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.

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

Abstract 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 (-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.

Resumo 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 (-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.

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

Abstract 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.

Resumo 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.

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.

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.

Ocorrência de anticorpos anti-Leishmania spp. em felinos em área endêmica do estado de São Paulo / Occurrence of anti-Leishmania spp antibodies in felines in an endemic area of the State of São Paulo

A leishmaniose visceral (LV) é uma zoonose de grande impacto em saúde pública. A infecção nos gatos tem sido relatada nos países onde a doença é endêmica. Seu papel como reservatório não está satisfatoriamente elucidado, embora a transmissão do parasito de um felino infectado para vetor tenha sido reportada por xenodiagnóstico. O objetivo do trabalho foi avaliar a presença de anticorpos anti-Leishmania spp. em animais da espécie felina em área endêmica para LV (Bauru-SP), por meio dos testes sorológicos de reação de imunofluorescência indireta (RIFI) e ensaio imunoenzimático (ELISA), e associá-los às variáveis: gênero, idade, raça e forma de criação. Foram testados soros de 276 felinos, dos quais 82 foram reagentes pelo método ELISA (29,71%), 17 pelo RIFI (6,15%) e 10 em ambos os testes (3,6%). Houve associação estatística significativa para a variável forma de criação, em que 100% dos animais errantes foram soropositivos a pelo menos um dos testes (P<0,005). Tal associação não foi encontrada para as demais variáveis analisadas (P>0,05). Não houve concordância entre o resultado dos testes, pois o método ELISA é mais sensível que o método RIFI.(AU)

Visceral leishmaniasis (VL) is a zoonosis with a great impact on public health. Infection in cats has been reported in countries where the disease is endemic. Its role as reservoir is not satisfactorily elucidated, although transmission of the parasite from an infected feline to vector has been reported by xenodiagnosis. The objective of this study was to evaluate the presence of anti-Leishmania spp antibodies in feline animals in an area endemic to LV (Bauru-SP), using the serological tests of Indirect Immunofluorescence Reaction (IFR) and ELISA and variables: gender, age, race and form of creation. Samples of 276 felines were tested, of which 82 were ELISA reagents (29,71%), 17 by IFR (6,15%) and 10 in both tests (3,6%). There was a significant statistical association for the variable form of breeding, where 100% of the wandering animals were seropositive to at least one of the tests (P <0,005). Such association was not found for the other variables analyzed (P >0,05). There was no concordance between the results of the tests, since the ELISA method is more sensitive than the RIFI method.(AU)

Analysis of uni and bi-parental markers in mixture samples: Lessons from the 22nd GHEP-ISFG Intercomparison Exercise.

Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.

The Comorbidity of Bipolar Disorder and Migraine: The Role of Inflammation and Oxidative and Nitrosative Stress.

Comorbid migraine in the course of bipolar disorder has been reported as highly prevalent and associated with increased morbidity. Patients with bipolar disorder and comorbid migraine tend to present with higher rates of rapid cycling, increased number of depressive episodes, more severe depression, and increased suicidality when compared to subjects with bipolar disorder alone. Both conditions display similar clinical features, such as relapsing-recovering presentation, and vulnerability to psychological and physical stress. Clinical implications of this association have been well established, however the biological underpinnings involved in both conditions remain poorly understood. Inflammation and oxidative and nitrosative stress seem to play a role as mediators in the cross-sensitization between bipolar disorder and migraine. Therefore, the present study aims to review the role of inflammation, oxidative and nitrosative stress as underlying mechanisms in the natural history of bipolar disorder comorbid with migraine.

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.

Identification of bacterial infections and clinical manifestation associated with cytomegalovirus in liver transplantation patients.

INTRODUCTION: Liver transplantation has become the most effective therapy for the treatment of patients with end-stage liver disease. With new immunosuppressive agents the incidence of acute rejection has been significantly reduced, but infection has become a serious problem. OBJECTIVE: Our objective was to correlate cytomegalovirus (CMV) positivity of antigenemia and polymerase chain reaction (PCR) with clinical manifestations and bacterial infections among patients undergoing liver transplantation. METHODS: This prospective study included patients monitored for 6 months for early detection of CMV infection. Sample collections were performed at the time of surgery and weekly until the second month followed by fortnightly in the third month, and monthly in the fourth to sixth month. CMV infection was defined by positive antigenemia (>3 cells) or 2 positive PCR tests associated or not with clinical symptoms. The methodology for the diagnosis of bacterial infection was through biochemical tests and the automated VITEK/bioMérieux (identification and antibiogram) using samples of urine and blood cultures. Chi-square test was used for dicotomic variables with significant differences when P < .05. RESULTS: Sixteen patients (32%) had CMV infections, including 13 (81%) with concomitant infections. Thirty-four patients (68%) did not have CMV infections and 8 of these (24%) had bacterial infection. There was a high correlation with bacterial infections among CMV-positive patients. CONCLUSION: Bacterial infections after liver transplantation were associated with CMV infection.

Isolation and in vitro culture of trypanosomes from Leptodactylus ocellatus from the Atlantic Forest in a new experimental culture medium.

The purpose of this study was to verify the in vitro development of Trypanosoma sp. isolated from Leptodactylus ocellatus frogs under a new protocol using a biphasic medium composed of Novy, McNeal, and Nicolle (NNN) blood agar medium as a solid phase and liver infusion, brain heart infusion, and tryptose (LIBHIT) medium as a liquid phase. Blood forms, collected by cardiac puncture or after the maceration of different organs, were inoculated in culture tubes containing the biphasic medium composed by NNN and LIBHIT. Trypanosomes were observed 4 days postinoculation; most bloodstream trypomastigotes had differentiated into epimastigotes and amastigotes by this time. Trypomastigotes were again observed in older cultures (7 days). Parasites were successfully subcultured for 8 mo in this medium and successfully cryopreserved. The present study provides a new protocol medium for the isolation and culture of anuran trypanosomes.

Detection of cytomegalovirus and human herpesvirus-6 DNA in liver biopsy specimens and their correlation with rejection after liver transplantation.

Cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6) reactivation after transplantation put patients at an increased risk of graft rejection mainly among those who receive organs that are positive in their donor biopsies. The aim of this study was to investigate the presence of CMV and HHV-6 DNA in liver biopsy specimens from the donors and from their grafts for correlation with rejection after transplantation. We followed 41 liver transplantation patients whose samples were evaluated using nested-polymerase chain reactions (N-PCR). Twenty-one (51%) of the 41 studied patients experienced rejection; 4/21 (19%) were CMV positive in the donor biopsy specimens and remained positive; another 5 subjects became positive. The patients who received organs from donors with biopsies positive for CMV demonstrated a trend to develop graft rejection after transplantation (Fisher's exact test [P = .0591] with significant results on univariate and multivariate analysis [P = .042]). Eight of the 21 who experienced rejection episodes were HHV-6 positive in the donor biopsy but there was no statistical significance CMV DNA diagnosed in liver donor biopsies remained positive posttransplantation in liver biopsy recipients; it was considered a tendency to develop acute cellular rejection after transplantation.

Co-infection and clinical impact of human herpesvirus 5 and 6 in liver transplantation.

BACKGROUND: Human herpesvirus (HHV) 5 and 6 remain latent after primary infection and can be reactivated after immunosuppression for organ transplantation. An association between HHV-5 and HHV-6 has been reported in liver transplant patients. The coinfection is associated with clinical manifestations and graft dysfunction. OBJECTIVE: The aim of this study was to monitor herpesviruses in liver transplant recipients to better understand issues involving coinfection with HHV-5/6 and correlations with acute cellular rejection episodes and bacterial infections. METHODS: Forty-five adult liver transplant patients of median age 47 years (range, 18-66), gave blood samples and liver biopsies in the first 6 months after their surgeries. Viremia was detected with the use of nested PCR and antigenemia; the Banff classification was used to detect allograft rejection. RESULTS: IgG positive for HHV-5 was observed in 94% of subjects whose main indication (67%) for transplantation was hepatitis C. Twenty-three (51.1%) displayed cytomeg virus (CMV) infections and 12 (26.7%) HHV-6 infection. There were 6 patients (13.3%) with HHV-5/6 coinfections. Eighteen of the 23 patients had CMV disease, showing a strong correlation between a positive test and CMV disease; 6 displayed an acute cellular rejection episode in the same period (χ(2) = 6.62; P < .03). Four out of 6 patients who displayed coinfections (HHV-5/6) had concomitant bacterial infections; 3/6 experienced graft rejection episodes. During follow-up, 1 patient had HHV-6 infection diagnosed as encephalitis followed by fever on the 24th day after surgery. The median 32 days for HHV-6 detection by nested PCR positivity was shorter than 38 days for HHV-5. CONCLUSIONS: HHV-5/6-infected patients displayed more allograft rejection episodes, coinfections, and concomitant bacterial infections, besides an higher risk for CMV disease.

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.

Cytomegalovirus, human herpesvirus-6, and human herpesvirus-7 in adult liver transplant recipients: diagnosis based on antigenemia.

Human herpesvirus (HHV)-6, HHV-7, and cytomegalovirus (CMV) that remain latent after primary infection can be reactivated during immunosuppression following organ transplantation in liver transplant recipients. The aim of this study was to monitor active infections for HHV-6, HHV-7, and CMV among adult liver transplantation recipients using antigenemia detected by an immunoperoxidase staining. Twenty-eight adult liver transplant patients were monitored using antigenemia in blood samples obtained at the time of transplantation, as well as weekly in the first month and once a month for 6 months. Of these patients, 28.5% showed positive CMV antigenemia; 39.2%, HHV-6 antigenemia; and 14.2%, HHV-7 antigenemia. The detection of the three viruses was considered to be independent of one another (P>.05). The results described above showed that few patients remain free of beta herpesviruses after liver transplantation. Most patients were infected sequentially and not concurrently. Antigenemia has been considered useful to detect active HHV-6 and HHV-7 infections. Antigenemia can be more efficiently interpreted when compared with polymerase chain reaction results, although other studies are necessary to establish the reference of HHV-6 and HHV-7 antigenemia.

Monitoring and detection of cytomegalovirus in liver transplant recipients.

Cytomegalovirus (CMV) is a ß-herpesvirus. CMV infections are a common complication contributing to morbidity and mortality after liver transplantation. Among organ transplant recipients, CMV can reactivate from latency during the first 6 months. This prospective study performed from February 2008 to December 2009 examined liver transplant recipients during the first 6 months. Two methods were performed to detect CMV infections: antigenemia (AGM) and nested (PCR). Ninety-four patients, including 72 men (76.6%) and 22 women (23.4%) underwent liver transplantation during this period. We analyzed 575 samples including 465 for AGM and PCR. Forty-three (9.25%) showed positive AGM as detected 2 to 179 days posttransplantation with a mean of 50 days and a median of 35 days, and 93/465 (20%) showed positive PCR at 0 to 186 days posttransplantation with a mean of 31 days and a median of 38 days. Among the 43 antigenemia patients, 38 samples were positive for up to 5 cells 18 of which were PCR-positive. Five samples were positive with more than 5 cells, including 3 that were PCR-positive. Only 4.51% had AGM and were PCR-positive in the same sample. Despite only 9.25% (43/465) showing AGM, the current study suggested the utility of routine monitoring to detect early CMV infection among liver transplantation patients seeking to reduce morbidity and mortality.

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.

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.

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.