Sublethal and lethal toxicity in juvenile Senegal sole (Solea senegalensis) exposed to copper: a preliminary toxicity range-finding test.

Static bioassay test for acute toxicity of copper in Senegal sole juveniles (Solea senegalensis) was conducted. The 96h LC(50) value of metal was found to be 0.32mg/L Cu. The intensity of histological alterations was increased gradually with the copper concentration and the exposure time. Numerous aneurysms, hypertrophy, hyperplasia and fusion of the lamellar epithelium in gills and a disorganization of the hepatic parenchyma and vacuolization in liver were common lesions in S. senegalensis juveniles exposed to copper. The results of the study indicate the sensitivity of juvenile S. senegalensis to copper (II), the persistence of sublethal effects and histology as a tool capable of revealing the sublethal effects of heavy metals on the environment and aquatic biota.

Ecotoxicity and biodegradability of an alkyl ethoxysulphate surfactant in coastal waters.

Alkyl ethoxysulphates (AES) are anionic surfactants widely used in numerous commercial and industrial applications. In spite of the high AES volume consumption a few data concerning the occurrence, fate and effects of AES in marine environments are reported in literature. The objective of this study is to evaluate the biodegradability and toxicity of AES in pristine sea water. Ultimate biodegradation was studied according to the guideline 835.3160 "Biodegradability in sea water" proposed by the United States Environmental Protection Agency (USEPA). Acute toxicity of AES was studied to the microalgae Nannochloropsis gaditana, Isochrysis galbana, Chaetoceros gracilis, Dunaliella salina and Tetraselmis chuii and the invertebrate Artemia franciscana, using culture growth inhibition and death, respectively, as effect criteria. During the degradative process two different stages were observed, which were better described with the first order and logistic kinetic models, respectively. Lag times were 3.3 (stage A) and 26.5 (stage B) days whereas half-lives were 18.6 (stage A) and 49.8 (stage B) days. AES inhibited the microalgae growth, with 96-h EC50 values ranging from 4.68 g L(-1) for D. salina to 24.02 mg L(-1) for I. galbana. Mean 48- and 72-h LC50 values for A. franciscana were 38.30 and 23.92 mg L(-1), respectively. The results indicate an extensive biodegradability of AES in sea water, although at a very slow rate. Acute toxicity was highly dependent on the species tested, being the green alga D. salina the most affected organism. The present study provides relevant data concerning the biodegradability and adverse effects of an AES surfactant on marine organisms, which are useful to establish water quality criteria in a regulatory framework.

Results of liver transplantation in patients with previous portosystemic shunts.

INTRODUCTION: Although liver transplantation is performed successfully in some patients with previous portosystemic shunts (PSS), these surgical procedures have been considered a relative contraindication for orthotopic liver transplantation (OLT). We aimed to determine whether a previous PSS worsens the prognosis of patients who undergo OLT. PATIENTS AND METHODS: Between March 1986 and October 2003, 520 liver transplants were performed in 467 patients in our center. Thirteen patients had undergone a PSS before OLT. The types of PSS were: portocaval (n = 8), splenorenal (n = 3), mesocaval (n = 1), and portoatrial (n = 1). We compared patients with previous PSS (cases) and the three patients with an OLT immediately before each case (controls). We analyzed the following variables: age, Child-Pugh stage, pretransplant liver disease, surgical times, transfusion requirements, infections, intensive care unit (ICU) stay, postoperative evolution, and survival. RESULTS: Age, Child-Pugh stage, and pretransplant liver disease were similar in both groups. There were no statistical differences in age, surgical times, ischemia time, anhepatic phase, transfusion requirements, ICU stay, infections, or hospital stay. The postoperative course was similar in both groups. Long-term survival was 84.62% in cases versus 78.5% in controls. CONCLUSIONS: Previous PSS should not be considered a contraindication for liver transplantation, even though this group of patients involves a special surgical challenge.

[Cerebral infarct secondary to carotid artery dissection]. / Infarto cerebral isquémico por disección de arteria carótida interna.

Dissection of the internal carotid artery is an important cause of ischemic stroke in children and young patients. Trauma and/or an underlying structural defect of the arterial wall have been suggested to be predisposing factors. The typical patient presents with ipsilateral headache or neck pain, ipsilateral Horner's syndrome and delayed ischemic symptoms. Diagnosis is given by ultrasound, transcranial Doppler, magnetic resonance imaging, magnetic resonance angiography and conventional angiography. Treatment of this type of injury includes anticoagulation therapy, antiplatelet therapy and surgery. We report a 14-year-old boy with internal carotid artery dissection who presented with ischemic stroke.

Infarto cerebral isquémico por disección de arteria carótida interna / Ischemic stroke secondary to carotid artery dissection

La disección de la arteria carótida interna es una causa importante de ictus isquémico en niños y pacientes jóvenes. En la patogenia se han implicado traumatismos y/o un posible defecto estructural de la pared arterial. Las manifestaciones clínicas típicas incluyen cefalea o dolor de cuello y síndrome de Horner en el lado de la disección, con la aparición después de síntomas isquémicos cerebrales. La ecografía, el Doppler transcraneal, la resonancia magnética (RM), la angiorresonancia y la angiografía proporcionan el diagnóstico. Las opciones de tratamiento comprenden anticoagulantes, antiagregantes plaquetarios y cirugía. Presentamos un adolescente de 14 años con un ictus isquémico secundario a disección de la arteria carótida interna. (AU)

Temporal bone dissection: a possible route for prion transmission?

The aim of this study was to determine whether neural tissue is present in the bone 'dust' given off during temporal bone drilling. Bone 'dust' from three temporal bone dissections was collected and examined. Evidence of neural tissue was present in two out of the three specimens. Neural tissue is present in the bone dust given off during temporal bone drilling. This poses the question as to the risk of prion transmission during such dissection.

Multiple basal cell carcinomas and malignant melanoma following radiotherapy for ankylosing spondylitis.

We present the case of a 53-year-old Caucasian woman with seven basal cell carcinomas and one malignant melanoma in situ along her back overlying her spine, which was irradiated in 1968 for ankylosing spondylitis. These lesions developed between 1997 and 1999. She has no other known risk factors for cutaneous malignancy, in particular no history of excessive sun exposure. She has skin type 2. Molecular studies of glutathione S-transferase and cytochrome P450 status showed her genotype not to constitute an overall increased inherited susceptibility. We therefore postulate that all her skin cancers have arisen as a consequence of her radiotherapy. To our knowledge this is the first case of multiple basal cell carcinoma in addition to a malignant melanoma following radiotherapy for benign disease.

Accuracy of staging in primary biliary cirrhosis.

AIMS: To evaluate sampling variability of liver biopsy in patients with primary biliary cirrhosis (PBC). METHODS: Sections from 50 PBC liver specimens obtained at transplantation were examined. The degree of fibrosis was assessed on a scale of 0-4 using two methods: (1) simulated needle biopsy in fields approximately the size of conventional needle biopsy; and (2) whole section scanning in areas with little and extensive fibrosis. RESULTS: Considerable variation in the range of stages of fibrosis was found when the whole section scanning method was used. Only 10 (20%) samples had a consistent degree of fibrosis in all sections scanned. By contrast, the same fibrosis stage was assigned in 30 (60%) specimens examined using the simulated needle biopsy method. When the results obtained by the two methods were compared, there was a discrepancy of one or two stages in 32 samples. This discrepancy was the result of discovering areas with a lesser degree of fibrosis in whole sections compared with the simulated needle biopsy specimens. CONCLUSION: There is considerable variation in the degree of fibrosis in the livers of patients with PBC, even when end stage specimens obtained at transplantation are examined. Consistent results were obtained when simulated needle biopsy specimens were examined. This, however, may be a reflection of the procedure applied when staging liver needle biopsy specimens, where the greatest degree of abnormality is used in determining the stage. The practice of staging of PBC in small needle biopsy specimens is valuable as long as the appearances are interpreted with caution, bearing in mind that there is considerable variability in the degree of fibrosis.

Superficial thrombophlebitis, dysplasia, and cholangiocarcinoma in primary sclerosing cholangitis.

Cholangiocarcinoma occurs in approximately 10% of patients with primary sclerosing cholangitis. Usually, liver failure, rapidly progressing jaundice, and an increase in alkaline phosphatase levels are suggestive diagnostic features. We report two cases of patients with primary sclerosing cholangitis who developed cholangiocarcinoma without jaundice and with no changes in their serum biochemistry. Both patients were taking ursodeoxycholic acid at the time of tumor diagnosis. Initial suspicion of malignancy was based on the development of superficial thrombophlebitis. Liver histology showed evidence of bile duct epithelial dysplasia in areas free from tumor in one patient, and in the other, bile duct epithelial dysplasia preceded the appearance of cholangiocarcinoma by at least 18 months. In one of the cases, the dysplastic epithelium stained positively for carcinoembryonic antigen. The histological finding of bile duct epithelial dysplasia in patients with primary sclerosing cholangitis may suggest either imminent or actual development of cholangiocarcinoma and may thus affect consideration of orthotopic liver transplantation. In addition, the development of superficial thrombophlebitis in patients with primary sclerosing cholangitis should arouse suspicion of the presence of cholangiocarcinoma even if there is no evidence of deterioration of the liver function or a dominant stricture on endoscopic retrograde cholangiography.

Monoclonal antibody JC1: new reagent for studying cell proliferation.

AIM: To characterise a newly developed mouse monoclonal antibody JC1 which recognises a nuclear antigen present in proliferating cells in normal tissues and neoplastic lesions, and which is absent in resting cells. METHODS: The methodology was established using a representative range of frozen sections from normal tissues and from certain tumours which were immunostained with antibodies Ki67 and JC1. The molecular weight of the antigen recognised by JC1 was obtained by western blot analysis and this was compared with that of Ki67. IM-9 cell lysates containing Ki67 derived plasmids were also tested with JC1 antibody. RESULTS: Biochemical investigation indicated that the antigen recognised by JC1 gives two molecular weight bands of 212 and 123 kilodaltons, which is distinct from the well characterised anti-proliferation monoclonal antibody Ki67 (395-345 kilodaltons). In addition recombinant Ki67 protein is not recognised by JC1. Immunohistological reactivity was seen in areas known to contain numerous proliferating cells such as lymphoid germinal centres, splenic white matter, cortical thymocytes and undifferentiated spermatogonia. In tumours many cells from adenocarcinomas, oat cell carcinomas, squamous cell carcinomas of lung, and seminomas were labelled by JC1 with a distribution and proportion similar to that seen with Ki67. In normal tissues the only apparent difference was in testis where JC1 stained a considerably greater number of cells than Ki67. In all cases studied the new antibody showed nuclear reactivity only. JC1 did not show any cytoplasmic crossreactivity with squamous cells as is frequently seen with Ki67. CONCLUSION: Antibody JC1, which recognises a nuclear antigen present in proliferating cells, should provide a useful adjunct to Ki67 as a marker of proliferation especially in those cases such as squamous cell carcinomas where a Ki67 index cannot be determined.

Intracellular free magnesium in lymphocytes from patients with congestive cardiac failure treated with loop diuretics with and without amiloride.

Cellular Mg2+ depletion has been reported in patients on long term diuretic therapy. We therefore measured lymphocyte free Mg2+ concentrations in patients with congestive cardiac failure treated with loop diuretics or a frusemide/amiloride mixture (Frumil) and compared them with control subjects. There was no correlation between lymphocyte free Mg2+ levels and plasma Mg2+ concentrations. Patients treated with loop diuretics did not show lymphocyte intracellular free Mg2+ depletion compared with normal controls, and those on Frumil showed higher intracellular free Mg2+ levels than normal.

Stimulation of the human leucocyte Na+/H+ antiport by D-glucose is mediated by protein kinase C.

Leucocyte Na+/H+ antiport activity is elevated in patients with essential hypertension and Type 1 diabetes with nephropathy. To examine the effects of hyperglycemia on the Na+/H+ antiport, normal leucocytes were incubated with 25 mmol l-1 D-glucose, L-glucose or glucose-6-phosphate for two days. Leucocyte Na+/H+ antiport activity was measured by a novel double ionophore fluorimetric method for controlling intracellular pH. Only incubation with D-glucose led to an increase in Na+/H+ antiport activity of about 31%. This effect was not due to non-enzymic glycation since glucose-6-phosphate, which glycates proteins faster than D-glucose, caused no significant difference in antiport activity. Also, osmotic effects could be excluded. Staurosporine (10 nmol l-1), a specific inhibitor of protein kinase C, prevented the rise in antiport activity due to incubation with D-glucose. As hyperglycaemia is known to increase protein kinase C activity, elevation of this kinase may be one mechanism for activation of the Na+/H+ antiport in Type 1 diabetes.

Intracellular free magnesium in human lymphocytes and the response to lectins.

1. Intracellular free [Mg2+] was measured in human peripheral blood lymphocytes using a fluorimetric method based on the dye furaptra. It was necessary to correct for the extracellular leakage of the dye by using either 10 mmol/l EDTA or 0.05 mmol/l Mn2+. 2. As the proliferative response of lymphocytes to mitogenic lectins has been linked to a dependence on extracellular Mg2+, the intracellular [Mg2+] was studied in lymphocytes stimulated with various mitogenic and non-mitogenic lectins. 3. Only lymphocytes treated with phytohaemagglutinin-L, a leucoagglutinin from Phaseolus vulgaris that binds to tri- and tetra-antennary complex glycoproteins, showed a marked increase in intracellular [Mg2+]. This effect was partially inhibited by N-acetylgalactosamine. The stimulation by different lectins of the incorporation of [3H]-thymidine into lymphocytes was not correlated to the changes in intracellular [Mg2+]. 4. The proliferative response of lymphocytes to lectins is therefore not wholly dependent on a rise in intracellular [Mg2+].

Leukocyte intracellular pH and Na/H antiporter activity in uraemia and type I diabetes mellitus.

The leukocyte Na/H antiporter has been studied in patients with end-stage renal failure on maintenance haemodialysis. Thirteen non-diabetic haemodialysis patients (CRF group) and eight haemodialysis patients with diabetic nephropathy (CRF-DM group) were investigated. Measurements were made using the pH-sensitive fluorescent dye bis (carboxyethyl) carboxyfluorescein (BCECF). The initial intracellular pH (pHi), intracellular buffering capacity, and Na/H antiporter Vmax (at pHi = 6.0) have been recorded in bicarbonate-free solutions. The mean initial intracellular pH in the CRF group was 7.34 (SD 0.05, P less than 0.004) and this was significantly less than the CRF-DM group (7.42, SD 0.07) and normal controls (7.43, SD 0.09, n = 25). The mean intracellular buffering capacity was normal in the CRF and CRF-DM groups. The mean Na/H antiporter Vmax was also normal in the CRF and CRF-DM groups (56.5, SD 9.9; and 56.8, SD 12.8, mmol/l per min respectively compared to 55.2, SD 8.8, mmol/l per min in controls). These data are discussed with reference to the reported high values of Na/H antiporter Vmax in diabetic patients with early nephropathy. This abnormality does not appear to be present in end-stage diabetic nephropathy.

Leucocyte Na+/H+ antiport activity in type 1 (insulin-dependent) diabetic patients with nephropathy.

The development of proteinuria in Type 1 (insulin-dependent) diabetic patients may depend on predisposition to essential hypertension in addition to poor glycaemic control. Previous work has shown increased leucocyte Na+/H+ antiport activity in essential hypertension and increased erythrocyte Li+/Na+ exchange in Type 1 diabetic patients with proteinuria. To test whether susceptibility to nephropathy in Type 1 diabetes was linked to abnormalities of leucocyte Na+/H+ antiport activity, we measured the intracellular pH and kinetics of the Na+/H+ antiport in 19 Type 1 diabetic subjects with, and 15 diabetic subjects without albuminuria and compared them to 25 matched normal control subjects. Intracellular pH (mean +/- SD 7.59 +/- 0.14) and maximal transport capacity of the antiport (Vmax 87.7 +/- 24.9 mmol.1-1.min-1) were higher in diabetic subjects with albuminuria compared to normotensive control subjects (pH 7.44 +/- 0.09; Vmax 55.6 +/- 10.3 mmol.l-1.min-1; p less than 0.001 for both), similar to the defect described in essential hypertension. These differences were not seen in diabetic subjects with normal urinary albumin/creatinine ratios (pH 7.46 +/- 0.09; Vmax 61.0 +/- 13.6 mmol.l-1.min-1). Buffering characteristics of the leucocytes at different pH in the Type 1 diabetic subjects with albuminuria differed from normal control subjects and diabetic subjects with normal urinary albumin/creatinine ratios. We conclude that increased leucocyte Na+/H+ antiport activity, a known marker of essential hypertension, is usually associated with nephropathy in Type 1 diabetes.

Effect of protein kinase C modulators on the leucocyte Na+/H+ antiport in type 1 (insulin-dependent) diabetic subjects with albuminuria.

It is uncertain why only one third of Type 1 (insulin-dependent) diabetic patients develop nephropathy. One suggestion is the inheritance of a predisposition to essential hypertension. We have previously found elevated Na+/H+ antiport activity and a raised intracellular pH in leucocytes from hypertensive and Type 1 diabetic subjects with albuminuria using a novel double ionophore fluorimetric technique. These changes are not found in Type 1 diabetic subjects without albuminuria. We wished to test the effect of a protein kinase C inhibitor staurosporine (100 nmol/l) on the elevated antiport activity, and the degree of stimulation achieved by exogenous diacyl glycerol. Raised leucocyte Na+/H+ antiport activity of Type 1 diabetic subjects with albuminuria (73.8 +/- 17.2 mmol.l-1.min-1) was restored to normal levels with staurosporine (54.9 +/- 17.9 mmol.l-1.min-1, p less than 0.001). The leucocyte Na+/H+ antiport activity of diabetic subjects without albuminuria fell significantly also with staurosporine but to a lesser extent (57.3 +/- 11.6 to 50.0 +/- 12.8 mmol/l, p less than 0.003). In contrast, leucocytes from normal control subjects showed no change in antiport activity with staurosporine (54.3 +/- 8.5 to 52.6 +/- 10.4 mmol.l-1.min-1). Dioctanoyl glycerol stimulated the leucocyte Na+/H+ antiport in normal subjects and diabetic patients without albuminuria, with significantly less stimulation in diabetic patients with albuminuria. We conclude that reversal by staurosporine of the elevated Na+/H+ antiport activity in Type 1 diabetic subjects with albuminuria could indicate a role for protein kinase C in activating the antiport. This hypothesis is supported by the reduced stimulation of the antiport by dioctanoyl glycerol in this group of patients.