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M Fabbi

Publications and source records attributed to M Fabbi.

At least 19 recordsLinked to original sources
PubMed1995

Taxol cytotoxicity on human leukemia cell lines is a function of their susceptibility to programmed cell death.

Taxol is the prototype of a class of antineoplastic drugs that target microtubules. It enhances tubulin-monomer polymerization and stabilizes tubulin polymers, increasing the fraction of cells in the G2 or M phase of the cell cycle. We report that treatment of HL-60 and U937 myeloid cell lines with 1-10 microM taxol induces DNA fragmentation and the appearance of morphological features consistent with the process of apoptosis. Taxol-induced apoptosis is inhibited neither by cycloheximide nor by actinomycin D and therefore appears to be independent of new protein synthesis. Taxol causes arrest in the G2 phase of the cell cycle and affects cell viability but does not induce DNA fragmentation in the K562 erythromyeloid cell line. Protein-synthesis inhibitors, colcemid, ionomycin, and starvation, known to trigger apoptosis, proved ineffective as well. These results suggest that the antineoplastic effect of taxol is mediated in susceptible cell lines by induction of the apoptotic machinery and that K562 partial resistance may depend upon the intrinsic inability of these tumor cells to undergo apoptosis.

Apoptosis↗
PubMed1995

Spontaneous apoptosis in human thymocytes.

Apoptosis seems to be involved in different stages of immune cell development. In particular, experimental evidence suggests that it is a major form of cell death in the thymus. The present analysis of human thymocytes reveals that a fraction of these cells, cultured in vitro, undergoes spontaneous apoptosis. This observation is based both on molecular (DNA fragmentation) and morphological (electron microscopic) investigations of the cells. The apoptotic thymocytes are CD3- or CD3lo, CD4lo, and CD8lo and do not express Bcl-2 protein. Furthermore, thymocytes die by apoptosis when exposed to pharmacological stimuli, such as tumor necrosis factor-alpha, dexamethasone, ATP, or Ca++ ionophore. Thus the apoptotic machinery in thymocytes can be triggered by an imbalance in growth factors in the in vitro culture media and can be modulated by various biochemical signals. The process of spontaneous apoptosis is independent of mRNA or protein synthesis, as actinomycin D and cycloheximide fail to inhibit this phenomenon. Furthermore, apoptosis seems to require active oxidative phosphorylation, as it is prevented by incubation of the cells with inhibitors of the respiratory chain.

Apoptosis↗
PubMed1992

IL-7 induces proliferation of CD3-/low CD4- CD8- human thymocyte precursors by an IL-2 independent pathway.

The proliferation potential of highly purified human CD3-CD4-CD8- (triple negative) and CD3low(lo)CD4-CD8- thymocyte precursors in response to various cytokines was investigated. High in vitro growth ability was observed in response to recombinant human IL-2 (rIL-2) and human rIL-7, both in the absence of any co-mitogen and in combination with phorbol 12-myristate 13-acetate (PMA). Furthermore, the proliferation of these thymocyte precursors in the presence of rIL-7, although accompanied by a significant increase of IL-2 receptor (IL-2R) p55 expression, appeared independent of that mediated by the autocrine IL-2 pathway, since mAbs to IL-2 and IL-2R p55 did not eliminate responsiveness to rIL-7. Synergism of rIL-7 with rIL-2 was also observed, while no cooperation was detectable with rIL-4 or rIL-6. Analysis of surface phenotype and cell cycle status of cells cultured in the presence of rIL-7, both plus and minus PMA, showed that CD3- as well as CD3lo cells readily proliferated to rIL-7. Upregulation of the levels of expression of CD3 antigen was also observed in these cultures. These results, together with the previous characterization of IL-7 as a human pre-B cell and mature T cell growth factor, identify IL-7 as a cytokine with biologic activities on a variety of target cells. They also suggest that IL-7, in analogy with the mouse system, might play a role in human T cell ontogeny.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Non-MHC-restricted target-cell lysis by a CD4-CD8- TCR alpha beta T-cell line, as well as by TCR gamma delta T-cell lines, results from lymphokine-activated killing.

A long-term CD4-CD8- TCR alpha beta human T-cell line, as well as similar CD4-CD8- TCR gamma delta T-cell lines for comparison, were generated from various tissues by negative selection using anti-CD4 and anti-CD8 monoclonal antibodies (MAbs) followed by positive selection with specific anti-TCR MAb and then repeated in vitro stimulation with interleukin-enriched media and lectin. These cell lines all demonstrated non-MHC-restricted cytolysis on a variety of human tumor cell lines. However, removal of lymphokines from the culture media for 24 hr abrogated most of the non-MHC-restricted target-cell lysis without affecting TCR alpha beta or TCR gamma delta cell viability or TCR function as determined by antibody-triggered redirected target-cell lysis. Subsequent re-exposure to lymphokines reconstituted non-MHC-restricted cytolysis by these cell lines. Thus, much of the non-specific, non-MHC-restricted cytolytic activity generated by CD4-CD8- TCR alpha beta or TCR gamma delta cells is secondary to lymphokine-activated killing (LAK) activity. These cells have potent LAK activity and may be prominent in LAK-cell populations. In addition, after lymphokine deprivation, both CD4-CD8- TCR alpha beta and TCR gamma delta cells showed residual activity against some tumor-cell targets, the nature of which remains to be defined.

Animals↗
PubMed1991

Comparison between specific immunoperoxidase staining and bacteriological culture in the diagnosis of renal leptospirosis of pigs.

Seventy-two pigs were examined for the presence of leptospires in the kidney by both bacteriological culture and an immunoperoxidase procedure performed on formalin-fixed, paraffin-embedded sections of tissue with a primary antibody raised in rabbits against serovar pomona. The methods were in accordance in 62 of 70 (89 per cent) of the specimens. Compared with culture the sensitivity of the immunoperoxidase procedure was 30 of 38 (78 per cent) and its specificity 100 per cent; the predictive value of a positive result was 100 per cent, of a negative result, 80 per cent. The major advantages of the immunoperoxidase procedure are specificity, speed of execution and the possibility of simultaneous visualisation of leptospiral antigen and microscopic lesion.

Animals↗
PubMed1990

Evidence for extrathymic changes in the T cell receptor gamma/delta repertoire.

The germline repertoire of variable genes for the TCR-gamma/delta is limited. This, together with the availability of several V delta-specific and a C delta-specific mAbs, has made it possible to assess differences in the TCR-gamma/delta repertoire in man. TCR-gamma/delta cells expressing particular V gene segments have been previously shown to be localized in different anatomical sites. In this study, analysis of TCR-gamma/delta V gene segment usage performed on subjects from the time of birth through adulthood revealed striking age-related changes in the TCR-gamma/delta repertoire in peripheral blood. V delta 1+ gamma/delta T cells predominated in thymus as well as in peripheral blood at birth and then persisted as a relatively constant proportion of CD3+ PBL. However, V delta 2+ gamma/delta T cells that constitute a small proportion of the CD3+ cells in thymus and in peripheral blood at birth, then expand and account for the major population of gamma/delta T cells in PBL in adults. No parallel postnatal expansion of V delta 2+ cells in the thymus was observed, even when paired thymus-peripheral blood specimens were obtained on subjects between the ages of 3 d and 8 yr. The subset of V delta 2+ lymphocytes that was expanded in peripheral blood expressed high levels of CD45RO suggesting prior activation of these cells, consistent with the possibility that their expansion might have resulted from exposure to foreign antigens or superantigens. In contrast, V delta 1+ T cells in PBL showed no comparable increase in relative numbers and were either negative or expressed only low levels of CD45RO. Consistent with evidence for extrathymic peripheral expansion of selective TCR-gamma/delta subsets, no link between MHC haplotype and differences in the TCR-gamma/delta V gene usage between individuals was apparent, and identical twins displayed TCR-gamma/delta variable gene segment phenotypes that were strikingly different from one another. The elements that determine the TCR-gamma/delta repertoire in individuals are not known. It is possible that both thymic selection and extrathymic factors may influence the peripheral repertoire. Recently, TCR-gamma/delta+ lymphocytes have been shown to expand markedly in peripheral lymphoid tissues and infectious lesions in response to mycobacterial antigens, and a correlation between mycobacterial responses and TCR-gamma/delta V gene usage has been shown in mice. The data presented here demonstrated peripheral age-related changes in the gamma/delta repertoire and point to the importance of extrathymic expansion of specific gamma/delta subsets in generating the human TCR-gamma/delta repertoire.

Adult↗
PubMed1990

Maturation or differentiation of human thymocyte precursors in vitro?

The differentiation or maturation potential of human thymocyte precursors has been studied by using a population of CD3/TCR-, CD4-, CD8- ("triple negative") thymocytes isolated by negative selection (TCR, T-cell receptor). This cell population, however, also contained 30-50% previously undescribed cells expressing very low levels of CD3/TCR gamma delta (CD3/TCR gamma delta low; approximately 60% of which expressed the variable region gene V delta 1). Correspondingly, TCR gamma and TCR delta gene rearrangements (predominantly V delta 1/joining region J delta 1) and full-length TCR gamma and TCR delta transcripts (but only immature TCR beta and no TCR alpha mRNAs) were found. These cells mobilized Ca2+ in response to ligation of CD3 but not following ligation of TCR gamma delta. When cultured in the presence of interleukin 7 or interleukin 2, these thymocytes gave rise to 30-60% CD3/TCR gamma delta medium and high cells (60-70% expressing V delta 1) seen as discrete populations. Thus, the proportion and V delta phenotype of in vitro generated CD3/TCR gamma delta cells closely resembled those of CD3/TCR gamma delta low cells in freshly isolated "thymocyte precursor" preparations. Small numbers of TCR alpha beta + cells also appeared. It is thus uncertain whether maturation, differentiation, or both account for the appearance of mature CD3/TCR+ thymocytes, although the former appears most likely.

Antibodies, Monoclonal↗
PubMed1989

Human lymphocytes bearing T cell receptor gamma/delta are phenotypically diverse and evenly distributed throughout the lymphoid system.

A direct quantitative and phenotypic cytofluorographic analysis of TCR-gamma/delta+ lymphocytes as well as an immunohistologic study of their tissue distribution and microanatomy was made possible by the availability of two mAbs (anti-TCR-delta 1 and anti-C gamma M1) specific for framework determinants on human TCR gamma and delta chains, respectively. TCR-gamma/delta+ lymphocytes, ranging between greater than 0.5 and 16% of CD3+ cells, were found in fetal and postnatal thymus, fetal and adult peripheral lymphoid organs, and adult peripheral blood. While TCR-gamma/delta+ lymphocytes comprised a small subpopulation of T cells (mean, approximately 4%) occasionally greater than 10-16% of CD3+ cells expressed TCR-gamma/delta. Virtually all TCR-gamma/delta+ thymocytes/lymphocytes expressed CD7, CD2, and CD5 but were heterogeneous with respect to their expression of CD1, CD4, CD8, CD28, CD11b, CD16, and Leu-7. Human TCR-gamma/delta+ cells populate both organized lymphoid tissues (thymus, tonsil, lymphnode, and spleen) as well as the gut- and skin-associated lymphoid systems at similar frequencies without obvious tropism for epithelial microenvironments. TCR-gamma/delta+ lymphocytes tend to be located within a given organ wherever TCR-alpha/beta+ lymphocytes are found. This study shows that TCR-gamma/delta+ lymphocytes constitute a small but numerically important, phenotypically diverse T cell population distributed throughout the body. These results support the concept that TCR-gamma/delta+ cells comprise a distinct, functionally heterogeneous, mature T cell sublineage that may substantially broaden the T cell repertoire at all immunologically relevant sites.

Antibodies, Monoclonal↗
PubMed1989

Double-negative (CD4-CD8-) lymphocytes bearing T-cell receptor alpha and beta chains in normal human skin.

Double-negative (CD4-CD8-) T-cell receptor alpha- and beta-chain (TCR alpha beta)-expressing lymphocytes have been identified in human skin. Two skin-derived lymphocyte cell lines (II-1 and II-2) expressing this phenotype were obtained. They each expressed full-length TCR alpha- and beta-chain transcripts and lacked TCR gamma- and delta-chain as well as CD4 and CD8 mRNAs. These polyclonal cell lines shared one predominant TCR beta-chain gene rearrangement. The CD3/TCR alpha beta complexes were functionally competent as evidenced by their capacity to transduce activation signals resulting in cell proliferation, lymphokine secretion, and cytolytic activity. The demonstration of CD4-CD8-TCR alpha beta lymphocytes as a subpopulation of the skin-associated immune system should contribute to understanding the biologic potential of this cell population as well as of this organ-specific immune system.

Adult↗
PubMed1988

Intermolecular complexes between three human CD1 molecules on normal thymus cells.

The first cluster of differentiation (CD1) defines at least three distinct human thymic cell-surface differentiation antigens-CD1a, CD1b, and CD1c. We looked for structural homology of the three CD1 heavy chains at their peptide level by two-dimensional peptide maps. We show here that the CD1a Mr 49,000 heavy chain and the CD1b Mr 45,000 heavy chain appear to be more homologous to each other than to the CD1c Mr 43,000 heavy chain and that only one tyrosil peptide is common to the three heavy chains. Study of the CD1 heavy chains from several individuals reveals a very limited polymorphism of these molecules. We also demonstrate here that CD1a or CD1a-like molecules and other CD1 molecules can form intermolecular complexes on the surface of normal thymus cells. Molecules that are structurally very similar to CD1a molecules are associated noncovalently either with CD1c molecules or with CD1b molecules, and only CD1a molecules can associate covalently with CD8 molecules. In contrast, we could not find these intermolecular complexes on the surface of leukemic T-cell lines in culture.

Antibodies, Monoclonal↗
PubMed1986

Generation of monoclonal antibodies blocking cytotoxic reactions by human NK clones: further characterization of a 40/80-kDa target cell receptor.

To study NK target antigen(s), mice were immunized with pooled cells from five human hematopoietic cell lines (K562, MOLT4, JM, CEM, U937) known to be susceptible to Natural Killer activity. Cells fusions were performed and 4 out of approximately 2000 hybridoma supernatants were selected because of their ability to block cytotoxic reactions between a human NK clone (termed JT9) and MOLT4 cells. Functional characterization of the four monoclonal antibodies (Mabs) indicated that individual treatment of each immunizing target cells resulted in a decreased cytotoxicity. This inhibition was very specific because it was exclusively observed when three clones, JT9, JT10, and JT11, which express the same clonotypic structure (NKTa), were used as effector cells. Parallel and sequential immunoprecipitations showed that the four reagents termed anti-TNKtar 1, 2, 3, and 4 were directed at the same 40/80-kDa heterodimeric structure previously identified by anti-TNKtar Mab. However, cross-blocking experiments indicated that TNKtar and TNKtar 1-4 represent two distinct epitopic clusters. Finally, it was shown that anti-TNKtar 1-4 recognition sites are either identical or closely related to that of an additional antibody termed 4F2. Together, the present data strengthen the hypothesis that the activation antigen recognized by these series of Mab serves as a target cell receptor for at least a minor population of NK active lymphocytes.

Animals↗
PubMed1986

The sheep erythrocyte receptor and both alpha and beta chains of the human T-lymphocyte antigen receptor bind the mitogenic lectin (phytohaemagglutinin) from Phaseolus vulgaris.

We have studied the interaction of mitogenic lectins such as phytohaemagglutinin (PHA) and concanavalin A (Con A) with both surface molecules which, by the use of monoclonal antibodies, are known to trigger T-cell mitogenesis. Monoclonal antibodies recognizing the T-lymphocyte receptor for antigen (Ti) and/or its associated structure, CD3, activate T cells. More recently, a second pathway of activation has been described which involves the sheep erythrocyte binding glycoprotein CD2, a surface molecule distinct from Ti-CD3. Lysates from surface-iodinated T-leukaemia cell lines were treated with lectin and affinity purified anti-lectin antibodies coupled to protein A-Sepharose. We have shown that eluates from Con A/anti-Con A or PHA/anti-PHA immunoprecipitates contained Ti, since a rabbit anti-T alpha serum, which recognizes the native and denatured forms of the constant region of the alpha chain, immunoprecipitated Ti from these eluates. Furthermore, Ti immunoprecipitated by anti-T alpha serum from lysates of surface iodinated E+ lymphocytes was binding to PHA after elution from the immunoprecipitate. When the purified Ti molecule was reduced and alkylated, allowing the permanent dissociation of its alpha and beta subunits, PHA interacted with both chains, whereas anti-T alpha serum immunoprecipitated the alpha chain only. Altogether, these results demonstrate that PHA interacts with both chains of the T cell receptor for antigen on human peripheral T lymphocytes. With the HPB-ALL tumour line, a similar approach showed that both alpha and beta chains of Ti bind to Con A and Ulex europaeus 1 but not Helix pomatia. Affinity chromatography on immobilized lectins and immunoprecipitation with lectin/anti-lectin antibodies were employed to test whether CD2 binds to PHA and Con A. The results show that CD2 from human peripheral T lymphocytes binds both lectins but with a lower affinity for PHA than Con A.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

The ontogeny, structure and function of the human T lymphocyte receptor for antigen and major histocompatibility complex.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90 kDa Ti heterodimer and the invariant 20 and 25 kDa T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunological competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate germline V, D, J and C segments which rearrange during intrathymic differentiation to form an active gene set. Triggering of the T3-Ti receptor complex induces a rapid increase in free cytoplasmic Ca2+ and gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous interleukin-2 production, release and subsequent binding to interleukin-2 receptors. The implications of these findings for understanding of human T cell growth and its regulation in disease states are discussed.

Amino Acid Sequence↗
PubMed1985

Production and characterization of antibody probes directed at constant regions of the alpha and beta subunit of the human T cell receptor.

To generate antibodies directed at constant regions of the human T cell receptor, purified alpha and beta subunits of a human T cell antigen/major histocompatibility complex receptor from the REX tumor (Ti-REX) were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and utilized to immunize rabbits. H36 (anti-alpha subunit) and H38 (anti-beta subunit) antisera were strongly reactive with the denatured subunits and also immunoprecipitated the Ti heterodimer from 125I surface-labeled lysates of REX, inducer, suppressor and cytotoxic T cell clones, peripheral T lymphocytes and thymocytes. Moreover, immunodepletion experiments showed that such antisera recognized antigenic determinant(s) shared by all Ti molecules expressed in the thymus. Several observations were made with these anticonstant region antibodies. First, peptide map analysis showed that the T cell receptor molecules recognized by the anti-clonotype and the anti-constant region heteroantisera on a given T cell clone are identical, thus supporting the view that the T cell receptor undergoes allelic exclusion. Second, since the individual antisera were weakly cross-reactive with the other denatured subunit, such subunits probably share conserved sequences. Third, the absence of antisera reactivity with intact cells implies that most of these constant region epitopes must be obscured by associated molecules, perhaps including one or more of the 20-25-kDa T3 subunits. Fourth, the extensive difference in two-dimensional peptide maps of Ti alpha subunits from clones of differing specificities makes it likely that the subunit contributes in a major way to antigen/major histocompatibility complex binding.

Antibody Formation↗
PubMed1985

The human T-cell receptor.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to the identification of the human T-cell antigen receptor as a surface complex comprised of a clonotypic 90-kD Ti heterodimer and the invariant 20- and 25-kD T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate genes. Moreover, the presence of unique peptides following proteolysis of different Ti molecules isolated by non-cross-reactive anticlonotypic monoclonal antibodies supports the notion that variable regions exist within both the alpha and the beta subunits. N-Terminal amino acid sequencing and molecular cloning of the Ti beta subunit further show that it bears an homology to the first V-region framework of immunoglobulin light chains and represents the product of a gene that rearranges specifically in T lymphocytes. Triggering of the T3-Ti receptor complex gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors. The implications of these findings for understanding human T-cell growth and its regulation in disease states are discussed.

Amino Acid Sequence↗