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In view of the heath problems induced by the presence into the environment of endocrine disruptors, laccase from Trametes versicolor was covalently immobilized on a chemically modified polypropylene membrane in order to remove phenol and its derivatives from polluted waters. Using phenol as substrate model the optimal immobilization conditions were determined. The immobilized laccase exhibited maximal enzyme activity at pH 5.5 and optimal temperature at 55 °C. These operative parameters have been compared with those obtained with the soluble laccase in order to ascertain the immobilization effect. When employed in a bioreactor operating under isothermal conditions the immobilized laccase was able to oxidize a wide range of phenolic substrates. In particular it was found that some phenol derivatives (2-CP, 3-CP, 4-CP, NP and chlorophene) were oxidized at a similar rate than phenol, other derivatives (paracetamol, 3-MP and chloroxyphenol) at a smaller rate, while others (2,4-DCP and BPA) at higher rate. When the catalytic membrane was employed in a non-isothermal reactor the reaction rate increased with the increase of the applied temperature difference. Practically the increase of the laccase oxidative power under the non-isothermal conditions followed the same sequence observed under isothermal conditions. Interesting enough, the percentage increase of enzyme reaction rate under non-isothermal conditions resulted higher in the cases in which the isothermal reaction rate was smaller. When the reduction of the production times by the presence of a temperature gradient is considered, the measured values strongly candidate the technology of non-isothermal bioreactors as a useful tool in processes of detoxification of waste waters polluted by endocrine disruptors of phenolic origin. © 2010 Elsevier B.V. All rights reserved.

Objective The aim of this study was to evaluate the effect of porins from Salmonella typhimurium on costimulatory molecules such as CD80/CD86 and CD28/CD152. The interactions between these molecules are able to influence the immune response through the regulation of cytokines release which, on their own, are able to regulate the immunological response by a feedback mechanism. Methods S. typhimurium strain SH5014 (a rough lipopolysaccharide (LPS) producing strain) was used as the source of porins and LPS. Peripheral blood mononuclear cells were obtained from healthy adult donors. THP1 cells were obtained from ATCC (Rockville, MD, USA). Immunofluorescence and flow cytometry were performed using a FACS IV (Becton–Dickinson, Mountain View, CA, USA). Results Our results show that porins of S. typhimurium increase the expression of CD86 and the expression of CD80 both on B lymphocytes and macrophages, while the expression of CD28 and CD152 on T lymphocytes was unaltered. The expression of CD80 and CD86 is dose-dependent and starts after 24 h post treatment, peaks at 48 h and goes back to the basal value after 72 h. Conclusions S. typhimurium porins are able to induce a high expression of costimulatory molecules (CD80 and CD86) on lymphocytes and macrophages.

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.

In this study, we examined the invasive capacity of Staphylococcus aureus and Salmonella typhi in human keratinocytes and monitored the number of viable intracellular bacteria at different post-infection times. The strains tested entered keratinocytes; both S. typhi and S. aureus were internalized within 30 min to 2 h after infection. No intracellular multiplication was observed, but S. typhi and S. aureus remained viable 72 h after infection. We also demonstrated that keratinocyte death following S. typhi and S. aureus invasion occurs by apoptosis as shown by DNA fragmentation. After 24 h of infection with S. typhi, the number of cells undergoing apoptosis were higher compared to infection with S. aureus. For prolonged infection times (48 h, 72 h) with both bacteria, there was no significant change in the number of cells undergoing apoptosis. The results demonstrated that viable intracellular S. typhi and S. aureus induced apoptosis in keratinocyte cells.

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.

From the experiments carried out in this study on mice kept on a 17.5% alcoholic diet (ethanol) for more than 90 days, a clear-out decrease was found in all the protective responses studied. In fact, the data show: (a) increased mortality with increased number of circulating bacteria; (b) diminished adherence and migrational capacity of polymorphonuclear leukocytes; (c) diminished phagocytosis index; (d) reduced release of cytokines from lymphocytes and monocytes; and (e) diminished response of lymphocytes to polyclonal stimuli (concanavalin A, lipopolysaccharide).

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#x0025; and 17#x0025;, respectively), while UV-inactivated microorganisms induced a lesser expression (20#x0025; and 11#x0025;, 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.

Octylphenol (OP) is an endocrine-disrupting chemical that accumulates in various organs. It has also been shown to exert noxious effects on the central nervous system. In the present study, we measured in Sprague–Dawley rats the degree of OP accumulation in different areas of the brain and investigated the effect of OP in pain modulation. Two groups of male Sprague–Dawley rats were treated for 20 days with 50 mg/kg BW/day of OP (group 1) or vehicle (group 2). At the end of the treatment, the formalin test was performed to evaluate the effect of OP exposure on pain. Soon after, rats were sacrificed, and the accumulation of OP in the cerebral cortex, hippocampus, hypothalamus, cerebellum, thalamus, striatum, mesencephalus and ventral hindbrain was measured by HPLC analysis. The results showed a greater accumulation of OP in the cerebral cortex compared to all the other areas; there was also more accumulation in the cerebellum compared to the mesencephalus and thalamus. No accumulation was found in the striatum. These results suggest that there is a preferential accumulation of OP in different areas of the brain with consequences to neural behaviour. On the contrary, experiments on facial grooming did not show significant effects of OP on pain.

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.

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.

Seminal vesicle protein IV (SV-IV) is a secretory anti-inflammatory, procoagulant, and immunomodulatory protein produced in large amounts by the seminal vesicle epithelium of the rat under the strict transcriptional control of androgen. In particular, this protein was shown to possess the ability to markedly inhibit in vivo the humoral and cell-mediated immune responses of mice to nonbacterial cellular antigens (sheep erythrocytes and spermatozoa). We report data that demonstrate that in mice treated with SV-IV and infected withSalmonella entericaserovar Typhimurium, SV-IV is able to downregulate some important immunological and biochemical parameters that serovar Typhimurium normally upregulates in these animals. This event did not correlate with a lower bacterial burden but was associated with a markedly increased one (300%). Furthermore, the treatment of mice with SV-IV alone also produced a significant increase in the rate of mortality among serovar Typhimurium-infected animals. The mechanism underlying these phenomena was investigated, and the strong immunosuppression produced by SV-IV in serovar Typhimurium-infected mice was suggested to be the basis for the increased rate of mortality. The SV-IV-mediated immunosuppression was characterized by a decrease in the humoral and cell-mediated immune responses, altered lymphocyte-macrophage interaction, downregulation of cytokine and inducible nitric oxide synthase gene expression, inhibition of macrophage phagocytosis and intracellular killing activities, and absence of apoptosis in the splenocyte population of SV-IV- and serovar Typhimurium-treated mice. The immunosuppressive activity of SV-IV was specific and was not due to aspecific cytotoxic effects. SV-IV-specific receptors (Kd= 10−8M) occurring on the macrophage and lymphocyte plasma membranes may be involved in the molecular mechanism underlying the SV-IV-mediated immunosuppression. Some results obtained by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay also revealed a functional impairment of mitochondria (a decrease in mitochondrial dehydrogenase activity), thus indicating the possible implication of these organelles in the immunosuppressive process.

Chlamydia pneumoniae is an obligate intracellular bacterium which frequently causes airway infection in humans and has been implicated in chronic inflammatory disease and atherosclerosis. Here we show that infection with C. pneumoniae protects THP-1 cells against the apoptosis which spontaneously occurs in macrophages in the absence of an activation signal. Analysis by flow cytometry at different post-infection times revealed that 50±7% of THP-1 cells were apoptotic at 48 h after onset of the experiments, whereas C. pneumoniae -infected cultures (multiplicity of infection, MOI=30) displayed only 18±4% of cells in apoptosis. At MOI=20 and MOI=10 the cells susceptible to apoptosis at 48 h were 28±5% and 35±6% respectively. Moreover, the results show that heat-inactivated bacteria do not give significant protection against apoptosis, even at higher MOI (MOI=30), while UV-treated Chlamydia did provide a degree of protection against apoptosis. These data suggest that the anti-apoptotic effect of C. pneumoniae requires a heat-labile component released during infection, and that the effect is not lipopolysaccharide-dependent.

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.

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.

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.

SUMMARY 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.

This study investigates the effect of some components of the Staphylococcus aureus cell wall [lipoteichoic acid (LTA), N-acetyl-muramyl-alanyl-d-isoglutamine (MD), muramic acid (MA) and protein A (PA)] in modulating expression of cell-surface adhesion molecules CD11a/CD18, CD11b/CD18 on monocytes qualitatively and quantitatively. Monocytes incubated with bacterial components presented different CD11b/CD18 expressions which were dose-dependent in contrast to controls. The results obtained demonstrated that lymphocytes incubated with bacterial components also increased the expression of CD11a/CD18. The modifications in activation of CD11a/CD18 and CD11b/CD18 expression are probably correlated with modifications of membrane fluidity measured as polarisation fluorescence (P).

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.

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.

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.

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.

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.

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.

The authors studied the production and effect of interleukin 2 obtained from T lymphocytes stimulated with Aspergillus niger on populations of LGL cells from the peripheral blood and spleen of infected and non-infected mice. The results show an increase in IL-2 production by T lymphocytes in the spleen. In addition, the data demonstrate an increase in LGL activity in the blood and spleen of infected mice. This was seen as the percentage of growth inhibition towards Aspergillus niger, around the tenth day of infection, and reached a maximum of activity on the fifteenth day. The NK activity of the LGL studied increases both in infected and non-infected mice when IL-2 from T lymphocytes of infected mouse blood and spleen is present.

A study has been made of the cellular response by T, K and NK cells in mice experimentally infected with Aspergillus niger. The tests were made on mice treated with 5 X 10(5) conidia. The lymphocyte populations in the blood, spleen and pulmonary lymphnodes have studied to identify possible modifications in immune response in time. The results obtained show that in experimentally infected mice with respect to uninfected mice there is an increase in T-lymphocytes, particularly in the spleen, a percentage increase in EA-RFC cells in the blood, spleen and lymphnodes, a percentage increase in LGL cells, with a rise in associated NK activity, in the blood and spleen in the first days of infection, but a delayed increase in the lymphnodes.

A study was conducted on the granulocytic phagocytosis of bacterial aggregates obtained under ideal environmental conditions. For the strains studied, aggregation was favored by low salt concentrations, low pH and temperatures between 30 degrees C and 40 degrees C. Our results show that the phagocytic capacity of granulocytes depends on the type and size of these aggregates. Those formed by a smaller number of cells are more easily phagocytized than the larger ones.

Granulocytes from diabetic subjects have impaired ability to engulf bacteria; the data obtained suggest that the alterations are correlated with an increase in surface hydrophobicity, as measured by contact angle.

Cell proliferation is significantly depressed in uremia; to assess the influence of PTH on it, normal lymphocytes were cultured in presence of uremic patients’ serum with low or high plasma PTH levels (Group A; PTH < 2.5 ng/ml; Group B: PTH > 12 ng/ml), and serum of normal subjects (Group C). Cell proliferation was lowered by serum from both groups (p A vs C < 0.004; p B vs C < 0.001). However, the depressing effect was more evident when group B serum was employed (p A vs B< 0.002).

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.

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.

The role of PTH in depressing polynuclear leucocyte (PMN) phagocytosis in uremia was investigated. The hydrophobicity and phagocytic activity of normal PMN was tested in presence of uremic patients’ serum with low (Group A) or high (Group B) levels of plasma PTH. The PMN phagocytic index was lowered by serum of both groups, but more in presence of Group B serum (p A vs B < 0.002). Similarly, the contact angle of cells was affected more in presence of serum of patients with high PTH levels (p B vs A < 0.003; p B vs C < 0.002).