Chemoradiotherapy in the treatment of cervical cancer - a single institution retrospective review.

BACKGROUND: The aim of this study is a retrospective analysis of treatment outcomes and toxicity in a group of patients with cervical cancer who underwent (chemo) radiotherapy at the Institute of Radiation Oncology in Bulovka University Hospital in Prague in the period 2014-2017. PATIENTS AND METHODS: During this period, 141 patients were treated, 105 (74.5%) of them underwent combined (chemo) radiotherapy with radical intent and palliative radiotherapy was performed in 36 (25.5%) cases. According to the International Federation of Gynecology and Obstetrics (FIGO) 2009 classification, the most numerous stages were IIB in 39 (27.7%) and IIIB in 64 (45.4%) cases; according to FIGO 2018, a significant number of newly established stages is evident: IIIC1 in 55 (39.0%) patients and IIIC2 in 22 (15.6%) cases. RESULTS: The median progression-free survival (PFS) and overall survival (OS) reached 31.3, resp. 40.1 months in the whole group. In the subgroup of patients treated with radical intent, the median PFS was 44.0 months and OS 48.8 months; in the palliative subgroup, the median PFS was 9.4 months and OS 14.8 months. In a radically treated subgroup, 7 (6.7%) patients had gastrointestinal or genitourinary manifestations of G3-4 toxicity, and overall acute toxicity (including skin and haematological reactions) of G3-4 occurred in 18 (17.1%) patients. Late toxicity of G3-4 was reported in 13 (12.4%) cases. Patients who underwent complete brachytherapy (BRT) showed significantly better survival compared to patients with a lower number of BRT fractions. The prognostic potential of PS (performance status) and anemia was confirmed; significantly longer overall survival was observed in patients in good general condition or in those without anemia. CONCLUSION: Our results confirmed the key role of BRT for the delivery of the curative dose to the target volume. The prognostic role of PS and anemia is evident. The side effects were in acceptable limits but we expect improvements because of the use of modern radiotherapy technologies.

The therapeutic effects of Agrimonia eupatoria L.

Agrimonia eupatoria L. is an herb of the Rosaceae family, widely used in traditional (folk) medicine for its beneficial effects. Its water extracts (infusions and decoctions) are used in the treatment of airway and urinary system diseases, digestive tract diseases, and chronic wounds. Phytochemical analyses of Agrimonia eupatoria L. identified a variety of bioactive compounds including tannins, flavonoids, phenolic acids, triterpenoids and volatile oils possessing antioxidant, immunomodulatory and antimicrobial activities. The authors review the available literature sources examining and discussing the therapeutic and pharmacological effects of Agrimonia eupatoria L. at the molecular level in vitro and in vivo.

The changes in FIGO staging for carcinoma of the cervix uteri.

INTRODUCTION: The carcinoma of the cervix uteri is the fourth most common cancer in women worldwide and more than 85% of these cases occur in developing countries. Altogether 822 new cases were found in the Czech Republic during 2016 which means the incidence 15,3 new diseases/100,000 women. OBJECTIVE: To provide an overview of changes in FIGO (International Federation of Gynecology and Obstetrics) staging for carcinoma of the cervix uteri with an incorporation of possible imaging methods and/or pathological findings, and clinical assessment of tumor size and extent. SETTINGS: Gynecologic Oncology Center, Department of Gynecology and Obstetrics, Hospital Na Bulovce and 1st Medical School of Charles University, Prague; Gynecologic Oncology Center, Department of Gynecology and Obstetrics, General Faculty Hospital and 1st Medical School of Charles University, Prague; Institute of Radiation Oncology, Hospital Na Bulovce, Prague. METHODS: For this review, we have used the results of studies, review articles, and guidelines of oncogynecologic organisations on the cervical cancer published in English. They were identified through a search of literature using PubMed, MEDLINE-Ovid, Scopus and Cochrane Library with the keywords. We summarize the new classification, main changes compared to the former one and their clinical impact. CONCLUSION: Lateral extension measurement is removed in the stage IA, the only criterion is the measured deepest invasion.

Current FIGO Staging for Carcinoma of the Cervix Uteri and Treatment of Particular Stages.

BACKGROUND: Here, we present a review of the revised FIGO (International Federation of Gynecology and Obstetrics) staging system for carcinoma of the cervix uteri, explaining the reasons for the changes and summarizing suitable diagnostic methods and treatment options for particular stages of disease according to current guidelines. AIM: The FIGO staging system has been revised as follows. Measurement of lateral extension has been removed from stage IA; the only criterion is a measurement for the deepest invasion of < 5.0 mm. Stage IB has been divided into three subgroups: IB1, tumors with a largest diameter measuring 5 mm and < 2 cm; IB2, tumors measuring 2-4 cm; IB3, tumors measuring 4 cm. Stage IIIC includes an assessment of retroperitoneal lymph nodes: IIIC1 if only pelvic lymph nodes are involved, and IIIC2 if para-aortic nodes are infiltrated. The revised staging system does not mandate the use of a specific imaging method or surgical assessment of the extent of the tumor. The method used to assign a stage should be recorded and reported. The European Society of Gynaecological Oncology, the European Society for Radiotherapy and Oncology, and the European Society of Pathology have developed clinically relevant and evidence-based guidelines to improve the quality of care for women with cervical cancer. These guidelines cover comprehensive staging, management, and follow-up for patients with cervical cancer. The guidelines are intended for use by gynaecologic oncologists, general gynaecologists, surgeons, radiation oncologists, pathologists, clinical oncologists, radiologists, general practitioners, palliative care experts, and other health professionals. CONCLUSION: We summarize the new FIGO classification system, including diagnostic methods and treatments for particular stages. We also discuss the main changes and their clinical impact. This work was supported by the Charles University project UNCE 204065. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

[Anticipated efficacy of HPV vaccination in prophylaxis against nongenital cancers]. / Predpokládaná úcinnost HPV vakcinace v profylaxi nongenitálních karcinomu

BACKGROUND: There is a considerable number of studies on the efficacy HPV (human papillomavirus) vaccination against different cancers but relevant information is scattered in diverse journals. This paper is a review summarizing current knowledge of the potential of HPV vaccination against all HPV related cancers. AIM: HPV infection is probably the most frequent sexually transmitted disease. At least 13 HPV genotypes are classified as carcinogenic or probably carcinogenic in respect to cervical cancer. Almost 100% of cervical cancers are linked to HPV infection. HPV 16 and HPV 18 are the most frequently involved genotypes and account together for approximately 70% of cervical cancer in the world. Persistent high risk HPV infection is responsible for a significant proportion of vulvar, vaginal, anal and penile carcinomas. The virus has also been implicated in oncogenesis of head and neck cancers, including oropharyngeal cancers. HPV infection can play an important role in cancerogenesis of lung, esophagus, breast, and colon and rectum. On the contrary, published results indicate that HPV infection is not associated with prostate oncogenesis. Strong predominance of HPV 16 has been reported for all HPV associated cancer sites. Generally, it is estimated that approximately 5.2% of all cancers are associated with oncogenic HPV infection. Currently, there are two vaccines on the market; quadrivalent Silgard® (Gardasil®) and bivalent CervarixTM. Large trials for both vaccines have shown efficacy against HPV related infection and disease. Efficacy has been very high in HPV naive subjects to vaccine related types. While HPV vaccination is currently approved for the prevention of cervical cancer, it also has the potential in the prevention of all HPV associated malignancies. The Czech republic belongs to countries that cover HPV vaccination of girls at the age of 13- 14 years by general health insurance. Overall impact of this vaccination remains to be evaluated. The new issues of the role of HPV in oncogenesis, as well as the potential effect of HPV vaccination against HPV related nongenital cancers are discussed. CONCLUSION: Approximately 5.2% of all human cancers are associated with oncogenic human papillomavirus infection. HPV vaccination against the most risky HPV oncotypes may cause a significant reduction of these cancers mainly in the HPV naive population.

[The reasons of changes in revised staging for carcinoma of the vulva]. / Duvody zmen ve stagingu karcinomu vulvy.

BACKGROUND: Review of revised staging system for vulva, explaining the changes of staging and their impact on the prognosis of disease is presented. AIM: The main objectives of a reliable staging system include an assessment of prognosis, planning treatment, and the evaluation of their outcomes. A good staging system must meet three basic characteristics: validity, reliability and practicality. Since medical research and practice in the field of oncology have shown explosive growth, the staging of vulvar cancer and some other cancers did not give a good spread of prognostic groupings. Changes based on new findings were proposed in 2008 by the FIGO Committee on Gynecologic Oncology, approved, and published a year later the changes in the staging system for carcinoma of the vulva. Stage 0 was deleted, since it represents preinvasive lesion. Stage IA remained unchanged and stage I and II were combined. The number and morphology of the involved nodes were taken into account, and the bilaterality of positive nodes has been discounted. CONCLUSION: The purpose of a good staging system is to offer a classification of the extent of gynecological cancer, in order to provide a method of conveying ones clinical experience to others for the comparison of different treatment methods. As a result of the explosion of medical research in the field of oncology, the staging of some of the gynecological cancers became outdated and did not give a good spread of prognostic groupings. According to the revised staging for carcimona of the vulva, patients are divided to groups with similar prognosis. Therefore, exchange of relevant information between oncological centers is facilitated, thus disseminating knowledge and stimulating research in other parts of the world.

[Current classification of malignant tumours in gynecological oncology--part II]. / Soucasná klasifikace zhoubných nádoru v onkogynekologii--cást II.

OBJECTIVE: Review of new staging systems for gynaecological cancers and their impact on prognosis and planning treatment. DESIGN: Review article. SETTING: Department of Gynaecology and Obstetrics, First Faculty of Medicine and University Hospital Na Bulovce, Charles University, Prague; Department of Radiotherapeutic Oncology, First Faculty of Medicine and University Hospital Na Bulovce, Charles University, Prague; Department of Pathology, University Hospital Na Bulovce, Prague. RESULTS: Every staging system should have 3 basic characteristics: it must be valid, reliable, and practical. Over the years, these staging classifications--with the exception of cervical cancer and gestational trophoblastic neoplasia--have shifted from a clinical to a surgical-pathological basis. Changes based on new findings were proposed in 2008 by the FIGO Committee on Gynecologic Oncology, approved in September 2008 by the FIGO Executive Board, and published in 2009. The greatest changes were made in the new staging system for carcinoma of the vulva and others in the new staging systems for carcinoma of the cervix and carcinoma of the endometrium. A new stanging system was also created for uterina sarcomas, based on the criteria used in other soft tissue sarcomas. A clinical staging system for carcinoma of cervix continues because surgical staging cannot be employed worldwide (especially in third world countries). Stage 0 has been deleted from the staging of all tumours, since it is pre-invasive lesion and it is not an invasive tumour. In the revised staging system for carcinoma of the endometrium, four fundamental changes have occurred, which will be discussed. Carcinosarcoma is still staged identically to carcinoma of the endometrium. A completely new staging system was created for adenosarcomas, along with an almost identical staging system for leiomyosarcoma and endometrial stromal sarcoma. The staging system for carcinoma of ovary and Fallopian tube remains without changes. CONCLUSION: Since medical research and practice in the field of oncology have shown explosive growth, the staging of some of the gynaecological cancers did not give a good spread of prognostic groupings. Therefore, revised FIGO and TNM staging system has been structured to represent major prognostic factors in predicting patients' outcomes and lending order to the complex dynamic behavior of gynaecological cancers. The purpose of good staging system is to offer a classification of the extent of gynaecological cancer in order to provide a method of conveying one's clinical experience to others for the comparison of treatment methods.

[Current classification of malignant tumours in gynecological oncology--part I]. / Soucasná klasifikace zhoubných nádoru v onkogynekologii--cást I.

OBJECTIVE: Review of new staging systems for gynaecological cancers and their impact on prognosis and planning treatment. DESIGN: Review article. SETTING: Department of Gynaecology and Obstetrics, First Faculty of Medicine and University Hospital Na Bulovce, Charles University, Prague. Department of Radiotherapeutic Oncology, First Faculty of Medicine and University Hospital Na Bulovce, Charles University, Prague. Department of Pathology, University Hospital Na Bulovce, Prague. RESULTS: The main objectives of any good staging system--essential to an evidence-based approach to cancer--include planning treatment, providing an assessement of prognosis and the evaluation of the results of treatment. With this approach, the exchange of relevant information between oncological centers is facilitated, thus disseminating knowledge and stimulating research in other parts of the world. A good staging system must have three basic characteristics: validity, reliability, and practicality. The first staging system for gynaecological cancers appeared around the turn of the 20th century and was applied to carcinoma of the cervix uteri. Classifications for the other gynaecological malignancies were not created until the 1950s. Over the years, these staging classifications--with the exception of cervical cancer and gestational trophoblastic neoplasia--have shifted from a clinical to a surgical-pathological basis. Some changes, brought about through new findings, were approved by the FIGO in 2008 and published in 2009. The greatest changes were made in the new staging system for carcinoma of the vulva, while others were made in the new staging systems for carcinoma of the cervix and carcinoma of the endometrium. A new stanging system was also created for uterine sarcomas, based on the criteria used in other soft tissue sarcomas. CONCLUSION: As a result of the explosion of medical research in the field of oncology, the staging of some of the gynaecological cancers became outdated and did not give a good spread of prognostic groupings. Therefore, revised FIGO and TNM staging system has been structured to represent major prognostic factors in predicting patients' outcomes and lending order to the complex dynamic behavior of gynaecological cancers. The purpose of a good staging system is to offer a classification of the extent of gynaecological cancer, in order to provide a method of conveying one's clinical experience to others for the comparison of different treatment methods.

Decisive role of lipopolysaccharide in activating nitric oxide and cytokine production by the probiotic Escherichia coli strain Nissle 1917.

Effects of Gram-negative probiotic E. coli strain Nissle 1917 (EcN) on the production of nitric oxide (NO) and cytokines were determined in cultures of resident peritoneal cells of rats. The cells (2 x 10(6)/mL) were cultured for 24 h in the presence of live EcN suspension (EcN-Susp), bacteria-free supernatant of this suspension (Sup-EcN), and LPS of EcN origin (LPS-EcN). The biosynthesis of NO was substantially enhanced using live bacteria counts as low as 10(3)/mL applied in the form of EcN-Susp. The same NO-enhancing effect was produced by the correspondingly diluted Sup-EcN. It was found that Sup-EcN contained relatively high amounts of LPS. Administration of the LPS-EcN mimicked the high NO-augmenting activities of both Sup-EcN and EcN-Susp. However, the activity of LPS-EcN was significantly less pronounced than were the activities of Sup-EcN and EcN-Susp containing identical amounts of LPS. The NO-stimulatory effects of the EcN preparations were completely inhibited by polymyxin B. All LPS-EcN and correspondingly diluted Sup-EcN and EcN-Susp stimulated the secretion of cytokines TNF-alpha, IL-1beta, IL-6, IL-10 and VEGF. Also these effects were abrogated by polymyxin B. In contrast to the effects on NO production, the cytokine-stimulatory effects were significantly less pronounced after the exposure of the cells to Sup-EcN and EcN-Susp than to the identical amounts of LPS-EcN. It may be concluded that the in vitro stimulatory effects of EcN on NO and cytokine production are mediated by LPS. It is suggested that the immunostimulatory activity of LPS is modulated by EcN-derived factor(s), the nature of which remains to be identified.

Current status and challenges of cytokine pharmacology.

The major concern of pharmacology about cytokines has originated from plentiful data showing association between gross changes in their production and pathophysiological processes. Despite the enigmatic role of cytokines in diseases, a number of them have become a subject of cytokine and anti-cytokine immunotherapies. Production of cytokines can be influenced by many endogenous and exogenous stimuli including drugs. Cells of the immune system, such as macrophages and lymphocytes, are richly endowed with receptors for the mediators of physiological functions, such as biogenic amines, adenosine, prostanoids, steroids, etc. Drugs, agonists or antagonists of these receptors can directly or indirectly up- and down-regulate secretion of cytokines and expression of cytokine receptors. Vice versa, cytokines interfere with drug pharmacokinetics and pharmacodynamics through the interactions with cytochrome P450 and multiple drug resistance proteins. The aim of the review is to encourage more intensive studies in these fields of cytokine pharmacology. It also outlines major areas of searching promising candidates for immunotherapeutic interventions.

Glucose release as a response to glucagon in rat hepatocyte culture: involvement of NO signaling.

Glucagon and alpha-adrenergic-induced glycogenolysis is realized via the agonist/adenylyl cyclase/cAMP/protein kinase signaling pathway or via the activation of phosphorylase kinase by the mobilized calcium that supports the inhibition of glycogen synthase, respectively. The role of nitric oxide (NO) in this process has not been extensively studied. The present work was directed to the question whether NO is produced during glucagon-induced glycogenolysis in rat hepatocyte in a similar way like alpha-adrenoceptor stimulation. Glycogen-rich hepatocyte cultures were used. NO production (NO(2)(-)) was assessed under the influence of glucagon, dibutyryl cyclic AMP (db-cAMP), forskolin, the nitric oxide synthase (NOS) inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, and the NO donor S-nitroso-N-acetyl penicillamine (SNAP). Inducible NOS (iNOS) mRNA was examined by reverse transcription-polymerase chain reaction. Glycogenolysis was followed up by estimation of medium glucose levels. The amount of glucose and NO(2)(-) released by glycogen-rich hepatocytes was increased as a result of glucagon, db-cAMP, forskolin and SNAP treatments. iNOS gene expression was upregulated by glucagon. Glycogenolysis that occurs through glucagon receptor stimulation involves NO production downstream of transduction pathways through an isoform of NO synthase. The present and previous studies document possible involvement of NO signaling in glycogenolytic response to glucagon and adrenergic agonists in hepatocytes.

In vivo study of the effect of antiviral acyclic nucleotide phosphonate (R)-9-[2-(phosphonomethoxy)propyl]adenine (PMPA, tenofovir) and its prodrug tenofovir disoproxil fumarate on rat microsomal cytochrome P450.

The total content of rat liver microsomal cytochrome P450 (CYP) significantly decreased after repeated i.p. administration of the antiviral agent tenofovir ((R)-9-[2-(phosphonomethoxy)propyl] adenine) and tenofovir disoproxil at a daily dose 25 mg/kg, although the content of liver microsomal protein did not change. The decrease of the CYP content was accompanied by concomitant increase of the amount of inactive CYP form, cytochrome P420. This effect was confirmed by a parallel study of the activities of selected CYP forms, CYP2E1 (p-nitrophenol hydroxylation) and CYP1A2 (7-ethoxyresorufin deethylation). The activity (expressed relatively to the protein content) of both CYP forms decreased significantly following the decrease of the total CYP. On the other hand, the CYP2E1 activity expressed relatively to the decreasing total CYP content remained unchanged. However, CYP1A2 activity also decreased when calculated relatively to the total native CYP content indicating lower stability of this form. Semiquantitative RT-PCR showed no significant changes in expression of major rat liver microsomal CYP forms after tenofovir treatment. In conclusion, repeated administration of tenofovir in higher doses led to significant decrease of the relative proportion of active liver microsomal CYPs accompanied by a conversion of these enzymes to the inactive form (CYP420) maintaining the sum of CYP proteins unchanged.

Thapsigargin, a selective inhibitor of sarco-endoplasmic reticulum Ca2+ -ATPases, modulates nitric oxide production and cell death of primary rat hepatocytes in culture.

Increased cytosolic calcium ([Ca2+]i) and nitric oxide (NO) are suggested to be associated with apoptosis that is a main feature of many liver diseases and is characterized by biochemical and morphological features. We sought to investigate the events of increase in [Ca2+]i and endoplasmic reticulum (ER) calcium depletion by thapsigargin (TG), a selective inhibitor of sarco-ER-Ca2+ -ATPases, in relation to NO production and apoptotic and necrotic markers of cell death in primary rat hepatocyte culture. Cultured hepatocytes were treated with TG (1 and 5 micromol/L) for 0-24 or 24-48 h. NO production and inducible NO synthase (iNOS) expression were determined as nitrite levels and by iNOS-specific antibody, respectively. Hepatocyte apoptosis was estimated by caspase-3 activity, cytosolic cytochrome c content and DNA fragmentation, and morphologically using Annexin-V/propidium iodide staining. Hepatocyte viability and mitochondrial activity were evaluated by ALT leakage and MTT test. Increasing basal [Ca2+]i by TG, NO production and apoptotic/necrotic parameters were altered in different ways, depending on TG concentration and incubation time. During 0-24 h, TG dose-dependently decreased iNOS-mediated spontaneous NO production and simultaneously enhanced hepatocyte apoptosis. In addition, TG 5 micromol/L produced secondary necrosis. During 24-48 h, TG dose-dependently enhanced basal NO production and rate of necrosis. TG 5 micromol/L further promoted mitochondrial damage as demonstrated by cytochrome c release. A selective iNOS inhibitor, aminoguanidine, suppressed TG-stimulated NO production and ALT leakage from hepatocytes after 24-48 h. Our data suggest that the extent of the [Ca2+]i increase and the modulation of NO production due to TG treatment contribute to hepatocyte apoptotic and/or necrotic events.

The role of adrenergic agonists on glycogenolysis in rat hepatocyte cultures and possible involvement of NO.

Certain liver metabolic diseases point to the presence of disturbances in glycogen deposition. Epinephrine raises the cAMP level that activates protein kinase A leading to the activation of phosphorylase and glycogen breakdown. In the present report, we sought to investigate whether NO is produced during adrenoceptor agonist-induced glycogenolysis in rat hepatocytes in cultures. Isolated glycogen rich rat hepatocytes in cultures were used. NO production (NO(2)(-)) was assessed under the effect of adrenergic agonists and adrenergic agonist/antagonist pairs, dibutyryl cyclic AMP sodium-potassium salt (db-cAMP), NO synthase (NOS) inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP). The inducible NO synthase (iNOS) mRNA was examined by the reverse transcription-polymerase chain reaction (RT-PCR). Glycogenolysis was quantified by glucose levels released into medium. The amount of glucose and NO(2)(-) released by hepatocytes was increased as a result of epinephrine, phenylephrine or db-cAMP treatments. The increase in glucose and NO(2)(-) released by epinephrine or phenylephrine was blocked or reduced by prazosin pretreatment and by NOS inhibitors aminoguanidine and L-NAME. iNOS gene expression was up-regulated by epinephrine. It can be concluded that glycogenolysis occurs through -adrenoceptor stimulation and a signaling cascade may involve NO production.

Tert-butyl hydroperoxide selectively inhibits mitochondrial respiratory-chain enzymes in isolated rat hepatocytes.

Sensitivity of various mitochondrial enzymes to oxidative damage was tested on isolated rat liver hepatocytes permeabilized by digitonin. In permeabilized hepatocytes normal respiratory control values were obtained and mitochondrial membranes remained intact. Respiratory rates of NADH-dependent (glutamate + malate, palmitylcarnitine + malate) and flavoprotein-dependent (succinate) substrates were determined in hepatocytes exposed for 5 min to 0.5-3 mM tert-butyl hydroperoxide before addition of digitonin. Our data showed that oxidation of NADH-dependent substrates is much more sensitive to oxidative stress than oxidation of flavoprotein-dependent ones, evidently due to the modification of iron-sulfur clusters or SH groups in the NADH dehydrogenase enzyme complex (Complex I).

Quantitative aspects of lipopolysaccharide and cytokine requirements to generate nitric oxide in macrophages from LPS-hyporesponsive (Lps(d)) C3H/HeJ mice.

Due to a gene defect (Lps(d)), C3H/HeJ mice are known to be hyporesponsive to the immunobiological potential of lipopolysaccharide (LPS). We studied dose requirements for LPS, IFN-gamma, and cytokines TNF-alpha and IL-10 to produce nitric oxide (NO) in peritoneal macrophages (Mphi) from these animals. In contrast to the Lps(n) C3H/HeN mice, high concentrations of LPS (up to 5 microg/mL) or IFN-gamma (up to 5 ng/mL) by themselves were unable to activate NO production in C3H/HeJ Mphi. The failure to produce NO could not be overcome by addition of L-arginine or tetrahydropterin. The high-output NO biosynthesis was dose-dependently stimulated by combined administration of varying concentrations of IFN-gamma (50-5000 pg/mL) and LPS (approximately 1 ng/mL) or to a lesser extent by IFN-gamma plus TNF-alpha or TNF-alpha/IL-10. Formation of NO in C3H/HeJ MCO triggered by high concentration of LPS (approximately 1 microg/mL) given together with IFN-gamma (0.2-5 ng/mL) reached the values typical for Lps(n) C3H/HeN mice. While Mphi from C3H/HeN mice secreted TNF-alpha, IL-10, and IL-10 upon contact with a low dose of LPS (1 ng/mL), C3H/HeJ Mphi required high concentration of LPS (5 microg/mL) to enhance the secretion of the cytokines. Yet, this dose remained ineffective to stimulate IFN-gamma in Mphi from C3H/HeJ mice. It can be presumed that one of the important factors influencing their deficient ability to form NO is a failure of Mphi to produce IFN-gamma upon LPS contact.

Effect of nitric oxide donors on isoprenaline-induced lipolysis in rat epididymal adipose tissue: studies in isolated adipose tissues and immobilized perfused adipocytes.

The present investigation was directed to study the effect of in vitro or ex vivo NO donors, sodium nitroprusside and molsidomine, using isolated sliced adipose tissue or in the form of immobilized and perfused adipocytes on the basal and isoprenaline-stimulated lipolysis. The results demonstrated that 1) in vitro application of sodium nitroprusside to perfused adipocytes or molsidomine to sliced adipose tissues affects isoprenaline-induced lipolysis in two ways, an increase in lipolysis at low isoprenaline concentrations (which means the sensitization of adipose tissues to adrenergic effect by NO) and decreased adrenergic agonist-stimulated lipolysis at higher concentration of isoprenaline (a decrease in the maximum lipolytic effect of isoprenaline), 2) low concentrations of molsidomine alone induced lipolysis from adipose tissue which attained more than 60% of that by isoprenaline (pD2 value for molsidomine = 11.2, while pD2 for isoprenaline = 8.17) while sodium nitroprusside did not affect the basal lipolysis significantly, 3) in vivo administration of molsidomine for 2 days reduced the maximum lipolytic effect of isoprenaline and (only non-significantly) increased the sensitivity to low doses of isoprenaline. In conclusion the present data demonstrate that NO plays an important role in adrenergic lipolysis in adipose tissues and further investigations are needed to unravel the exact role of NO in lipolysis.

Potential application of immobilized and perfused hepatocytes in environmental toxicology studies.

Conventional cellular models have contributed significantly to the understanding of many aspects of cell physiology and molecular biology. In these models cells are metabolically less active, due to the inefficient oxygenation and waste product buildup. Therefore perfusion methods for the cells are expected to improve cell activities. Cells have to be fixed in or on an appropriate inert carrier or support, which enables cellular perfusion, maintains integrated cellular functions and makes a bioreactor. Since isolated hepatocytes are extensively used in biomedical studies including those dealing with environmental pollutants or toxins and in xenobiotic biotransformation investigations, an efficient hepatocyte perfusion model has to be available for researchers. This research article is focusing on the value of hepatocyte immobilization as a laboratory bioreactor model and is shedding light on its potentiality in research related to public health. We demonstrate the application of this cellular model as a means to study representative phase I and phase II biotransformation reactions using hexobarbital hydroxylation and 7-ethoxycoumarin deethylation and 4-chloro 2-dinitrobenzene glutathione. Both phase I and phase II drug biotransformation in hepatocytes was demonstrated in this study non-destructively to the cells and in an efficient way. In spite of the aforementioned advantages, immobilized hepatocytes yet have relatively limited applications compared to conventional hepatocyte cellular systems. Reasons for this discrepancy are discussed. This cellular system may become popular due to the better performance of immobilized hepatocytes as compared to conventional hepatocyte culture and due to economic and ethical reasons. Naturally its applicability will cover several biomedical areas including basic research in environmental toxicology and other public health issues.

Modulatory effect of cyclosporin A on tert-butyl hydroperoxide-induced oxidative damage in hepatocytes.

In the present work, we followed an in vitro protective action of cyclosporin A (CsA) against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in hepatocytes. Various parameters (cell viability, cytosolic calcium level, rhodamine 123 accumulation as indicator of mitochondrial membrane potential and alanine-aminotransferase leakage from cells) were measured as an index of cytotoxicity. Tert-butyl hydroperoxide (1 mM) significantly increased cytosolic Ca2+ and affected mitochondrial membrane potential. Pretreatment with cyclosporin A (0.5 microM) reduced t-BHP-induced cytosolic Ca2+ increase and ALT (alanine-aminotransferase) leakage, but had no protective effect on t-BHP-induced changes of mitochondrial membrane potential. Our data thus suggest that the mechanism of cytoprotection of CsA on the cytosolic Ca2+ changes and ALT leakage induced by t-BHP, does not directly correlate with protection of t-BHP-induced changes of mitochondrial membrane potential.

Cyclosporin A modifies cytoplasmic calcium levels in isolated hepatocytes exposed to oxidative stress due to tert-butyl hydroperoxide.

Within the framework of our studies on hypertension in various rat strains, we have examined the effect of cyclosporin A (CsA) on intracellular calcium signaling under conditions of oxidative stress. For these preliminary experiments, we have chosen isolated hepatocytes of normotensive rats as a model system for the study of the role of intracellular calcium. We used tert-butyl hydroperoxide (t-BHP, 1 mmol x l(-1)) as an prooxidant agent. When compared to the controls, we found increased levels of cytosolic free calcium concentration (Ca2+i) during 120 min incubation. The preincubation of hepatocytes with CsA in the concentration of 0.5 micromol x l(-1)] did not change the physiological level of cytosolic calcium. However, a dual action of CsA on elevated Ca2+i was observed during oxidative injury of hepatocytes: while in the first period of incubation CsA increased Ca2+i, CsA reduced the effect of t-BHP on Ca2+i during the next period of incubation. This indicates the ability of CsA to modify oxidative stress, but further studies are necessary to explain these findings.