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M Ferrarini

Publications and source records attributed to M Ferrarini.

At least 73 records · Page 4Linked to original sources
PubMed1996

Analysis of Epstein-Barr virus (EBV) type and variant in spontaneous lymphoblastoid cells and Hu-SCID mouse tumours.

Epstein-Barr virus (EBV) type and strain variations were examined using both lymphoblastoid cell lines (LCLs), spontaneously derived in vitro from peripheral blood mononuclear cells (PBMC) of 15 HIV-1-seropositive individuals; and SCID mouse tumours induced by inoculation of PBMC from 11 healthy human donors (Hu-SCID tumours). Polymerase chain reaction (PCR) analysis disclosed that all but one of the 26 EBV + samples harboured EBV nuclear antigen (EBNA) 2 and 3C type A virus. On the other hand, single strand conformation polymorphism (SSCP) analysis using Epstein-Barr encoded RNA (EBER) specific primers detected an AG876-like (type B) band pattern in 21 of the 26 EBV + samples. Three Hu-SCID tumours scored as B95.8-like (type A), and two showed neither a type A nor a type B SSCP migration pattern. Sequence analysis of the amplified EBER fragments confirmed the PCR-SSCP findings; moreover, additional mutations were present not only in the two EBV + samples with anomalous SSCP pattern, but also in two other samples with a standard SSCP profile. Thus, EBER analysis did not correlate with EBNA typing, and appeared to be unsuitable for EBV type assessment. Latent membrane protein (LMP) analysis disclosed, on the whole, sever size variants: as expected, the differences were due to the variable numbers of a 33-bp repeat in the amplified fragment, as assessed by direct sequencing. The broader variability detected by LMP analysis should prove more useful than typing for assessing the presence of single and/or mixed variants resulting from EBV reactivation and/or reinfection.

Animals↗
PubMed1996

B cells and their fate in health and disease.

A recent meeting discussed advances in our understanding of germinal centers, B-cell malignancies and B-cell signal transduction. Such developments have yielded a more refined view of the role of B-cells in health and disease.

B-Lymphocytes↗
PubMed1995

Daudi lymphoma killing triggers the programmed death of cytotoxic V gamma 9/V delta 2 T lymphocytes.

A common feature of human V gamma 9/V delta 2 gamma delta T lymphocytes is their ability to kill Daudi lymphoma cells. We show here that during this killing a substantial fraction of cytotoxic gamma delta T cells dies as well. Their death has morphologic, cytometric, and biochemical features of apoptosis and depends on TCR triggering. We suggest that interaction with the target physiologically induces the programmed death of the cytotoxic gamma delta effector. The death of activated gamma delta T cells upon challenge with the relevant Ag may represent a negative feedback mechanism and contribute to explain the limited time span of gamma delta T cell expansions during infectious diseases, even when pathogens are not eliminated and persist in the host.

Apoptosis↗
PubMed1995

Somatic diversification and selection of immunoglobulin heavy and light chain variable region genes in IgG+ CD5+ chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes. Most studies have found that these leukemic CD5+ B cells, like their normal counterparts, use immunoglobulin (Ig) variable (V) region genes that exhibit minimal, if any, somatic diversity. These and other observations have suggested that CD5+ B cells may be incapable of generating Ig V gene diversity, and therefore may not be able to develop higher affinity binding sites that could be selected by antigen. However, most of the studies of CLL and normal CD5+ B cells have focused on IgM-producing cells. Since somatic mutations are most often seen in B cells that have undergone an isotype class switch, we analyzed the Ig heavy (H) and light (L) chain variable region genes of seven IgG+CD5+ CLL B cells to determine if somatic diversification and antigen selection had occurred. The data derived provide evidence for skewed use, somatic diversification, and antigenic selection of the Ig V region genes. Nonrandom use of both H and L chain V region genes was manifested by an overrepresentation of VH4 and VKI family genes and the underrepresentation of the JH4 gene segment. Furthermore, VH4 gene use was restricted to only two family members (4.21 and 4.18). In four of the seven cases, the VH and VL genes displayed > or = 5% difference from the most homologous known germline counterparts. Polymerase chain reaction and Southern blot analyses performed in two of these patients demonstrated that their unique VH CDR2 and adjacent sequences were not present in their germline DNA. In addition, a significant level of diversity was seen in the rearranged DJH segments and at the VL-JL junctions of every patient that occurred both at the time of recombination and subsequently. The localization of replacement changes to complementarity determining regions of some patients suggested that antigen selection had occurred. Furthermore, the mutations identified in the VH and VL genes of each individual patient were strikingly similar, both in number and location. Collectively, the data indicate that a subset of CD5+ CLL B cells can display Ig V region gene mutations. In addition, they are consistent with the notions that in some cases antigen selection of these mutations may have occurred, and that antigen stimulation may be a promoting factor in the evolution of certain CLL clones.

Amino Acid Sequence↗
PubMed1995

Transfection of the c-myc oncogene into normal Epstein-Barr virus-harboring B cells results in new phenotypic and functional features resembling those of Burkitt lymphoma cells and normal centroblasts.

Activated c-myc gene was introduced into the cells of three normal Epstein-Barr virus (EBV)-positive lymphoblastoid B cell lines (LCL). The cells were monitored for the appearance of new phenotypic and functional features compared with the control LCL cells transfected with plasmid that did not contain the c-myc gene. The LCL-expressing c-myc constitutively did not arrest growth in low serum concentration. However, the cell number in the cultures failed to increase because of substantial cell death. Death was due to apoptosis as demonstrated by flow cytometric analysis of propidium iodide-stained cells, by typical DNA laddering in gel electrophoresis, and by the inspection of Giemsa-stained cell smears. Apoptosis was also induced by exposing the transfected cells to antibodies directed to the immunoglobulin mu chain (a-mu-ab) irrespective of the serum concentration in the culture. Exposure of the cells to CD40 ligand (CD40L) or CD40 monoclonal antibody prevented cell apoptosis. Upon transfection with c-myc, the LCL cells acquired a vacuolated morphology that was never observed in control cells. Moreover, the expression of CD10 and CD38 was upregulated, while that of CD39 and especially CD23 was downregulated. Unlike that observed in certain Burkitt lymphoma (BL) cell lines that share the same surface phenotype (CD10+CD38+CD23-CD39-), the c-myc-transfected cells expressed lymphocyte function-associated (LFA) 1, LFA-3, and intercellular adhesion molecule 1 and grew in large clumps rather than single-cell layers. Expression of CD10 and CD38 was particularly evident on the cells undergoing apoptosis, thus suggesting a correlation between the presence of these markers and the apoptotic process. Cells placed in conditions favoring in vitro apoptosis displayed downregulation of Bcl-2 protein. Bcl-2 expression was, however, upregulated when the cells were exposed to CD40L. These data indicate that the B cells expressing c-myc constitutively acquire some of the features of normal centroblasts and of BL cells, including the expression of CD10 and CD38, and the propensity to undergo apoptosis, which can be prevented by exposure to CD40L. Therefore, these cells can serve as a model system to study both BL lymphomagenesis as well as the process of B cell selection occurring in the germinal centers.

ADP-ribosyl Cyclase↗
PubMed1995

Production of inflammatory cytokines by Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines spontaneously originated from the peripheral blood of patients with human immunodeficiency virus (HIV) infection.

In this study we have raised spontaneous Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines (LCL) from the peripheral blood of human immunodeficiency virus (HIV)-infected individuals and of control patients with primary EBV infections. These LCLs were also raised in the presence of the viral inhibitor phosphonoformate (PFA); under these conditions, the in vitro infection of bystander B lymphocytes with EBV released in culture by in vivo infected B cells is inhibited. Thus, the latter LCLs are likely to represent the progeny of B cells latently infected by EBV in vivo. The LCLs raised in the presence or absence of PFA had the same phenotypic features, type of EBV latency, and growth pattern irrespective of whether they had been raised from HIV-seropositive individuals or patients with primary EBV infections or had been generated by infecting normal B cells in vitro. Studies on the production of inflammatory cytokines were conducted by Northern blotting or by determining the cytokine concentrations in the cell supernatants by immunoassays or bioassays. Three of eight LCLs from HIV-seropositive patients released TNF alpha and 5/5 released TNF beta, IL6 was present in the supernatants of 1/8 LCLs, and IL1 alpha and IL1 beta were not detected in any culture supernatant. No differences were noticed in the patterns of cytokine secretion among the LCLs from HIV-seropositive patients and in those raised from patients with primary EBV infections or obtained by infecting normal B cells in vitro with EBV. It is tempting to speculate that abnormally expanded EBV-harboring B cells in HIV-seropositive patients may participate in the pathogenesis of certain clinical manifestations by releasing inflammatory cytokines; some of these cytokines might also contribute to the in vivo spreading of HIV infection. However, the spontaneous LCLs from HIV-seropositive individuals do not display abnormal features compared to latently EBV-infected LCLs from other sources despite the high frequency of EBV-driven lymphoproliferative disorders observed in AIDS patients.

Adult↗
PubMed1995

Butyric acid, a metabolic end product of anaerobic bacteria, inhibits B-lymphocyte function.

The effect on B-lymphocytes of butyric acid, a metabolic by-product formed by selected anaerobic bacteria of the subgingival plaque and present in mM amounts in the crevicular fluid of patients affected by adult periodontitis was investigated. A dose-dependent inhibition of both mitogenesis and Ig production in B-cells challenged with formalinized Staphylococcus aureus, strain Cowan I (SAC), was observed. A role of this molecule in the downregulation of the local immune response in periodontal patients is proposed.

Adult↗
PubMed1994

Constitutive expression of the heat shock protein 72 kDa in human melanoma cells.

Heat shock proteins (hsp) are a family of proteins characteristically produced under stress conditions in normal cells. Overexpression of hsp 70 kDa protects tumoral cells from tumor necrosis factor cytotoxicity and is related to drug resistance. In this study we investigated whether hsp are abnormally expressed in melanoma cells in vivo. Antibodies directed against hsp 72 and hsp 90 were used to identify hsp on non-stressed neoplastic cells derived from surgical specimens of two primary and four metastatic melanomas. Hsp 72 and hsp 90 were always present at high level in the cytoplasm of melanoma cells. It is possible that the activation of hsp genes during neoplastic transformation confers a selective advantage to tumoral cells in vivo, allowing them to escape the immunological surveillance.

Cells, Cultured↗
PubMed1994

Spontaneous production of granulocyte colony-stimulating factor in vitro by human B-lineage lymphocytes is a distinctive marker of germinal center cells.

The ability of human B lymphocytes to produce granulocyte (G)-CSF in vitro was investigated. Highly purified tonsillar B cells were fractionated into large and small cells by a Percoll density gradient, cultured, and tested for G-CSF gene expression. Large B cells spontaneous produced G-CSF mRNA and protein, whereas small B cells did not, even after incubation with various stimuli. Immunophenotypic analyses showed that large B lymphocytes contained approximately 60 to 70% of cells with the characteristic surface markers of germinal center (GC) B cells (CD38+, CD10+, and surface IgG+). The remaining cells expressed CD39, CD23, and surface IgD and were presumably in vivo-activated follicular mantle zone B cells. Fractionation of the large B lymphocytes into CD39+, surface IgD+, and CD39-, surface IgD- cells showed that the latter, but not the former, cell type produced G-CSF spontaneously in culture. Stimulation of purified (CD39-, surface IgD-) GC B cells with a CD40 mAb alone or in combination with IL-4 increased G-CSF production. Because these stimuli rescued a large fraction of GC cells (up to 50%) from spontaneous apoptosis in vitro, the finding may suggest that prevention of apoptotic death resulted in an increased G-CSF production or that CD40 mab and/or IL-4 increased G-CSF gene expression in G-CSF-producing GC B cells. Malignant B cells purified from the invaded lymph nodes of three patients with follicular center cell lymphoma and three Burkitt lymphoma cell lines, which had an immunophenotype identical with that of normal GC B cells, spontaneously produced G-CSF in vitro, thus confirming the GC origin of the cytokine. Incubation of normal purified GC B cells with rG-CSF resulted in the rescue of GC B cells from apoptosis, suggesting that G-CSF may be used by GC B cells in an autocrine manner. This autocrine loop of production and response to G-CSF by GC B cells may be activated by stimuli such as those delivered via the surface CD40 molecule, that participate in the rescue of GC B cells from apoptosis.

Adenosine Triphosphatases↗
PubMed1994

A potential role for antigen selection in the clonal evolution of Burkitt's lymphoma.

Burkitt's lymphoma (BL) is a monoclonal lymphoproliferative disorder characterized by the presence of specific chromosomal translocations that involve the c-myc proto-oncogene. Two subtypes of BL exist (endemic and sporadic) that differ in the prevalence of EBV genome expression. Although EBV infection may promote cellular proliferation in endemic BL, little is known about the forces that drive clonal expansion and evolution in the majority of EBV-negative sporadic BL. This study on an EBV-negative sporadic BL cell line derived from an AIDS patient provides evidence that antigenic stimulation may play a role in the development and/or expansion of such tumors. This cell line (BRG-P) contained a series of cellular clones that elaborated both IgM and IgA. Southern blot analyses of the line and its sublines indicated that both the IgM+ and IgA+ cells had identical c-myc and Ig JH gene rearrangements, indicating that they were derived from a common precursor, some of which eventually underwent an isotype switch. Ig VH gene sequence analyses of 21 molecular clones derived from the parental BRG BL line and two of its sublines demonstrated that all the clones used the same VH3 gene. Five unique intraclonal variants were identified at four distinct nucleotide positions (125, 161, 355, 375), which undoubtedly represented somatic mutations. Four of these five mutations occurred within complementary determining regions; all resulted in amino acid replacements. Moreover, an identical G to A nucleotide substitution that resulted in an identical amino acid change occurred at two distinct points in clonal evolution that were separated by the isotype class switch. Thus, the locations and types of the VH gene mutations, together with the occurrence of an isotype switch, are highly suggestive of an ongoing role for Ag stimulation and selection in the evolution of the malignant clone.

Adult↗
PubMed1994

Distinct pattern of HSP72 and monomeric laminin receptor expression in human lung cancers infiltrated by gamma/delta T lymphocytes.

We have previously demonstrated that gamma/delta T lymphocytes may participate in the host immune response against lung adenocarcinomas. Here we show that, in about one-fourth of human lung cancers, gamma/delta T cells represented a significant proportion of freshly isolated tumor-infiltrating lymphocytes. Moreover, these cells selectively expand in vitro upon culture in the presence of IL-2, thus suggesting a prior activation in vivo. Finally, when we evaluated the expression of heat shock proteins and of a panel of tumor-associated antigens in lung cancers infiltrated by gamma/delta vs. alpha/beta T cells, we found that the former displayed a distinct antigenic pattern, characterized by over-expression of HSP72 and of the 67-kDa high-affinity laminin receptor, which might account for gamma/delta T-cell recognition.

Adenocarcinoma↗
PubMed1994

CD38 signaling by agonistic monoclonal antibody prevents apoptosis of human germinal center B cells.

The present study demonstrates that an agonistic anti-CD38 monoclonal antibody (mAb) (IB4) is capable of preventing apoptosis of human tonsillar germinal center (GC) B cells as measured by either morphological methods on Giemsa-stained cytospin preparations or flow cytometry on propidium iodide-stained cells. Two other anti-CD38 mAb (Leu-17 and OKT10) consistently failed to prevent apoptosis in the same cells, even when tested over a wide range of concentrations. Furthermore, exposure of GC B cells to IB4 mAb up-regulates the bcl-2 proto-oncogene product in a manner similar to that observed with CD40 ligand (CD40L). The ability of IB4 mAb to prevent apoptosis of GC B cells was inferior to that of both anti-CD40 mAb and CD40L. No synergistic or additive effects were observed when IB4 mAb was used together with CD40L. Unlike anti-CD40 mAb or CD40L, IB4 mAb neither induced a proliferation of GC B cells nor increased their proliferative response to anti-CD40, CD40L or recombinant interleukin-4, used alone or in combination. The present results are consistent with the recent findings on either the feature of the CD38 molecules to deliver activation signals and on the mechanisms of selection of B cells that operates in the GC.

ADP-ribosyl Cyclase↗
PubMed1994

Expression of CD5 and CD38 by human CD5- B cells: requirement for special stimuli.

In this study the mode of expression of CD5 by human tonsillar CD5- B cells after stimulation with different agents was investigated. Resting B cells were separated into CD5+ and CD5- cells and the two cell fractions exposed to phorbol 12-myristate 13-acetate (PMA). CD5- B cells expressed CD5 and maximum CD5 expression was achieved after approximately 60 h of culture. Based upon the proportions of cells that express CD5 as well as those of the cells surviving in culture, it was calculated that 15-25% of the total CD5- B cells were induced to express CD5. Unlike CD5- B cells, CD5+ B cells proliferated vigorously in response to PMA as assessed by [3H]thymidine incorporation and cell cycle analysis in vitro. However, the expression of CD5 by CD5- B cells was not related to the selective expansion of some CD5+ B cells left over as contaminant cells since this occurred in the absence of cell proliferation. Upon exposure to PMA, CD5- B cells remained in the G0-G1 phases of the cell cycle and did not express the Ki67 antigen or incorporate [3H]thymidine. Furthermore, mitomycin C treatment of the CD5- B cells did not prevent CD5 expression. Phenotypic studies disclosed that CD5+ B cells but not CD5- B cells expressed CD39. This finding offered the opportunity to carry out an additional control experiment. Separation of the two populations according to the expression of CD39 confirmed the finding obtained by fractionating the cells into CD5+ and CD5- B cells. The cells induced to express CD5 also expressed CD38 that was not detected on resting CD5- B cells. In this respect, the CD5- B cells that converted into CD5+ cells (inducible CD5+ B cells) resembled the cells from the CD5+ B cell fractions that up-regulated CD5 and also expressed CD38 upon exposure to PMA alone. Another example of coordinate expression of these two antigens was the finding that exposure to PMA in the presence of recombinant interleukin-4 (rIL-4) resulted in inhibition of the expression of CD5 and CD38. Although virtually all of the tonsillar CD5- B cells expressed the CD69 activation marker, no cells other than those co-expressing CD5 and CD38 were induced to express CD5 by PMA alone. Resting CD5- B cells failed to express CD5 and/or CD38 when cultured with PMA in the presence of EL4 T cells and IL-4-free T cell supernatants.(ABSTRACT TRUNCATED AT 400 WORDS)

ADP-ribosyl Cyclase↗
PubMed1994

HIV-1 induces down-regulation of bcl-2 expression and death by apoptosis of EBV-immortalized B cells: a model for a persistent "self-limiting" HIV-1 infection.

Interactions between HIV-1 and EBV were studied in HIV-1-infected EBV-positive lymphoblastoid B cells. Following in vitro exposure of B cells to HIV-1, the number of infected cells reached a plateau (25-35%) in approximately 20 days and remained fairly stable thereafter, despite the presence of infectious virus in culture supernatants. HIV-1-positive (gp120+) were separated from HIV-1-negative (gp120-) cells, and the two fractions were further characterized for EBV antigens, bcl-2 expression, and growth capacity in vitro. Compared to gp120- cells, EBNA 1, EBNA 2, and LMP 1 were down-regulated, and the episomal form of EBV-DNA was dramatically decreased in the gp120+ cells. When plated in culture gp120+, but not gp120-, cells died; BZLF1 antigen was not expressed, thus ruling out a reactivation of the EBV lytic cycle. Cytofluorometric, morphological, and molecular analyses disclosed that gp120+ cell death was due instead to apoptosis; evidence of bcl-2 down-regulation in these cells was consistent with this finding. gp120+ cell apoptosis contributed to keeping the level of HIV-1-infected cells at a steady state in the unfractionated culture, where persistent infection was maintained by HIV-1 transmission to B cells newly arising from the proliferation of HIV-1-uninfected cells.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Sensitivities and predictive values of paraclinical tests for diagnosing multiple sclerosis.

The sensitivities and predictive values of visual, somatosensory, and brain auditory evoked potentials (EPs), cerebrospinal fluid oligoclonal banding (CSF-OB) and magnetic resonance imaging (MRI) were evaluated for the early diagnosis of clinically definite multiple sclerosis (CDMS). Paraclinical evidence of asymptomatic lesions allows a diagnosis of CDMS. Eighty-two patients in whom MS was suspected but diagnosis of CDMS was not possible entered the study prospectively. Paraclinical examinations were performed at entry. Patients were examined and underwent EPs every 6 months, and MRI yearly. After a mean follow-up of 2.9 years, 28 patients (34%) had developed CDMS (McDonald-Halliday criteria). The initial MRI was strongly suggestive of MS in 19 of these (68%), while 27 (96%) had at least one MS-like abnormality in the initial MRI. CSF-OB and EPs had lower sensitivities. CDMS developed during follow-up in 19 of the 36 patients (53%) who had an initial MRI strongly suggestive of MS but in only 1 of the 25 who had normal MRI when first studied. These results support previous conclusions that MRI is the most sensitive test for detecting white matter asymptomatic lesions, and the most predictive for the diagnosis of CDMS.

Adolescent↗