Biomarker discovery by plasma proteomics in familial Brugada Syndrome.

Brugada Syndrome (BS) is a polygenic inherited cardiac disease characterized by life-threatening arrhythmias and high incidence of sudden death. In this study, two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (LC-MS/MS) was used to investigate specific changes in the plasma proteome of BS patients and family members sharing the same gene mutation (SCN5AQ1118X), with the aim to identify novel disease biomarkers. Our data demonstrate that the levels of several proteins were significantly altered in BS patients compared with controls. In particular, apolipoprotein E, prothrombin, vitronectin, complement-factor H, vitamin-D-binding protein, voltage-dependent anion-selective channel protein 3 and clusterin were considerably increased in plasma sample of BS patients, whereas alpha-1-antitrypsin, fibrinogen and angiotensinogen were considerably decreased; moreover, post-translational modifications of antithrombin-III were detected in all affected individuals. On the light of these results, we hypothesize that these proteins might be considered as potential markers for the identification of disease status in BS.

Induction of VEGF and MMP-9 expression by toll-like receptor 2/4 in human endothelial cells infected with Chlamydia pneumoniae.

Matrix metalloproteinases (MMP) are a family of host-derived enzymes involved in the turnover of extracellular matrix (ECM) molecules, and, in particular, it is demonstrated that the 92 KDa gelatinase MMP-9 is often expressed in atherosclerotic plaques by macrophages and smooth muscle cells. Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerosis lesions. In this study, we analyzed the TLR2/TLR4 expression in HUVEC infected with C. pneumoniae and correlated it to the production of VEGF and MMP-9. The results obtained showed an increased VEGF and MMP-9 production correlated with a time-dependent increase in cellular proliferation in HUVEC infected with C. pneumoniae at a multiplicity of infection (MOI) of 2 IFU/cell. HUVEC preincubated with VEGF antibody did not release MMP-9, as detected by zymography assessment and ELISA assay. In addition, we demonstrated that TLR2/TLR4 are expressed in HUVEC infected with viable microorganisms (25% and 17%, respectively), while UV-inactivated microorganisms induced a lesser expression (20% and 11%, respectively) compared to control cells and HUVEC exposed to heat-killed bacteria showed a percentage of TLR-expressing cells similar to the control cells. In addition, the cells preincubated for 60 min with TLR2/TLR4 neutralizing antibodies showed a decrease in C. pneumonia-induced VEGF and MMP-9 production.

Expression of IL-23, VEGF and TLR2/TLR4 on mononuclear cells after exposure to Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative, aerobic bacillus causing infections of the respiratory and other organ systems in susceptible hosts. Although it does not cause pulmonary infections in immunocompetent individuals, P. aeruginosa causes chronic lung infection in individuals with cystic fibrosis and nosocomial pneumonia resulting in significant morbidity and mortality. Exogenous administration of an important P. aeruginosa virulence factor, lipase, present in P. aeruginosa culture supernatant, induces potent mononuclear cell activation leading to the production of numerous proinflammatory cytokines. In particular, P. aeruginosa culture supernatant stimulated increased proliferation of THP-1 cells and monocytes (MN). The addition of culture supernatant to THP-1 cells and MN also induced Interleukin (IL)-23 and vascular endothelial growth factor (VEGF) release in a time-dependent manner. To investigate whether any compounds present in the supernatant lipase contributed to releasing IL-23 and VEGF, the culture supernatant from P. aeruginosa containing lipase was treated with hexadecylsulfonylfluoride (AMSF). The AMSF-treated culture supernatant (CS) did not show any induction on the IL-23 and VEGF release compared to the cells treated with CS without AMSF. We also showed that Toll-like receptors (TLR)2/TLR4 are expressed in THP-1 cells and MN treated with P. aeruginosa CS in a time-dependent fashion. Flow cytometry analysis revealed a higher TLR4 and a lower TLR2 expression at 48 and 72 h of treatment. The treatment of cells with TLR4 neutralizing antibody, and to a lesser extent with TLR2 neutralizing antibody, resulted in a decrease in P. aeruginosa CS-induced IL-23 and VEGF production.

Relationship between Chlamydia pneumoniae infection, inflammatory markers, and coronary heart diseases.

Chlamydia pneumoniae is an intracellular pathogen and an important cause of respiratory tract infections in humans and more recently it has been associated with chronic diseases such as atherosclerosis. Numerous studies have been performed to show the "infectious" hypothesis of atherosclerosis by direct detection of the organisms within atheromatous plaques by seroepidemiological estimation and by animal, immunological and antibiotic interventional studies. In this work we investigated the relation between chronic chlamydial infection, inflammatory markers, Interleukin 7 (IL-7) production and coronary heart disease. We studied 60 patients with coronary heart diseases (CHD), 45 of whom were men and 15 women, with a mean age of 65+/-5 years, and a control group of 20 healthy subjects, 15 men and 5 women, with a mean age of 60+/-7 years. Detailed histories including symptoms, risk factors and demographic data were obtained from patients and healthy subjects by administering a standardized questionnaire. Our results demonstrate that the enzyme-linked immunoassay (ELISA) test appears to have a greater sensitivity than the microimmunofluorescence (MIF) technique. 80% of patients had positive IgG to C. pneumoniae and 58% positive IgA to C. pneumoniae with ELISA, while the MIF test showed 68% and 55% positive IgG and IgA to C. pneumoniae, respectively. The control subjects showed 55% positive IgG and 10% IgA to C. pneumoniae by ELISA and 35% positive IgG and 5% IgA to C. pneumoniae by MIF. The combination of positive IgG and IgA to C. pneumoniae was present more frequently than in the control group. Serum levels of IL-7 measured by ELISA were also significantly higher in patients compared to healthy subjects. In conclusion, our study shows that C. pneumoniae IgG and IgA seropositivity, inflammatory markers such as IL-7, fibrinogen, C-reactive protein were significantly correlated with CHD.

Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in antiretroviral-naive HIV patients in Campania, Italy.

The aim of our study was to determine the prevalence of genotypic resistance to nucleoside analogues and protease inhibitors before and after 1997, the year of introduction of Highly Active Antiretroviral Therapy (HAART) in Campania (Italy). Forty-eight plasma HIV-RNA positive patients who had not been previously treated for HIV infection (naïve) were enrolled in two Divisions of Infectious Diseases. The main demographic characteristics were collected for each subject and the primary mutant genotypes were sought only in HIV-RNA positive patients with viral loads higher than 10,000 copies/ml. The diagnosis of HIV infection dated back to before 1996 for 21 out of 48 patients and to after 2000 for the other 27. INNO-Line Probe Assay (LiPA) HIV-RT and INNO-LiPA HIV protease (Innogenetics, Italy) were used to detect mutations conferring resistance to zidovudine, didanosine, zalcitabine, lamivudine, stavudine, saquinavir, indinavir, rotonavir, nelfinavir and amprenavir. No mutations associated with primary resistance to nucleoside analogues and protease inhibitors were detected in the 21 patients who had acquired HIV infection before 1996, whereas one or more mutations were seen in three of the 27 (11.1%) patients with HIV infection diagnosed after 2000. This study confirms that LiPA is a suitable tool for epidemiological surveys of HIV genotypic primary resistance. Drug-resistant HIV-1 genotypes, resistant both to nucleoside analogues and protease inhibitors, were detected only in subjects who had acquired HIV infection after 2000, most of whom had zidovudine-resistant mutants. These data suggest that the introduction of HAART has brought about the circulation of drug-resistant HIV genotypes.

Defense mechanisms of IFN-gamma and LPS-primed murine microglia against Acanthamoeba castellanii infection.

In the central nervous system (CNS), cytokine-primed microglia play a central role in host's defense against Acanthamoeba castellanii infection. In this study, the effect of recombinant interferon (rIFN)-gamma and Salmonella enterica serovar enteritidis lipopolysaccharide (LPS), both inflammatory stimuli, on A. castellanii infection in murine microglia was examined. Priming of microglia with rIFN-gamma and LPS synergistically triggered, in a dose-dependent manner, amebastatic activity in these cells. More than 52%, 88% or 95% of this function was then abrogated by anti-IL-1beta (but not anti-IL-1alpha), IL-6 or TNF-alpha neutralizing antibodies, suggesting that these endogenously produced cytokines may participate in the antimicrobial capacity. Consistent with these findings, the priming of microglia with rIFN-gamma and LPS elicited the release of proinflammatory interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. Since L-canavanine affected amebastatic activity only during the priming process but not during the infection process, NO-dependent pathway appears to be not the sole antiparasitic mechanism involved in this function. These data suggest that rIFN-gamma and LPS, likely through a proinflammatory network, up-regulate the release of IL-beta, IL-6 and TNF-alpha, which could trigger antimicrobial activity against A. castellanii infection in the brain.

Apoptosis modulation by mycolic acid, tuberculostearic acid and trehalose 6,6'-dimycolate.

The object of our study is to demonstrate that some components of M. tuberculosis, such as cord factor or mycolic acid or whole bacteria can prolong cell survival compared to controls. The cells treated with cord factor or mycolic acid at a concentration of 5 microg/ml were 65+/-8% viable reaching 70+/-8% at a concentration of 10 microg/ml. The cells treated with heat killed mycobacteria were 70+/-8% viable; while control cells exhibited a viability 50+/-7%. Conversely, tuberculostearic acid induced early cell death. The results also demonstrated a dose-dependent effect on the viability or induction of macrophage apoptosis. We also showed that prolonged viability of the treated cells with mycolic acid or cord factor (+20+/-4% and +25+/-5%, respectively) was correlated with a significant increase in Bcl-2 expression. The treated cells with whole bacteria presented a Bcl-2 expression of 40+/-6%, while Fas expression was not changed compared to controls. This study confirm that at the site of mycobacterial infection, necrosis, apoptosis or prolonged survival of the cells depend on the quantity and quality of the molecules expressed by the mycobacteria; whether necrosis or apoptosis or prolonged survival is more or less favorable to the host likely depends on several factors regarding the inflammatory and immune response, both markedly stimulated by mycobacteria.

Effects of cytokines and prolactin on the replication of Toxoplasma gondii in murine microglia.

In the central nervous system, cytokine-activated microglia play a crucial role in host defence against Toxoplasma gondii infections. In this study, the effect of recombinant tumor necrosis factor (rTNF)-alpha and prolactin (PRL) on T. gondii infection in microglia was examined. Pretreatment of microglia with rTNF-alpha and PRL induced toxoplasmastatic activity, the intracellular killing of T. gondii and the release of interleukin (IL)-1 beta IL-3 and IL-6: 50% of the intracellular killing was abrogated by anti-ICAM-1 monoclonal antibodies, whereas more than 54 or 87% of toxoplasmastatic activity was reversed by anti-IL-3 or IL-6 monoclonal antibodies. In addition, the treatment of microglia with either rIL-3 or rIL-6, in the absence or presence of rTNF-alpha significantly limited T. gondii replication. Inasmuch as either NMA or S-M-ITU affected cytokine-activated toxoplasmastatic activity during the infection phase, the NO-dependent pathway itself appears not to be directly involved in the parasitostatic activity. These findings suggest that TNF-alpha and PRL up-regulate the expression of ICAM-1 and the production of endogenous IL-6 and IL-3 by microglia, which could induce anti-parasitic functions against T. gondii infection in the brain.

HSP and apoptosis in leukocytes from infected or vaccinated animals by Brucella abortus.

The production of hsp and apoptosis of leukocytes in the peripheral blood of animals naturally infected with Brucella spp or treated with the vaccine Brucella abortus 19 have been investigated in this study. Cytokines able to induce phagocytic activity in macrophages of non treated healthy animals were found in the supernatant of bovine leukocytes cultivated in vitro. A long-lasting antibody response against hsp 60 kDa and 27 kDa, which lasts a long time, is induced in naturally infected animals, while in animals vaccinated with B. abortus 19 we detected an antibody response against hsp 60 and 70 kDa which is much shorter, disappearing in two months. During the early phase of infection, lymphocytes and monocytes of naturally infected animals show a delay of apoptosis in vitro compared to the same cells coming from healthy controls and vaccinated animals.

The effect of dietary lipid manipulation on murine splenic lymphocytes apoptosis and heat shock protein over expression.

In this study, we kept BALB/c mice on a hyperlipidic diet for 120 days and then assessed the predisposition to apoptosis and the appearance of heat shock protein (Hsp) on splenic lymphocytes. By immunoblot analysis, bands corresponding to Hsp 60 and Hsp 70 in cells from mice kept on a saturated fatty acid diet showed a greater expression already after 1 month while two other bands, which correspond to Hsp 25 and Hsp 27, were slightly present after 1 month of treatment. In cells from mice kept on a diet rich in unsaturated fatty acid, there was a marked expression of Hsp 25 and Hsp 27 after only 30 days of treatment, which was maintained constant for up to 4 months; while for bands corresponding to Hsp 60 and Hsp 70, a significant minor signal was only detectable after 2-4 months from the beginning of the treatment. Splenic lymphocytes from animals kept on a lipidic diet containing saturated fatty acids were more susceptible to death by apoptosis, while cells of animals treated with unsaturated fatty acid were shown to be more resistant.

Effect of transforming growth factor beta on experimental Salmonella typhimurium infection in mice.

We have investigated the effect of the in vivo administration of recombinant transforming growth factor beta (rTGF-beta) on the pathogenic mechanisms involved in Salmonella typhimurium experimental infection in mice. The protective response elicited by macrophages was induced by rTGF-beta1 by 2 days after experimental infection, as demonstrated by an increased NO production, while the humoral protective effect began with cytokine mRNA expression 2 days after the challenge and continued after 5 days with cytokine release and lymphocyte activation. We demonstrated that all mice who received rTGF-beta1 survived 7 days after infection. The number of bacteria recovered in the spleens and in the livers of rTGF-beta1-treated mice 2 and 5 days after infection was significantly smaller than that found in the same organs after phosphate-buffered saline (PBS) inoculation. Furthermore, 2 and 5 days after infection, splenic macrophages from rTGF-beta1-treated mice showed a greater NO production than did those from PBS-treated mice. The effect of rTGF-beta1 on S. typhimurium infection in mice was correlated with the expression of cell costimulatory CD28 molecules. Five days after S. typhimurium infection, the percentage of CD28(+)-expressing T cells in splenic lymphocytes from rTGF-beta1-treated mice increased with respect to that from control mice. Gamma interferon (IFN-gamma) mRNA was present in a greater amount in spleen cells from rTGF-beta1-treated mice after 2 days, although the intensity of the band decreased 5 days after the challenge. A similar pattern was obtained with the mRNAs for interleukin-1alpha (IL-1alpha), IL-6, TGF-beta, and inducible nitric oxide synthase, which showed greater expression in cells obtained from rTGF-beta1-treated and S. typhimurium-infected mice 2 days after challenge. The treatment with rTGF-beta1 induced an increase in IL-1alpha and IFN-gamma release in the supernatant of splenocyte cultures 5 days after the experimental infection with S. typhimurium. Moreover, we demonstrated that 5 days after infection, the IFN-gamma titer was significantly greater in the sera of rTGF-beta-treated mice than in those of PBS-treated mice. Also, hsp60 showed greater expression 2 days after the challenge in splenocytes from rTGF-beta1-treated mice. The role played by proinflammatory and immunoregulatory cytokines and by CD28 is discussed.

Polyclonal T cell elimination by prolonged immunostimulation in an experimental model.

An experimental model of immunological deficiency obtained by treating mice for 6 months with serum of human blood drawn from different healthy individuals has been studied. The results show that an alteration of a circulating lymphocyte population with alterations of the ratio CD4+/CD8+ appeared in mice stimulated for a long period with immunogens. Mice treated for 2-4 months showed an increase in B lymphocytes and a decrease in the total number of T lymphocytes, with a decrease in CD4+ lymphocytes and an increase in CD8+ lymphocytes. After 4 months, the CD8+ lymphocyte population started to decrease, with a ratio of CD4+/CD8+ reaching almost 1. In animals treated for 2-3 months, the mean survival time (MST) following experimental infection with Salmonella typhimurium presented a decrease to 5 days, and after 5-6 months of treatment presented a decrease to 3-2.5 days. The bacteraemia was modified in comparison with controls. Prolonged exposure to antigens also induced lymphocyte apoptosis: cells of animals treated for 4-6 months presented increased levels of apoptosis with a percentage that reached 30-35%. A semiquantitative evaluation of the level of heat shock protein (hsp) in splenic lymphocytes showed an increase in the presence of hsp60 and hsp70 in the first 3 months of treatment, which then remained constant for up to 6 months.

Effects of benzodiazepines on immunodeficiency and resistance in mice.

Our results indicate that benzodiazepine (Bz) treatment time, greater than 2-3 months, induce a decrease of both specific and nonspecific responses. Mice treated for different times with diazepam or chlordemethyldiazepam showed decreased survival to experimental Salmonella typhimurium infections after three months of treatment. Adherence, expressed as the polymorphonuclear cells (PMN) capacity to attach to nylon wool, was impaired after 7 days of treatment. Longer treatments further increase this impairment. PMN from mice treated with Bz for 90 days also demonstrate on impaired chemotaxis and phagocytosis for Saccharomyces cerevisiae. Monocytes from mice treated for 7 days secreted more IL-1 alpha then controls; the antibody titer in mice given to prolonged treatment progressively diminished compared to controls. Con A or LPS stimulated lymphocytes showed an increase of H3-thymidine incorporation from mice treated for a short time and conversely a decreased incorporation when taken from mice that underwent longer treatments. Benzodiazepines were therefore found to affect PMN chemotaxis and phagocitosis, general immunity and survival of mice to infections.

In vivo inhibition of cell-mediated and humoral immune responses to cellular antigens by SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

Microgram amounts of protein SV-IV, a major secretory protein produced by adult rat seminal vesicle epithelium, markedly decrease the mouse humoral immune response to cellular xenogeneic or allogeneic antigens (sheep red blood cells (SRBC) or mouse epididymal spermatozoa). The significant reduction in the total number of splenocytes and their main cell subsets in SRBC-immunized mice, the dramatic decrease in the number of Ia+ splenic T cells and the marked inhibition of splenocyte ability to respond in vitro to polyclonal mitogen stimuli suggest that the macrophage accessory cells are the primary target of the SV-IV immunosuppressive activity in vivo. Moreover, the infection of SV-IV-treated mice with Salmonella typhimurium produced an increased mortality of the experimental animals associated with a marked decrease of the phagocytic and intracellular killing activities of their peritoneal macrophages.

HLA class II antigens and interleukin-1 in patients affected by type-II diabetes mellitus and hyperlipemia.

In order to evaluate the influence of hyperlipemia on the specific cell defence reaction in type-II diabetes mellitus in humans, 20 diabetics were recruited in this study. They were divided into two groups on the basis of the absence or coexistence of abnormal serum lipid pattern. The lymphocyte and monocyte cells drawn from the type-II diabetic patients with abnormally elevated serum levels of cholesterol and triglycerides showed a decreased expression of major histocompatibility complex (MHC) class-II antigens and an impaired secretion of interleukin (IL-1). Values of MHC class-II antigen expression in diabetics without lipid metabolic alterations were not significantly different from those found in healthy subjects. In conclusion, abnormalities of lipid metabolism often found in type-II diabetes mellitus may play a key role in the impaired specific cell reaction toward infectious diseases of these patients.

Cytotoxic activity and IL-1 production in mice infected with Aspergillus niger.

In this report, we demonstrate an interaction between macrophages and T lymphocytes during A. nigr infection. Supernatants obtained after 48 hrs adherence of infected peritoneal macrophages were able to increase the cytotoxicity of T lymphocytes. Our results also indicate that macrophage supernatant (MS) from mice, in the first 5 days after challenge, is more active on T cell than MS produced later. Splenic T cells activated by IL-1 from mice at 5 days of infection show a significantly increased cytotoxicity, at 10 days after challenge, the cytotoxicity of T cells activated by IL-1 did not significantly differ from non-activated T cells.

Autoreactive cytotoxic cells in rabbits after prolonged immunostimulation.

Rabbits immunized for 6 months with different amounts of sterile human serum showed weight loss and a decrease in leukocytes. The lymphocyte population reacting to ConA contained autoreactive cells capable of causing 51Cr release from labeled cells from a culture consisting of splenic and peripheral lymphocytic cells from the same animal.

Iak antigen and interleukin-1 secretion by lymphocytes and monocytes of mice fed a lipid-rich diet.

Female mice were maintained on a lipid-rich diet. After 33 days, the mice showed a decrease in the cell population bearing MHC class II molecules, and in the production of interleukin-1 (IL-1). After 60 days, we reported a further decrease of the cell population with MHC class II molecules and the secretion IL-1.

Further characterization of the impaired protective function in mice fed with lipid diet.

Female mice were maintained on lipid diet for 20 days. The nonspecific and immunological defense capability was determined by in vitro and in vivo methods. It was found that mice held mostly on a lipid diet demonstrate an all-round lowered response. Following 20 days of lipid diet the splenocytes exhibit: (1) an inversed lipid-protein ratio; (2) an inability to respond to sheep erythrocytes; (3) a reduction in [3H] thymidine incorporation in splenocytes stimulated with lipopolysaccharide (LPS) or with concanavalin A; (4) a reduction in the number of cells bearing surface immunoglobulins in splenocytes stimulated with LPS; (5) an inhibition of phagocytosis and intracellular killing in macrophages; (6) a lowering in granulocyte chemotaxis and adherence capacity; (7) a higher mortality to LPS after loading with galactosamine; and (8) a lowered complement activity even following LPS activation.