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Biomedical subjects

M Fraziano

Publications and source records attributed to M Fraziano.

At least 19 recordsLinked to original sources
PubMed2006

Lysophosphatidic acid enhances antimycobacterial activity both in vitro and ex vivo.

Lysophosphatidic acid (LPA) is a polar lipid metabolite which is involved in a wide range of biological processes, including cell proliferation and migration, wound healing, and increase of endothelial permeability. The present study reports evidences showing that LPA is able to enhance the antimicrobial activity of human macrophages and of bronchoalveolar lavage cells from tuberculosis patients leading to intracellular growth control of Mycobacterium tuberculosis. Such antimicrobial activity is mediated by the activation of phospholipase D which in turn induces acidification of M. tuberculosis containing phagosomes and is associated with the enhanced expression of Cathepsin D. These results suggest the possible protective role of this lysophospholipid in the activation of innate antimycobacterial response.

Adjuvants, Immunologic↗
PubMed2004

Expansion of CCR5+ CD4+ T-lymphocytes in the course of active pulmonary tuberculosis.

Nonsyncytium inducing, macrophage tropic HIV strains predominate in the course of active tuberculosis (TB). The present study assesses the expression of CCR5 in CD4+ T-lymphocytes from blood and bronchoalveolar lavage (BAL) of TB patients, non-TB lung disease controls and healthy controls. Memory (CD45RO+), recently activated (CD69+), proliferating (Ki67+) CCR5+ or CCR3+ CD4+ T-lymphocytes were determined by multiparametric flow cytometry analysis. Results show that BAL CD4+ T-lymphocytes expressing CCR5 or CCR3 were significantly increased when compared to peripheral blood both in patients and in healthy controls. However, the data show that the proportions of peripheral blood CCR5+ CD4+ and CCR3+ CD4+ T-lymphocytes and BAL CCR5+ CD4+ T-lymphocytes, but not BAL CCR3+ CD4+ T-lymphocytes, were significantly increased in TB patients. Furthermore, the observation that BAL CCR5+ CD4+ T-lymphocytes from TB patients expressed early activation markers, were not proliferating and showed down-regulation of CCR5 expression suggests recruitment and trapping at the site of disease. Altogether, these results suggest that the lower respiratory tract mucosa may provide cellular targets accessible for efficient transmission of macrophage tropic HIV-1 variants and that tuberculosis may enhance this phenomenon.

Adult↗
PubMed2003

Diagnosis of tuberculosis: available technologies, limitations, and possibilities.

Rapid diagnosis and treatment are important for preventing transmission of Mycobacterium tuberculosis. However, the diagnosis of tuberculosis continues to pose serious problems, mainly because of difficulties in differentiating between patients with active tuberculosis and those with healed lesions, normal mycobacterium boris BCG (Bacillus Calmette Guerin) vaccinated individuals, and unvaccinated Manteux positives. Physicians still rely on conventional methods such as Ziehl-Neelsen (ZN) staining, fluorochrome staining, sputum culture, gastric lavage, and other non-traditional methods. Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult because sensitization with nontuberculous mycobacteria leads to false-positive tests. There have been numerous unsuccessful attempts to develop clinically useful serodiagnostic kits for tuberculosis. A number of proteinaceous and nonprotein antigens (such as acyltrehaloses and phenolglycolipids) have been explored from time to time for the development of such assays but they have not proved to be clinically useful. It has been difficult to develop an ELISA utilizing a suitable antigen because M. tuberculosis shares a large number of antigenic proteins with other microorganisms that may or may not be pathogenic. With the advent of molecular biology techniques, there have been significant advances in nucleic acid-based amplification and hybridization, which are helping to rectify existing flaws in the diagnosis of tuberculosis. The detection of mycobacterial DNA in clinical samples by polymerase chain reaction (PCR) is a promising approach for the rapid diagnosis of tuberculous infection. However, the PCR results must be corrected for the presence of inhibitors as well as for DNA contamination. In the modern era of genetics, marked by proteomics and genomics, the day is not far off when DNA chip-based hybridization assays will instantly reveal mycobacterial infections.

Antigens, Bacterial↗
PubMed2003

Batimastat reduces Mycobacterium tuberculosis-induced apoptosis in macrophages.

In this study, we report evidences that Mycobacterium tuberculosis (MTB)-induced apoptosis in macrophages is reduced by a broad-spectrum hydroxamic acid-based matrix metalloproteinase (MMP) inhibitor, Batimastat (BB-94). In particular, we show that BB-94 administration to MTB-infected macrophages inhibits apoptosis and the downmodulation of membrane CD14 expression. Moreover, the addition of broad spectrum matrix metalloproteinase inhibitor to cell culture, during MTB infection, decreases the release of soluble TNF-alpha and leads to a simultaneous increase of membrane TNF-alpha. These results show that MTB-induced apoptosis in macrophages is reduced by a MMP inhibitor and most probably is related to TNF-alpha release. This identifies BB-94 as a simultaneous anti-apoptotic and anti-inflammatory molecule during MTB infection.

Annexin A5↗
PubMed2003

Detailed analysis of the effects of Glu/Lys beta69 human leukocyte antigen-DP polymorphism on peptide-binding specificity.

The polymorphism at position beta69 of the human leukocyte antigen (HLA)-DP molecule has been associated with susceptibility to several immune disorders and alloreactivity. Using molecular modeling, we have predicted a detailed structure of the HLA-DP2 molecule (carrying Glubeta69) complexed with class II associated invariant chain derived peptide (CLIP) and compared it with the form carrying Lys at beta69 (HLA-DP2K69). Major changes between the two models were observed in the shape and charge distribution of pocket 4 and of the nearby pocket 6. Consequently, we analyzed in detail the peptide-binding specificities of both HLA-DP molecules expressed as recombinant proteins. We first determined that the minimum peptide-binding core of CLIP for both HLA-DP2 and DP2K69 is represented by nine aminoacids corresponding to the sequence 91-99 of invariant chain (Ii). We then assessed the peptide-binding specificities of the two pockets and determined the role of position beta69, using competition tests with the Ii-derived peptide CLIP and its mutated forms carrying all the aminoacidic substitutions in P4 and P6. Pocket 4 of HLA-DP2 showed high affinity for positively charged, aromatic, and polar residues, whereas aliphatic residues were disfavored. Pocket 4 of the DP2K69 variant showed a reduced aminoacid selectivity with aromatic residues most preferred. Pocket 6 of HLA-DP2 showed high affinity for aromatic residues, which was increased in DP2K69 and extended to arginine. Finally, we used the experimental data to determine the best molecular-modeling approach for assessing aminoacid selectivity of the two pockets. The results with best predictive value were obtained when single aminoacids were evaluated inside each single pocket, thus, reducing the influence of the overall peptide/ major histocompatibility complex interaction. In conclusion, the HLA-DPbeta69 polymorphism plays a fundamental role in the peptide-binding selectivity of HLA-DP. Furthermore, as this polymorphism is the main change in the pocket 4 area of HLA-DP, it could represent a supertype among HLA-DP molecules significantly contributing to the selection of epitopes presented in the context of this HLA isotype.

Amino Acid Sequence↗
PubMed2003

Role of macrophage phospholipase D in natural and CpG-induced antimycobacterial activity.

The present study addresses the differential ability of macrophages to control intracellular growth of non-pathogenic Mycobacterium smegmatis (Msm) and pathogenic M. tuberculosis (MTB). Results reported herein show that 3 h post infection, intracellular Msm, but not MTB, was significantly killed by macrophages. As the role of human macrophage phospholipase D (PLD) in the activation of antimicrobial mechanisms has been documented, we hypothesised the role of such enzyme in antimycobacterial mechanisms. To this aim, macrophage PLD activity was analysed at different times after exposure with either pathogenic MTB or non-pathogenic Msm. Results showed that, starting from 15 min after mycobacterial exposure, MTB did not induce macrophage PLD activity, whereas the environmental non-pathogenic Msm stably increased it. The direct contribution of PLD in intracellular mycobacterial killing was also analysed by inhibiting enzymatic activity with ethanol or calphostin C. Results show that PLD inhibition significantly increases intracellular Msm replication. In order to see whether the innate PLD-mediated antimicrobial mechanisms against MTB are also induced after CpG ODN stimulation, the role of PLD has been analysed in the course of CpG-mediated intracellular MTB killing. CpG DNA increased PLD activity in both uninfected and MTB-infected macrophages, and the inhibition of PLD activity resulted in a significant reduction of CpG-induced MTB killing. Taken together, our data suggest a relationship between host PLD activation and the macrophage ability to control intracellular mycobacterial growth.

Cell Line↗
PubMed2003

Differential sensitivity of human monocytes and macrophages to ANP: a role of intracellular pH on reactive oxygen species production through the phospholipase involvement.

Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present work was to study the effect of ANP on the intracellular pH (pHi) of human monocytes and macrophages and to investigate whether pHi changes could play a role on phospholipase activities and reactive oxygen species (ROS) production. Human macrophages isolated by peripheral blood mononuclear cells and THP-1 monocytes, which were shown to express all three natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C), were treated with physiological concentrations of ANP. A significant decrease of pHi was observed in ANP-treated macrophages with respect to untreated cells; this effect was paralleled by enhanced phospholipase activity and ROS production. Moreover, all assessed ANP effects seem to be mediated by the NPR-C. In contrast, no significant effect on pHi was observed in THP-1 monocytes treated with ANP. Treatment of macrophages or THP-1 monocytes with 5-(N-ethyl-N-isopropyl)amiloride, a specific Na(+)/H(+) antiport inhibitor, decreases pHi in macrophages and monocytes. Our results indicate that only macrophages respond to ANP in terms of pHi and ROS production, through diacylglycerol and phosphatidic acid involvement, pointing to ANP as a new modulator of ROS production in macrophages.

Amiloride↗
PubMed2002

Proinflammatory cytokines in the course of Mycobacterium tuberculosis-induced apoptosis in monocytes/macrophages.

Mycobacterium tuberculosis (MTB) can induce apoptosis in monocytes/macrophages both in vitro and in vivo, and this phenomenon is associated with mycobacterial survival. The present study addresses the possibility that apoptotic and inflammatory pathways could coexist through a caspase-1-mediated mechanism. In this context, a caspase-1 inhibitor (YVAD), but not caspase-3 (DEVD) or caspase-4 (LEVD) inhibitors, was able to strongly inhibit MTB-induced apoptosis. Moreover, caspase-1 activity was confirmed by the increased maturation of interleukin (IL)-1beta. Of interest, IL-1beta and tumor necrosis factor (TNF)-alpha were produced massively in the course of infection, and both were inhibited by YVAD pretreatment. To determine whether TNF-alpha was produced actively by apoptotic cells, the intracytoplasmatic cytokine content and apoptotic phenotype were analyzed at the single-cell level. Results showed a progressive increase of TNF-alpha production in annexin V-positive cells. These results indicate that MTB-induced apoptosis is associated with proinflammatory cytokine production.

Apoptosis↗
PubMed2002

Atrial natriuretic factor inhibits mitogen-induced growth in aortic smooth muscle cells.

Atrial natriuretic factor (ANF) is a polypeptide able to affect cardiovascular homeostasis exhibiting diuretic, natriuretic, and vasorelaxant activities. ANF shows antimitogenic effects in different cell types acting through R(2) receptor. Excessive proliferation of smooth muscle cells is a common phenomenon in diseases such as atherosclerosis, but the role of growth factors in the mechanism which modulate this process has yet to be clarified. The potential antimitogenic role of ANF on the cell growth induced by growth factors appears very intriguing. Aim of the present study was to investigate the possible involvement of ANF on rat aortic smooth muscle (RASM) cells proliferation induced by known mitogens and the mechanism involved. Our data show that ANF, at physiological concentration range, inhibits RASM cell proliferation induced by known mitogens such as PDGF and insulin, and the effect seems to be elicited through the modulation of phosphatidic acid (PA) production and MAP kinases involvement.

Adrenergic beta-Antagonists↗
PubMed2001

Macrophage response to Mycobacterium tuberculosis during HIV infection: relationships between macrophage activation and apoptosis.

Human macrophages represent the first line of defense for the containment of Mycobacterium tuberculosis infection. After phagocytosis, macrophages express activation surface markers and produce proinflammatory cytokines and chemokines whose main role is to control pathogen spreading by recruiting peripheral lymphocytes and monocytes at the site of inflammation. However, in the case of a concomitant human immunodeficiency virus (HIV) infection, these signals strongly enhance the susceptibility to viral infection both at the viral entry and replication levels. Under these conditions, viral expansion extends beyond tissue macrophages to T cells and vice-versa, according to the emerging viral phenotype. In absence of an efficient immune response, Mycobacterium tuberculosis can replicate in macrophages in an uncontrolled fashion culminating in macrophage death by apoptosis. As a consequence, a more severe form of immunedepression, involving both innate and specific immune responses, could be responsible for both ematogenous mycobacterial dissemination and extrapulmonary form of tuberculosis in HIV-infected patients.

Apoptosis↗
PubMed2000

Mycobacterium tuberculosis-induced apoptosis in monocytes/macrophages: early membrane modifications and intracellular mycobacterial viability.

Apoptosis has been observed in monocytes/macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis (MTB) infection. In order to define the early events of MTB-induced apoptosis, membrane CD14 expression and the exposure of Annexin V-binding sites in MTB-infected monocytes/macrophages have been monitored. Moreover, the role of MTB-induced apoptosis was further analyzed in vitro in terms of mycobacterial viability. Results show that monocyte/macrophage apoptosis is a very early event that is strictly dependent on the MTB amount, and this apoptosis is associated with a selective down-regulation of surface CD14 expression. Furthermore, no statistically significant decrease in mycobacterial viability was observed, which indicates that the apoptotic pathway triggered by high doses of MTB is associated with parasite survival rather than with killing of the parasite.

Apoptosis↗
PubMed1999

Expression of CCR5 is increased in human monocyte-derived macrophages and alveolar macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis infection.

Human immunodeficiency virus (HIV) replicates more efficiently in Mycobacterium tuberculosis (MTB)-infected macrophages than in uninfected controls. We investigated whether this may be partly explained by changes in expression of CCR5 in the course of mycobacterial infection, as this molecule has been shown to be a coreceptor for HIV entry. Since the lung is the preferential organ of HIV replication in the course of tuberculosis, we preliminarily analyzed beta-chemokine receptor expression in alveolar macrophages from patients with active tuberculosis, using flow cytometry based on an MIP-1alpha ligand-biotin/avidin-FITC detection system. Increased MIP-1alpha receptor (MIP-1alphaR) expression in alveolar macrophages from infected patients was observed whereas no detectable expression could be revealed in uninfected controls. Since MIP-la can also bind CCR1 and CCR4, the presence of CCR5 mRNA was investigated in bronchoalveolar lavage (BAL) cells and detected in alveolar macrophages from tuberculosis patients only. The study was then extended to in vitro MTB-infected macrophages. Monocyte-derived macrophages (MDMs) were left to differentiate for 7 days before MTB H37Rv infection, and CCR5 expression was monitored, by using a specific monoclonal antibody, on days 1, 6, and 11 after infection. Increased CCR5 expression in MTB-infected macrophages was observed, with a peak on day 6 (64% in MTB-infected versus 33% in control cultures) and a decrease by day 11 (25% in MTB infected versus 13% in control cultures). These results show that CCR5 expression is enhanced in the course of in vitro MTB infection and during active pulmonary tuberculosis.

Cells, Cultured↗
PubMed1999

Nitric oxide inhibits the HIV-1 reverse transcriptase activity.

Nitric oxide (NO) is a polypotent regulatory molecule involved in a variety of activities, such as the modulation of the catalytic activity of cysteine-containing enzymes. The present study reports the modulation of the HIV-1 reverse transcriptase activity by NO, released by the NO-donors 3, 3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), 4-(phenylsulfonyl)-3-((2-(dimethylamino) ethyl)thio)furoxan oxalate (SNO-102), and sodium nitroprusside (SNP). NO inhibits dose-dependently the HIV-1 reverse transcriptase activity, likely due to oxidation of Cys residue(s). Present results, representing a new insight into the modulation mechanism of the HIV-1 reverse transcriptase activity, may be relevant to develop new strategies for inhibition of HIV-1 replication.

Catalysis↗
PubMed1999

Nitric oxide inhibits HIV-1 replication in human astrocytoma cells.

Astroglial cells represent a target for HIV infection in the central nervous system. In astrocytes, HIV infection is poorly productive, being characterized by a persistent state of viral latency. However, activation of the nuclear factor NF-kappaB and its binding to HIV long terminal repeat (LTR) can induce HIV replication. Moreover, nitric oxide (NO) can affect NF-kappaB activation in glial cells. Therefore, we hypothesize that NO may reduce HIV replication in human astroglial cells by inhibiting HIV-1 LTR transcriptional activity. In this respect, we show that NO donors reduce viral replication in HIV-1-infected human astrocytoma T67 cells, taken as an astroglial model. Furthermore, using transfected T67 cells, we demonstrate that NO donors inhibit HIV-1 LTR transcriptional activity. These results suggest that the use of NO-releasing drugs may represent a potential, novel approach in inhibiting HIV replication in the central nervous system.

Astrocytoma↗
PubMed1999

Molecular bases for the anti-HIV-1 effect of NO. Commentary.

In infected human cells, nitric oxide (NO) has been shown to inhibit the replication of the human immunodeficiency virus-1 (HIV-1), the etiological agent of AIDS. Evidence suggests that NO may regulate HIV-1 replication by affecting the sulphydryl redox state. In this respect, it has been very recently demonstrated that NO-donors inactivate the HIV-1-encoded protease and reverse transcriptase in vitro. Further viral and host NO targets may be envisaged. Although no data are available on the anti-HIV-1 effect of NO in vivo, NO-releasing drugs, clinically used in the treatment of cardiovascular disorders, may represent a novel class of molecules for decreasing virus replication. Here, the possible molecular bases for the anti-HIV-1 effect of NO are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1997

The presence of antibodies against HIV peptides in the sera of alloimmune mice and thalassemic patients is due to a polyclonal activation mechanism.

This paper analyses the HIV-1 gp120 epitope specificity and activation mechanisms (i.e., polyclonal versus oligoclonal) of antibodies present in the sera of alloimmune mice and humans. Sera from CBA mice engrafted with C57BL/6 lymphoid cells significantly reacted against the gp120-derived peptide as 261-270, which shares high homology with the membrane-proximal domain of HLA class II beta-chains (HLA/ gp120) and against the HIV gp120 V3 loop-derived peptides DP32 (HIV-1 MN-derived as 302-334) and C53 (HIV-1 IIIB-derived as 304-318). The same sera also reacted against the HIV-unrelated peptide necdin. Moreover, sera from BALB/c mice injected with LPS presented antibodies reacting against both HIV-related and -unrelated peptides, suggesting that similar mechanisms are shared in alloimmune and LPS-treated mice. A similar analysis was then performed on the sera of patients affected with beta-thalassemia major, receiving at least 10 blood transfusions/year. In particular, 15 of 58 (26%) sera from HIV-uninfected thalassemic patients showed a significantly reactivity against the HLA/gp 120-derived peptides. Moreover, 22 of 58 (38%) sera from the same cohort showed a significant reactivity against DP32 peptide. This reactivity was related to a polyclonal activation mechanism since the DP32-reactive sera significantly bound a panel of HIV-unrelated peptides, as observed by testing 22 sera against necdin, 21 against HSP65 kDa, 21 against amyloid-1, and 17 against MAGE-1 peptides. Moreover, a significant increase of IgG concentration was also observed in all thalassemic sera, when compared to healthy controls, without regard to the anti-gp120 antibody reactivity. Taken together, these results indicate that (i) allogeneic stimuli may induce anti-gp120 antibodies in CBA and in 38% of polytransfused patients and (ii) this reactivity is related to a polyclonal activation mechanism but not to a heightened concentration of IgG.

Amyloid↗