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L D'Apice

Publications and source records attributed to L D'Apice.

9 recordsLinked to original sources
PubMed2001

Design of cassette vectors permitting cloning of all types of human TCR variable alpha and beta regions.

T cell clones are an irreplaceable asset for the study of immune responses relevant to human pathologies. Such cells, however, cannot always be maintained in long-term culture. In order to reconstitute functional human T cell receptors (TCRs) into stable and fast growing hybridoma T cells, we developed a general approach based on a versatile cassette system, which allows cloning of all types of human T cell receptor variable alpha and beta region genes fused to murine constant regions. These chimeric constructs are easily excised and transferred into expression vectors that can be used to transfect a human CD4-expressing murine T cell hybridoma recipient. The resulting transfectants are highly stable both in terms of T cell receptor-CD3 expression and IL-2 response to the specific antigenic stimulus. Using these cassette vectors, we reconstituted the original HLA-restricted antigen specificity for two human T cell clones, one recognizing an immunodominant epitope of HIV-1 gp120, and the other recognizing an immunodominant epitope of HIV-1 reverse transcriptase. We found that the reconstituted hybridomas maintain the ability of the original T cell clones to recognize the appropriate epitope in the context of the relevant MHC either as a synthetic peptide or after processing. Their unlimited growth capacity makes them particularly suited for in vitro studies.

Animals↗
PubMed2000

Identification of a new subset of cells exhibiting dendritic phenotypes in patients affected by breast proliferative disorders.

We report that a subset of circulating cells reacting with a monoclonal antibody raised against a protein marker is significantly increased in the peripheral blood of women carrying benign or malignant breast diseases, particularly in patients under 55 years of age with ductal mammary carcinomas. These cells were statistically (confidence level of 99%) less represented in a control population including healthy women or women carrying carcinomas of origin other than breast. Double staining analysis showed that they harbor markers of dendritic cells and exhibit endo- cytic activity, as determined by their ability to internalize FITC-dextran particles. Their dendritic morphology was further demonstrated by electron microscopy of sorted antibody-positive cells. However, expression of surface molecules, such as CD34 and CD14, usually not present in differentiated populations of dendritic cells was also observed. Adherent cells of patients with breast ductal carcinoma including mostly cells of this new subset were efficient stimulators of mixed lymphocyte reaction, attaining maximal stimulatory activity attained after TNFalpha treatment. In conclusion, we have shown that a subset of cells characterized by a phenotype suggestive of a yet undescribed stage of maturation of the dendritic cell lineage is accumulated in the blood of patients affected by breast proliferative disorders.

Adult↗
PubMed1999

Recognition of HIV-derived B and T cell epitopes displayed on filamentous phages.

The amino acid sequence of HIV reverse transcriptase (RT) from residue 248 to residue 262 was expressed on the surface of filamentous phage fd, fused to the major coat protein gVIIIp. The chimeric phage was used to assess the ability of anti-RT (248-262) human T cell lines and clones to become activated by the phage-displayed peptide. The RT peptide displayed on phage was recognized by the T-cells and induced production of Abs. However, not all T cells raised against the synthetic RT (248-262) peptide could respond. Lack of recognition did not depend on differences in the ability of different APCs to present the phage, but was apparently determined by the TCR specificity. The results presented here may be relevant to the design of recombinant protein-based subunit vaccines.

Animals↗
PubMed1998

Simultaneous identification of antibodies to Brucella abortus and Staphylococcus aureus in milk samples by flow cytometry.

Two flow cytometric assays are described herein. The single cytometric test (SCT) detects antibodies to either Brucella abortus or Staphylococcus aureus in the serum or milk of a cow or water buffalo. The double cytometric test (DCT) detects both anti-B. abortus and anti-S. aureus antibodies concurrently. In the SCT, the sample to be tested is incubated in succession with the antigen (either B. abortus or S. aureus) and the proper secondary antiserum (fluorescein isothiocyanate-labelled rabbit anti-cow immunoglobulin antiserum or rabbit anti-water buffalo immunoglobulin antiserum). In the DCT, the sample to be tested is incubated first with B. abortus and S. aureus antigens and then with the secondary antiserum. The B. abortus antigen used in the DCT is covalently bound to 3-microm-diameter latex particles. The difference in size between B. abortus and S. aureus permits the establishment of whether the antibodies are directed against one, the other, or both antigens. When compared to the complement fixation test, the SCT and DCT each show a specificity and a sensitivity of 100%. The SCT has been used previously to detect anti-S. aureus antibodies. Here its use is extended to the detection of anti-B. abortus antibodies. The DCT is described here for the first time. The DCT appears to be useful for large-scale brucellosis eradication programs. It offers the possibility of using one test to identify animals that are serologically positive for both B. abortus and S. aureus.

Animals↗
PubMed1997

DNA content differences in laboratory mouse strains determined by flow cytometry.

The nuclear DNA content of seven mouse laboratory strains has been measured by flow cytometry. The differences observed between strains as well as those between sexes within the strain were all statistically significant. The highest DNA content (approximately 6.4 pg/female nucleus) was found in the Balb/c strain; the lowest (approximately 5.7 pg/male nucleus) in the C3H/he strain. The difference between sexes varied from 1.6% (in CD-1 mice) to 6.3% (in nude mice). The interest of these results is twofold. First, the mouse can now be used to study the adaptive significance of genome size variation, so far studied only in plants. Second, DNA content analysis can become a quick method for mouse strain identification.

Animals↗
PubMed1997

Identification by flow cytometry of seiridin, one of the main phytotoxins produced by three Seiridium species pathogenic to cypress.

Seiridin (SE), one of the main phytotoxins produced in vitro by Seiridium species pathogenic to cypress, was oxidized and the corresponding ketone derivative covalently linked to bovine serum albumin (BSA). The conjugate (SE-BSA) was used to prepare an antiserum to SE. The antibodies were absorbed with BSA and their specificity was assayed by ELISA and flow cytometry against SE, iso-seiridin (ISE), a structural isomer of SE, and some derivatives of these two metabolites. The antibodies tested in a competitive indirect ELISA did not show any binding activity to SE, ISE and their derivatives. The cytometry test, instead, was successful. SE-BSA and SE showed the highest binding activity with the antibodies. SE derivatives having a shift on the adjacent carbon, oxidation, or acetylation of the hydroxy group of the heptyl side chain at C-4 or conversion of the gamma-lactone in the corresponding planar furane ring reacted less than SE. The 2'-dansylhydrazoneSE and the 3,4-dihydroSE having a bulky group attached to the heptyl side chain and a saturated lactone ring, respectively, showed a weak reactivity. SE derivatives in which the gamma-lactone ring was destroyed and ISE derivatives presenting the shift of the hydroxy group at C-3' and another structural modification had no binding activity.

Animals↗
PubMed1997

Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry.

The simultaneous detection is described of cucumber mosaic virus (CMV), potato virus Y (PVY) and tomato mosaic virus (ToMV) by flow cytometry. Extracts from leaves of healthy and CMV or PVY infected plants were incubated with latex particles, each with a diameter of 3 microm. Extracts from ToMV infected or uninfected plants, however, were incubated with particles, each with a diameter of 6 microm. Beads were washed and incubated in succession with primary and secondary antibodies, the latter labeled with phycoerythrin (PE) or fluorescein (FITC). CMV and PVY were distinguished on the basis of the fluorescence emitted by FITC and PE; ToMV was distinguished from CMV and PVY on the basis of the different diameter (6 microm) of the particles on which it was adsorbed. The three viruses were detected also by another approach. Latex particles with a diameter of 3, 6 and 10 microm were separately sensitized with antibodies specific for CMV, PVY and ToMV. An equal number of sensitized particles was mixed and incubated with the plant extracts containing the three viruses and then with anti-CMV, anti-PVY and anti-ToMV antibodies labeled with FITC. The study describes also a virus purification method based on the use of antibody coated latex particles. The method is simple technically and applicable to the purification of large as well as minute amounts of different viruses (CMV, PVY and ToMV).

Antibodies, Viral↗
PubMed1996

Detection of antibodies to Staphylococcus aureus in water buffalo milk by flow cytometry.

An assay has been developed to detect antibodies to Staphylococcus aureus in water buffalo milk by flow cytometry. The method was the protein A-deficient strain Wood 46 of S aureus incubated with milk samples and fluorescein-labelled rabbit anti-water buffalo antiserum. The assay can detect antibodies when the pathogen is not detectable by bacterial tests and can determine the antibody titre directly on undiluted samples.

Animals↗