Andrzej Plewka

Ozonotherapy in an Induced Septic Shock. I. Effect of Ozonotherapy on Rat Organs in Evaluation of Free Radical Reactions and Selected Enzymatic Systems

The confirmed advantageous effects of oxygen/ozone therapy in several clinical conditions stimulated experimental studies on effects of the therapy in rats with an induced septic shock. The studies were conducted on adult male rats of Wistar strain. Four groups of the animals, each of 15 rats, included: I—control group, (C); II—animals intraperitoneally administered with O2/O3 (CO), III—rats given of Escherichia coli endotoxin (lipopolysaccharide—LPS) (CL), IV—rats administered with the lipopolysaccharide plus administered with the oxygen/ozone mixture (OL). Activities of catalase and superoxide dismutase and of free radical reactions were estimated. The exposure to LPS augmented activities of SOD and of catalase in liver, lungs and heart. In all the examined organs LPS induced significant changes in levels of free radicals. Except of the lungs, parallel administration of the rats with LPS and ozone/oxygen revoked development of the alterations. The obtained results point to a strong, stabilizing and regenerative effect of ozonotherapy.

Immunohistochemical localization of selected pro-inflammatory factors in uterine myomas and myometrium in women of various ages

Uterine myomas represent one of the most frequently manifested benign tumors in women. They originate from smooth muscle cells of myometrium or its blood vessels. Many studies suggest that inflammation and pro-inflammatory factors may play a role in the carcinogenesis with an involvement of the transcription factor NF-kappaB which activity can be controlled by various environmental factors, including many cytokines. The aim of the study was to investigate the expression of NF-B, interleukin-1β (IL-1β), tumor necrosis factor a (TNF-α), cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) in myometrium and uterine myomas of women of various age. The expression of NF-kappaB, selected cytokines and enzymes was estimated in women of reproductive or perimenopausal age by semiquantitative immunohistochemistry. The expression of the examined proteins was higher in myomas than in control myometrium and was dependent on the size of myomas and the age of women. However, the expression of the cytoplasmic NF-kappaB observed in uterine myomas was independent on the size of myomas and no significant differences were observed in the number of stained nuclei between control and myoma groups. Thus, the expression of proinflammatory factors in myomas was not accompanied by the nuclear activation of NF-kappaB p65. The results of our study indicate that the examined factors may be involved in the pathogenesis of benign tumors and not only malignant diseases.

Daily and circadian rhythms in the activity of mixed function oxidases system in rats of different age

Abstract An age‐associated alterations in daily and circadian changes of cytochrome P‐450‐linked monooxygenases system activity were studied using 1‐, 6‐ and 12‐months old male Wistar rats, in winter and in spring season. Cytochrome P‐450 and NADPH‐cytochrome P‐450 reductase showed 12h rhythm in all investigated age groups of animals. Cytochrome b5 and NADH‐cytochrome b5 reductase were characterized by a 12h daily rhythm in 1‐month old rats, but in older ones 24h circadian rhythm was found. There was not significant changes of the rhythm pattern in the activity of investigated MFO system ingredients in rats of different age, between spring and winter.

Ozone Therapy in Induced Endotoxemic Shock. II. The Effect of Ozone Therapy Upon Selected Histochemical Reactions in Organs of Rats in Endotoxemic Shock

Mitochondria and lysosomes were evaluated by assessment of changes in activity of selected enzymes: lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), adenosinetriphosphatase (ATPase), acid phosphatase (AcPase) and β-glucuronidase (BG) in rats under profound hypoxia induced by endotoxemic shock. The study was conducted on adult male Wistar rats. The animals formed the following four groups of 15 rats each: control animals (C);—rats receiving intraperitonally O2/O3 (CO), rats receiving of Escherichia coli toxin (LPS) (CL); rats receiving LPS plus oxygen–ozone mixture (OL). Histoenzymatic examinations of liver, kidney, lungs, and heart muscle were performed. Lipopolysaccharide suppressed activities of all the enzymes except for LDH, the activity of which as high as a fourfold increase. The results demonstrated potent, stabilizing and regenerative effects of ozone therapy on body enzymatic processes in course of induced endotoxemic shock in rats, which might prove to be of clinical significance.

Effects of age, phenobarbital, β-naphthoflavone and dexamethasone on rat hepatic heme oxygenase

There is a wide range of change in both microsomal heme oxygenase activity and cytochrome P-450 level in the livers of rats of various ages. We tried to investigate the phases of heme oxygenase activity, both spontaneous and caused by typical MFO inducers in the lifetime of the rat. Wistar male rats aged 0.5, 1, 2, 4, 8, 12, 20 and 28 months received phenobarbital (50 mg/kg) twice, 3 and 2 days before being killed. ß-Naphthoflavone and dexamethasone were given three times: 3, 2 and 1 day before decapitation 20 mg/kg and 10 mg/kg, respectively). The highest heme oxygenase activity is observed in intact 2-week-old animals (1.16±0.038 nM/h per mg protein). Before maturity this activity decreases slightly up to the 2nd month of life. Then it stabilizes and remains virtually unchanged till the 8th month of life (1.02±0.03). Afterwards HO activity tends to increase until the 28th month of life (1.10±0.06), but does not reach the level observed in the 2-week-old animals. We have found that some typical MFO inducers can modify HO activy. While phenobarbital stimulates HO activity only in premature animals (1.42±0.056; 1.30±0.059 and 1.13±0.035, respectively in 0.5-, 1- and 2-month-old animals), ß-naphthoflavone enhances HO activity in all the groups studied. Dexamethasone, as a physiological inducer of the MFO system, modifies HO activity very characteristically. It induces this activity until the 2nd month of life and then its inducibility appears to remain unchanged. Correlations between HO activity, ALAS activity and cytochrome P-450 levels still need to be investigated to elucidate these problems.

Immunohistochemical visualization of pro-inflammatory cytokines and enzymes in ovarian tumors

Epithelial ovarian cancer represents one of the most deadly gynaecological neoplasms in developed countries and is a highly heterogeneous disease. Epidemiological studies show that anti-inflammatory drugs reduce the incidence and mortality of several types of cancer, indicating the potential role of pro-inflammatory factors in carcinogenesis. The expression of pro-inflammatory factors in various cancer types, including ovarian cancer, was assessed in many studies, yielding in consistent results, often due to the histological heterogeneity of various cancers. The aim of the study was to investigate the expression of IL-1, IL-6, TGF-β, TNF-α, COX-2, iNOS, and NF-kB in serous and mucinous ovarian cancers. Ninety cases of ovarian tumors classified into mucous and serous type (45 patients in each group) were selected. Each group was classified into subgroups according to the three stages of tumor differentiation, i.e. into (i) benign, (ii) borderline and (iii) malignant tumors. The presence of proteins of interest in paraffin sections was analysed by immunohistochemistry. The expression of most of the studied factors depended on the histological tumor subtype and the degree of malignancy. Expression of NF-κB appears to be related to the level of the neoplastic differentiation only in the group of serous tumors, while the presence of IL-6 in the mucinous tumor subtype was observed only in the case of benign lesions. Expression of IL-1, TNF-α and COX-2 increased with the stage of the disease in both serous and mucinous tumors. The highest level of TGF-β expression was observed in serous borderline tumors. The different levels of iNOS immunoreactivity between the groups of serous and mucinous tumors were observed only in borderline tumors. The results of our study may be helpful in designing therapeutic strategies depending on the type of ovarian cancer.

Expression of selected cytochrome P450 isoforms and of cooperating enzymes in colorectal tissues in selected pathological conditions.

The current interest in CYP expression in the colon results from its uniqueness as a target organ for cancer. To date, the CYP expression profiles in the colon have not yet been subject of comprehensive research. In this study, we investigated 40 patients with Crohns disease, 40 with ulcerative colitis, and 40 healthy subjects as a control group. Colon tissues were fixed, dehydrated, cleared in xylene and embedded in paraffin. Sections were prepared from paraffin blocks for immunohistochemical staining with specific antibodies. We used antibodies to the human CYP1A1, CYP2B6, CYP2C9, CYP2E1 and CYP3A4 isoforms, as well as antibodies to the human glycoprotein P, glutathione-S transferase and antibody to the UDP-glucuronosyltransferase. The sections were stained immunohistochemically and examined using light microscopy. Cellular localization was determined, and computer image analysis was used. In all cases with Crohns disease, the proteins studied showed at least a twofold expression. Ulcerative colitis showed a much weaker influence regarding the expression of the proteins studied but in case of CYP2C9 and UDP-glucuronosyltransferase, a decrease of expression was observed.

Processes of apoptosis and cell proliferation in uterine myomas originating from reproductive and perimenopausal women

We studied uterine myomas originating from females of reproductive age and from females of perimenopausal age. Uterine myomas represent benign tumors of the myometrium, and they develop frequently in women of reproductive age. The frequency of uterine myomas increases with age until women reach the menopause. The study included patients with a myomatous uterus, in the reproductive age or peri-menopausal age, independently evaluating small and large myomas. Myometrial alterations in their direct vicinity were evaluated independently of the myomas. The study included evaluation of immunolocalization of two index proteins which participate in myoma cells growth control: Ki-67 nuclear antigen and caspase 3. In women of reproductive age, both in small and large myomas, elevated immunostaining of Ki-67 was noted in parallel to low levels of caspase 3 staining, which indicated the ongoing process of proliferation. In women of peri-menopausal age with small or large myomas, no Ki-67 immunostaining was detected, while staining of caspase 3 manifested low levels. Proliferation in reproductive age women myomas is higher than in the peri-menopausal age.

Immunohistochemical localization of telomerase in myomas and in uterine myometrium

Telomerase activity could be a potential marker for the neoplastic process, because it is absent in normal cells and present in tumor cells. Immunohistochemical studies were conducted using samples obtained from 32 uterine myomas, each sample having a size of 3-4 cm and obtained from women between 35 and 45 years of age. These studies also concentrated on fragments of macroscopically unaltered myometrium, collected 3-4 cm from a uterine tumor. Immunohistochemistry was performed using antibody to the catalytic unit of telomerase (hTERT; clone 44F12, NCL-L-hTERT, Novocastra Laboratories, UK). This study aimed at detecting a possible presence of potentially neoplastic cells in the margins of healthy tissue, which was removed together with the primary tumor. The results were classified according to the number of telomerase-positive cells. Tumors of the first group had up to 50% telomerase-positive cells, while their content in the second group exceeded 50%. Our study demonstrated an almost two-fold increase in the number of telomerase-positive tumor cells compared with myometrial cells 3-4 cm from the tumor. Hopefully, investigating the presence of telomerase in both uterine myometrium and myoma could facilitate the diagnosis of the neoplastic process.

The relation between plasma α-synuclein level and clinical symptoms or signs of Parkinson's disease

INTRODUCTION Parkinson disease (PD) is the common neurodegenerative disease. α-Synuclein (ASN), main aggregating protein in neural cells of CNS in PD, was found in peripheral fluids. Testing ASN in plasma is potential test for diagnose PD, but previous studies are controversial. The aim of this study was to investigate if plasma ASN level may be a valuable biomarker, is the level of plasma ASN concentration different in various motor subtypes of diseases, is there a relation between the level of plasma ASN and the severity of motor symptoms. METHODS Patients with PD hospitalized in Neurology Department, Medical College were performed sequencing the 8th and 9th exon of GBA gene. Next plasma ASN level was tested in 58 patients with sequenced GBA gene and in 38 healthy volunteers (HV), matched by the age (respectively 68.43 vs. 64.57 years of age) and sex (female %, respectively: 43.10 vs.44.74). Patients were assessed with the scales: UPDRS (II, III, IV), Hoehn-Yahr (HY) and qualified to PIGD or TD subtype. For homogeneity of the group patients with GBA mutation were excluded from the analysis. RESULTS The ASN level did not differ between patients and HV (respectively: 4.53 vs. 3.73ng/ml) and between patients with different subtypes. There was inverse correlation between ASN and HY in PIGD subtype. CONCLUSIONS Plasma ASN level is not valuable marker of the disease. It does not differ in subtypes of the disease. There is relation between plasma ASN level and the severity of the disease in PIGD subtype.