TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production.

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.

Costimulator B7-DC attenuates strong Th2 responses induced by Nippostrongylus brasiliensis.

The caliber and magnitude of T cell responses are regulated by costimulatory molecules following the engagement of TCRs and MHC molecules. B7-DC has the highest homology with B7-H1 in the B7 family, and both of them bind an immunoregulatory molecule, programmed death 1. Previous studies have demonstrated that B7-DC stimulates T cell proliferation and CTL generation, which sharply contrasts the inhibitory role of B7-H1. Th2 cytokines prompt B7-DC expression, which in turn enhances Th1 responses. In this study, we used an intestinal nematode, Nippostrongylus brasiliensis, to induce strong Th2 responses and to evaluate B7-DC function under Th2-polarizing conditions in vivo. By either blocking B7-DC expression during N. brasiliensis infection or by examining N. brasiliensis-infected B7-DC knockout mice, we observed enhanced eosinophilia, the overproduction of serum IgE, and increased Th2 cytokine production along with decreased Th1 cytokine production (particularly IFN-gamma production), indicating that B7-DC inhibits Th2 responses. Our results further demonstrate that the inhibition of Th2 responses by B7-DC occurs independently of programmed death 1 but conceivably acts through an as yet unknown alternative receptor that enhances Th1 responses. Although the deficiency of B7-DC expression that enhanced the production of IL-13 paradoxically resulted in better protection against N. brasiliensis infection, our results show that B7-DC plays an important role in bolstering a robust Th1 response that is required for effective antiviral and anticancer immunity, even under a strong Th2-polarizing environment induced by N. brasiliensis infection.

Antigen-specific immunity and cross-priming by epithelial ovarian carcinoma-induced CD11b(+)Gr-1(+) cells.

Both innate and adaptive immune systems are considered important for cancer prevention, immunosurveillance, and control of cancer progression. It is known that, although both systems initially eliminate emerging tumor cells efficiently, tumors eventually escape immune attack by a variety of mechanisms, including differentiation and recruitment of immunosuppressive CD11b(+)Gr-1(+) myeloid suppressor cells into the tumor microenvironment. However, we show that CD11b(+)Gr-1(+) cells found in ascites of epithelial ovarian cancer-bearing mice at advanced stages of disease are immunostimulatory rather than being immunosuppressive. These cells consist of a homogenous population of cells that morphologically resemble neutrophils. Moreover, like dendritic cells, immunostimulatory CD11b(+)Gr-1(+) cells can strongly cross-prime, augmenting the proliferation of functional CTLs via signaling through the expression of costimulatory molecule CD80. Adoptive transfer of these immunostimulatory CD11b(+)Gr-1(+) cells from ascites of ovarian cancer-bearing mice results in the significant regression of s.c. tumors even without being pulsed with exogenous tumor Ag prior to adoptive transfer. We now show for the first time that adaptive immune responses against cancer can be augmented by these cancer-induced granulocyte-like immunostimulatory myeloid (CD11b(+)Gr-1(+)) cells, thereby mediating highly effective antitumor immunity in an adoptive transfer model of immunity.

Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication.

We recently demonstrated that human p38 mitogen-activated protein kinase (MAPK) inhibitors reduced in vitro and in vivo replication of the protozoan parasites Toxoplasma gondii and Encephalitozoon cuniculi. In this study, we assessed the efficacy of five p38 MAPK inhibitors to block the replication of Plasmodium falciparum in human erythrocytes cultured ex vivo and demonstrate that the pyridinylimidazole RWJ67657 and the pyrrolobenzimidazole RWJ68198 reduced P. falciparum replication, yielded trophozoites that were greatly diminished in size at 24h, and that these two agents interfered with stage differentiation. Interestingly, the chloroquine-resistant strain W2 was significantly more sensitive to these drugs than was the chloroquine-sensitive strain HB3. These results suggest that pyridinylimidazoles and pyrrolobenzimidazoles designed to inhibit human p38 MAPK activation can be developed to treat malaria.

IFNα Augments Clinical Efficacy of Regulatory T-cell Depletion with Denileukin Diftitox in Ovarian Cancer.

PURPOSE: Immunotherapy treats some cancers, but not ovarian cancer. Regulatory T cells (Tregs) impede anti-ovarian cancer immunity but effective human Treg-directed treatments are lacking. We tested Treg depletion with denileukin diftitox (DD) ± IFNα as ovarian cancer immunotherapy. PATIENTS AND METHODS: Mice with syngeneic ID8 ovarian cancer challenge were treated with DD, IFNα, or both. The phase 0/I trial tested one dose-escalated DD infusion for functional Treg reduction, safety, and tolerability. The phase II trial added IFNα2a to DD if DD alone failed clinically. RESULTS: DD depleted Tregs, and improved antitumor immunity and survival in mice. IFNα significantly improved antitumor immunity and survival with DD. IFNα did not alter Treg numbers or function but boosted tumor-specific immunity and reduced tumor Treg function with DD by inducing dendritic cell IL6. DD alone was well tolerated, depleted functional blood Tregs and improved immunity in patients with various malignancies in phase 0/I. A patient with ovarian cancer in phase 0/I experienced partial clinical response prompting a phase II ovarian cancer trial, but DD alone failed phase II. Another phase II trial added pegylated IFNα2a to failed DD, producing immunologic and clinical benefit in two of two patients before a DD shortage halt. DD alone was well tolerated. Adding IFNα increased toxicities but was tolerable, and reduced human Treg numbers in blood, and function through dendritic cell-induced IL6 in vitro. CONCLUSIONS: Treg depletion is clinically useful but unlikely alone to cure ovarian cancer. Rational treatment agent combinations can salvage clinical failure of Treg depletion alone, even when neither single agent provides meaningful clinical benefit.

5S clavam biosynthetic genes are located in both the clavam and paralog gene clusters in Streptomyces clavuligerus.

The Streptomyces clavuligerus clavam gene cluster was examined to identify genes specifically involved in 5S clavam biosynthesis. A reduction/loss of 5S clavam production was seen in cvm2 and cvm5 gene mutants, and a clavam metabolite not previously observed, 2-carboxymethylideneclavam, accumulated in the cvm5 mutant. Disruption of additional genes from the region of the clavam cluster did not have any effect on 5S clavam production. Examination of the paralog gene cluster region for 5S clavam biosynthetic genes led to the identification of cvm6P and cvm7P, which encode a putative aminotransferase and a transcriptional regulator, respectively. Mutants defective in cvm6P and cvm7P were completely blocked in 5S clavam but not clavulanic acid production. The loss of 5S clavam production in cvm7P mutants suggests that this gene encodes a transcriptional regulator specific for 5S clavam metabolite biosynthesis.

Trends in immunoconjugate and ligand-receptor based targeting development for cancer therapy.

Many agents used to treat cancer are toxic to normal tissues. Thus, treatments delivering drug specifically to tumour, while minimising exposure to normal tissue, may be advantageous over non-targeted treatments. The exquisite specificity of the immune system has been used successfully to help develop targeted anticancer agents. The most common (and successful) tissue-specific targeting strategies rely on antibody conjugates, but additional approaches, including targeting through cytokines, peptides and recombinant viruses, have also been used successfully. This review summarises the agents exploiting the immunological principles of target specificity to help maximise delivery to tumour while minimising collateral damage to normal tissues. Such targeted molecules are collectively referred to as immunoconjugates.

Identification of a novel mitogen-activated protein kinase in Toxoplasma gondii.

Toxoplasma gondii is an Apicomplexan parasite causing significant morbidity and mortality in immunocompromised hosts. Mitogen activated protein kinases regulate diverse biologic processes including proliferation, differentiation, survival and stress responses. We searched a new T. gondii genomic database to identify a 1.6 kilobase pair (kbp) coding region with features suggesting a mitogen activated protein kinase. This gene is predicted to encode a 58kDa protein with a threonine, aspartic acid, tyrosine (TDY) activation loop, similar to parasite and plant mitogen activated protein kinases, but distinct from mammalian mitogen activated protein kinases (with threonine, glycine, tyrosine (TGY) motifs). The predicted protein shares 45% amino acid identity with human stress-activated p38alpha mitogen activated protein kinase. Expression of the cloned gene in Escherichia coli produced a protein with an apparent molecular weight of 63kDa and which exhibited kinase activity. Following osmotic stress, the abundance of the mRNA encoding this T. gondii mitogen activated protein kinase, which we name TgMAPK-1, increased in tachyzoites. Its expression rescued hog1-deficient yeast grown under osmotic stress. These data confirm that the gene product is a stress-response mitogen activated protein kinase. Upon conversion of T. gondii tachyzoites to the latent bradyzoite form in vitro, tgMAPK-1 transcript accumulation increased, suggesting a role in parasite proliferation or stage differentiation. We previously demonstrated that pyridinylimidazole p38 mitogen activated protein kinase inhibitors block T. gondii replication. These inhibitors also blocked TgMAPK-1 autophosphorylation, suggesting that TgMAPK-1, or other parasite mitogen activated protein kinases are novel drug development targets.

Genetic diversity among Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis strains using repetitive element polymorphism-PCR.

Repetitive element polymorphism-PCR (REP-PCR) is one of the tools that has been used to elucidate genetic diversity of related microorganisms. Using the MB1 primer, REP-PCR fingerprints from 110 Bacillus strains within the "B. cereus group" have identified eighteen distinct categories, while other more distantly related bacterial species fell within six additional categories. All Bacillus anthracis strains tested were found to be monomorphic by fluorophore-enhanced REP-PCR (FERP) fingerprinting using the MB1 primer. In contrast, other non- B. anthracis isolates displayed a high degree of polymorphism. Dendrogramic analysis revealed that the non- B. anthracis strains possessing the Ba813 chromosomal marker were divided into two clusters. One of the clusters shared identity with the B. cereus strains examined.

Mitigating age-related immune dysfunction heightens the efficacy of tumor immunotherapy in aged mice.

Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4(+)CD25(hi) regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti-Gr-1 antibody was immunologically and clinically more efficacious than anti-Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti-Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved.

Parasite mitogen-activated protein kinases as drug discovery targets to treat human protozoan pathogens.

Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs) as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known.

A simple method to detect Toxoplasma gondii-specific cytotoxic T cells in vivo.

Cytotoxic T cells (CTLs) are an important component of adaptive immunity. The study of antigen-specific CTLs in vivo is desirable yet difficult. Identification of the class I-restricted peptide used by CTLs for target recognition is often required for detailed studies, but is generally not known for most antigens. Toxoplasma gondii is a medically important, obligate intracellular parasite and is often used as a model for studies of parasite immunology. No class I-restricted peptides for CTLs are known. We show here a new and convenient method to detect T. gondii-specific CTLs in vivo. We engineered T. gondii tachyzoites to express the model antigen ovalbumin, for which many useful reagents and transgenic mice are available. Using ovalbumin-transgenic T. gondii tachyzoites, antigen-specific CTLs were detected in vivo, and at much earlier time points post-infection than previously reported. This new method has several additional advantages over current methods to detect T. gondii-specific CTLs.

Altering regulatory T cell function in cancer immunotherapy: a novel means to boost the efficacy of cancer vaccines.

Cancers express tumor associated antigens that should elicit immune attack, but spontaneous immune rejection of established cancer is rare. Recent data demonstrate that specific and active tumor-mediated mechanisms hinder host anti-tumor immunity. CD4+CD25+ T regulatory cells (Tregs) are important mediators of active immune evasion in cancer. Disrupting tumor-mediated mechanisms hindering host immunity is a novel approach to tumor immunotherapy. Treg depletion improves endogenous anti-tumor immunity and the efficacy of active immunotherapy in animal models for cancer, suggesting that inhibiting Treg function could also improve the limited successes of human cancer immunotherapy. We have identified five strategies to block Treg activity: depletion, interference with trafficking, inhibition of differentiation, blockade of function or raising the effector T cell threshold for suppression. Discovery of additional regulatory cell populations expands the potential targets for these approaches. The fusion toxin denileukin diftitox (Ontak) reduces Treg numbers and function in the blood of some patients with cancer. We discuss specific strategies to block Treg activity and present some of our preliminary data in this area. Combining Treg depletion with active vaccination and other approaches poses additional challenges that are discussed.

An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance.

The HER2-targeted therapy trastuzumab is widely used for the treatment of patients with metastatic breast tumors overexpressing HER2. However, an objective response is observed in only 12% to 24% of patients treated with trastuzumab as a single agent and initial responders regress in <6 months (1-3). The reason for the clinical failure of trastuzumab in this setting remains unclear. Here we show that local lymph node-positive disease progression in 89% of breast cancer patients with HER2-positive tumors involves the HER2 oncogenic variant HER2Delta16. We further show that ectopic expression of HER2Delta16, but not wild-type HER2, promotes receptor dimerization, cell invasion, and trastuzumab resistance of NIH3T3 and MCF-7 tumor cell lines. The potentiated metastatic and oncogenic properties of HER2Delta16 were mediated through direct coupling of HER2Delta16 to Src kinase. Cotargeting of HER2Delta16 and Src kinase with the single-agent tyrosine kinase inhibitor dasatinib resulted in Src inactivation, destabilization of HER2Delta16, and suppressed tumorigenicity. Activated Src kinase was also observed in 44% of HER2Delta16-expressing breast carcinomas underscoring the potential clinical implications of coupled HER2Delta16 and Src signaling. Our results suggest that HER2Delta16 expression is an important genetic event driving trastuzumab-refractory breast cancer. We propose that successful targeted therapeutics for intervention of aggressive HER2-positive breast cancers will require a strategy to suppress HER2Delta16 oncogenic signaling. One possibility involves a therapeutic strategy employing single-agent tyrosine kinase inhibitors to disengage the functionally coupled oncogenic HER2Delta16 and Src tyrosine kinase pathways.

Toxoplasma gondii expresses two mitogen-activated protein kinase genes that represent distinct protozoan subfamilies.

All eukaryotes express mitogen-activated protein kinases (MAPKs) that govern diverse cellular processes including proliferation, differentiation, and survival. Even though these proteins are highly conserved throughout nature, MAPKs from closely related species often possess distinct signature sequences, making them well suited as drug discovery targets. Based on the central amino acid in the TXY dual phosphorylation loop, mammalian MAPKs are classified as extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), or p38 stress-response MAPKs. The presence of MAPKs in nonmetazoan eukaryotes suggests significant evolutionary conservation of these important signalling pathways. We recently cloned a novel stress-response MAPK gene (tgMAPK1) from Toxoplasma gondii, an obligate intracellular human parasite that can cause life-threatening infections in immunocompromised patients, and we now present data on a second T. gondii MAPK gene (tgMAPK2) that we cloned. We show that tgMAPK1 and tgMAPK2 are members of two distinct and previously unknown protozoan MAPK subfamilies that we have named pzMAPKl/pzMAPK3 and pzMAPK2. Our phylogenetic analysis of a collection of protozoan and metazoan MAPK genes in relation to ERK8-like genes demonstrates that an ERK8-like family, which includes the pzMAPK2 subfamily, is represented across a large variety of eukaryotic kingdoms and is evolutionarily very distant from other MAPK families.

Drugs designed to inhibit human p38 mitogen-activated protein kinase activation treat Toxoplasma gondii and Encephalitozoon cuniculi infection.

We recently showed that the pyridinylimidazoles SB203580 and SB202190, drugs designed to block human p38 mitogen-activated protein kinase (MAPK) activation, also inhibited replication of the medically important intracellular parasite Toxoplasma gondii in cultured human fibroblasts through a direct effect on the parasite. We now show that additional pyridinylimidazole and imidazopyrimidine p38 MAPK inhibitors inhibit intracellular T. gondii replication in vitro and protect mice against fatal T. gondii infection. Mice surviving infection following treatment with p38 MAPK inhibitors were resistant to subsequent T. gondii challenge, demonstrating induction of protective immunity. Thus, drugs originally developed to block human p38 MAPK activation are useful for treating T. gondii infection without inducing significant immunosuppression. MAPK inhibitors combined with either of the approved anti-Toxoplasma drugs sulfadiazine and pyrimethamine resulted in improved survival among mice challenged with a fatal T. gondii inoculum. A MAPK inhibitor also treated mice infected with the Microsporidium parasite Encephalitozoon cuniculi, suggesting that MAPK inhibitors represent a novel class of agents that may have a broad spectrum of antiparasitic activity. Preliminary studies implicate a T. gondii MAPK homologue as the target of drug action, suggesting possibilities for more-selective agents.