Marcelo Vidigal Caliari

Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection

Background Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. Methods and Findings We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-γ, TNF-α and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. Conclusion This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.

An Oral Formulation of Angiotensin-(1-7) Produces Cardioprotective Effects in Infarcted and Isoproterenol-Treated Rats

In this study we evaluated the cardiac effects of a pharmaceutical formulation developed by including angiotensin (Ang)-(1-7) in hydroxypropyl &bgr;-cyclodextrin (HP&bgr;CD), in normal, infarcted, and isoproterenol-treated rats. Myocardial infarction was produced by left coronary artery occlusion. Isoproterenol (2 mg/kg, IP) was administered daily for 7 days. Oral administration of HP&bgr;CD/Ang-(1-7) started immediately before infarction or associated with the first dose of isoproterenol. After 7 days of treatment, the rats were euthanized, and the Langendorff technique was used to analyze cardiac function. In addition, heart function was chronically (15, 30, 50 days) analyzed by echocardiography. Cardiac sections were stained with hematoxylin/eosin and Masson trichrome to evaluate cardiac hypertrophy and damage, respectively. Pharmacokinetic studies showed that oral HP&bgr;CD/Ang-(1-7) administration significantly increased Ang-(1-7) on plasma whereas with the free peptide it was without effect. Oral administration of HP&bgr;CD/Ang-(1-7) (30 &mgr;g/kg) significantly reduced the deleterious effects induced by myocardial infarction on systolic and diastolic tension, ±dT/dt, perfusion pressure, and heart rate. Strikingly, a 50% reduction of the infarcted area was observed in HP&bgr;CD/Ang-(1-7)–treated rats. Furthermore, HP&bgr;CD/Ang-(1-7) attenuated the heart function impairment and cardiac remodeling induced by isoproterenol. In infarcted rats chronically treated with HP&bgr;CD/Ang-(1-7), the reduction of ejection fraction and fractional shorting and the increase in systolic and diastolic left ventricular volumes observed in infarcted rats were attenuated. Altogether, these findings further confirm the cardioprotective effects of Ang-(1-7). More importantly, our data indicate that the HP&bgr;CD/Ang-(1-7) is a feasible formulation for oral administration of Ang-(1-7), which can be used as a cardioprotective drug.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-γ, TNF-α, and low IL-10 production during the acute infection phase.

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions.

Trypanosoma cruzi high infectivity in vitro is related to cardiac lesions during long-term infection in Beagledogs

Trypanosoma cruzi is a hemoflagelate parasite associated with heart dysfunctions causing serious problems in Central and South America. Beagle dogs develop the symptoms of Chagas disease in humans, and could be an important experimental model for better understanding the immunopathogenic mechanisms involved in the chagasic infection. In the present study we investigated the relation among biological factors inherent to the parasite (trypomastigote polymorphism and in vitro infectivity) and immunoglobulin production, inflammation, and fibrosis in the heart of Beagle dogs infected with either T. cruzi Y or Berenice-78 strains. In vitro infectivity of Vero cells as well as the extension of cardiac lesions in infected Beagle was higher for Y strain when compared to Berenice-78 strain. These data suggested that in vitro infectivity assays may correlate with pathogenicity in vivo. In fact, animals infected with Y strain, which shows prevalence of slender forms and high infectivity in vitro, presented cardiomegaly, inflammation, and fibrosis in heart area. Concerning the immunoglobulin production, no statistically significant difference was observed for IgA, IgM or IgG levels among T. cruzi infected animals. However, IgA together IgM levels have shown to be a good marker for the acute phase of Chagas disease.

Peptides containing T cell epitopes, derived from Sm14, but not from paramyosin, induce a Th1 type of immune response, reduction in liver pathology and partial protection against Schistosoma mansoni infection in mice

Sm14 and paramyosin are two major Schistosoma mansoni vaccine candidate antigens. Recently, we have identified Sm14 and paramyosin epitopes that are recognized by T cells of resistant individuals living in endemic areas for schistosomiasis. Herein, mice were immunized with these peptides separately or in association in order to evaluate their vaccine potential. Immunization of mice with Sm14 peptides alone or mixed with paramyosin peptides was able to induce 26%-36.7% or 28%-29.2% of worm burden reduction, 67% or 46% of intestinal eggs reduction and also 54%-61% or 43%-52% of liver pathology reduction, respectively. Protection was associated with a Th1 type of immune response induced by Sm14 peptide immunization. In contrast, paramyosin peptide vaccination did not engender protective immunity or liver pathology reduction and immunization was associated with a Th2 type of immune response.

Comparison of Trypanosoma cruzi infection in dogs inoculated with blood or metacyclic trypomastigotes of Berenice-62 and Berenice-78 strains via intraperitoneal and conjunctival routes

This paper aimed to verify the influence of the inoculum source (blood or metacyclic trypomastigote) and the route of inoculation (intraperitoneal or conjunctival) on the course of T. cruzi infection in dogs, using comparatively the T. cruzi strains Berenice-62 and Berenice-78. All dogs inoculated intraperitoneally became infected independently of the T. cruzi strain and source of trypomastigotes used. High level of infectivity was also observed when metacyclic trypomastigotes of both strains were inoculated by conjunctival route. However, when blood trypomastigotes were inoculated by conjunctival route the percentages of infectivity were significantly lower in dogs inoculated with both strains. Parasitaemia was significantly higher in animals infected with metacyclic trypomastigotes via the conjunctival route independently of the T. cruzi strain used. All animals infected with Berenice-78 strain showed severe acute myocarditis. On the other hand, animals infected with Berenice-62 showed severe acute myocarditis only when infected with metacyclic trypomastigote, via the intraperitoneal route. The results suggest that the source of the inoculum and the route of inoculation remarkably influence the evolution of the infection for the T. cruzi in the vertebrate host even when the same strain of the parasite is used.

Mesenchymal stem cells associated with porous chitosan–gelatin scaffold: A potential strategy for alveolar bone regeneration

Tissue engineering has emerged as a novel treatment for replacement of lost bone tissue. This study evaluated the effects of a chitosan-gelatin scaffold seeded with bone marrow mesenchymal stem cells (BMMSCs) in the healing process of tooth sockets in rats. BMMSCs isolated from transgenic rats expressing enhanced green fluorescent protein (eGFP) were expanded and seeded on a chitosan-gelatin scaffold. These constructs were cultured for three days and characterized by scanning electronic microscopy (SEM) and energy dispersion spectroscopy (EDS). Receptor rats received the implant in the left sockets, after upper first-molar extraction. Right alveoli served as control. Animals were sacrificed at days 5, 21, and 35 post-graft for examination. Morphometry demonstrated increased bone mineralization after 21 and 35 days in transplanted sockets. Migration, differentiation, and fate of eGFP-labeled BMMSCs were monitored by immunohistochemistry. Tartrate-resistant acid phosphatase staining (TRAP) was carried out at 21 days, to identify the involvement of osteoclastic cells in the scaffold resorption. The biomaterial was resorbed by TRAP-negative giant cells in a typical foreign body reaction. Immunohistochemical findings showed that BMMSCs contributed to bone, epithelial, and vascular repair. Together, results indicate that BMMSCs loaded in the chitosan-gelatin scaffold is a strategy for tissue development in bone engineering.

Effects of single wall carbon nanotubes and its functionalization with sodium hyaluronate on bone repair

AIMS Sodium hyaluronate (HY) accelerates the repair of bone defects. However, the weak stability of HY formulations in aqueous environments has hindered its wide utilization. The functionalization of carbon nanotubes (SWCNT) with HY (HY-SWCNT) results in a reinforced hydrogel with an increased stability. Nevertheless, the biological effects of HY-SWCNT have not been explored. Thus, our objective was to evaluate whether this biomaterial preserves the bioactivity of the HY. MAIN METHODS Wistar rats were subjected to molar extraction and the sockets were treated with SWCNT (50-400 microg/mL), 1% HY, HY-SWCNT (50-400 microg/mL) or carbopol (vehicle). After seven days of surgery, histological and morphometric analyses were performed to evaluate the trabecular bone formation and the number of cell nuclei in the sockets. Expression of collagen types I and III was determined by immunohistochemistry. KEY FINDINGS Treatment with SWCNT did not alter the bone deposition, as well as the cell nuclei counting. Additionally, no significant evidence of toxicity was observed in SWCNT-treated sockets. Contrastingly, both HY and HY-SWCNT induced a marked increase in the bone formation (HY: 10.10+/-1.99%; HY-SWCNT 100 microg/mL: 10.90+/-1.13%; control: 3.69+/-1.17%) and decreased the cell nuclei amount in the sockets. Moreover, collagen type I expression was more pronounced in HY- and HY-SWCNT-treated sockets. No significant differences were viewed in the expression of collagen type III. SIGNIFICANCE Our results indicate that SWCNT is a feasible material to deliver HY to bone defects. Importantly, the functionalization of SWCNT with HY preserved the beneficial biological properties of HY in the healing process, thereby suggesting that HY-SWCNT scaffolds are potentially useful biomaterials for the restoration of bone defects.

Quantitative analysis of cardiac lesions in chronic canine chagasic cardiomyopathy.

Lesions observed in chronic chagasic cardiopathy frequently produce electrocardiographic alterations and affect cardiac function. Through a computerized morphometrical analysis we quantified the areas occupied by cardiac muscle, connective and adipose tissues in the right atrium of dogs experimentally infected with Trypanosoma cruzi. All of the infected dogs showed chronic myocarditis with variable reduction levels of cardiac muscle, fibrosis and adipose tissue replacement. In the atrial myocardium of dogs infected with Be78 and Be62 cardiac muscle represented 34 and 50%, fibrosis 28 and 32% and adipose tissue 38 and 18%, respectively. The fibrosis observed was both diffuse and focal and mostly intrafascicular, either partially or completely interrupting the path of muscle bundles. Such histological alterations probably contributed to the appearance of electrocardiographic disturbances verified in 10 out 11 dogs which are also common in human chronic chagasic cardiopathy. Fibrosis was the most important microscopic occurrence found since it produces rearrangements of collagen fibers in relation to myocardiocytes which causes changes in anatomical physiognomy and mechanical behavior of the myocardium. These abnormalities can contribute to the appearance of cardiac malfunction, arrythmias and congestive cardiac insufficiency as observed in two of the analyzed dogs. Strain Be78 caused destruction of atrial cardiac muscle higher than that induced by strain Be62.

Sm21.6 a novel EF-hand family protein member located on the surface of Schistosoma mansoni adult worm that failed to induce protection against challenge infection but reduced liver pathology.

Schistosomiasis continues to be a significant public health problem that affects 200 million people worldwide. This is one of the most important parasitic diseases, and one whose effective control is unlikely in the absence of a vaccine. In this study, we have isolated a cDNA clone encoding the Schistosoma mansoni Sm21.6 protein that has 45% and 44% identity with Sm22.6 and Sj21.7 EF-hand containing antigens, respectively. Confocal microscopy analysis revealed that Sm21.6 is a membrane-associated protein localized on the S. mansoni adult worm. Mouse immunization with rSm21.6 induced a mixed Th1/Th2 cytokine profile and no protection against infection. However, vaccination with rSm21.6 reduced by 28% of liver granuloma numbers, 21% of granuloma area and 34% of fibrosis. Finally, rSm21.6 was recognized by sera from individuals resistant to reinfection compared with patients susceptible to reinfection and this molecule should be further studied as potential biomarker for disease resistance. In conclusion, Sm21.6 is a new tegument protein from S. mansoni that plays an important role in reducing pathology induced by parasite infection.