Biomedical subjects

S Fiorentini

Publications and source records attributed to S Fiorentini.

At least 37 records · Page 2Linked to original sources

PubMed1997

Humanization of an antibody recognizing a breast cancer specific epitope by CDR-grafting.

BACKGROUND: Muc1-H23 is a cell surface mucin that is expressed on normal breast luminal epithelial cells and over-expressed in most breast tumors. In addition, Muc-1 expressed by malignant cells is glycosylated differently than Muc-1 expressed by normal cells. This difference in glycosylation exposes a peptide epitope on malignant cells which is not exposed on normal cells. Murine monoclonal antibody H23 recognizes this epitope and stains 91% of breast cancers, but only 1/56 non-malignant breast tissue samples. OBJECTIVE: To create a human antibody that was equivalent to H23 for potential uses in imaging and/or the therapy of breast cancer. STUDY DESIGN: We decided to humanize H23 by CDR-grafting using overlap PCR, and to this end, designed and constructed a bacterial expression vector that would allow V-regions, cloned via unique restriction sites, to be expressed as Fab fragments. In this way, we hoped to be able to rapidly evaluate Fab constructs for binding to Muc-1 and to cells and tissue sections that expressed the antigen. RESULTS: A fully humanized Fab fragment was able to bind Muc-1 peptide, as well as breast cancer cells known to express the epitope and tissue sections, generally showing the same reactivity as the native antibody. In addition, an analysis of sFab expressed with a [His]6 tag preceded by a factor Xa proteolytic cleavage site suggested that E. coli periplasmic signal peptidase was able to cleave the factor Xa site, thereby removing the [His]6 tag. CONCLUSION: We have generated a human antibody that is capable of recognizing a tumor specific epitope expressed by 91% of breast cancers.

Antigens, Neoplasm↗

PubMed1996

Evidence for distinct contributions of heavy and light chains to restriction of antibody recognition of the HIV-1 principal neutralization determinant.

We have used phage Ab display technology to analyze two mAbs to HIV-1 envelope proteins gp120 and gp41. From the data obtained we are able to demonstrate that the recognition of the principal neutralization determinant of different strains of HIV-1 by neutralizing mAb M77 is restricted by its heavy and light chains in different ways. Native M77 is able to recognize and neutralize HIV-1 strain IIIB through binding to the gp120 V3 loop. M77 is unable to recognize strains of HIV-1 that differ on either the left or right side of the V3 loop tip. A chain-switched Fab fragment containing the M77 Fd fragment and a different light chain was able to recognize HIV-1 strains that differ from IIIB on the left side but not the right side of the V3 loop tip.

Amino Acid Sequence↗

PubMed1996

Heat-killed Bacillus subtilis inhibits T-cell proliferative response to mitogens and recall antigens.

Heat-killed vegetative forms of Bacillus subtilis were found to impair considerably the capacity of human T-lymphocytes to secrete interleukin-2 (IL-2) and to proliferate (in terms of [3H]thymidine incorporation) after phytohaemagglutinin (PHA) stimulation. B. subtilis was also found to interfere with T-cell proliferation induced by concanavalin A (Con A) and the recall antigen tetanus toxoid (TT). The suppressive activity was dependent on bacterial concentration, and was not ascribed to mitogen, medium-nutrient absorption or cell killing. Moreover, B. subtilis did not interfere with mitogen-induced IL-2 receptor expression on the T-cell surface. On the other hand, B. subtilis did not interfere with T-cell proliferation induced by phorbol myristate acetate (PMA) and ionomycin stimulation. All data obtained suggest the binding of B. subtilis subcomponents to- or very close to-the T-cell receptor (TCR). Identification and purification of the basic structure(s) or component(s) of B. subtilis with TCR antagonist activity in vitro will help to exploit different aspects of T-cell activity and development, and possibly, will provide a means of specific control or modification of the immune response.

Bacillus subtilis↗

PubMed1996

IFN-gamma restores HIV- and non-HIV-specific cell mediated immune response in vitro and its activity is neutralized by antibodies from patients with AIDS.

The addition of IFN-gamma to cultures of peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic HIV-infected patients increased cell proliferation in response to HIV envelope synthetic peptides (Env), influenza A virus (VIRUS), and allogeneic lymphocytes (ALLO) but not to phytohaemagglutinin (PHA) stimulation. F(Ab)2 fragments of IgG purified from the sera of HIV-seropositive patients specifically interfered with IFN-gamma-induced cell proliferation in response to recall antigens. Neutralization of the lymphokine activity was found to be sustained by specific IFN-gamma antibodies. Data obtained demonstrate that IFN-gamma can restore the cell-mediated immunity of a number of asymptomatic HIV+ individuals in vitro, while IFN-gamma antibodies present in sera of patients with AIDS interfere with the activity of the lymphokine.

Acquired Immunodeficiency Syndrome↗

PubMed1995

A monoclonal antibody to the NH2-terminal region of human interferon-gamma inhibits its antiproliferative activity without affecting its internalization.

MAb IGMB-15, an anti-hIFN-gamma MAb, neutralizes the antiproliferative activity of hIFN-gamma without affecting that of hIFN-alpha or hIFN-beta. The neutralizing capacity of MAb IGMB-15 is wide: it has been assessed on cell lines whose origin and sensitivity to hIFN-gamma differ. The binding of hIFN-gamma to its receptor and its subsequent internalization into the target cell were not influenced by the antibody. MAb IGMB-15 has been found to interact with hIFN-gamma in solution but not when the lymphokine was associated with its cell surface receptor, showing that the growth of certain cell lines can be inhibited at the cell membrane level. This finding is consistent with the existence of an accessory factor responsible for the antiproliferative activity of hIFN-gamma.

Antibodies, Monoclonal↗

PubMed1995

V-region and class specific RT-PCR amplification of human immunoglobulin heavy and light chain genes from B-cell lines.

We have designed and tested primers that amplify complete human kappa and lambda light chain genes, and human Fd fragments from gamma, mu and alpha heavy chain genes. These primers were tested for efficiency and specificity on monoclonal sources of human immunoglobulin RNA, obtained from human B-cell lines of known immunoglobulin gene expression. Analysis of the sequences derived from these B-cells confirms the specificity of the PCR primers and the extent of somatic mutation seen in different B-cell malignancies supports existing concepts for differing aetiologies in the tumours concerned.

Amino Acid Sequence↗

PubMed1994

Development of a flow cytometric assay for the detection and measurement of neutralizing antibodies against human immunodeficiency virus.

Cells infected with human immunodeficiency virus type 1 (HIV-1) develop viral antigens which can be detected by immunofluorescence. We developed a flow cytometric immunofluorescence assay (FIFA) which provides a quantitative analysis of HIV-1 p24 using a monoclonal antibody (mAb) as a specific reagent. The reduction of HIV p24 antigen expression in viral infected cells was then used to determine HIV neutralizing antibody titers in human sera. Results obtained by FIFA for detecting neutralizing antibodies against HIV-1, when compared with results obtained by indirect immunofluorescence assay (IFA), showed an overall index of agreement of 94.1%. The correlation between the neutralizing antibody titers obtained with each method was found to be highly significant (ro = 0.8; p < 0.01). The simple methodology and the adaptability of this FIFA to various assay conditions make it suitable for routine purposes and for assessing the efficacy of vaccination strategies.

Antibodies, Monoclonal↗

PubMed1969