Sulodexide counteracts endothelial dysfunction induced by metabolic or non-metabolic stresses through activation of the autophagic program.

OBJECTIVE: Endothelial dysfunction (ED) predisposes to venous thrombosis (VT) and post-thrombotic syndrome (PTS), a long-term VT-related complication. Sulodexide (SDX) is a highly purified glycosaminoglycan with antithrombotic, pro-fibrinolytic and anti-inflammatory activity used in the treatment of chronic venous disease (CVD), including patients with PTS. SDX has recently obtained clinical evidence in the "extension therapy" after initial-standard anticoagulant treatment for the secondary prevention of recurrent deep vein thrombosis (DVT). Herein, we investigated how SDX counteracts ED. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVEC) were used. Metabolic and non metabolic-induced ED was induced by treating with methylglyoxal (MGO) or irradiation (IR), respectively. Bafilomycin A1 was used to inhibit autophagy. The production of reactive oxygen species (ROS), tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, Real-time PCR and Western blot analysis for gene and protein expression were used. RESULTS: SDX protected HUVEC from MGO- or IR-induced apoptosis by counteracting the activation of the intrinsic and extrinsic caspase cascades. The cytoprotective effects of SDX resulted from a reduction in a) ROS production, b) neo-synthesis and release of pro-inflammatory cytokines (TNFα, IL1, IL6, IL8), c) DNA damage induced by MGO or IR. These effects were reduced when autophagy was inhibited. CONCLUSIONS: Data herein collected indicate the ability of SDX to counteract ED induced by metabolic or non-metabolic stresses by involving the intracellular autophagy pathway. Our experience significantly increases the knowledge of the mechanisms of action of SDX against ED and supports the use of SDX in the treatment of CVD, PTS and in the secondary prevention of recurrent DVT.

Allergic conjunctivitis: a cross-sectional study.

BACKGROUND: Ocular allergy is a common disease in daily practice. OBJECTIVES: A cross-sectional study was conducted to evaluate clinical aspects of and therapeutic approaches to ocular allergy in Italy. METHODS: Of the 3685 patients affected by ocular allergy and enrolled by 304 ophthalmologists nationally, 3545 were eligible to be included in the statistical analysis. A questionnaire was administered in office to record demographic data, comorbidities, trigger factors, number of conjunctivitis episodes, and past treatments. Signs and symptoms were graded according to their severity, frequency, and duration. RESULTS: Mean age of enrolled patients was 38 ± 19 years. Seasonal allergic conjunctivitis (55% of patients) was equally distributed among the different age groups, while perennial allergic conjunctivitis (18%) increased with age and vernal keratoconjunctivitis (9%) was more frequent under the age of 18. Itching and redness were reported in 90% and 85%, respectively; lid skin involvement was observed in 22% of cases and keratitis in 11%. Pollen sensitivities were indicated as the most frequent triggers; however, exposure to non-specific environmental conditions, pollutants, and cigarette smoke was frequently reported. Only 35% of patients underwent a diagnostic evaluation of specific allergic sensitization, with positive allergy tests found in 82% of this subset. With regard to treatment, topical decongestants were used in 43% of patients, corticosteroids in 41%, antihistamines in 29%, systemic antihistamines in 27%, and mast cell stabilizers in 15%. CONCLUSION: This survey provided useful epidemiological information regarding the clinical characteristics and treatment options of a large cohort of patients affected by different forms of ocular allergy. CLINICAL RELEVANCE: An understanding of ocular allergic disease, its incidence, demographics, and treatment paradigms provides important information towards understanding its pharmacoeconomics and burden on the national health system.

[Topical treatment of vaginal infections by the association of metronidazole-clotrimazole]. / L'attualità del trattamento topico delle infezioni vaginali con l'associazione metronidazolo-clotrimazolo.

Vaginal infections are one of the most gynecological frequently diseases observed and with significant psychological and clinical implications. Their pharmacological treatment may require different options, but even today, scientific literature and international guidelines recommend the use of metronidazole for the treatment of bacterial vaginosis (BV) and trichomoniasis, and the clotrimazole for fungal infections from Candida (VVC). In this contest, the topical association of clotrimazole-metronidazole (vaginal pessaries, cream and douches) represents a current reference treatment for these types of infections with a number of important pharmacological properties. This combination allows an effective activity against to a broad spectrum of pathogens (bacterial, fungal and protozoan), a feature particularly relevant in the case of mixed infections. Furthermore it allows a synergistic action that improve the therapeutic abilities of the individual components, a reduction of the spontaneous resistance of some microorganisms and the activity against symptoms and signs of vaginal inflammation with maintaining the vaginal ecosystem, since they have no activity against endogenous lactobacilli. Finally, recent studies have shown the ability of the topical association of metronidazole-clotrimazole to inhibit the in vitro phenotypic switching of Candida albicans, and its effectiveness against Recurrent Vulvovaginal Candidiasis (RVVC).

The IFN-alpha-inducing capacity of Sendai Virus in human leukocytes is independent from the hemagglutinating and hemolytic activities and from the viral proteins of the Sendai Virus preparations.

About 30 Sendai Virus (SV) preparations, examined for their capacity to induce natural human interferon alpha from fresh human leukocytes (Le-IFN-alpha) of healthy donors, were characterized for hemagglutinating (HA) and hemolytic (HemA) activities and for SDS-PAGE proteic pattern. The SV preparations were produced by a single passage or by serial propagations through eggs in different conditions of multiplicity of infection (m.o.i.). The produced SV subpopulations showed variable IFN-inducing capacity, the values of which are distributed over a 6-fold range resembling a Gaussian distribution. No detectable difference was observed comparing the SV preparation obtained by serial propagations with those obtained by a single passage. The variability of the measured HA activity and HemA activity and as well as the SDS-PAGE proteic pattern of the SV preparations did not correlate with the induced amount of IFN per cell. In the same experimental conditions to produce Le-IFN-alpha, u.v.-treated SV preparations were unable to induce interferon depending on the u.v.-treatment. So it can be concluded that the distinct nHu-IFN-alpha-inducing capacity of SV subpopulation could be mainly associated with divergent compositions of the viral RNAs rather than with a different contents of viral proteins, among those detectable in SDS-PAGE and those responsible for HA activity and for HemA activity.

Variations in the interferon-inducing capacity of Sendai virus subpopulations.

Several Sendai virus (SV) preparations, propagated through eggs from the same viral seed, exhibited significantly different capacities to induce interferon (IFN) in human leukocytes (nHu-IFN-alpha). The amount of induced IFN and the numbers of SV IFN-inducing particles (IFP) per cell were determined in dose (SV concentration)-response (IFN yield) curves, kinetics of IFN production, and coinfection experiments with SV preparations that differed in IFN-inducing capacities. The possible role of leukocyte sources and the quality of the SV preparations and of allantoic fluids in affecting the IFN-inducing capacity of SV populations also were tested. The data indicate that different SV preparations induced different amounts of IFN per leukocyte and contained approximately the same concentrations of IFN-inducing particles. There was no apparent correlation between the IFN induced and the apparent quality of the SV preparations examined (EID50, HAU, and EID50/HAU). The leukocyte source and the allantoic impurities of SV preparations did not have any influence on the magnitude of the IFN yield. Similar shapes of the dose-response curves, the absence of any lag in the kinetics of IFN production, and the ability of a viral preparation that induced low yields of IFN to suppress partially a high-yielding inducer suggest that a common mechanism of induction is always present. Hence, propagation of SV in eggs from low multiplicity produced virus stocks that differed significantly in their inducing capacity, suggesting that genetic bottlenecks may be operative.

Sendai virus and herpes virus type 1 induce apoptosis in human peripheral blood mononuclear cells.

Recent reports suggest that several viruses, besides human immunodeficiency virus, induce apoptosis in infected cells. We report here that Sendai virus or Herpes simplex virus type 1 (HSV-1), two potent inducers of interferon-alpha, caused cell death in a consistent number of human peripheral blood mononuclear cells. A careful analysis of infected cells by different techniques, such as optical and electron microscopy, DNA agarose gel electrophoresis, and cytofluorimetric analysis of DNA content, showed that cell death was of apoptotic type. Sendai virus was more apoptogenic than HSV-1, and it was further studied to understand the mechanism(s) by which it induced apoptosis. Physical (uv and heat) and chemical (beta-propiolactone) inactivation reduced or abolished the apoptogenic power of Sendai virus. The use of a novel technique, which allows the study of mitochondrial membrane potential (MMP) in intact cells by flow cytometry, showed that a decrease of MMP is concomitant with the appearance of the hypodiploid peak. These results suggest that Sendai virus and HSV-1 can be added to the list of viruses causing apoptosis, which appears to be a general mechanism occurring during viral infection.

Potentiation of interferon-alpha in vitro antiviral activity by interferon-gamma is not abrogated by antibody to interferon-gamma.

The addition of minute amounts of recombinant interferon-gamma (IFN-gamma) to recombinant IFN-alpha 2 significantly enhances its antiviral activity. Although this potentiating action of IFN-gamma is destroyed by treatment with acid, it is not significantly affected by treatment with antibodies to IFN-gamma, suggesting that IFN-gamma molecules can interact with cells to potentiate IFN-alpha activity, even in the presence of neutralizing antibodies. Implications of this phenomenon are multifaceted, including the possibility that other IFN activities can be altered synergistically by combinations of different IFN types, even in the presence of specific antibodies, which have been described to occur in vivo in several pathological conditions.

Acid lability is not an intrinsic property of interferon-alpha induced by HIV-infected cells.

Human immunodeficiency virus (HIV)-infected cells induce acid-labile interferon-alpha (al-IFN-alpha) in cultures of mononuclear cells from peripheral human blood. We have investigated the physiochemical properties of such preparations to elucidate the reasons for acid-lability of this IFN. Al-IFN-alpha is a mixture of both glycosylated and unglycosylated molecules as shown by separation on Concanavalin-A Sepharose. Acid-lability is associated only with glycosylated molecules. Upon chromatography of the glycosylated fraction on Sepharose coupled to IFN-alpha-specific antibody, the portion of the IFN that is retained and eluted with guanidine-HCl is acid-stable, whereas the excluded antiviral activity is acid-labile, and is partially neutralized by antibodies to either IFN-alpha or IFN-gamma. Also, upon further purification of the unglycosylated fraction on the same antibody column, all antiviral activity remains indistinguishable from conventional IFN-alpha. Reconstitution experiments showed that glycosylated material excluded from the anti-IFN-alpha column potentiates antiviral activity of the IFN that is specifically retained by the column. This potentiation is abolished by acid treatment. Similar results are obtained with al-IFN-alpha from the serum of acquired immunodeficiency syndrome (AIDS) patients, indicating that its acid-lability is also the consequence of an acid-labile component that is capable of enhancing the antiviral activity. The potentiation of antiviral activity obtained by combining recombinant preparations of IFN-alpha and IFN-gamma suggests that the cooperating molecule is IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of glycosilation in the susceptibility of "acid labile" interferon alpha to acid treatment.

Mononuclear cells from blood of healthy donors produce acid-labile interferon (IFN) alpha when stimulated with HIV-infected cells. A large proportion of this IFN appears to be glycosilated, as treatment with neuraminidase causes a shift of the isoelectric point (IP) from pH = 5.2-5.4 to pH = 5.8-6.2. To assess the role of glycosilation in determining the instability of antiviral activity after exposure to acid (pH lower than 4) peripheral blood mononuclear cells (PBMC) were induced to produce IFN with HIV-infected cells in the presence of tunicamycin, an inhibitor of glycosilation. The IFN produced under such experimental conditions (tu-IFN) was acid-stable. Tu-IFN was compared to a standard acid-labile IFN by affinity chromatography on Con A-sepharose. The elution pattern showed that tu-IFN does not bind to the gel, whereas the acid-labile IFN is eluted in two fractions, one unbound, which is stable at pH2, and one bound, which retains the initial acid-lability. These results suggest that acid-labile IFN alpha is largely glycosilated, and that the presence of glycosilated molecules contribute to render the IFN molecule unstable at acidic pH. It is to be determined whether some glycosilated molecule present in the IFN preparation, or glycosilation of the IFN molecule per se, is responsible for acid-lability of the antiviral activity.

Membrane interactions involved in the induction of acid labile interferon alpha.

Human normal peripheral blood mononuclear cells (PBMC) produce acid-labile interferon (IFN) alpha when stimulated in vitro with HIV-infected cells fixed with glutaraldehyde. The cells responsible for IFN production are mainly B lymphocytes. The present study was aimed to further elucidate the cellular source of this IFN and to analyze the membrane interactions involved in the induction process. To this purpose PBMC were stimulated with inducers of acid labile IFN alpha in the presence or absence of a panel of monoclonal antibodies (MoAbs) against antigens of the lymphocyte membrane, namely HLA Class I and II and CD4. The results indicate that both HLA Class II and CD4 antigens are involved in the induction process. Conversely B cell lines seem capable of producing conventional alpha IFN but they fail to produce acid labile IFN alpha even in the presence of cooperating CD4 positive T cell lines. Furthermore PBMC cultured for more than 20 hours prior to stimulation lose the ability to produce acid labile IFN alpha, while remaining fully capable of producing conventional IFN alpha and gamma. It remains to be established whether this phenomenon reflects the disappearance of some membrane structure necessary for acid labile IFN alpha induction, or whether it is due to some early appearing functional alteration of B cells.