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M Amicosante

Publications and source records attributed to M Amicosante.

At least 19 recordsLinked to original sources
PubMed2006

Accuracy of an immune diagnostic assay based on RD1 selected epitopes for active tuberculosis in a clinical setting: a pilot study.

A previous case-control study reported that an in-vitro interferon (IFN)-gamma response to early secreted antigenic target (ESAT)-6 selected peptides was associated with active tuberculosis (A-TB). The objective of the present pilot study was to evaluate the diagnostic accuracy of this assay for TB disease in a clinical setting. An IFN-gamma ELISPOT assay was performed on samples from patients with suspected A-TB using two peptides selected from ESAT-6 protein and three peptides selected from culture filtrate 10 (CFP-10) proteins. The results were compared with those obtained by two commercially available assays approved for diagnosis of TB infection (T SPOT-TB and QuantiFERON-TB Gold) which use ESAT-6/CFP-10 (RD1) overlapping peptides. Sensitivity to the RD1 selected peptides was 70% (positive for 16 of 23 patients with microbiologically diagnosed A-TB) and specificity was 91% (positive for three of 32 controls). In contrast, the sensitivity and specificity were 91% and 59%, respectively, for T SPOT-TB, and were 83% and 59%, respectively, for QuantiFERON-TB Gold. The RD1 selected peptides assay had the highest diagnostic odds ratio for A-TB. Thus, the results suggest that an assay based on RD1 selected peptides has a higher diagnostic accuracy for A-TB in a clinical setting compared with commercially available assays based on RD1 overlapping peptides.

Adult↗
PubMed2004

Evaluation of the BDProbeTec strand displacement amplification assay in comparison with the AMTD II direct test for rapid diagnosis of tuberculosis.

The BDProbeTec MTB assay for direct detection of Mycobacterium tuberculosis was evaluated in comparison with the AMTD-II assay on 94 samples from different patients with clinical suspicion of tuberculosis. Using a combination of culture on Lowenstein-Jensen medium (with or without preculture in BACTEC 9000) and clinical diagnosis as the standard, BDProbeTec MTB showed high sensitivity and specificity (96.1% and 100%, respectively), similar to AMTD-II (96.1% and 97.1%, respectively), with significantly higher sensitivity than the Ziehl-Neelsen stain for acid-fast bacilli (73%, p < 0.05).

Culture Media↗
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↗
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

HLA-DP-unrestricted TNF-alpha release in beryllium-stimulated peripheral blood mononuclear cells.

Berylliosis is a granulomatous disorder of the lung caused by inhalation of beryllium (Be) and dominated by the accumulation of CD4+ T-helper (Th)1 memory T-cells proliferating in response to Be in the lower respiratory tract. Two gene markers have been associated with susceptibility to berylliosis: 1) the human leucocyte antigen (HLA)-DP gene whose allelic variants, carrying glutamate in position 69 of the beta-chain (HLA-DPGlu69), can bind Be directly and present it to interferon (IFN)-gamma releasing Th1 T-cell clones from patients with berylliosis; and 2) the cytokine gene tumour necrosis factor (TNF)-alpha which has been shown to increase berylliosis risk independent of HLA-DPGlu69. In order to determine whether TNF-alpha release was triggered by Th1 T-cell activation by Be stimulation in the context of HLA-DPGlu69 molecules, the proliferation of BeSO4-stimulated blood mononuclear cells and the release of IFN-gamma, TNF-alpha, RANTES (regulated on activation normal T-cell expressed and secreted), granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4, IL-6, IL-8, IL-10 and IL-12 by BeSO4-stimulated blood mononuclear cells was quantified in 11 individuals with berylliosis using an anti-HLA-DP antibody as a probe for HLA-DP restricted T-cell activation. While proliferation and IFN-gamma release were completely abrogated by HLA-DP inhibition (inhibition with anti-HLA-DP monoclonal antibody (mAb): 88+/-16 and 77+/-16%, respectively; anti-HLA-DR: 29+/-38 and 14+/-10%, respectively), the release of TNF-alpha was not (inhibition with anti-HLA-DP mAb: 8.9+/-7.8%). No other cytokine was detected at significant levels. Moreover, Be was able to induce TNF-alpha production in healthy control subjects not exposed to Be in the absence of T-cell proliferation and IFN-gamma production. In conclusion, these data suggest that the tumour necrosis factor-alpha response of mononuclear cells is independent of the activation of beryllium-specific human leucocyte anitgen-DP restricted T-cells, which is consistent with the finding that the tumour necrosis factorA2 and the human leucocyte anitgen-DPGlu69 genetic markers are independently interacting in increasing berylliosis risk.

Adult↗
PubMed2001

Beryllium binding to HLA-DP molecule carrying the marker of susceptibility to berylliosis glutamate beta 69.

Berylliosis is a chronic granulomatous disorder caused by inhalation of Be dusts that is driven by the accumulation of Be-specific CD4+ Th1-cells at disease sites. Susceptibility to berylliosis has been associated with the supratypic variant of HLA-DP gene coding for glutamate at position beta69 (HLA-DPbetaGlu69). The aim of this study was to test the hypothesis that the HLA-DPbetaGlu69 residue plays a role in the interaction with Be. To this end, soluble HLA-DP2 molecule (carrying betaGlu69) and its mutated form carrying lysine at position beta69 (HLA-DP2Lys69) were produced in Drosophila melanogaster and then used in a Be binding assays. BeSO4 (1-1000 microM) was used to compete for the binding of the biotinilated invariant chain-derived peptide CLIP (50 microM). BeSO4 was capable of compete out biotin-CLIP binding from the HLA-DP2 (IC50%: 4.5 microM of BeSO4 at pH 5.0 and 5.5 microM of BeSO4 at pH 7.5), but not from the HLA-DP2Lys69 molecule (IC50%: 480 microM of BeSO4 at pH 5.0 and 220 microM of BeSO4 at pH 7.5). Moreover, the binding of NFLD.M60, a MoAb recognizing an epitope in the HLA-DP peptide binding region, to the HLA-DP2, but not to the HLA-DP2Lys69 soluble molecules was inhibited BeSO4. NFLD.M60 binding to HLA-DP2, but not to HLA-DP2Lys69 stably transfected murine cells was also inhibited by Be both at pH 5.0 and at pH 7.5. The data indicate a direct interaction of Be with the HLA-DPGlu69 molecule, in the absence of antigen processing.

Amino Acid Sequence↗
PubMed2001

Beryllium disease.

Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of insoluble beryllium (Be) dusts and characterized by the accumulation of CD4+ T cells and macrophages in the lower respiratory tract. In response to Be inhalation, noncaseating granuloma formation and, eventually, fibrosis. The immunopathogenic process is maintained by Be-specific lung CD4+ T-lymphocytes. Consistent with the disease immunopathology, these Be-specific T cells have a T-helper 1 phenotype producing interleukin-2 and interferon-gamma, the macrophage-activating cytokine driving the granulomatous reaction. Previous studies have demonstrated that the glutamic acid in position 69 of the human leukocyte antigen class II b chain is strongly associated with increased susceptibility to Be in exposed workers, suggesting that human leukocyte antigen gene markers may be used as epidemiological probes to identify population groups at higher risk.

Amino Acid Sequence↗
PubMed2001

Major histocompatibility locus genetic markers of beryllium sensitization and disease.

Hypersensitivity to beryllium (Be) is found in 1-16% of exposed workers undergoing immunological screening for beryllium disease using the beryllium lymphocyte proliferation test (BeLPT). However, only approximately 50% of BeLPT-positive workers present with lung granulomas (i.e. berylliosis). As berylliosis is associated with the human leukocyte antigen (HLA)-DP supratypic marker DPGlu69, the authors asked whether this marker is differentially associated with disease presentation. A population of 639 workers from a beryllium factory undergoing BeLPT screening was evaluated in a nested case-control study for the prevalence of HLA-DPGlu69, the HLA-DPB1, HLA-DQ and HLA-DR alleles and of the biallelic tumour necrosis factor (TNF)-alpha polymorphism TNF-alpha-308 in 23 individuals presenting as "sensitized" (i.e. BeLPT-positive without lung granulomas) and in 22 presenting as "diseased" (i.e. BeLPT-positive with granulomas in the lung biopsy). The HLA-DPGlu69 marker was associated with "disease" (odds ratio (OR) 3.7, p=0.016, 95% confidence interval (CI) 1.4-10.0), whilst the high TNF-alpha production-related TNF-alpha-308*2 marker was associated with both a positive BeLPT (OR 7.8, corrected p<0.0001, 95% CI 3.2-19.1) with no difference between "sensitization" and "disease". Furthermore, the HLA-DRArg74 marker was associated with "sensitization" without disease (OR 3.96, p=0.005, 95%, CI 1.5-10.1). The data indicate that tumour necrosis factor-alpha, human leukocyte antigen-DR and human leukocyte antigen-DP markers play different roles in beryllium sensitization and granuloma formation in beryllium-exposed workers.

Adult↗
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↗
PubMed2000

Evaluation of a western blot test as a potential screening tool for occupational exposure to Mycobacterium tuberculosis in health care workers.

Health care workers (HCWs) have a higher than average risk for contracting Mycobacterium tuberculosis (MTB) infection and tuberculosis (TB). No markers of MTB-exposure are available, and TB risk assessment is performed by tuberculin screening, identifying individuals with acquired MTB infection. This study evaluated a western blot (WB) anti-M. bovis A60 complex antibody as a MTB-exposure marker. WB reactivity was evaluated on 127 exposed and 28 non-exposed HCWs from four divisions of the Policlinico Hospital of Modena, and 140 non-exposed bacille Calmette-Guérin-vaccinated controls. Excess of occupational TB risk according to the Occupational Safety and Health Administration (OSHA) was calculated in each division. WB-positivity (%) was: (1) significantly higher in exposed HCWs compared with non-exposed (72% vs 25%, P < 0.00001), (2) highly related (r = 0.99) to OSHA risk excess in all divisions, (3) higher than non-exposed in HCWs with short (< 5 years) MTB-exposure (purified protein derivative [PPD], P > 0.18; WB, P < 0.04). PPD-positivity (%) was higher than controls only in HCWs with longer (> 5 years) MTB-exposure. The study suggests that the WB antibody might represent a more sensitive biological marker of MTB contact among exposed HCWs, related to the level of TB risk and detectable earlier than the PPD skin test, thus providing new tools for TB risk assessment in health care facilities.

Adult↗
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

Sensitivity and specificity of a multi-antigen ELISA test for the serological diagnosis of tuberculosis.

A new serological assay (DETECT-TB, BioChem ImmunoSystems), using three recombinant proteins and two synthetic peptides for the detection of the anti-Mycobacterium tuberculosis IgG, was evaluated using a panel of serum specimens collected from 100 tuberculosis (TB) patients and 270 controls, in comparison with a homemade ELISA test using purified protein derivative (PPD) as antigen. DETECT-TB presented a higher sensitivity (75%) compared to PPD-ELISA (56%; P < 0.01), while the specificity of each assay was similar (DETECT-TB 97%; PPD-ELISA 100%; P > 0.80). Receiver operating characteristics (ROC) analysis obtained with these data confirmed the higher level of performance of DETECT-TB in comparison with PPD-ELISA. Considering the rapidity, cost-effectiveness and simplicity of this assay, its use may provide useful clinical information aiding in the rapid diagnosis of difficult TB cases.

Adult↗
PubMed1998

Immunogenetic basis of environmental lung disease: lessons from the berylliosis model.

The role of genetic factors has been hypothesized in the pathogenesis of a number of chronic inflammatory lung diseases. The genes of the major histocompatibility complex (MHC) locus on human chromosome 6 have been identified as important determinants in diseases caused both by inorganic and organic compounds such as beryllium, gold, acid anhydrides, isocyanates and grass pollens. Since many environmental factors are the determinants of the immunopathogenesis of asthma, pulmonary granulomatous disorders, hypersensitivity pneumonitis and fibrotic lung disorders, an understanding of the interaction between environmental factors is crucial to epidemiology, prevention and treatment of these disorders. Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of beryllium dusts. A human leukocyte antigen class II marker (HLA-DP Glu69) has been found to be strongly associated with the disease. In in vitro studies, the gene has been shown to play a direct role in the immunopathogenesis of the disease. In human studies, the gene has been shown to confer increased susceptibility to beryllium in exposed workers, thus suggesting that HLA gene markers may be used as epidemiological probes to identify population groups at higher risk of environmental lung diseases, to identify environmental levels of lung immunotoxicants that would be safe for the entire population and to prevent disease risk associated with occupation, manufactured products and the environment. Studies on the associations between human leukocyte antigens and chronic inflammatory lung disorders are reviewed in the context of the berylliosis model.

Animals↗
PubMed1997

Serological markers predicting tuberculosis in human immunodeficiency virus-infected patients.

SETTING: Human immunodeficiency virus (HIV)-positive patients retrospectively identified at the Hospital of Bari, Italy, with diagnosis of tuberculosis (TB) (n = 30) or non-tuberculous pneumonia (n = 29). Serum samples drawn at the time of diagnosis and one year before. Anti-purified protein derivative (PPD) and anti-diacyltrehalose (DAT) serum antibodies quantified by ELISA assay. OBJECTIVE: Since TB patients with HIV infection may present with elevated serum antibodies against Mycobacterium tuberculosis, we hypothesized that TB-specific antibody markers might predict TB in these subjects. DESIGN: A retrospective study was designed to assess the presence of M. tuberculosis-specific antibodies in HIV-positive patients developing TB. RESULTS: Of 30 HIV-positive TB patients, 24 (80%) had anti-PPD or anti-DAT antibodies at the time of TB diagnosis, and 20 (67%) one year before. In a sub-population of 16 of the 30 HIV-positive subjects, positivity for anti-PPD or anti-DAT antibodies one year before TB diagnosis was higher (11/16, 69%) than for the PPD skin test (4/16, 25%, P < 0.01). Antibody tests were specific for TB since positivity rates were lower both in patients with non-tuberculous pneumonia (P < 0.01) and in those with M. avium infection (P < 0.05). CONCLUSION: Antibody markers may predict TB in HIV-positive subjects, including those with negative PPD skin test.

AIDS-Related Opportunistic Infections↗
PubMed1996

Evaluation of a western blot serum test for the diagnosis of Mycobacterium tuberculosis infection.

This study was designed to evaluate the possibility of monitoring Mycobacterium tuberculosis infection using a serological assay. A discriminant score comprising antigen fractions of 38, 28, 24 and 19 kDa, identified in western blots using the Mycobacterium bovis bacille Calmette-Guérin (BCG) A60 antigen complex was established in a sample of 57 purified protein derivative (PPD)-negative and 47 PPD-positive individuals. It was then tested in a group of 140 subjects undergoing BCG vaccination as a model of tuberculosis complex infection and in a group of human immunodeficiency virus (HIV)-infected individuals as a model of cell-mediated immunodeficiency-related risk of tuberculosis. The discriminant score identified 57 out of 57 (100%) PPD-positives and none (0%) of the 47 PPD-negatives. In the BCG vaccinated subjects, 1.4% tested positive before vaccination and 90% after vaccination. In the HIV-positive subjects, 90% of the PPD-positive and 5% of the PPD-negative subjects had a positive score. This study suggests that the western blot discriminant score is an accurate test to survey M. tuberculosis infection in serum samples.

Adult↗
PubMed1995

Evaluation of a novel tuberculosis complex-specific 34 kDa protein in the serological diagnosis of tuberculosis.

Tuberculosis (TB) serological testing with antigen complexes, although very sensitive, is not always as specific due to reactive serum antibodies in patients with inactive TB or nontuberculous infections. Since the use of recombinant M. tuberculosis proteins may enhance specificity, this study was designed to evaluate a novel 34 kDa tuberculosis complex-specific protein as a component of an antigen panel of recombinant proteins. Seventy patients with active TB (41 positive and 29 negative for acid-fast bacilli (AFB) in sputum) were evaluated, in comparison with 30 tuberculin purified protein derivative skin test positive (PPD+) and 30 PPD- normals, 20 subjects with inactive TB and 20 PPD+ subjects with nontuberculous pneumonia as controls. Serum antibody levels were quantified using enzyme linked immunosorbent assay (ELISA) tests with MS2-34, a fusion protein comprising the NH2-terminal 16 kDa of the 34 kDa protein, a recombinant 38 kDa protein (p38), and PPD. Using MS2-34 and p38 as an antigen panel in active TB patients yielded higher sensitivity and negative predictive value (sensitivity 86%; negative predictive value 91%) than using PPD (sensitivity 66%; negative predictive value 81%). Importantly, the MS2-34+p38 panel yielded a higher sensitivity (83%) than PPD (66%) in the subset of AFB- active TB patients. Thus, this novel protein increases sensitivity and specificity of serological testing for TB when used in panels of recombinant proteins.

Adolescent↗
PubMed1994

Use of discriminant analysis to assess disease activity in pulmonary tuberculosis with a panel of specific and nonspecific serum markers.

Several cell activation markers, acute phase reactants, enzymes, and antituberculous antibody serum levels have been proposed as possible markers to monitor disease activity in patients with tuberculosis. They have all shown limited sensitivity or specificity. The authors therefore attempted to generate a canonical variable using discriminant analysis, including sensitive and specific parameters, to be a reliable marker in classifying patients correctly during the course of pulmonary tuberculosis. The following parameters were selected: two soluble cell activation markers (soluble interleukin-2 receptor and sCD8); the levels of immunoglobulin (Ig) G and IgM antibodies against the A60 antigen complex; and the presence of specific antibodies directed to eight different A60 components, revealed by Western blot analysis. The tests were performed on sera from three groups of patients with pulmonary tuberculosis. The first group comprised 25 patients with onset tuberculosis, clinically active (OTCA), evaluated at the time of admission. The second group included 28 patients with chemotherapy-treated tuberculosis, clinically active (CTCA), 2 months after therapy had begun. The third group included 20 patients with tuberculosis, nonclinically active (TNCA), who had had at least 1 year of effective therapy. The authors obtained an 80.9% rate of correct classification for the three groups and a rate of 100% when OTCA and TNCA were compared. The patients with CTCA were scarcely differentiated and tended to be distributed into the two other groups. To improve the separation between patients with CTCA and those with OTCA, a second canonical variable was generated with a 91.7% rate of correct classification, as compared with 71% obtained using the sCD8 as the best single variable. The mean values of the last canonical variable were statistically different (Mann-Whitney test, P = .049) when stratified for acid fast bacilli-positive or negative CTCA patients (microscopic detection). Three patients, followed during the entire course their disease, were, as expected, correctly positioned with respect to the subsequent disease phases.

Adult↗