An antigenic domain of the Leishmania amazonensis nucleoside triphosphate diphosphohydrolase (NTPDase 1) is associated with disease progression in susceptible infected mice.

An antigenic conserved B domain was previously identified within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, the r-potDomain B, a 6× His-tag polypeptide belonging to the conserved B domain from the potato apyrase, and synthetic peptides LbB1LJ and LbB2LJ derived from the B domain from Leishmania NTPDase 1 were used as molecular tools for studies of the Leishmania amazonensis NTPDase 1. Widespread subcellular location of the specific NTPDase 1 was detected by Western blots of promastigote fractions and ultrastructural immunocytochemical microscopy using immune sera raised against these biomolecules. In addition, the L. amazonensis-infected BALB/c mice were evaluated at 12 to 120 days after infection, which progresses showing typical nodular lesion. High antibody reactivity with either r-potDomain B, LbB1LJ, or LbB2LJ was found in L. amazonensis-infected BALB/c mice indicating the antigenicity of the B domain from NTPDase 1 isoform. The IgG1 antibody reactivity significantly increased at 90-120 days postinfection, 18- to 24-fold when compared to the 12th day, and remained elevated even at 120th after infection, coinciding with the most active stage of the disease. In contrast, significantly higher IgG2a antibody reactivity with each biomolecule was observed at 40th day, about two- to fourfold higher than those found at 12th or 20th day, and decreased along 120-day period. Apparently, the conserved B domain is capable to induce IgG2a production in early disease stages. All together, these results suggest that r-potDomain B or synthetic peptides could be molecular starting points in experimental protocols of immunotherapy and/or vaccination for leishmaniasis.

Correlation of cytomegalovirus and human herpesvirus 7 with CD3+ and CD3+ CD4+ cells in chronic periodontitis patients.

BACKGROUND AND OBJECTIVE: Human chronic periodontitis is an inflammatory process characterized by dense accumulation of immune cells in the periodontal tissue. The periodontitis can lead to loss of teeth in the patient and the pathogenesis of this disease is not completely known. This study tested the hypothesis that chronic periodontitis-affected sites can harbor betaherpesviruses and that viruses are linked to a profile of the inflammatory infiltrate. MATERIAL AND METHODS: Biopsies of periodontal tissue were taken from periodontitis-affected patients and from healthy subjects. Immunohistochemistry was performed to count CD19(+) B cells, CD3(+) total T cells, T-CD4(+) and T-CD8(+) cell subsets, and PCR was performed to detect cytomegalovirus and human herpesvirus 6 and 7 in the samples. One slide of each sample was stained with Giemsa for histopathological examination and to evaluate the quality of the cellular infiltrate. RESULTS: As expected, tissues collected from healthy subjects presented no significant level of inflammatory infiltration and were therefore excluded from immunostaining procedures. Results showed that CD19(+) B cells were in higher number than CD3(+) T cells in the periodontitis-affected tissue, but this was not statistically significant. The T-CD4(+) lymphocyte subset was significantly higher than the T-CD8(+) lymphocyte subset (p = 0.004) in the samples. Cytomegalovirus and human herpesvirus 7 were found at periodontitis-affected sites, but not in tissue collected from healthy subjects (p = 0.04 and p = 0.04, respectively). Human herpesvirus 6 was rarely detected. We found a correlation between cytomegalovirus and lower CD19(+) /CD3(+) ratios (ratio < 0.9, p = 0.003) and between human herpesvirus 7 and lower CD19(+) /CD3(+) ratios (ratio < 0.9, p = 0.003) and higher CD4(+) /CD8(+) ratios (ratio > 1.1, p = 0.002). CONCLUSION: This study shows that cytomegalovirus and human herpesvirus 7 can be present at periodontitis-affected sites but are uncommon at healthy periodontal sites. Moreover, our data suggest that cytomegalovirus can be related to an inflammatory infiltrate with predominance of CD3(+) T cells, whereas human herpesvirus 7 can be associated with an infiltrate with predominance of T-CD4(+) cells. However, further studies are necessary to support this hypothesis. Herpesviruses could play a role in human chronic periodontitis by modulation of the T cell response.

Reproductive status effects of pair-housed male pigs on natural, agonistic and sexual behaviours.

Boar rearing, which avoids pain and suffering caused by surgical castration, provides better performance, a greater deposition of muscle tissue and leaner carcasses and thus has beneficial effects on both animal welfare and the product. Some countries that do not slaughter boars must consider their boar taint and aggressive and sexual behaviours. Considering that pigs are housed in large groups, which may complicate the formation of social hierarchies and increase fighting and mounting behaviours, some studies have conducted research with reduced numbers of pigs per pen, but these behaviours continued to be observed. However, a study of the reproductive status of pair-housed male pigs has yet to be reported. The aim of this study was to determine whether the reproductive status of uncastrated, immunocastrated and surgically castrated pair-housed male pigs alters their natural, agonistic and sexual behaviours. A total of 48 male pigs from Agroceres PIC™ genetics were assigned to three groups: surgically castrated (barrows), immunocastrated and uncastrated (boars). Natural, aggressive and sexual behaviours of the pigs were assessed by direct observations during four periods of 12 h each (six, five and three weeks before slaughter and the slaughter week). The pigs were housed in pairs from the growing phase until slaughter. Animal behaviour was observed from the finishing phase to slaughter. Carcass lesions were assessed according to five different classes (one: no injury; two to five: severely injured). Overall, boars spent more time lying and less time eating and drinking than barrows. In total of all the periods (48 h), boars expressed more aggressive and sexual behaviours than barrows, whereas immunocastrated pigs displayed similar behaviours to boars, before and after the second vaccine dose. No differences in carcass lesions between treatments and no prevalence of carcasses with severe injuries were observed. In conclusion, the reproductive status of pair-housed male pigs did not change the natural behaviour of boars, immunocastrated pigs or barrows. The agonistic and sexual behaviours of boars and barrows remained unchanged. When housing pigs in pairs, immunocastrated pigs presented similar agonistic and sexual behaviours to boars before and after the second immunocastration vaccine dose. The use of pair-housed uncastrated male pigs has generated welfare benefits for these animals, as the number of carcasses with injuries did not differ from barrows and immunocastrated pigs.

Evaluation of antioxidant, photoprotective and antinociceptive activities of Marcetia macrophylla extract: potential for formulation of sunscreens.

The antioxidant, photoprotective and antinociceptive Marcetia macrophylla active extract was investigated as an active ingredient in a sunscreen cream formulation. Thus, the M. macrophylla extract showed IC50 of 3.43 mg/ml of the antioxidant (DPPH∙ scavenging test) and Sun Protection Factor of 20.25 (SPF/UV-B, at 250 µg/ml) and UV-A of 78.09% (photobleaching trans-resveratrol test). The antinociceptive activity was superior to all standards tested using the in vivo acetic acid-induced writhing test (99.14% at the dose of 200 mg/kg) and the high-performance liquid chromatography coupled with diode array detector and mass spectroscopy multi-stage (HPLC-DAD-MS/MS) enabled the structural characterization of the quercetin-3-O-hexoside, quercetin-3-O-pentoside and quercetin-3-O-desoxihexoside. The pharmaceutical formulation containing the Marcetia macrophylla crude active extract was prepared and the physicochemical tests (organoleptic characteristics, pH analysis and centrifugation), the in vitro UVB (sun protection factor, SPF) and UVA (ß-carotene) using the spectroscopic method were investigated. The formulation showed satisfactory results concerning the physicochemical parameters evaluated and active against the UV test. Thus, M. macrophylla showed biological activities with potential use in pharmaceutical preparations.

Leishmania (Leishmania) infantum/chagasi: Histopathological aspects of the skin in naturally infected dogs in two endemic areas.

In the New World, visceral leishmaniasis (VL), which is a progressive disease and frequently fatal, is caused by Leishmania (Leishmania) infantum/chagasi. It is endemic in many regions of Brazil and occasionally occurs in non-endemic regions when dogs from an endemic area are introduced. The aim of the present study is to compare different skin infection patterns of dogs from two leishmaniasis endemic areas. A histological analysis of dogs from Campo Grande, Mato Grosso do Sul state, a region where epidemic episodes are currently taking place, showed dermic inflammatory infiltrates, composed of numerous vacuolated parasitized macrophages, few lymphocytes, plasma cells and many degranulated mast cells. In the other region of the study, São Luís, Maranhão state, the skin of dogs presented a remarkable inflammatory reaction composed mainly of plasma cells, lymphocytes and very few parasites. We concluded that there is a difference in the skin lesion patterns of dogs with leishmaniasis that is directly related to the endemic area where the animals live.

The emerging human pathogen Photorhabdus asymbiotica is a facultative intracellular bacterium and induces apoptosis of macrophage-like cells.

Photorhabdus species are gram-negative entomopathogenic bacteria of the family Enterobacteriaceae. Among the different members of the genus, one species, Photorhabdus asymbiotica, is a pathogen of both insects and humans. The pathogenicity mechanisms of this bacterium are unknown. Here we show that P. asymbiotica is a facultative intracellular pathogen that is able to replicate inside human macrophage-like cells. Furthermore, P. asymbiotica was shown for the first time in an intracellular location after insect infection. We also demonstrated that among Australian and American clinical isolates, only the Australian strains were able to invade nonphagocytic human cells. In cell culture infection experiments, Australian clinical isolates as well as cell-free bacterial culture supernatant induced strong apoptosis of a macrophage cell line at 6 h postinfection. American isolates also induced cellular death, but much later than that induced by Australian ones. Mammalian cultured cells analyzed for key features of apoptosis displayed apoptotic nuclear morphology, activation of the initiator caspases 8 and 9 and the executioner caspases 3 and 7, and poly(ADP-ribose) polymerase proteolysis, suggesting activation of both the intrinsic and extrinsic apoptotic pathways.

Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner [1]. To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

Human herpesvirus-7 in Brazilian liver transplant recipients: a follow-up comparison between molecular and immunological assays.

Human herpesvirus-6 and -7 (HHV-6, HHV-7) remain latent after primary infection and can reactivate after transplantation. HHV-6 active infection has been related to some clinical manifestation, but the role of HHV-7 remains unclear. The clinical significance of HHV-7 DNAemia is not completely known and the immune response against HHV-7 has been poorly studied in transplantation. In this study, we investigated HHV-7 DNAemia in liver transplant recipients and evaluated the immunoglobulin (Ig) G and IgM response against HHV-7. A total of 22 adult liver transplant recipients were followed up for 90 days. HHV-7 DNAemia was detected by nested polymerase chain reaction (PCR) in DNA extracted from sera. IgG and IgM detection was performed by immunofluorescent assay using HHV-7-infected cord blood mononuclear cells. A significant virus antibody response was defined as either a positive IgM or a > or =4-fold rise in the virus IgG antibody. All patients had pre-transplant HHV-7-positive serostatus. Nine of 22 (40.9%) patients presented HHV-7 DNAemia during follow-up. All these patients had anti-HHV-7-positive IgM and/or significant increase in IgG titers with concurrent or subsequent DNAemia. In patients without DNAemia and low persistent IgG antibody titers, IgM was not detected. Correlation between nested PCR and IgM detection was statistically significant (P=0.01). Our study indicates that nested PCR in DNA extraction from serum can be useful to detect and monitor HHV-7 active infection in liver transplant recipients. IgM antibody detection also can be useful as a first immunological technique to detect active infection, especially if combined with PCR.

A Leishmania (L.) amazonensis ATP diphosphohydrolase isoform and potato apyrase share epitopes: antigenicity and correlation with disease progression.

A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.

Evaluation of antioxidant, photoprotective and antinociceptive activities of Marcetia macrophylla extract: potential for formulation of sunscreens / Avaliação da atividade antioxidante, photoprotetora e antinociceptiva de Marcetia macrophylla: potencial formulação fotoprotetora

The antioxidant, photoprotective and antinociceptive Marcetia macrophylla active extract was investigated as an active ingredient in a sunscreen cream formulation. Thus, the M. macrophylla extract showed IC50 of 3.43 mg/ml of the antioxidant (DPPH∙ scavenging test) and Sun Protection Factor of 20.25 (SPF/UV-B, at 250 µg/ml) and UV-A of 78.09% (photobleaching trans-resveratrol test). The antinociceptive activity was superior to all standards tested using the in vivo acetic acid-induced writhing test (99.14% at the dose of 200 mg/kg) and the high-performance liquid chromatography coupled with diode array detector and mass spectroscopy multi-stage (HPLC-DAD-MS/MS) enabled the structural characterization of the quercetin-3-O-hexoside, quercetin-3-O-pentoside and quercetin-3-O-desoxihexoside. The pharmaceutical formulation containing the Marcetia macrophylla crude active extract was prepared and the physicochemical tests (organoleptic characteristics, pH analysis and centrifugation), the in vitro UVB (sun protection factor, SPF) and UVA (β-carotene) using the spectroscopic method were investigated. The formulation showed satisfactory results concerning the physicochemical parameters evaluated and active against the UV test. Thus, M. macrophylla showed biological activities with potential use in pharmaceutical preparations.

O extrato bruto de Marcetia macrophylla mostrou atividade antioxidante, fotoprotetora e antinociceptiva, sendo em seguida investigado como ingrediente ativo em uma formulação fotoprotetora. Assim, o extrato de M. macrophylla apresentou atividade antioxidante com IC50 de 3,43 mg/mL (teste de sequestro do DPPH∙) e Fator de Proteção Solar de 20,25 (FPS/UV-B, 250 µg/mL) e UV-A de 78,09% (teste de fotobranqueamento do trans-resveratrol). A atividade antinociceptiva usando o teste in vivo de contorções abdominais induzidas por ácido acético foi superior a todos os padrões testados (99,14% na dose de 200 mg/Kg). A análise por cromatografia líquida de alta eficiência acoplada a detector de fotodiodos e espectroscopia de massas multi-estágio (CLAE-DAD-EM/EM) possibilitou a caracterização dos flavonoides quercetina-3-O-hexosídeo, quercetina-3-O-pentosídeo e quercetina-3-O-desoxihexosídeo. A formulação farmacêutica contendo o extrato ativo bruto de Marcetia macrophylla foi preparada e os testes físico-químicos (características organolépticas, análise de pH e centrifugação), o UVB in vitro (fator de proteção solar, FPS) e UVA (β-caroteno) foram investigados. A formulação apresentou resultados satisfatórios frente aos parâmetros físico-químicos avaliados e ativos contra UV. Assim, M. macrophylla apresentou atividades biológicas com potencial uso em preparações fitofarmacêuticas.