20S proteasome in the blood plasma of boys with cryptorchidism.

PURPOSE: To evaluate the concentration of 20S proteasome in the blood plasma of boys with cryptorchidism. METHODS: Patients-50 boys aged 1-4 years (median = 2.4 years) with unilateral cryptorchidism. The control group-50 healthy, age-matched boys (aged 1-4 years, median = 2.1 years), admitted for planned herniotomy. In our study, we used a novel technique Surface PLASMON RESONANCE Imaging. RESULTS: The median concentration of 20S proteasome in the blood plasma of boys with cryptorchidism was 2.5-fold higher than in boys with inguinal hernia. We noticed statistically significant difference between 20S proteasome levels in boys with cryptorchidism up to 2 years old and above 2 years old. CONCLUSIONS: We believe that the 20S proteasome concentrations in the blood plasma of boys with cryptorchidism reflect the heat-induced apoptosis of germ cells.

Immunoproteasome in the blood plasma of children with acute appendicitis, and its correlation with proteasome and UCHL1 measured by SPR imaging biosensors.

The aim of this study was to determinate the immunoproteasome concentration in the blood plasma of children with appendicitis, and its correlation with circulating proteasome and ubiquitin carboxyl-terminal hydrolase L1 (UCHL1). Twenty-seven children with acute appendicitis, managed at the Paediatric Surgery Department, were included randomly into the study (age 2 years 9 months up to 14 years, mean age 9·5 ± 1 years). There were 10 girls and 17 boys; 18 healthy, age-matched subjects, admitted for planned surgeries served as controls. Mean concentrations of immunoproteasome, 20S proteasome and UCHL1 in the blood plasma of children with appendicitis before surgery 24 h and 72 h after the appendectomy were higher than in the control group. The immunoproteasome, 20S proteasome and UCHL1 concentrations in the blood plasma of patients with acute appendicitis were highest before surgery. The immunoproteasome, 20S proteasome and UCHL1 concentration measured 24 and 72 h after the operation decreased slowly over time and still did not reach the normal range (P < 0·05). There was no statistical difference between immunoproteasome, 20S proteasome and UCHL1 concentrations in children operated on laparoscopically and children after classic appendectomy. The immunoproteasome concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation may thus be helpful in the assessment of the efficacy of treatment. The method of operation - classic open appendectomy or laparoscopic appendectomy - does not influence the general trend in immunoproteasome concentration in children with appendicitis.

Correlation between circulating proteasome activity, total protein and c-reactive protein levels following burn in children.

AIM OF THE STUDY: To characterize burn-induced changes following burn in children by analyzing circulating proteasome (c-proteasome) activity in the plasma in correlation with total protein and c-reactive protein levels in the plasma, and the severity of the burn. METHODS: Fifty consecutive children scalded by hot water who were managed at the Department of Pediatric Surgery after primarily presenting with burns in 4-20% TBSA were included into the study. The children were aged 9 months up to 14 years (mean age 2.5±1 years). Patients were divided into groups according to the pediatric injury severity score used by American Burns Association. Plasma proteasome activity was assessed using Suc-Leu-Leu-Val-Tyr-AMC peptide substrate, 2-6 h, 12-16 h, 3 days, 5 days, and 7 days after injury. 20 healthy children consecutively admitted for planned inguinal hernia repair served as controls. RESULTS: Statistically significant elevation of plasma c-proteasome activity was noted in all groups of burned children 12-16 h after the injury. We found a strong negative correlation of c-proteasome activity with total protein levels, and positive correlation with CRP levels 12-16 h after burn. We also found stronger correlation between c-proteasome activity and severity of burn, than CRP level and severity of burn 12-16 h, and 3 days after the burn. Correlations were statistically significant. CONCLUSIONS: This study characterized circulating 20S proteasome activity levels after burn. C-proteasome activity elevate after burn and correlate negatively with plasma total protein level, thus plasma 20S proteasome activity could be additional biomarker of tissue damage in burn in pediatric population.

Brown-Séquard syndrome in a 11-year-old girl due to penetrating glass injury to the thoracic spine.

Injuries in children are one of the most frequent causes of high morbidity and mortality, and they present a challenge to the treating physician. Fortunately, spinal trauma in pediatric patient is relatively rare. Brown-Séquard syndrome is a rare form of incomplete spinal cord injury consisting of ipsilateral upper motor neuron paralysis (hemiplegia) and loss of proprioception with contralateral pain and temperature sensation deficits resulting from hemisection or lateral injury to the spinal cord. A 11-year-old girl was admitted to our Pediatric Trauma Emergency Department after she had suffered a penetrating back injury. Neurological examination demonstrated left lower extremity paresis and moderate spastic paralysis of the right lower extremity. The examination showed loss of temperature sensation contralateral to and below the lesion. The examination of the pain sensation was difficult because the patient was in pain shock, but it was diminished on the side opposite to the damage. Multislice spiral computed tomography (MSCT) demonstrated a triangular foreign body in spinal canal at the level of the Th11-Th12. After a Th11-L2 laminectomy and retrieval of foreign bodies, dura repair was performed. Patient was discharged from the hospital with partial recovery. Operative decompression of the neural elements in case of spinal canal compromise is the treatment of choice. Indication for surgical intervention in existing cerebrospinal fluid fistula includes closure of the dura and reducing neural elements compression and lowering the risk of infectious complications by removing bone or foreign body fragments. Patients with Brown-Séquard syndrome have good prognosis for functional recovery.

Mast cells in peritoneal fluid in rats with experimentally induced peritoneal adhesions.

Mast cells (MC) produce, store and release many biologically active substances, especially inflammatory factors, chemotactic substances for neutrophiles, cytokines and prostaglandins. They play very important role in fibrinosis and they are an important factor in peritoneal adhesions formation and lysis. In this study we tried to evaluate role of mast cells in peritoneal adhesions formation. We estimated number of mast cells in peritoneal fluid in rats with experimentally developed peritoneal adhesions. The number of mast cells per ml was counted in flow cytometry in specimens of peritoneal fluid taken from operated rats. The fluid was taken in standardized conditions the same for each group at the first operation and during reoperation. Peritoneal cavity was washed with 0.9% Saline solution. MC were visualized using indirect immunohistochemical method LSAB with mouse antibody. The animals were divided into 4 groups. 1 st group was control group (n=20) on which the abdomen was opened and closed without any manipulations, and the reoperation was done after 72 hours. The other groups (2, 3, 4; n=20 for each group) were operated and scarification of the partial peritoneum and serosa was performed. The rats were brought back to conscious and then were reoperated respectively after 24, 72 and 168 hours after first surgery. After the laparotomy and damage of the peritoneum we observed formation of the peritoneal adhesions between intestine loops and between intestines and damaged parietal peritoneum. Also the higher number of mast cells was observed in the groups of animals with damaged peritoneum. The highest number of peritoneal adhesions was observed in the group of animals reoperated after 72 hours. After 72 and 168 hours the higher number of MC and neutrophils was observed. The difference was statistically significant. The percentage of mast cells was increasing during the experiment duration. It was different from other cells populations which decreased after 168 hours. The MC and neutrophils were cell population which changed significantly after manipulations in peritoneal cavity. It is very probable that they play an important role in peritoneal adhesions formation.

Use of synaptophysin immunohistochemistry in intestinal motility disorders.

The purpose of this study was to describe synaptophysin (SY) immunoreactivity in colonic specimens from patients with Hirschsprung's disease (HD), chronic constipation (CC), or anal atresia (AA). This membrane protein is specific for the synaptic vesicles in the central and peripheral nervous system and responsible for neurotransmission. Biopsy specimens of the intestinal wall were obtained from 18 patients (age range, 2 days to 7 years). Immunohistochemistry was performed using rabbit anti-human antibodies specific for synaptophysin (DAKO). In the ganglionic colon of HD patients and others the immunoreactivity of SY-positive synapses was abundantly present in the smooth muscle layers. Distinct immunoreactivity showed ganglion cells and nerve fibers inside circular and longitudinal muscle layers. In some non-HD patients' colonic specimens SY-positive synapses were present in the muscularis mucosae. In the aganglionic colonic segment of HD-patients no immunoreactivity of synapses and ganglions was seen. In the transition zone, where ganglion cells appeared sporadically, synapses were very rarely present. In two patients from the CC group the amount of visualized synapses was clearly smaller and the concentration of ganglion cells within ganglions in these cases was much lower than usual (but still within normal ranges). In the AA group in the distal part of the atretic rectum (at the place where the fistula was cut) SY-positive synapses were present in smooth muscle layers and small dysplastic ganglions were seen in the submucosal and muscular region, but not in large numbers. These patients had a normal distribution of ganglion cells and synapses at the place of colostomy. Synaptophysin immunohistochemistry is an indirect labeling method with a high detection rate for intestinal ganglion cells by demonstrating their synapses. Changed intestinal distributions of SY-positive synaptic vesicles usually accompany colonic ganglion cell disorders. The pattern of SY-positive synapses distribution in circular and longitudinal colonic muscles and intermuscular ganglions can reflect functional disturbances of large bowel motility and could be helpful in the description of the innervation status of colonic specimens in HD patients.

Oxyuriasis-induced intestinal obstruction in a child--case report.

PURPOSE: The presentation of an unusual case of the tumor of ileum wall induced by pinworm infection in a 5-years-old child. MATERIAL AND METHODS: The record of a 4-years-old boy treated in the department of pediatric surgery was analyzed concerning the diagnostic difficulties. After 6 month from an episode of ileo-cecal intusussception successfully treated with a barium colon enema, the diagnosis of lymphoma was made and the resection of distant segment of small intestine was performed. RESULTS: No clinical and laboratory features of oxyuriasis could be stated before the onset of disease, during hospitalization and in the follow-up period. The hypertrophied and activated lymphatic tissue with a non-specific inflammatory reaction to the pinworms were seen in the wall of ileum, appendix and mesenteric lymph nodes. No neoplastic cells were found in the microscopic study of ileum, appendix, mesenteric lymph nodes and peritoneal lavage fluid. CONCLUSIONS: The proper diagnosis of oxyuriasis may be difficult when the course is atypical. The enterobius vermicularis infestation as an etiologic factor should be taken into account in any case of abdominal pathology. However, the methods routinely used in "acute abdomen" including examinations of blood, urine and stool, repeated ultrasound and CT, are not reliable. As the infestation may mimic neoplasm, the surgical treatment and microscopic examination can be necessary for the final diagnosis in some cases.

Suitability of selected markers for identification of elements of the Intestinal Nervous System (INS).

The possibility of identifying and characterising elements of the enteric nervous system based on their contents of cathepsin D, chromogranin A, neuronal specific enolase and S-100 protein was studied in colorectal specimens (operative full-thickness, seromuscular and mucosomuscular biopsies) obtained from 15 children, aged 2 days to 10 years. Nine patients suffered from Hirschsprung's disease, and two from chronic constipation. Four neonates with imperforate anus or meconium ileus composed the control group. All markers were identified immunohistochemically by antibodies against human antigens with appropriate detection methods. Chromogranin A staining was not always adequate to identify all neuronal cell bodies and other nervous elements. However, it proved superior to the other methods in the depiction of neuroendocrine cells in the intestinal mucosa. Cathepsin D antibodies stained normal and abnormal neural cells with different intensity; nerve fibres were not stained. This marker did not allow an unequivocal differentiation of ganglion cells from macrophages within the submucosa; the latter exhibited exceptionally strong marking and in some cases represented the predominant elements in this area. Neuronal specific enolase was distinctly expressed in nerve cells and fibres of the intestinal wall. Atrophic and hypoplastic features could be identified, suggesting that this method may give some insight into functional aspects. Continuous connections between ganglions were also observed. S-100 protein antibodies resulted in a negative image of unstained ganglion cells surrounded by extensively marked Schwann cells and neural fibres. With respect to clinical application, all these markers may provide supplementary information for the differential diagnosis of intestinal motility disorders.

The evaluation of murine pleural lavage fluid cellular composition in experimental hemorrhagic shock with special regard to mast cells morphometry.

In the course of a hemorrhagic shock, pathological changes occur, which result in intensifying the insufficiency of various vital organs. It can also lead to the development of the multiorgan dysfunction syndrome (MODS) that is the cause of high posthemorrhagic mortality. As a result of the ischemia in the lung there appear proinflammatory factors that mobilize and activate mast cells, inducing degranulation in them. The aim of the study was the analysis of cellular composition and cytomorphometric evaluation of mast cells present in the lavage fluid from the pleural cavity of rats in a sham operated group and in the group presenting hemorrhagic shock. The results revealed an increase of the total cell count in the lavage fluid from the pleural cavity. In the cytological smears a statistically significant accumulation of inflammatory cells was present, especially neutrophils. The increase in mast cells and eosinophils number was not statistically significant. There was not a change in the morphometric parameters of mast cells except the circularity index. A decline of the circularity index indirectly may suggest the degranulation of mast cells, which reflects an inflammatory process in the lungs.

Hemorrhagic shock activates mast cells in the rat stomach wall.

The activation of mast cells (MC) and liberation of their mediators can play an essential role in initiating and controlling inflammatory processes in the wall of the gastrointestinal tract (GIT) due to ischemia. The role of MC in changes induced by hemorrhagic shock (HS) remains unknown. Heparin provided by MC seems to inhibit local inflammation and prevent DIC. The aim of this study was to evaluate the morphometric changes and biochemical activity of MC in the stomach wall after 75 minutes of hemorrhagic shock. The MC in mucosal, submucosal, muscular and serosal compartments of the various stomach wall regions were examined in shocked rats and in the control group. Additionally, the contents of glycosaminoglycans (GAG), measured as uronic acids concentration, as well as anticoagulative activity in the stomach wall were assessed. HS resulted in an evident increase in the number of mast cells detected in the stomach mucosa and serosa, in slight alterations in number of MC in the submucosal and muscular layers, a significant increase in size and changes of the shape of the MC. The elevation of the width, area, and circular shape of MC in all layers were noted. No clear and significant differences between various stomach regions in MC numbers and MC sizes could be shown. No reaction of other inflammatory cells at this stage of shock was observed. Highly significant increases in GAG concentration, and anticoagulative activity in the stomach wall due to shock were noted. The morphometric and biochemical data may indicate MC activation, especially in mucosa and serosa. The shock-induced migration of MC settled in the stomach wall seems to be possible. The results suggest an essential role of MC reaction in the stomach wall in the early phase of hemorrhagic shock.

BN52021 stabilizes the oxidant-antioxidant equilibrium in peritoneal lavage fluid in experimental hemorrhagic shock.

The overproduction of highly reactive oxygen metabolites initiates and contributes to the damage to abdominal organs in hemorrhagic shock (HS). Peritoneal environment including free cells located in peritoneal cavity may interact with the inflammatory processes occurring in abdominal organs during HS. Peritoneal lavage was carried out in 48 rats divided into following groups: (1) control, (2) untreated HS for 75 minutes, (3) HS + restoration of blood volume with polyelectrolyte solution (PES) 60 minutes after blood withdrawal, and (4) HS + platelet activating factor (PAF) receptor antagonist BN52021 directly after bleeding + PES after 60 minutes of HS. Peritoneal lavage fluid (PLF) was examined for Cu-, Zn-superoxide dismutase (SOD) activity, sulfhydryl compound (-SH) concentration, and malondialdehyde (MDA) level measured as thiobarbituric acid reactive substances (TBARS). The untreated shock (group 2) as well as HS + PES (group 3), resulted in significant increase in cell numbers in PLF. In groups 2 and 3, the SOD activities were not detected while -SH group levels were significantly higher, than those in the control. The group of shocked rats after blood volume restoration with PES was the only group where the MDA in PLF was found. The highest -SH group concentrations and detectable SOD activities were recorded in shocked rats treated with BN52021 and PES. Systemic hemorrhage may cause significant alterations in the oxidant-antioxidant (O-A) balance in peritoneal cavity, accompanied by significant elevation of number of cells lavaged from peritoneal cavity. There is an escalation of disturbances in O-A balance in peritoneal lavage fluid due to restoration of blood volume. BN52021 may exert beneficial effects stabilizing peritoneal antioxidant system in the hemorrhagic shock.

BN52021 inhibits activation of peritoneal mast cells caused by hemorrhage and blood volume restoration.

An extensive blood loss activates generalized inflammatory response. Abdominal organs and especially intestines are very sensitive to the ischemia-reperfusion insults due to hemorrhagic shock (HS) and blood volume restoration. Previously obtained results suggest that studies on peritoneal lavage fluid (PLF) can contribute to elucidation of inflammatory processes in abdominal organs in HS. Histamine (H) levels, total cell, and mast cell (MC) numbers, and MC ultrastructure in the fluid lavaged from peritoneal cavity were compared in the following groups of rats: control (gr. 1), sham operation (gr. 2), untreated hemorrhagic shock (gr. 3), shock treated with blood volume restoration with lactated Ringer's solution (LR) (gr. 4), shock treated with platelet activating factor (PAF)-receptor antagonist Ginkgolide B (BN52021), and LR (gr. 5). A shock-related significant increase in total cell numbers, MC numbers, MC degranulation, and histamine levels in PLF were observed. The restoration of blood volume caused further elevation of the above phenomena (gr. 4) while BN52021 seemed to inhibit peritoneal MC mobilization and degranulation as well as to attenuate increase in peritoneal H level (gr. 5). The peritoneal cavity is a place of rapid and strong reaction to hemorrhage. Evaluation of peritoneal histamine levels might be helpful in the monitoring of shock dependent intra-abdominal processes. Peritoneal MC mobilization and degranulation, and increase in histamine level is inhibited by BN52021.

Hemorrhagic shock-induced changes in the cathepsin D activity in the intestinal wall and blood serum in rats.

Hemorrhagic shock causes release of lysosomal proteolytic enzymes which contribute to intestinal wall destruction and can be moved into the circulation as well as into the gut lumen. The aim of the study was to examine the activity of cathepsin D in relation to the intestinal wall injury after 60 minutes of untreated hemorrhagic shock in rats. The total cathepsin D activity was investigated in duodenum, jejunum, ileum, cecum and colon, as well as in systemic and mesenteric blood serum, and the biochemical results were compared with morphological changes in the respective segments including immunohistochemical expression of cathepsin D. We found an increase in cathepsin D activity in duodenum and significant decrease in other parts of the gut in shocked rats. The enzyme activity increased also in blood serum, especially systemic (p < 0.05) and insignificantly in mesenteric blood. However, sham-operated animals (one-side carotid artery occlusion) revealed a significant increase in cathepsin D activity measured in mesenteric blood. The shock resulted in lowering protein concentration in the intestinal wall and its increase in mesenteric blood. The contents of peptides and amino nitrogen, as potential proteolytic reaction products, changed in different ways in various segments of intestine. Morphologically, the most intensive destruction was observed in ileum, duodenum and jejunum. Lifting of epithelial layer from lamina propria was the most frequently observed lesion of the intestinal wall after 60 minutes of shock. More advanced lesions, such as denuded mucosa with disintegration of lamina propria, occurred rarely and were not observed in colon and rectum. By means of polyclonal antibodies against cathepsin D, we found that the strong expression of this enzyme was in epithelial layer--the part of intestinal wall which was partially detached into gut lumen due to hemorrhagic shock. The changes of cathepsin D activity after 60 minutes of hemorrhagic shock were correlated with signs of morphological damage to the intestinal wall. Cathepsin D liberation in the intestinal wall during shock indicates the lysosomal membranes impairment and can confirm involvement of proteases in the damage to the intestinal tissue. We conclude that liberation of intestinal cathepsin D is an early phenomenon during hemorrhagic shock which may contribute to the local wall disintegration and activation of systemic inflammatory response.

Platelet-activating factor receptor-antagonist (BN 52021) stabilizes the oxidative-antioxidative balance and attenuates the morphological changes in the gastrointestinal tract in experimental hemorrhagic shock.

The influence of the platelet-activating factor receptor-antagonist (BN 52021) and polyelectrolyte solution (PES) on the morphology and oxidant-antioxidant equilibrium in the gastrointestinal wall in hemorrhagic shock was examined in rats. The animals were divided into five groups: I - control, II - sham operation, III - untreated hemorrhagic shock lasting 75 minutes, IV - shock treated with PES, and V - shock treated with BN 52021 and PES. The most expressed morphological changes were found in the small intestine in group IV (shock treated with PES) and in group III (untreated shock). Morphological changes were well correlated with the malondialdehyde (MDA) level reflecting oxidative tissue injury and with the antioxidative potential: sulfhydryl compounds (-SH) level, and superoxide dismutase (SOD) activity. The signs of morphological and biochemical injury to the gastrointestinal tract in animals from group V (treated with BN 52021 and PES) were significantly less expressed than those in groups III and IV. The results suggest an essential role of PAF in oxygen radicals-mediated gastrointestinal injury and a beneficial effect of the early application of BN 52021 in hemorrhagic shock.

Ultrastructure of bone marrow megakaryocytes in experimental haemorrhagic shock in rats. I. Correlation between ultrastructure of MK nuclei and DNA ploidy.

The correlation was described between the ultrastructural picture and DNA mass content of MK nuclei in experimental haemorrhagic shock in rats. Significant disproportions were revealed between the morphological pictures and the ploidy of MK nuclei in the successive phases of the shock. Abnormalities of the maturation of marrow MK nuclei were found in the first hours of the shock, being most pronounced in the 24th hour.

[Coexistence of some diseases and analysis of death causes based on autopsy examinations carried out in liver cirrhosis patients based on autopsy observations in 1976-1990]. / Wspólistnienie niektórych schorzen oraz analiza przyczyn zgonów u osób z marskoscia watroby w swietle obserwacji autopsyjnych w latach 1976-1990.

The 19,094 autopsy examinations carried out between 1976-1990 revealed 698 (3.65%) case of cirrhosis, of which 64.6% were men. During the last 5 years the percentage of coexistance of hepatoma (hepatocellular carcinoma) with cirrhosis was higher 5-year periods (5.8%; 5.4%). Moreover, the same changing interrelation was observed for other malignancies and cirrhosis-higher (15%) in the last period than in the proceeding years (11.1%; 11.3%). The severity of atherosclerotic changes and coexistance of peptic ulcers, gall bladder disease and productive pulmonary tuberculosis in cirrhotic patients were also assessed. Finally the direct causes of these patients' death were discussed.

Peripheral blood megakaryocytes in experimental hemorrhagic shock.

The number and morphological types of megakaryocytes (MK) in the rat inferior cava vein blood were evaluated in untreated hemorrhagic shock lasting 60 min and in 6 to 48 hours after treatment with a standard polyelectrolyte solution (PES). The rats were bled through carotid artery. MK were isolated using the 5 microns filters. The results were compared with those found in the control animals not subjected to surgical manipulation and subjected to sham operation (cervical incision only, and cervical incision + carotid artery cannulation). The most considerable increase in the circulating MK occurred in 12 hours after the PES treatment. The slight increase in the number of MK was also observed in rats with carotid artery cannulation without hemorrhage. Increase in the MK number was accompanied by a shift in their morphological types.

[Histomorphometry of megakaryocytes in bone marrow during acute hemorrhagic shock in rats]. / Histomorfometryczna ocena megakariocytów szpiku kostnego w doswiadczalnym wstrzasie krwotocznym u szczurów.

Quantitation and morphometric parameters of bone marrow MK in rats during acute haemorrhagic shock were analysed. The results were compared with group of normal rats and rats after surgical manipulation. In examining animals statistical significant differences were observed in localization, size, form factor, N/C ratio, forming cluster forms and phenomena of emperipolesis. These disturbances of MK were often observed in rats after surgical manipulation. The results indicate that the changes of MK have just observed after 60 min. from indication of haemorrhagic shock.

[Circulating megakaryocytes in blood during experimental acute hemorrhagic shock in rats]. / Megakariocyty krazace we krwi w doswiadczalnym wstrzasie krwotocznym u szczurów.

The circulating megakaryocytes (MK) in the central venous blood of rats during haemorrhagic shock have been studied. Nucleopore polycarbonate membranes with a pore size of 5 microns were used to isolate circulating MK. Different morphologic types of MK were analysed. The results showed an increased number of MK migrating from the bone marrow to blood and confirm that circulation MK are a normal physiologic component of blood. The number of the rise during haemorrhagic shock, especially large MK and MK with scant cytoplasm ("naked nuclei" MK).