Department of Biophysics, Biological Faculty, Lomonosov Moscow State University, Moscow, Russia
Mast cells are known to be among the most important effector immunocompetent cells participating in various biological responses. The description of the mast cell in connective tissue is ascribed to Ehrlich. Ehrlich made the important discovery that the mast cell stained well and specifically with certain dyes of the aniline family, the specific metachromatic staining reaction. Ehrlich also proposed the relation of mast cells with inflammation, blood vessels and neural tissue. Mast cell develop from progenitor cells that in turn arise from uncommited hemopoietic stem cells in the bone marrow, they undergo terminal differentiation in tissues. It was determined that IgE molecules have a high affinity for specific receptors on mast cells and that the reaction of cell-bound IgE molecules with multivalent antigens or divalent anti-IgE antibody induces the release of a variety of chemical mediators from the cells, that testify a central role for the mast cell in immunological reactions. More recently the role of mast cells in inflammatory disease and host defense was established. Following activation, these cells express mediators such as histamine, leukotrienes and prostaglandins, as well as proteases, and many cytokines and chemokines, pivotal to the genesis of an inflammatory response. Mast cells have been shown to play roles in allergic inflammation, and more recently, they have been shown to modulate coagulation cascades, host defense and tissue remodeling. Although mast cells were discovered long ago they are certainly no less interesting today and their history is far from complete
Augmenting Endogenous Nitric Oxide Production to Kill Multiresistant Pseudomonas aeruginosa in Cystic Fibrosis Lung Disease. GRASEMANN H Hospital for Sick Children Research Institute, and Division of Respiratory Medicine, The Hospital for Sick Children, University of Toronto, Toronto, Canada
Cystic fibrosis (CF) is among the most common of the fatal genetic diseases. CF affects multiple organs but lung disease is the major determinant of morbidity and mortality. Chronic inflammation and infection are the hallmarks of CF lung disease.
Nitric oxide (NO) is involved in multiple aspects of lung biology including broncho-motor control and antimicrobial defense. Reduction of NO formation in CF is associated with airway obstruction and increased susceptibility to lung infections.
P. aeruginosa is capable of robust anaerobic growth by respiration using the NO metabolites nitrate (NO3-) or nitrite (NO2-) as terminal electron acceptors. Still, during anaerobic growth, P. aeruginosa must control the level of toxic NO by synthesis of protective NO reductase (NOR) and nitrite reductase (NIR).
As CF lung disease progresses mucoid P. aeruginosa strains emerge which are inherently resistant to antibiotics. The mechanisms behind the phenotypic switch to the mucoid form are incompletely understood, however the best characterized mechanism of mucoid conversion in CF isolates is via mutations in the mucA gene. NIR and NOR activity are remarkable low in mucA mutant P. aeruginosa, making this particular pathogen extraordinarily susceptible to NO-mediated killing. MucA mutant bacteria also have a markedly reduced capacity to remove NO generated aerobically from S-nitrosoglutathione. In addition, it was shown that 15 mM NO2- kills mucA mutant P. aeruginosa in CF airways at pH 6.5 under anaerobic conditions. In vitro experiments on the dose-effect relationship between NO2- concentration and killing of mucoid P. aeruginosa showed that the LD50 was approximately 3 mM NO2- after 24 hours. Previous studies demonstrated that overproduction of NO by anaerobic P. aeruginosa biofilms resulted in metabolic suicide of these bacteria, an event that was preventable by an NO scavenger. In vitro enhancement of antibiotic susceptibility of P. aeruginosa in biofilms had also been reported with L-arginine, NO3- or NO2- supplementation; presumably through an NO mediated mechanism.
Clinical and animal studies are currently ongoing to evaluate the therapeutic effects of L-arginine supplementation on pulmonary function and Pseudomonas-killing in the CF airways.
Delivery of specific targeted drugs into the cells by TAT-technology
GRDISA M, MIKECIN A-M
Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka cesta 54, 10 000 Zagreb, Croatia.
Background: The importance of drug delivery is pivotal in the wide area of pharmacological research and it has not been solved yet. The main goal of every drug delivery system is the delivery of a precise amount of a drug to the desired location in order to achive the necessary drug concentration in the targeted organ for effective treatment. Proteins and peptides are useful research and therapeutic tools, however their applications are limited because delivery to the desired location is not easily achiveable. Process of protein transduction, using TAT-technology, allows the delivery of drugs and genetic materials inside the cells. This process occurres in a rapid, concentration-dependent fashion that appears to be independent of receptors and transporters. It has a broad implications in experimental systems for regulating intracellular processes and has the potential to be used in the development of new therapeutic strategies for cancer, infectious diseases, and development of vaccines.
Hyper proliferation of cancer cells is associated with deregulation of cell cycle progression, which is driven by the activities of CDKs. A key regulator of their activities is protein p27. It has significant role in cancer progression and antitumor drug response.
Results: To examine p27 as specific target molecule and its role in tumor cells apoptosis, transduction of TAT-p27, TAT-ptp27 and TAT-N'p27 was performed. It was shown that different forms of TAT-p27 protein can modulate the cell cycle of cultured cell lines and induced apoptosis, depending on the concentration and type of the cells. Also was shown that different signal transduction pathways were involved in induction of apoptosis.
Conclusions: Extraxellular p27 could be use for induction of apoptosis in tumor cells. Protein transduction therefore could give an opportunity for delivering of drugs into cells with emphasis on specific target molecules.
The “antiinflammatory” 5-ASA, “immune-modulators” azathioprine, 6-MP, methotrexate & thalidomide and “immune-suppressants” Cyclosporine A, Rapamycin & Tacrolimus are all unsuspected “Magic Bullets” the inhibit M. avium subspecies paratuberculosis growth in culture GREENSTEIN RJ VAMC Bronx NY USA
Background: Without known mechanisms of action, “anti-inflammatories” (5-ASA,) “immune-modulators” (azathioprine, 6-MP, methotrexate & thalidomide) and “immune-suppressants” (Cyclosporine A, Rapamycin & Tacrolimus) are used to treat a variety of non-malignant “idiopathic” and “autoimmune” diseases. M. avium subspecies paratuberculosis (MAP) causes a chronic wasting diarrheal disease in ruminants called Johne’s disease, that is evocative of human inflammatory bowel disease (IBD). MAP may be zoonotic. We hypothesized that, aside from their well-documented effects on eukaryotic cells, these agents may additionally be “Magic Bullets” that inhibit prokaryotes, particularly MAP. We herein report on the effects of these agents on MAP in culture.
Methods: We studied growth kinetics of four MAP strains (three human isolates, “Ben”, “Dominic” & UCF-4) a bovine MAP isolate ATCC 19698 and three mycobacterial controls (M. avium & BCG,) using the radiometric 14CO2 (Bactec®) system. Growth is quantified as arbitrary Growth Index (GI) units and inhibition as “percent decrease in cumulative GI” (%-cGI.)
Results: (Most published at www.PLoSOne.org Thalidomide data Unpublished)
Our negative controls do not inhibit MAP. The test agents cause dose dependent inhibition of MAP. The most potent is methotrexate (89%-cGI at 4µg/ml: “Dominic”) followed by 6-MP, azathioprine, Cyclosporine A, Rapamycin, Tacrolimus and 5-ASA (46%-cGI at 64µg/ml: ATCC 19698.) Thalidomide comprises two components. Phthalimide causes no inhibition, whereas the piperidine 2,6 dione moiety inhibits MAP (46%-cGI at 64µg/ml: “Dominic”.)
Conclusions: 1) We show in culture heretofore-undescribed inhibition of MAP growth by a variety of agents that are used, simply because of empirical efficacy, to treat several “inflammatory” and “autoimmune” diseases. 2) These data show that these agents effect prokaryotic, in addition to eukaryotic cells. 3) We conclude that, unknowingly, the medical profession has been treating MAP infections since 1942, when Nanna Svartz introduced sulfasalazine. 4) We posit that MAP may be responsible for multiple “inflammatory” and “autoimmune” diseases that have been empirically treated with these agents.
Authors’ disclosure statement The author has submitted patents based on the hypotheses tested in these studies
Verapamil Reverts Acute Renal Functional Impairment Induced by Angiotensin II Converting Enzyme Inhibitors ALVAREZ GREGORI JA1,2, BOUCHOUTROUCH Y2, MACIAS NUÑEZ JF2 1 CS. Casto Prieto. UDMFYC. SACYL. Salamanca; 2 Faculty of Medicine. University of Salamanca
Background: Antihypertensive agents have been found effective in arresting glomerulosclerosis. Initially, it was thought that the healthy effect of these drugs was exclusively due to their hemodynamic effects. However, it has become clear that nonhemodynamic actions of these agents are an important component of their beneficial effects. Among thepharmac ological agents that may have a favorable
influence in the course of renal failure, angiotensin converting enzyme (ACE) inhibitors, and calcium channel blockers (CCB) have generated the most interest.
Angiotensin-converting enzyme inhibitors (ACEI) have proven to be effective drugs for the treatment of hypertension and represent a major therapeutic breakthrough in the management of hypertension and renal function preservation in diabetic and nondiabetic nephropathies. In some patients, ACEI may induce a rapid deterioration of renal function, assessed as an increase in serum creatinine (SCr), which can be reversed by withdrawing the drug.In these cases, maintaining the renal protection due to ACEI, instead of withdrawing these drugs could be desirable.
Calcium antagonists are a heterogeneous group of agents with diverse effects in terms of nephroprotection. Some of these differences relate to their effects on renal microcirculation. Dihydropyridine agents appear to act only on the afferent arteriole, increasing intraglomerular pressure, and albumin excretion rate. In contrast, nondihydropyridine agents like verapamil, may dilate efferent arterioles in addition to afferent arterioles and with normalization of the systemic blood pressure, verapamil may reduce intraglomerular pressure, and proteinuria. However, some other non hemodynamic protective effects of CCB could be explained by its capacity to inhibit the extracellular calcium influx, an important signal for the proliferative effect of mesangial cells mitogens, its influence in the decrease in mesangial entrapment of macromolecules and, possibly, its effect as free radical scavenger
Münter et al. describe that nephroprotective effects of ACEI/CCB combination can occur at doses, which do not significantly alter systemic blood pressure in the stroke-prone SHR. We have described that the combined therapy with these agents provide in the remnant kidney model a synergistic effect in preventing renal injury, independently of their effects on blood pressure.In a preliminary study, we have demonstrated in a small group of patients that nondihydropiridin (nonDHP) CCB are able to revert renal function reduction associated to ACEI treatment.
The main purpose of this study was to assess the efficacy and safety of low doses of verapamil (180 mg/day) added to the previous ACEI treatment for reverting decreased glomerular filtration rate observed in patients treated with ACEI. A secondary purpose was to test the ability of the fixed combination Verapamil-SR 180 mg plus Trandolapril 2 mg in attaining BP control and maintaining there renal function throughout the study.
Methods: This was a multicenter, nonrandomized, prospective, open study developed in five Spanish Hospitals. All patients were referred from the outpatients Departments of Internal Medicine and Nephrology. The Institutional Review Boards approved the study protocol and each patient entering the study signed a written informed consent.
Eligible patients presented a previous diagnosis of hypertension and an increase in SCr of 20% or 45 mmol/L from last values, in the course of ACEI treatment for more than four weeks. Exclusion criteria were renal insufficiency, defined as SCr >354 mmol/L (4 mg/dL), stroke, AMI in the last three months, unstable angina, cardiac failure, other causes of renal hypo-perfusion, or low volume syndrome such as dehydration, vomiting, diarrhea, laxative, and known hypersensitivity to verapamil.
In the selected group, a clinical evaluation was carried out, including clinical history, physical examination, BP measurement, ECG, and biochemistry (Visit 1). Patients fulfilling the inclusion/exclusion criteria were enrolled in the study.
[Regrettably not enough space for this long abstract.]
Molecular Modelling of Inhibitor-Kinase Interactions. 'Icy', Highly Polarized Water Molecules Can Tip the Relative Energy Balances of Competing Inhibitors. de COURCY B1, PIQUEMAL JP2, GARBAY C1, GRESH N1 1Laboratoire de Pharmacochimie Moléculaire et Cellulaire, U648 INSERM, UFR Biomédicale,Université Paris Descartes, 45, rue des Saints-Pères, 75006 Paris, France, 2Laboratoire de Chimie Théorique, UMR 7616 CNRS, Université Pierre-et-Marie-Curie, 4, place Jussieu, F75252 Paris
Background. Fak (Focal Adhesion Kinase) kinase is the target for the development of antitumor drugs. We perform molecular modelling computations in order to compare the relative energy balances for the binding to FAK kinase of two competing inhibitors in the pyrrolopyrimidine series, which were designed by the Novartis firm (Hao et al., Bioorg. Med. Chem. Letts, 2006, 16, 2809). The first, denoted as 16i, has micromolar affinities while the second, 32, has nanomolar affinities.
Methods. The protein-ligand interaction energies are computed by the SIBFA (Sum of Interactions Between Fragments Ab initio computed) procedure, an anisotropic, polarisable molecular mechanics procedure formulated an calibrated on the basis of ab initio Quantum Chemistry (Review paper: Gresh et al., J. Chem. Theory. Comput., 2007, 3, 960).
Results. The energy balances encompassing the contribution of continuum solvation favour 16i, contrary to the experimental results. Including a limited number of five 'discrete', highly polarized, water molecules in the inhibitor-FAK recognition site results in an inversion of the energy balances, now in favour of 32.
Conclusions. These findings imply that: 1) some structural, ‘discrete’ water molecules found in the recognition sites of protein can affect dramatically the comparative energy balances of structurally related, competing inhibitors. These water molecules undergo extremely large increases in their dipole moments amenable to quantum chemistry and polarisable molecular mechanics computations; 2) it is essential that the protein-inhibitor-water intermolecular interactions be computed with accurate potential energy functions, embodying the non-additive polarization contribution.
Cancer-testis antigens: effective molecules for developing successful immunotherapeutic strategies in the light of cancer complexity GRIZZI F1 1Laboratories of Quantitative Medicine, Istituto Clinico Humanitas IRCCS, Rozzano, Milan, Italy.
Background: Despite advances in our molecular knowledge, human cancer remains one of the major public health problems throughout the world. Although a number of cancer-testis (CT) antigens have been discovered and it has been suggested that they could be useful in the immunological treatment of cancer, the complexity of human beings leads us to reflect on the need to establish new criteria for validating their applicability. Aims: 1) To discuss cancer as a complex dynamical disease. 2) To discuss CT antigens as effective molecules for developing immunotherapeutic strategies in the light of cancer complexity.
Methods: This study investigate the immunolocalization of Sperm protein 17, a labeled CT antigen in multiple myeloma and ovarian carcinoma, in a panel of natural tissues of unrelated histologic origin and their tumoral counterparts. After deparaffination and rehydration, two-micrometers thick sections were placed in a bath for antigen retrieval, incubated with H2O2 to quench endogenous peroxidase activity, and then treated with monoclonal primary antibodies raised against Sp17 (BD Biosciences). Mouse IgG1 was used as negative control. This was followed by incubation with the DAKO Envision system. 3,3-diaminobenzidine tetrahydrochloride was used as a chromogen to yield brown reaction products. Countarstained slides were analyzed under a light microscope.
Results: Sp17 was found in human germinal cells of the testis (except for spermatogonia), and in the ciliated epithelia of the respiratory airways and in both the male and female reproductive systems. Sp17 has also been recognized in ovarian inclusion cysts, melanophages of cutaneous melanocytic lesions, as well as in a proportion of primary nervous system and liver tumors, a subset of esthesioneuroblastomas and a high number of pituitary adenomas.
Conclusions: 1) Sp17 is more widely distributed in the human body than originally thought. 2) The expression of CT antigens is mainly studied at the level of gene expression and gene level measurement by RT-PCR analysis and the quantitative RT-PCR technology. However, the information provided by these approaches is limited by the fact that the phenomena observed at each level of anatomical organization have properties that do not exist at a lower or higher level. 3) A multidisciplinary system-level approach, which takes into account the human being as a complex hierarchical system, provides a different way of investigating human cancer, thus promising a more widely applicability of CT antigens for developing effective immunotherapeutic strategies.
Ascorbic acid and dietary selenium bioavailability in rats GROSICKI A, KOWALSKI B.
National Veterinary Research Institute, Department of Radiobiology,
24-100 Pulawy, Poland.
Background: Vitamin C is a nutrient that may affect the bioavailability of both trace (copper and iron) and undesirable (lead, mercury, and cadmium) elements. It is also quite possible that vitamin C may interfere with other trace elements. For example it is suggested that vitamin C may reduce dietary selenium to an elementary form that cannot be utilized by the body. Taking into account a very narrow range of the save selenium intake the purpose of this study was to evaluate if moderate dietary vitamin C supplements affect selenium retention in selected organs of rats
Methods: Studies involved 135 male Wistar rats, 212 ± 11 assigned into three groups each of 45 animals: group 1 (controls), and groups 2 and 3 exposed to 1g/L and 2 g/L of vitamin C via drinking water, respectively. All rats received traces of sodium selenite (labeled with selenium-75) by a stomach tube for 28 days. Selenium-75 activity was measured in the duodenum, blood, liver, kidneys, spleen, muscles, heart, brain, and testicles within 28 days postdosing. Results were compared using Student’s t-test at P < O.05.
Results: No differences were noted in tap water (about 29 mL/rat/d) and feed intake, organ to body ratio, and body weight gains among the groups tested. The retention of selenium –75 in selected organs was shown as the AUC values. In groups 2 and 3 the AUC values were compared to those in the controls that were considered as 100 per cent. Data were analyzed statistically using Student’s t-test at P<0.05.
Vit. C in water
Blood
Liver
Kidneys
Skeletal muscles
Testicles
AUC
AUC
AUC
AUC
AUC
0 g/L
100 (in %)
100 (in %)
100% (in %)
100% (in %)
100% (in %)
1 g/L
151*
152*
131*
124
134*
2 g/L
121
132*
125*
110
119
Conclusions: 1) Diet supplemented with moderate doses of vitamin C may enhance selenium retention in the organs especially those revealing a high rate of selenium metabolism. 2) The ability to raise selenium retention seems to be inversely related to increasing doses of vitamin C when a dosage is higher than 1 mg/L. 3) It is suggested that vitamin C may improve selenium bioavailability when this element consumption is low or not adequate; however, it should be stressed that too high intake of vitamin C may be undesirable when selenium intake sufficient.
