Expressão de transtirretina no sistema gastrointestinal do murganho

A transtirretina (TTR) é uma proteína plasmática constituída por quatro subunidades idênticas de aproximadamente 14KDa e de massa molecular de 55 KDa (Blake et al., 1978). A TTR é responsável pelo transporte de tiroxina (T4) (Andrea et al., 1980) e retinol (vitamina A), neste último tipo de transporte através da ligação à proteina de ligação ao retinol (RBP) (Kanai et al., 1968). É sintetizada principalmente pelo fígado e secretada para o sangue (Murakami et al., 1987) e também sintetizada pelas células epiteliais do plexo coróide e secretada para o líquido cefaloraquidiano (LCR) (Aleshire et al., 1983). Existem outros locais que expressam TTR mas em menor quantidade, nomeadamente: a retina do olho (Martone et al., 1988), o pâncreas (Kato et al., 1985), o saco vitelino visceral (Soprano et al., 1986) o intestino (Loughna et al., 1995); o estômago, coração, músculo e baço (Soprano et al., 1985). A TTR é uma proteína, do ponto de vista filogenético, extremamente conservada o que já de si é um indicador da sua importância biológica (Richardson, 2009) O objectivo deste trabalho foi avaliar a expressão de transtirretina ao longo do sistema gastrointestinal do murganho, nos seguintes órgãos esófago, estômago, duodeno, cólon e também bexiga, com cerca de 3 meses de idade. O segundo objectivo foi identificar as células responsáveis por essa expressão, nos órgãos em estudo. Foi possível verificar que apenas o estômago apresenta valores de expressão normalizada de TTR diferente de zero, expressão essa muito inferior à do fígado, tal como se esperava. Por imunohistoquímica/imunofluorescência foi possível determinar que as células que expressam TTR são pouco abundantes e estão presentes na região glandular do estômago do murganho e também do humano. Para além disto, verificou-se que a TTR co-localiza com somatostatina e que as células que sintetizam TTR correspondem às células D, responsáveis pela secreção de somatostatina

Linfangiogénese no tecido adiposo em modelos de doença inflamatória crónica (asma e obesidade): uma abordagem por imunohistoquímica e lipidómica

O tecido adiposo é um órgão endócrino dinâmico, secretando factores importantes na regulação do metabolismo, fluxo vascular sanguíneo e linfático, e função imunológica, entre outros. Em caso de acumulação de tecido adiposo por ingestão de uma dieta gorda, ou por disfunção metabólica, os adipócitos podem desencadear uma reacção inflamatória por falha na drenagem linfática, acumulando-se mediadores inflamatórios, os quais potenciam a propagação da reacção. Assim, questiona-se uma potencial associação entre o aumento de tecido adiposo na obesidade, hipóxia adipocitária e estimulação da linfangiogénese. Além disso, a expressão de adipocinas varia de acordo com a distribuição do tecido adiposo (subcutâneo, TAS e visceral, TAV). Deste modo, pretende-se com este estudo contribuir para o aumento do conhecimento sobre os complexos mecanismos moleculares subjacentes à linfangiogénese. Ensaios com ratinhos da estirpe C57Bl/6J (modelo de obesidade) e BALB/c (modelo de asma e obesidade), divididos em grupos submetidos a dieta normal e dieta rica em gordura. Avaliação semi-quantitativa da expressão tecidular de LYVE-1 (marcador da linfangiogénese) por imunohistoquímica em material embebido em parafina, no TAS e TAV, e cromatografia líquida de ultra-performance acoplada de espectrometria de massa (UPLC-MS) para análise da expressão plasmática de ceramida e esfingosina-1-fosfato (S1P). No modelo de obesidade observou- -se diminuição do número de vasos linfáticos e expressão de LYVE-1 ao longo do tempo no TAV, e aumento de ambos os parâmetros e hipertrofia adipocitária no TAS. As concentrações de ceramida e S1P corroboram a existência de um processo inflamatório nos ratinhos em estudo, ainda que numa fase muito inicial. No modelo de asma e obesidade, após 17 semanas de tratamento, observou-se incremento da linfangiogénese no TAV, mas não no TAS. A resposta inflamatória avaliada através dos diferentes parâmetros permite afirmar que num estadio inicial de obesidade a proliferação linfática poderá estar a ser retardada pela hipertrofia adipocitária. A libertação de adipocinas será observada apenas numa fase posterior, desencadeando todo o processo inflamatório que incrementará a proliferação linfática. Adicionalmente, é possível sugerir que a maior pressão à qual o TAV se encontra sujeito não favorece a proliferação linfática, pelo menos num estadio incial.

Boulder deposition during major tsunami events

A remarkable accumulation of marine boulders located above the present spring tide level has occurred in two coastal lowlands of the Algarve (Portugal). The size-interval of the particles studied here is seldom reported in the literature in association with extreme events of coastal inundation, thus making this study of relevance to many other coasts worldwide. The spreads of boulders extend several hundred meters inland and well beyond the present landward limit of storm activity. The marine origin of the boulders is demonstrated by well-developed macro-bioerosion sculpturing and in situ skeletal remains of endolithic shallow marine bivalves. The good state preservation of the fossils within the boulders indicates that abrasion duringtransport and redeposition was not significant. We envisage boulder deposition as having taken place during the Lisbon tsunami of ad 1755 through the simultaneous landward entrainment of coarse particles from nearshore followed by rapid shoreward suspended-dominated transport and non-graded redeposition that excluded significant sorting by weight or boulder dimensions. We use numerical hydrodynamic modeling of tsunami (and storm) waves to test the observational data on boulder dimensions (density, size, distribution) on the most likely processes of sediment deposition. This work demonstrates the effectiveness of the study of boulder deposits in tsunami reconstruction. Copyright (C) 2011 John Wiley & Sons, Ltd.

Imunorreatividade em material histológico arquivado durante um, quatro e sete anos

Quando aplicada no âmbito da Anatomia Patológica, a imuno-histoquímica tem constituído um poderoso meio de identificação/caracterização de várias estruturas histológicas, permitindo delinear prognóstico e terapêutica para várias patologias. Tendo em conta que as amostras histológicas analisadas podem ser conservadas ao longo de vários anos, interessa avaliar a manutenção da antigenicidade ao longo do tempo, de forma a garantir a qualidade final da técnica quando aplicada em material de arquivo. Assim, o principal objetivo deste trabalho foi comparar a imunorreatividade do material histológico arquivado durante um, quatro e sete anos. Foi utilizado material histológico de próstata, pulmão e mama, no qual se procedeu à imunomarcação de citoqueratinas (Clones AE1/AE3), CD34 e proteína p63, por método de multímero/HRP no sistema Ventana BenchMark Ultra®. Foi realizado um ensaio com recuperação antigênica por alta temperatura (RAAT) e outro sem esta etapa. As imunomarcações (n=162) foram classificadas por três avaliadores independentes num escore quantitativo final (escala 0-100). O par média/desvio-padrão do escore final para os casos com sete anos foi de 69,06/19,05, para os casos com quatro anos foi de 66,47/20,73 e para os casos com um ano foi de 69,08/19,35, não se tendo encontrado diferenças estatisticamente significativas. Os casos sem RAAT obtiveram um par média/desvio-padrão de 54,90/17,00, enquanto os casos com RAAT obtiveram 81,50/11,60, o que revelou diferenças estatisticamente significativas (p=0,000). Para os casos em estudo conclui-se que o fator “tempo de arquivo” não está associado a alterações da imunorreatividade. A importância da RAAT na obtenção de imunomarcação de qualidade sai fortemente realçada. ABSTRACT - When applied within the framework of Pathology, immunohistochemistry has been a powerful means of identification/characterization of various histological structures, allowing to outline prognosis and therapy for various diseases. Given that the analyzed histological samples can be preserved for several years, it is interesting to assess the retention of antigenicity over time in order to ensure the quality of the final technique, when applied to stored material. Thus, the main objective of this study was to compare the immunoreactivity of the histological material archived for one, four and seven years. It was used histological material from prostate, lung and breast, in which it was performed the immunostaining of cytokeratins (clones AE1/AE3), CD34 and p63 protein by the method of multimer/HRP system on a Ventana BenchMark Ultra®. It was conducted a test with heat induced epitope retrieval (HIER) and another one without this step. The stained slides (n=162) were classified by three independent assessors using a quantitative score (scale 0-100). The pair mean/standard deviation of the score for cases with seven years was 69,06/19,05, for cases with four years was 66,47/20,73 and for cases with one year was 69,08/19,35, which did not revealed any statistically significant differences. The cases without HIER had a couple mean/standard deviation of 54.90/17.00 while the cases with HIER obtained 81.50/11.60, which revealed statistically significant differences (p=0.000). For this case study it was concluded that the factor archive period is not associated with changes in immunoreactivity. The importance of HIER in obtaining high quality immunostaining comes out strongly highlighted.

Avaliação da expressão do gene BDNF em doenças neurodegenerativas

O Factor Neurotrófico Derivado do Cérebro (BDNF) está associado a processos de crescimento, diferenciação e sobrevivência das células neuronais. A expressão diferencial do BDNF, particularmente no hipocampo, está relacionada com a manifestação clínica de algumas doenças do foro psiquiátrico e cognitivo como a doença de Huntington, Alzheimer, depressão e esquizofrenia. Este trabalho pretende dar conhecimento das técnicas utilizadas para avaliar a expressão do gene BDNF. As técnicas de ELISA, IHC e Western blot, por permitirem a avaliação precisa da expressão de BDNF, são úteis para uma melhor compreensão, diagnóstico e tratamento de algumas doenças neurodegenerativas.

In vivo and in vitro effects of RAD001 on bladder cancer

Objective: To evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines. Methods: ICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms. Results: The incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G0/G1 phase arrest, as well as a statistically significant induction of apoptosis (P 0.001), was only observed in the 5637 cell line. Conclusion: RAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.

Neurokinin-1 receptor, a new modulator of lymphangiogenesis in obese-asthma phenotype

Aims Obesity and asthma are widely prevalent and associated disorders. Recent studies of our group revealed that Substance P (SP) is involved in pathophysiology of obese-asthma phenotype in mice through its selective NK1 receptor (NK1-R). Lymphangiogenesis is impaired in asthma and obesity, and SP activates contractile and inflammatory pathways in lymphatics. Our aim was to study whether NK1-R expression was involved in lymphangiogenesis on visceral (VAT) and subcutaneous (SAT) adipose tissues and in the lungs, in obese-allergen sensitized mice. Main methods Diet-induced obese and ovalbumin (OVA)-sensitized Balb/c mice were treated with a selective NK1-R antagonist (CJ 12,255, Pfizer Inc., USA) or placebo. Lymphatic structures (LYVE-1 +) and NK1-R expression were analyzed by immunohistochemistry. A semi-quantitative score methodology was used for NK1-R expression. Key findings Obesity and allergen-sensitization together increased the number of LYVE-1 + lymphatics in VAT and decreased it in SAT and lungs. NK1-R was mainly expressed on adipocyte membranes of VAT, blood vessel areas of SAT, and in lung epithelium. Obesity and allergen-sensitization combined increased the expression of NK1-R in VAT, SAT and lungs. NK1-R antagonist treatment reversed the effects observed in lymphangiogenesis in those tissues. Significance The obese-asthma phenotype in mice is accompanied by increased expression of NK1-R on adipose tissues and lung epithelium, reflecting that SP released during inflammation may act directly on these tissues. Blocking NK1-R affects lymphangiogenesis, implying a role of SP, with opposite physiological consequences in VAT, and in SAT and lungs. Our results provide a clue for a novel SP role in the obese-asthma phenotype.

Imunohistoquímica no diagnóstico da glomerulonefrite membranosa: estudo comparativo de binómios cromogênio + coloração de contraste

A glomerulonefrite membranosa faz parte das doenças glomerulares que provocam glomerulonefrite crônica, apresentando-se como uma das causas da doença renal terminal. As técnicas de imunofluorescência são o gold standard no estudo imunológico desta patologia em biópsia renal, através da deteção de imunocomplexos (e.g. IgG e C3) e do seu padrão de distribuição granular característico. No entanto, a imunofluorescência não permite uma contextualização histológica e os fluorocromos utilizados possuem um reduzido tempo de atividade, ao contrário das técnicas imunoenzimáticas que utilizam cromogénios coloridos precipitados que permitem a obtenção de uma marcação permanente e a sua contextualização histológica por via da utilização de eficientes colorações de contraste. Com a finalidade de contribuir para a qualidade do diagnóstico da glomerulonefrite membranosa, em biópsias renais, procurou-se, com esta pesquisa, identificar uma técnica imunoenzimática, através da conjugação entre diferentes cromogênios e colorações de contraste, que permita a deteção de depósitos de IgG e C3, com padrão granular. Foram constituídos diferentes binômios cromogênio + coloração, com os cromogênios 3,3›- Diaminobenzidine Tetrahydrochloride e 3-Amino-9-ethylcarbazole e as colorações Periodic Acid Schiff, Periodic Acid Methenamine Silver e Hematoxilina. Foram utilizadas 72 secções de tecido provenientes de seis de casos de biópsias renais com diagnóstico de glomerulonefrite membranosa, fixados em formalina a 10% e incluídos em parafina. A recolha de dados foi realizada por observação microscópica com preenchimento de uma grelha de classificação dos parâmetros: preservação da morfologia, intensidade da marcação específica, quantidade relativa de estruturas marcadas, marcação inespecífica/fundo, contraste e padrão da marcação, que permitiu a classificação dos binómios estudados num score quantitativo de 0-100 pontos. O binômio que apresentou melhores resultados foi 3-Amino-9-ethylcarbazole + Hematoxilina (score 71,81) e o binômio 3,3›- Diaminobenzidine Tetrahydrochloride+Periodic Acid Methenamine Silver (score 7,81), apresentou os piores resultados. O resultado do teste Kruskal-Wallis indica-nos a presença de diferenças estatísticas entre os binómios em estudo (p=0,000). A Hematoxilina pode ser considerada a coloração mais eficaz, pois cumpriu a sua função de auxiliar e facilitar a observação do tipo de padrão com os dois cromogênios utilizados. O cromogênio 3-Amino-9-ethylcarbazole apresentou resultados semelhantes aos produzidos pelo 3,3›-Diaminobenzidine Tetrahydrochloride, no entanto, permitiu identificar em todos os casos o padrão granular de imunomarcação, ao contrário do que aconteceu com este último.

Influência do peróxido e do uso de diferentes substâncias de combate à dor na inflamação e na expressão de neuropeptídeos pró-inflamatórios após o tratamento clareador

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Macrophages as biomarkers in BCG treatment response in bladder cancer

Bladder cancer is a common urologic cancer and the majority has origin in the urothelium. Patients with intermediate and high risk of recurrence/progression bladder cancer are treated with intravesical instillation with Bacillus Calmette-Guérin, however, approximately 30% of patients do not respond to treatment. At the moment, there are no accepted biomarkers do predict treatment outcome and an early identification of patients better served by alternative therapeutics. The treatment initiates a cascade of cytokines responsible by recruiting macrophages to the tumor site that have been shown to influence treatment outcome. Effective BCG therapy needs precise activation of the Th1 immune pathway associated with M1 polarized macrophages. However, tumor-associated macrophages (TAMs) often assume an immunoregulatory M2 phenotype, either immunosuppressive or angiogenic, that interfere in different ways with the BCG induced antitumor immune response. The M2 macrophage is influenced by different microenvironments in the stroma and the tumor. In particular, the degree of hypoxia in the tumors is responsible by the recruitment and differentiation of macrophages into the M2 angiogenic phenotype, suggested to be associated with the response to treatment. Nevertheless, neither the macrophage phenotypes present nor the influence of localization and hypoxia have been addressed in previous studies. Therefore, this work devoted to study the influence of TAMs, in particular of the M2 phenotype taking into account their localization (stroma or tumor) and the degree of hypoxia in the tumor (low or high) in BCG treatment outcome. The study included 99 bladder cancer patients treated with BCG. Tumors resected prior to treatment were evaluated using immunohistochemistry for CD68 and CD163 antigens, which identify a lineage macrophage marker and a M2-polarized specific cell surface receptor, respectively. Tumor hypoxia was evaluated based on HIF-1α expression. As a main finding it was observed that a high predominance of CD163+ macrophage counts in the stroma of tumors under low hypoxia was associated with BCG immunotherapy failure, possibly due to its immunosuppressive phenotype. This study further reinforces the importance the tumor microenvironment in the modulation of BCG responses.

The predominance of M2-polarized macrophages in the stroma of low-hypoxic bladder tumors is associated with BCG immunotherapy failure

OBJECTIVE: Bacillus Calmette-Guérin (BCG) immunotherapy is the gold standard treatment for superficial bladder tumors with intermediate/high risk of recurrence or progression. However, approximately 30% of patients fail to respond to the treatment. Effective BCG therapy needs precise activation of the type 1 helper cells immune pathway. Tumor-associated macrophages (TAMs) often assume an immunoregulatory M2 phenotype and may directly interfere with the BCG-induced antitumor immune response. Thus, we aim to clarify the influence of TAMs, in particular of the M2 phenotype in stroma and tumor areas, in BCG treatment outcome. PATIENTS AND METHODS: The study included 99 patients with bladder cancer treated with BCG. Tumors resected before treatment were evaluated using immunohistochemistry for CD68 and CD163 antigens, which identify a lineage macrophage marker and a M2-polarized specific cell surface receptor, respectively. CD68+ and CD163+ macrophages were evaluated within the stroma and tumor areas, and high density of infiltrating cells spots were selected for counting. Hypoxia, an event known to modulate macrophage phenotype, was also assessed through hypoxia induced factor (HIF)-1α expression. RESULTS: Patients in whom BCG failed had high stroma-predominant CD163+ macrophage counts (high stroma but low tumor CD163+ macrophages counts) when compared with the ones with a successful treatment (71% vs. 47%, P = 0.017). Furthermore, patients presenting this phenotype showed decreased recurrence-free survival (log rank, P = 0.008) and a clear 2-fold increased risk of BCG treatment failure was observed in univariate analysis (hazard ratio = 2.343; 95% CI: 1.197-4.587; P = 0.013). Even when adjusted for potential confounders, such as age and therapeutic scheme, multivariate analysis revealed 2.6-fold increased risk of recurrence (hazard ratio = 2.627; 95% CI: 1.340-5.150; P = 0.005). High stroma-predominant CD163+ macrophage counts were also associated with low expression of HIF-1α in tumor areas, whereas high counts of CD163+ in the tumor presented high expression of HIF-1α in tumor nests. CONCLUSIONS: TAMs evaluation using CD163 is a good indicator of BCG treatment failure. Moreover, elevated infiltration of CD163+ macrophages, predominantly in stroma areas but not in the tumor, may be a useful indicator of BCG treatment outcome, possibly owing to its immunosuppressive phenotype.

Neurokinin-1 receptor, a new modulator of lymphangiogenesis in obese-asthma phenotype

Aims: Obesity and asthma are widely prevalent and associated disorders. Recent studies of our group revealed that Substance P (SP) is involved in pathophysiology of obese-asthma phenotype in mice through its selective NK1 receptor (NK1-R). Lymphangiogenesis is impaired in asthma and obesity, and SP activates contractile and inflammatory pathways in lymphatics. Our aim was to study whether NK1-R expression was involved in lymphangiogenesis on visceral (VAT) and subcutaneous (SAT) adipose tissues and in the lungs, in obeseallergen sensitized mice. Main methods: Diet-induced obese and ovalbumin (OVA)-sensitized Balb/c mice were treated with a selective NK1-R antagonist (CJ 12,255, Pfizer Inc., USA) or placebo. Lymphatic structures (LYVE-1+) and NK1-R expression were analyzed by immunohistochemistry. A semi-quantitative score methodology was used for NK1-R expression. Key findings: Obesity and allergen-sensitization together increased the number of LYVE-1+ lymphatics in VAT and decreased it in SAT and lungs. NK1-R was mainly expressed on adipocyte membranes of VAT, blood vessel areas of SAT, and in lung epithelium. Obesity and allergen-sensitization combined increased the expression of NK1-R in VAT, SAT and lungs. NK1-R antagonist treatment reversed the effects observed in lymphangiogenesis in those tissues. Significance: The obese-asthma phenotype in mice is accompanied by increased expression of NK1-R on adipose tissues and lung epithelium, reflecting that SP released during inflammation may act directly on these tissues. Blocking NK1-R affects lymphangiogenesis, implying a role of SP, with opposite physiological consequences in VAT, and in SAT and lungs. Our results provide a clue for a novel SP role in the obese-asthma phenotype.

Amplificação em imunohistoquímica: estudo comparativo de sistemas de polímeros

Desde o início da utilização da imunohistoquímica em anatomia patológica, um dos objetivos tem sido detetar as quantidades mais ínfimas de antigénio, tornando-o visível ao microscópio ótico. Vários sistemas de amplificação têm sido aplicados de forma a concretizar este objetivo, tendo surgido um grupo genérico de métodos simples e que apresentam uma amplificação superior: são os denominados métodos do polímero indireto. Tendo em conta a variedade de métodos disponíveis, o autor propõe-se a comparar a qualidade de quatro sistemas de amplificação, que recorrem ao método do polímero indireto com horseradish peroxidase (HRP). Foram utilizadas lâminas de diferentes tecidos, fixados em formol e incluídos em parafina, nos quais se procedeu à identificação de 15 antigénios distintos. Na amplificação recorreu-se a quatro sistemas de polímero indireto (Dako EnVision+ System – K4006; LabVision UltraVision LP Detection System – TL-004-HD; Leica NovoLink – RE7140-k; Vector ImmPRESS Reagent Kit – MP-7402). A observação microscópica e classificação da imunomarcação obtida foram feitas com base num algoritmo que enquadra intensidade, marcação específica, marcação inespecífica e contraste, num score global que pode tomar valores entre 0 e 25. No tratamento dos dados, para além da estatística descritiva, foi utilizado o teste one-way ANOVA com posthoc de tukey (alfa=0.05). O melhor resultado obtido, em termos de par média/desvio-padrão, dos scores globais foi o do NovoLink (22,4/2,37) e o pior foi o do EnVision+ (17,43/3,86). Verificou-se ainda que existe diferença estatística entre os resultados obtidos pelo sistema NovoLink e os sistemas UltraVision (p=.004), ImmPRESS (p=.000) e EnVision+ (p=.000). Concluiu-se que o sistema que permitiu a obtenção de melhores resultados, neste estudo, foi o Leica NovoLink.

Serological and infection statuses of dogs from a visceral leishmaniasis-endemic area

OBJECTIVE This study investigated the serological status of dogs living in a visceral leishmaniasis-endemic area and its correlation with the parasitological condition of the animals.METHODS Canine humoral response was evaluated using the sera of 134 dogs by enzyme-linked immunosorbent assay and immunohistochemistry to detect parasites in the skin, lymph node, and spleen of the animals. The specific antibodies investigated were IgG, IgG1, IgG2, and IgE.RESULTS According to the parasitological, laboratory, and clinical findings, the dogs were placed into one of four groups: asymptomatic with (AP+, n = 21) or without (AP-, n = 36) Leishmania tissue parasitism and symptomatic with (SP+, n = 52) or without (SP-, n = 25) parasitism. Higher IgG and IgE levels were positively correlated with the infection condition and parasite load, but not with the clinical status. In all groups, total IgG was the predominant antibody, which occurred at the expense of IgG2 instead of IgG1. Most of the infected dogs tested positive for IgG (SP+, 98.1%; AP+, 95.2%), whereas this was not observed with IgE (SP+, 80.8%; AP+, 71.2%). The most relevant finding was the high positivity of the uninfected dogs for Leishmania-specific IgG (SP-, 60.0%; AP-, 44.4%), IgE (SP-, 44.0%; AP-, 27.8%), IgG1 (SP-, 28.0%; AP-, 22.2%), and IgG2 antibodies (SP-, 56.0%; AP-, 41.7%).CONCLUSIONS The serological status of dogs, as determined by any class or subclass of antibodies, did not accurately distinguish dogs infected with L. (L.) infantum chagasifrom uninfected animals. The inaccuracy of the serological result may impair not only the diagnosis, but also epidemiological investigations and strategies for visceral leishmaniasis control. This complex serological scenario occurring in a visceral leishmaniasis-endemic area highlights the challenges associated with canine diagnosis and points out the difficulties experienced by veterinary clinicians and coordinators of control programs.

Répartition des foraminifères benthiques dans les gisements de surface du Miocène d'Aquitaine (SW de la France)

The Aquitaine Basin (southwestern France) is known since long ago for its richness in marine miocene deposits of various facies. A few stratotypes concerning this period have been described in the investigated area. The stratigraphical framework has been recently revised and the study of new exposures completes our knowledge on these levels. In the present work, the authors produce a biostratigraphical distribution of about 160 species (larger and smaller foraminifera), found in the surface exposures of Aquitaine, from the topmost Oligocene (Chattian) through to Middle Miocene (including Serravallian). As a rule, the common species without significant ranges have not bcen mentioned. The microfaunas of several exposures have been thoroughly revised, which has allowed to precise the distribution of many species and induced a few modifications of the results previously produced. Synonymy problems and new taxonomical revisions have been taken into account. Of course, this work will be probably submitted to some changes according to new research on the already known exposures or other more recently discovered.