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The role of cytokines in Leishmania amazonensis experimental infection has not been as well studied as in Leishmania major infection model. Here we investigated the role of interleukin (IL)-4 and PGE(2) in L. amazonensis infection of susceptible BALB/c mice. IL-4 deficient (-/-) or wild-type (+/+) BALB/c mice were infected with different inocula of L. amazonensis. Two weeks after infection with 5x10(6) promastigotes/footpad, the production of interferon (IFN)-gamma upon L. amazonensis antigen stimulation was significantly higher in lymph node cell cultures of IL-4-/- mice than in IL-4+/+ mice. The levels of anti-leishmania IgG2a antibodies were also significantly higher in serum from IL-4-/- mice. In contrast, the levels of IgG1 antibodies were increased in IL-4+/+ mice and almost undetectable in IL-4-/- mice. Despite the increased Th1 response, lesions of IL-4-/- BALB/c mice progressed similarly to those of IL-4+/+ mice upon infection with the 5x10(6) inoculum. However, IL-4-/- mice developed smaller lesions upon infection with 10(5), 10(4) or 10(3) parasites than IL-4+/+ mice. The resistance of IL-4-/- correlated with higher Th1 response, compared to IL-4+/+ upon infection with 10(4)L. amazonensis. IL-4+/+ mice treated with indomethacin, an inhibitor of PGE(2) synthesis, during the first 3weeks of infection developed smaller lesions and lower parasitic load when compared to the control group. The lesions of indomethacin-treated groups contained mostly macrophages without vacuoles and small or absent necrotic areas. These results indicate that IL-4 and PGE(2) are susceptibility factors to L. amazonensis infection.

Mice chronically infected with Schistosoma mansoni develop one of two anatomical forms of hepatic lesions: severe, periportal (pipestem) fibrosis or milder, isolated granulomas. The pathogenesis of periportal fibrosis is poorly understood. In this work we compared mice with either periportal fibrosis or isolated granulomas to identify specific markers of severe pathology. BALB/c or Swiss Webster mice were infected with 30 cercarie, once or six times, and liver biopsies were performed to classify the animals into two pathological groups 16 weeks later. Sixty percent of the animals sacrificed at 20 or 24 weeks had periportal fibrosis, 15–20% had isolated granulomas and the remainder had indeterminate pathology. There was no correlation between frequency of infections or egg burden (eggs/g liver) at 20 and 24 weeks post-infection and the development of periportal fibrosis. Livers with periportal fibrosis at 20 or 24 weeks of infection were characterized by larger areas of fibrotic tissue and greater vascularization compared to livers of mice with isolated granulomas. Plastic casts of the portal vein system showed marked changes in vascular structure in mice with periportal fibrosis, including collateral vessels sprouting from the main portal branches, amputation of the more delicate peripheral ramifications, and distortions of the medium and small branches. Vascular changes were not observed in mice with isolated granulomas. These results suggest that the interaction between schistosome eggs and portal vascular changes is of paramount importance in the development of pipestem fibrosis.

Physalis angulata is a solanaceae widely used in folk medicine in various tropical countries in the world. We have previously described that seco-steroids (physalins) purified from P. angulata are potent inhibitors of macrophage activation, blocking the production of pro-inflammatory cytokines and LPS-induced lethality. Herein we investigated the immunomodulatory activities of these substances in lymphocyte proliferation and cytokine production and in transplantation. The addition of physalins B, F or G to concanavalin A-activated splenocyte cultures induced a concentration-dependent inhibition of proliferation. Physalin B also inhibited IL-2 production by Con A-activated spleen cells. The addition of 2 mug/ml physalin B to mixed lymphocyte reaction (MLR) caused a 100% inhibition of proliferation. More importantly, treatment of mice with physalin B, F or G prevented the rejection of allogeneic heterotopic heart transplant. Our results demonstrate the suppressive activity of physalins B, F and G in lymphocyte function and indicate the potential use of physalins as immunosuppressive agents for treatments of pathologies in which inhibition of immune responses is desired.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:45:32Z No. of bitstreams: 1 S0074-02762005000600001.pdf: 2193761 bytes, checksum: d66ab3ce25c0d2df6bd09d883eb0e4cf (MD5) Made available in DSpace on 2013-08-22T18:45:32Z (GMT). No. of bitstreams: 1 S0074-02762005000600001.pdf: 2193761 bytes, checksum: d66ab3ce25c0d2df6bd09d883eb0e4cf (MD5) Previous issue date: 2005-10-01 Made available in DSpace on 2013-09-30T19:34:38Z (GMT). No. of bitstreams: 2 S0074-02762005000600001.pdf: 2193761 bytes, checksum: d66ab3ce25c0d2df6bd09d883eb0e4cf (MD5) S0074-02762005000600001.pdf.txt: 179481 bytes, checksum: 49f2bb8910ccb63dffa8cd07f4b9798b (MD5) Previous issue date: 2005-10-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:54:59Z No. of bitstreams: 2 S0074-02762005000600001.pdf: 2193761 bytes, checksum: d66ab3ce25c0d2df6bd09d883eb0e4cf (MD5) S0074-02762005000600001.pdf.txt: 179481 bytes, checksum: 49f2bb8910ccb63dffa8cd07f4b9798b (MD5) Made available in DSpace on 2014-05-20T13:54:59Z (GMT). No. of bitstreams: 2 S0074-02762005000600001.pdf: 2193761 bytes, checksum: d66ab3ce25c0d2df6bd09d883eb0e4cf (MD5) S0074-02762005000600001.pdf.txt: 179481 bytes, checksum: 49f2bb8910ccb63dffa8cd07f4b9798b (MD5) Previous issue date: 2005-10-01 The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity. Pontifícia Universidade Católica do Rio Grande do Sul Faculdade de Biociências Centro de Pesquisas em Medicina Tropical UFRGS Centro de Biotecnologia Laboratório de Fisiologia Vegetal UFCe Departamento de Química Orgânica e Inorgânica Unesp Laboratório de Biologia Estrutural e Zooquímica UFSC Departamento de Farmacologia Universidade do Amazonas Programa de Pós-Graduação em Biotecnologia Fundação Gonçalo Moniz-Fiocruz Pontifícia Universidade Católica do Rio Grande do Sul Faculdade de Farmácia Centro de Pesquisas em Biologia Molecular e Funcional Unesp Laboratório de Biologia Estrutural e Zooquímica

Reperfusion of an ischaemic tissue is associated with an intense inflammatory response and inflammation-mediated tissue injury. Physalins, a group of substances with secosteroidal chemical structure, are found in Physalis angulata stems and leaves. Here, we assessed the effects of physalins on the local, remote and systemic injuries following intestinal ischaemia and reperfusion (I/R) in mice and compared with the effects of dexamethasone. Following I/R injury, dexamethasone (10 mg kg(-1)) or physalin B or F markedly prevented neutrophil influx, the increase in vascular permeability in the intestine and the lungs. Maximal inhibition occurred at 20 mg kg(-1). Moreover, there was prevention of haemorrhage in the intestine of reperfused animals. Dexamethasone or physalins effectively suppressed the increase in tissue (intestine and lungs) and serum concentrations of TNF-alpha. Interestingly, treatment with the compounds was associated with enhancement of IL-10. The anti-inflammatory effects of dexamethasone or physalins were reversed by pretreatment with the corticoid receptor antagonist RU486 (25 mg kg(-1)). The drug compounds suppressed steady-state concentrations of corticosterone, but did not alter the reperfusion-associated increase in levels of corticosterone. The IL-10-enhancing effects of the drugs were not altered by RU486. In conclusion, the in vivo anti-inflammatory actions of physalins, natural steroidal compounds, appear to be mostly due to the activation of glucocorticoid receptors. Compounds derived from these natural secosteroids may represent novel therapeutic options for the treatment of inflammatory diseases.

A cardiopatia chagásica crônica é ainda uma das maiores causas de óbito por insuficiência cardíaca na América Latina e para a qual não há nenhum tratamento eficaz até o momento. Enquanto a população de indivíduos com doença de Chagas aguarda o desenvolvimento de novos quimioterápicos mais eficientes e de menor toxicidade para a eliminação do Trypanosoma cruzi, uma nova estratégia surgiu na tentativa de reparar ou diminuir os danos causados ao miocárdio de pacientes com forma crônica cardíaca. Trata-se do transplante de células de medula óssea obtidas do próprio indivíduo a ser tratado, que pode levar à melhora funcional e da qualidade de vida dos pacientes, como ocorreu em estudos utilizando esta abordagem para o tratamento de pacientes com insuficiência cardíaca de etiologia isquêmica. Os possíveis efeitos de terapias celulares e sua utilização em pacientes cardiopatas chagásicos crônicos são discutidos no presente artigo.Chronic chagasic cardiomyopathy remains a major cause of death due to heart failure in Latin American countries and for which there is currently no effective treatment. While chagasic patients wait for the development of more efficient and less toxic chemotherapeutics for the elimination of Trypanosoma cruzi, a new strategy has appeared in an attempt to repair or ameliorate the damage caused to the myocardium of patients with chronic chagasic cardiomyopathy. This therapy, involving the transplant of bone marrow cells obtained from the patient to be treated, may lead to improvement in heart function and in life quality of patients with severe chagasic cardiopathy, similar to that obtained with this approach in the treatment of patients with heart failure of ischemic etiology. The possible effects of cell therapies and its applications in chronic chagasic cardiopaths are discussed in the present report.

Trypanosoma cruzi is an obligate intracellular protozoan parasite that actively penetrates into non-phagocytic mammalian cells. To accomplish this, the parasite relies on the binding of cell surface ligands. It is reported herein that the T. cruzi trans-sialidase (TS), which is exposed on the parasite surface, binds to mouse heart cells, and should therefore be further studied as a possible cell penetration-related ligand. In addition, as has been proposed elsewhere, the binding of T. cruzi to tissues may turn them into targets for parasite-specific immune reactions. Washed heart sections from T. cruzi-infected mice were subjected to immunoenzymatic staining with antisera against whole T. cruzi and with polyclonal or monoclonal antibodies against TS. The anti-TS antibodies stained both parasites and uninfected heart cells in the vicinity of T. cruzi nest remains/trypomastigotes. On the other hand, an anti-T. cruzi serum, which did not recognize TS, only stained the parasites. In addition, normal heart sections from uninfected nude mice were shown to react with both enzymatically active and inactive recombinant TS molecules, probably through their amino-terminal region, since a recombinant TS lacking this region failed to bind.

Apresentado o primeiro caso de transplante de célula de medula óssea para o miocárdio de um portador de insuficiência cardíaca de etiologia chagásica. Homem, 52 anos, portador de insuficiência cardíaca crônica, em classe funcional III da NYHA, apesar de terapêutica clínica otimizada. Como procedimento, foi aspirado 50 ml de medula óssea através de punção da crista ilíaca, seguidos de filtragem, separação das células mononucleares, ressuspensão e injeção intracoronariana. A fração de ejeção do ventrículo esquerdo em repouso, medida pela ventriculografia radioisotópica com hemácias marcadas, antes do transplante, era de 24% e, após 30 dias, aumentou para 32% sem alterar o esquema medicamentoso. Foram avaliados, antes e 30 dias após o procedimento, respectivamente, o diâmetro diastólico final do ventrículo esquerdo (82 mm; 76 mm); escore de qualidade de vida de Minnesota (55; 06); distância caminhada no teste de 6min (513 m; 683 m). Achados demonstraram ser possível realizar a injeção intracoronariana de célula de medula óssea, sugerindo que este procedimento é potencialmente seguro e efetivo em pacientes com insuficiência cardíaca chagásica.We report the first case of bone marrow cell transplantation to the myocardium of a patient with heart failure due to chagas' disease. The patient is a 52-year-old man with chronic heart failure, NYHA functional class III, despite the optimized clinical therapy. The procedure consisted of aspiration of 50 mL of bone marrow through puncture of the iliac crest, followed by filtration, separation of the mononuclear cells, resuspension, and intracoronary injection. The left ventricular ejection fraction at rest, measured using radionuclide ventriculography with labeled red blood cells prior to transplantation, was 24%, and, after 30 days, it increased to 32% with no change in the medicamentous schedule. The following measurements were assessed before and 30 days after transplantation: left ventricular end diastolic diameter (82 mm and 76 mm, respectively); Minnesota living with heart failure questionaire score (55 and 06, respectively); and distance walked in the 6-minute walking test (513 m and 683 m, respectively). Our findings show that intracoronary injection of bone marrow cells may be performed, suggesting that this is a potentially safe and effective procedure in patients with due to Chagas' disease heart failure.

The protozoan Trypanosoma cruzi causes chronic Chagas’ disease myocarditis (CCDM) in infected mammals. The pathogenesis of CCDM, however, is still unclear. Indirect evidence for either parasite- or heart-specific immune responses playing a pathogenic role is available. In this work, the participation of autoimmunity in the development of CCDM is demonstrated in mice in which immunological tolerance to heart antigens was induced or strengthened prior to their infection by T. cruzi. Tolerance was induced by heart antigen administration in the presence of complete Freund’s adjuvant and anti-CD4 antibodies. Tolerized mice developed less intense CCDM than control nontolerized animals that had received only anti-CD4 and adjuvant. This result confirms the important notion that tolerance to self, and in particular to heart antigens, may be reinforced/induced in normal animals, and raises the possibility that analogous interventions may prevent the development of CCDM in millions of T. cruzi-infected human beings.

The administration of stem cells holds promise as a potential therapy for spinal cord injury (SCI). Mesenchymal stem cells have advantages for clinical applications, since they can be easily obtained, are suitable for autologous transplantation and have been previously shown to induce regeneration of the spinal cord in experimental settings. Here we evaluated the feasibility, safety and potential efficacy of autologous transplantation of mesenchymal stem cells in subjects with chronic complete SCI. We conducted a phase I, non-controlled study in 14 subjects of both genders aging between 18 to 65 years, with chronic traumatic SCI (>6 months), at thoracic or lumbar levels, classified as American Spinal Injury Association (ASIA) A - complete injury. Baseline somatosensory evoked potentials (SSEP), spinal magnetic resonance imaging (MRI) and urodynamics were assessed before and after treatment. Pain rating was performed using the McGill Pain Questionnaire and a visual analogue score scale. Bone marrow-derived mesenchymal stem cells were cultured and characterized by flow cytometry, cell differentiation assays and G-band karyotyping. Mesenchymal stem cells were injected directly into the lesion following laminectomy and durotomy. Cell transplantation was an overall safe and well-tolerated procedure. All subjects displayed variable improvements in tactile sensitivity and eight subjects developed lower limbs motor functional gains, principally in the hip flexors. Seven subjects presented sacral sparing and improved American Spinal Injury Association impairment scale (AIS) grades to B or C – incomplete injury. Nine subjects had improvements in urologic function. One subject presented changes in SSEP 3 and 6 months after mesenchymal stem cells transplantation. Statistically significant correlations between the improvements in neurological function and both injury size and level were found. Intralesional transplantation of autologous mesenchymal stem cells in subjects with chronic, complete spinal cord injury is safe, feasible, and may promote neurological improvements. ClinicalTrials.gov NCT01325103 – Registered 28 March 2011

Stem cells are being investigated by their potential use in regenerative medicine. There is currently a lack of effective clinical therapy for a number conditions, such as severe spinal cord injury (SCI). SCI can lead to chronic paraplegia, considered an irreversible condition. The administration of stem cells has been tested as a potential therapy for SCI. In the present study we evaluated the feasibility, safety and potential efficacy of autologous mesenchymal stem cells (MSC) transplantation in patients with chronic complete SCI. We conducted a phase I/II, non-controlled study in 14 patients of both genders aging between 18-65 years, with ASIA A classified chronic traumatic SCI. Baseline somatosensory evoked potentials (SSEP), spinal magnetic resonance imaging (MRI) and urodynamics were assessed before and after treatment. Pain rating was performed using the McGill Pain Questionnaire and a visual analogue score scale. Bone marrow-derived MSC were cultured and characterized by flow cytometry, cell differentiation assays and G-band karyotyping, showing morphological and phenotypic characteristics of MSC, as well as genetic stability. MSC were injected directly into the lesion following laminectomy and durotomy. Transplantation of bone marrow derived MSC was an overall safe procedure. All of the patients were discharged within 48 hours after surgery. Only one patient developed a post-operatory complication, evolving a liquoric fistula that was treated by an additional surgical procedure. Nine patients had improvements in urologic function. All patients displayed variable improvements in sensitivity and 8 patients developed lower limbs motor functional gains, principally in the hip flexors. All patients had variable improvements in ASIA global scores. Additionally, 6 patients had changes in the AIS score to grade B and one to grade C. A direct correlation between motor gain and lesion level and an inverse correlation between light touch gain and lesion volume were found 6 months after transplantation. One patient presented changes in SSEP 3 and 6 months after MSC transplantation. Although there is an abundance of studies describing the natural history of neurologic functional gains during the first year post-SCI, there is a lack of data regarding the degree of neurological recovery that may naturally occur during prolonged follow up investigation. In the present study, we enrolled only patients with chronic and complete spinal cord injury (ASIA A) whom had previously been subjected to decompressive surgery and lengthy rehabilitation protocols without acquiring significant motor or sensory gains. Although this was not a controlled study, based on the patient profiles and the expected spontaneous gains, our study showed potential benefits of MSC transplantation treatment, in variable degrees of motor and sensory improvements, clinical pain measures and urodynamics parameters. The use of MSC presents several advantages, such as isolation from bone marrow aspirates and large-scale expansion in cell culture procedures, which are feasible to be performed in autologous transplantations. We conclude that intralesional transplantation of autologous MSC in spinal cord injury patients is safe and feasible, and may provide some progressive benefits. We are currently beginning new clinical trials based on MSC therapy for SCI.

Chagas' disease is a major health problem in Latin America, where it constitutes one of the leading causes of heart failure. About one fourth of Trypanosoma cruzi-infected individuals develop chronic chagasic cardiomyopathy (CChC), the most severe form of the disease. CChC is histologically characterized by the presence of multifocal inflammatory infiltrates in the heart, composed mainly by mononuclear cells, usually adhered to myocytes and leading to myocytolysis, and frequently by interstitial fibrosis. The pathogenesis of CChC is still unclear, despite intense investigations both in human beings and in animal models of the disease. Although tissue parasitism is rare in the chronic phase of infection, an immune response targeted to persistent parasites or parasite antigens is suggested, by some authors, as the pathogenic mechanism of CChC. Other researchers affirm that the lack of correlation between tissue parasitism and intensity of inflammation suggests, along with the presence of autoreactive immune responses, that CChC results from the action of an autoimmune response. Herein we review reports from the literature and our own data, which together indicate, on one hand, the participation of parasite-specific immune responses and, on the other hand, clearly demonstrate the participation of heart-specific immune responses in the pathogenesis of CChC. Moreover, multiple factors may determine whether an individual in the indeterminate form of the disease will develop CChC. The mechanisms by which T. cruzi breaks immunological tolerance to heart antigens are also discussed.A doença de Chagas constitui um grave problema de saúde pública na América Latina, onde é uma das principais causas de problemas cardíacos. A cardiopatia chagásica crônica (CChC), forma mais grave da doença, manifesta-se em cerca de 25% dos indivíduos infectados pelo Trypanosoma cruzi, e é caracterizada, a nível histopatológico, pela presença de infiltrados multifocais de células mononucleares, freqüentemente aderidas a miócitos e induzindo miocitólise, sendo também bastante comum o achado de fibrose intersticial. Embora a doença tenha sido intensamente estudada, tanto em seres humanos como em modelos animais, o mecanismo de patogênese da CChC ainda é discutido. Apesar da escassez de parasitas na fase crônica da doença, uma possível resposta imune contra os parasitas persistentes seria, para alguns, o mecanismo de patogênese da CChC. Por outro lado, a falta de correlação entre o parasitismo tecidual e intensidade de inflamação sugere, juntamente com a presença de respostas autoreativas, que a CChC resulte da ação de um mecanismo autoimune. Neste artigo, revisamos dados da literatura e dados obtidos em nossos laboratórios, que sugerem a participação de mecanismos efetores ativados em resposta a antígenos parasitários e demonstram um papel de respostas autoreativas contra antígenos cardíacos na patogênese da CChC. Múltiplos fatores devem contribuir para a progressão da forma indeterminada para a CChC. Os possíveis mecanismos ativados pela infecção por T. cruzi que levam à quebra de tolerância imunológica a antígenos cardíacos são discutidos.

Chronic chagasic cardiomyopathy (CChC) is characterized by an inflammatory reaction which may eventually lead to heart enlargement, arrythmia, and death. As described herein, interleukin-4-deficient mice mount increased specific T helper (Th) 1 immune responses when infected with Trypanosoma cruzi, as compared to wild-type mice. Interestingly, these mice had reduced parasitism and mortality and exacerbated inflammation in their hearts, demonstrating a clear dissociation between inflammation and parasite load. The modulation of these phenomena so as to maximize host and parasite survivals may depend on a fine balance between Th responses, in which a Th1 response will, on one hand, control parasitism and, on the other hand, enhance heart inflammation throughout the course of the infection.

To study the role of autoreactive T cells in the pathogenesis of cardiomyopathy in Chagas' disease, we generated a cell line by repeated in vitro antigenic stimulation of purified splenic CD4+ T lymphocytes from a chronically Trypanosoma cruzi-infected mouse. Cells from this line were confirmed to be CD4+ CD8- and proliferated upon stimulation with soluble heart antigens from different animal species, as well as with T. cruzi antigen, in the presence of syngeneic feeder cells. In vitro antigen stimulation of the cell line produced a Th1 cytokine profile, with high levels of IFNgamma and IL-2 and absence of IL-4, IL-5 and IL-10. The cell line also terminated the beating of fetal heart clusters in vitro when cocultured with irradiated syngeneic normal spleen cells. In situ injection of the cell line into well established heart transplants also induced the cessation of heart beating. Finally, adoptive transfer of the cell line to heart-immunized or T. cruzi-infected BALB/c nude mice caused intense heart inflammation.

The mechanisms by which Trypanosoma cruzi causes cardiomyopathy and induces neuronal destruction are discussed in this paper. The results suggest that autoimmunity in the chronic phase is the main cause of the progressive cardiac destruction, and that autoreactivity is restricted to the CD4+ T cell compartment. During the acute phase, the neuronal and cardiac fiber destruction occurs when ruptured parasite nests release T. cruzi antigens that bind to the cell surface in the vicinity which become targets for the cellular and humoral immune response against T. cruzi. The various factors involved in the genesis of autoimmunity in chronic T. cruzi infection include molecular mimicry, presentation of self-antigens and imbalance of immune regulation.

Uma paciente com síndrome de imunodeficiência adquirida (SIDA) e doença de Chagas, com xenodiagnóstico positivo, estava em uso prolongado de cetoconazol com o objetivo de suprimir a parasitemia e prevenir a reativação da doença de Chagas. O cetoconazol foi suspenso inadvertidamente após 6 meses de uso. Um mês após, a paciente foi internada com febre, cefaléia, vômitos, taquicardia e hepatoesplenomegalia. Tanto o xenodiagnóstico como o exame de sangue a fresco demonstraram a presença de Trypanosoma cruzi. O tratamento com benzonidazol foi instituído, com supressão da parasitemia. A paciente desenvolveu concomitantemente uma provável neurotoxoplasmose, evoluindo para o óbito em septicemia. À necropsia, não foram encontrados parasitas.

Summary Neonates and young C3H/HeJ mice were highly susceptibleto lethal infection with Leptospira interrogans serovar icterohaemorrhagiae. The main pathological changes were seen by light microscopy in the lung and kidneys of 3-week old mice at 11 days after inoculation. Lung histological lesions included small and medium-sized vasculitis with fibrinoid changes, hemorrhages, moderate infiltrate of mononuclear inflammatory cells and fibrin thrombi. In the kidney there was mild to severe acute tubular necrosis associated with interstitial nephritis. Repair of damaged tubules in surviving mice was observed within 17 days after inoculation. Pathological findings of CD4+ and CD8+ cell-depleted mice were clearly more severe than that seen in untreated animals by 17 days after inoculation. Comparatively, CD4+/CD8+ cell-depleted mice had more marked lung and kidney lesions than in the CD8+ or CD4+ cell-depleted mice. A very high level of tubular alterations was seen in the kidneys of all treated groups. Increased degrees of interstitial nephritis also reflected the T-cell subsets depletion related events. Leptospires were clearly demonstrated by immunoperoxi dase close to the sites of histological damage in all infected mice. C3H/HeJ mice represent a useful model for further studies in pathogenicity of leptospires and natural resistance of the host.

The use of stem cells in injury repair has been extensively investigated. Here, we examined the therapeutic effects of autologous bone marrow mesenchymal stem cells (MSC) transplantation in four dogs with natural traumatic spinal cord injuries. MSC were culturedin vitro, and proliferation rate and cell viability were evaluated. Cell suspensions were prepared and surgically administered into the spinal cord. The animals were clinically evaluated and examined by nuclear magnetic resonance. Ten days after the surgical procedure and MSC transplantation, we observed a progressive recovery of the panniculus reflex and diminished superficial and deep pain response, although there were still low proprioceptive reflexes in addition to a hyperreflex in the ataxic hind limb movement responses. Each dog demonstrated an improvement in these gains over time. Conscious reflex recovery occurred simultaneously with moderate improvement in intestine and urinary bladder functions in two of the four dogs. By the 18th month of clinical monitoring, we observed a remarkable clinical amelioration accompanied by improved movement, in three of the four dogs. However, no clinical gain was associated with alterations in magnetic resonance imaging. Our results indicate that MSC are potential candidates for the stem cell therapy following spinal cord injury.

Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Engenharia Tecidual e Imunofarmacologia. Salvador, BA, Brasil Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Engenharia Tecidual e Imunofarmacologia. Salvador, BA, Brasil Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Engenharia Tecidual e Imunofarmacologia. Salvador, BA, Brasil Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Engenharia Tecidual e Imunofarmacologia. Salvador, BA, Brasil Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Engenharia Tecidual e Imunofarmacologia. Salvador, BA, Brasil Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil INTRODUCTION: New therapeutic options are necessary for patients with chronic Chagas disease, a leading cause of heart failure in Latin American countries. Stem cell therapy focused on improving cardiac function is a promising approach for treating heart disease. Here, we evaluated the therapeutic effects of cardiac mesenchymal stem cells (CMSCs) in a mouse model of chronic Chagas disease. METHODS: CMSCs were isolated from green fluorescent protein (GFP) transgenic C57BL/6 mouse hearts and tested for adipogenic, osteogenic, chondrogenic, endothelial, and cardiogenic differentiation potentials evaluated by histochemical and immunofluorescence techniques. A lymphoproliferation assay was performed to evaluate the immunomodulatory activity of CMSCs. To investigate the therapeutic potential of CMSCs, C57BL/6 mice chronically infected with Trypanosoma cruzi were treated with 106 CMSCs or saline (control) by echocardiography-guided injection into the left ventricle wall. All animals were submitted to cardiac histopathological and immunofluorescence analysis in heart sections from chagasic mice. Analysis by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed in the heart to evaluate the expression of cytokines involved in the inflammatory response. RESULTS: CMSCs demonstrated adipogenic, osteogenic, and chondrogenic differentiation potentials. Moreover, these cells expressed endothelial cell and cardiomyocyte features upon defined stimulation culture conditions and displayed immunosuppressive activity in vitro. After intramyocardial injection, GFP+ CMSCs were observed in heart sections of chagasic mice one week later; however, no observed GFP+ cells co-expressed troponin T or connexin-43. Histopathological analysis revealed that CMSC-treated mice had a significantly decreased number of inflammatory cells, but no reduction in fibrotic area, two months after treatment. Analysis by qRT-PCR demonstrated that cell therapy significantly decreased tumor necrosis factor-alpha expression and increased transforming growth factor-beta in heart samples. CONCLUSIONS: We conclude that the CMSCs exert a protective effect in chronic chagasic cardiomyopathy primarily through immunomodulation.

Glycolipoprotein (GLP) cytotoxin was extracted from Leptospira interrogans serovar canicola. The silver staining profile of GLP subjected to SDS-PAGE under denaturing conditions showed a number of bands in the mol. weight range of 14-66 kDa. Mouse Monoclonal Antibodies (MAbs) IgG3 recognizing a band near to 24 kDa of leptospiral GLP were produced (clone number MGLP-01). The agglutinating property of MAbs was established by microscopic agglutination test (MAT) using 25 different serovars as antigens. Only the homologous serovar was agglutinated by MAbs suggesting that the recognized epitope is a specific surface-exposed antigen. The MAbs were applied to demonstration of leptospiral antigens in tissue damage by avidin-biotin immunoperoxidase staining. Golden hamsters were experimentally infected with a virulent strain of L. interrogans serovar canicola. Histologically kidneys stained by routine hematoxylin and eosin showed changes characterized by injury of tubular epithelial cells leading to acute tubular necrosis (ATN). Typical, well-defined morphologic leptospires or finely granular deposits were found by immunoperoxidase staining near to blood vessels, within inflammatory infiltrates and intraluminal in proximal and distal parts of the nephron. Binding of leptospiral antigens to capillary endothelial cells, tubular epithelial cells and macrophages were also demonstrated. This entails a basis for further studies either in research or in diagnostic histopathology.

SUMMARYWe previously observed that physalins have immunomodulatory properties, as well as antileishmanial and antiplasmodial activities. Here, we investigated the anti-Trypanosoma cruziactivity of physalins B, D, F and G. We found that physalins B and F were the most potent compounds against trypomastigote and epimastigote forms ofT. cruzi. Electron microscopy of trypomastigotes incubated with physalin B showed disruption of kinetoplast, alterations in Golgi apparatus and endoplasmic reticulum, followed by the formation of myelin-like figures, which were stained with MDC to confirm their autophagic vacuole identity. Physalin B-mediated alteration in Golgi apparatus was likely due toT. cruziprotease perturbation; however physalins did not inhibit activity of the trypanosomal protease cruzain. Flow cytometry examination showed that cell death is mainly caused by necrosis. Treatment with physalins reduced the invasion process, as well as intracellular parasite development in macrophage cell culture, with a potency similar to benznidazole. We observed that a combination of physalins and benznidazole has a greater anti-T. cruziactivity than when compounds were used alone. These results indicate that physalins, specifically B and F, are potent and selective trypanocidal agents. They cause structural alterations and induce autophagy, which ultimately lead to parasite cell death by a necrotic process.

Emphysema is a chronic obstructive pulmonary disease characterized abnormal dilatation of alveolar spaces, which impairs alveolar gas exchange, compromising the physical capacity of a patient due to airflow limitations. Here we tested the effects of G-CSF administration in pulmonary tissue and exercise capacity in emphysematous mice. C57Bl/6 female mice were treated with elastase intratracheally to induce emphysema. Their exercise capacities were evaluated in a treadmill. Lung histological sections were prepared to evaluate mean linear intercept measurement. Emphysematous mice were treated with G-CSF (3 cycles of 200 mg/kg/day for 5 consecutive days, with 7-day intervals) or saline and submitted to a third evaluation 8 weeks after treatment. Values of run distance and linear intercept measurement were expressed as mean SD and compared applying a paired t-test. Effects of treatment on these parameters were analyzed applying a Repeated Measures ANOVA, followed by Tukey’s post hoc analysis. p < 0.05 was considered statistically significant. Twenty eight days later, animals ran significantly less in a treadmill compared to normal mice (549.7 181.2 m and 821.7 131.3 m, respectively; p < 0.01). Treatment with G-CSF significantly increased the exercise capacity of emphysematous mice (719.6 200.5 m), whereas saline treatment had no effect on distance run (595.8 178.5 m). The PCR cytokines genes analysis did not detect difference between experimental groups. Morphometric analyses in the lung showed that saline-treated mice had a mean linear intercept significantly higher (p < 0.01) when compared to mice treated with G-CSF, which did not significantly differ from that of normal mice. Treatment with G-CSF promoted the recovery of exercise capacity and regeneration of alveolar structural alterations in emphysematous mice.

Stem cell-based therapy has been investigated in a number of degenerative and traumatic diseases, including spinal cord injury. In the present study, we investigated the use of autologous mesenchymal stem cells in the functional rehabilitation of a domestic cat presenting a compressive L1-L5 fracture. Bone marrow cells collected by puncture of the iliac crest were cultured to obtain mesenchymal stem cells three weeks before surgery. Hemilaminectomy was performed, followed by injection of the mesenchymal stem cells in the injured area. Clinical evaluation of the animal prior to surgery showed absence of pain, muscular tonus, and panniculi reflexes. Seven days after surgery and cell transplantation the examination revealed a progressive recovery of the panniculus reflexes and of the responses to superficial and deep pain stimuli despite the low proprioceptive and hyperreflexic ataxic hind limbs. Physiotherapy protocols were applied for clinical rehabilitation after surgery. The cat’s first steps, three-minute weight-bearing, and intestine and urinary bladder partial reestablishment were observed 75 days post-surgery. Our results indicate the therapeutic potential of mesenchymal stem cells in chronic spinal cord injuries.

In this study we investigated the involvement of inflammatory cells in the pleural accumulation of eosinophils induced by lipopolysaccharide (LPS). Intrathoracic (i.t.) injection of LPS (250 ng/cavity) into rats induced a significant eosinophil accumulation that developed within 24 h, was maximal at 48 h, and returned to control values within 120 h. This eosinophil influx was preceded by a huge neutrophil influx within 4 h and accompanied by a mononuclear cell accumulation between 24 and 48 h. Pretreatment with an antineutrophil monoclonal antibody (RP-3, 2 ml per animal) selectively reduced the number of circulating neutrophils within 8 h but failed to alter the LPS-induced eosinophilia. Similarly, platelet depletion with an anti-rat platelet antiserum did not alter the LPS-induced eosinophil accumulation. Cyclosporine (50 mg/kg, 12 and 2 h before) partially inhibited (51%) the LPS-induced pleural eosinophilia, whereas the eosinophilia was not changed by prior degranulation of pleural mast cells with polymyxin B (10 μg/cavity, 24 h before). Moreover, selective depletion of T lymphocytes using an anti-Thy 1.0 monoclonal antibody significantly inhibited the eosinophilia triggered by LPS. The i.t. injection of liposomes containing dichloromethylene diphosphonate significantly reduced (65%) the number of resident macrophages after 5 days. Under this condition, the eosinophil infiltration induced by LPS was completely inhibited. Accordingly, the i.t. injection of supernatant from macrophage monolayers, obtained from the pleural cavities of LPS-injected rats, into naive recipient animals led to a twofold increase in the number of pleural eosinophils. In conclusion, our data suggest an important role for resident macrophages and T lymphocytes in the eosinophil accumulation induced by LPS. J. Leukoc. Biol. 56: 151–158; 1994.

Cirrhosis, end-stage liver disease, is caused by different mechanisms of injury, associated with persistent inflammation. Galectin-3 is an important regulator of fibrosis that links chronic inflammation to fibrogenesis. We investigated the role of bone marrow cell (BMC) transplantation in chronic inflammation and hepatic fibrosis.Liver cirrhosis was induced by administration of carbon tetrachloride and ethanol to wild-type C57BL/6 or bone marrow chimeric mice. Bone marrow chimeras were generated by lethal irradiation and transplantation with BMC obtained from green fluorescent protein (GFP(+) )donors. Wild-type cirrhotic mice were transplanted with BMC without irradiation. Livers from chimeras and cirrhotic transplanted mice were obtained for evaluation of inflammation, fibrosis and regulatory factors [galectin-3, matrix metallopeptidase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1 and transforming growth factor (TGF)-β].The development of cirrhosis was associated with increased expression of galectin-3 by F4/80(+) cells and intense migration of BMC to the liver. Furthermore, when transplanted after the establishment of cirrhosis, BMC also migrated to the liver and localized within the fibrous septa. Two months after BMC therapy, cirrhotic mice had a significant reduction in liver fibrosis and expression of type I collagen. We did not find any difference in levels of TGF-β, TIMP-1 and MMP-9 between saline and BMC groups. However, the numbers of inflammatory cells, phagocytes and galectin-3(+) cells were markedly lower in the livers of cirrhotic mice treated with BMC.Our results demonstrate an important role for BMC in the regulation of liver fibrosis and that transplantation of BMC can accelerate fibrosis regression through modulatory mechanisms.

Recent advances in image technology, including significant gains in spatial resolution, have made realtime sequential ovarian evaluations possible in small rodents, allowing longitudinal (continued) studies of the ovarian cycle and reducing the required number of experimental animals. The aim of this study was to evaluate exogenous stimulated follicular growth in mice using high-resolution ultrasound technology. Female mice (n = 15) received a 5 IU intraperitoneal injection of equine chorionic gonadotropin (eCG) and 48 h later a 5 IU injection of human chorionic gonadotropin (hCG), and were allowed to mate thereafter. In experiment 1, animals (n = 7) were evaluated every 6 h, from 3 to 51 h after eCG injection, with an ultrasound biomicroscopy (UBM) equipped with a realtime 45 MHz microvisualization probe (RMV 707b). The ovaries were identified and follicular population quantified, and follicles were classified according to the diameter as small (≤449 µm) or large (≥450 µm). A significant change in the distribution of follicle population according to category was observed only 45 h after eCG injection (P < 0.05). In experiment 2, animals (n = 8) were evaluated every 2 h, from 2 h to 10 h after hCG treatment. The largest follicles reached a maximum size (596.7 ± 106.0 µm) 5.8 ± 2.3 h after hCG injection. As expected, the population of large follicles decreased thereafter, indicating the progress of ovulations, but large follicles were still detected late after treatment (10.1 ± 1.1 h). In conclusion, UBM can be used to evaluate follicle dynamics in superstimulated mice (C57BL/6 and BALB/c); significant changes in follicle distribution only occur at later stages after eCG stimulation; and hCG-induced ovulations may not occur synchronously in mice.

FUNDAMENTO: Insuficiência cardíaca (IC) causada por Doença de Chagas (DC) é uma cardiomiopatia inflamatória progressiva que afeta milhões de pessoas na América Latina. Estudos com modelos de camundongo de IC devido à DC indicam que o transplante de células mononucleares derivadas da medula óssea (TCDMO) pode reduzir a inflamação, fibrose e melhorar a função miocárdica. OBJETIVO: O propósito desse estudo foi avaliar, pela primeira vez em seres humanos, a segurança e a eficácia de TCDMO no miocárdio de pacientes com IC devido à DC. MÉTODOS: Um total de 28 pacientes com IC devido à DC (média de idade de 52,2 ± 9,9 anos) com classe funcional NYHA III e IV foram submetidos à TCDMO através de injeção coronariana. Os efeitos na fração de ejeção do ventrículo esquerdo (FEVE), capacidade funcional, qualidade de vida, arritmias e parâmetros bioquímicos, imunológicos e neuro-humorais foram avaliados. RESULTADOS: Não houve complicações diretamente relacionadas ao procedimento. A FEVE foi 20,1 ± 6,8% e 28,3 ± 7,9%, p < 0,03 a nível basal e 180 dias após o procedimento, respectivamente. No mesmo período, melhoras significantes foram observadas na classe funcional NYHA (3,1 ± 0,3 para 1,8 ± 0,5; p < 0,001), qualidade de vida (50,9 ± 11,7 para 25,1 ± 15,9; p < 0,001), e no teste de caminhada de seis minutos (355 ± 136 m para 437 ± 94 m; p < 0,01). Não houve alterações nos marcadores de ativação imune ou neurohormonais. Nenhuma complicação foi registrada. CONCLUSÃO: Nossos dados sugerem que a injeção intracoronariana de células derivadas da medula óssea é segura e potencialmente efetiva em pacientes com IC devido à DC. A extensão do benefício, entretanto, parece ser discreta e precisa ser confirmada em estudos clínicos maiores, randomizados, duplo-cegos, controlados com placebo. BACKGROUND: Heart failure due to Chagas' disease (HFCD) is a progressive inflammatory cardiomyopathy that affects millions of individuals in Latin America. Studies using mice models of HFCD indicate that bone marrow mononuclear cell transplantation (BMCT) may reduce inflammation, fibrosis, and improve myocardial function. OBJECTIVE: The purpose of this study was to evaluate, for the first time in humans, the safety and efficacy of BMCT to the myocardium of patients with HFCD. METHODS: A total of 28 HFCD patients (mean age 52.2 ± 9.9 years) with NYHA class III and IV were submitted to BMCT through intracoronary injection. Effects on the left ventricle ejection fraction (LVEF), functional capacity, quality-of-life, arrhythmias, biochemical, immunological, and neuro-humoral parameters, were evaluated. RESULTS: There were no complications directly related to the procedure. LVEF was 20.1 ± 6.8% and 28.3 ± 7.9%, p < 0.03 at baseline and 180 days after the procedure, respectively. In the same period, significant improvements were observed in the NYHA class (3.1 ± 0.3 to 1.8 ± 0.5; p < 0.001), quality-of-life (50.9 ± 11.7 to 25.1 ± 15.9; p < 0.001), and in the six-minute walking test (355 ± 136 m to 437 ± 94 m; p < 0,01). There were no changes in markers of immune or neurohormonal activation. No complications were registered. CONCLUSION: Our data suggest that the intracoronary injection of BMCT is safe and potentially effective in patients with HFCD. The extent of the benefit, however, appears to be small and needs to be confirmed in a larger randomized, double blind, placebo controlled clinical trial. FUNDAMENTO: La insuficiencia cardíaca (IC), causada por la enfermedad de Chagas (EC), es una cardiomiopatía inflamatoria progresiva que afecta a millones de personas en Latinoamérica. Estudios con modelos experimentales de IC en razón de la EC, nos indican que el transplante de células mononucleares derivadas de la médula ósea (TCMO), puede reducir la inflamación y la fibrosis, mejorando así la función miocárdica. OBJETIVO:El objetivo de este estudio fue evaluar, por primera vez en seres humanos, la seguridad y la eficacia del TCMO en el miocardio de pacientes con IC debido a la EC. MÉTODOS:Fueron estudiados un total de 28 pacientes con IC debido a la EC (con edad promedio 52,2 ± 9,9 años), en clases funcionales III y IV (NYHA), al TCMO por medio de una inyección coronaria. Se evaluaron los efectos en la fracción de eyección del ventrículo izquierdo (FEVI), capacidad funcional, calidad de vida, arritmias y parámetros bioquímicos, inmunológicos y neurohumorales. RESULTADOS:No se registraron complicaciones relacionadas directamente con el procedimiento. La FEVI pasó de 20,1 ± 6,8% para 28,3 ± 7,9%, p < 0,03, cuando se comparó con el período basal y 180 días después del procedimiento, respectivamente. En el mismo período, también se observaron mejorías en la clase funcional NYHA promedio (3,1 ± 0,3 para 1,8 ± 0,5; p < 0,001), puntuación de calidad de vida de Minnesota (50,9 ± 11,7 para 25,1 ± 15,9; p < 0,001), y en el test de esfuerzo de seis minutos (355 ± 136 m para 437 ± 94 m; p < 0,01). No hubo alteraciones en los marcadores de activación inflamatoria o neurohormonales. Ninguna complicación fue registrada. CONCLUSIÓN:Nuestros datos sugieren que la inyección intracoronaria de las células derivadas de la médula ósea es segura y potencialmente efectiva en pacientes con IC debido a la EC. La extensión del beneficio, sin embargo, parece ser discreta, y necesita ser confirmada en los ensayos clínicos randomizados, doble ciegos, controlados con placebo.

The authors have previously shown that epithelioid cells isolated from mice secrete a factor, called macrophage deactivating factor (MDF), that promptly deactivates superoxide release by activated macrophages and neutrophils. In this paper some biological properties of a polyclonal rat antiserum directed to MDF and other substances secreted by these cells are described. The immunoglobulin fraction of this antiserum reacted, by immunocytochemical methods, with epitopes in the cell membrane of macrophages adherent to coverslips subcutaneously implanted for 14 days; but not for 5 days. It also reacted with antigens within and outside cells in BCG-induced granulomas. This antiserum blocked completely the macrophage deactivating activity of epithelioid cell culture supernatants. Anti-IL-10 monoclonal antibody, did not block MDF activity. The administration of the immunoglobulin fraction from immunized rats to C5deficient mice bearing BCG-induced granulomatas in the footpad, significantly reduced the size of the lesions. A marked necrosis of inflammatory cells and mononuclear cells phagocyting debris of necrotic cells were observed in these lesions.

Objective: Several studies have demonstrated that human dental pulp is a source of mesenchymal stem cells. To better understand the biological properties of these cells we isolated and characterized stem cells from the dental pulp of EGFP transgenic mice. Methods: The pulp tissue was gently separated from the roots of teeth extracted from C57BL/ 6 mice, and cultured under appropriate conditions. Flow cytometry, RT-PCR, light microscopy (staining for alkaline phosphatase) and immunofluorescence were used to investigate the expression of stem cell markers. The presence of chromosomal abnormalities was evaluated by G banding. Results: The mouse dental pulp stem cells (mDPSC) were highly proliferative, plasticadherent, and exhibited a polymorphic morphology predominantly with stellate or fusiform shapes. The presence of cell clusters was observed in cultures of mDPSC. Some cells were positive for alkaline phosphatase. The karyotype was normal until the 5th passage. The Pou5f1/Oct-4 and ZFP42/Rex-1, but not Nanog transcripts were detected in mDPSC. Flow cytometry and fluorescence analyses revealed the presence of a heterogeneous population positive for embryonic and mesenchymal cell markers. Adipogenic, chondrogenic and osteogenic differentiation was achieved after two weeks of cell culture under chemically defined in vitro conditions. In addition, some elongated cells spontaneously acquired a contraction capacity. Conclusions: Our results reinforce that the dental pulp is an important source of adult stem cells and encourage studies on therapeutic potential of mDPSC in experimental disease models

Rutaceae is a taxon with species very well distributed in Brazilian semi-arid area, commonly used in folk medicine. Species from this genus have diverse biological activity described in literature. In this work, immunomodulatory and bactericidal activity are described for chloroform and ethyl acetate extracts of three of them (Esenbeckia grandiflora Mart., Pilocarpus spicatus A.St.-Hil. and Galipea simplicifolia Schult.). Initially all the samples had their cytotoxicity evaluated, aiming to determine the LC50. The immunomodulatory potential was evaluated in cultures of murine splenocytes stimulated or not with concanavalin A and in a mixed lymphocyte reaction (MLR) using splenocytes from BALB/c (H-2d) mice immunized with splenocytes from C57Bl/6 (H-2b) mice. Four samples had higher values of lymphoproliferation inhibition in concanavalin A-stimulated cultures and were evaluated in MLR. The antibacterial activity of extracts was also evaluated and the minimal inhibitory concentrations (MIC) for two active samples were 1.0 and 5.0 mg/ml for extracts from Esenbeckia grandiflora Mart. and Galipea simplicifolia Schult., respectively. Thus, our results reinforce data of literature relating biological activity for many species of the Rutaceae family and encourage studies with these species aiming to discover active compounds, candidates to new medicines. A família Rutaceae apresenta espécies vegetais muito bem distribuídas no Semi-Árido Brasileiro e comumente usadas em medicina popular. Espécies dessa família tem diversas atividades biológicas descritas na literatura. Neste trabalho, atividades imunomoduladora e bactericida são descritas para o extrato acetato de etila e clorofórmico de três espécies da família (Esenbeckia grandiflora Mart., Pilocarpus spicatus A.St.-Hil. e Galipea simplicifolia Schult.). Todas as amostras foram inicialmente avaliadas quanto à sua citotoxicidade, com objetivo de determinar a LC50. O potencial imunomodulador foi avaliado em culturas de esplenócitos murinos estimulados ou não com concanavalina A e também em reação mista linfocitária (RML), usando também esplenócitos de camundongos BALB/c (H-2d) imunizados com esplenócitos de camundongos C57Bl/6 (H-2b). Quatro amostras tiveram os mais elevados valores percentuais de inibição da proliferação de linfócitos ativados pela concanavalina A e foram avaliados em RML. A atividade antibacteriana dos extratos foi também avaliada e a concentração mínima inibitória (CMI) para duas amostras ativas foi de 1.0 e 5.0 mg/mL, respectivamente para as espécies Esenbeckia grandiflora Mart. and Galipea simplicifolia Schult. Assim, os dados aqui apresentados reforçam informações da literatura científica relacionados à atividade biológica para muitas espécies da família Rutaceae e incentivam outros estudos com estas visando descobrir substâncias ativas, potenciais candidatas a novos fármacos.

Heart failure due to Chagas' disease (HFCD) is a progressive inflammatory cardiomyopathy that affects millions of individuals in Latin America. Studies using mice models of HFCD indicate that bone marrow mononuclear cell transplantation (BMCT) may reduce inflammation, fibrosis, and improve myocardial function.The purpose of this study was to evaluate, for the first time in humans, the safety and efficacy of BMCT to the myocardium of patients with HFCD.A total of 28 HFCD patients (mean age 52.2 ± 9.9 years) with NYHA class III and IV were submitted to BMCT through intracoronary injection. Effects on the left ventricle ejection fraction (LVEF), functional capacity, quality-of-life, arrhythmias, biochemical, immunological, and neuro-humoral parameters, were evaluated.There were no complications directly related to the procedure. LVEF was 20.1 ± 6.8% and 28.3 ± 7.9%, p0.03 at baseline and 180 days after the procedure, respectively. In the same period, significant improvements were observed in the NYHA class (3.1 ± 0.3 to 1.8 ± 0.5; p0.001), quality-of-life (50.9 ± 11.7 to 25.1 ± 15.9; p0.001), and in the six-minute walking test (355 ± 136 m to 437 ± 94 m; p0,01). There were no changes in markers of immune or neurohormonal activation. No complications were registered.Our data suggest that the intracoronary injection of BMCT is safe and potentially effective in patients with HFCD. The extent of the benefit, however, appears to be small and needs to be confirmed in a larger randomized, double blind, placebo controlled clinical trial.

The effects of physalin F (1), a steroid derivative purified from Physalis angulata, were investigated in models of collagen-induced arthritis in DBA/1 mice and allergic airway inflammation in BALB/c mice. Oral treatment with 1 or dexamethasone caused a marked decrease in paw edema and joint inflammation when compared to vehicle-treated arthritic mice. In contrast, treatment with 1 had no effect in mice with allergic airway inflammation caused by ovalbumin immunization, whereas dexamethasone significantly reduced the number of inflammatory cells and eosinophils in the broncoalveolar lavage fluid and in lung sections of challenged mice. To further demonstrate that 1 acts through a mechanism different from that of glucocorticoids, a nuclear translocation assay was performed of the glucocorticoid receptor (GR) using COS-7 cells transfected with a plasmid encoding for a yellow fluorescent protein (YFP)-GR fusion protein. Untreated or treated cells with 1 had YFP staining mainly in the cytoplasm, whereas in dexamethasone-treated cells the YFP staining was concentrated in the nuclei. It is concluded that the mechanism of the immunosuppressive activity of physalin F is distinct from that of the glucocorticoids.

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries. About 30% of Trypanosoma cruzi-infected individuals develop this severe symptomatic form of the disease, characterized by intense inflammatory response accompanied by fibrosis in the heart. We performed an extensive microarray analysis of hearts from a mouse model of this disease and identified significant alterations in expression of approximately 12% of the sampled genes. Extensive up-regulations were associated with immune-inflammatory responses (chemokines, adhesion molecules, cathepsins, and major histocompatibility complex molecules) and fibrosis (extracellular matrix components, lysyl oxidase, and tissue inhibitor of metalloproteinase 1). Our results indicate potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets in chronic Chagas disease.

Objectives In the present study we investigated the antinociceptive, anti-inflammatory and antipyretic effects of 7-hydroxycoumarin (7-HC) in animal models. Methods The effects of oral 7-HC were tested against acetic acid-induced writhing, formalin test, tail flick test, complete Freund's adjuvant (CFA)-induced hypernociception, carrageenan-induced paw oedema, lipopolysaccharide-induced fever and the rota rod test. Key findings 7-HC (3–60 mg/kg) produced a dose-related antinociception against acetic acid-induced writhing in mice and in the formalin test. In contrast, treatment with 7-HC did not prevent thermal nociception in the tail flick test. A single treatment with 7-HC, 60 mg/kg, produced a long-lasting antinociceptive effect against CFA-induced hypernociception, a chronic inflammatory pain stimulus. Notably, at 60 mg/kg per day over 4 days the administration of 7-HC produced a continuous antinociceptive effect against CFA-induced hypernociception. 7-HC (30–120 mg/kg) produced anti-inflammatory and antipyretic effects against carrageenan-induced inflammation and lipopolysaccharide-induced fever, respectively. Moreover, 7-HC was found to be safe with respect to ulcer induction. In the rota rod test, 7-HC-treated mice did not show any motor performance alterations. Conclusions The prolonged antinociceptive and anti-inflammatory effects of 7-HC, in association with its low ulcerogenic activity, indicate that this molecule might be a good candidate for development of new drugs for the control of chronic inflammatory pain and fever.

The distribution of bone marrow cells in brain areas during the acute period after pilocarpine-induced status epiepticus (SE) was investigated here. To achieve this, we generated chimeric mice by engrafting bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic mice. GFP(+) bone marrow-derived cells were found throughout the brain, predominantly in the hippocampus. As expected, these cells exhibited the characteristics of microglia. The pattern of distribution, proliferation, and differentiation of GFP(+)cells changes as a function of intensity and time following SE. This pattern is also a consequence of the inflammatory response, which is followed by the progressive neuronal damage that is characteristic of the pilocarpine model.

International audience; The ferrocenyl diphenol 1 [1,1-bis(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene] displays strong cytotoxic effects against a variety of cancer cells. In the present study we have evaluated the immunomodulatory and antiparasitic activities of compound 1 and its protected dipalmitate analogue 2. We have furthermore compared the antiparasitic results of 1 and 2 with the organic analogue, 3 [1,1-bis(4-hydroxyphenyl)-2-phenyl-but-1-ene], where the ferrocenyl group has been replaced by a phenyl ring. When assayed against normal (noncancerous) splenocytes, all compounds were considered nontoxic. Compound 1 inhibited NO production by macrophages, inhibited concanavalin A-induced lymphoproliferation, and was active against Leishmania amazonensis and Trypanosoma cruzi. Compound 2 had lower activity in all assays performed. Surprisingly, compounds 1 and 2 exhibited similar and significant activity against Plasmodium falciparum, with IC(50) values of 3.50 and 1.33 mu M, respectively. Compound 3 showed an inverse activity profile, being active against T. cruzi but far less active against P. falciparum. Drug Dev Res 71:69-75, 2010. (C) 2009 Wiley-Liss, Inc.

Liver failure is one of the main causes of death worldwide and is a growing health problem. Since the discovery of stem cell populations capable of differentiating into specialized cell types, including hepatocytes, the possibility of their utilization in the regeneration of the damaged liver has been a focus of intense investigation. A variety of cell types were tested both in vitro and in vivo, but the definition of a more suitable cell preparation for therapeutic use in each type of liver lesions is yet to be determined. Here we review the protocols described for differentiation of stem cells into hepatocytes, the results of cell therapy in animal models of liver diseases, as well as the available data of the clinical trials in patients with advanced chronic liver disease.

Chagas disease which is caused by the parasite Trypanosoma cruzi is an important cause of cardiomyopathy in Latin America. In later stages chagasic cardiomyopathy is associated with congestive heart failure which is often refractory to medical therapy. In these individuals heart transplantation has been attempted. However, this procedure is fraught with many problems attributable to the surgery and the postsurgical administration of immunosuppressive drugs. Studies in mice suggest that the transplantation of bone-marrow-derived cells ameliorates the inflammation and fibrosis in the heart associated with this infection. Cardiac magnetic resonance imaging reveals that bone marrow transplantation ameliorates the infection induced right ventricular enlargement. On the basis of these animal studies the safety of autologous bone marrow transplantation has been assessed in patients with chagasic end-stage heart disease. The initial results are encouraging and more studies need to be performed.

The recent highlights on the biochemical pathways of Trypanosoma cruzi have allowed a significant improvement in the development of new strategies for drug design and also in the understanding of the mechanisms of action of new trypanocidal agents. Several biochemical pathways of fundamental importance and validated targets (e.g. cysteine protease cruzain, trypanothione reductase, trans-sialidase) of T. cruzi have proved usefulness for drug development in many examples of new candidates to anti-T. cruzi drugs. This review will focus on some approaches used for the design of new potential trypanocidal agents, exploring modern concepts of medicinal chemistry such as bioisosterism, molecular hybridization, bioinspired design in lead compounds, as well as the complexation of transition metals with bioactive ligands. The examples discussed in this article may serve as lessons for the antitrypanosomal drug design.

Despite many advances made in disease mechanisms knowledge and drug discovery and development processes, the election of promising lead compounds continues to be a challenge. Efficient techniques are required for lead selection of hit compounds selected through in vitro pharmacological studies, in order to generate precise low cost throughput data with minimal amount of compound to support the right decision making. In this context, the selection of lead compounds with physicochemical parameters that will benefit orally bioavailable drugs are crucial for patients compliance and cost effectiveness, as well as for successful pharmacology. A concept based in Lipinski's rules point out the importance of analyzing these informations in early stages. A hepatocyte screening system may provide data on many processes such as drug-drug interaction, metabolite formation, drug toxicity and ADME profile of a hit. Drug-induced liver injury is the most frequent reason for the withdrawal of an approved drug from the market and hepatocytes have a central role in the metabolism of xenobiotics. Cytotoxicity screening assays can also give some information about toxicity early drug discovery process. A set of goals in lead compound selection must be shared between all areas involved so the chances of success can be improved in translational research.

Chagas disease which is caused by the parasiteTrypanosoma cruziis an important cause of cardiomyopathy in Latin America. In later stages chagasic cardiomyopathy is associated with congestive heart failure which is often refractory to medical therapy. In these individuals heart transplantation has been attempted. However, this procedure is fraught with many problems attributable to the surgery and the postsurgical administration of immunosuppressive drugs. Studies in mice suggest that the transplantation of bone-marrow-derived cells ameliorates the inflammation and fibrosis in the heart associated with this infection. Cardiac magnetic resonance imaging reveals that bone marrow transplantation ameliorates the infection induced right ventricular enlargement. On the basis of these animal studies the safety of autologous bone marrow transplantation has been assessed in patients with chagasic end-stage heart disease. The initial results are encouraging and more studies need to be performed.

The therapeutic effects of umbelliferone (30, 60 and 90 mg/kg), a coumarin isolated from Typha domingensis (Typhaceae) were investigated in a mouse model of bronchial asthma. BALB/c mice were immunized and challenged by nasal administration of ovalbumin. Treatment with umbelliferone (60 and 90 mg/kg) caused a marked reduction of cellularity and eosinophil numbers in bronchoalveolar lavage fluids from asthmatic mice. In addition, a decrease in mucus production and lung inflammation were observed in mice treated with umbelliferone. A reduction of IL-4, IL-5, and IL-13, but not of IFN-gamma, was found in bronchoalveolar lavage fluids of mice treated with umbelliferone, similar to that observed with dexamethasone. The levels of ovalbumin-specific IgE were not significantly altered after treatment with umbelliferone. In conclusion, our results demonstrate that umbelliferone attenuates the alteration characteristics of allergic airway inflammation. The investigation of the mechanisms of action of this molecule may contribute for the development of new drugs for the treatment of asthma.

Este artigo apresenta a técnica de codificação PBIW. Essa técnica é baseada na fatoração de grupos de operações escalonadas em instruções codificadas e padrões. Uma instrução codificada não contém dados redundantes e é armazenada em uma cache de instruções. Os padrões são armazenados em uma cache de padrões. Foi realizado um estudo de caso desta técnica sobre esquemas de codificação de instruções denominados 2D-VLIW e EPIC em uma arquitetura de alto desempenho chamada 2D-VLIW. A técnica PBIW foi avaliada com os benchmarks MediaBench, SPECint e SPECfp. Os resultados revelam que a técnica PBIW produz programas até 81% menores que 2D-VLIW e até 46% menores que EPIC, além de programas até 96% mais rápidos que 2D-VLIW e até 69% mais rápidos que EPIC.

To investigate the potential of bone marrow mononuclear cells (BM-MCs) in the regeneration of hepatic lesions induced by Schistosoma mansoni (S.mansoni) chronic infection.Female mice chronically infected with S.mansoni were treated with BM-MCs obtained from male green fluorescent protein (GFP) transgenic mice by intravenous or intralobular injections. Control mice received injections of saline in similar conditions. Enzyme-linked immunosorbent assay (ELISA) assay for transforming growth factor-beta (TGF-beta), polymerase chain reaction (PCR) for GFP DNA, immunofluorescence and morphometric studies were performed.Transplanted GFP(+) cells migrated to granuloma areas and reduced the percentage of liver fibrosis. The presence of donor-derived cells was confirmed by fluorescence in situ hybridization (FISH) analysis for detection of cells bearing Y chromosome and by PCR analysis for detection of GFP DNA. The levels of TGF-beta, a cytokine associated with fibrosis deposition, in liver fragments of mice submitted to therapy were reduced. The number of oval cells in liver sections of S.mansoni-infected mice increased 3-4 fold after transplantation. A partial recovery in albumin expression, which is decreased upon infection with S.mansoni, was found in livers of infected mice after cellular therapy.In conclusion, transplanted BMCs migrate to and reduce the damage of chronic fibrotic liver lesions caused by S.mansoni.

Malaria is one of the most important tropical diseases and mainly affects populations living in developing countries. Reduced sensitivity of Plasmodium sp. to formerly recommended antimalarial drugs places an increasing burden on malaria control programs as well as on national health systems in endemic countries. The present study aims to evaluate the antimalarial activity of betulinic acid and its derivative compounds, betulonic acid, betulinic acid acetate, betulinic acid methyl ester, and betulinic acid methyl ester acetate. These substances showed antiplasmodial activity against chloroquine-resistant Plasmodium falciparum parasites in vitro, with IC(50) values of 9.89, 10.01, 5.99, 51.58, and 45.79 microM, respectively. Mice infected with Plasmodium berghei and treated with betulinic acid acetate had a dose-dependent reduction of parasitemia. Our results indicate that betulinic acid and its derivative compounds are candidates for the development of new antimalarial drugs.

Heart failure due to Chagas cardiomyopathy (HFCC) differs from failure with other etiologies because of the occurrence of intense inflammatory infiltrate and right ventricle compromise. This article investigates correlations of B-type natriuretic peptide (BNP) levels with parameters of severity in HFCC. Twenty-eight patients and 8 normal controls underwent heart catheterization and clinical and laboratory analyses. BNP levels were higher in patients with HFCC (P