Biomedical subjects

Maria Bettinotti

Publications and source records attributed to Maria Bettinotti.

7 recordsLinked to original sources

PubMed2006

Identification of HLA-A33-restricted CMV pp65 epitopes as common targets for CD8(+) CMV-specific cytotoxic T lymphocytes.

OBJECTIVE: To identify an immunodominant HLA-A33-restricted epitope within the CMV matrix phosphoprotein 65 (pp65) that could be used to elicit CMV-specific CTLs. METHODS: A computer algorithm was used to identify pp65 peptides that were expected to bind to HLA-A33. The peptides were screened for their ability to reactivate memory T lymphocytes from CMV-seropositive HLA-A33 donors by using quantitative real-time RT-PCR. The most promising peptides were then tested for their ability to expand a CD8(+) population of HLA-A33 CTLs that produced interferon-gamma (IFN-gamma) and were cytotoxic to either peptide-loaded or CMV-infected target cells. RESULTS: Sixteen out of 250 peptides were selected using a computer algorithm and peptide stimulation by 8 out of the 16 induced a significant quantity of IFN-gamma mRNA transcript from CMV-seropositive HLA-A33 peripheral blood mononuclear cells. All the eight peptides induced consistent T-cell reactivation. One specifically, the peptide pp65(91-100) (SVNVHNPTGR), proved to be more active. T cells in vitro sensitized for 2 or 3 weeks with pp65(91-100) were cytotoxic to both HLA-A33 peptide-loaded and CMV-infected target cells. CONCLUSIONS: CMV pp65(91-100) is a new HLA-A33-restricted peptide that broadens the list of antigenic determinants that can be used for CMV adoptive immunotherapy.

Amino Acid Sequence↗

PubMed2006

The gene overexpressed in polycythemia rubra vera, PRV-1, and the gene encoding a neutrophil alloantigen, NB1, are alleles of a single gene, CD177, in chromosome band 19q13.31.

BACKGROUND: PRV-1 mRNA is overexpressed by neutrophils from polycythemia vera patients and is homologous to NB1 a gene overexpressed in reactive neutrophilia. STUDY DESIGN AND METHODS: These investigations were designed to confirm searches of genome databases suggesting that PRV-1 and NB1 are alleles of the same gene, CD177, and confirm a pseudogene adjacent to CD177. Methods included polymerase chain reaction (PCR), cloning, sequencing, and fluorescent hybridization studies. RESULTS: The coding region of PRV-1 was PCR-amplified from human fetal RNA, cloned, and used to screen the RPCI-11 bacterial artificial chromosome (BAC) library. Five BACs were reactive with the PRV-1 probe. PCR analysis of the BACs with primers encompassing PRV-1 exons, containing four known single-nucleotide polymorphisms, followed by sequencing rendered amplicons identical to PRV-1 in all five BACs. Analysis of all five by restriction digestion yielded fragments possible only if both the gene and the pseudogene are present. End sequencing of the BACs localized them to the same chromosome region. G-banding and fluorescence in situ hybridization at the 400- and 850-band levels of resolution mapped one BAC to chromosome band 19q13.2 and sublocalized the BAC to band 19q13.31, respectively. CONCLUSION: PRV-1 and NB1 are alleles of the same gene now referred to as CD177. Changes in CD177 expression may be a marker of increased or decreased myelopoiesis and are therefore an effect of, rather than a cause of, myeloproliferative disorders.

Alleles↗

PubMed2004

CD177: A member of the Ly-6 gene superfamily involved with neutrophil proliferation and polycythemia vera.

Genes in the Leukocyte Antigen 6 (Ly-6) superfamily encode glycosyl-phosphatidylinositol (GPI) anchored glycoproteins (gp) with conserved domains of 70 to 100 amino acids and 8 to 10 cysteine residues. Murine Ly-6 genes encode important lymphocyte and hematopoietic stem cell antigens. Recently, a new member of the human Ly-6 gene superfamily has been described, CD177. CD177 is polymorphic and has at least two alleles, PRV-1 and NB1. CD177 was first described as PRV-1, a gene that is overexpressed in neutrophils from approximately 95% of patients with polycythemia vera and from about half of patients with essential thrombocythemia. CD177 encodes NB1 gp, a 58-64 kD GPI gp that is expressed by neutrophils and neutrophil precursors. NB1 gp carries Human Neutrophil Antigen (HNA)-2a. Investigators working to identify the gene encoding NB1 gp called the CD177 allele they described NB1. NB1 gp is unusual in that neutrophils from some healthy people lack the NB1 gp completely and in most people NB1 gp is expressed by a subpopulation of neutrophils. The function of NB1 gp and the role of CD177 in the pathogenesis and clinical course of polycythemia vera and essential thrombocythemia are not yet known. However, measuring neutrophil CD177 mRNA levels has become an important marker for diagnosing the myeloproliferative disorders polycythemia vera and essential thrombocythemia.

Journal Article↗

PubMed2004

CD177 polymorphisms: correlation between high-frequency single nucleotide polymorphisms and neutrophil surface protein expression.

BACKGROUND: Human neutrophil antigen-2a (NB1) is located on NB1 glycoprotein, which is expressed on subpopulations of neutrophils. PRV-1 is a gene that is over-expressed in neutrophils from patients with polycythemia rubra vera. The gene encoding NB1 differs from PRV-1 at four nucleotides. The purpose of this study was to determine if PRV-1 and NB1 are alleles of the same gene or two separate genes; and, moreover, if they are alleles, to explore potential correlations to NB1 glycoprotein expression. STUDY DESIGN AND METHODS: Primer pairs were used to amplify WBC DNA in the regions surrounding the four NB1 polymorphic sites. The four resulting amplicons were sequenced. The size of the neutrophil population in each donor that stained brightly with CD177 antibody was assessed by flow cytometry. RESULTS: If PRV-1 and NB1 are separate genes, then all people should be heterozygous for the PRV-1/NB1 polymorphisms. Because 11 of 23 donors were homozygous for PRV-1 polymorphisms at all four sites, PRV-1 and NB1 are alleles of the same gene, CD177. When the sequenced exons were compared with PRV-1, 13 single nucleotide polymorphisms (SNPs) that result in amino acid changes were found. The G42C NB1 polymorphism, found in 10 donors, was the most common SNP. The size of the CD177 bright-staining neutrophil population was greater in 42C/C donors than in 42G/G donors (66 +/- 20% vs. 41 +/- 21%, p = 0.004). CONCLUSIONS: PRV-1 and NB1 are alleles of the polymorphic gene CD177. The most common SNP in bp 42 predicted an amino acid change that may have an effect on protein expression.

Black or African American↗

PubMed2003

Expression of human neutrophil antigen-2a (NB1) is increased in pregnancy.

BACKGROUND: The expression of human neutrophil antigen-2a (HNA-2a) or NB1 is highly variable. This study investigated variations in neutrophil expression of HNA-2a by comparing the expression of epitopes recognized by three HNA-2a-specific CD177 antibodies among healthy adults and pregnant women. STUDY DESIGN AND METHODS: Flow cytometry was used to compare the neutrophil reactions of three CD177 antibodies (MEM-166, TAG4, and 7D8) among 52 healthy adults. Because HNA-2a expression is greater in women than men, neutrophils were studied from 21 women early in pregnancy and 23 women late in pregnancy. RESULTS: All three CD177 antibodies reacted with two populations of neutrophils: a brightly staining population and a dimly staining population. Among the 52 healthy adults, there was no difference in the mean size of the brightly reactive population of neutrophils recognized by each antibody (46 +/- 23% for MEM-166, 49 +/- 23% for TAG4, and 49 +/- 23% for 7D8), and all three antibodies were specific for HNA-2a. For all three antibodies the size of the antigen bright neutrophil population was greater on neutrophils collected both early and late in pregnancy than in healthy adults. There was no difference in the size of the antigen bright population among neutrophils tested early and late in pregnancy. CONCLUSION: HNA-2a antigen expression increases in pregnancy. Some of the variations in neutrophil expression of HNA-2a among individuals is likely due to differences in gene regulation or differences in post-translational protein modifications rather than gene polymorphisms.

Adult↗

PubMed2003

The matrix protein pp65(341-350): a peptide that induces ex vivo stimulation and in vitro expansion of CMV-specific CD8+ T cells in subjects bearing either HLA-A2402 or A0101 allele.

BACKGROUND: The stimulation of PBMNCs with HLA Class I restricted synthetic peptides derived from CMV phosphorylated matrix protein 65 (pp65) evokes CMV-specific cytotoxic T lymphocyte (CTL) activity, a necessary condition for initiating adoptive immunotherapy against CMV-related diseases in immune-compromised patients. It was previously demonstrated that the CMV decamer (10-mer) peptide pp65(341-350), QYDPVAALFF, was able to induce CMV-specific CTLs in HLA-A*2402 CMV-seropositive individuals. STUDY DESIGN AND METHOD: We investigated the ability of the peptide pp65(341-350) to reactivate memory CD8+ T cells in CMV-seropositive subjects bearing either the HLA-A24 or A1 allele. CTL responses were measured by IFN-gamma mRNA expression and IFN-gamma protein production as well as cytotoxic activity. RESULTS: In this study it was found that peptide pp65(341-350) induced a specific reactivation of memory CD8+ T cells from CMV-seropositive donors expressing either HLA-A*2402 and/or HLA-A*0101. Moreover, a pp65(341-350)-specific selection and expansion using PBMNCs of CMV-seropositive donors bearing both HLA-A*2402 and HLA-A*0101 alleles produced cytotoxic CTLs to both HLA-A24 and A1 peptide-pulsed and autologous CMV-infected target cells. CONCLUSION: The results demonstrate that pp65(341-350) induced a specific CTL activity at both molecular and protein levels and that the peptide is specifically processed, presented, and recognized by subjects bearing HLA-A*2402 and/or A*0101. These findings suggest that it may be possible to use this single immune dominant peptide to induce and expand CMV-reactive CTLs for the treatment of individuals with both HLA-A24 and A1 types.

Alleles↗

PubMedDate not supplied

Identification of immune dominant cytomegalovirus epitopes using quantitative real-time polymerase chain reactions to measure interferon-gamma production by peptide-stimulated peripheral blood mononuclear cells.

The identification of HLA restricted immune dominant cytotoxic T cell (CTL) epitopes limits immune therapy. Cytomegalovirus (CMV) disease remains a significant cause of morbidity after allogeneic stem cell transplantation. Adoptive immune therapy using CTLs stimulated with immune dominant CMV pp65 peptides may be effective in preventing CMV disease, but immune dominant CMV peptides have been identified for only a few HLA class I molecules. The purpose of this study was to use a novel molecular system to establish a rapid and precise method to identify new HLA-restricted CMV epitopes. Cytomegalovirus pp65 peptides expected to bind to the HLA-24 molecule were identified with a computer algorithm. Five candidate peptides were screened by direct ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) from CMV-seropositive HLA-A*2402 individuals, and quantitative real time PCR (qRT-PCR) was used to evaluate CTL responses by measuring interferon-gamma (IFN-gamma) transcripts. One of the five candidate peptides, pp65341-350 (QYDPVAALFF), induced significant quantities of IFN-gamma mRNA production after 3 hours. PBMCs from CMV-seropositive HLA-A*2402 individuals sensitized in vitro with pp65341-350 also recognized CMV-infected targets. In conclusion, the measurement of IFN-gamma mRNA by qRT-PCR can be used to detect CTL responses 3 hours after peptide stimulation of a small quantity of PBMCs. This method has an advantage over other methods used to identify immune dominant epitopes in that it does not require in vitro expansion of CTLs with cytokines or virally infected targets. As a result, this method measures naturally induced immune reactions.

Antigens, Viral↗