Long-term protective immune response elicited by vaccination with an expression genomic library of Toxoplasma gondii

Immunization of BALB/c mice with an expression genomic library of Toxoplasma gondii induces a Th1-type immune response, with recognition of several T. gondii proteins (21 to 117 kDa) and long-term protective immunity against a lethal challenge. These results support further investigations to achieve a multicomponent anti-T. gondii DNA vaccine.     

Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration

The maintenance of a benign chronic Toxoplasma gondii infection is mainly dependent on the persistent presence of gamma interferon (IFN-gamma) in the central nervous system (CNS). However, IFN-gamma-activated microglia are paradoxically involved in parasitism control and in tissue damage during a broad range of CNS pathologies. In this way, nitric oxide (NO), the main toxic metabolite produced by IFN-gamma-activated microglia, may cause neuronal injury during T. gondii infection. Despite the potential NO toxicity, neurodegeneration is not a common finding during chronic T. gondii infection. In this work, we describe a significant down-modulation of NO production by IFN-gamma-activated microglia in the presence of conditioned medium of T. gondii-infected astrocytes (CMi). The inhibition of NO production was paralleled with recovery of neurite outgrowth when neurons were cocultured with IFN-gamma-activated microglia in the presence of CMi. Moreover, the modulation of NO secretion and the neuroprotective effect were shown to be dependent on prostaglandin E(2) (PGE(2)) production by T. gondii-infected astrocytes and autocrine secretion of interleukin-10 (IL-10) by microglia. These events were partially eliminated when infected astrocytes were treated with aspirin and cocultures were treated with anti-IL-10 neutralizing antibodies and RP-8-Br cyclic AMP (cAMP), a protein kinase A inhibitor. Further, the modulatory effects of CMi were mimicked by the presence of exogenous PGE(2) and by forskolin, an adenylate cyclase activator. Altogether, these data point to a T. gondii-triggered regulatory mechanism involving PGE(2) secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia. This may reduce host tissue inflammation, thus avoiding neuron damage during an established Th1 protective immune response.     

The inflammatory lesion of T cell line transferred experimental autoimmune encephalomyelitis of the Lewis rat: distinct nature of parenchymal and perivascular infiltrates

We have investigated the T cell receptor (TCR) repertoire in the inflammatory infiltrates of T line-transferred experimental autoimmune encephalomyelitis (EAE) of the Lewis rats. Using a panel of TCR V-specific monoclonal antibodies (mAbs) and immunocytochemistry, we studied the nature of the T cells entering the central nervous system (CNS) after transfer of either myelin basic protein (MBP)-reactive, or MBP-reactive but non-encephalitogenic T cell lines. All the MBP-specific T cell lines predominantely used the V8.2 TCR chain. T cell lines specific for the tuberculin purified protein derivative (PPD), using TCR V genes different from V8.2, served as controls. We first studied the time course of T cells entering the CNS. In all recipient rats, small, but significant numbers of -TCR-expressing infiltrate cells appeared in the CNS within the first 24 h after T cell transfer. In animals injected with either type of MBP-reactive T cells, the early infiltrate cells were preferentially located within the parenchyma of the spinal cord, while in PPD T lineinjected rats, the lymphocytes were mostly found in the meninges. TCR V gene usage was examined on the peak of clinical disease. Six days after T cell transfer, the TCR repertoire used by infiltrating lymphocytes in general seemed to be highly diverse. None of the V isotypes examined (i.e. V8.2, V8.5 or V10) was used by a major population of the -TCR-positive T cells. A more detailed, quantitative analysis of individual infiltrate compartments revealed, however, a preferential accumulation of V8.2-positive T cells within the parenchyma. In contrast, perivascular infiltrating cells used V genes randomly. Our results confirm first that activated T lymphocytes enter the brain rapidly irrespective of their antigen specificity. Second, the data show that most of the perivascular infiltrate T cells in the acute EAE lesion are host-derived, recruited presumably from the recirculating T cell pool, while the encephalitogenic, V8.2-positive T cells preferentially persist within the parenchyma.Abbreviations EAE experimental autoimmune encephalomyelitis - MBP myelin basic protein - TCL T cell line Supported by the Brazilian Research Council (CNPq)     

Chronic Chagas' disease in rhesus monkeys (Macaca mulatta): evaluation of parasitemia,serology, electrocardiography,echocardiography, and radiology

Severe chronic damage to the heart and gastrointestinal tract in patients with Chagas' disease are often observed 10-20 years after the acute phase. The course of long-lasting infection with the Colombian strain of Trypanosoma cruzi was studied in seven rhesus monkeys infected for 15-19 years. Subpatent parasitemia was detected in all studied animals, using hemoculture (two of seven), artificial xenodiagnosis (three of seven), and a polymerase chain reaction PCR (six of six). High titers of specific IgG antibody to T. cruzi persisted throughout the chronic phase of infection. Abnormal electrocardiographic (three of six) and echocardiographic (one of six) patterns detected in the T. cruzi-infected monkeys were possibly related to parasite-triggered myocardial damage. The results suggest that rhesus monkeys experimentally infected with T. cruzi, besides reproducing the acute phase of Chagas' disease, also develop chronic chagasic cardiomyopathy.     

Unraveling the lethal synergism between Trypanosoma cruzi infection and LPS: a role for increased macrophage reactivity

Various infections sensitize to lethal shock by promoting hyperactivation of macrophages to LPS stimulation. Although macrophages are thought to be deactivated upon contact with apoptotic cells during Trypanosoma cruzi infection, T. cruzi infection also sensitizes mice to endotoxemia. Herein, we studied the mechanisms of sensitization to endotoxemia in T. cruzi-infected mice in order to solve the paradox. Live (but not fixed) trypomastigotes from various stocks sensitized mice to endotoxemia. Mice deficient in glycolipid recognition (TLR2(-/-) and CD1d(-/-)) were sensitized by infection to challenge with LPS. Infected mice hyperproduced TNF and IL-10 upon LPS challenge. Infected TNF-R1(-/-), macrophage migration inhibitory factor (MIF)(-/-) and IFN-gamma(-/-) mice were lethally sensitized, but infected TNF-R1(-/-) mice administered anti-MIF survived shock with LPS. Macrophages from infected mice hyperproduced TNF in response to LPS stimulation and displayed increased expression of TLR4 compared to non-infected controls. Treatment with the PGE(2) synthesis inhibitor acetylsalicylic acid (AAS) in vivo reduced parasitemia and enhanced LPS-stimulated production of TNF by macrophages, but the effect was less in infected mice than in normal mice. Nevertheless, AAS treatment did not increase the susceptibility of infected mice to sublethal shock with LPS. Our results point to independent MIF and TNF/TNF-R1 lethal pathways and suggest a role for hyperactivated macrophages in T. cruzi-sensitized LPS-induced shock.     

Immunogenicity of Plasmodium vivax merozoite surface protein-9 recombinant proteins expressed in E. coli

Merozoite surface protein-9 of Plasmodium vivax (PvMSP-9) is highly conserved and present in several malaria species. Here, we present the immunogenic properties of two recombinant glutathione S-transferase (GST) fusion proteins comprising the N-terminus (PvMSP-9-Nt) and the second block of tandem repeats (PvMSP-9-RepII) of PvMSP9. These recombinants proteins were used to immunize BALB/c mice. The specificity and subtyping of the antibodies and the cellular immune responses were evaluated by enzyme-linked immunosorbent assay (ELISA) and ELISPOT, respectively, using the recombinant proteins as antigens. Our results demonstrate that both the N-terminal and the tandem repeat regions of MSP9 are immunogenic in mice. The ELISA antibody titers elicited by PvMSP-9-Nt were significantly higher (1:819,200) than the antibody titers elicited by PvMSP-9-RII (1:409,600). Analysis of IgG subclasses showed that both recombinant proteins induce similar antibody patterns where IgG1, IgG2a and IgG2b were most predominant. Moreover, all sera from mice immunized with either PvMSP-9-Nt or PvMSP-9-RII, which were positive by ELISA showed reactivity with P. vivax, P. cynomolgi, P. knowlesi and P. coatneyi schizonts by immunofluorescence assays (IFA). Similar results were observed in western immunoblot analyses using parasite extracts. Furthermore, immunization of mice with the PvMSP-9-Nt upon stimulation with PvMSP-9-Nt secreted IFN-gamma and IL-5. We have also used the two PvMSP-9 recombinant constructs to show that individuals exposed to P. vivax infections in an endemic area of Brazil had IgG antibodies reactive with the recombinant proteins.     

Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera

Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4⁺ and CD8⁺ PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development.     

Microglial involvement in experimental autoimmune inflammation of the central and peripheral nervous system

Microglial cells form a network of potential antigen presenting cells throughout the nervous system. Much progress has recently been made towards a better understanding of their immunological properties. This study examines their activation in 2 models of T cell-mediated autoimmune inflammation of the nervous system, experimental autoimmune encephalomyelitis (EAE) and its peripheral counterpart, experimental autoimmune neuritis (EAN), induced by the transfer of antigen-specific T cell lines. In both models microglial activation occurs at early stages of the disease. Activated microglial cells show an increased expression of MHC class I and II antigens. In EAE ultrastructural analysis revealed that MHC antigen expression is pronounced on perivascular microglial cells, suggesting this cell population may be important for antigen presentation at a site close to the blood-brain barrier. In contrast to EAE, the microglial reaction in EAN occurs at sites remote from the inflammatory response in the peripheral nerve, not only in the spinal cord but also in the terminal projection fields of primary sensory neurons in the lower brainstem. This early microglial activation in EAN suggests that a rapid and remote signaling mechanism can operate following peripheral inflammation. Immuno-electron microscopy revealed that activated microglial cells are also involved in the synaptic deafferentation of spinal cord motoneurons during autoimmune reactions. The rapid involvement of microglial cells in experimental autoimmune inflammation of the nervous system further points to their role as the main intrinsic immuneffector cell population of the central nervous system.     

Ectopic Thyroid Presenting as a Submandibular Mass

Although extremely rare, the presence of ectopic thyroid tissue in the submandibular region should be considered in the differential diagnosis of tissue masses in the cervical region. Diagnosis is confirmed by fine-needle aspiration biopsy and exclusion of malignancy should be confirmed by histopathologic analysis of the lesion. In general, surgery is the treatment of choice. A rare case of ectopic thyroid in the right submandibular region is reported; it was diagnosed after total thyroidectomy and successfully treated through surgery.     

HLA class II and antibody responses to circumsporozoite protein repeats of P. vivax (VK210, VK247 and P. vivax-like) in individuals naturally exposed to malaria

We studied the seroreactivity against the circumsporozoite protein (CSP) repeats of Plasmodium vivax variants in individuals living in malaria-endemic area of the Brazilian Amazon region (Candeias do Jamari - RO). The prevalence of IgG antibodies for at least one of the P. vivax CSP repeats was 49%. Among these positive individuals, 34.2% were positive for the standard repeat sequence VK210, 24% for the VK247 and 31.5% for the P. vivax-like sequence. HLA typing showed an association between antibody responses to the CS repeats of VK247 and the presence of HLA-DR16 and between HLA-DR7 and the absence of antibody responses to the CS repeats of VK210. We also investigated the potential relationship between HLA-DQB1 allele profile and antibody response to the CSP repeats of P. vivax but no segregation with responding profile was evidenced. The observed findings indicate that antibody responses to the CSP repeats of P. vivax variants appear to be modulated by HLA class II molecules in malaria naturally exposed individuals.