[The role of plasmapheresis in prevention of complications after thoracoscopic thymectomy in patients with myasthenia combined with thymic hyperplasia]. / Rol' plazmafereza v profilaktike oslozhnenii videotorakoskopicheskoi timektomii u patsientov s miasteniei v sochetanii s giperplaziei vilochkovoi zhelezy.

Surgical indications for thymectomy include thymus tumour of different genesis as well as myasthenia. Minimally invasive methods such as thorascopy and robot-assisted thymectomy have been widely used lately as well as standard surgical methods namely sternotomy and thoracotomy. Regardless the type of access in myasthenia patients specific crisis conditions of respiratory failure can appear in postoperative period that usually requires mechanical ventilation. This study was aimed to estimate the efficacy of pre- and postoperative plasmapheresis in prevention of specific myasthenia-associated complications.

[Surgery for cardio-esophageal cancer].

The experience of surgery of 369 patients with cardio-esophageal cancer treated in the Kazan Republic Clinical Oncology Center is presented. The patients are divided into 3 groups respective of the type of adenocarcinoma (classification by J.R. Siewert). Thus, the first group consists of 45 (12.1%) patients, the second--172 (46.6%) and the third--152 (41.3%) patients. Each group is divided into subgroups according to the performed operation: transhiatal esophagoplasty with esophagogastroanastomosis on the neck, transthoracal esophagoplasty (operations by Lewis and Gerlock) with intrapleural esophadogastroanastomosis and gastrectomies with high resection of esophagus. High productivity of the differentiated approach to surgical treatment of cardio-esophageal cancer depending on the type of adenocarcinoma is shown. Such approach allows the surgical treatment to be more radical, to reduce quantity of the early and remote complications and to raise the survival. So, it is revealed that performance of transhiatal and transthoracal esophagoplasty is possible in cases of type I adenocarcinoma. Gastrectomies in cases of type II adenocarcimoma are of poor prognosis.

[The program of accelerated rehabilitation after esophagoplasty (fast track surgery) in esophageal cancer surgery].

Esophagectomy with simultaneous plasty in patient with esophageal cancer is still associated with a high incidence of postoperative complications and long-stay patient in the clinic. The purpose of our report is to inform the use of the program of accelerated rehabilitation after esophagectomy in a prospective study of 13 patients during the period from 2010 to 2011 year and the role of the anesthesiologist in its implementation. Methods aimed at the preoperative examination, minimally invasive surgery, thoracic epidural anesthesia/analgesia with local anesthetics as a component of anesthesia and postoperative analgesia, early extubation and mobilization of the patient with the implementation of breathing exercises, early enteral feeding, and the planned short postoperative stay in resuscitation and hospital were used. Postoperative complications were observed in 3 (23/1%) patients: one patient (7/7%) had right-side pneumonia, two patients (15/4%) had right-side pneumothorax requiring emergency re drainage. The average intensive care stay was 2 (1-4) days, postoperative hospital stay--9 (7-12) days. Further monitoring of the patients did not show any long-term complications. The results confirm that it is possible to optimize the healing perioperative process in patients after esophagectomy with simultaneous plasty by using of accelerated rehabilitation program without the risk of increasing the frequency of postoperative complications. it will provide the reduction of length of hospital stay. In view of multifaceted and controversial issue the following researches in this direction are necessary.

[SOME ASPECTS OF DIAGNOSIS AND COMPREHENSIVE TREATMENT OF BARRETT'S ESOPHAGUS].

OBJECTIVE: Assess the value of routine endoscopy in the diagnosis of Barrett's esophagus (BE) in patients with gastroesophageal reflux disease (GERD) and to show the feasibility of a comprehensive treatment algorithm that includes antireflux surgery as an essential component. MATERIALS AND METHODS: The study was conducted on a sample of 171 patients with Barrett's esophagus who underwent antireflux surgery. In evaluating the operating characteristics of endoscopy were recruited another 675 patients with GERD without BE. On the diagnostic phase was used endoscopy with double chromoscopy and biopsy followed by histological examination. At the stage of treatment, patients received conservative therapy by PPl after which underwent antireflux surgery and argon-plasma coagulation (in some cases). RESULTS: Endoscopy of the esophagus with a double chromoscopy in identification BE in patients with GERD, has a moderate sensitivity (71.9%, 95% Cl 64.6%-78.5%) and high specificity (100%, 95% Cl 99.5%-100%). A comprehensive treatment approach that includes antireflux surgery as an essential component has allowed to achieve excellent and satisfactory immediate results of treatment in 88.9% of patients (95% Cl 83.2%-93.2%). Excellent and satisfactory long-term results were achieved in 89.5 % of patients (95% Cl 83.9%-93.6 %). CONCLUSION: The results indicate the need for biopsy and histological examination in all patients with GERD, who has no suspicion of BE according to endoscopy, and prove high efficiency of complex treatment algorithm that includes antireflux surgery as an essential component.

[The ruptures of the trachea with the intubation tube in patients with esophagus cancer].

Postintubation tracheal ruptures is a rare but serious complication with high risk for the patient's life. The preliminary diagnosis is usually made after occurrence of subcutaneous emphysema, blood spitting, respiratory insufficiency, pneumothorax and/or pneumomediastinum. The suspected rupture of the trachea should be verified by fiber-optic bronchoscopy. The decision about necessity of surgical or conservative treatment is based on the compilation of clinical, radiologic and endoscopic data. We present 9 cases (7 women and 2 men) of postintubation tracheal ruptures, occurred during the esophageal (6), lung (2) and mammary gland (1) surgery.

[Endoscopic diagnosis and complex treatment of Barrett's esophagus complicated by hernia of esophageal foramen of the diaphragm].

A protocol is suggested of complex diagnosis and treatment of Barrett's esophagus using sparing endoscopic removal of Barrett's epithelium in combination with surgery and medicinal antireflux therapy. Eighty-three patients were diagnosed and treated for hernia of esophageal foramen of the diaphragm and gastro-esophageal reflux complicated by Barrett's esophagus. Ninety-two percent of patients receiving our four-component treatment were cured; no recurrent esophageal adenocarcinoma was reported during the 56.7 +/- 2.4 month follow-up. Conversely, in patients receiving three-component treatment, efficacy was 56%; esophageal adenocarcinoma was reported in 3 (12%).

[An integrated approach to diagnosis and treatment of Barrett's esophagus].

Barrett's oesophagus is a condition when the oesophagus adenocarcinoma risk increases. There are different ways of diagnostic and treatment for this disease abroad and our country. We offer a complex method for Barrett's oesophagus treatment. Our method reveals Barrett's oesophagus effectively. We also take antireflux treatment and Barrett's epithelium elimination using miniinvasive surgery with drugs therapy. We have experience of curing 48 patients from Barrett's oesophagus. Considering obtained results our tactic for clinical practice is recommended.

Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells.

In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.

Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor.

Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter.

To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the reporter gene luciferase and the murine preproendothelin-1 promoter. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9-kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per microgram of protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. The further demonstrate the targeting capability of this system, we overexpressed the lipid-peroxidating enzyme, human 15-lipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localization in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins.

Cloning and characterization of a murine macrophage lipoxygenase.

We have isolated a murine macrophage cDNA encoding a 12-lipoxygenase, that represents the homolog of the human 15-lipoxygenase. The predicted amino acid sequence of this lipoxygenase is highly similar to the rat 12-lipoxygenase isolated from brain and human 15-lipoxgenase. The recombinant enzyme expressed in Cos-7 cells oxidizes arachidonic acid to 12- and 15-HETE with a profile similar to that obtained from peritoneal macrophages. A polyclonal antibody generated against a putative peptide recognizes a 75 kDa protein in cell extracts from mouse peritoneal macrophages and transfected Cos-7 cells. The lipoxygenase cDNA hybridizes to a 2.5 kb mRNA present in peritoneal macrophages, lung, spleen, heart and liver. RT-PCR analysis indicates that the same lipoxygenase is expressed in mouse reticulocytes. A partial genomic clone for this lipoxygenase has also been characterized. Southern blot analysis of mouse genomic DNA indicates that this is a single copy gene.

Characterization and sequence of an additional 15-lipoxygenase transcript and of the human gene.

15-lipoxygenase is a lipid-peroxidating enzyme that oxidizes fatty acids, such as those esterified to cellular membranes. It has been implicated in the oxidative modification of low-density lipoprotein and is thus thought to contribute to the development of atherosclerosis. The enzyme has also been shown to be specifically induced by interleukin-4 in human blood monocytes. Two 15-lipoxygenase-hybridizing messages were detected in these cells; one (2.7 kb) corresponds to the previously isolated cDNA for 15-lipoxygenase, while the other (4 kb) was of unknown origin. We have isolated and characterized this 4 kb transcript. Our experiments show that it has 1.2 kb additional sequence in its 3' untranslated region, and that it is generated from genomic sequences through differential polyA site selection. We present studies to address the functional significance of the extended 3'UTR. Selection of an upstream polyadenylation signal results in production of the 2.7 kb transcript. In addition, we present here for the first time the cloning and sequence of the human 15-lipoxygenase gene, as well as the identification of regulatory elements in the promoter region of this gene.

Overexpression, purification and characterization of human recombinant 15-lipoxygenase.

Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system. Catalytically active enzyme was readily purified (90-95% pure) from cytosolic fractions by anion-exchange chromatography on a Mono Q column with approx. 95% recovery of enzymatic activity. Routinely, a yield of 25-50 mg of pure enzyme per L of culture and a specific activity of 7.1-21 mumol 13-hydroxyoctadecadienoic acid (13-HODE)/mg.min (turnover rate of 8.4-25 s-1) were obtained. Both the specific activity and the enzyme's iron content was significantly increased by the addition of ferrous ions to either the purified enzyme or to the insect cell culture medium during production. An isoelectric point of 5.85 was determined and the N-terminal amino acid sequence was found to be identical to that predicted from the cDNA. The purified recombinant enzyme exhibits a dual positional specificity with arachidonic acid (formation of 15S- and 12S-hydroxyeicosatetraenoic acid (12S-HETE) in a ratio of 12:1). Double oxygenation products 14R,15S- and various 8,15-DiHETE isomers were also identified. With linoleic acid as substrate, a pH-optimum of 7.0 and a KM of 3 microM were determined. The enzyme undergoes suicidal inactivation during fatty acid oxygenation, is sensitive to standard lipoxygenase inhibitors, and oxygenates phospholipids, cholesterol esters, biomembranes and human low-density lipoprotein. Contrary to prior studies on the rabbit enzyme, no glycosylation was detected.

12/15-Lipoxygenase gene disruption attenuates atherogenesis in LDL receptor-deficient mice.

BACKGROUND: Human 15-lipoxygenase (LO) and its murine analogue 12/15-LO are capable of directly oxidizing esterified fatty acids in lipoproteins and phospholipids. Because these oxidized products possess atherogenic properties, it was suggested that LOs may be involved in enhancing atherogenesis. Previous in vivo tests of the role of LOs in atherogenesis animal models, however, have yielded conflicting results. METHODS AND RESULTS: Aiming to study the role of the 12/15-LO in murine atherogenesis, we crossed LDL-receptor-deficient mice (LDL-R(-/-)) with 12/15-LO-knockout mice and evaluated plaque formation 3 to 18 weeks after initiation of a high-fat diet. Atherosclerotic lesions were considerably reduced in the LDL-R/12/15-LO-double-knockout mice compared with LDL-R(-/-) mice at 3, 9, 12, and 18 weeks, at the aortic root as well as throughout the aorta. The cellular composition of plaques from mice deficient in 12/15-LO did not differ with respect to macrophage and T-lymphocyte content compared with plaques from 12/15-LO littermates. CONCLUSIONS: 12/15-LO plays a dominant role in promoting atherogenesis in LDL-R(-/-) mice.

Overexpression of 15-lipoxygenase in vascular endothelium accelerates early atherosclerosis in LDL receptor-deficient mice.

To study the possible role of the human lipid-oxidizing enzyme 15-lipoxygenase (15-LO) in atherosclerosis, we overexpressed it specifically in the vascular wall of C57B6/SJL mice by using the murine preproendothelin-1 promoter. The mice overexpressing 15-LO were crossbred with low density lipoprotein (LDL) receptor-deficient mice to investigate atherogenesis. High levels of 15-LO were expressed in the atherosclerotic lesion in the double-transgenic mice as assessed by immunohistochemistry. The double-transgenic, 15-LO-overexpressing, LDL receptor-deficient mice (LDLR-/-/15LO) developed significantly larger atherosclerotic lesions at the aortic sinus compared with lesions in the LDL receptor-deficient (LDLR-/-) mice after 3 and 6 weeks (107,000 versus 28,000 microm(2) [P:<0.001] and 121,000 versus 87,000 microm(2) [P:<0.05], respectively) of an atherogenic diet. LDL from the LDLR-/-/15LO mice was more susceptible to oxidation than was the LDL from the control LDLR-/- mice, as shown by a shorter lag period for copper-induced conjugated diene formation. On the other hand, no differences were found in the levels of serum anti-oxidized LDL antibodies between the study groups. There were also no differences with respect to the density of macrophages and T lymphocytes infiltrating the lesions in both experimental groups. Taken together, these results support the hypothesis that 15-LO overexpression in the vessel wall is associated with enhanced atherogenesis.

Applied genomics: integration of the technology within pharmaceutical research and development.

Multiple novel technologies have recently been developed to improve the analysis of genetic sequences, to rapidly assess RNA or protein levels in relevant tissues, and to validate function of potential new drug targets. The challenge facing pharmaceutical research is one of effective integration of these new technologies in ways that can maximally affect the discovery and development pipeline. Although database mining and transcriptional profiling clearly have increased the number of putative targets, the current focus is to assign function to new gene targets in a high-throughput manner. This requires a restructuring of the classical linear progression from gene identification, functional elucidation, target validation and screen development. New approaches are called for that can make this process non-linear and high-throughput.

A possible role for 15-lipoxygenase in atherogenesis.

It is well accepted that high levels of low-density lipoprotein (LDL) cholesterol in the plasma are associated with increased risk of atherosclerosis. The cellular and molecular mechanisms linking the two however, have not been fully resolved. One of the processes involved in atherogensis that has been intensively studied in this regard is the oxidation of LDL. Oxidation may convert LDL into an atherogenic form, which incites an inflammatory and proliferative response characteristic of the atherosclerotic lesion. One of the potential mediators in this process is the lipid peroxidating enzyme 15-lipoxygenase, which has been shown to be induced in the atherosclerotic lesion and is capable of oxidizing LDL. In this article, we review the motivation for looking at mechanisms of LDL oxidation and the proposed involvement of 15-lipoxygenase in the pathogenesis of the disease.

Interleukin (IL)-4 deficiency does not influence fatty streak formation in C57BL/6 mice.

Abundant data is present to implicate oxidatively modified low-density lipoprotein (oxLDL) in enhanced atherogenesis. Among the factors involved in LDL oxidation, an important role has been attributed to human 15-lipoxygenase (LO) and its murine analog 12-LO. The expression of these peroxidizing enzymes is under the control of cytokines, the principal of which is IL-4. In the present study we tested the hypothesis that knocking out the IL-4 gene from C57BL/6 mice would result in suppression of fatty streaks. For this purpose, we have fed 45 female IL-4 transgenic knockout (IL-4T KO) and 45 wild-type (WT) mice an atherogenic diet for 15 weeks. Consecutive determinations of the lipid profile from both study groups were performed at monthly intervals, and fatty streak formation was assessed at the aortic sinus level, upon sacrifice. The two study groups did not differ significantly with respect to the lipid profile or the uptake and degradation of iodinated oxLDL by their peritoneal macrophages. We found that the endogenous deficiency of IL-4 did not confer protection from early atherosclerosis in the IL-4T KO as compared to their WT littermates (determined at the aortic sinus). Immunohistochemical studies, Western blots and 12/15-LO activity assays revealed the presence and activity of 12/15-LO in macrophages of WT mice as well as in IL-4T KO mice. Both did not differ significantly between the study groups. The data from this study imply that deficiency in IL-4 does not affect early atherosclerosis in C57BL/6 mice fed a high-cholesterol diet.

The effects of N-6 polyunsaturated fatty acid supplementation on the lipid composition and atherogenesis in mouse models of atherosclerosis.

Despite numerous studies, the precise role of dietary n-6 polyunsaturated fatty acids in the pathogenesis of atherosclerosis remains controversial. It has been shown that feeding an n-6-enriched diet resulted in decreased atherosclerosis in African green monkeys and was associated with a reduction in LDL levels. However, other authors reported that n-6 supplementation increased the oxidative stress and the susceptibility of LDL to undergo in vitro oxidation, thus potentially enhancing atherosclerosis. The present study was designed to investigate the effect of dietary supplementation of n-6 polyunsaturated fats (safflower oil), as compared with a saturated fat-rich diet (Paigen), on the blood lipid profile and atherosclerosis in two mouse models. In the first experiment, female C57BL/6 mice (n=23-30 per group) were fed a cholate containing Paigen diet, a safflower oil-rich diet (with cholate), or normal chow for 15 weeks. No significant differences between the high fat diet groups were evident with respect to total cholesterol, LDL, HDL or triglyceride levels. The extent of aortic sinus fatty streaks did not differ significantly between the two groups. In the second experiment, LDL-receptor-deficient (LDL-RD) mice (n=20-30 per group) were randomized into similar dietary regimens. Mice consuming a safflower oil-enriched diet developed significantly less atherosclerosis, in comparison with Paigen diet-fed mice. A reduction in LDL levels, although not of a similar magnitude as the reduction in atherosclerosis, was evident in the safflower oil-fed mice when compared to the Paigen diet-fed littermates. In both mouse models of atherosclerosis, LDL isolated from the plasma of mice on the n-6 polyunsaturated diet was rendered slightly more susceptible to oxidation in vitro, as indicated by a shorter lag period for diene formation. Thus, the effects of n-6 fatty acids on the lipoprotein composition and other potential influences may have contributed to the anti-atherogenic effect in the LDL-RD mouse model.

On the positional specificity of 15-lipoxygenase.

The lipoxygenases comprise a family of fatty acid dioxygenases that are involved in a variety of inflammatory conditions. Various approaches have been taken in order to understand the different regiospecificities of the different lipoxygenases. Here we have reviewed the current knowledge of the structural features of the substrate and of the enzyme that form the basis of the regiospecificity of 15-lipoxygenase. Earlier experiments on the structural features of the substrate were reviewed, as well as more recent results of site-directed mutagenesis studies. The structure of the soybean lipoxygenase isoform-1 was also briefly reviewed.