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.

N-cadherin, beta-catenin and connexin 43 expression in astrocytic tumours of various grades.

INTRODUCTION: Astrocytic tumors are the most common primary brain tumors, but little is known about their etiology and prognostic factors. N-cadherin and beta-catenin are adhesive proteins, and are often overexpressed in many types of cancers, including breast or colorectal cancer, resulting in better prognosis. Connexin 43 is a gap junction protein involved in cell-cell signaling pathway taking part in the process of carcinogenesis. The aim of the study was to evaluate N-cadherin, beta-catenin and connexin 43 expression in astrocytic tumors of various grades. MATERIALS AND METHODS: We examined 131 cases of astrocytic tumors, including 26 cases of diffuse astrocytoma (group I), 44 anaplasic astrocytomas (group II) and 61 glioblastoma cases (group III)--primary and secondary. To evaluate N-cadherin, beta-catenin and connexin 43 expression, we used immunohistochemical reaction with specific antibodies (Santa Cruz Biotechnology). The obtained results were correlated with clinical and morphological features. RESULTS: Beta-catenin expression was observed in 69.3% of diffuse astrocytomas, 75% of anaplastic astrocytomas, and 82% of glioblastoma cases. N-cadherin expression was observed in 92.3% of diffuse astrocytomas, 90.1% of anaplastic astyrocytomas, and in all glioblastoma cases. Connexin 43 was observed in 76.9% of diffuse astrocytomas, and in all cases of anaplastic astrocytomas and glioblastomas. Beta-catenin expression was significant within the nucleus of neoplastic cells in groups I and II. In group III, staining was observed only in the cellular membranes. N-cadherin and connexin 43 expression was observed only in the cells' membranes. In glioblastomas, both primary and secondary, all protein expression was significant within the cells surrounding the necroses and blood vessels and weak or absent in the tumor's margins. CONCLUSION: Our study shows that beta-catenin nuclear expression in group of diffuse astrocytomas and anaplastic astrocytomas is evidence for transcriptional function of beta-catenin in those groups. Strong N-cadherin and connexin 43 expression in those groups may be evidence for their role in tumor formation and progression. However, in glioblastomas a very important role of all examined proteins is generating intracellular connections to facilitate the escape of tumor cells from the effects of hypoxia or their accumulation around the blood vessels rather than tumor invasion into the brain parenchyma.

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.

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.

Neutrophil function in chemical plant workers employed in the production of dust pesticides.

In two divisions of a chemical plant producing dust pesticides, employees exposed to dust, containing 28%-65% SiO2, were examined. In the first division (group I, 38 males and 35 females), the average air dust concentration was 5.0 mg/m3, and the active substances were: chlorinated hydrocarbons, captan, carbamates and dodine. In the other division (group II, 26 males and 33 females), the air dust concentration was 4.8-5.2 mg/m3, and the active substances included: carbamates, triazine compounds, cupric oxychloride, captan, lindane, carboxine. Spirographic investigations showed signs of pulmonary emphysema (RV/TLC) in 65.8% males and 60% females of group I and 38.5% males and 40.6% females of group II. In leucocyte concentrate smears, the cytochemical reactions to beta-glucuronidase, acid phosphatase and myeloperoxidase, as well as the nitroblue tetrazolinum (NBT)-dye reduction of neutrophils were performed. The random migration and chemotaxis of isolated neutrophils, washed or incubated in 10% autologous serum, their phagocytic activity and tube adherence test were also investigated. Compared to the controls, the reaction to beta-glucuronidase as well as the NBT reduction were increased, whereas the acid phosphatase and myeloperoxidase reactions were lowered. Impairment of neutrophil chemotaxis stimulated with zymozan-activated serum was observed in all groups of workers; random migration was enhanced in workers of group I and lowered in male workers of group II. Higher phagocytosis of latex particles occurred in workers of group I and in males of group II, while tube adhesion was impaired in group I and enhanced in males of group II.

Tumour related inhibition of macrophage chemotaxis in patients with colon cancer.

The chemotactic migration in vitro of peripheral blood, intestinal mucosal, and mesenteric lymph node mononuclear cells has been assessed in patients with colorectal carcinoma. Peripheral blood mononuclear cells of patients exhibited normal chemotaxis. For control patients with non-malignant, non-inflammatory intestinal disease, the chemotaxis of mucosal mononuclear cells was similar to that of autologous peripheral blood mononuclear cells. The chemotactic migration of mucosal mononuclear cells, however, isolated distant from a colon cancer was less than that of autologous peripheral blood mononuclear cells. Chemotactic migration was progressively impaired with increasing closeness to the tumour itself. Chemotaxis of mucosal mononuclear cell was independent of the site of tumour and the Dukes' grading. Mononuclear cells from mesenteric lymph nodes, however, exhibited impaired migration only in patients with Dukes' C tumours. Supernatants of the collagenase digestion of either tumour or adjacent mucosa contained macrophage directed inhibitors of chemotaxis and these inhibitors were not produced by tumour mononuclear cells. The presence of such inhibitors in the digestion supernatants and the demonstration that proximity to the tumour was associated with impaired mononuclear cell motility suggest that the production of macrophage directed chemotactic inhibitors is by colon cancer cells and that this may be occurring in vivo.

The effect of levamisole on the maintenance of remission of ulcerative colitis. A 2-year double-blind study.

The results of our study of 50 patients show that levamisole is ineffective in the treatment of patients with ulcerative colitis. The data presented here support our hypothesis that the only effective drugs in ulcerative colitis act as suppressors of both acquired and natural immunity.

Function of phagocytes in experimentally induced colitis in rabbits. I. The Arthus phenomenon in the colon.

Adhesion, random and chemotactic migration, phagocytosis and nitroblue tetrazolium (NBT) dye reduction of peritoneal exudate polymorphonuclear leukocytes (PMN) and macrophages were examined in 110 rabbits with immune complex-mediated colitis and in controls. We may conclude that Arthus-like colitis in rabbits is accompanied by an impairment of adhesion and phagocytosis, and an increase in chemotactic and random migration of both PMN and macrophages. The examined phagocytes were obtained from the peritoneal cavity, and the changes in their function are related much more directly to the presence of circulating immune complexes than to the colitis. The mentioned impairment in their function may be responsible for prolonged persistence of immune complexes in the serum of rabbits with colitis; however, it does not prolong the time of healing of the ulcers.

Function of phagocytes in experimentally induced colitis in rabbits. II. The Shwartzman phenomenon in the colon.

Adhesion, phagocytosis, chemotactic and random migration, nitroblue tetrazolium dye reduction of peritoneal exudate neutrophils and macrophages, fibrinogen level, gelation of soluble fibrin and serial dilution protamine sulfate test were investigated in 115 New Zealand white rabbits with experimentally induced Shwartzman phenomenon in the colon, and in control animals. The results presented in this report demonstrated impairment of chemotactic migration of phagocytes in the presence of endotoxin. The depression was dose-dependent and less marked when neutrophils were stimulated with monocyte-derived chemotactic factor or with casein, than with complement-derived chemotactic factor. The prolonged depression of chemotactic migration of neutrophils and macrophages in rabbits with colitis, however, did not affect the healing time of the ulcers in the colon.

Normal chemotactic migration of polymorphonuclear leukocytes stimulated with mononuclear-derived chemotactic factor in ulcerative colitis.

The migration of polymorphonuclear leukocytes preincubated with autologous or heterologous serum was examined in 100 patients with untreated ulcerative colitis (UC) and in 100 age- and sex-matched healthy controls. The activity of complement-derived chemotactic factors and mononuclear-derived chemotactic factor (MDCF) was also investigated in the same group of patients. No significant difference was found in random and chemotactic migration of patients or control leukocytes preincubated in different concentrations (10, 50 and 100%) of autologous or heterologous serum. Defective chemotaxis of leukocytes stimulated with complement-derived chemotactic factors was found in UC and was more marked in patients in remission than with active UC independently of whether complement was activated by the alternative or the classical pathway. However, the random migration of neutrophils was enhanced in both groups of UC patients. The leukocytes of patients stimulated with MDCF (mononuclear cells were activated with lipopolysaccharide from Escherichia coli O55:B5) show normal chemotaxis. Our data suggest an impairment of neutrophil receptors for complement-derived chemotactic factor in UC. The decreased response of neutrophils to these factors and normal response to MDCF suggest that the main way in which cells are attracted to the site of inflammation in UC may be a factor produced by stimulated mononuclear cells.

Effect of long-term therapy with sulphasalazine, levamisole, corticosteroids and ascorbic acid and of disease activity on polymorphonuclear leukocyte function in patients with ulcerative colitis.

Adhesion, random migration and chemotactic migration, phagocytosis, and spontaneous nitroblue tetrazolium-dye reduction (NBT) capacity of polymorphonuclear leukocytes (PMNs) were analysed in 38 patients with ulcerative colitis before and during therapy with sulphasalazine (12 months), corticosteroids (6 months), levamisole (12 months), ascorbic acid (6 months) and sulphasalazine plus levamisole (12 months). Remission was achieved by levamisole in as many patients as by sulphasalazine and by sulphasalazine and levamisole used together. Nevertheless, the only function of PMNs affected by levamisole therapy was a depression of chemotaxis, whereas other functions as well as chemotaxis were altered by sulphasalazine, whether used alone or in combination with levamisole. Of patients treated with corticosteroids, all were in remission at 6 months and the most marked decrease in chemotaxis was seen. No clinical benefits were observed after 6 months of treatment with ascorbic acid and PMNs function was unaltered. The depression of chemotaxis occurred whether or not remission was achieved, suggesting a direct drug-induced effect. The apparent discrepancies of the "in vivo" drug effects in comparison with previously reported findings may reflect abnormal reactivity of PMNs in UC.

Isolation of intestinal mononuclear cells: factors released which affect lymphocyte viability and function.

Whether toxic or immunomodulatory factors are released during the collagenase digestion phase of the isolation of mononuclear cells from human intestinal mucosa was investigated by assessing the effect of the collagenase supernatant on the viability and natural killer activity of normal peripheral blood mononuclear cells. Three hours' incubation in collagenase supernatant suppressed natural killer activity by 25 +/- 4% and decreased the viability of peripheral blood mononuclear cells by 11 +/- 2%. The ability of collagenase supernatants to kill 51Cr-labelled peripheral blood mononuclear cells over four hours was assessed in 16 collagenase supernatants, eight of which produced lysis of 20 +/- 4%. There was no ultrastructural evidence of early degenerative changes in the viable intestinal mononuclear cells fresh from the isolation process or in peripheral blood mononuclear cells incubated in collagenase supernatant. Because prostaglandins are known to inhibit natural killer activity, PGE was measured in 20 collagenase supernatants by radioimmunoassay and found to be high at 27.5 +/- 4.0 ng/ml. Addition of indomethacin to the collagenase medium, however, failed to abolish the inhibitory effect of collagenase supernatant on natural killer activity and did not increase the natural killer activity of isolated intestinal mononuclear cells. The release of cytotoxic and immunomodulatory factors during the isolation of intestinal mononuclear cells indicates the necessity for careful assessment of the potential effects of the isolation process on any function being examined and casts doubt upon the relationship between in vitro findings and in vivo functional capabilities.

Neutrophil function and infectious disease in workers occupationally exposed to phosphoorganic pesticides: role of mononuclear-derived chemotactic factor for neutrophils.

Neutrophil function and the prevalence of infections in 85 workers occupationally exposed to phosphoorganic pesticides were examined and compared to those of age- and sex-matched healthy controls. Activity of cholinesterase (ChE) in red blood cells (RBC) and in serum was used as marker of organophosphate toxicity. A decrease of CHE activity was observed in all groups of workers, both in serum and in RBC, and was dependent on the degree of exposure to the pesticides. A marked impairment of neutrophil chemotaxis stimulated with zymosan-activated serum (from workers or controls) was observed in all groups of workers. There was no significant change of polymorphonuclear neutrophil (PMN) migration after their preincubation in 10% autologous serum or washed cells. However, neutrophil chemotaxis was normal after stimulation with specific mononuclear-derived chemotactic factor (MDCF). The area of random migration increased in those working directly in the production of organophosphates. A decrease of adhesion and an increase of nitroblue tetrazolium-dye reduction was also found. The frequency of upper respiratory tract infection (in 68.8%) was greater in workers than in controls and similarly to their recurrence was dependent upon the time of exposure to the insecticides. A normal frequency of infections localized outside of the upper respiratory tract was observed. It may be concluded that (1) impairment of chemotaxis in the workers is not dependent on serum factors; (2) stimulation of neutrophil chemotaxis by MDCF occurs by a different mechanism to their stimulation by zymosan-activated serum; (3) the increase of morbidity in the workers is probably caused by prolonged inhalation of both the insecticides and solvents; (4) the impaired function of neutrophils may be not so important as expected. However, the local effects of the pesticides should be explored.

Factors affecting the spontaneous cell-mediated cytotoxicity of intestinal mononuclear cells.

This study was performed to determine what factors related to the enzymatic isolation technique and assay conditions may influence the measurement of spontaneous cell-mediated cytotoxicity (SCMC) of mononuclear cells (MNC) isolated from human intestinal mucosa. In 18 studies, the SCMC of freshly isolated cells was 1.8 +/- 0.4% but increased to 12 +/- 3% following 24 hr culture without a change in the proportion of cells with the NK phenotype (Leu-7+). The SCMC tended to plateau with more prolonged culture. Culturing peripheral blood (PB) MNC for 24 hr did not alter SCMC nor the proportion of Leu-7+ cells. However, the suppression of the SCMC of PBMNC preincubated with the supernatant of the collagenase digestion of intestinal mucosa was completely reversed by 24 hr culture. Intestinal MNC were found to suppress the SCMC of autologous PBMNC in mixing experiments. However, 24 hr culture did not affect the degree of suppression and the proportion of T cells of the suppressor-cytotoxic phenotype (Leu-2a+) was also unchanged. It is concluded that the SCMC of intestinal MNC may be accurately assessed following 24 hr culture of the cells to allow recovery from the suppressive influences of the isolation process and that this does not introduce other artefactual problems. However, suppression of cytotoxicity within the assay may result in an underestimation of the SCMC of intestinal MNC when compared to that of PBMNC.

Inhibition of human leukocyte locomotion by tilorone hydrochloride and by RMI 9563 DA measured in vitro.

The effect of tilorone hydrochloride and RMI 9563 DA on neutrophil locomotion was studied in experimental conditions. It was shown that depression of random and stimulated migration and of chemotaxis increased with rising concentration of these drugs.