Altered cellular membrane fluidity levels and lipid peroxidation during experimental pancreas transplantation.

Although the pathogenesis of ischemia reperfusion (IR) injury is based on complex mechanisms, free radicals play a central role. We evaluated membrane fluidity and lipid peroxidation during pancreas transplantation (PT) performed in 12 pigs (six donors and six recipients). Fluidity was measured by fluorescence spectroscopy, and malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations were used as an index of lipid oxidation. Pancreatic tissues were collected as follows: (A) donor, immediately before vascular clamping; (B) graft, following perfusion lavage with University of Wisconsin preservation fluid; (C) graft, after 16 h of cold ischemia; and (D) recipient, 30 min vascular postreperfusion. Fluidity and MDA and 4-HDA concentrations were similar in cases A, B, and C. However, there was significant membrane rigidity and increased lipid peroxidation after reperfusion (D). These findings suggest that reperfusion exaggerates oxidative damage and may account for the rigidity in the membranes of allografts during PT.

Predictive ultrasound factors of lymphatic invasion in rectal cancer: "extra-corporeal" study.

OBJECTIVE: the accuracy of preoperative endorectal ultrasound in the status evaluation of lymph nodes is around 50-70%, with a lack of eco-morphological patterns of clinical use. Since, accurate local staging is of great value in prognosis and decision-making we decided to analyze the referenced eco-morphological parameters in a try to find a proper predictive tool of clinical help that could improve the accuracy of rectal ultrasound. MATERIAL AND METHOD: the resected specimens of 24 patients that were operated on by radical surgery because rectal cancer, without preoperative radiotherapy were suspended in warm water and ultrasound scanned (360º circular probe with a transducer of 10 Mhz). All suspicious nodes were recorded and marked for the definitive histological report. RESULTS: from the 24 specimens, 318 nodes were imaged(210 benign and 100 involved). All ultrasound parameters analysed were significant but only lobulation, echogenicity and hilar reflection were independent values. An score system was design with the addition of all parameters that showed a sensitivity of 98%and specificity of 99,1%. CONCLUSIONS: our study shows that a careful study of ultra-sound lymph node images can get a high level of accuracy and better help in tailoring the treatment of any particular case.

A prospective study about functional and anatomic consequences of transanal endoscopic microsurgery.

INTRODUCTION: transanal endoscopic microsurgery (TEM) was developed in 1983 by Büess as a minimally invasive technique to manage rectal villous adenomas and early rectal adenocarcinomas. Many studies have been published worldwide about its excellent results in morbidity and recidive rate, but there are few studies addressing functional results. The objective of this study is to analyze the effect of this technique in the anal anatomy and compare with the manometric results. MATERIAL AND METHODS: we devised a prospective study of 40 patients. 39% female, 61% male. All of them filled an incontinence questionnaire (Pescatori scale) and endoanal ultrasonography and manometry was carried out preoperatively, third month postoperative and at sixth month only if incontinence appeared. RESULTS: 32 patients (80%) had villous adenomas and 8 patients (20%) had adenocarcinomas (uT1). Three patients complained of flatus incontinence at 3rd postoperative month that disappeared with normal continence at 6th month. Anorectal manometric values: mean anal resting pressure (ARP) decreased at 3rd month (from 87.2 mmHg to 70.1 mmHg), as it was for maximal squeeze pressure (MSP) from 152.5 mmHg preoperatively to 142.2 mmHg at 3rd month. Ultrasonography demonstrated internal anal sphincter (IAS) rupture in 3 patients, with a full integrity of the external anal sphincter in all patients. CONCLUSIONS: during TEM, a significant anal dilatation occurs, because of rectoscopy (40 mm wide), what can produce a rupture of IAS, with the consequent decreasing in ARP, and a dilatation without rupture of external sphincter what produces a decreasing of MSP. The fall of anal pressures had minima clinical repercussion when sphincter is intact, but when IAS is broken a temporal incontinence develops.

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.

Sequential treatment for proctalgia fugax. Mid-term follow-up.

INTRODUCTION: Proctalgia fugax (PF) is a benign, self-limiting disease characterized by episodes of intense anorectal pain at frequent intervals in the absence of organic proctological disease. Even though PF was described more than a century ago, its etiology remains unclear. Currently there is no information available. Few papers quoting many ways of management have been published. The aim of this study was to investigate patients complaining of this condition and to treat them with sequential therapy. PATIENTS AND METHODS: We devised a descriptive, prospective study of patients complaining of acute perianal pain--duration less than 30 minutes--without organic disease or previous perianal surgery since 1996 to 2002 in our Department. We treated these patients using a three-step treatment (1: information, hip bath, benzodiazepines; 2: sublingual nifedipine 10 mg, or topic 0.1% nitroglycerin on demand; 3: internal anal sphincterotomy if hypertrophy of the internal anal sphincter was demonstrated by anal ultrasonography and no improvement was confirmed with the previous steps of treatment). We defined remarkable improvement as a decrease in the number of episodes by half or in pain intensity by 50%. RESULTS: Fifteen patients with an average follow-up of 4 years. Anal endosonography confirmed a grossly thickened internal anal sphincter (IAS) in 5 cases. After the first step of treatment 7 patients improved and 1 patient was cured; after the second step of treatment 3 patients improved and 1 was cured; the third step was applied to 3 patients with a thickened IAS; 1 patient improved and 1 patient was cured. CONCLUSION: A total resolution of PF is not always possible, but we may improve symptoms and their frequency. Almost 50% of patients in our series improved with the first step of treatment; 30% of our patients had IAS hypertrophy. Anal endosonography can help in the diagnosis of organic diseases or IAS hypertrophy, for which we can perform an internal anal sphincter myectomy.

Relationship between DNA-reactivity and cytostatic effect of two novel bile acid-platinum derivatives, Bamet-UD2 and Bamet-D3.

BACKGROUND AND AIMS: Several platinum(II)-bile acid derivatives, named Bamets, have been previously synthesized. Their ability to interact with DNA, their cytostatic activity and their liver organotropic properties have been characterized. Two new compounds of this family, with particular structural properties, have been developed. Bamet-UD2 was formed by two ursodeoxycholic acid moieties bound by the carboxylate groups to cisplatin. In contrast, in Bamet-D3, glycine and a polyamine were used as tandem spacer elements to separate a cholic acid moiety from the platinum(II) atom. The aim of this work was to evaluate how these changes affect the ability of these compounds to interact with DNA and reduce tumour cell growth. MATERIALS AND METHODS: Drug reactivity with DNA was determined by changes in the electrophoretic mobility of the pUC18 plasmid test and by the ethidium bromide (EthBr) displacement assay. Cytostatic activity was measured against two mouse-derived cell lines from lymphocytic leukemia (L1210) and sarcoma (S-180-II). RESULTS: Bamet-UD2, and more markedly Bamet-D3, induced changes in the electrophoretic mobility of pUC18, suggesting the formation of DNA-drug interactions. Bamet-UD2 displaced EthBr from its binding to DNA. This effect was stronger in the case of Bamet-D3. Scatchard plots revealed that pre-incubation with both Bamet-UD2 and Bamet-D3 decreased the number of DNA sites available and their ability to bind EthBr. In spite of the enhanced DNA-reactivity of Bamet-D3, its ability to reduce tumour cell growth was much weaker than that of Bamet-UD2, which was seen to exert a very strong cytostatic effect. CONCLUSION: Although the distance between the platinum atom and the bile acid moiety affects the in vitro Bamet reactivity with DNA, other factors determine the overall cytostatic activity of these compounds.

Liver organotropism and biotransformation of a novel platinum-ursodeoxycholate derivative, Bamet-UD2, with enhanced antitumour activity.

BACKGROUND/AIMS: Several members of a novel family of bile acid derivatives with cytostatic and virostatic activity have been synthesized and characterized. The aim of this work was to investigate the liver organotropism and biotransformation of two novel compounds with enhanced DNA-reactivity: Bamet-D3, in which a glycine-polyamine tandem was used as a spacer to separate the glycocholic acid moiety from the platinum(II) atom, and Bamet-UD2, in which cisplatin was directly bound to the carboxylate group of two ursodeoxycholic acid moieties. METHODS: Drug uptake and "in vitro" toxicity were investigated using rat hepatocytes in primary culture. Following i.v. administration of 0.5 mumol cisplatin, Bamet-D3 or Bamet-UD2, bile output, urinary and fecal excretion, organ distribution and pharmacokinetic parameters were determined in short-term (3 h) and long-term (14 days) experiments carried out on anaesthetized and conscious rats, respectively. Liver biotransformation was investigated by HPLC analysis of bile samples. Total platinum was measured by flameless atomic absorption spectroscopy. Using Nude mice, antitumour activity was investigated in subcutaneously implanted Hepa 1-6 mouse hepatoma cells. RESULTS: Uptake by rat hepatocytes was Bamet-UD2 (11.3 nmol/mg protein) > Bamet-D3 (5.6 nmol/mg protein) > cisplatin (2.1 pmol/mg protein). Bamet-UD2 induced "in vitro" cell toxicity, which was not observed for Bamet-D3 or cisplatin. On the contrary, no toxicity "in vivo" for Bamet-UD2 was found which was observed for cisplatin and Bamet-D3. This may be related with the fact that bile output of Bamet-UD2, which occurs with no major biotransformation, was > 10 fold higher than that of cisplatin and 3-fold higher than that of Bamet-D3, which was previously transformed into at least three different metabolites. Fecal excretion was Bamet-UD2 > Bamet-D3 > cisplatin, whereas urinary output was Bamet-D3 > cisplatin > Bamet-UD2. Accordingly, a marked liver- and a reduced kidney-vectoriality for Bamet-UD2, but not for Bamet-D3, was observed. Bamet-UD2 and cisplatin, but not Bamet-D3, were efficient in inhibiting tumour growth whereas, only Bamet-UD2 significantly prolonged survival time. CONCLUSIONS: There results indicate that Bamet-UD2 is a cisplatin-ursodeoxycholate derivative with strong antitumour activity, marked hepatobiliary organotropism, and reduced toxic side-effects as compared to the parent drug cisplatin.

[The mechanisms of satiation]. / Mecanismos de saciedad.

There are many published experimental studies which attempt to explain certain aspects of satiety, but only very few treat this problem as a whole, synthesizing concepts. At present, the understanding of the mechanisms of satiety is extremely interesting for the study and application of medical or surgical anti-obesity treatments.

[Endocrine metabolic and arterial pressure changes in morbidly obese patients treated with vertical gastroplasty]. / Modificaciones endocrino-metabólicas y de la presión arterial en obesos morbidos tratados mediante gastroplastia vertical.

When overweight surpasses 100% of the ideal weight, morbid obesity, the obese patients is condemned to a complete inability to work, social and sexual inability, and shall suffer from an increase in its morbidity and mortality. This depends to a large degree on the additions to the obesity of insulin resistance, carbohydrates intolerance, hypertriglyceridemia, hypercholesterolemia, and arterial hypertension, all of which is enveloped in a atmosphere of neuroendocrine alterations. An efficient method of treating this syndrome is weigh loss. Medical treatments have not achieved prolonged weight losses during long periods in morbid obese patients, which is a reason for surgery to try and propose new lines of treatment for these patients. The purpose of our study is to examine the effect of weight loss in 100 patients treated with vertical gastroplasty, on the metabolic disorders (triglycerides, cholesterol, glucose) and the arterial hypertension, which are considered to be risk factors in the mortality associated with morbid obesity. Our results indicate that the weight loss modified the metabolic conditions of the patients, with there being a decrease of the levels of triglycerides, cholesterol, glucose, and arterial pressure, after 6 to 12 months after the weigh loss.

Endoluminal ultrasography for rectal tumors: efficacy, sources of error and limitations.

OBJECTIVES: endorectal ultrasound (EUS) is currently accepted as the best technique for the preoperative study of patients with rectal tumors, and surgical decisions depend increasingly on EUS staging. The main pitfalls in staging rectal tumors are over- or understaging as well as errors in imaging lymph nodes. Being aware of such errors and their causes may help to improve the overall results. The aim of the present study was to evaluate the accuracy of EUS in staging rectal neoplasms, and to study potential sources of error. METHODS: from May 1996 to December 1998, 120 patients with rectal tumors were studied preoperatively by EUS. The uTNM classification described by Hildebrandt and coworkers was used. The EUS findings were compared prospectively with the results of pathological examination. When there was no correlation, both the specimen and the EUS findings were carefully reviewed to look for potential sources of error. RESULTS: 41 out of 120 patients were classified as uT1, 10 as uT2, 60 as uT3 and 9 out of 120 as uT4. 31 patients had positive lymph nodes (uN1). On comparing these data with the results of the pathological report, we found 90% accuracy in staging rectal wall penetration, and 70% accuracy in the diagnosis of lymph nodes. Errors were due basically to technical problems, characteristics of the tumor itself, and difficulties in staging lymph nodes. CONCLUSIONS: it is important to identify the potential source of errors as well as the current limitations of EUS to improve the overall results with this technique.

Anal endosonography for the study of anal canal anatomy: normal images and sonographic variants.

OBJECTIVE: To present normal images and sonographic variants of the anal canal to be used as reference for the study of sphincter and anal canal abnormalities. MATERIAL AND METHODS: Sixty subjects without known anal canal disease were studied by means of anal endosonography. Subject were divided according to age in two groups (up to 50 years and more than 50 years). All of them underwent an outpatient study with B&K medical ultrasound 2,003 scanner and 1,850 multifrequency transducer. RESULTS: Four layers can be sonographically identified in the anal canal: an inner hyperechoic layer which is the submucosa, a second hypoechoic layer which is the internal sphincter, a third one which is a longitudinal muscle and the outer hyperechoic layer which is the external sphincter and the only to be found in the low anal canal. In people older than 50 years, both sphincters were significantly thicker (0.3-0.5 mm). At the high anal canal 40% of women presented an anterior gap in the external anal sphincter. CONCLUSIONS: Anal endosonography allows an easy division in high-, mid-, and low anal canal. In some women there is a gap at the mid-high anal canal that must be taken into account in order to avoid diagnostic errors. An internal sphincter thickness greater than 3.5 mm should be considered abnormal at any sex or age.

Transanal endoscopic surgery for rectal tumors.

OBJECTIVE: To report our results with local excision by transanal endoscopic microsurgery (TEM) to treat 42 cases of rectal lesions (29 adenomas and 13 carcinomas). METHODS: Prospective, descriptive study. Sex distribution: 55% men, 45% women, mean age 65 years (range: 17-84 years). SYMPTOMS: rectal bleeding 67%, diarrhea 23%. SURGICAL TECHNIQUE: mucosectomy 6 cases, full-thickness excision 36 cases. Average follow-up: 11 months (range: 1-36 months). RESULTS: We analyzed operating time (average 85 min; range: 25-180 min), bleeding (average 100 ml, range 10-350 ml), distance of the tumor from the anal verge (lower tumor margin: mean, 8.8 cm; range, 1-20 cm; distal tumor margin: mean, 12.9 cm; range, 5-22 cm), tumor size (mean, 3.9 cm; range, 2-10 cm), postoperative hospital stay (average, 4 days; range, 2-15 days), morbidity (hemorrhage 1 case; perforation, 1 case), mortality (0) and follow-up (temporary incontinence to flatus in 6 cases, 1 recurrence of carcinoma treated with abdominoperineal resection, 2 recurrences of adenoma and 2 new adenomas). CONCLUSIONS: TEM is a safe technique for the treatment of rectal lesions. Low morbidity and recurrence rates and short hospital stays make TEM a procedure of choice when local rectal surgery is indicated.

[Graves' disease with associated thyroid nodules (nodular Graves' disease). Clinical, diagnostic and therapeutic considerations]. / Enfermedad de Graves-Basedow con nódulos tioideos asociados (enfermedad de Graves-Basedow nodular). Consideraciones clínicas, diagnósticas y terapéuticas.

BACKGROUND: Sometimes Graves disease (GD) can appear in association with thyroid nodules, which seems to increase the risk of carcinoma. In this article, we try to establish clinical characteristics, diagnostic means and appropriate treatment for Graves patients with co-existent nodules. METHOD: A retrospective study was made of 153 consecutive patients who underwent operation for GD between 1967 and 2000. Each patient was subject to a regular protocol including physical examination, diagnostic test, total or subtotal thyroidectomy and follow-up in the long term with the purpose of making a valuation of the postsurgical morbidity, evolution and relapses. Data were processed through computing in order to get the statistical information. RESULTS: 28.1% of GD had thyroid nodules and carcinoma was diagnosed in four patients (9.3%), all of them belonging to papillary variety. Surgery consisted of 57 subtotal thyroidectomies (37.3%) and 94 total thyroidectomies. Parathyroid and recurrent morbidity was established in 4.6 and 3.9%, respectively, a year later since the operation, though it had a strong tendency to decrease from 1980. 96% of cases showed no relapse. CONCLUSIONS: Nodular GD is very common in our setting, especially in Graves patients with late beginning who wait for ages until they are undergone surgery. Initial treatment should be by means of braking therapy with antithyroid drugs and clinical, cytologic and ultrasonographic control. Surgery would be advised, from the outset or during the follow-up, in view of either any suspicion about cancer or presence of local growth. The procedure of choice is total thyroidectomy performed with low morbidity.

PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity.

BACKGROUND AND PURPOSE: PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that is currently in phase I clinical development for the treatment of solid tumours. In this study we have characterized the interactions of PM01183 with selected DNA molecules of defined sequence and its in vitro and in vivo cytotoxicity. EXPERIMENTAL APPROACH: DNA binding characteristics of PM01183 were studied using electrophoretic mobility shift assays, fluorescence-based melting kinetic experiments and computational modelling methods. Its mechanism of action was investigated using flow cytometry, Western blot analysis and fluorescent microscopy. In vitro anti-tumour activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the in vivo activity utilized several human cancer models. KEY RESULTS: Electrophoretic mobility shift assays demonstrated that PM01183 bound to DNA. Fluorescence-based thermal denaturation experiments showed that the most favourable DNA triplets providing a central guanine for covalent adduct formation are AGC, CGG, AGG and TGG. These binding preferences could be rationalized using molecular modelling. PM01183-DNA adducts in living cells give rise to double-strand breaks, triggering S-phase accumulation and apoptosis. The potent cytotoxic activity of PM01183 was ascertained in a 23-cell line panel with a mean GI(50) value of 2.7 nM. In four murine xenograft models of human cancer, PM01183 inhibited tumour growth significantly with no weight loss of treated animals. CONCLUSIONS AND IMPLICATIONS: PM01183 is shown to bind to selected DNA sequences and promoted apoptosis by inducing double-strand breaks at nanomolar concentrations. The potent anti-tumour activity of PM01183 in several murine models of human cancer supports its development as a novel anti-neoplastic agent.

Effect of maternal cholestasis on the kinetics of bile acid transport across the canalicular membrane of infant rat livers.

A reversible impairment in the ability of the liver to secrete cholephilic compounds has been reported to exist in infant rats born from mothers with surgically induced complete cholestasis during the last third of the pregnancy. Canalicular plasma membranes (CPM) were purified from livers obtained from 4 and 8 week-old offspring of healthy or cholestatic rats. Using radiolabelled glycocholic acid (GC) and a rapid filtration technique, bile acid transport by CPM vesicles in the presence of 3 mM ATP plus an ATP-regenerating system was measured at varying substrate concentrations. Kinetic parameters were calculated by nonlinear regression analysis. Similar values for the apparent affinity constant (Kt) were found in all experimental groups (approximately 350 microM). The value of the maximal velocity of the transport (Vmax) was similar for CPM obtained from control animals at 4 or 8 weeks of age (approximately 1.5 nmol/20 s/mg protein). In the offspring of cholestatic mothers the Vmax value was not different from that found in control animals as far as 4 week-old rats were concerned. However, Vmax in the 8 week-old group from cholestatic mothers was two-fold higher than that found in the rest of the experimental groups. Thus, the efficiency of transport, defined as Vmax/Kt, was very similar in all experimental groups, except in the group of 8 week-old offspring of cholestatic mothers, where this value was 60% higher. Isolated livers obtained from this group were able to secrete a tracer dose of radiolabelled GC (11.25 nmol) into bile significantly faster than isolated livers obtained from control animals of the same age (8 weeks). In sum, these results indicate that, in young infant rats (4 week-old), in which the maximal secretion rate for bile acids was reduced by maternal cholestasis during pregnancy, the kinetics of ATP-dependent bile acid transport across the canalicular membrane were not affected. By contrast, in older infant rats (8 week-old), in which the overall ability of the liver to secrete bile acids seems to be restored to normality, the efficiency of the canalicular transport system was actually enhanced. This suggests the existence of compensation at the level of the canalicular membrane transfer and thus that there is another hitherto unidentified mechanism involved in bile acid secretion.