development of the immune system and that, in ontogenesis, the propensity to develop autoimmunity is altered by the absence of this MMP.

NH2- and COOH-terminal truncations of murine granulocyte chemotactic protein-2 augment the in vitro and in vivo neutrophil chemotactic potency.

Chemokines are important mediators of leukocyte migration during the inflammatory response. Post-translational modifications affect the biological potency of chemokines. In addition to previously identified NH2-terminally truncated forms, COOH-terminally truncated forms of the CXC chemokine murine granulocyte chemotactic protein-2 (GCP-2) were purified from conditioned medium of stimulated fibroblasts. The truncations generated 28 natural murine GCP-2 isoforms containing 69-92 residues, including most intermediate forms. Both NH2- and COOH-terminal truncations of GCP-2 resulted in enhanced chemotactic potency for human and murine neutrophils in vitro. The truncated isoform GCP-2(9-78) was 30-fold more potent than intact GCP-2(1-92)/LPS-induced CXC chemokine (LIX) at inducing an intracellular calcium increase in human neutrophils. After intradermal injection in mice, GCP-2(9-78) was also more effective than GCP-2(1-92)/LIX at inducing neutrophil infiltration. Similar to human IL-8 and GCP-2, murine GCP-2(9-78) and macrophage inflammatory protein-2 (MIP-2) induced calcium increases in both CXCR1 and CXCR2 transfectants. Murine GCP-2(9-78) could desensitize the calcium response induced by MIP-2 in human neutrophils and vice versa. Furthermore, MIP-2 and truncated GCP-2(9-78), but not intact GCP-2(1-92)/LIX, partially desensitized the calcium response to human IL-8 in human neutrophils. Taken together, these findings point to an important role of post-translationally modified GCP-2 to replace IL-8 in the mouse.

Enhanced autoimmune arthritis in IFN-gamma receptor-deficient mice is conditioned by mycobacteria in Freund's adjuvant and by increased expansion of Mac-1+ myeloid cells.

Induction of experimental autoimmune diseases often relies on immunization with the organ-specific autoantigens in CFA, which contains heat-killed mycobacteria. In several of these models, including collagen-induced arthritis, endogenous IFN-gamma acts as a disease-limiting factor in the pathogenesis of the disease. Here we show that in collagen-induced arthritis the protective effect of IFN-gamma depends on the presence of mycobacteria in the adjuvant. Omission of mycobacteria inverts the role of endogenous IFN-gamma to a disease-promoting factor. Thus, the mycobacterial component of CFA opens a pathway by which endogenous IFN-gamma exerts a protective effect that supersedes its otherwise disease-promoting effect. Extramedullary hemopoiesis and expansion of the Mac-1+ cell population accompanied the accelerated and more severe disease course in the IFN-gamma receptor knockout mice immunized with CFA. Treatment of such mice with Abs against the myelopoietic cytokines IL-6 or IL-12 inhibited both disease development and the expansion of the Mac-1+ population. We postulate that mycobacteria in CFA stimulate the expansion of the Mac-1+ cell population by a hemopoietic process that is restrained by endogenous IFN-gamma. These results have important implications for the validity of animal models of autoimmunity to study the pathogenesis and to evaluate cytokine-based therapy of autoimmune diseases.

Role of endogenous interleukin-12 (IL-12) in induced and spontaneous relapses of experimental autoimmune encephalomyelitis in mice.

Actively induced, chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was studied in SJL/J and in Biozzi ABH mice. In Biozzi ABH mice, relapses occurred spontaneously with high frequency. In SJL/J mice, spontaneous relapses occurred infrequently; however they could be induced reproducibly by reimmunization. In both models, moderately increased levels of serum IL-12(p40) were consistently found shortly before primary attacks, but irregularly at later times. Injections of anti-IL-12 antibody inhibited disease development in both SJL/J and in Biozzi ABH mice. The time window during which treatment needed to be initiated in order to be effective, ranged from before induction until shortly before the symptoms of primary attacks emerged. Such treatment inhibited not only the first attack but also the spontaneous or induced relapses. Most significantly, anti-IL-12 antibody given during remission of primary disease inhibited actively re-induced relapses in SJL/J, but not spontaneous relapses in Biozzi ABH mice. These results indicate that endogenous IL-12 favours EAE development by crucially affecting the active induction process, but that a second burst of IL-12 production may not be necessary for triggering spontaneous relapses.

Vaccination of mice with a combination of BCG and killed Leishmania promastigotes reduces acute Trypanosoma cruzi infection by promoting an IFN-gamma response.

The combination of BCG with killed Leishmania promastigotes, demonstrated to be efficient in the cure of patients suffering American cutaneous leishmaniasis and in the induction of a long-term immune response in healthy vaccinated volunteers, was tested in BALB/c mice infected with Trypanosoma cruzi, in comparison to BCG or Leishmania alone, and a vehicle (PBS) control. BCG-Leishmania vaccination, applied intra-peritoneally 10 and 3 days before T. cruzi trypomastigote inoculation, prolonged the survival, and reduced blood parasitaemia of infected animals. Proliferation studies indicated that splenocytes of mice vaccinated with BCG-Leishmania and harvested in the acute phase of T. cruzi infection displayed stimulation indices higher than cells from PBS-treated mice when stimulated with PHA mitogen, PPD, Leishmania or T. cruzi antigens. Injections of a monoclonal antibody able to neutralise IFN-gamma into BCG-Leishmania vaccinated mice increased parasitaemia to levels similar to those of control animals (treated with PBS) and reversed the beneficial effect of vaccination on the proliferative response to T. cruzi antigen. These results show that vaccination of mice with BCG plus killed Leishmania promastigotes delayed acute T. cruzi infection, stimulated a T-cell response to T. cruzi antigen and promoted IFN-gamma production.

Differential nitric oxide and TNF-alpha production of murine Kupffer cell subfractions upon priming with IFN-gamma and TNF-alpha.

AIMS/BACKGROUND: We have previously shown a striking heterogeneity of naive murine Kupffer cells (KC) that depends on cell size. METHODS: In the present study, we demonstrate a shift in response of KC fractions separated on cell size by countercurrent elutriation upon priming with tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma). RESULTS: Whereas unprimed large KC are most active in the production of TNF-alpha and nitric oxide (NO), after priming of KC with TNF-alpha predominantly small and intermediate sized KC produce TNF-alpha in response to bacteria. Priming with IFN-gamma enhanced NO production in all KC. A strong synergy, with respect to production of NO, was observed when KC subfractions were exposed to a combination of TNF-alpha and IFN-gamma. Concerning TNF-alpha production, priming of KC subfractions seemed to induce a shift of activity from large KC to smaller KC. CONCLUSIONS: The present data demonstrate a clear heterogeneity among murine KC with respect to immunologic response to stimuli. These results demonstrate that KC have different functions in immunologic reactions that seem to be related to size.

Anti-IL-12 antibody prevents the development and progression of collagen-induced arthritis in IFN-gamma receptor-deficient mice.

In several models of inflammation, including collagen-induced arthritis (CIA), the disease-promoting effect of IL-12 has been attributed to its well-known ability to produce IFN-gamma. However, IFN-gamma receptor knockout (IFN-gammaR KO) mice of the DBA/1 strain have been reported to be more susceptible to CIA than corresponding wild-type mice, indicating the existence of an IFN-gamma-mediated protective pathway in this model. In the present study the development of CIA was found to be completely prevented by pretreatment with a neutralizing anti-IL-12 antibody, not only in wild-type, but significantly also in IFN-gammaR KO mice. In both strains of mice, the protective effect of anti-IL-12 was associated with lower production of anti-collagen type II antibodies. In vivo stimulation with anti-CD3 antibody in arthritic IFN-gammaR KO mice resulted in production of higher levels of circulating IFN-gamma, TNF and IL-2 than in corresponding control mice that had not received the arthritis-inducing immunization. This was not the case in arthritis-developing wild-type mice. Furthermore, the protective effect of anti-IL-12 antibody in mutant, but not in wild-type mice, was associated with lower circulating IFN-gamma, TNF and IL-2 and higher IL-4 and IL-5 cytokine levels following an anti-CD3 challenge. The data indicate that IL-12 promotes the development of arthritis independently of its ability to induce or favor production of IFN-gamma. In fact, any IFN-gamma produced in the course of the disease process rather exerts a protective effect. Furthermore, our study suggests that, in the absence of a functional IFN-gamma system, endogenous IL-12 exerts its disease-promoting effect by favoring production of other Th1-associated cytokines (IL-2 and TNF), by inhibiting development of IL-4- and IL-5-producing T cells and by stimulating production of anti-collagen autoantibodies.

Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation.

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.

Involvement of T cells in enhanced resistance to Klebsiella pneumoniae septicemia in mice treated with liposome-encapsulated muramyl tripeptide phosphatidylethanolamine or gamma interferon.

We have previously shown that prophylactic administration of the liposome-encapsulated immunomodulating agents muramyl tripeptide phosphatidylethanolamine (MTPPE) and gamma interferon (IFN-gamma) results in strongly increased survival of mice from a normally lethal septicemia with Klebsiella pneumoniae. It was anticipated that the treatment acts on macrophages and nonspecifically augments host resistance to various infections. In the present study, we provide evidence for a key role for T cells in host defense potentiation by the liposomal immunomodulators toward K. pneumoniae septicemia. It is shown that both CD4 and CD8 cells are important in immunomodulation, most likely due to production of IFN-gamma. Depletion of circulating IFN-gamma resulted in strong reduction of the antimicrobial host defense activation. Administration of interleukin-10 resulted in decreased antimicrobial host defense activation by liposomal immunomodulators. Moreover, administration of liposomal immunomodulators was shown to induce predominantly T-helper type 1 (Th1) cell populations in the spleen. These findings indicate that immunomodulation with liposomal MTPPE and IFN-gamma favors Th1 and NK cell activation.

In vitro and in vivo oncogenic potential of bovine leukemia virus G4 protein.

In addition to the genes involved in the structure of the viral particle, the bovine leukemia virus (BLV) genome contains a region called X which contains at least four genes. Among them, the tax and rex genes, respectively, are involved in transcriptional and posttranscriptional regulation of viral transcription. Two other genes, R3 and G4, were identified after cloning of the corresponding mRNAs from BLV-infected lymphocytes. Although the function of the two latter genes is still unknown, they appear to have important roles, since deletion of them restricts viral propagation in vivo. In order to assess the oncogenic potential of the R3 and G4 proteins, we first analyzed their ability to immortalize and/or transform primary rat embryo fibroblasts (Refs). In this assay, the G4 but not the R3 protein cooperated with the Ha-ras oncogene to induce tumors in nude mice. It thus appears that G4 exhibited oncogenic potential in vitro. To extend these observations in vivo, the pathology induced by recombinant viruses with mutations in G4 and in R3 and G4 was next evaluated with the sheep animal model. Viral propagation, as measured by semiquantitative PCR, appeared to be reduced when the R3 and G4 genes were deleted. These observations confirm and extend our previous data underlining the biological function of these genes. In addition, we present the results of a clinical survey that involves 39 sheep infected with six different BLV recombinants. Over a period of 40 months, 83% of the sheep infected with a wild-type virus developed leukemias and/or lymphosarcomas. In contrast, none out of 13 sheep infected with viruses with mutations in G4 or in R3 and G4 developed disease. We conclude that in addition to its oncogenic potential in vitro, G4 is required for pathogenesis in vivo. These observations should help us gain insight into the process of leukemogenesis induced by the related human T-cell leukemia virus type 1.