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V Colizzi

Publications and source records attributed to V Colizzi.

At least 73 records · Page 4Linked to original sources
PubMed1992

Recognition and killing of tumour cells expressing heat shock protein 65 kD with immunotoxins containing saporin.

The expression of heat shock proteins (HSP) of the 65 kD family (groEL) has been observed by flow cytometry using murine monoclonal antibody (MoAb) anti-HSP 65 kD (ML30) on the surface of B (Daudi) or T (H9) lymphoma cells, on a monocyte cell line (U937) and also on a primary culture of a human pancreatic carcinoma (HPC). Moreover, the MoAb ML30 was coupled to Saporin 6, a ribosome-inactivating protein recovered from the seeds of Saponaria officinalis, to kill HSP-expressing cells with a specific immunotoxin. An indirect method using first MoAb ML30 and then anti-mouse IgG1 immunotoxin was also performed. With this method a human serum positive for HSP65-antibodies was tested using anti-human IgG1 or IgM immunotoxins. All cell lines were inhibited when preincubated with the specific immunotoxin directed to HSP65 (ML30 SO6), although H9 cells were susceptible to immunotoxin only after thermal stress. Daudi and HPC cells were inhibited both after long-term culture and when freshly explanted from SCID mice. Proliferation of the U937 monocytic cell line, that constitutively expresses high levels of HSP65 on the surface (as determined by flow cytometry), was completely inhibited (100% inhibition) by the ML30 SO6. However, not all tumour cells constitutively express high levels of surface HSP65, as determined by cytometric analysis. For this reason it was not always possible to obtain complete inhibition of cellular proliferation.

Animals↗
PubMed1992

A gp120 HIV peptide with high similarity to HLA class II beta chains enhances PPD-specific and autoreactive T cell activation.

The recent report that anti-gp120 antibodies can be induced by allogeneic stimuli in experimental animals in the absence of HIV, has focused attention on the structural similarities between gp120 and MHC. Here we report that some HIV+ individuals develop antibodies which similarly react with the gp120 HIV sequence (aa 254-263) and with the HLA-DR beta chains (aa 142-151). As these two peptides share a high level of similarity, we have investigated the role of this gp120 region on HLA class II mediated T cell recognition. The synthetic peptide corresponding to the gp120 HIV sequence aa 254-263 has been tested on T cell line (TCL) activation. Both the PPD-specific and the self-HLA reactive TCL proliferation increased in the presence of this peptide. Prepulsing experiments indicate that this enhancing effect carried out by HIV peptide is exerted at the level of antigen presentation. Moreover, the specificity of this interaction is supported by the fact that a MoAb specific for this HIV peptide blocked the autoreactive TCL proliferation, similarly to the inhibition carried out by anticlass II antibody. These data support the hypothesis that the functional homology between the HIV peptide and the HLA beta chain described may be involved in the pathogenesis of AIDS.

Amino Acid Sequence↗
PubMed1992

Cytotoxic activity of intestinal lamina propria lymphocytes on human immunodeficiency virus (HIV)-infected cells.

The phenotype and cytotoxic activity of lamina propria lymphocytes (LPL) from the colorectal mucosa have been investigated primarily to analyse the role of LPL in human immunodeficiency virus (HIV) infection. The results reported here show that LPL strictly required a proliferative stimulus [either interleukin-2 (IL-2) or phytohaemaglutinin (PHA) to develop strong in vitro cytotoxicity, since freshly isolated LPL do not exhert cytotoxicity against either natural killer (NK)-sensitive or NK-resistant target cells. The cytotoxicity of activated LPL against a large panel of myeloid tumours or colorectal carcinoma target cells shows the irrelevance of the tissue origin of target cells. Moreover, activated LPL lysed HIV-infected H9 cells more efficiently than peripheral blood lymphocytes (PBL), and were susceptible to HIV infection. In contrast, unstimulated LPL failed to be cytotoxic and susceptible to HIV. Thus, we strongly suggest that for the lymphocytes of the colorectal mucosa expression of cytotoxic activity and susceptibility to HIV-infection show two faces of the same coin, and therefore may be relevant in understanding the mechanisms and paths of transmission of HIV infection.

Cells, Cultured↗
PubMed1992

Surface expressed heat-shock proteins by stressed or human immunodeficiency virus (HIV)-infected lymphoid cells represent the target for antibody-dependent cellular cytotoxicity.

The expression of heat-shock proteins (HSP) of the 72,000 MW family by Daudi and H9 lymphoma cells has been investigated by flow cytometry. It has been found that both heat-stressed and chronically human immunodeficiency virus (HIV)-infected lymphomas show an increased expression of heat-shock proteins (HSP). Moreover, murine monoclonal antibody (mAb) against 72,000 MW HSP was able to mediate antibody-dependent cellular cytotoxicity (ADCC) using peripheral blood lymphocytes (PBL) as effector cells. All target cells used in these experiments were efficiently lysed in the presence of anti-HSP antibody suggesting a role of membrane HSP in the elimination of stressed or infected cells.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1991

The combined treatment of human peripheral blood mononuclear cells with thymolymphotropin and interleukin 2 increases PPD-driven T-cell proliferation and IL-2 induced cellular cytotoxicity against HIV-infected cells.

Two in vitro systems (the DNA synthetic response to mycobacterial antigens and cytotoxicity against lymphoid cells) were used to analyse the effect of thymolymphotropin (TLT) on peripheral blood mononuclear cells (PBMC). Purified protein derivative of mycobacteria (PPD)-driven T-cell proliferation in low-responder donors was increased by the combined treatment with TLT and suboptimal doses of recombinant interleukin 2 (IL-2). Similarly, the activities of natural killer (NK) cells and lymphokine-activated killer (LAK) cells have been enhanced in PBMC cultures pretreated with TLT. Also, TLT showed an enhancing effect on the development of LAK cells capable of lysing Epstein-Barr virus (EBV)-transformed B-lymphocytes infected or uninfected with the human immunodeficiency virus (HIV).

Cytotoxicity, Immunologic↗
PubMed1991

Identification of a specific Mycobacterium tuberculosis protein able to activate T lymphocytes.

The recent advances in the field of immunology and molecular biology allow to consider not distant the identification of protective antigens to be used in new prophilactic and diagnostic tools. In our laboratory we have analysed a M. tuberculosis expression library by murine monoclonal antibodies, human sera and human T lymphocytes. We have identified a 35 Kd protein present only on the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum). This 35 Kd protein is also detected, by immunohistological analysis, inside the alveolar macrophage of pulmonary tuberculosis patients. Both recombinant beta-galactosidase and MS2 polymerase-fused proteins have been produced and used to perform western blotting and T cell proliferation assay. As the cloned fragment contains an internal EcoRI restriction site, it was possible to identify two fragments of 1.7 and 2.2 kb respectively. Southern blot analysis showed that both the fragments hibridized with the genomic DNA from M. tuberculosis complex but not with the DNA from other mycobacterial isolates from 12 pulmonary tuberculous patients hibridized with the 1.7 kb fragment. The possible use of this insert for the molecular diagnosis of tuberculosis is under investigation.

Animals↗
PubMed1990

Regulation of self-major histocompatibility complex reactive human T-cell clones.

The proliferative response of human T-lymphocyte clones, (TLC) specific for self-major histocompatibility complex (MHC) products either alone or associated with PPD epitopes are inhibited in vitro by dexamethasone (DEX) and by a non-specific inhibitory factor(s) (nsINH) produced by PPD-activated T-cells. The inhibiting effect has been investigated by preincubating autoreactive and PPD-specific TLC with nsINH or DEX. Results obtained indicate that T-lymphocytes are the target of these two immunoregulatory molecules. Moreover, the addition of exogenous recombinant interleukin 2 (rIL-2) substantially reverses the inhibition observed in both nsINH- or DEX-treated cultures.

Antigens, Bacterial↗
PubMed1990

Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.

Antigens, Bacterial↗
PubMed1990

Increased autoreactive T cell frequency in tuberculous patients.

The development of putative self-MHC-reactive T cells and their precursor frequency was estimated in peripheral blood lymphocyte cultures stimulated in vitro with PPD. The role of foreign antigen in the generation of self-MHC-reactive T cells in vivo was analyzed by comparing the frequency of autoreactive T cells in the peripheral blood of tuberculous patients with that observed in healthy individuals. It was found that PPD in vitro and Mycobacterium tuberculosis infection in vivo increased substantially the generation of autoreactive T cells. Autoreactive T cell clones were shown (1) to recognize self MHC class II products; (2) to release gamma interferon in the absence of exogenous antigen, and (3) to express autocytotoxic activity. All these findings suggest that self-MHC-reactive T cells may be involved in the inflammatory response to M. tuberculosis.

Cells, Cultured↗
PubMed1989

Limiting dilution analysis of T cell unresponsiveness to mycobacteria in advanced disseminated tuberculosis.

Peripheral blood mononuclear cells from patients with advanced disseminated tuberculosis (Dis-TB) do not respond to purified protein derivative (PPD) measured as cell proliferation, lymphokine production and interleukin (IL)-2 receptor (Tac antigen) expression. Limiting dilution analysis revealed "multi-hit" curves and low frequencies of PPD-reactive T cells in cultures of Dis-TB, and "single-hit" curves and high frequencies of PPD-reactive T cells in cultures of patients with localized form of pulmonary tuberculosis. Moreover, a strict relationship between Tac antigen expression and ability of exogenous IL-2 to enhance bulk culture cell proliferation was observed in Dis-TB patients.

Humans↗
PubMed1989

Immunohistologic analysis of mycobacterial antigens by monoclonal antibodies in tuberculosis and mycobacteriosis.

Four monoclonal antibodies (MoAbs), 60.15, 61.3, 105.10, and 2.16, directed to different proteins of Mycobacterium tuberculosis were used by an indirect avidin-biotin complex peroxidase-antiperoxidase method to detect mycobacterial antigens in lung, lymph node, and joint tissue specimens of tuberculous patients. Using MoAb 60.15, which recognizes a broad range of cross-reactive mycobacterial proteins with a molecular mass of 28 kilodaltons (kD), scattered materials (mycobacterial in origin) were observed, many of which were located within phagocyte cytoplasm. With MoAb 61.3, which reacts with a 35 kD protein present in M tuberculosis, Mycobacterium africanum, and Mycobacterium bovis, many clumped particles similar in size and shape to acid-fast bacilli were observed within the phagocyte cytoplasm (lung tissue) and positive macrophages with lysosomes were distributed throughout the cytoplasm (bronchoalveolar lavage). The specificity of this MoAb (61.3) was confirmed by the negative staining of positive lymph node specimens obtained from a patient infected with Mycobacterium kansasii. MoAbs 105.10 and 2.16 bind to the cross-reactive 65 kD heat shock protein that is present in mycobacteria and stain scattered particles and dark clumps of bacilli within the phagocyte cytoplasm. On the basis of this study, immunohistochemical detection of mycobacterial antigens appears to be useful in establishing the mycobacterial etiology of caseating granulomas and in avoiding the false-negative results obtained by traditional staining methods.

Antibodies, Monoclonal↗
PubMed1989

Analysis of Mycobacterium tuberculosis genoma and production of a recombinant protein containing specific B and T cell antigenic determinants--new approaches to second generation antituberculosis vaccines.

Tuberculosis is still a major health problem in almost all over the world. Thus, new directions in basic and applied research of tuberculosis are under investigation in several laboratories. In this paper, we provide recent data obtained in our laboratory with the recombinant DNA technology which allow a systematic survey of the microbial genome. Screening of the M. tuberculosis genomic DNA library in the phage lambda gt11 expression vector, using E. coli as surrogate host, has evidenced the possibility of producing recombinant M. tuberculosis proteins recognized by sera from tuberculosis patients and by specific monoclonal antibodies. Using this technology, we have isolated a recombinant protein (molecular weight 130 kilodaltons) which is identified by a murine monoclonal antibody recognizing a mycobacterial cell wall antigenic determinant present on a mycobacterial protein. This protein is only present on the mycobacterial species related to the tuberculosis complex (M. tuberculosis, M. bovis, M. africanum) and does not crossreact with nonpathogenic Mycobacteria. Since the immune response to mycobacterial infections is cell-mediated, the question arises about the use of M. tuberculosis-specific T lymphocytes to screen this gene bank. Thus, the recombinant mycobacterial protein isolated by antibodies has been then used to stimulate the proliferation of T lymphocytes from patients with tubercular pleuritis. This experiment indicates that the recombinant protein contains antigenic determinants recognized by T cells. Moreover, such protein is able to elicit delayed type hypersensitivity skin reaction in mice immunized or infected with M. tuberculosis and M. bovis. Finally, gene mapping and hybridization studies with native M. tuberculosis DNA confirme the mycobacterial nature of the recombinant DNA insert. Thus a good candidate for the prophylaxis and the immunodiagnosis of tuberculosis has been identified. The identification and selection of genes encoding antigenic determinants recognized by mycobacteria-specific T cells with protective functions will allow in the near future the construction by genetic engineering of recombinant vaccines effective in the control of this disease. This paper will briefly discuss the present strategy used in our laboratory to reach this goal.

Animals↗
PubMed1988

A alpha and A beta class II I-A determinants of antigen-specific T-helper factor and its antigen-nonbinding chain.

Antigen-specific T-helper factor (ThF) of CBA (H-2k) origin in the picryl (TNP) contact sensitivity system (Mr 60-70 kDa) was reduced with dithiothreitol under mild conditions. Affinity chromatography on antigen yielded an antigen-binding chain (Mr 20-30 kDa) and an antigen-nonbinding chain (Mr 40-50 kDa). Both chains were glycoproteins and were bound by lentil lectin. Affinity chromatography on anti-I-A monoclonal antibodies showed that I-A determinants occurred on the complete molecule and on the antigen-nonbinding, but not on the antigen-binding, chain. In contrast, five different monoclonal antibodies to I-E alpha failed to absorb ThF. Moreover, the complete molecule and the I-A+ antigen-nonbinding chains had determinants of the alpha and beta chains of I-A and conformational determinants which are based on both chains. Sequential absorption and elution showed that A alpha and A beta determinants occurred on the same molecular complex. These data suggest a minimal model of ThF as a two-chain disulfide-bonded structure with an antigen-binding chain and a separate I-A+ antigen-nonbinding chain which behaves as a single unit in phosphate-buffered saline and has elements of both A alpha and A beta.

Animals↗
PubMed1988

IL-2 influences the balance between immunity and unresponsiveness in the picryl (TNP) contact sensitivity system by blocking the development or action of an Lyt-2+, I-J+ T suppressor cell.

Mice injected with antigen (picrylated spleen cells) intravenously fail to develop contact sensitivity. However, contact sensitivity occurs if these mice are injected with IL-2. This effect of IL-2 was reproduced in vitro by taking spleen cells 2 days after injecting antigen intravenously and culturing them with either 150 u/ml recombinant IL-2 for 2 days or by pulsing with 600-1200 u/ml IL-2 at 4 degrees C for 1 hr. After 2 days in culture these antigen-exposed cells transfer contact sensitivity to naive recipients in a 24-hr experiment. However, the ability of antigen-exposed cells, pulsed with IL-2, to transfer contact sensitivity is abolished when they are incubated with unpulsed antigen-exposed cells and as few as 1/16 of their number have a significant effect. This phenomenon is specific, as normal cell or cells from mice injected with oxazolonated cells intravenously have no effect. The suppressor cells were Thy-1+, Lyt-1-, 2+, I-J+ T cells. It was concluded that IL-2 prevents the development/action of antigen-specific T suppressor cells.

Animals↗