Iron oxides in human spleen.

Iron is an essential element for fundamental cell functions and a catalyst for chemical reactions. Three samples extracted from the human spleen were investigated by scanning (SEM) and transmission electron microscopy (TEM), Mössbauer spectrometry (MS), and SQUID magnetometry. The sample with diagnosis of hemosiderosis (H) differs from that referring to hereditary spherocytosis and the reference sample. SEM reveals iron-rich micrometer-sized aggregate of various structures-tiny fibrils in hereditary spherocytosis sample and no fibrils in hemochromatosis. Hematite and magnetite particles from 2 to 6 µm in TEM with diffraction in all samples were shown. The SQUID magnetometry shows different amount of diamagnetic, paramagnetic and ferrimagnetic structures in the tissues. The MS results indicate contribution of ferromagnetically split sextets for all investigated samples. Their occurrence indicates that at least part of the sample is magnetically ordered below the critical temperature. The iron accumulation process is different in hereditary spherocytosis and hemosiderosis. This fact may be the reason of different iron crystallization.

A novel way of liver preservation improves rat liver viability upon reperfusion.

BACKGROUND/AIM: Currently, the liver is cold-preserved at 0 approximately 4 degrees C for experimental and clinical purposes. Here, we investigated whether milder hypothermia during the initial phase of the preservation period was beneficial for liver viability upon reperfusion. METHODS: In the first set of experiments, rat livers were preserved either conventionally in clinically used histidine-trypthopan-ketoglutarate (HTK) solution (Group A: 45 min and Group B: 24 h) or by slow cooling HTK solution (from 13 degrees C to 3 degrees C) during the initial 45 min of preservation (Group C: 24 h). In the second set of experiments, additional groups of livers were evaluated: Group BB--preservation according to Group B and Group CC--preservation according to Group C. Further, some livers were preserved at 13 degrees C for 24 h. Livers were then reperfused using a blood-free perfusion model. RESULTS: Bile production was approximately 2-fold greater in Group C compared to Group B. Alanine transaminase (ALT) and aspartate transaminase (AST) release into perfusate were 2 approximately 3-fold higher in Group B compared to Group C. No significant differences were found in ALT and AST release between Group C and Group A. Livers in Group CC compared to Group BB exhibited significantly lower portal resistance, greater oxygen consumption and bromosulfophthalein excretion into bile and lower lactate dehydrogenase (LDH) release into perfusate. Histological evaluation of tissue sections in Group BB showed parenchymal dystrophy of hepatocytes, while dystrophy of hepatocytes was absent in Group CC. Livers preserved at 13 degrees C for 24 h exhibited severe ischemic injury. CONCLUSION: These results suggest that the conventional way of liver preservation is not suitable at least for rat livers and that slow cooling of HTK solution during the initial phase of cold storage can improve liver viability during reperfusion.

The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor--an alternative gene therapy.

BACKGROUND: Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer). Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis. In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease. However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation. AIM: To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis. METHODS AND RESULTS: Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF121, VEGF164, VEGF189). Selection of recombinants was achieved by cultivating E. coli cells on ampicillin-added medium. The expression of target genes in the T7 expression system was induced by isopropyl-beta-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms. Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy. CONCLUSION AND HYPOTHESIS: In summary, E. coli strain expressing VEGF was prepared and its biological effect confirmed. Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use. The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine. The main advantage of the described approach lies in the enhanced regulation control--bacterial expression can be regulated positively (induction by exogenous low molecular weight agents) and negatively (application of antibiotics). The hypothesis of alternative gene therapy should be proved in further studies.

Effects of anabolic steroids and antioxidant vitamins on ethanol-induced tissue injury.

Various mechanisms are involved in the process of ethanol-induced tissue impairment. Oxidative stress and its effects are among the most important. We compared the effects of antioxidant vitamins (vitamin C and E in combination) and steroids (testosterone and nandrolone separately) on the toxicity of ethanol in rats. Animals (male Wistar rats, n = 48) were randomised into following groups-Control, Ethanol, Testosterone, Ethanol + Testosterone, Ethanol + Nandrolone, Ethanol + Vitamins. Alcohol was given daily by gavage in a dose of 5 g/kg of body weight. On the 27th day of the study the animals were sacrificed by decapitation and tissue samples were taken. Metabolic status, parameters of the hepatic metabolism, hormone levels (testosterone, ACTH, corticosterone), lipoperoxidation markers (malondialdehyde and conjugated diens in forebrain cortex and in cerebellum) and advanced glycation end-products were analysed. Tissue samples underwent histological examination. Histological outcomes showed a protective effect of antioxidants on hepatic and cerebellar injury caused by chronic ethanol intake. Anabolic steroids protected especially the central nervous tissue against the toxicity of alcohol. Both, antioxidant vitamins and anabolic steroids protect against the ethanol-induced toxicity, however, this effect is tissue specific.

Collagen type IV in epithelial tumours of colon.

Collagen type IV in the lamina propria mucosae is one of the main components of the basement membrane of normal and transitional colon mucosa. The aim of the present study was to assess the use of anti-collagen type IV antibodies in the evaluation of biological activity of epithelial tumours of the colon. Formalin-fixed and paraffin-embedded specimens of polyps and carcinomas of the colon from 14 patients were analyzed. In transitional mucosa around epithelial tumours, only minor deformities of the evenly thick collagen type IV-containing basement membranes were found. This pattern was different in polyps where collagen type IV-positive basement membrane components extended between basolateral membranes of epithelial cells. Local changes of collagen type IV positivity in basement membranes of polyps were observed. Positivity of epithelial basement membranes disappeared in adenocarcinomas but there was an increased positivity in fibrillar components of stroma. Basement membranes of microvessels in lamina propria mucosae were also positive for collagen type IV. Similar observations were made in the stroma of polyps. Our results indicate that loss of collagen type IV in basement membranes of adenocarcinomas is related to loss of differentiation and the malignant potential of epithelial tumours of colon.

Fluorescence of hematoxylin and eosin-stained histological sections of the human spleen.

A major problem in the morphometric evaluation of human spleen is the simple but reliable determination of the border between T-cell and B-cell dependent areas, and other structures of the spleen. It was investigated whether cryostat sections of frozen surgical specimens of the human spleen and sections of paraffin-embedded specimens could be used for this purpose after being stained with haematoxylin and eosin and mounted in autofluorescence-free medium for fluorescence microscopical evaluation. Comparison was made with sections that were immunohistochemically-stained for fibronectin and collagens type II and type IV. Both in cryostat sections and paraffin sections, fluorescence was found in circumferential reticulum of periarterial lymphatic sheets, arterial terminals, arterial walls and walls of red pulp sinuses in the spleen. Evaluation was hindered by fluorescence of erythrocytes in paraffin sections but not in cryostat sections. Results were similar as those obtained with immunohistochemical fibronectin staining and are sufficient for morphometric evaluation or orientation in the tissue in case of neoplasia.