The Ruthenium Complex cis-(Dichloro) Tetraammineruthenium(III) Chloride Presents Immune Stimulatory Activity on Human Peripheral Blood Mononuclear Cells

Ruthenium compounds in general are well suited for medicinal applications. They have been investigated as immunosuppressants, nitric oxide scavengers, antimicrobial agents, and antimalarials. The aim of this study is to evaluate the immunomodulatory activity of cis-(dichloro) tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) on human peripheral blood mononuclear cells (PBMC). The cytotoxic studies performed here revealed that the ruthenium( III) complex presents a cytotoxic activity towards normal human PBMC, only at very high concentration. Results also showed that cis-[ RuCl(2)(NH(3))(4)] Cl presents a dual role on PBMC stimulating proliferation and interleukin-2 (IL-2) production at low concentration and inducing cytotoxicity, inability to proliferate, and inhibiting IL-2 production at high concentration. The noncytotoxic activity of cis-[RuCl(2)(NH(3))(4)] Cl at low concentration towards PBMC, which correlates with the small number of annexin V positive cells and also the absence of DNA fragmentation, suggest that this compound does not induce apoptosis on PBMC. For the first time, we show that, at low concentration (10-100 mu g L(-1)), the cis-[ RuCl(2)(NH(3))(4)] Cl compound induces peripheral blood lymphocytes proliferation and also stimulates them to IL-2 production. These results open a new potential applicability of ruthenium(III) complexes as a possible immune regulatory compound acting as immune suppressor at high concentration and as immune stimulator at low concentration.

Volume-activated chloride channels in mice Leydig cells

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume- activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of similar to 75 mOsm. These currents display the typical biophysical signature of volume- activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(-)>Cl(-)>F(-)). Staurosporine (200 nM) did not block the activation of I(Cl), swell. The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 mu M), SITS (200 mu M), ATP (500 mu M), pyridoxalphosphate-6- azophenyl-2`,4`-disulfonate (PPADS; 100 mu M), and Suramin (10 mu M) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(d)(0) and fractional distance of the binding site (delta) of 334 mu M and 47%, 880 mu M and 35%, 2,100 mu M and 49%, 188 mu M and 27%, and 66.5 mu M and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that ICl, swell in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Physiological implications of the regulation of vacuolar H+-ATPase by chloride ions

Vacuolar H+-ATPase is a large multi-subunit protein that mediates ATP-driven vectorial H+ transport across the membranes. It is widely distributed and present in virtually all eukaryotic cells in intracellular membranes or in the plasma membrane of specialized cells. In subcellular organelles, ATPase is responsible for the acidification of the vesicular interior, which requires an intraorganellar acidic pH to maintain optimal enzyme activity. Control of vacuolar H+-ATPase depends on the potential difference across the membrane in which the proton ATPase is inserted. Since the transport performed by H+-ATPase is electrogenic, translocation of H+-ions across the membranes by the pump creates a lumen-positive voltage in the absence of a neutralizing current, generating an electrochemical potential gradient that limits the activity of H+-ATPase. In many intracellular organelles and cell plasma membranes, this potential difference established by the ATPase gradient is normally dissipated by a parallel and passive Cl- movement, which provides an electric shunt compensating for the positive charge transferred by the pump. The underlying mechanisms for the differences in the requirement for chloride by different tissues have not yet been adequately identified, and there is still some controversy as to the molecular identity of the associated Cl--conducting proteins. Several candidates have been identified: the ClC family members, which may or may not mediate nCl-/H+ exchange, and the cystic fibrosis transmembrane conductance regulator. In this review, we discuss some tissues where the association between H+-ATPase and chloride channels has been demonstrated and plays a relevant physiologic role.

Fed-batch cultivation of Arthrospira (Spirulina) platensis: Potassium nitrate and ammonium chloride as simultaneous nitrogen sources

Arthrospira platensis was cultivated in minitanks at 13 klux, using a mixture of KNO(3) and NH(4)Cl as nitrogen source. Fed-batch daily supply of NH(4)Cl at exponentially-increasing feeding rate allowed preventing ammonia toxicity and nitrogen deficiency, providing high maximum cell concentration (X(m)) and high-quality biomass (21.85 mg chlorophyll g cells(-1); 20.5% lipids; 49.8% proteins). A central composite design combined to response surface methodology was utilized to determine the relationships between responses (X(m), cell productivity and nitrogen-to-cell conversion factor) and independent variables (KNO(3) and NH(4)Cl concentrations). Under optimum conditions (15.5 mM KNO3; 14.1 mM NH(4)Cl), X(m) was 4327 mg L(-1), a value almost coincident with that obtained with only 25.4 mM KNO(3), but more than twice that obtained with 21.5 mM NH(4)Cl. A 30%-reduction of culture medium cost can be estimated when compared to KNO(3)-batch runs, thus behaving as a cheap alternative for the commercial production of this cyanobacterium. (C) 2010 Elsevier Ltd. All rights reserved.

Influence of ammonium chloride feeding time and light intensity on the cultivation of Spirulina (Arthrospira) platensis

This study dealt with the influence of both the feeding time and light intensity on the fed-batch culture of the cyanobacterium Spirulina (Arthrospira) platensis using ammonium chloride as a nitrogen source. For this purpose, a 2 2 plus star central composite experimental design combined with response surface methodology was employed, and the maximum cell concentration (X-m), the cell productivity (P-X), and the yield of biomass on nitrogen (Y-X/N) were selected as the response variables. The optimum values of X-m (1,833 mgL(-1)) and Y-X/N (5.9 gg(-1)) estimated by the model at light intensity of 13 klux and feeding time of 17.2 days were very close to those obtained experimentally under these conditions (X-m = 1,771 +/- 41 mg L-1; Y-X/N = 5.7 +/- 0.17 gg(-1)). The cell productivity was a decreasing function of the ammonium chloride feeding time and a quadratic function of the light intensity. The protein and lipid contents of dry biomass collected at the end of cultivations were shown to decrease with increasing light intensity.

The Ruthenium Complex cis-(Dichloro)tetraammineruthenium(III) Chloride Presents Selective Cytotoxicity Against Murine B Cell Lymphoma (A-20), Murine Ascitic Sarcoma 180 (S-180), Human Breast Adenocarcinoma (SK-BR-3), and Human T Cell Leukemia (Jurkat) Tumor Cell Lines

The aim of present study was to verify the in vitro antitumor activity of a ruthenium complex, cis-(dichloro)tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) toward different tumor cell lines. The antitumor studies showed that ruthenium(III) complex presents a relevant cytotoxic activity against murine B cell lymphoma (A-20), murine ascitic sarcoma 180 (S-180), human breast adenocarcinoma (SK-BR-3), and human T cell leukemia (Jurkat) cell lines and a very low cytotoxicity toward human peripheral blood mononuclear cells. The ruthenium(III) complex decreased the fraction of tumor cells in G0/G1 and/or G2-M phases, indicating that this compound may act on resting/early entering G0/G1 cells and/or precycling G2-M cells. The cytotoxic activity of a high concentration (2 mg mL(-1)) of cis-[RuCl(2)(NH(3))(4)]Cl toward Jurkat cells correlated with an increased number of annexin V-positive cells and also the presence of DNA fragmentation, suggesting that this compound induces apoptosis in tumor cells. The development of new antineoplastic medications demands adequate knowledge in order to avoid inefficient or toxic treatments. Thus, a mechanistic understanding of how metal complexes achieve their activities is crucial to their clinical success and to the rational design of new compounds with improved potency.

Mineral trioxide aggregate with or without calcium chloride in pulpotomy

The aim of this study was to evaluate the reaction of the pulp tissue against mineral trioxide aggregate (MTA) with or without 10% calcium chloride (CaCl2). Pulpotomies were performed in 4 canines and 8 premolars of two 8-month-old dogs. MTA with or without CaCl2 was applied on the pulp tissue. The animals were killed after 90 days, and the specimens were processed for the microscopic analysis. Pulp tissue response was similar for MTA with and without CaCl2 Pulp vitality was present in all specimens, along with pulp repair with formation of mineralized tissue bridging. The addition of CaCl2 to MTA did not change its biologic properties in formation of mineralized barrier after pulpotomy.

Effects of Cobalt Chloride in Junctional Epithelium and Reduced Enamel Epithelium of the Rat Maxillary First Molar

Cobalt is one of the main components of cast metal alloys broadly used in dentistry. It is the constituent of 45 to 70% of numerous prosthetic works. There are evidences that metal elements cause systemic and local toxicity. The purpose of the present study was to evaluate the effects of cobalt on the junctional epithelium and reduced enamel epithelium of the first superior molar in rats, during lactation. To do this, 1-day old rats were used, whose mothers received 300mg of cobalt chloride per liter of distilled water in the drinker, during lactation. After 21 days, the rat pups were killed with an anesthetic overdose. The heads were separated, fixed in ""alfac"", decalcified and embedded in paraffin. Frontal sections stained with hematoxylin and eosin were employed. Karyometric methods allowed to estimate the following parameters: biggest, smallest and mean diameters, D/d ratio, perimeter, area, volume, volume/area ratio, eccentricity, form coefficient and contour index. Stereologic methods allow to evaluate: cytoplasm/nucleus ratio, cell and cytoplasm volume, cell number density, external surface/basal membrane ratio, thickness of the epithelial layers and surface density. All the collected data were subjected to statistic analysis by the non-parametric Wilcoxon-Mann-Whitney test. The nuclei of the studied tissues showed smaller values after karyometry for: diameters; perimeter, area, volume and volume/area ratio. Stereologically, it was observed, in the junctional epithelium and in the reduced enamel epithelium, smaller cells with scarce cytoplasm, reflected in the greater number of cells per mm3 of tissue. In this study, cobalt caused epithelial atrophy, indicating a direct action on the junctional and enamel epithelium.

Pulp tissue from primary teeth: new source of stem cells

SHED (stem cells from human exfoliated deciduous teeth) represent a population of postnatal stem cells capable of extensive proliferation and multipotential differentiation. Primary teeth may be an ideal source of postnatal stem cells to regenerate tooth structures and bone, and possibly to treat neural tissue injury or degenerative diseases. SHED are highly proliferative cells derived from an accessible tissue source, and therefore hold potential for providing enough cells for clinical applications. In this review, we describe the current knowledge about dental pulp stem cells and discuss tissue engineering approaches that use SHED to replace irreversibly inflamed or necrotic pulps with a healthy and functionally competent tissue that is capable of forming new dentin.

Effects of light-curing time on the cytotoxicity of a restorative composite resin on odontoblast-like cells

This in vitro study evaluated the cytotoxicity of an experimental restorative composite resin subjected to different light-curing regimens. METHODS: Forty round-shaped specimens were prepared and randomly assigned to four experimental groups (n=10), as follows: in Group 1, no light-curing; in Groups 2, 3 and 4, the composite resin specimens were light-cured for 20, 40 or 60 s, respectively. In Group 5, filter paper discs soaked in 5 µL PBS were used as negative controls. The resin specimens and paper discs were placed in wells of 24-well plates in which the odontoblast-like cells MDPC-23 (30,000 cells/cm²) were plated and incubated in a humidified incubator with 5% CO2 and 95% air at 37ºC for 72 h. The cytotoxicity was evaluated by the cell metabolism (MTT assay) and cell morphology (SEM). The data were analyzed statistically by Kruskal-Wallis and Mann-Whitney tests (p<0.05). RESULTS: In G1, cell metabolism decreased by 86.2%, indicating a severe cytotoxicity of the non-light-cured composite resin. On the other hand, cell metabolism decreased by only 13.3% and 13.5% in G2 and G3, respectively. No cytotoxic effects were observed in G4 and G5. In G1, only a few round-shaped cells with short processes on their cytoplasmic membrane were observed. In the other experimental groups as well as in control group, a number of spindle-shaped cells with long cytoplasmic processes were found. CONCLUSION: Regardless of the photoactivation time used in the present investigation, the experimental composite resin presented mild to no toxic effects to the odontoblast-like MDPC-23 cells. However, intense cytotoxic effects occurred when no light-curing was performed.

Determination of the maximum inhibitory dilution of cetylpyridinium chloride-based mouthwashes against staphylococcus aureus: an in vitro study

The aim of this in vitro study was to determine the maximum inhibitory dilution (MID) of four cetylpyridinium chloride (CPC)-based mouthwashes: CPC+Propolis, CPC+Malva, CPC+Eucaliptol+Juá+Romã+Propolis (Natural Honey®) and CPC (Cepacol®), against 28 Staphylococcus aureus field strains, using the agar dilution method. Decimal dilutions ranging from 1/10 to 1/655,360 were prepared and added to Mueller Hinton Agar. Strains were inoculated using Steers multipoint inoculator. The inocula were seeded onto the surface of the culture medium in Petri dishes containing different dilutions of the mouthwashes. The dishes were incubated at 37ºC for 24 h. For readings, the MID was considered as the maximum dilution of mouthwash still capable of inhibiting microbial growth. The obtained data showed that CPC+Propolis had antimicrobial activity against 27 strains at 1/320 dilution and against all 28 strains at 1/160 dilution, CPC+Malva inhibited the growth of all 28 strains at 1/320 dilution, CPC+Eucaliptol+Juá+Romã+Propolis inhibited the growth of 2 strains at 1/640 dilution and all 28 strains at 1/320 dilution, and Cepacol® showed antimicrobial activity against 3 strains at 1/320 dilution and against all 28 strains at 1/160 dilution. Data were submitted to Kruskal-Wallis test, showing that the MID of Cepacol® was lower than that determined for the other products (p<0.05). In conclusion, CPC-mouthwashes showed antimicrobial activity against S. aureus and the addition of other substances to CPC improved its antimicrobial effect.

Repair of critical-size defects with autogenous periosteum-derived cells combined with bovine anorganic apatite/collagen: an experimental study in rat calvaria

The aim of this study was to evaluate the bone repair using autogenous periosteum-derived cells (PDC) and bovine anorganic apatite and collagen (HA-COL). PDC from Wistar rats (n=10) were seeded on HA-COL discs and subjected to osteoinduction during 6 days. Critical-size defects in rat calvarias were treated with blood clot (G1), autogenous bone (G2), HA-COL (G3) and HA-COL combined with PDC (G4) (n=40), and then analyzed 1 and 3 months after surgeries. Radiographic analysis exhibited no significant temporal change. G1 and G2 had discrete new marginal bone, but the radiopacity of graft materials in G2, G3 and G4 impaired the detection of osteogenesis. At 3 months, histopathological analysis showed the presence of ossification islets in G1, which was more evident in G2, homogeneous new bone around HA-COL in G3 and heterogeneous new bone around HA-COL in G4 in addition to moderate presence of foreign body cells in G3 and G4. Histomorphometric analysis showed no change in the volume density of xenograft (p>0.05) and bone volume density in G2 was twice greater than in G1 and G4 after 3 months (p<0.05), but similar to G3. The PDC did not increase bone formation in vivo, although the biomaterial alone showed biocompatibility and osteoconduction capacity.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results

PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

Biological activity of metal-edds (ethylenediaminedisuccinate) complexes in K562 and PBMC cells

The effect of S,S-ethylenediaminedisuccinic acid (edds) on the quenching of metal-catalyzed (metal = Mn, Fe, Co, Ni, Cu, Zn) oxidation of ascorbic acid was tested in vitro via oxidation of the fluorescent probe 1,2,3-dihydrorhodamine dihydrochloride. The pro-oxidant activity of iron was not fully suppressed, even at a four-fold molar excess of the ligand. The effect of serum on the toxicity to peripheral blood mononuclear cells (PBMC) and K562 cells was investigated. The cytotoxic effect of Fe-edds was abrogated in the presence of Trolox or serum proteins. The probable pathways of cell toxicity were investigated through blocking of the monocarboxylate transporters (MCT) in association with cell cycle studies by flow cytometry. Cells treated with metal complexes and alpha-cyano-4-hydroxycinnamic acid, a known MCT inhibitor, showed recovery of viability, suggesting that MCT proteins may be involved in the internalization of metal-edds complexes. The free acid induced cell cycle arrest in G0/G1 (PBMC) and S (K562) phases, suggesting direct DNA damage or interference in DNA replication.

Protector mechanisms of the association between gastroesophageal reflux disease and asthma: experimental study in rats

BACKGROUND: It is well known the association between gastroesophageal reflux disease and asthma. The hyperreactivity of the airways is a characteristic of an asthmatic. Many studies associate the increase of the airways reactivity with gastroesophageal reflux disease. AIM: In this study we have evaluated the effect of the intraluminal exposition to gastric juice of trachea on the reactivity to methacholine from rats submitted to a pulmonary allergic inflammation. METHODS: Group of rats were sensitized and challenged with ovalbumin. After 24 hours the animals were sacrificed, and their tracheae were removed to be cultured with gastric juice. The gastric juice was obtained from a donor rat. Subsequently the segments were placed into plastic plates with RPMI-1640 for incubation, under suitable atmosphere and time. After the period of incubation the segments were put into chambers for the analysis of the contractile response to methacholine. RESULTS: We observed reduction in the contractile response of trachea cultured with gastric juice from allergic rats. This result was confirmed by the pharmacological treatments with compound 48/80 and dissodium cromoglicate (mast cells blockade), L-NAME (nitric oxide inhibitor, NO), capsaicin (neuropeptides depletion) and indomethacin (ciclooxigenase inhibitor). CONCLUSIONS: Our results highlight to the existence of a complex interaction between pulmonary allergy and gastric juice in the airways. The involvement of the non-adrenergic non-cholinergic system, NO, prostanoids and mast cells are directly related to this interaction. We suggest that the reduced contractile response observed in vitro may represent a protector mechanism of the airways. Despite its presence in the human body it can not be observed due to the predominant effects of excitatory the non-adrenergic non-cholinergic system.