Prospective, randomized and controlled study of mesh displacement after laparoscopic inguinal repair: fixation versus no fixation of mesh.

INTRODUCTION: Repair of inguinal hernia is one of the most common elective operations performed in general surgery practice. Mesh hernia repair became the gold standard because of its low recurrence rate in comparison with non-tension-free repair. Laparoscopic approach seems to have potential advantages over open techniques, including faster recovery and reduced acute and chronic pain rate. Laparoscopic mesh fixation is usually performed using staples, which is associated with higher cost and risk for chronic pain. Recently, the role of mesh fixation has been questioned by several surgeons. AIM: To evaluate mesh displacement in patients undergoing laparoscopic inguinal hernia repair comparing mesh fixation with no fixation. METHODS: From January 2012 to May 2014, 60 consecutive patients with unilateral inguinal hernia were randomized into two groups: control group--10 patients underwent totally extraperitoneal (TEP) repair with mesh fixation; NO FIX group-50 patients underwent TEP repair with no mesh fixation. Mesh was marked with three 3-mm surgical clips at its medial inferior, medial superior and lateral inferior corners. Mesh displacement was measured by comparing an initial X-ray, performed in the immediate postoperative period, with a second X-ray obtained 30 days later. RESULTS: The mean displacement of all three clips in control group was 0.1-0.35 cm (range 0-1.2 cm), while in NO FIX group was 0.1-0.3 cm (range 0-1.3 cm). The overall displacement of control and NO FIX group did not show any difference (p = 0.50). CONCLUSION: Fixation of the mesh for TEP repair is unnecessary. TEP repair with no mesh fixation is safe and is not associated with increased risk of mesh displacement.

Consanguinity and founder effect for Gaucher disease mutation G377S in a population from Tabuleiro do Norte, Northeastern Brazil.

Gaucher's disease (GD) is caused by a ß-glucocerebrosidase deficiency, leading to the accumulation of glucocerebroside in the reticuloendothelial system. The prevalence of GD in Tabuleiro do Norte (TN) (1:4000) is the highest in Brazil. The purpose of this study was to present evidence of consanguinity and founder effect for the G377S mutation (c.1246G>A) among GD patients in TN based on enzyme, molecular and genealogical studies. Between March 2009 and December 2010, 131 subjects at risk for GD (GC in dried blood ≤2.19 nmol/h/ml) and 5 confirmed GD patients from the same community were submitted for molecular analysis to characterize the genetic profile of the population. Based on the enzymatic and molecular analysis, the subjects were classified into three categories: affected (n = 5), carrier (n = 20) and non-carrier (n = 111). All carriers were (G377S/wt). Affected subjects were homozygous (G377S/G377S). The identification of a single mutation in carriers and homozygotes from different generations, the history of the community and the genealogy study suggest that the high prevalence of GD in this population may be due to a combination of consanguinity and founder effect for the G377S mutation.

LAMP2 as a marker of EBV-mediated B lymphocyte transformation in the study of lysosomal storage diseases.

Following the degradative pathway, vesicles loaded with extracellular material, eventually, dock and fuse with lysosomes, acquiring specific membrane markers of these organelles and acid hydrolases responsible for digest their content. The lysosomal-associated membrane protein 2 (LAMP-2), the best characterized lysosomal membrane protein, is found in late stages of endosome maturation and may be used as a marker of lysosome-associated membranes. Lysosomal storage disorders (LSDs) are described by the absence or deficiency in hydrolase activity leading to substrate accumulation within lysosomal components and to the onset of several diseases. It is known that lymphocytes infected by Epstein-Barr virus (EBV) are able to form cytoplasmic vacuoles, which work as a storage compartment for lysosomal acidic hydrolases. At the present study, we validate the EBV as a transforming agent of B lymphocytes in stability studies of long-term stored samples, since the methods used to keep samples in liquid nitrogen and thaw them have all proven to be efficient in samples frozen for up to 2 years. To confirm and investigate some of the most prevalent LSDs in the South of Brazil-Pompe, Fabry and Gaucher diseases-we first measured the enzymatic activity of α-glicosidase, α-galactosidase, and ß-glicosidase in those cytoplasmic-formed vacuoles and then looked to LAMP-2 immunoreactivity by employing confocal microscopy techniques.

Effect of one year of cryopreservation on the activity of lysosomal hydrolases from EBV-transformed lymphocytes.

BACKGROUND: The Epstein-Barr virus (EBV) was used as an agent of B lymphocyte proliferation for subsequent diagnosis of lysosomal storage disease. Due to the constant handling of long-preserved samples in our cell bank, we decided to observe the behavior and then compare cultured and frozen samples for at least one year's cryopreservation. METHODS: Twenty-five samples from healthy individuals were used to assess the possible changes in activity of enzymes ß-galactosidase, ß-glucosidase, α-iduronidase, α-galactosidase, and α-glucosidase. Transmission electron microscopy was used to confirm cell transformation of B lymphocytes into EBV-infected cells, generating lymphoblastoid cell lines. RESULTS: Transmission electron microscopy findings confirmed previous reports in the literature that is, significant and evident morphological changes in the nucleus occur after day 12 and the consequent cell transformation into EBV-infected cells. After thawing and subsequent treatment with the five enzymes utilized, we observed no significant changes in samples cryopreserved for more than one year, as compared to samples cultured for 12 days.

The effect of Mycoplasma and mycoplasma removal agent on the hydrolase activity in fibroblasts of patients with lysosomal diseases.

This study was designed to evaluate the effect of mycoplasma contamination on acid hydrolase activity and the action of the mycoplasma removal agent (MRA), in cultures of human fibroblasts from individuals with lysosomal diseases. For this purpose, we measured the activity of the b-galactosidase, arylsulphatase B (ASB), hexosaminidase A and a-glucosidase enzymes. The activity of the above mentioned enzymes in fibroblasts contaminated by mycoplasma was measured before and after the addition of the MRA. The results were then compared to the enzymatic activity in contamination-free cultures. Only the ASB enzyme showed significant alteration in activity both in the presence of mycoplasma and MRA. The remaining enzymes did not suffer significant interference by the presence of the two agents. Of the four enzymes tested, three did not suffer significant alterations by the presence of the mycoplasma nor from the MRA. However, the activity measured in the ASB enzyme increased significantly in the presence of mycoplasma and MRA and could lead to a doubtful diagnosis. Therefore, we suggest that contamination should be prevented by using aseptic techniques as well as the MRA in those fibroblast cultures that cannot be discarded.

Variable ultrasound echogenicity in flowing blood.

Real-time ultrasound imaging of large abdominal veins revealed bloodstream echogenicity of variable intensity. This variability is largely due to the entrance and persistance of tributary blood currents that show different echogenicity. Red cell aggregation is probably an important cause of bloodstream echoes and their variable intensity.

Biliary excretion of technetium-99m-sestamibi in wild-type dogs and in dogs with intrinsic (ABCB1-1Delta mutation) and extrinsic (ketoconazole treated) P-glycoprotein deficiency.

P-glycoprotein (P-gp), the product of ABCB1 gene, is thought to play a role in the biliary excretion of a variety of drugs, but specific studies in dogs have not been performed. Because a number of endogenous (ABCB1 polymorphisms) and exogenous (pharmacological P-gp inhibition) factors can interfere with normal P-gp function, a better understanding of P-gp's role in biliary drug excretion is crucial in preventing adverse drug reactions and drug-drug interactions in dogs. The objectives of this study were to compare biliary excretion of technetium-99m-sestamibi ((99m)Tc-MIBI), a radio-labelled P-gp substrate, in wild-type dogs (ABCB1 wild/wild), and dogs with intrinsic and extrinsic deficiencies in P-gp function. Dogs with intrinsic P-gp deficiency included ABCB1 mut/mut dogs, and dogs with presumed intermediate P-gp phenotype (ABCB1 mut/wild). Dogs with extrinsic P-gp deficiency were considered to be ABCB1 wild/wild dogs treated with the P-gp inhibitor ketoconazole (5 mg/kg PO q12h x 9 doses). Results from this study indicate that ABCB1 mut/mut dogs have significantly decreased biliary excretion of (99m)Tc-MIBI compared with ABCB1 wild/wild dogs. Treatment with ketoconazole significantly decreased biliary excretion of (99m)Tc-MIBI in ABCB1 wild/wild dogs. P-gp appears to play an important role in the biliary excretion of (99m)Tc-MIBI in dogs. It is likely that concurrent administration of a P-gp inhibitor such as ketoconazole will decrease P-gp-mediated biliary excretion of other substrate drugs as well.

Epstein-Barr virus-induced transformation of B cells for the diagnosis of genetic metabolic disorders--enumerative conditions for cryopreservation.

Epstein-Barr virus (EBV) infection in vitro causes transformation of B cells and generates B lymphoblastoid cell lines (LCLs). These LCLs have been widely used for the diagnostic of several genetic metabolic disorders. However, up to now, efficiency of LCL generation has been based on misleading subjective analysis. In this study, quantitative analyses have been performed to indicate efficiency of B-cell transformation to measuring human lysosomal acid hydrolases associated with: GM1-gangliosidosis type I, Gaucher disease and mucopolysaccharidosis type I. Peripheral blood mononuclear cells were isolated from 13 subjects, and LCLs were produced by culturing them with EBV for 12 days. Activities of the enzymes beta-galactosidase, beta-glucosidase and alpha-iduronidase were measured before and after cryopreservation in liquid nitrogen for 30 days. Efficiency of the B-cell transformation was screened every 4 days by the enumeration of cell proliferation, cell counts and changes in granularity estimated by flow cytometry. We observed the generation of 13 LCLs. Cell transformation was confirmed by the gradual increase of cellular clusters, cell size and granularity. In addition, we determined that the activity of the enzymes mentioned above did not change following cryopreservation. These data suggest that our enumerative approach for screening of EBV-LCLs is efficient for the enzymatic determination of human lysosomal acid hydrolases and may thus replace misleading subjective analyses.

Effect of plasma proteins and temperature on echogenicity of blood.

An explanation is proposed for the echogenicity to ultrasound scanning at 5 mHz and above of unclotted blood under conditions of stasis. In vitro experiments using blood from normal subjects and from patients with myeloma revealed that: 1) lysis of red cells prevented echogenicity, 2) echogenicity increased with increasing hematocrit, fibrinogen and other macromolecules, and temperature, and 3) blood from myeloma patients showed increased echogenicity and rouleau formation, a form of aggregation seen on peripheral smears. From these experiments it was concluded that red cell aggregation is a major cause of echogenicity of unclotted blood, requiring both intact red cells and conditions which are known to enhance red cell aggregation, such as the presence of macromolecules and increased temperature.

Detection of experimental arterial defects by portable and serial biplanar operative arteriography.

The accuracy of arteriography in detecting vascular defects similar to those encountered during vascular surgery was evaluated experimentally. Intimal flaps, thrombi, and strictures were created in dog aortas and studied under circumstances resembling operating room conditions by single-view portable and serial biplanar contrast arteriography. The specificity of both types of arteriography was comparable and high for detecting vascular defects. The sensitivity for diagnosing strictures was also comparable and high. However, both radiographic techniques were relatively insensitive in the detection of small intimal flaps and thrombi. In the detection of these two types of defects, serial biplanar arteriography was significantly more sensitive than portable arteriography. The degree of the superiority of serial bi-plane and the possible clinical insignificance of small defects indicates that portable arteriography in the operating room may still be an adequate methodology.

Ultrasonography of blood during stasis and coagulation.

A series of in vitro and animal experiments were performed to determine ultrasonic features of blood during stasis and coagulation. Liquid whole blood became echogenic within a few seconds to 3 minutes following the onset of stasis. This occurred in citrated human blood allowed to stand and in occluded segments of vena cava and aorta of dogs. Mechanical agitation of the blood in stasis caused a disappearance of echoes. This echogenicity occurred with gray-scale and real-time ultrasonography using 7.5-mHz transducers, but was not observed with 3.5-mHz transducer used in real-time scanning. Whole blood clot was echogenic both with 7.5- and 3.5-mHz scanning. Fibrin clot was sonolucent. Echogenicity with stasis did not occur with plasma, serum, or packed erythrocytes. The development of echogenicity required the presence of fibrinogen or its products plus erythrocytes. The echogenicity of blood during stasis detectable by high-frequency ultrasonography probably was related to physical layering of blood products. The disappearance of reflective echoes following agitation suggested that the layering could be readily disrupted.

A new method for correlating pancreatic and biliary duct pressures and sphincter of Oddi electromyography.

Myoelectric activity of the sphincter of Oddi and duodenum was correlated with pancreatic and biliary duct pressures in eight opossums, in both the fasted and fed states. Four bipolar electrode pairs were implanted in the sphincter of Oddi and duodenum. A polyethylene T tube was placed in the pancreatic duct. The common duct was cannulated through a small bile duct. This method allowed pressure recording for several weeks and avoided interference with the flow of bile or pancreatic juice into the duodenum. The frequency of slow waves was the same in the sphincter of Oddi and duodenum (19 per minute). The variation in the frequency of spike potentials in the sphincter of Oddi correlated to that of the migrating myoelectric complex in the duodenum. The average frequency of slow waves that have superimposed spike potentials in the sphincter of Oddi and duodenum was 3.0 and 0 in phase 1, 4.7, and 6.2 in phase II, 6.1 and 15.1 in phase III, and 3.4 and 6.3 in phase IV, respectively. The average duration of a migrating myoelectric complex cycle was 92 minutes. After feeding, the interdigestive phases of the migrating myoelectric complex were abolished and substituted by a feeding activity pattern that was characterized by an average number of sphincter of Oddi and duodenum spikes of 6.6 and 10.7, respectively. The mean fasting pressure in the pancreatic and biliary duct was 15 and 13 mmHg, respectively. Pressure changes were of two types--synchronous with respiratory movements and with each sphincter of Oddi spike potential. There was no variation in the baseline pressure during the migrating myoelectric complex phases and the fed state. It is concluded that the sphincter of Oddi of the fasting opossum exhibits cyclic changes in the number of spike potentials that correlate with the migrating myoelectric complex in the duodenum. However, the number and amplitude of spike potentials are different in the sphincter of Oddi and duodenum. There is no change in the baseline pressure during fasting and feeding states, and a temporary pressure elevation synchronic with each sphincter of Oddi spike potential was observed.

The role of biliary obstruction in the pathogenesis of acute pancreatitis in the opossum.

Pancreatitis was induced in the opossum by occluding the common bile duct above or below the entrance of the pancreatic duct. The common channel theory was tested by evaluating the effect of preligation of the pancreatic duct to prevent the reflux of bile after ligation of the distal common duct. The severity of the disease was determined by histologic grading of the degree of pancreatic tissue necrosis. Serum amylase, lipase, and calcium were determined. Concomitant obstruction of the biliary and pancreatic ducts produced severe necrotizing pancreatitis whether or not bile reflux was present. Pancreatic ductal obstruction alone was associated with acinar atrophy and mild interstitial pancreatitis. Biliary obstruction alone above the entrance of the pancreatic duct resulted in marked hyperemia of the gland but without histologic evidence of pancreatic inflammation. A positive bacterial culture of the pancreas was obtained in only four of 36 opossums in a distribution to suggest random contamination. There was an inverse correlation between calcium levels and the degree of tissue necrosis. This study demonstrates that biliary obstruction rather than bile reflux into the pancreas is a requisite for the pathogenesis of severe biliary pancreatitis in this model.

Estimation of hepatic blood flow by hydrogen gas clearance.

The hydrogen gas clearance technique was evaluated to estimate regional hepatic blood flow. Initially, the H2 gas method was compared to the indocyanine green clearance in mini pigs. The blood flow measured by the H2 gas method (0.49 +/- 0.03 ml/min/gm) was only 39% of the calculated blood flow by the indocyanine green method (1.26 +/- 0.14 ml/min/gm). The blood flow determined by H2 gas decreased to 0.06 +/- 0.04 ml/min/gm after ligation of the hepatic artery. No difference in blood flow was found by simultaneous measurements on the left (0.89 +/- 0.1 ml/min/gm) and right (0.96 +/- 0.13 ml/min/gm) liver lobes in rats. After ligation of the left hepatic artery in the rats, a delayed response to H2 application was present and the determined blood flow (0.14 +/- 0.02 ml/min/gm) was significantly reduced (p less than 0.001) compared with the right liver lobe H2 determined blood flow as a control. No current was generated after ligation of the left hepatic artery and the portal vein indicating that there was no blood flow measured by the gas H2 method. The results indicate that the H2 gas clearance technique may be a method that assesses hepatic arterial liver perfusion rather than estimates total hepatic blood flow. The safety and simplicity of the H2 gas clearance technique may make it suitable for clinical application in serial measurement of hepatic arterial blood flow in man.

Gallbladder contractility and mucus secretion after cholesterol feeding in the prairie dog.

The purpose of our study was to evaluate changes in gallbladder contractility and mucus secretion in vitro during the early stages of gallstone formation in prairie dogs. Thirty-two animals were divided into five groups. Control animals were fed a trace cholesterol diet. Experimental animals were fed a high-cholesterol diet for 3, 6, 8, and 14 days, respectively. Muscle stress was measured in response to cholecystokinin octapeptide in each of the groups. The maximal stresses in the 8-day diet (68 +/- 7 gm/cm2) (mean +/- SEM) and 14-day diet animals (83 +/- 7 gm/cm2) were found to be significantly lower than those of the control animals (137 +/- 12 gm/cm2). The stress in 3-day diet animals was significantly greater (224 +/- 23 gm/cm2). A significant increase in mucus secretion was observed only in 14-day diet animals (11.0 +/- 0.5 X 10(6) dpm/gm dry wt) compared with the control animals (6.4 +/- 1.0 X 10(6) dpm/gm). The decrease in contractility may be the initial event in cholesterol stone formation, and the prolonged exposure of the gallbladder epithelium to crystals may stimulate the release of mucus into the bile.

Effects of gastrointestinal hormones on Oddi's sphincter and duodenal myoelectric activity and pancreatobiliary pressure. Studies in the opossum.

Myoelectric activity of Oddi's sphincter and duodenum was correlated with common duct and pancreatic duct pressures in conscious opossums after infusion of the following substances: cholecystokinin, pentagastrin, glucagon, secretin, and acetylcholine. Cholecystokinin and pentagastrin increased and glucagon and secretin decreased the frequency of spike potentials in Oddi's sphincter and the duodenum. Acetylcholine was associated with the largest increase in spike potentials in both Oddi's sphincter and the duodenum. Although there was variation in the number of pressure elevations related to Oddi's sphincter spike potentials, the baseline pressure remained constant during administration of the hormones. Acetylcholine infusion increased the biliary and pancreatic pressures to 29 and 31 mm Hg, respectively. We conclude that gastrointestinal hormones may have an important role in regulating the excretion of bile and pancreatic juice into the duodenum.

The effect of preoperative internal and external biliary drainage on mortality of jaundiced rats.

Mortality following abdominal infection induced by cecal ligation and puncture was studied in rats with obstructive jaundice and after relief of the obstruction by preoperative internal or external biliary drainage. Four groups of adult Sprague-Dawley rats were used: common bile duct ligation (BDL), BDL followed by internal drainage after two weeks, BDL followed by external drainage after two weeks, and sham operation. The serum bilirubin concentration was significantly increased in the BDL group and returned to normal following internal and external drainage. Mortality in the sham group was 16.5% and increased to 83.3% following BDL, but it decreased significantly (25%) to near-normal values after preoperative internal drainage. However, after external drainage no significant reduction in mortality (63%) was found. These findings confirm the poor results of preoperative external drainage in previous studies and establish the efficacy of internal biliary drainage in a well-controlled experimental model that has to be evaluated in the clinical situation.

Ultrasonic imaging during vascular surgery.

Real-time B-mode ultrasound scanning was used during arterial reconstruction surgery to supplement the information of preoperative arteriography and to detect vascular defects immediately after restoration of blood flow. At 55 operations, 100 sites were examined. At three operations, ultrasound provided new information that, in two patients, directly affected the choice of operation. Intraoperative postreconstruction scanning detected vascular defects in over 20% of the patients. Intimal flaps were the commonest defects observed. In most instances in which defects were seen, the vessels were not reentered because the defects were considered to be too small or located in a nonstrategic site or both. One relatively large intimal flap in a common carotid artery prompted reentry. Ultrasound during vascular operations proved to be a highly sensitive diagnostic tool to supplement preoperative arteriography and to detect vascular defects resulting from reconstructive surgery.

Ultrasonic assistance during surgery for pancreatic inflammatory disease.

Real-time B-mode ultrasound scanning was used during 35 operations for inflammatory disease of the pancreas. In 21, ultrasonography was considered helpful in operative management of pancreatic pseudocysts, abscesses, and chronic pancreatitis. Assistance consisted of establishing a diagnosis not made during preoperative testing, more precisely localizing pancreatic abnormalities, and excluding the presence of pseudocyst, abscess cavities, and dilated ducts. Operative ultrasound is a safe and simple technique that may reduce the use of tissue dissection, contrast material injections, and radiation.

Detection of pancreatic tumors by ultrasound during surgery.

Real-time B-mode ultrasonic scanning was performed during 14 operations for pancreatic tumors. All operations were for adenocarcinoma, except in one patient with a Zollinger-Ellison tumor. The ultrasonic tissue appearance of pancreatic carcinoma was not specific. However, ultrasonic signs of pancreatic duct dilation, striction or invasion of the superior mesenteric veins, and common bile duct involvement may help to establish the diagnosis of malignancy. A triad of ultrasound signs indicative of malignant obstruction of the common bile duct consisted of (1) dilation, (2) absence of biliary stones, and (3) a distinctive termination pattern of the duct. Operative ultrasound was used to guide a biopsy needle to obtain pancreatic tissue samples. The Zollinger-Ellison tumor of the pancreas produced a sonolucent appearance that clearly distinguished it from the surrounding tissue.