Biomedical subjects

Massimo Amicosante

Publications and source records attributed to Massimo Amicosante.

14 recordsLinked to original sources

PubMed2006

Molecular epidemiology: HIV-1 and HCV sequences from Libyan outbreak.

In 1998, outbreaks of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infection were reported in children attending Al-Fateh Hospital in Benghazi, Libya. Here we use molecular phylogenetic techniques to analyse new virus sequences from these outbreaks. We find that the HIV-1 and HCV strains were already circulating and prevalent in this hospital and its environs before the arrival in March 1998 of the foreign medical staff (five Bulgarian nurses and a Palestinian doctor) who stand accused of transmitting the HIV strain to the children.

Child↗

PubMed2006

T cell recognition in chronic beryllium disease.

Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by beryllium exposure in the workplace and is characterized by the accumulation of beryllium-specific CD4(+) T cells. Depending on genetic susceptibility and the nature of the exposure, CBD occurs in up to 20% of exposed workers. Genetic susceptibility has been associated with particular HLA-DP alleles, especially those possessing a negatively charged glutamic acid residue at the 69th position of the beta-chain. The mechanism for this association lies in the ability of these HLA-DP molecules to bind and present beryllium to pathogenic CD4(+) T cells. Large numbers of effector memory, beryllium-specific CD4(+) T cells are recruited to the lung of these subjects and secrete Th1-type cytokines upon beryllium recognition. The presence of circulating beryllium-specific CD4(+) T cells directly correlates with the severity of lymphocytic alveolitis. With the presence of a known antigenic stimulus, CBD serves as an important model of immune-mediated, organ destruction. Thus, our findings in CBD have important implications for studies in autoimmune diseases, in particular those with an unknown inciting antigen and an inaccessible target organ.

Antigen Presentation↗

PubMed2005

Identification of HLA-DRPhebeta47 as the susceptibility marker of hypersensitivity to beryllium in individuals lacking the berylliosis-associated supratypic marker HLA-DPGlubeta69.

BACKGROUND: Susceptibility to beryllium (Be)-hypersensitivity (BH) has been associated with HLA-DP alleles carrying a glutamate at position 69 of the HLA-DP beta-chain (HLA-DPGlu69) and with several HLA-DP, -DQ and -DR alleles and polymorphisms. However, no genetic associations have been found between BH affected subjects not carrying the HLA-DPGlu69 susceptibility marker. METHODS: In this report, we re-evaluated an already described patient populations after 7 years of follow-up including new 29 identified BH subjects. An overall population 36 berylliosis patients and 38 Be-sensitization without lung granulomas and 86 Be-exposed controls was analysed to assess the role of the individual HLA-class II polymorphisms associated with BH-susceptibility in HLA-DPGlu69 negative subjects by univariate and multivariate analysis. RESULTS: As previously observed in this population the HLA-DPGlu69 markers was present in higher frequency in berylliosis patients (31 out of 36, 86%) than in Be-sensitized (21 out of 38, 55%, p = 0.008 vs berylliosis) and 41 out of 86 (48%, p < 0.0001 vs berylliosis, p = 0.55 vs Be-sensitized) Be-exposed controls.However, 22 subjects presenting BH did not carry the HLA-DPGlu69 marker. We thus evaluated the contribution of all the HLA-DR, -DP and -DQ polymorphisms in determining BH susceptibility in this subgroup of HLA-Glu69 subjects. In HLA-DPGlu69-negatives a significant association with BH was found for the HLA-DQLeu26, for the HLA-DRB1 locus residues Ser13, Tyr26, His32, Asn37, Phe47 and Arg74 and for the HLA-DRB3 locus clusterized residues Arg11, Tyr26, Asp28, Leu38, Ser60 and Arg74. HLA-DRPhe47 (OR 2.956, p < 0.05) resulting independently associated with BH. Further, Be-stimulated T-cell proliferation in the HLA-DPGlu69-negative subjects (all carrying HLA-DRPhe47) was inhibited by the anti-HLA-DR antibody (range 70-92% inhibition) significantly more than by the anti-HLA-DP antibody (range: 6-29%; p < 0.02 compared to anti-HLA-DR) while it was not affected by the anti-HLA-DQ antibody. CONCLUSION: We conclude that HLA-DPGlu69 is the primary marker of Be-hypersensitivity and HLA-DRPhe47 is associated with BH in Glu69-negative subjects, likely playing a role in Be-presentation and sensitization.

Adult↗

PubMed2005

Function associated transforming growth factor-beta gene polymorphism in chronic beryllium disease.

Chronic beryllium disease (CBD) is a rare occupational, granulomatous lung disease clinically resembling sarcoidosis. The immune response to beryllium is thought to depend on genetic susceptibility. Although a glutamic acid in position 69 of the human leukocyte antigen-DP beta chain (HLA-DPB1-Glu69) is associated with the development of CBD, it cannot fully explain susceptibility. It is likely that additionally other genes are involved in regulating the immune and inflammatory response in the pathogenesis of this disease. Functional gene polymorphisms (PMs) of the tumor necrosis factor (TNF)A and transforming growth factor (TGF) beta(1) genes are suspected to modify the course of granulomatous disorders. We analyzed the TGF-beta(1) (codon 25) PM in 59 patients with CBD and 164 matched healthy controls, from two groups of European/Israeli and United States origin. Additionally, patients were genotyped for HLA class II gene variants and the TNFA (-308) PM. The most significant results were found for the TGF-beta(1) (codon 25) PM with a shift towards the low producing non-GG genotypes in the subgroup of European and Israeli patients with CBD (62.50% vs. 13.82% in healthy controls; P<0.001). This phenomenon was not observed in the group from the United States. Moreover, TGF-beta(1) (codon 25) PM genotype frequencies from United States CBD patients differed significantly from those of European and Israeli patients. In contrast, increased frequencies for the high producing TNFA2 allele were found only in the patients from the United States (28.20% vs. 8.96% in healthy controls; P<0.005) but not in the group of Europe and Israel. In conclusion, the increase in TGF-beta(1) (codon 25) PM genotype frequency associated with a low TGF-beta release suggests that immunoregulatory cytokines such as TGF-beta are involved in the pathogenesis of CBD. Moreover, based on the interaction of gene PMs associated with the control of the immune response, such as TNF-alpha and TGF-beta(1), with a specific immune response gene such as HLA-DPB1-Glu69 or other HLA-class II PMs driving the immune response to Be, the present data suggest that a combination of different genetic backgrounds determine susceptibility for the same immunopathological reaction and disease.

Adult↗

PubMed2005

Selected RD1 peptides for active tuberculosis diagnosis: comparison of a gamma interferon whole-blood enzyme-linked immunosorbent assay and an enzyme-linked immunospot assay.

We recently set up a gamma interferon (IFN-gamma) enzyme-linked immunospot assay (ELISPOT), using selected early secreted antigenic target 6 (ESAT-6) peptides, that appears specific for active tuberculosis (A-TB). However, ELISPOT is difficult to automate. Thus, the objective of this study was to determine if the same selected peptides may be used in a technique more suitable for routine work in clinical laboratories, such as whole-blood enzyme-linked immunosorbent assay (WBE). For this purpose, 27 patients with A-TB and 41 control patients were enrolled. Our WBE, using the already described selected peptides from ESAT-6 plus three new ones from culture filtrate protein 10, was performed, and data were compared with those obtained by ELISPOT. Using our selected peptides, IFN-gamma production, evaluated by both WBE and ELISPOT, was significantly higher in patients with A-TB than in controls (P < 0.0001). Statistical analysis showed a good correlation between the results obtained by WBE and ELISPOT (r = 0.80, P < 0.001). To substantiate our data, we compared our WBE results with those obtained by QuantiFERON-TB Gold, a whole-blood assay based on region of difference 1 (RD1) overlapping peptides approved for TB infection diagnosis. We observed a slightly higher sensitivity with QuantiFERON-TB Gold than with our WBE (89% versus 81%); however, our test provided a better specificity result (90% versus 68%). In conclusion, results obtained by WBE based on selected RD1 peptides significantly correlate with those generated by ELISPOT. Moreover, our assay appears more specific for A-TB diagnosis than QuantiFERON-TB Gold, and thus it may represent a complementary tool for A-TB diagnosis for routine use in clinical laboratories.

Antigens, Bacterial↗

PubMed2005

Role of the berylliosis-associated HLA-DPGlu69 supratypic variant in determining the response to beryllium in a blood T-cells beryllium-stimulated proliferation test.

BACKGROUND AND AIM OF THE WORK: Exposure to beryllium (Be) compounds may cause sensitization as revealed by blood T-cell proliferation against Be in a standardized Be-stimulated T-cell proliferation test (BeLPT). Further, susceptibility to Be hypersensitivity has been associated with the expression of HLA-DP allelic variants carrying a glutamate residue at position 69 of the beta-chain (HLA-DPGlu69) in more than 80% of affected subjects and, at lower frequency, with other HLA-DP, -DQ and -DR alleles/polymorphisms. The aim of this study was to assess whether heterozygous or homozygous carriage of the HLA-DPGlu69 marker may dictate the intensity of the T-cell response to Be in vitro. METHODS: The results of the blood Be-LPT, performed in a single laboratory on 165 subjects (86 Be-exposed controls, 38 Be-sensitized without lung granulomas and 41 berylliosis cases) identified at a single large Be production factory, were analyzed for their realtionship with the HLA-DPGlu69 status as determined by high resolution HLA-DP typing. RESULTS: Be-sensitized subjects carrying HLA-DPGlu69 presented a significantly higher T-cell response to Be (mean SI: 24.6 +/- 38.7) than the HLA-DPGlu69-negative subjects (mean SI: 11.8 +/- 6.6, p = 0.021). Furthermore, HLA-DPGlu69-positive subjects presented a higher frequency of positive Be-LPT tests (mean frequency 0.36 +/- 0.23) compared to the HLA-DPGlu69-negatives (mean frequency: 0.22 +/- 0.15; p = 0.002). HLA-DPGlu69 homozygosity was not associated with an increased response to Be in the BeLPT CONCLUSIONS: These results indicate that the expression of HLA-DPGlu69 determines higher T-cell proliferation rates and more consistent resposes to Be in vitro in the BeLPT test, both in the homozygous and the eterozygous state, possibly leading to an underestimation of the numbers of HLA-DPGlu69-negative sensitized subjects within exposed populations.

Adult↗

PubMed2004

Use of a T cell-based assay for monitoring efficacy of antituberculosis therapy.

Monitoring the efficacy of antituberculosis therapy is crucial both for the individual patient and for better control of the spread of tuberculosis. We studied 18 patients with microbiologically confirmed tuberculosis, both at the time of diagnosis and 3 months after they started therapy, using an in vitro assay that detects T cell-mediated interferon- gamma response to selected peptides of Mycobacterium tuberculosis-specific early secretory antigenic target 6 (ESAT-6) protein. All patients had positive results at diagnosis; however, 3 months later, the response to ESAT-6 peptides was still detectable only in the 5 patients with microbiological isolation and/or absence of clinical improvement after treatment. On the basis of these data, we conclude that our assay is a useful tool in monitoring the efficacy of antituberculosis therapy.

Adult↗

PubMed2004

Analysis of TNF-alpha promoter polymorphisms in the susceptibility to beryllium hypersensitivity.

BACKGROUND AND AIM OF THE WORK: In susceptible individuals beryllium (Be)-exposure may cause Be-hypersensitivity (BH), leading to a spectrum of immune abnormalities ranging from the systemic responsiveness to Be to the CD4+ T-cell dominated chronic granulomatous pneumonitis known as berylliosis. Two gene markers have been previously associated with berylliosis: HLA-DP allelic variants carrying glutamate in position 69 of the beta-chain and the high TNF-alpha production-associated TNF-alpha promoter allele TNFA2. Since a number of TNF-alpha promoter sequence variants have been associated with higher or lower gene-expression, the entire TNF-alpha promoter region was screened for BH-associated variants. METHODS: Denaturating High Performance Liquid Chromatography (DHPLC) analysis followed by DNA sequence analysis of the heteroduplex observed was performed on a DNA bank obtained from a Be-exposed population composed of 73 subjects with BH and 43 Be-exposed controls. RESULTS: The data show that the TNF-alpha TNFA2 variant (genotypic frequency: BH 26.7%, Be-exposed controls 5.8%; p < 0.0001), the -857T variant (BH 19.7%, Be-exposed controls 10.5%; p = 0.045) are significantly associated with BH and there is no linkage disequilibrium between them. Further, 64.4% of BH subjects carried at least one of higher TNF-alpha production-associated polymorphisms (Be-exposed controls 34.8%; p = 0.0036). CONCLUSIONS: The finding that TNF-alpha production-associated polymorphisms are carried at higher frequency by BH-affected compared to Be-exposed controls suggests that the TNF-alpha may play a central role in the determination of susceptibility to BH.

Adult↗

PubMed2003

Levels of interleukin-15 in plasma may predict a favorable outcome of structured treatment interruption in patients with chronic human immunodeficiency virus infection.

Structured treatment interruption (STI) may help to alleviate the problems associated with long-term antiretroviral therapy (ART) in human immunodeficiency virus (HIV)-infected patients. We analyzed the role that baseline levels of cytokines in plasma play as markers of a favorable outcome of STI. Two groups of patients were defined: STI responders and STI nonresponders. STI responders showed a higher baseline concentration of interleukin (IL)-15 in plasma than did STI nonresponders and showed lower levels of tumor necrosis factor (TNF)-alpha during STI. No differences were observed in levels of IL-2, IL-7, or interferon-alpha in plasma. Our data show that (1) levels of TNF-alpha in plasma correlate with HIV viremia and (2) monitoring baseline levels of IL-15 in plasma allows for the identification of a favorable outcome of STI.

Adult↗

PubMed2003

Quantifying recruitment of cytosolic peptides for HLA class I presentation: impact of TAP transport.

MHC class I ligands are recruited from the cytosolic peptide pool, whose size is likely to depend on the balance between peptide generation by the proteasome and peptide degradation by downstream peptidases. We asked what fraction of this pool is available for presentation, and how the size of this fraction is modulated by peptide affinity for the TAP transporters. A model epitope restricted by HLA-A2 and a series of epitope precursors with N-terminal extensions by single residues modifying TAP affinity were expressed in a system that allowed us to monitor and modulate cytosolic peptide copy numbers. We show that presentation varies strongly according to TAP affinities of the epitope precursors. The fraction of cytosolic peptides recruited for MHC presentation does not exceed 1% and is more than two logs lower for peptides with very low TAP affinities. Therefore, TAP affinity has a substantial impact on MHC class I Ag presentation.

ATP-Binding Cassette Transporters↗

PubMed2003

Functional analysis of HLA-DP polymorphism: a crucial role for DPbeta residues 9, 11, 35, 55, 56, 69 and 84-87 in T cell allorecognition and peptide binding.

The information available on the specific function of HLA-DP and the structure-function relationships is very limited. Here, single amino acid substitutions of HLA-DPB1*02012 have been used to analyze the role of polymorphic residues of the DPbeta1 domain on DP-mediated T cell allorecognition and peptide binding. Using a panel of specific anti-HLA-DP mAb, we identified the HLA-DP residues involved in the recognition by these mAb, with a crucial role for DPbeta56 for most of the mAb assayed. Individual substitutions at residues 9, 11, 35, 55, 56 and 69 completely abrogated T cell recognition mediated by two different HLA-DPw2-allospecific T cell clones (8.3 and 8.9). Interestingly single changes at positions 9, 11, 35 and 55 of HLA-DPbeta also altered the binding of peptides AAII(12-27) and IIP(53-65), natural ligands of the HLA-DPB1*02012 molecule. Individual changes at residues located in pocket 1 (84, 85, 86 and 87 from HLA-DPbeta) led to a partial reduction in cytotoxic T lymphocyte-mediated lysis and also partially affected peptide binding. However, the simultaneous substitution of these positions completely abolished both T cell allorecognition and peptide binding, suggesting a major role for polymorphisms at pocket 1 in HLA-DP function. Molecular modeling, used to predict changes induced by amino acid substitutions, supported the functional data. Taken together, these results strongly suggest that polymorphic residues 84, 85, 86 and 87 at pocket 1, residues 9, 35 and 55 at pocket 9, and residues 11 and 69 at pockets 6 and 4 respectively play a key role in HLA-DP function, probably by modifying the way the peptide is bound within the groove of HLA-DP2 and determining changes in the conformation of the MHC-peptide complex recognized by the TCR.

Antibodies, Monoclonal↗

PubMed2002

Expansion of pre-terminally differentiated CD8 T cells in chronic HIV-positive patients presenting a rapid viral rebound during structured treatment interruption.

OBJECTIVE: The influence of structured treatment interruption on effector/memory CD8 T cell dynamics was analysed in chronic HIV-infected patients showing a rapid or delayed viral rebound. DESIGN: Structured treatment interruption consisted of at least one month of discontinuation, followed by highly active antiretroviral therapy (HAART) resumption. Two groups of HIV structured treatment interruption patients were selected on the basis of plasma viral HIV-RNA value (> 30 000 copies/ml, branched DNA): group A (n = 14), patients with a rapid viral rebound (within one month) and group B (n = 6), patients with a delayed or no viral rebound (after a minimum of 4 months). METHODS: A clinical and immunological follow-up was performed at HAART suspension (t 0), one month from suspension (t 1), at HAART resumption (t 2), and 30 days from resumption (t 3). RESULTS: A sustained viral rebound was observed in group A patients, showing a rapid expansion of circulating CD8 T lymphocytes. In this group, the frequencies of CD8 T cells releasing IFN-gamma after mitogen-induced or Gag-specific stimulation were highly increased after HAART discontinuation. Nevertheless, these CD8 T lymphocytes were mainly composed of pre-terminally differentiated cytotoxic T lymphocytes (CTL) expressing a CCR7 CD27 CD45RA phenotype and a reduced amount of perforin. In contrast, group B patients showed no significant changes in immunological parameters during a prolonged drug-free period. CONCLUSION: These data indicate that monitoring CD8 T cell dynamics during structured treatment interruption could be clinically relevant, and new therapeutic strategies should aim qualitatively to restore CTL effector functions.

Adult↗

PubMed2002

Computer-based design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte assay for monitoring HIV-specific immunity.

BACKGROUND: Human immunodeficiency virus (HIV)- specific CD8-positive cytotoxic T-lymphocytes (CTL) play a key role in controlling HIV infection. Monitoring CTL response could be clinically relevant during structured therapy interruption (STI), HIV exposure, and vaccine trials. However, HLA patients' restriction and HIV variability limited the development of a CTL assay with broad specificity. MATERIALS AND METHODS: We designed an HLA-class I/HIV-1 clade independent assay for assessing HIV- specific CTL by using a computer-assisted selection ofthe CTL epitopes. Twenty-eight 15-mers were selected by peptide-binding motifs analysis using different databases (HIV-Immunology Database, SYFPEITHI, BIMAS). Altogether they putatively bind to more than 90% of HLA haplotypes in different populations, with an overall HIV-1 variability below 9%. The peptide pool was used as an antigen in an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CTL response. RESULTS: The test can be performed using both fresh and cryopreserved peripheral blood mononuclear cells (PBMC), whereas GAG protein as antigen works only on fresh PBMC. A significantly higher CTL response with respect to HIV-negative controls was detected in all HIV-1 infected subjects of two groups of patients with different ethnicities (Caucasians and Africans) and coming from areas with different HIV-1 clade prevalences (clade B and A/G, respectively). In Caucasian patients, after month of STI, the number of HIV-1 specific CTL (2,896 +/- 2,780 IFN-gamma specific CD8 cells/ml) was significantly higher than that found at enrolment (2,125 +/- 4,426 IFN-gamma specific CD8 cells/ml, p< 0.05). CONCLUSIONS: These data indicate that this CTL assay is broadly specific and could represent a useful clinical tool for HIV immunodiagnostic independent of HLA-haplotype and HIV-clade variabilities.

Adult↗

PubMedDate not supplied

Identification of early secretory antigen target-6 epitopes for the immunodiagnosis of active tuberculosis.

The early secretory antigenic target (ESAT)-6 purified protein and peptides from Mycobacterium tuberculosis were evaluated as antigens for the immunodiagnosis of tuberculosis (TB). Because the control of TB requires improved diagnostic procedures, efforts have increased to identify Mycobacterium tuberculosis-specific epitopes for the immunodiagnosis of active TB and to discriminate between active and latent states of infection. Two multiepitopic peptides from ESAT-6 protein were selected by computational analysis. Patients with active TB (7 HIV(+) and 20 HIV(-)) and control patients (17 HIV(+) and 28 HIV(-)) were enrolled. Enzyme-linked immunospot assay analysis for interferon-g expression by peripheral blood mononuclear cells was quantified after stimulation with selected ESAT-6 peptides, purified protein derivative, or the intact ESAT-6 protein. During active TB, 20 of 27 patients responded in vitro to ESAT-6 peptides and 23 of 27 patients to purified protein derivative. None of the controls without active TB, including individuals with latent TB infection, recognized ESAT-6 peptides. By contrast, latently infected individuals did respond in vitro to both intact ESAT-6 protein and purified protein derivative. Thus, high T-cell response frequencies to ESAT-6 peptides are present only during active TB and can be used to discriminate between active and latent forms of infection.

Adult↗