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1.
Diagnóstico (Perú) ; 49(4): 177-179, oct.-dic. 2010. graf
Article in Spanish | LILACS, LIPECS | ID: lil-590810

ABSTRACT

La carga de las infecciones del tracto urinario (ITU) es alta en el mundo entero. Debido a que los tratamientos causales de estas infecciones frecuentemente recurrentes tienen límites sustanciales, las medidas preventivas merecen la prioridad. Entre ellas, la inmunoestimulación con extractos bacterianos ha demostrado que previene las ITUs y que es muy segura. Esta revisión enfoca el inmunoestimulante OM-89, un extracto liofilizado de Escherichia coli producido mediante biotecnología, formulado para la administración por vía oral a partir de este germen frecuentemente responsable de las ITU, y cubrirá sus efectos farmacológicos y clínicos con respecto a eficacia y seguridad. Se pondrá énfasis especial en los estudios clínicos realizados en niños, adultos y pacientes especiales así como en un estudio que evaluó el efecto de OM-89 sobre la calidad de vida de los pacientes.


Subject(s)
Anti-Bacterial Agents/pharmacology , Urologic Diseases/immunology , Urologic Diseases/therapy , Escherichia coli
2.
Diabetes ; 59(10): 2637-45, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20627932

ABSTRACT

OBJECTIVE: Calcium dobesilate (CaD) has been used in the treatment of diabetic retinopathy in the last decades, but its mechanisms of action are not elucidated. CaD is able to correct the excessive vascular permeability in the retina of diabetic patients and in experimental diabetes. We investigated the molecular and cellular mechanisms underlying the protective effects of CaD against the increase in blood-retinal barrier (BRB) permeability induced by diabetes. RESEARCH DESIGN AND METHODS: Wistar rats were divided into three groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with CaD. The BRB breakdown was evaluated using Evans blue. The content or distribution of tight junction proteins (occludin, claudin-5, and zonula occluden-1 [ZO-1]), intercellular adhesion molecule-1 (ICAM-1), and p38 mitogen-activated protein kinase (p38 MAPK) was evaluated by Western blotting and immunohistochemistry. Leukocyte adhesion was evaluated in retinal vessels and in vitro. Oxidative stress was evaluated by the detection of oxidized carbonyls and tyrosine nitration. NF-κB activation was measured by enzyme-linked immunosorbent assay. RESULTS: Diabetes increased the BRB permeability and retinal thickness. Diabetes also decreased occludin and claudin-5 levels and altered the distribution of ZO-1 and occludin in retinal vessels. These changes were inhibited by CaD treatment. CaD also inhibited the increase in leukocyte adhesion to retinal vessels or endothelial cells and in ICAM-1 levels, induced by diabetes or elevated glucose. Moreover, CaD decreased oxidative stress and p38 MAPK and NF-κB activation caused by diabetes. CONCLUSIONS: CaD prevents the BRB breakdown induced by diabetes, by restoring tight junction protein levels and organization and decreasing leukocyte adhesion to retinal vessels. The protective effects of CaD are likely to involve the inhibition of p38 MAPK and NF-κB activation, possibly through the inhibition of oxidative/nitrosative stress.


Subject(s)
Calcium Dobesilate/pharmacology , Diabetes Mellitus, Experimental/physiopathology , Diabetic Retinopathy/physiopathology , Leukocytes/physiology , Retina/physiology , Tight Junctions/physiology , Animals , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/physiology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Claudin-5 , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/prevention & control , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/physiology , Endothelium/drug effects , Endothelium/physiology , Hemostatics/pharmacology , Leukocytes/drug effects , Male , Membrane Proteins/drug effects , Membrane Proteins/metabolism , Occludin , Permeability/drug effects , Phosphoproteins/drug effects , Phosphoproteins/metabolism , Rats , Rats, Wistar , Retina/drug effects , Retina/physiopathology , Tight Junctions/drug effects , Zonula Occludens-1 Protein
3.
Vaccine ; 26(1): 108-18, 2007 Dec 21.
Article in English | MEDLINE | ID: mdl-18063445

ABSTRACT

In this study, we tested two triacylated pseudo-dipeptidic molecules, OM-197-MP-AC and OM-294-BA-MP as candidate adjuvants for allergy vaccines. Both molecules induce human dendritic cell (h-DC) maturation and polarize naïve T cells toward the Th1 type with IFNgamma production. Only OM-294-BA-MP induces IL10 gene expression both in monocyte-derived DCs and CD4+ naïve T cells. Sublingual administration of OM-294-BA-MP plus the antigen enhances tolerance induction in BALB/c mice with established asthma to ovalbumin with an impact on both airways hyperresponsiveness and lung inflammation. Given its Th1/Treg polarizing properties, OM-294-BA-MP is a valid candidate for sublingual allergy vaccines.


Subject(s)
Adjuvants, Immunologic/pharmacology , Desensitization, Immunologic/methods , Dipeptides/pharmacology , Immune Tolerance , T-Lymphocytes, Regulatory/immunology , Th1 Cells/immunology , Administration, Sublingual , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/physiology , Cell Polarity , Dendritic Cells/drug effects , Dendritic Cells/physiology , Female , Humans , Interferon-gamma/biosynthesis , Mice , Mice, Inbred BALB C
4.
Eur J Pharmacol ; 563(1-3): 1-17, 2007 Jun 01.
Article in English | MEDLINE | ID: mdl-17383632

ABSTRACT

Liver or lung metastases usually relapse under chemotherapy. Such life-threatening condition urgently needs new, systemic anticancer compounds, with original and efficient mechanisms of action. In B16 melanoma mice treated with cyclophosphamide, D'Agostini et al. [D'Agostini, C., Pica, F., Febbraro, G., Grelli, S., Chiavaroli, C., Garaci, E., 2005. Antitumour effect of OM-174 and Cyclophosphamide on murine B16 melanoma in different experimental conditions. Int. Immunopharmacol. 5, 1205-1212.] recently found that OM-174, a chemically defined Toll-like receptor(TLR)2/4 agonist, reduces tumor progression and prolongs survival. Here we review 149 articles concerning molecular mechanisms of TLR2/4 agonists, alone or in combination with chemotherapy. It appears that TLR2/4 agonists induce a well controlled tumor necrosis factor-alpha (TNF-alpha) secretion, at plasma levels known to permeabilize neoangiogenic tumor vessels to the passage of cytotoxic drugs. Moreover, TLR2/4 agonists induce inducible nitric oxide synthase (iNOS) expression, and nitric oxide is able to induce apoptosis of chemotherapy-resistant tumor cell clones. Finally, TLR2/4-stimulation activates dendritic cell traffic and its associated tumor-specific, cytotoxic T-cell responses. Therefore, parenteral TLR2/4 agonists seem promising molecules to prolong survival in cancer patients who relapse under chemotherapy.


Subject(s)
Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , Signal Transduction/drug effects , Toll-Like Receptor 2/agonists , Toll-Like Receptor 4/agonists , Animals , Antineoplastic Agents/immunology , Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , BCG Vaccine/immunology , Chemotherapy, Adjuvant , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Drug Resistance, Neoplasm , Enzyme Induction/drug effects , Humans , Immunotherapy/methods , Interleukin-10/metabolism , Lipid A/analogs & derivatives , Lipid A/pharmacology , Lipopolysaccharides/pharmacology , Neoadjuvant Therapy , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms/therapy , Nitric Oxide Synthase Type II/biosynthesis , Recurrence , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/metabolism , Toll-Like Receptor 2/immunology , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolism , Treatment Outcome , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology
5.
Immunobiology ; 211(10): 767-77, 2006.
Article in English | MEDLINE | ID: mdl-17113914

ABSTRACT

Triggering the maturation of dendritic cells (DC) with toll-like receptor (TLR) agonists is a favored strategy for the development of vaccine adjuvants. The triacyl pseudo-dipeptidic agent OM-197-MP-AC mimicking the lipid A structure of endotoxin induces the maturation of human monocyte-derived DC. In this study we investigated the signaling pathway by which this molecule activates DC. The ability of OM-197-MP-AC to induce maturation of human and mouse DC and macrophages was dependent on TLR4, not TLR2. Ovalbumin-specific humoral and T helper cell responses were significantly augmented by OM-197-MP-AC treatment. Taken together these results indicate that OM-197-MP-AC is a TLR4 agonist inducing DC maturation and represents a novel class of vaccine adjuvants devoid of the known pyrogenic effects associated with classical LPS derivatives.


Subject(s)
Antigen-Presenting Cells/immunology , Epitopes/immunology , Lipid A/analogs & derivatives , Lipid A/metabolism , Phospholipids/immunology , Signal Transduction/physiology , Toll-Like Receptor 4/physiology , Acylation , Animals , Antigen-Presenting Cells/metabolism , Cells, Cultured , Dendritic Cells/immunology , Humans , Lipid A/chemical synthesis , Lipid A/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
6.
Int J Radiat Oncol Biol Phys ; 66(5): 1473-80, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17056198

ABSTRACT

PURPOSE: To explore whether antitumor immunoadjuvant OM-174 can stimulate immune cells to produce interferon-gamma (IFN-gamma) and thereby radiosensitize tumor cells. METHODS AND MATERIALS: Splenocytes from BALB/c mice were stimulated by OM-174 at plasma-achievable concentrations (0.03-3 mug/mL), and afterward analyzed for the expression and secretion of IFN-gamma by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Stimulated splenocytes were used as a source of IFN-gamma to radiosensitize hypoxic EMT-6 tumor cells through the cytokine-inducible isoform of nitric oxide synthase (iNOS). RESULTS: OM-174 activated the production of IFN-gamma at high levels that reached 70 ng/mL in normoxia (21% oxygen) and 27 ng/mL in tumor-relevant hypoxia (1% oxygen). This caused up to 2.1-fold radiosensitization of EMT-6 tumor cells, which was associated with the iNOS-mediated production of the radiosensitizing molecule nitric oxide, as confirmed by accumulation of its oxidative metabolite nitrite, Western blot analysis, and reverse transcriptase-polymerase chain reaction. Both iNOS activation and radiosensitization were counteracted by neutralizing antibodies against IFN-gamma. The same mechanism of radiosensitization through the IFN-gamma secretion pathway was identified for IL-12 + IL-18, which are known to mediate IFN-gamma responses. Hypoxia displayed a dual effect on the immune-tumor cell interaction, by downregulating the expression of the IFN-gamma gene while upregulating iNOS at transcriptional level. CONCLUSION: Immunoadjuvant OM-174 is an efficient radiosensitizer of tumor cells through activation of the IFN-gamma secretion pathway in immune cells. This finding indicates a rationale for combining immunostimulatory and radiosensitizing strategies and extends the potential therapeutic applications of OM-174.


Subject(s)
Adjuvants, Immunologic/pharmacology , Interferon-gamma/metabolism , Lipopolysaccharides/pharmacology , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide/biosynthesis , Radiation Tolerance , Animals , Cell Hypoxia/physiology , Cell Line, Tumor , Down-Regulation , Enzyme Activation , Immunity, Cellular , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/genetics , Interleukin-12/metabolism , Interleukin-18/metabolism , Mice , Mice, Inbred BALB C , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/genetics , RNA, Messenger/metabolism , Radiation Tolerance/drug effects , Radiation Tolerance/immunology , Spleen/cytology , Up-Regulation
7.
Am J Ther ; 13(3): 236-47, 2006.
Article in English | MEDLINE | ID: mdl-16772766

ABSTRACT

Ethamsylate (2,5-dihydroxy-benzene-sulfonate diethylammonium salt) is a synthetic hemostatic drug indicated in cases of capillary bleeding. This review covers more than 40 years of intensive clinical and fundamental research with ethamsylate. First, we summarize the large medical literature concerning its clinical efficacy. Of these, well-controlled clinical trials clearly showed the therapeutic efficacy of ethamsylate in dysfunctional uterine bleeding, with the magnitude of blood-loss reduction being directly proportional to the severity of the menorrhagia. Other well-controlled clinical trials showed therapeutic efficacy of ethamsylate in periventricular hemorrhage in very low birth weight babies and surgical or postsurgical capillary bleeding. Second, we review the numerous investigations performed to elucidate the mechanism of action of ethamsylate. Ethamsylate acts on the first step of hemostasis by improving platelet adhesiveness and restoring capillary resistance. Recent studies showed that ethamsylate promotes P-selectin-dependent, platelet adhesive mechanisms. Finally, we compare ethamsylate with other recent hemostatic agents. It is suggested that the place of ethamsylate as a hemostatic agent is that of a mild but well-tolerated drug, particularly useful in dysfunctional uterine bleeding when contraception is not needed.


Subject(s)
Blood Loss, Surgical/prevention & control , Ethamsylate/therapeutic use , Hemorrhage/drug therapy , Hemostatics/therapeutic use , Menorrhagia/drug therapy , Adult , Animals , Child, Preschool , Clinical Trials as Topic , Ethamsylate/adverse effects , Ethamsylate/metabolism , Female , Hemostatics/adverse effects , Hemostatics/metabolism , Humans , Infant, Newborn
8.
BioDrugs ; 20(3): 141-9, 2006.
Article in English | MEDLINE | ID: mdl-16724862

ABSTRACT

Extracts of lysed pathogenic bacteria were developed approximately 4 decades ago as oral vaccines in order to stimulate efficient specific immune and proinflammatory responses in patients experiencing recurrent infections, the ultimate aim being to rid the patient of the pathogen responsible for the infections. OM-89, a lysate of Escherichia coli, is clinically effective in patients who experience recurrent urinary tract infections by activating both innate and adaptive immunity. If immune activation is necessary to combat infectious pathogens, it may appear at first sight to be detrimental in patients with autoimmune diseases. However, OM-89 has also shown clear efficacy in patients with rheumatoid arthritis or with undifferentiated spondyloarthropathies, probably through oral tolerance and the long-term activation of regulatory cells. These phenomena may be explained by a hypothesis that immune exclusion and oral tolerance, both key functions of the gut, may be boosted by adjuvant-like molecules within orally administered OM-89.


Subject(s)
Antigens, Bacterial/administration & dosage , Arthritis, Rheumatoid/therapy , Immune Tolerance , Immunosuppressive Agents/administration & dosage , Urinary Tract Infections/therapy , Animals , Arthritis, Rheumatoid/immunology , Bacteria , Cell Extracts , Escherichia coli/immunology , Humans , Urinary Tract Infections/immunology
9.
Diabetes ; 55(1): 179-85, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16380491

ABSTRACT

The onset of type 1 diabetes in NOD mice is delayed by oral administration of a bacterial extract (OM-85) and can be completely prevented by its intraperitoneal administration. Optimal prevention is observed when starting treatment at 3 or 6 weeks of age, and some effect is still observed with treatment at 10 weeks of age. Using genetically deficient mice and cytokine-neutralizing monoclonal antibodies, we demonstrate here that the therapeutic effect does not involve T-helper type 2 cytokines (interleukin [IL]-4 and -10) but is tightly dependent on transforming growth factor (TGF)-beta. Natural killer T-cells also participate in the therapeutic effect because CD1d(-/-) NOD mice are partially resistant to the protective effect of OM-85. The question remains of the specificity of the protective effect of OM-85, which may include proinflammatory components. It will thus be important to further characterize the molecular components that afford protection from type 1 diabetes. Lipopolysaccharide is excluded, but other Toll-like receptor (TLR) agonists could be involved because OM-85 stimulated dendritic cells and induced TGF-beta production by splenocytes in a TLR-2-, TLR-4-, and MyD88-dependent fashion.


Subject(s)
Cell Extracts/pharmacology , Diabetes Mellitus, Type 1/drug therapy , Diabetes Mellitus, Type 1/metabolism , Killer Cells, Natural/metabolism , Transforming Growth Factor beta/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Aging , Animals , Antigens, CD1/genetics , Bacteria , Cell Extracts/therapeutic use , Diabetes Mellitus, Type 1/pathology , Dose-Response Relationship, Drug , Female , Gene Deletion , Interleukin-4/genetics , Killer Cells, Natural/drug effects , Ligands , Mice , Mice, Inbred NOD , Myeloid Differentiation Factor 88 , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolism , Transforming Growth Factor beta/pharmacology
10.
Treat Endocrinol ; 4(4): 221-32, 2005.
Article in English | MEDLINE | ID: mdl-16053339

ABSTRACT

The incidence of diabetic retinopathy is still increasing in developed countries. Tight glycemic control and laser therapy reduce vision loss and blindness, but do not reverse existing ocular damage and only slow the progression of the disease. New pharmacologic agents that are currently under development and are specifically directed against clearly defined biochemical targets (i.e. aldose reductase inhibitors and protein kinase C-beta inhibitors) have failed to demonstrate significant efficacy in the treatment of diabetic retinopathy in clinical trials. In contrast, calcium dobesilate (2,5-dihydroxybenzenesulfonate), which was discovered more than 40 years ago and is registered for the treatment of diabetic retinopathy in more than 20 countries remains, to our knowledge, the only angioprotective agent that reduces the progression of this disease. An overall review of published studies involving calcium dobesilate (CLS 2210) depicts a rather 'non-specific' compound acting moderately, but significantly, on the various and complex disorders that contribute to diabetic retinopathy. Recent studies have shown that calcium dobesilate is a potent antioxidant, particularly against the highly damaging hydroxyl radical. In addition, it improves diabetic endothelial dysfunction, reduces apoptosis, and slows vascular cell proliferation.


Subject(s)
Calcium Dobesilate/therapeutic use , Diabetic Retinopathy/drug therapy , Animals , Antioxidants , Blood-Retinal Barrier , Calcium Dobesilate/adverse effects , Calcium Dobesilate/pharmacokinetics , Diabetic Retinopathy/prevention & control , Diabetic Retinopathy/surgery , Evidence-Based Medicine , Humans , Oxidative Stress , Rats
11.
Int Immunopharmacol ; 5(7-8): 1205-12, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15914325

ABSTRACT

We report that OM-174, a purified water soluble diphosphorylated and triacetylated lipid A derived from E. coli, is able to reduce tumour progression and to prolong the survival of mice in the B16 melanoma experimental model. At the doses employed in our study OM-174 had an antitumour activity comparable to that of a single high dose of CY. More striking effects were achieved by means of the combination of the two agents in a protocol consisting of a single administration of CY (200 mg/kg) followed by five injections of OM-174 (1 mg/kg). Immunological studies of treated and control mice revealed that the antitumour activity of OM-174, alone or in combination with CY, is mediated by the stimulation of natural killer (NK) and cytotoxic T lymphocyte (CTL) responses as well as by a significant increase in the absolute number of NK1.1, CD4 and CD8 positive cells. OM-174 is thus candidate for association with cytostatic drugs in chemo-immunotherapy protocols.


Subject(s)
Antineoplastic Agents/therapeutic use , Cyclophosphamide/therapeutic use , Lipopolysaccharides/therapeutic use , Melanoma, Experimental/drug therapy , Animals , CD4-CD8 Ratio , Killer Cells, Natural/immunology , Male , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , T-Lymphocytes, Cytotoxic/immunology
12.
Eur J Pharmacol ; 510(1-2): 149-56, 2005 Mar 07.
Article in English | MEDLINE | ID: mdl-15740736

ABSTRACT

Calcium dobesilate reduces vascular endothelial growth factor (VEGF) over-expression in diabetic rat retina, but its effect on intraocular angiogenesis is unknown. Therefore, we tested calcium dobesilate for its in vitro and ex vivo effects on choroidal explant angiogenesis in spontaneously diabetic Goto-Kakizaki (GK) rats. Choroidal explants were cultured in gels of collagen. Budded microvessels numbers and VEGF formation were taken as markers of angiogenesis. Ex vivo studies were performed in GK rats orally given 100 mg/kg/day calcium dobesilate for 10 days. In vitro, calcium dobesilate dose- and time-dependently inhibited both microvessel formation and VEGF production, at concentrations >or=25 mug/ml (i.e. >or=60 microM), with complete inhibition at 100 microg/ml. Oral treatment of diabetic GK rats with calcium dobesilate induced a significant reduction of choroidal angiogenesis ex vivo (38.8% after 3 days of culture). In conclusion, calcium dobesilate inhibited choroidal explant angiogenesis both in vitro and ex vivo. This effect may be due, at least in part, to inhibition of VEGF production. Antiangiogenesis by calcium dobesilate can be involved in its therapeutic benefit in diabetic retinopathy.


Subject(s)
Calcium Dobesilate/pharmacology , Choroidal Neovascularization/prevention & control , Diabetes Mellitus, Type 2/complications , Animals , Choroid/blood supply , Choroid/drug effects , Choroid/metabolism , Choroidal Neovascularization/etiology , Choroidal Neovascularization/metabolism , Dose-Response Relationship, Drug , Female , Male , Rats , Rats, Wistar , Time Factors , Tissue Culture Techniques , Vascular Endothelial Growth Factor A/metabolism
13.
Immunobiology ; 209(1-2): 67-77, 2004.
Article in English | MEDLINE | ID: mdl-15481142

ABSTRACT

Dendritic cells (DC) play a pivotal role in linking innate and adaptive immunity. Only mature DC are able to initiate adaptive immune responses by sensitising naive antigen-specific T cells. For clinical immunotherapeutic applications, safe and efficient clinical grade maturation factors of DC are required. Here, we investigated the impact of OM-197-MP-AC (OM-197), a synthetic lipid A analogue pseudo-dipeptide derived from amino acids linked to three fatty acid chains, on the maturation of human monocyte-derived-DC (Mo-DC) and leukemia-derived DC generated in serum-free conditions. After culture with clinical grade GM-CSF and IL-13, OM-197 at 20 microg/ml efficiently induced CD83+ Mo-DC. In comparison to immature Mo-DC that were derived by culture with GM-CSF and IL-13 only, CD40, CD80, CD86, HLA-ABC and HLA-DR molecules were up-regulated upon OM-197 or LPS treatment similarly. In MLR, OM-197-matured Mo-DC were found to be as potent stimulators as LPS-matured Mo-DC for CD4+ T cell proliferation. No significant difference in IFN-gamma quantification was shown between naive CD4+ T cells stimulated by LPS- or OM-197-Mo-DC suggesting that OM-197-Mo-DC can drive naive T cells towards a Th1 response profile that was mainly independent of IL-12 secretion. Similarly, CD8+ T cells could be efficiently polarized into IFN-gamma-secreting-cells by OM-197-Mo-DC, and activated polyclonal pp65-cytomegalovirus-specific CD8+ T lymphocytes. Finally, myeloid leukemic blasts were able to differentiate in vitro into mature functional DC-like cells upon OM-197 treatment in our culture model. Overall, the in vitro effects of clinical grade adjuvant OM-197, showed that it represents a potent inducer of both normal and leukemic-DC maturation, and is likely a good candidate for adjuvant immunotherapy in DC-based vaccines.


Subject(s)
Dendritic Cells/drug effects , Leukemia, Myeloid/pathology , Phospholipids/pharmacology , Adult , Aged , Cell Differentiation , Culture Media , Culture Media, Serum-Free , Cytokines/biosynthesis , Female , Humans , Male , Middle Aged , Phenotype , T-Lymphocytes/immunology , Tumor Cells, Cultured
14.
Fundam Clin Pharmacol ; 18(4): 423-30, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15312148

ABSTRACT

The hemostatic agent ethamsylate enhances membrane expression of P-selectin in human platelets, but whether this promotes platelet-leukocyte aggregate formation is unknown. Here we investigated this point by flow cytometry determination of human platelet-leukocyte aggregates under basal conditions and after whole-blood perfusion through a damaged rabbit aorta segment. Actions of ethamsylate on adhesive molecules of platelets and leukocytes were investigated in parallel. Under basal conditions, ethamsylate was unable to modify whole-blood platelet-leukocyte aggregation, but following whole-blood perfusion through a damaged vessel, ethamsylate produced a modest, but significant increase in platelet-leukocyte aggregates (48+/-21 and 45+/-26% above control levels at ethamsylate 20 and 40 microm respectively). In isolated leukocyte plasma membranes, 14C-ethamsylate specifically bound up to an amount of 660 pmol/mg protein. Moreover, at concentrations > or =1 microm, ethamsylate induced an important (100-200%) and significant increase in the P-selectin glycoprotein ligand 1 (PSGL-1) fluorescence signal in isolated leukocytes and was unable to significantly modify the percentage of CD11b-positive cells. However, no significant changes in aggregate formation were found when ethamsylate was incubated with isolated leukocytes and blood was reconstituted and perfused. In isolated platelet cell membranes, anti-P-selectin antibody and the anti-integrin RGD-containing pentapeptide (GRDGS) were unable to displace 14C-ethamsylate binding. In conclusion, ethamsylate specifically binds to plasma membranes of leukocytes, enhances membrane PSGL-1 expression and promotes leukocyte-platelet aggregation in whole-blood perfused through a damaged vascular segment. These results together with the previously observed enhancement of platelet P-selectin membrane expression [Thromb. Res. (2002)107:329-335] confirms and extends the view that ethamsylate acts on the first step of hemostasis, by improving platelet homo- and heterotypic adhesiveness.


Subject(s)
Cell Membrane/metabolism , Ethamsylate/pharmacology , Hemostatics/pharmacology , Membrane Glycoproteins/metabolism , Platelet Aggregation/drug effects , Animals , Ethamsylate/metabolism , Flow Cytometry , Hemostatics/metabolism , Humans , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/injuries , Rabbits
15.
Eur J Pharmacol ; 495(2-3): 217-24, 2004 Jul 14.
Article in English | MEDLINE | ID: mdl-15249173

ABSTRACT

Calcium dobesilate stabilizes blood-retinal barrier in patients with diabetic retinopathy and possesses antioxidant properties in the retinas of rats with streptozotocin-induced diabetes, exposed ex vivo to ischemia-reperfusion. Here we investigated the action of calcium dobesilate on retinal albumin leakage in streptozotocin-diabetic rats, together with relevant in vivo retinal antioxidant and permeability markers, i.e., carboxymethyl-lysine-advanced glycation end product (CML-AGE) formation and vascular endothelial cell growth factor (VEGF) overexpression. Twenty days after streptozotocin administration, diabetic rats were treated for 10 days with calcium dobesilate (100 mg/kg/day per os) or vehicle. Retinal albumin leakage, CML-AGE formation, and VEGF overexpression were evaluated by immunohistochemistry of frozen eye sections. Diabetic rats exhibited dramatic increases in: (i) retinal albumin leakage (31% of positive vessels vs. 0.2% in nondiabetic rats, P<0.008), (ii) CML-AGE retinal occurrence (40+/-3% vs. undetectable positive vessels), and (iii) retinal VEGF protein expression (14.6+/-1.1 vs. 3.5+/-0.5 VEGF-positive spots/field, P<10(-4)). Calcium dobesilate significantly reduced: (i) retinal albumin leakage (by 70%, P<0.008), (ii) retinal CML-AGEs contents (by 62%, P<0.008), and (iii) retinal VEGF expression (by 69.4%, P<0.008). In conclusion, calcium dobesilate orally given to diabetic rats markedly reduced retinal hyperpermeability, CML-AGE contents, and VEGF overexpression. These results strongly suggest that calcium dobesilate stabilizes blood-retinal barrier in diabetic retinopathy via an in situ antioxidant action. Further studies in patients are required to confirm such view.


Subject(s)
Albumins/metabolism , Antioxidants/pharmacology , Blood-Retinal Barrier/drug effects , Calcium Dobesilate/pharmacology , Diabetic Retinopathy/prevention & control , Animals , Capillary Permeability/drug effects , Diabetes Mellitus, Experimental/complications , Diabetic Retinopathy/etiology , Diabetic Retinopathy/metabolism , Glycation End Products, Advanced/metabolism , Hemostatics/pharmacology , Immunohistochemistry , Lysine/analogs & derivatives , Lysine/metabolism , Male , Rats , Rats, Wistar , Retina/chemistry , Retina/drug effects , Retina/metabolism , Vascular Endothelial Growth Factor A/biosynthesis
16.
Eur J Pharmacol ; 466(1-2): 199-205, 2003 Apr 11.
Article in English | MEDLINE | ID: mdl-12679157

ABSTRACT

Arachidonic acid was investigated for its vascular permeabilizing potential in the rat peritoneal cavity and for its mechanism of action. The antagonistic potential of antioxidants (vitamin E, vitamin C and troxerutin) was also evaluated. Vascular permeability was equated to the rate of extravasation of Evans blue dye from plasma into the peritoneal cavity. Baseline permeability was linear up to 2 h, with a rate constant (k) of 0.0031+/-0.0007 h(-1). Intravenous arachidonate (from 30 microg/kg to 3 mg/kg) induced an immediate, dose-related and significant increase in permeability (ranging from 80% to 150%), which was comparable to the effect induced by similar doses of serotonin. Aspirin (10 mg/kg) reduced the arachidonate-induced permeability by 75%, but interestingly neither the stable thromboxane A(2) receptor agonist U46619 (prostaglandin H(2) endoperoxide epoxymethane) nor prostacyclin was able to increase peritoneal vascular permeability. In contrast, the permeabilizing action of arachidonic acid was very sensitive to antioxidant agents. Thus, vitamin C and the flavonoid compound troxerutin (100 mg/kg) fully abolished arachidonate-induced permeability, whereas vitamin E had only a partial effect (40-100% inhibition). In conclusion, intravenous administration of arachidonic acid strongly enhanced peritoneal vascular permeability in the rat, apparently via free radical generation. This rat peritoneal model can be used to evaluate the in vivo antinflammatory potential of antioxidant drugs.


Subject(s)
Antioxidants/pharmacology , Arachidonic Acid/pharmacokinetics , Capillary Permeability/drug effects , Hydroxyethylrutoside/analogs & derivatives , Animals , Antioxidants/administration & dosage , Arachidonic Acid/administration & dosage , Ascorbic Acid/administration & dosage , Ascorbic Acid/pharmacology , Capillary Permeability/physiology , Coloring Agents , Disease Models, Animal , Evans Blue , Extravasation of Diagnostic and Therapeutic Materials , Hydroxyethylrutoside/administration & dosage , Hydroxyethylrutoside/pharmacology , Injections, Intravenous , Male , Peritoneal Cavity/pathology , Rats , Rats, Wistar , Reactive Oxygen Species , Time Factors , Vitamin E/administration & dosage , Vitamin E/pharmacology
17.
Eur J Pharmacol ; 466(1-2): 207-12, 2003 Apr 11.
Article in English | MEDLINE | ID: mdl-12679158

ABSTRACT

The hemostatic agent, ethamsylate, inhibits arachidonic acid metabolism by a mechanism independent of cyclooxygenase activity and blocks carrageenan-induced rat paw edema. Here, ethamsylate was investigated for (i) in vivo actions on the free radical-dependent, permeabilizing responses to arachidonic acid and (ii) its antioxidant potential in vitro. Vascular permeability was equated to the extravasation rate of Evans blue from plasma into the rat peritoneal cavity. Antioxidant potential was investigated by classical in vitro tests for superoxide radicals, hydroxyl radicals (OH(.)), and nitric oxide. Intravenous ethamsylate induced a very important and significant reduction of permeability responses to arachidonate, both when given preventively and cumulatively. Thus, (i) ethamsylate significantly reversed arachidonate-induced permeabilization, even at the lowest dose tested (44+/-5% at 10 mg/kg) and (ii) a maximal reversal (about 70%) was reached between 50 and 200 mg/kg ethamsylate. In contrast, ethamsylate (100 mg/kg) was unable to antagonize the vascular permeabilization induced by serotonin (5-HT). In antioxidant assays, ethamsylate showed scavenging properties against hydroxyl radicals generated by the Fenton reaction (H(2)O(2)/Fe(2+)) even at 0.1 microM (-20+/-3%). OH(.) scavenging by ethamsylate reached 42+/-8% at 10 microM and 57+/-7% at 1 mM and was comparable to that of reference compounds (vitamin E, troxerutin, and mannitol). Conversely, ethamsylate was a poor scavenger of superoxide and nitric oxide radicals. In conclusion, intravenous ethamsylate potently antagonized the peritoneal vascular permeabilization induced by arachidonate, an action likely due to its antioxidant properties, particularly against hydroxyl radical. Such a mechanism can explain previous observations that ethamsylate inhibits carrageenan-induced rat paw edema. Whether it also participates in the hemostatic action of ethamsylate deserves further investigation.


Subject(s)
Antioxidants/pharmacology , Arachidonic Acid/pharmacokinetics , Capillary Permeability/drug effects , Ethamsylate/pharmacology , Hydroxyethylrutoside/analogs & derivatives , Peritoneal Cavity/pathology , Animals , Antioxidants/chemistry , Arachidonic Acid/administration & dosage , Capillary Permeability/physiology , Coloring Agents , Ethamsylate/chemistry , Evans Blue , Extravasation of Diagnostic and Therapeutic Materials , Hydroxyethylrutoside/pharmacology , Hydroxyl Radical/chemistry , Hydroxyl Radical/metabolism , Injections, Intravenous , Nitric Oxide/chemistry , Nitric Oxide/metabolism , Rats , Superoxides/chemistry , Superoxides/metabolism , Vitamin E/pharmacology
18.
Vaccine ; 21(9-10): 836-42, 2003 Feb 14.
Article in English | MEDLINE | ID: mdl-12547591

ABSTRACT

The aim of this study was to test the capacity of the novel adjuvant OM-174, a lipid A analog, to induce the migration and the maturation of murine dendritic cells (DC) in vivo, a step which is considered as the initiation of the adaptive immune response. BALB/c mice were injected intravenously or subcutaneously with OM-174. The spleen and popliteal lymph nodes were harvested, and analyzed for DC localization and phenotype. The data presented here clearly show that, OM-174 induces the migration of DC from the periphery to the T cell areas of lymphoid organs, and their maturation into cells expressing high levels of MHC class II and co-stimulatory molecules, with a potency close to that of Escherichia coli lipopolysaccharide (LPS).


Subject(s)
Adjuvants, Immunologic/pharmacology , Dendritic Cells/cytology , Dendritic Cells/drug effects , Lipopolysaccharides/pharmacology , Adjuvants, Immunologic/administration & dosage , Animals , Antigen-Presenting Cells/classification , Antigen-Presenting Cells/cytology , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Cell Differentiation/drug effects , Cell Movement/drug effects , Dendritic Cells/classification , Dendritic Cells/immunology , Female , Injections, Intravenous , Injections, Subcutaneous , Langerhans Cells/classification , Langerhans Cells/cytology , Langerhans Cells/drug effects , Langerhans Cells/immunology , Lipopolysaccharides/administration & dosage , Lymph Nodes/cytology , Lymph Nodes/drug effects , Lymph Nodes/immunology , Mice , Mice, Inbred BALB C , Spleen/cytology , Spleen/drug effects , Spleen/immunology
19.
Thromb Res ; 107(6): 329-35, 2002 Sep 15.
Article in English | MEDLINE | ID: mdl-12565720

ABSTRACT

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p < 0.05). Immunofluorescence showed a sizable (39%) and significant (p < 0.01) enhancement of P-selectin expression at the lowest concentration of ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance.


Subject(s)
Blood Platelets/metabolism , Endothelium, Vascular/metabolism , Ethamsylate/pharmacology , Hemostatics/pharmacology , P-Selectin/metabolism , Platelet Activation/drug effects , Animals , Blood Platelets/drug effects , Cell Line , Cells, Cultured , Flow Cytometry , Humans , Microscopy, Fluorescence , Rats
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