Molluscicidal potential of Heterorhabditis baujardi (Rhabditida: Heterorhabditidae), strain LPP7, on Lymnaea columella (Gastropoda: Pulmonata): An alternative for biological control of fasciolosis.

This study elucidated for the first time, under laboratory conditions, the susceptibility of Lymnaea columella to infective juveniles of Heterorhabditis baujardi LPP7. Exposure to the nematodes induced an average mortality rate of 66.66% in the population of L. columella, with the highest values attained from the second week after exposure onward. In addition, all the reproductive parameters analyzed (total number of eggs, number of egg masses, number of eggs laid/snail, embryo hatching rate and content of galactogen stored in the albumen gland) changed as a result of the infection. The results indicate the occurrence of the phenomenon of parasitic castration in L. columella infected by H. baujardi LPP7, probably through depletion of energy reserves such as galactogen, necessary to meet the intense metabolic demands of the nematode's larval stages. Finally, histopathological analysis demonstrated an intense process of cell disorganization, characterized by the occurrence of granulomatous inflammatory reactions in tissues of exposed snails, induced by the spoliative action of the bacteria/nematode. The results suggest the use of H. baujardi LPP7 as an alternative for biological control of the population of this intermediate host, and thus of the diseases in whose epidemiological chain it participates, especially fasciolosis, in line with the recommendations of the World Health Organization (WHO).

Treatment of experimental esophagogastric myotomy with bone marrow mesenchymal stem cells in a rat model.

BACKGROUND: Over the last 15 years, many studies demonstrated the myogenic regenerative potential of bone marrow mesenchymal stem cells (BM-MSC), making them an attractive tool for the regeneration of damaged tissues. In this study, we have developed an animal model of esophagogastric myotomy (MY) aimed at determining the role of autologous MSC in the regeneration of the lower esophageal sphincter (LES) after surgery. METHODS: Syngeneic BM-MSC were locally injected at the site of MY. Histological and functional analysis were performed to evaluate muscle regeneration, contractive capacity, and the presence of green fluorescent protein-positive BM-MSC (BM-MSC-GFP(+) ) in the damaged area at different time points from implantation. KEY RESULTS: Treatment with syngeneic BM-MSC improved muscle regeneration and increased contractile function of damaged LES. Transplanted BM-MSC-GFP(+) remained on site up to 30 days post injection. Immunohistochemical analysis demonstrated that MSC maintain their phenotype and no differentiation toward smooth or striated muscle was shown at any time point. CONCLUSIONS & INFERENCES: Our data support the use of autologous BM-MSC to both improve sphincter regeneration of LES and to control the gastro-esophageal reflux after MY.

Coexistence of primary sclerosing cholangitis in a patient with myasthenia gravis.

Myasthenia gravis (MG) is an immune-mediated disease that compromises the postsynaptic membrane of the neuromuscular junction. Primary sclerosing cholangitis (PSC) is considered an immune-mediated cholestatic liver disease. Both MG and PSC include an autoimmune pathogenesis, so there is some evidence that patients with MG or PSC have a higher risk of developing autoantibodies and other immune disorders than normal controls, but the coexistence of these two disorders has never been documented. We report a 40-year-old woman who presented with MG when she was 20 years old and developed PSC 20 years after a thymectomy. Liver biochemistry revealed cholestasis. Magnetic resonance imaging showed multifocal strictures and beads involving the intrahepatic bile ducts. A liver biopsy confirmed sclerosing cholangitis. Serological analysis demonstrated positive autoantibodies (Anti-nuclear antibodies, anti-smooth muscle antibodies). Repetitive stimulation had a decremental response, and antibodies to acetylcholine receptors were detectable. To our knowledge, this is the first case of PSC in a patient with MG. The main characteristics of both MG and PSC combination are discussed.

Molecular characterisation of congenital myasthenic syndromes in Southern Brazil.

OBJECTIVE: To perform genetic testing of patients with congenital myasthenic syndromes (CMS) from the Southern Brazilian state of Parana. PATIENTS AND METHODS: Twenty-five CMS patients from 18 independent families were included in the study. Known CMS genes were sequenced and restriction digest for the mutation RAPSN p.N88K was performed in all patients. RESULTS: We identified recessive mutations of CHRNE in ten families, mutations in DOK7 in three families and mutations in COLQ, CHRNA1 and CHRNB1 in one family each. The mutation CHRNE c.70insG was found in six families. We have repeatedly identified this mutation in patients from Spain and Portugal and haplotype studies indicate that CHRNE c.70insG derives from a common ancestor. CONCLUSIONS: Recessive mutations in CHRNE are the major cause of CMS in Southern Brazil with a common mutation introduced by Hispanic settlers. The second most common cause is mutations in DOK7. The minimum prevalence of CMS in Parana is 0.18/100 000.

Lymphatic vessels in human eyelids: an immunohistological study in dermatochalasis and chalazion.

We investigated lymphatic morphology and expression of endothelin (ET-1) axis molecules in human eyelids affected by an inflammatory state (chalazion) and an age-related degenerative condition (dermatochalasis). Lymphatics were immunohistologically detected by D2-40/LYVE-1 staining. Absorbing lymphatic vessels were localized in papillary dermis and around skin appendages with distinctive morphology. In chalazion, D2-40 reactive flattened lymphatic profiles were compressed by inflammatory infiltrate; in dermatochalasis, large fully opened lymphatics were observed, with a significantly wider total area (lymphatic lumina/200x field; p < 0.05). The lymphatic density (number/200x field) in the two groups was within the same range. Lymphatic dilation is possibly dependent on reduction and fragmentation of the dermal elastic network as well as of oxytalanic fibers in the papillary dermis of dermatochalasis, as shown by Weigert's reaction. Multifunctional peptide ET-1, involved in vasomotion, inflammation and connective proliferation, was faintly and discontinuously localized on lymphatics, as was its type A receptor. In contrast, the consistent expression of type B receptor indicates that lymphatic endothelium is a physiological target for ET-1, whose effects are modulated by multiple pathophysiological conditions. Thus, vasoactive factors play a role in the physiology of richly vascularized eyelids, and therefore, morphofunctional characterization of lymphatic vessels may be useful in suggesting treatment options.

Hamartoma fibrolipomatoso de nervo mediano: relato de caso / Fibrolipomatous hamartoma of the median nerve: case report

Fibrolipomatous hamartoma is a rare benign neoplasm that in some cases is associated with macrodactylia. We describe a 31-year-old man who had a tissue enlargement in the wrist, second and third fingers of the left hand since infancy. At 23-years-old he began with continuous, progressive and high intensity pain that occurred more frequently at night, localized in the left hand. It was associated with paraesthesias and hypostesias predominantly at the fingers described above. Investigation with X-ray, ultrasonography, electrodiagnosis, magnetic resonance image of the left wrist and hand showed carpal tunnel syndrome with macrodactylia by fibrolipomatous hamartoma of the median nerve. The patient did not a have good response to clinical therapy, so he was submitted to a surgical decompression of the left carpal tunnel, and after three months of follow up is asymptomatic.

O hamartoma fibrolipomatoso é neoplasia benigna rara que em alguns casos esta associada com macrodactilia. Descrevemos o caso de homem de 31 anos que apresentava desde o nascimento aumento de volume em região de punho, segundo e terceiro quirodáctilos da mão esquerda. Aos 23 anos iniciou dor contínua, de forte intensidade, predominante no período noturno e de evolução progressiva em mão esquerda. Associada à dor havia hipoestesia e parestesias de predomínio nos segundo e terceiro quirodáctilos esquerdos. A investigação complementar com radiografia, ultrassonografia, estudo eletrofisiológico e ressonância magnética de mão e punho esquerdos confirmaram a suspeita de síndrome do túnel do carpo secundária a macrodactilia com hamartoma fibrolipomatoso do nervo mediano. O paciente foi submetido à descompressão cirúrgica do túnel do carpo esquerdo devido a ausência de resposta ao tratamento clínico e evoluiu com melhora dos sintomas em avaliação após três meses do procedimento.

Estudo da condução nervosa motora na doença de McArdle: relato de caso / Motor nerve conduction study in McArdle disease: case report

McArdle disease (glycogenosis type V) is a metabolic myopathy with symptoms of exercise intolerance caused by deficiency of the enzyme myophosphorylase. In these patients, the motor nerve conduction studies after a short period of maximal voluntary muscle contraction or repetitive stimulation reveals characteristic findings of the disease. A 37-year-old man presented symptoms of exercise intolerance, muscular fatigue and cramps in the beginning of the physical activity with [quot ]second wind[quot ] phenomenon. The motor nerve conduction studies after a voluntary contraction of 30 and 90 seconds presented decrease in the amplitude of the compound muscle action potential in median, ulnar and deep peroneal nerves; and decrement after 200 stimulation at the 40 Hz in deep peroneal nerve. The electromyography presented myopathic pattern and during the ischemic exercise electric silence was not proven. The characteristic of electrophysiological studies are discussed with emphasis at the importance of the motor nerve conduction studies in the patients with suspicion of metabolic myopathy.

A doença de McArdle (glicogenose tipo V) é miopatia metabólica com sintomas de intolerância ao exercício, causados pela deficiência da enzima miofosforilase. Nesses pacientes, o estudo da condução nervosa motora após período de esforço muscular máximo ou ao estímulo repetitivo pode revelar achados característicos da doença. Descrevemos o caso de um homem de 37 anos com sintomas de intolerância aos exercícios, fadiga muscular e cãibras no início da atividade física com a presença do fenômeno de "second wind". O estudo da condução nervosa motora apresentava redução na amplitude do potencial de ação muscular composto após esforço de 30 e 90 segundos em nervos mediano, ulnar e fibular profundo e decremento após 200 estímulos a 40 Hz em nervo fibular profundo. A eletromiografia de agulha apresentava padrão miopático e durante o exercício isquêmico não se evidenciou silêncio elétrico. Discutimos as características eletrofisiológicas enfatizando a importância do estudo da condução nervosa motora e teste de estimulação repetitiva nos pacientes com suspeita de miopatia metabólica.

Copper biosorption by Auricularia polytricha.

AIMS: The objective of the present study was to determine the optimum conditions for copper (Cu) biosorption by Auricularia polytricha mycelium in view of its immobilization in polyvinyl alcohol (PVA). METHODS AND RESULTS: The adsorption of Cu(II) onto A. polytricha was studied in batch with respect to initial pH, temperature, adsorption time, initial metal ion and biomass concentration. At optimal adsorption conditions, biomass was immobilized in PVA in column and a biosorption capacity of about 90% was obtained. CONCLUSIONS: Auricularia polytricha strain could successfully be used as Cu biosorbent. SIGNIFICANCE AND IMPACT OF THE STUDY: The low cost and simplicity of the technique make it suitable for the detoxification of contaminated effluents before their environmental discharge.

Mechanisms regulating c-met overexpression in liver-metastatic B16-LS9 melanoma cells.

Liver selected B16-LS9 melanoma cells show a dramatic overexpression of the proto-oncogene c-met, the cellular receptor for hepatocyte growth factor/scatter factor. As a consequence, c-met becomes constitutively active, and the cells become more responsive to hepatocyte growth factor stimulation. We have investigated the molecular mechanisms regulating c-met expression in both the parental line B16-F1, which has low expression levels, and the liver-specific B16-LS9, overexpressing c-met. Overexpression is observed at the protein and mRNA levels, however without further evidence of gene amplification or rearrangement. c-met promoter activity was higher in B16-LS9 than B16-F1 cells, and also a nuclear run-off showed higher transcription levels in B16-LS9 cells. Moreover, we found that c-met mRNA had a longer half-life in B16-LS9 cells, thus indicating also the involvement of post-transcriptional regulation mechanisms. Finally, we found evidence that autonomous activation of the melanocortin receptor-1 (MCR-1) is at least partially responsible for c-met upregulation in B16-LS9 cells, since treatment of the cells with a potent MSH antagonist (the agouti peptide) has strong down-regulatory effects.

A novel monoclonal antibody specific for lymphatic endothelium.

The difficulty of identifying and differentiating lymphatic and blood microvessels in tissue sections can be overcome by a monoclonal antibody specific for lymphatic endothelium. Unfortunately, the only known antibody also reacts with the endothelium of some blood vessels. The technique of double immunization (passive, with an antiserum to blood endothelium, and active, with a suspension of lymphatic endothelial cells) was, therefore, used to increase the chances of recognizing specific lymphatic antigens by the mouse immune system. The monoclonal antibody obtained, LyMAb, a G1 immunoglobulin, reacted strongly with the endothelium of bovine thoracic duct, mesenteric collecting vessels and lymphatic vessels of gallbladder and lymph nodes and moderately with those of the intestinal wall. Blood vessels (intercostal arteries, azygos vein and blood microvessels of all organs tested) were consistently negative. The antibody was species-specific and did not react with formalin-fixed, paraffin-embedded sections. Cross-reactivity was limited to some connective tissue fibres and scattered cells in the lymph node parenchyma, intestinal villi and hepatic lobules.

Regulation of c-met expression in B16 murine melanoma cells by melanocyte stimulating hormone.

B16 murine melanoma cells selected in vivo for enhanced liver metastatic ability (B16-LS9) show on the one hand an increased expression and constitutive activation of the proto-oncogene c-met (the receptor for hepatocyte growth factor/scatter factor), and on the other hand a more differentiated phenotype, when compared to the parental cell line, B16-F1. Following this observation, we have tried to establish whether there is a direct relationship between differentiation and c-met expression in B16 melanoma cells. Treatment of these cells with differentiating agents indicated that c-met expression was strongly induced by melanocyte stimulating hormone, while retinoic acid had almost no influence. c-met induction was triggered by engagement of the melanocortin receptor, cAMP elevation and PKA/PKC(&agr;) activation, as respectively shown by the effects of ACTH, cAMP elevating agents and specific PK inhibitors. Regulation of c-met expression via the melanocortin receptor and cAMP raises the intriguing possibility that autocrine and/or paracrine mechanisms acting in vivo on this circuit might influence (through c-met expression and activation) the metastatic behavior of these tumor cells, which we have shown to be dependent on their c-met expression.

Hepatocyte growth factor/scatter factor and hepatocytes are potent downregulators of tyrosinase expression in B16 melanoma cells.

Reiterated selection in vivo of B16 murine melanoma cells for enhanced liver metastatic ability yielded a cel line (B16-LS9) dramatically overexpressing a constitutively active hepatocyte growth factor/scatter factor (HGF/SF) receptor, the product of the c-met proto-oncogene. Most likely because of their overexpressing c-met, B16-LS9 cells appear to be more responsive than parental B16-F1 cells to HGF stimulation, in terms of motility, invasion, and growth. They are also more pigmented, and express higher levels of tyrosinase as compared to parental B1 6-F1 cells. Therefore, we set out to explore whether HGF/SF and the liver might influence the differentiation state of B1 6 cells. We found that HGF/SF and MSH, two factors which reportedly have a strong influence on the phenotype and the malignant behavior of melanoma cells, may act at different levels, and with opposite results, on the regulation of gene expression. In fact, while MSH induces, at the transcriptional level, an increase in the production of both c-met and tyrosinase, HGF/SF, in contrast, promotes a decrease in the expression of both c-met and tyrosinase, however at a posttranscriptional level. These two opposite effects can counter-balance each other, when the cells are treated with both factors at the same time, apparently through a mechanism involving MAP kinase activation. The effects were, however, additive when morphological changes were considered. Most intriguingly, we also describe a very strong downregulatory activity, limited to tyrosinase expression, by hepatocytes in coculture with B16 cells. This activity, also at the posttranscriptional level, is much stronger than that exerted by HGF/SF, and appears to be due to a labile soluble factor produced by the hepatocytes.

Influence of hepatocyte growth factor/scatter factor on the metastatic phenotype of B16 melanoma cells.

B16 melanoma cells selected in mice for liver-specific metastasis (B16-LS9) overexpress a constitutively active form of the hepatocyte growth factor/scatter factor receptor (HGF/SFr), the product of the c-met proto-oncogene. HGF/SF can affect both invasion and growth of receptive cells. In fact, we show that overexpression of c-met in B16-LS9 cells results in a higher inducibility of two different proteolytic activities (uPA and gelatinase), in correlation with a stronger invasive and motility response to HGF/SF treatment. However, HGF/SF treatment inhibits growth of B16 cells, which might appear in contradiction with the observation that c-met overexpression and constitutive activation seems to be required for efficient liver colonization. However, this apparent discrepancy is resolved by the finding that liver-derived, but not lung-derived factor(s), can efficiently rescue B16-LS9 cells from the growth inhibitory effects of HGF/SF, while not changing their motility response. Therefore, overexpression of c-met in B16-LS9 cells might give a specific advantage in liver colonization, because specifically at this site B16-LS9 cells can take full advantage of the positive effects exerted by HGF/SF stimulation on motility and invasion, while the negative effects on growth are counteracted by other paracrine factor(s).

C-met activation is necessary but not sufficient for liver colonization by B16 murine melanoma cells.

Metastasis to the liver is a frequent event in clinical oncology, the molecular mechanisms of which are not fully understood. We have recently reported a consistent overexpression of c-met in B16 melanoma cells selected in vivo for enhanced liver metastatic ability. In this study we address the question as to whether constitutive activation of c-met is a necessary and sufficient condition for enhanced liver colonization by B16 melanoma cells. Different levels of c-met expression and/or activation in B16 cells were achieved by subcloning, or by c-DNA transfection with either HGF/SF or the oncogenic form of c-met (tpr-met). Metastatic ability of the different populations was then evaluated in vivo by the lung colonization (experimental metastasis) assay. Results indicate that c-met (but not tpr-met) activation in B16 melanoma cells may increase their liver colonizing potential, probably by enhancing motility and invasion in response to paracrine interactions with its ligand. C-met expression per se, however, is not able to change the organ specificity of the cells. C-met activation appears instead to be required at later stages of liver colonization by B16 melanoma cells, in order to enhance their site-specific metastatic ability.

Human bone cells stimulate the growth of human breast carcinoma cells.

Human breast cancer cells were cultured together with their metastatic target, bone tissue, to analyze possible growth promotion effects. The coculture of human osteosarcoma cells (TE-85) with human mammary carcinoma cells (ZR-75.1) resulted in up to 8.4-fold stimulation of proliferation of the breast tumor cells. Cell contact of the two cultures was permitted through the channels of Nuclepore filters. However, physical contact turned out not to be necessary, since the proliferative stimulus was also mediated by a bone-derived diffusible factor. Conditioned medium (CM), collected from human primary bone cultures, enhanced the rate of proliferation of several breast tissue cell lines (ZR-75.1, BT-20, HBL-100), while some lines were not affected by osteoblast CM. Breast tissue lines responding to bone CM express low to intermediate levels of the c-erbB-2 gene, in contrast to nonstimulated lines, which overexpress the gene. Recent observations of metastatic spread in breast cancer patients suggest a distinctive pattern of secondary tumor distribution in association with c-erbB-2 protein expression. Bone tissue seems to be a preferential target for metastases of c-erbB-2-negative breast tumors.

Glioma cells induce gamma-glutamyl transpeptidase activity in cultured blood but not lymphatic endothelial cells.

Glial cells have been shown to induce enzyme activities, which are characteristically high in brain capillaries, in cerebral and non cerebral endothelial cells in culture. We evaluated the activity of gamma-glutamyl transpeptidase (gamma GT) in freshly isolated (ex vivo) bovine blood (BEC) and lymphatic (LEC) endothelial cells. We also tested the effect of C6 glioma cell conditioned medium (GCM) on gamma GT activity of BEC and LEC in primary culture. Ex vivo BEC had a high gamma GT activity, only 9% of which was retained in culture. After exposure to GCM, however, gamma GT activity of cultured BEC was twofold higher than with control medium. By contrast gamma GT activity was extremely low in ex vivo LEC and did not significantly increase in cultured LEC exposed to GCM. These data show that the basal levels of gamma GT are markedly different in BEC and LEC and also that, unlike BEC, LEC are not capable of producing more gamma GT in response to glial stimulation.

Constitutive activation of c-Met in liver metastatic B16 melanoma cells depends on both substrate adhesion and cell density and is regulated by a cytosolic tyrosine phosphatase activity.

Serial selection in vivo for liver colonization of B16 murine melanoma cells consistently resulted in cell lines expressing elevated amounts of the hepatocyte growth factor/scatter factor receptor (c-Met), which is constitutively activated in the absence of its cognate ligand. In this paper we present evidence suggesting that c-Met constitutive activation in liver-specific B16 melanoma cells depends on both receptor concentration on the cell surface and a cytosolic tyrosine phosphatase activity. In fact, c-Met constitutive activation is suddenly lost upon detachment of the cells from the substrate and is dramatically decreased in adherent cells plated at low density. The loss of tyrosine phosphorylation of c-Met in suspension appears to depend, at least partly, on an increased cytosolic tyrosine phosphatase activity. Instead, lower activation of c-Met at low density mostly results from a decrease in receptor concentration on the membrane. Moreover, we show that c-Met activation does not occur homogeneously on the surface of adherent cells. In fact, receptor concentration and activation appear to be higher on the ventral surface (adherent to the substrate) than on the apical surface. Upon detachment, compartmentalization is lost, leading to a decrease in average receptor density on the plasma membrane and hence to a lower activation.

Expression of constitutively activated hepatocyte growth factor/scatter factor receptor (c-met) in B16 melanoma cells selected for enhanced liver colonization.

The murine melanoma B16-LS9 cell line was obtained after repeated passages in vivo through the liver of syngeneic mice, and shows an enhanced ability to colonize the liver after intravenous inoculation when compared to its parental, unselected counterpart B16-F1. We have previously shown that paracrine growth effects mainly account for better growth of B16-LS9 in the liver than at other sites, and more recently we reported hepatic transferrin as a factor contributing to efficient growth in the liver. Here we show that increased expression of constitutively activated c-met (the receptor for Hepatocyte Growth Factor/Scatter Factor) consistently occurs during selection of B16 cells through the liver. Constitutive activation of c-met seems to follow its own overexpression and not to depend on an autocrine mechanism. As a consequence, liver-selected B16 melanoma cells have higher tyrosine-kinase activity and higher amounts of tyrosine-phosphorylated proteins than parental B16-F1 or lung-specific B16-F10 cells. Overexpression of constitutively activated c-met enhances motility and invasiveness of B16-LS9 cells, presumably favoring their colonization efficiency in vivo. However, whether levels of c-met expression also determine the organ-specificity of B16 melanoma cells needs further clarification.

Influence of soil characteristics on the clastogenic activity of maleic hydrazide in root tips of Vicia faba.

The clastogenic activity of the herbicide maleic hydrazide (MH) in Vicia faba seedlings grown in different soils was studied. In the first series of experiments the seedlings were treated with MH in nine soils of different selected properties. The second experimental protocol provided for a growth step in each of the nine soils under study and then for the treatment of the seedlings with MH in a sandy soil. The results obtained show that less clastogenic damage (induction of micronuclei and aberrant anatelophases) was observed in the seedlings treated in the soils with a high content of organic and clay-type colloids. A growth step carried out in the organic soils, before the treatment with MH, was observed to reduce the genotoxic effects of the herbicide when the seedlings were treated in a sandy soil. These findings suggest that some components of soil organic matter, such as fulvic and humic acids, may be absorbed by the plant roots and so carry out an antimutagenic activity against MH within the plant.