Zdeněk Zídek

The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases

Metagenomic approaches are currently being used to decipher the genome of the microbiota (microbiome), and, in parallel, functional studies are being performed to analyze the effects of the microbiota on the host. Gnotobiological methods are an indispensable tool for studying the consequences of bacterial colonization. Animals used as models of human diseases can be maintained in sterile conditions (isolators used for germ-free rearing) and specifically colonized with defined microbes (including non-cultivable commensal bacteria). The effects of the germ-free state or the effects of colonization on disease initiation and maintenance can be observed in these models. Using this approach we demonstrated direct involvement of components of the microbiota in chronic intestinal inflammation and development of colonic neoplasia (i.e., using models of human inflammatory bowel disease and colorectal carcinoma). In contrast, a protective effect of microbiota colonization was demonstrated for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. Interestingly, the development of atherosclerosis in germ-free apolipoprotein E (ApoE)-deficient mice fed by a standard low-cholesterol diet is accelerated compared with conventionally reared animals. Mucosal induction of tolerance to allergen Bet v1 was not influenced by the presence or absence of microbiota. Identification of components of the microbiota and elucidation of the molecular mechanisms of their action in inducing pathological changes or exerting beneficial, disease-protective activities could aid in our ability to influence the composition of the microbiota and to find bacterial strains and components (e.g., probiotics and prebiotics) whose administration may aid in disease prevention and treatment.

A novel and efficient one-pot synthesis of symmetrical diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates and evaluation of their biological activities

A novel and efficient method for the one-pot synthesis of diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates, starting from free phosphonic acids or phosphonate diesters is reported. The approach from phosphonate diesters via their bis(trimethylsilyl) esters is highly convenient, eliminates isolation and tedious purification of the phosphonic acids, and affords the corresponding bis-amidates in excellent yields (83-98%) and purity. The methodology has been applied to the synthesis of the potent anticancer agent GS-9219, and symmetrical bis-amidates of other biologically active phosphonic acids. Anti-HIV, antiproliferative, and immunomodulatory activities of the compounds are discussed including the bis-amidate prodrugs 14 and 17 that exhibited anti-HIV activity at submicromolar concentrations with minimal cytotoxicity.

IFN‐γ‐induced TNF‐α is a prerequisite for in vitro production of nitric oxide generated in murine peritoneal macrophages by IFN‐γ

The effect of IFN‐γ to stimulate formation of nitric oxide (NO) by normal murine peritoneal macrophages (Mϕ) has been found to be completely dependent on the ability of IFN‐γ to activate secretion of TNF‐α. The NO‐stimulatory effect of IFN‐γ was abolished by anti‐TNF‐α antibodies, the inhibitory intervention of which could be fully reversed by exogenously supplied TNF‐α. Accordingly, the failure of Mϕ from C3H/HeJ mice to secrete TNF‐α upon stimulation with IFN‐γ was associated with their complete incapability to generate NO, unless they were simultaneously treated with IFN‐γ + TNF‐α. Collectively, the data document that similar to the NO up‐regulatory action of other cytokines, the effect of IFN‐γ is not independent, but depends on a synergistic cooperation with the self‐produced TNF‐α. The findings thus indicate that a widespread opinion claiming that IFN‐γ per se is able to stimulate biosynthesis of NO needs revision.

Resveratrol attenuates lipopolysaccharide-induced hepatitis in D-galactosamine sensitized rats: role of nitric oxide synthase 2 and heme oxygenase-1.

The goal of study was directed to investigate the effects of resveratrol (RES) pretreatment on the enhancing action of D-galactosamine (D-GalN; 800 mg/kg) on lipopolysaccharide (LPS; 0.5 microg/kg) inducing liver failure in rats. Liver function was assessed by determination of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alpha-glutathione S-transferase (alpha GST) and bilirubin (BILI). Plasma NO(2)(-) was assessed by NO(2)(-)/NO(3)(-) colorimetric kit. The estimation of nonenzymatic and enzymatic antioxidants (glutathione and catalase) was performed in plasma and liver homogenate. Lipid peroxidation was evaluated by the thiobarbituric acid reacting substances (TBARS) and the conjugated dienes (CD). Morphological examinations using light and electron microscopy were performed. Observations related to pharmacological increases of inducible nitric oxide synthase (NOS-2)/nitric oxide (NO) and inducible heme oxygenase (HO-1) in fulminant hepatic failure and modulation by resveratrol were followed up by real-time reverse transcription PCR (RT-PCR) in liver tissue. In the present study we found that among the mechanisms responsible for the hepatoprotective effect of resveratrol in the LPS/D-GalN liver toxicity model are reduction in NO, downregulation of NOS-2, modification of oxidative stress parameters and modulation of HO-1 which led to overall improvement in hepatotoxic markers and morphology after the hepatic insult.

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.

Hepatoprotective effect of curcumin in lipopolysaccharide/-galactosamine model of liver injury in rats: Relationship to HO-1/CO antioxidant system

This work studied a relationship between HO-1/CO system and lipid peroxidation with consequent effects on liver functions and NOS-2. We focused on curcumin pretreatment in rat toxic model of d-galactosamine and lipopolysaccharide. Hepatocyte viability, lipid peroxidation, antioxidant status, ALT and AST were evaluated. HO-1 and NOS-2 expressions and respective enzyme activity were determined. Curcumin caused decreases in ALT and AST levels as well as in lipid peroxidation. Furthermore, curcumin pretreatment increased liver HO-1 (2.4-fold, p=0.001), but reduced NOS-2 (4.1-fold, p=0.01) expressions. In conclusion, the tuning of CO/NO pathways is important in shedding light on curcumins cytoprotective effects in this model.

Review Article Erratic behavior of nitric oxide within the immune system: illustrative review of conflicting data and their immunopharmacological aspects

The literature data assembled in this article document the variation of immunobiological effects of nitric oxide (NO). A number of factors are obviously responsible for the diversity, ranging from inactivity, alleviation, but not rarely to exacerbation of certain pathogenetic processes. A better understanding of NO interactions with the immune system can only be reached if more complex experimental designs to study the effects of reactive nitrogen species are adopted in the future. They should integrate major participating variables and take into account pharmacodynamic/kinetic aspects of NO production in triggering the ultimate effects. If manipulation of NO in the organism by means of recently developed NO inhibitors and NO donors is to become a rational tool of immunopharmacological strategies, detailed knowledge of their pharmacologies and toxicologies is urgently needed in order to differentiate between the effects of NO and other side effects. Hopefully, this approach could improve the predictability of the clinical outcomes of NO manipulation.

EFFECTS OF RESVERATROL PRETREATMENT ON TERT-BUTYLHYDROPEROXIDE INDUCED HEPATOCYTE TOXICITY IN IMMOBILIZED PERIFUSED HEPATOCYTES: INVOLVEMENT OF INDUCIBLE NITRIC OXIDE SYNTHASE AND HEMOXYGENASE-1

The aim of this work was to study the effects of resveratrol (RES) as compared to silymarin (SM) pretreatments on tert-butylhydroperoxide (tBH) induced apoptotic/necrotic markers in hepatocytes. Hepatocyte in cultures (48 h) and in perifused immobilized agarose threads (5h) were used as cellular systems. Hepatocyte apoptosis was estimated morphologically using Annexin-V combined with propidium iodide, or toluidine blue staining. Hepatocyte viability and functionality were evaluated by ALT and urea synthesis. Nitric oxide (NO) and carbon monoxide involvements were also examined. Resveratrol and silymarin reduced tBH-induced hepatocyte toxic effects in short term experiments (5h) as measured by a significant reduction in ALT and NO increase produced by tBH. Both inducible nitric oxide synthase (NOS-2) and hemoxygenase-1 (HO-1) gene expression were increased by tBH and reduced by both RES and SM pretreatments. Morphologically, there were ameliorations in both apoptotic and necrotic markers under RES treatment and were similar to biochemical findings. In addition, RES improved hepatocyte stability in both cellular systems. It may be concluded that resveratrol and sylimarin ameliorative effects on tBH hepatocyte toxicity are comparable; involve NOS-2 and HO-1 expression and should be re-evaluated in various in vitro and in vivo experimental conditions.

Antiretroviral agent (R)-9-(2-phosphonomethoxypropyl)adenine stimulates cytokine and nitric oxide production

The immunomodulatory properties of (R)-enantiomer of 9-(2-phoshonomethoxypropyl)adenine ((R)-PMPA), one of the most potent acyclic nucleotide analogs effective against human immunodeficiency virus (HIV), were investigated under in vitro conditions using murine peritoneal macrophages. Remaining without influence on interferon-gamma and interleukin-2 expression, (R)-PMPA dramatically stimulated in a concentration- and time-dependent manner the secretion of tumor necrosis factor alpha (TNF-alpha) and interleukin-10. It also substantially augmented the production of nitric oxide (NO) induced by exogenous interferon-gamma. Inhibitory experiments using neutralizing antibodies against TNF-alpha and/or interleukin-10 demonstrated that these two cytokines are major factors responsible for triggering the underlying mechanism(s) leading to enhanced NO production. The novel findings on the immunomodulatory potential of acyclic nucleotide analogs are discussed in the context of their possible implication in antiviral therapeutic efficacy.

Macrophage activation by antiviral acyclic nucleoside phosphonates in dependence on priming immune stimuli

Acyclic nucleoside phosphonates (ANPs) are potent broad-spectrum antivirals, also effective against immunodeficiency viruses and hepatitis viruses. Effects of several ANPs on in vitro cytokine gene expression and nitric oxide (NO) production by murine peritoneal macrophages were investigated. Included in the study were 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; Adefovir), 9-(R)-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; Tenofovir], 9-(S)-[2-(phosphonomethoxy)propyl]adenine; (S)-PMPA), 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP), 9-(R)-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine (PMPDAP), and 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG). Some of them, i.e. (R)-PMPA, (S)-PMPA, and PMEG, stimulate secretion of TNF-alpha and IL-10 in a concentration-dependent manner, and enhance the IFN-gamma-induced secretion of TNF-alpha. Although unable to activate production of nitric oxide (NO) on their own, these compounds substantially augment NO formation induced by IFN-gamma. Analysis of the expression of inducible NO synthase mRNA indicates that the NO-enhancing effect of ANPs is mediated posttranscriptionally. In contrast to IFN-gamma, production of NO triggered by lipopolysaccharide (LPS) alone, or synergistically by LPS+IFN-gamma, remains unaltered by ANPs. The immunomodulatory effects have been differentially expressed in distinct genotypes of inbred strains of mice, including the low NO-responders Balb/c and high NO-responders C3H/HeN. Although less effectively, PMEG and (R)-PMPA also increase production of TNF-alpha and NO by the IFN-gamma- but not LPS-co-stimulated macrophages from C3H/HeJ mice, which are otherwise hypo-responsive to major immune stimuli provided by IFN-gamma and LPS. It can be concluded that the expression of immunomodulatory properties of ANPs depends on the immune state of cells and its activation by distinct priming signals.