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G B Ferrara

Publications and source records attributed to G B Ferrara.

At least 19 recordsLinked to original sources
PubMed2001

Investigation of HLA class I downregulation in breast cancer by RT-PCR.

Downregulation of HLA class I antigen expression has been reported in a significant proportion of primary breast carcinomas suggesting an escape mechanism from CTL mediated lysis leading to tumor dissemination and metastasis. We have previously reported the biochemical and immunohistochemical analysis of HLA total class I (W6/32 mAb), alpha-chain (Q1/28,TP25.99 mAbs) and beta(2)-microglobulin (Namb-1 mAb) subunits expression in 25 primary breast carcinomas. This study at protein level resulted in the observation of three different HLA class I expression patterns by both techniques: high, low, and absent downregulation patterns. To better characterize the HLA class I antigens downregulation we extended such analysis also at RNA level by RT-PCR using HLA-A, HLA-B, HLA-C, and beta(2)-microglobulin specific primers either in breast cancer or normal tissues derived from the same patient. None (100%) of the alpha-chain genes analyzed in patient tumor tissues showed significant reduction of expression. In 10 patients out of 25 (40%) the beta(2)-microglobulin gene showed complete loss of expression compared with the corresponding normal tissue counterpart, which showed a constitutive expression, whereas in 2 patients (12.5%) its expression was comparable with the normal counterpart. Sequence analysis at genomic level revealed no defects affecting beta(2)-microglobulin gene in those patients showing lack of expression. Also TAP1 and TAP2 genes expression were investigated in order to confirm or exclude involvement of the MHC class I molecules assembling machinery. The RT-PCR approach mainly confirmed our beta(2)-microglobulin biochemical analysis indicating that in breast cancer specimens it is possible to address the HLA class I gene downregulation as a phenomenon occurring at post-transcriptional level mainly affecting the beta(2)-microglobulin gene expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Patients with neoplastic and nonneoplastic hematologic diseases acquire CTLA-4 antibodies after blood transfusion.

BACKGROUND: The presence of antibodies to CTLA-4, a negative regulator of T-cell activation, was investigated in multiply transfused patients with malignant and non- malignant hematologic diseases. A previous study showed that, in multiply transfused patients, an immune response against nuclear matrix proteins can be induced by WBCs undergoing apoptosis during RBC unit storage. This study evaluated whether the same phenomenon could be involved in the induction of CTLA-4 antibodies in the patients analyzed. STUDY DESIGN AND METHODS: Patient sera were tested for binding to the recombinant full-length CTLA-4 beta-galactosidase fusion protein by an ELISA. Immuno-fluorescence stainings were performed to analyze the CTLA-4 epitopes recognized by the antibodies and to detect such epitopes in the apoptotic cells present in the RBC units. RESULTS: CTLA-4 antibodies were found in multiply transfused patients with beta-thalassemia (40%) and with other hemolytic diseases (33%) including leukemias (42%). A higher incidence of CTLA-4 antibodies was found in patients receiving non-WBC-reduced blood (88%) than in those receiving WBC-reduced blood (26%). Immunofluorescence staining showed that WBCs undergoing apoptosis in the RBC unit expressed CTLA-4 epitopes. CONCLUSIONS: The apoptotic WBCs present in the RBC units, after cold storage, express CTLA-4 epitopes. These epitopes can be released and induce formation of CTLA-4 antibodies with profound implications in the development of autoimmune disorders and in facilitating tumor dissemination and metastasis.

Abatacept↗
PubMed2000

Analysis of CIITA encoding AIR-1 gene promoters in insulin-dependent diabetes mellitus and rheumatoid arthritis patients from the northeast of Italy: absence of sequence variability.

Qualitative and/or quantitative alterations in the expression of the MHC class II molecules affect the onset and maintenance of the immune response and may be the basis of a wide variety of disease states, such as autoimmunity and immunodeficiency.CIITA is a major physiological regulator of the expression of MHC class II genes. The availability of CIITA ap- pears generally essential for MHC class II gene expression, and hence its own transcriptional regulatory mechanisms result of fundamental importance for a correct homeostasis of the immune response. Therefore, it is possible to hypothesize that variability at the CIITA-encoding locus, AIR-1, could constitute an additional source of susceptible traits to autoimmune diseases. Mutations at AIR-1/CIITA promoters could modulate expression of CIITA. Variations in CIITA expression could influence the qualitative and quantitative expression of MHC class II molecules at cell surface. We have analyzed sequence variation at AIR-1/CIITA promoters by PCR-SSCP in 23 IDDM and 30 RA patients compared to a sample of 19 unaffected normal controls and 16 unaffected IDDM family members, for a total of 88 Caucasian subjects from the Northeast of Italy. No sequence difference was found at the four AIR-1/CIITA promoters between autoimmune patients and normal controls. Moreover, the promoters resulted invariant within the entire group of 88 subjects analyzed, comprising patients and controls. This finding suggests a possible selective advantage in maintaining CIITA upstream regulatory sequences invariant.

Arthritis, Rheumatoid↗
PubMed2000

HLA-G, -E, -F preworkshop: tools and protocols for analysis of non-classical class I genes transcription and protein expression.

Non-classical MHC class I HLA-E, -F, and -G molecules differ from classical class I histocompatibility antigens by specific patterns of transcription, protein expression, and immunological functions. Restriction of the expression pattern of these non-classical antigens may play a key role in modulation of immune responses during pregnancy and diseases but remains to be additionally defined. A specific component of the second International Conference on HLA-G and the 13th HLA-G Histocompatibility Workshop will be dedicated to the analysis of transcription and expression of non-classical class I genes in normal and pathological tissues. In a first step, referred to as the preworkshop, we here report the analysis and conclusions of a working group which was constituted to gather and validate optimal reagents and protocols allowing RT-PCR analysis of HLA-E, -F, -G transcript levels and flow cytometry and immunochemistry analysis of HLA-G expression in cells and tissues. As a result of this work, use of specific primers and probes detecting alternative transcripts of HLA-E, -F, and G have been validated in transfected cells expressing differential pattern of HLA class I antigens. Analysis of the specificity and affinity of collected antibodies has allowed definition of reagents to be proposed for immunochemistry and flow cytometry analysis of HLA-G expression in normal and pathological tissues during the workshop. This work has allowed constitution of an extended workshop group which is now initiating analysis of non-classical class I transcription and expression in various cells and tissues, a collective contribution that will additionally refine our view of the expression of these antigens in normal and pathological situations.

Antibodies, Monoclonal↗
PubMed2000

Biochemical analysis of HLA class I subunits expression in breast cancer tissues.

The expression of HLA class I alpha-chain and beta(2)-m subunits was studied at the protein level by a semiquantitative Western blot (WB) approach, in 25 primary breast tumors. The results indicated three pathways of alterations defined comparing the tumor WB gel band with the corresponding PBL gel band: (i) high downregulation pattern (the tumor WB gel band was < or =50% relative to the PBL band), which was found in 44% and 36% of tumors for alpha-chain and beta(2)-m, respectively; (ii) low downregulation pattern (the tumor gel band was between 51% and 75%), which was found in 24% and 20% of tumors for alpha-chain and beta(2)75%), which was found in 32% and 44% of tumors for alpha-chain and beta(2)-m, respectively. The concordance rate with immunohistochemistry (IHC) performed on the same tissue samples was 72% for alpha-chain and 64% for beta(2)-m. This study shows that the use of a semiquantitative WB technique can well define the levels of HLA class I antigens in an autologous setting allowing the biochemical analysis of HLA class I downregulation directly in solid tumor tissues. In addition, the WB technique can be a valuable tool to objectively support the IHC method.

Blotting, Western↗
PubMed2000

Molecular characterization and applications of recombinant scFv antibodies to CD152 co-stimulatory molecule.

Recombinant human monoclonal antibodies against CD152 have been generated by selecting a synthetic phage scFv library with purified CD152-Ig fusion protein. Sixteen scFv fragments were isolated which specifically react with CD152 by enzyme-linked immunoabsorbent assay (ELISA) and Western blot resulting in their clustering into two groups recognizing different antigenic determinants. One group of scFvs (#3, #13, #40, #44, #47, #51, #57, #80 #83) recognized an epitope on CD152 dimer whereas another group (#15, #18, #31, #35, #54, #72, #81) recognized an epitope on both dimeric and monomeric CD152 molecule suggesting their possible use in understanding the subunit structure of CD152 which is still controversial. Sequencing of the VH genes revealed that all the scFvs belonged to the VH3 gene family but they were different in CDR3 length and composition. It was possible to correlate specific CDR3 sequences with reactivity of the two groups of scFvs. Four scFvs, #3, #40, #81 and #83, each representative of one specific CDR3, were selected for further analysis. Competition ELISA experiments showed that they recognize CD152 in its native configuration and bound to different epitopes from the CD80/CD86 interaction site. The scFvs were able to stain human T lymphocytes stimulated either with anti-CD3 and CD28 antibodies or PHA, PMA and ionomycin by cytofluorimetry suggesting that they can be useful reagents for monitoring the kinetics of surface-bound and intracellular CD152.

Abatacept↗
PubMed2000

Novel associations among HLA-DQA1 and -DQB1 alleles, revealed by high-resolution sequence-based typing (SBT).

Althought it is a valuable tool for the identification of HLA alleles, sequence-based typing (SBT) presents difficulties when used to determine HLA-DQA1 and -DQB1 alleles. Specifically, some HLA-DQA1 alleles have a three-base deletion at codon 56 of exon 2 that interferes with the sequencing read. Moreover, the frequently used primers for HLA-DQB1 may co-amplify the HLA-DQB2 pseudogene. To overcome these problems, we amplified DQA1 exon 2 using five group-specific polymerase chain reactions (PCRs) which allowed separation of deleted from non-deleted DQA1 alleles. DQB1 exon 2 was amplified using two group-specific amplifications. To increase typing resolution, we also analyzed DQA1 exons 1, 3 and 4 and DQB1 exon 3 by PCR using sequence-specific primers (PCR-SSP) or SBT analysis. Using this method we found some important associations between DQA1 and DQB1 alleles: DQA1*05011 and DQB1*0201, DQA1*0505 and DQB1*03011, DQA1*01021 and DQB1*06, DQA1*01022 and DQB1*0502.

Alleles↗
PubMed2000

Melanomas and melanoma cell lines do not express HLA-G, and the expression cannot be induced by gammaIFN treatment.

HLA-G is an effective ligand of natural killer (NK) inhibitory receptors, HLA-G transcripts have been detected in several human tumors, and cytokines like gamma interferon (IFN) enable HLA-G molecules to be expressed. These findings are particularly upsetting in case of melanomas: IFN treatment is frequently included in melanoma therapeutic protocols, and downregulation of classical class I molecules occurs in nearly half of these tumors. Therefore, a melanoma cell downregulating classical class I and de novo expressing HLA-G, either constitutively or upon IFN treatment, is probably a stealthy target for the immune system, having inhibited both the cytotoxic T lymphocyte (CTL) and the NK activity. To elucidate this point we have investigated the expression of HLA-G molecules in 45 melanoma cell lines before and after gammaIFN treatment. Analysis was performed by immunofluorescence and flow cytometry, using the anti-HLA-G MoAbs 87G and G233, by Western blot, using the anti-HLA-G MEM/G1 MoAb and PAG1 antiserum, and by RT-PCR analysis. In addition, 8 melanoma tissues from patients free from therapy and 6 nevi were studied by immunohistochemistry using the 87G MoAb. No evidence was gathered of HLA-G expression, neither constitutive nor, in cell lines, after gammaIFN treatment. We therefore conclude that HLA-G expression is an uncommon event in melanomas, and that a therapy including IFNs cannot harm the patient by inducing the de novo expression of HLA-G molecules at least in its G1 isoform.

Antibodies, Monoclonal↗
PubMed2000

Identification of a new DRB3*02 allelic variant (DRB3*0209) by high-resolution sequence-based typing.

The HLA-DRB3/B4/B5 sequence-based typing method developed in this study in combination with PCR-SSP, enabled us to identify a new DRB3*02 allele, that was named as DRB3*0209 (GenBank accession number AF148518). This name has been officially assigned by the WHO Nomenclature Committee in May 1999. The new allele differs from DRB3*0207 by one substitution in codon 51 from AGG to ACG and another in codon 60 from TAC to TCC, resulting in aminoacid changes from Arg-->Thr (codon 51) and from Tyr-->Ser (codon 60). The DRB3*0209 allele was discovered in two related North Italian families. The fact that it was present in an hemizygous situation in three members of the paternal family and in one member of the secondary related family enabled us to isolate and sequence the new DRB3 allele without cloning, to identify its association with the DRB1 locus, and to generate an Epstein-Barr virus (EBV)-transformed cell line, now present in our ECBR (European Collection for Biomedical Research) Cell Line Bank.

Alleles↗
PubMed2000

HLA class II alleles associations of anticardiolipin and anti-beta2GPI antibodies in a large series of European patients with systemic lupus erythematosus.

The objective of this study was to determine the HLA class II associations of the anticardiolipin (aCL) and anti-beta2GPI (abeta2GPI) antibodies in a large series of European patients with systemic lupus erythematosus (SLE). A cohort of 577 European SLE patients was enrolled. aCL and abeta2GPI were measured by ELISA methods. Molecular typing of HLA-DRB1, DRB3, DRB4, DRB5, DQA1 and DQB1 loci was performed by the polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) method. aCL of IgG, IgM and IgA isotypes were detected in 22.8%, 14% and 13.9% of patients, respectively. IgG and IgM abeta2GPI were detected in 20% of patients. aCL showed positive association with HLA DRB1*04, DRB1*0402, DRB1*0403, DRB1*07, DRB3*0301, DQA1*0201, DQA1*0301, DQB1*0302, and negative association with DQA1*0501, DRB3*0202. abeta2GPI showed positive association with DRB1*0402, DRB1*0403, DQB1*0302. DRB1*0402 carried the highest relative risk for the presence of both aCL (RR=8. 1) and abeta2GPI (RR=4.6). Our results confirm the already described associations of aCL with HLA DR4 and DR7, but also demonstrate that, among the alleles at the DRB1*04 locus, the *0402 was most represented both in aCL and in abeta2GPI positive patients. In addition, HLA class II associations of abeta2GPI are for the first time extensively examined in a large cohort of European SLE patients.

Adolescent↗
PubMed2000

Anti-ganglioside antibodies in a large cohort of European patients with systemic lupus erythematosus: clinical, serological, and HLA class II gene associations. European Concerted Action on the Immunogenetics of SLE.

OBJECTIVE: To assay anti-ganglioside antibodies (aGM1) in sera of a large cohort of European patients with systemic lupus erythematosus (SLE) to define the prevalence of these autoantibodies in SLE; to evaluate the association of aGM1 with clinical manifestations and other autoantibodies found in SLE; and to search for aGM1 association with HLA class II alleles. METHODS: Four hundred forty-eight patients with SLE were consecutively enrolled in 8 centers from 6 European countries. All sera were tested for antinuclear antibodies by immunofluorescence on HEp-2 cells as substrate, anti-dsDNA, aGM1, aCL, abeta2-glycoprotein I (abeta2-GPI) antibodies by ELISA, and antineutrophil cytoplasmic antibodies (ANCA) by immunofluorescence and by ELISA. Genomic typing for HLA class II loci was performed by polymerase chain reaction-sequence specific oligonucleotide probe method. Clinical assessment was done at the time of enrolment. RESULTS: We found 41.9% of patients with clinical signs of neuropsychiatric involvement; 15.5% of patients were positive for aGM1, 8% of the IgG isotype and 8.6% of the IgM isotype; aGM1-IgG were associated with neuropsychiatric manifestations (NPM) (RR = 3.7), with migraine (RR = 2.4), with OBS (RR = 7.3), and with peripheral neuropathy (RR = 8.5). aGM1-IgM were associated with NPM (RR = 4) and with depression (RR = 3.4). Furthermore, the genetic study showed that aGM1-IgG were associated with HLA-DQB1*0404 (RR = 7.2) while aGM1-IgM were associated with HLA-DQB1*0605 (RR = 33.3). No associations were found between aGM1 and anti-dsDNA, aCL, abeta2GP1, or ANCA. CONCLUSION: Our results show aGM1 can be found in patients with SLE. aGM1 may play a pathogenetic role for some NPM in this condition.

Adolescent↗
PubMed1999

Low prevalence of selective human leukocyte antigen (HLA)-A and HLA-B epitope losses in early-passage tumor cell lines.

The down-regulation of human leukocyte antigen (HLA) class I molecules, especially the selective down-regulation of certain allelic products, is believed to represent a major mechanism of tumor escape from immune surveillance. In the present report, an original approach is described to precisely evaluate and classify HLA class I epitope losses in 30 cancer patients with malignant melanoma and lung, breast, endometrium, ovary, and colon carcinoma tumors. Early-passage tumor cell lines were established in culture from the corresponding metastatic tumor lesions obtained in each patient. Both the cell lines and the tumor lesions were compared, in their HLA-A and -B expression, to the peripheral blood mononuclear cells (PBMCs) obtained from the same patient (autologous PBMCs). On the basis of HLA-genotyping data, the appropriate monoclonal antibodies identifying mono- and poly-morphic HLA-A and HLA-B epitopes were selected from a panel of 34 antibodies for a total of 24 testable alleles. The selected antibodies were used not only in immunohistochemical assays on cryostatic tumor sections and cytospins of PBMCs but also in quantitative, sensitive flow cytometry assays on early-passage tumor cells and PBMC suspensions. With this latter method, a low overall HLA expression was detected in 26 tumor cell explants and a complete, generalized HLA-A, HLA-B, HLA-C loss in the remaining 4 cases. However, no complete, selective loss of any of the 45 tested HLA-A and HLA-B allomorphs was observed. Sequences from all of the HLA class I alleles could be detected at the genomic DNA level in tumor cells and tissues. At variance from the literature and the results of immunohistochemical experiments performed in parallel on the corresponding tumor lesions, the relative proportions of the various HLA epitopes were relatively preserved in each early-passage cell line/PBMC pair, and selective increases, rather than decreases, in the expression of polymorphic HLA epitopes had the highest prevalence and greatest magnitude. Our data suggest an alternative tumor stealth strategy in which up- and down-regulation are equally important. This alternative model of tumor-host interaction better fits the available models of tumor cell recognition by CTLs and natural killer cells bearing activatory and inhibitory receptors for HLA-A, HLA-B, HLA-C molecules.

Antibodies, Monoclonal↗
PubMed1999

Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients.

Bone marrow transplant (BMT) relies on the engraftment of donor hemopoietic precursors in the host marrow space. Colony forming units-fibroblasts (CFU-f), the precursor compartment for the osteogenic lineage, are essential to hemopoietic stem cell survival, proliferation and differentiation. We have studied CFU-f in donors (aged 5 months to 62 years) and in patients who had received allogeneic BMT (aged 2 months to 63 years). In donor marrows we found an inverse correlation between CFU-f frequency and age. In BMT recipients CFU-f frequencies were reduced by 60%-90% (p < 0.05) and the numbers did not recover up to 12 years after transplant. Stromal reconstitution to normal levels was found only in patients < 5 years old. In all patients studied CFU-f post-BMT were of host origin. Patients with low CFU-f levels displayed also a decreased bone mineral density (p < 0.05) and significantly reduced levels of long-term culture-initiating cells (LTC-IC) (p < 0.05). Our study demonstrates that the marrow stromal microenvironment is seriously and irreversibly damaged after BMT. Donor cells do not contribute to reconstitute the marrow microenvironment, whose residual CFU-fs remain of host origin.

Adolescent↗
PubMed1999

HLA-B locus sequence-based typing.

We describe an approach for HLA-B high-resolution typing. A single locus-specific amplification generates a 1-kb fragment useful for direct sequencing. Four internal primers are necessary for exon 2 and 3 cycle-sequencing in both directions. Fluorescent dye-labelled nucleotides are incorporated during cycle-sequencing and reaction products are analyzed in an automated DNA sequencer. At present, software programs allow automatic assignment of exon 2 only; analysis of exon 3 is not automatic. In the future, the development of more sophisticated software will improve allele assignment. The approach described in this work offers a precise and efficient identification of known allele sequences and at the same time can differentiate new alleles. Furthermore, it may be applied as a model for the development of similar molecular typing approaches for other polymorphic HLA loci.

Alleles↗
PubMed1999

Biosynthesis of HLA-C heavy chains in melanoma cells with multiple defects in the expression of HLA-A, -B, -C molecules.

Recent investigations have shown that malignant transformation may down-regulate the expression of class I HLA molecules, beta2-microglobulin (beta2m) and members of the antigen-processing machinery. In the present study, we HLA-genotyped and identified at a biochemical level the three (HLA-A25, -B8, -Cw7) class I alleles expressed by the previously described [D'Urso CM et al (1992) J Clin Invest 87: 284-292] beta2m-defective human melanoma FO-1 cell line and tested their ability to interact with calnexin, calreticulin and the TAP (transporter associated with antigen processing) complex. All these alleles were found to bind calnexin, but not calreticulin or the poorly expressed TAP complex, both in parental and beta2m-transfected FO-1 cells, demonstrating a complex defect of class I expression in FO-1 cells. In these conditions, Cw7 heavy chains interacted with calnexin more strongly than A25 and B8, and preferentially accumulated in the endoplasmic reticulum, in both a calnexin-associated and a calnexin-free form. In addition, they could be transported to the cell surface at low levels even in the absence of beta2m, without undergoing terminal glycosylation. These results establish a parallel between HLA-C and the murine Db and Ld molecules which have been found to be surface expressed and functional in beta2m-defective cells. They also demonstrate distinctive features of HLA-C molecules. We propose that the accumulation of several assembly intermediates of HLA-C might favour the binding of peptide antigens not readily bound by HLA-A and -B molecules in neoplastic cells with suboptimal class I expression.

Alleles↗
PubMed1999

HLA-A, -B, -C genotyping and expression in human nonlymphoid tumor cell lines.

A combination of molecular genotyping and protein biochemistry methods was used to assess the HLA-A, -B, -C genotyping and expression of six tumor cell lines. Four cell lines had been previously HLA typed by conventional serologic methods. Two could not be typed by serology because deficient in the surface expression of HLA-A, -B, -C molecules. As shown herein, all the 25 alleles carried by the six tested cell lines were typed at the DNA level. In addition, discrepancies between the previous serologic and the present DNA typing results were detected in 9 of the 21 tested serologic specificities. Typing at the protein level by isoelectric focusing and allele-specific monoclonal antibodies confirmed the DNA typing data. Our results exemplify the limits of the serologic typing procedures and demonstrate that molecular methods are highly desirable to conduct functional experiments and identify HLA losses in neoplastic cells at single allele level.

Alleles↗