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M Vitale

Publications and source records attributed to M Vitale.

At least 127 records · Page 7Linked to original sources
PubMed1994

Loss of polarity and de novo expression of the beta 1 family of integrins in thyroid tumors.

The expression and cell-membrane distribution of the beta 1 family of integrins (very-late-activation antigens, VLA) were investigated in benign and malignant human thyroid tumors. We compared tissue samples of normal glands, nodular goiters, adenomas and carcinomas. We also examined 3 thyroid-carcinoma cell lines cultured in vitro. The expression of subunits of the beta 1 family of integrins was assessed by flow cytometry and specific antibodies in dispersed single-cell suspensions and by immunofluorescence on frozen tissue sections. In contrast to the heterogeneity of the expression of beta 1 integrins observed in other tumors, thyroid neoplastic lesions showed a remarkably constant VLA profile. In all tumors, benign as well as malignant, and in carcinoma cell lines, all sub-units of beta 1 integrins were expressed at high levels. While sub-units alpha 1, alpha 3, alpha 5, alpha 6 and occasionally alpha 2 were also present in a cell sub-set of normal glands and nodular goiters, expression of alpha 4 was restricted to neoplastic lesions; this integrin can be therefore considered an antigen associated with thyroid tumors. It has been reported that in normal glands and in nodular goiters, the expression of beta 1 integrins is restricted to the basal-cell membrane. Immunofluorescence on tissue sections showed instead that, in adenomas and carcinomas, the polarized distribution of these integrins on the cell membrane is lost.

Carcinoma, Papillary, Follicular↗
PubMed1994

Evidence for a globin promoter-specific silencer element located upstream of the human delta-globin gene.

We describe the negative regulatory activity of a 1.7 kilobase (kb) region (R) in the human beta-globin locus located between 4.0 and 2.3 kb upstream of the delta-globin gene capsite, using a transient assay with the chloramphenicol acetyltransferase (CAT) reporter gene in mouse erythroleukemia (MEL) cells. The R region is deleted in most cases of deletion hereditary persistence of fetal hemoglobin (HPFH), but is unaffected in most delta beta zero-thalassemias. However, no experiments addressing its function in globin gene expression have been reported to date. We show that R inhibits CAT expression of constructs containing a fetal (gamma) or adult (beta) globin gene promoter, but does not affect expression of similar constructs using a non-globin (SV40) promoter. The inhibitory effect on the beta-globin promoter can be localized to a 651 bp sub-region of R. For the gamma-globin promoter, no sub-region of R can reproduce the level of inhibition associated with the entire region.

Animals↗
PubMed1994

A subset of human CD34+ hematopoietic progenitors express low levels of CD4, the high-affinity receptor for human immunodeficiency virus-type 1.

We investigated the expression of CD4 antigen in normal bone marrow (BM) samples, enriched in CD34+ hematopoietic progenitor cells. At flow cytometry, a significant fraction (ranging from 25% to 65%) of CD34+ cells also showed low levels of CD4 antigen on their surface. The CD4 receptor densities on the surface of hematopoietic progenitors was approximately 50% that of peripheral blood monocytes and 5% of peripheral blood T lymphocytes. In immunoprecipitation experiments, the CD4 antigen expressed by BM hematopoietic progenitors appeared to be the same form expressed by mature peripheral blood CD4+ cells and appeared to be a potentially functional receptor for human immunodeficiency virus-type 1, because it specifically bound recombinant envelope gp120. Moreover, BM samples, highly enriched in CD34+ cells, showed the presence of CD4 mRNA at reverse transcription-polymerase chain reaction examination. In experiments of complement-mediated cytotoxicity with Leu3-a+Leu3b anti-CD4 monoclonal antibody, a significant reduction in the number of both classes of megakaryocyte (burst-forming unit-meg [BFU-meg] and colony-forming unit-meg [CFU-meg]) and granulocyte/macrophage (CFU-GM) progenitors was observed, whereas erythroid (BFU-E) progenitors were only slightly affected. Moreover, purified CD4+ BM cells obtained by immunomagnetic selection, using high concentrations of Leu3a+Leu3b, showed a colony-forming ability of megakaryocyte and granulocyte/macrophage progenitors comparable with that of CD4- BM cells. In conclusion, the present data show that immature hematopoietic progenitor cells express low levels of CD4, the high-affinity receptor of human immunodeficiency virus-type 1.

Antibodies, Monoclonal↗
PubMed1994

Human natural killer cell receptors for HLA-class I molecules. Evidence that the Kp43 (CD94) molecule functions as receptor for HLA-B alleles.

GL183 or EB6 (p58) molecules have been shown to function as receptors for different HLA-C alleles and to deliver an inhibitory signal to natural killer (NK) cells, thus preventing lysis of target cells. In this study, we analyzed a subset of NK cells characterized by a p58-negative surface phenotype. We show that p58-negative clones, although specific for class I molecules do not recognize HLA-C alleles. In addition, by the use of appropriate target cells transfected with different HLA-class I alleles we identified HLA-B7 as the protective element recognized by a fraction of p58-negative clones. In an attempt to identify the receptor molecules expressed by HLA-B7-specific clones, monoclonal antibodies (mAbs) were selected after mice immunization with such clones. Two of these mAbs, termed XA-88 and XA-185, and their F(ab')2 fragments, were found to reconstitute lysis of B7+ target cells by B7-specific NK clones. Both mAbs were shown to be directed against the recently clustered Kp43 molecule (CD94). Thus, mAb-mediated masking of Kp43 molecules interferes with recognition of HLA-B7 and results in target cell lysis. Moreover, in a redirected killing assay, the cross-linking of Kp43 molecules mediated by the XA185 mAb strongly inhibited the cytolytic activity of HLA-B7-specific NK clones, thus mimicking the functional effect of B7 molecules. Taken together, these data strongly suggest that Kp43 molecules function as receptors for HLA-B7 and that this receptor/ligand interaction results in inhibition of the NK-mediated cytolytic activity. Indirect immunofluorescence and FACS analysis of a large number of random NK clones showed that Kp43 molecules (a) were brightly expressed on a subset of p58-negative clones, corresponding to those specific for HLA-B7; (b) displayed a medium/low fluorescence in the p58-negative clones that are not B7-specific as well as in most p58+ NK clones; and (c) were brightly expressed as in the p58+ clone ET34 (GL183-/EB6+, Cw4-specific). Functional analysis revealed that Kp43 functioned as an inhibitory receptor only in NK clones displaying bright fluorescence. These studies also indicate that some NK clones (e.g., the ET34) can coexpress two distinct receptors (p58 and Kp43) for different class I alleles (Cw4 and B7). Finally, we show that Kp43 molecules function as receptors only for some HLA-B alleles and that still undefined receptor(s) must exist for other HLA-B alleles including B27.

Alleles↗
PubMed1994

In vitro exposure to human immunodeficiency virus type 1 induces apoptotic cell death of the factor-dependent TF-1 hematopoietic cell line.

In this study, we evaluated the effect of a short-term exposure (2 hours) to two different lymphocytotropic strains of human immunodeficiency virus type 1 (HIV-1; HIVIIIB and ICR-3) on the survival of a factor-dependent CD34+ hematopoietic progenitor cell line (TF-1). At flow cytometry analysis, a significant (P < .05) increase in the frequency of apoptotic cell death was observed in HIV-1-treated TF-1 cells, supplemented with low doses of either interleukin-3 (IL-3; 0.02 to 1 ng/mL) or granulocyte-macrophage colony-stimulating factor (GM-CSF; 0.02 to 0.2 ng/mL) with respect to mock-treated cells. On the other hand, higher doses of both cytokines or combinations of suboptimal concentrations of IL-3 plus GM-CSF (eg, 0.2, plus 0.2 ng/mL) completely reversed the HIV-1-induced increase of apoptosis. Remarkably, no signs of productive or latent virus replication were ever observed in HIV-1-treated TF-1 cells up to 16 days of liquid culture. In parallel experiments, the in vitro exposure to HIVIIIB induced a significant and progressive increase of apoptotic death in purified bone marrow CD34+ cells, seeded in liquid cultures in the presence of 1 ng/mL IL-3. The HIV-1-induced apoptosis of TF-1 cells was likely triggered by the simple interaction of HIV-1 envelope glycoprotein gp120 with CD4 receptor, which was expressed at a low level on the surface of TF-1 cells. In fact, treatment of TF-1 cells with recombinant gp120 plus a polyclonal anti-gp120 antibody or with anti-CD4 monoclonal antibody plus rabbit antimouse IgG significantly increased the percentage of apoptotic death. These data suggest that HIV-1, and perhaps also free gp120 in the presence of anti-gp120 antibody; could play a direct role in the pathogenesis of peripheral blood cytopenias in acquired immunodeficiency syndrome patients by inducing apoptotic death of hematopoietic progenitor cells without the need of a direct infection.

Antigens, CD↗
PubMed1994

Self class I molecules protect normal cells from lysis mediated by autologous natural killer cells.

The surface expression of given HLA class I alleles protects target cells from lysis mediated by natural killer (NK) clones specific for these (or related) alleles. We could define two groups of NK clones specifically recognizing either Cw4 and related C alleles ("group 1") or Cw3 and related C alleles ("group 2"), respectively. Monoclonal antibodies (mAb) to class I molecules should interfere with the interaction between NK receptors and class I molecules, thus resulting in lysis of protected target cells. However, none of the numerous available mAb to class I molecules had this effect. Therefore, we attempted to select new mAb on the basis of their ability to induce lysis of Cw4- or Cw3-protected lymphoblastoid cell lines by "group 1" or "group 2" NK clones, respectively. From mice immunized with phytohemagglutinin (PHA)-activated lymphocytes expressing either Cw3 or Cw4 alleles, two mAb were selected, the 6A4 (IgG1) and the A6-136 (IgM), on the basis of their ability to induce lysis of protected target cell. Both mAb immunoprecipitated molecules which, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gave two bands of 45 and 12 kDa, typical of the class I heavy chain and beta 2 microglobulin, respectively. It has been proposed (but not proven), that self major histocompatibility complex class I molecules protect normal cells from autologous NK cell lysis. Thus, we used the 6A4 and A6-136 mAb to assess this possibility directly. Cw4-specific ("group 1") and Cw3-specific ("group 2") NK clones were isolated from donors expressing the corresponding (or related) protective C alleles. None of these clones lysed autologous PHA-induced blasts, used as target cells. However, addition of the F(ab')2 of 6A4 mAb or the A6-136 mAb resulted in lysis of autologous target cells by "group 1" or "group 2" NK clones, respectively. These data provide direct evidence that the expression of class I molecules protects normal cells from lysis by autologous NK cells.

Animals↗
PubMed1994

The human natural killer cell receptor for major histocompatibility complex class I molecules. Surface modulation of p58 molecules and their linkage to CD3 zeta chain, Fc epsilon RI gamma chain and the p56lck kinase.

The natural killer cell (NK)-specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between p58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183-EB6+ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4+ C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183+/EB6+ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the zeta family (i.e. CD3 zeta and Fc epsilon RI gamma chains). Two-dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58+ NK cells indicated a preferential association with CD3 zeta chains either in the form of covalently linked zeta-zeta homodimers or in the form of zeta-gamma heterodimers, while gamma-gamma homodimers were detectable in low amounts. However, p58+ clones displaying a unique association with gamma-gamma homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti-p56lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via p58 molecules induced increments of p58/p56lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 zeta chains, no tyrosine phosphorylation was detected in the p58-associated CD3 zeta chains. In these in vivo experiments neither anti-CD16 nor anti-p58 mAb could induce tyrosine phosphorylation of the gamma chains. Finally, the anti-p58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompanied by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 zeta chains.

Antibodies, Monoclonal↗
PubMed1994

Nuclear pores in the apoptotic cell.

During apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models. Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas. In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cisternae. A possible functional significance of this pore behaviour during apoptosis is discussed.

Animals↗
PubMed1994

The behaviour of nuclear domains in the course of apoptosis.

Programmed cell death is activated, by different stimuli and in many cell types, to regulate cell population balance during tissue proliferation and embryogenesis. Its initial event seems to be, in most cases, the activation of a Ca(2+)-dependent endonuclease, causing DNA cleavage into nucleosomic fragments. Its morphological expression is characterized by deep nuclear changes, consisting of typical cap-shaped chromatin marginations, followed by nuclear fragmentation and final formation of numerous micronuclei. Cytoplasmic damage appears in a very late stage of the process and the greatest part of the phenomenon appears to take place despite good preservation of the plasma membrane and organellar component. In the present study we analyzed apoptosis in camptothecin-treated HL60 leukaemia cells, and in freshly isolated mouse thymocytes treated with dexamethasone. The process was first quantified and time monitored by flow cytometry. Subsequently the specimens were processed for morphological examination in order to investigate the behaviour of the different nuclear domains. To follow DNA and RNA localization, we utilized osmium ammine and DNase-colloidal gold cytochemical reactions. The concentration of most DNA in the cap-shaped structures was demonstrated by these reactions. Confocal microscopy of cells processed by in situ nick-translation suggested that DNA was firstly cleaved and subsequently condensed in cup-shaped structures. Despite the strong nuclear modifications, nucleoli could be clearly recognized until the late apoptotic stages.

Animals↗
PubMed1994

High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy.

HeLa metaphase chromosomes were examined by means of "in lens" field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.

Chromosomes, Human↗
PubMed1994

Age-related events in active T lymphocyte subpopulation. A morphological study.

The incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of active T lymphocytes from healthy aged donors was evaluated after in vitro PHA stimulation by means of light microscopy, electron microscopy and flow cytometry. The percentage of BrdU-labelled cells differed markedly between aged and young donors after 48 h of PHA stimulation, due to an enlarged early S phase compartment. In contrast, after 72 h the percentage of positive cells was quite similar in both age groups and no significant differences in the distribution within S phase nor changes in the patterns of ultrastructural localization of DNA replicon sites were observed. Our study provides evidence that an altered synchronization and a substantial delay in the in vitro cell proliferation occur in this peculiar T subpopulation as a consequence of the ageing process.

Adult↗
PubMed1994

Evidence for an early and transient involvement of nuclear inositol lipids in subcellular signalling events related to DNA repair processes.

The involvement of nuclear inositol lipids in the processes related to DNA repair upon ionizing radiation has been investigated in Murine Erythroleukaemia cells. Early changes in the in vitro phosphatidylinositol-bisphosphate phosphorylation in isolated nuclei were found to precede transiently the marked increase in DNA synthesis occurring after irradiation. Such an increase detected by anti-BrdU monoclonal antibodies has been found to be related mainly to DNA polymerase beta activity as revealed by the kinetic analysis of in vitro DNA synthesis. The results here presented allow us to speculate on a possible involvement of nuclear inositol lipids in the cascade of the early events leading to the regulation of DNA repair in the nucleus.

Animals↗
PubMed1994

In vitro growth of human fetal CD34+ cells in the presence of various combinations of recombinant cytokines under serum-free culture conditions.

CD34+ cells were purified from midtrimester human fetal blood and adult bone marrow samples and seeded in serum-free fibrin-clot cultures in order to evaluate the number and the responsiveness to recombinant cytokines of pluripotent (CFU-GEMM), erythroid (BFU-E), megakaryocyte (BFU-meg and CFU-meg) and granulocyte/macrophage (CFU-GM) haemopoietic progenitor cells. The number of the different haemopoietic progenitors/1 x 10(3) CD34+ cells, except CFU-meg, was significantly higher in fetal blood than in adult bone marrow in cultures stimulated by any combination of cytokines including interleukin-3 (IL-3), granulocyte/macrophage colony stimulating factor (GM-CSF) or stem cell factor (SCF) plus erythropoietin (Epo). Nevertheless, whereas adult BFU-E showed a maximal growth in the presence of Epo plus IL-3 or Epo plus SCF, fetal BFU-E showed an optimal growth in the presence of Epo alone, the sensitivity of fetal BFU-E to suboptimal concentrations of Epo being approximately 10-15-fold higher than that of adult BFU-E. Addition of optimal concentrations of IL-3, GM-CSF or SCF, alone or in various combinations, to Epo-containing cultures induced a significant increase in both the number and size of fetal CFU-GEMM, and CFU-GM, and a parallel decrease of fetal BFU-E. Finally, SCF potently synergized with IL-3 in increasing the growth of both classes of fetal megakaryocyte progenitors, BFU-meg and CFU-meg.

Adult↗
PubMed1994

Ultrastructural features of apoptosis.

Apoptosis is a gene-directed physiological and programmed process of cell deletion aimed at the regulation of tissue and organ development. It affects different cell types and is triggered by a variety of stimuli all inducing closely comparable structural changes. Despite the deeply different morphology and metabolism of the cell models and the various inducers and their initial effects, a convergence seems to take place in a common metabolic pathway that, in most cases, involves the activation of a Ca2+ dependent endonuclease. A growing body of data is now available on the molecular events that lead to DNA damage. DNA cleavage in nucleosomic or oligonucleosomic fragments is related to the appearance of unusual and very characteristic ultrastructural changes. The nucleus is especially affected, and shows chromatin rearrangements consisting of cup-shaped marginations, sharply separated from diffuse chromatin areas. Nuclear fragmentation subsequently appears, finally followed by the formation of numerous micronuclei. Cytoplasmic damage appears at a very late stage and the process takes place despite good preservation of plasma membrane and cytoplasm.

Animals↗
PubMed1994

Biochemical markers of acromegalic activity.

Against a clinical score we compared the effectiveness of three biochemical markers of acromegalic activity: a) growth hormone response to i.v. thyrotropin-releasing hormone, b) growth hormone suppression one hour after an oral glucose load, c) basal plasma IGF-I levels. In 21 patients we obtained 39 observations comprising the four parameters. According to the clinical score, patients were divided into three groups: group I = patients before surgery; group II = improved but still clinically active; group III = clinically inactive. After i.v. thyrotropin-releasing hormone, abnormal observations increased although the patients improved clinically, so that this test was excluded from statistical analysis. Abnormalities in plasma IGF-I levels and in GH suppression after oral glucose were similarly frequent in the groups studied: both 100% in group I, both 71.4% in group II, 27.3% vs. 18.2% in group III (p > 0.05). Both tests made exactly the same contribution to the other's capacity to detect acromegalic activity: 3 out of 39 observations (5.1%). We conclude that basal plasma IGF-I is the single best biochemical marker to detect acromegalic activity, since it is statistically at least as useful as growth hormone suppression, and is far more simple for the patient and the laboratory.

Acromegaly↗
PubMed1994

Lymphocyte binding to K562 cells: effect of target cell irradiation and correlation with ICAM-1 and LFA-3 expression.

Tumor irradiation induces modifications in the interaction of surviving target cells with immune cells. This interaction is mediated by adhesion molecules, whose expression can be strongly altered by radiation treatment. Here the probably of K562 tumor cells for lymphocyte binding was studied after exposure of target cells to different doses of gamma-radiation. Results were correlated to the expression of ICAM-1 and LFA-3 adhesion molecules on target cells. Radiation treatment enhanced the expression of both ICAM-1 and LFA-3 on the surface of target cells in a dose and time of culture-dependent fashion, reaching a maximum 24 hrs postirradiation, when also lymphocyte binding was increased. 10-30 Gy irradiation of K562 cells in vitro induces after 24 hrs, an up-regulation of ICAM-1 and LFA-3 expression that, in turn, increase lymphocyte binding, making tumor cells more exposed to cytotoxic attack. The progressive morphological damage induced by radiation, documented by the scattering singlas in flow cytometry and by electron microscopy analysis of irradiated K562 cells, induced, particularly at delayed times of culture in high doses irradiated cells, alterations of the target cell surface that might prevent the correct interaction with immune cells.

Adult↗
PubMed1994

Negative signalling via the P58/NKR for HLA.C alleles.

Recent studies have demonstrated that the interaction between HLA class I alleles and specific NK receptors results in negative signals which inhibit NK-mediated cytotoxicity. Such NK receptors have been identified by GL183 and EB6 mAbs which recognize distinct members of a molecular family involved in the recognition of two groups of HLA.C alleles. Now we describe a new allospecific NK group (group 0) which recognize all HLA.C alleles and we demonstrate that, on these clones, the p58 molecules EB6 and GL183 act independently to recognize Cw4 (and related alleles) and Cw3 (and related alleles) respectively. Finally we investigate whether the inhibitory signal mediated by the NK-receptor for HLA.C induce a temporary turn off on the cytolytic activity.

Alleles↗