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Biomedical subjects

G Cattoretti

Publications and source records attributed to G Cattoretti.

At least 37 records · Page 2Linked to original sources
PubMed1996

Distinctive expression pattern of the BCL-6 protein in nodular lymphocyte predominance Hodgkin's disease.

The BCL-6 gene encoding a nuclear-located Kruppel-type zinc finger protein is rearranged in about 30% diffuse large B-cell lymphomas and is expressed predominantly in normal germinal center B cells and related lymphomas. These findings suggest that BCL-6 may play a role in regulating differentiation of normal germinal center B cells and that its deregulated expression caused by rearrangements may contribute to lymphomagenesis. This prompted us to investigate the expression of the BCL-6 protein in Hodgkin's disease (HD), focusing on the nodular lymphocyte predominance subtype (NLPHD), which differs from classical HD by virtue of the B-cell nature of the malignant cell population (so-called L&H cells) and its relationship with germinal centers. Forty-one HD samples (19 NLPHD, 12 nodular sclerosis, and 10 mixed cellularity) were immunostained with the monoclonal antibodies PG-B6 and PG-B6p that react with a fixative-sensitive and a formalin-resistant epitope on the aminoterminal region of the BCL-6 gene product, respectively. Strong nuclear positivity for the BCL-6 protein was detected in tumor (L&H) cells in all cases of NLPHD. In contrast, BCL-6 was expressed only in a small percentage of Hodgkin and Reed-Sternberg cells in about 30% of classical HD cases. Notably, the nuclei of reactive CD3+/CD4+ T cells nearby to and rosetting around L&H cells in NLPHD were also strongly BCL-6+, but lacked CD40 ligand (CD40L) expression. This staining pattern clearly differed from that of classical HD, whose cellular background was made up of CD3+/CD4+ T cells showing the BCL-6-/CD40L+ phenotype. These results further support the concept that NLPHD is an histogenetically distinct, B-cell-derived subtype of HD and suggest a role for BCL-6 in its development.

Animals↗
PubMed1995

BCL-6 protein is expressed in germinal-center B cells.

Structural alterations of the 5' noncoding region of the BCL-6 gene have been found in 40% of diffuse large cell lymphoma (DLCL) and 5% to 10% of follicular lymphomas (FL), suggesting that deregulated BCL-6 expression may play a role in lymphomagenesis. Nucleotide sequencing of BCL-6 cDNA predicted a protein containing six zinc-finger domains, suggesting that it may function as a transcription factor. Using antisera raised against N- and C-terminal BCL-6 synthetic oligopeptides in immunoprecipitation, immunoblot, and immunocytochemical assays, this study identifies the BCL-6 gene product as a 95-kD nuclear protein. Western blot analysis of human tumor cell lines representative of various hematopoietic lineages/stages of differentiation showed that the BCL-6 protein is predominantly expressed in the B-cell lineage where it was found in mature B cells. Immunohistochemical analysis of normal human lymphoid tissues indicated that BCL-6 expression is topographically restricted to germinal centers including all centroblasts and centrocytes. The BCL-6 protein was also detectable in inter- and intra-follicular CD4+ T cells, but not in other follicular components including mantle-zone B cells, plasma cells, dendritic cells, and macrophages. Immunohistochemical analysis of DLCL and FL biopsy samples showed that the BCL-6 protein is detectable in these tumors independent of the presence of BCL-6 gene rearrangements. These results indicate that the expression of the BCL-6 gene is specifically regulated during B-cell differentiation and suggest a role for BCL-6 in germinal center development or function. Because DLCL derive from germinal-center B cells, deregulated BCL-6 expression may contribute to lymphomagenesis by preventing postgerminal center differentiation.

Amino Acid Sequence↗
PubMed1995

Immuno-electron microscopy characterization of human bone marrow stromal cells with anti-NGFR antibodies.

Human bone marrow stromal cells have been examined with an immuno-electron microscopy technique in order to better define their structure and function in normal hematopoiesis. Bone marrow fragments from normal donors, after mild permeabilization and glutaraldehyde prefixation were labeled with the Me20.4 Mab, which recognizes the low affinity nerve growth factor (NGFR) and was recently described as specifically identifying fibroblastic-like bone marrow stromal cells. Five nm gold immuno-conjugates served as markers. NGFR+ cells were showing either a star-shape, with long and convoluted dendritic projections, and branching with each other to form a complex system of lacunae upon which hematopoietic cells were arranged. Other NGFR+ cells had an elongated spindle-like morphology. NGFR+ dendrites were seen in close contact with each other and with the different hematopoietic cells, although definite junctions were never noticed. NGFR+ dendrites were also observed surrounding mature plasma cells, in close apposition with adipocytes or surrounding bone marrow sinusoids. These findings may give some clues about the function of the bone marrow stromal cells, which are known to be involved in the homing and recirculation of hemopoietic cells; in addition, the presence and distribution of NGFR in the bone marrow stroma may support the recent evidence of a co-stimulatory effect of NGF in early hematopoiesis.

Bone Marrow↗
PubMed1995

Correlation between presence of clonal rearrangements of immunoglobulin heavy chain genes and B-cell antigen expression in Hodgkin's disease.

Southern blot analysis of Hodgkin's disease (HD), although often compromised by the small number of abnormal cells present in the tissue, have tended to favor a B-cell derivation of the Hodgkin's and Reed-Sternberg (HRS) cells in cases of nodular sclerosis (NS) and mixed cellularity (MC) Hodgkin's disease. Eighteen frozen and 29 paraffin-embedded sections of lymph node specimens from 29 patients with pretreatment HD (22 NSHD and 7 MCHD) were studied by molecular analysis and immunohistochemistry to determine the phenotype of HRS cells. All cases were reviewed and showed typical morphology and CD45-, CD30+, CD15+, BLA.36+ HRS cells. In 11 of 29 (38%) cases, HRS cells were reactive with at least one B-cell marker (CD20, CD79a, MB2), 7 of 29 (24%) cases showed reactivity with the T-cell marker CD3, and 11 of 29 (38%) cases displayed a "null" phenotype. By using a polymerase chain reaction (PCR) and consensus primers for the V and J regions of the immunoglobulin heavy chain (IgH) gene, the authors were able to detect B-cell clonality in 9 of 18 (50%) frozen samples of HD analyzed. IgH gene rearrangement was present in 8 of 15 (53%) NSHD and in 1 of 3 (33%) MCHD. In five of nine (56%) of these cases, HRS cells were reactive with at least one B-cell marker, whereas one case expressed the T-cell marker CD3. The other three cases with IgH gene rearrangement showed a "null" immunophenotype. IgH gene analysis was negative in all remaining CD3+ cases and in two other cases that expressed B-cell markers by immunohistology. Southern blotting failed to detect rearrangement of the T-cell receptor beta-chain gene and immunoglobulin heavy and light genes in any of these cases. The results show that PCR represents a specific and sensitive technique for the detection of IgH gene rearrangements in cases of Hodgkin's disease. The results also suggest a lymphoid B-cell derivation of HRS cells in a high proportion of the cases.

Antigens, Neoplasm↗
PubMed1995

A specific monoclonal antibody (PG-B6) detects expression of the BCL-6 protein in germinal center B cells.

The BCL-6 gene is frequently involved in translocations occurring at the 3q27 locus and is rearranged in approximately 30% of diffuse large cell lymphomas and in a small fraction of follicular lymphomas. The BCL-6 gene encodes for a Kruppel-type zinc-finger protein, the cell/tissue expression and function of which is unknown. In this study, we describe a new monoclonal antibody (PG-B6) that is specifically directed against a fixative-sensitive epitope on the amino-terminal region of the BCL-6 protein. By immunocytochemical analysis, BCL-6 localizes in the nucleus where PG-B6 staining gives a microgranular/diffuse pattern with exclusion of the nucleoli. The main reactivity of PG-B6 in tonsil and spleen is with the nuclei of germinal center B cells, whereas B cells within the mantle and marginal zones do not express BCL-6. No other lymphoid cells in the tonsil express BCL-6 except for a subset of CD3+/CD4+ intrafollicular and interfollicular T cells. A few lymphoid cells of unknown phenotype express BCL-6 in the thymus. Extra-lymphoid BCL-6 expression includes a weak nuclear positivity of epithelia. In non-Hodgkin's lymphomas, BCL-6 expression parallels that observed in normal lymphoid compartments, eg, expression in germinal center-derived tumors (follicular and diffuse large cell lymphomas), but not in mantle cell and marginal zone lymphomas. In most diffuse large cell lymphomas, the BCL-6 protein is expressed at high levels in cases with or without BCL-6 gene rearrangements. These findings indicate that BCL-6 expression is specifically regulated during B lymphocyte development and suggest that BCL-6 may play a role during B cell differentiation in the germinal center.

Animals↗
PubMed1994

Bcl-2 protein expression in carcinomas originating from the follicular epithelium of the thyroid gland.

The bcl-2 product has been related to the block of programmed cell death (apoptosis) both in lymphoid and in epithelial cells. The pathological expression of bcl-2 has been investigated mainly in haematological malignancies. Here we have investigated bcl-2 expression in a model of epithelial tumours represented by the spectrum of carcinomas arising from the follicular epithelium of the human thyroid gland. The analysis was carried out by immunocytochemistry on archival material using monoclonal antibodies against bcl-2 and thyroglobulin (Tg) on consecutive sections of 94 well-differentiated carcinomas (WDCs), 19 poorly differentiated carcinomas (PDCs), and 22 undifferentiated carcinomas (UCs) of the thyroid gland. In a subset of 5 cases of UC showing a differentiated component (UC-D), the expression of p53 protein was also investigated. As controls, fetal and adult normal thyroid glands and adenomas were analysed. bcl-2 expression was detected in 74 of 94 cases (78.7 per cent) of WDC, 16 of 19 cases (84.2 per cent) of PDC, and 3 of 22 cases (13.6 per cent) of UC. Simultaneous expression of bcl-2 protein and Tg was observed in 74 of 94 cases (78.7 per cent) of WDC, 13 of 19 cases (68.4 per cent) of PDC, and in no case of UC. bcl-2 and Tg immunostaining was detected in all fetal and normal thyroid glands as well as in the adenoma specimens examined. In the subset of UC-D, mutual exclusion of bcl-2 and p53 expression was observed in the undifferentiated and differentiated components (p53 but not bcl-2 expressed in the former).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

Terminal deoxynucleotidyl transferase staining in acute leukemia and normal bone marrow in routinely processed paraffin sections.

Terminal deoxynucleotidyl transferase (TdT) is a nuclear protein widely used as a marker for the diagnosis and classification of acute leukemia. The usual methods for detecting TdT require smears, imprints, or cryostat sections of unfixed tissue. A polyclonal rabbit anti-TdT serum was used to immunostain 54 routinely processed bone marrow sections from patients with acute leukemic disorders, using a recently described antigen-unmasking technique based on microwave oven heating. The specificity of this method of TdT analysis was confirmed by comparing the results obtained with conventional TdT analysis by indirect immunofluorescence. Terminal deoxynucleotidyl transferase reactivity was also evaluated in 44 nonmalignant and normal bone marrow specimens. All cases that were TdT-positive by immunofluorescence (41 of 42 "pre-B" and T-cell acute lymphoblastic leukemia, 2 of 5 acute myeloid leukemia, and 1 of 5 chronic myeloid leukemia in blast crisis) were also positive in paraffin sections. The percentage fluorescence positivity correlated with the percentage of immunoperoxidase stained cells in 44 of 45 cases. The remaining nonneoplastic and normal bone marrow biopsy specimens were TdT-negative. These results show that TdT immunoperoxidase staining of conventionally processed bone marrow specimens can be readily achieved by the use of a simple antigen-unmasking technique and may provide useful diagnostic information particularly in cases in which fresh tissue samples are unavailable.

Acute Disease↗
PubMed1994

A novel panel of antibodies that segregates immunocytochemically poorly differentiated carcinoma from undifferentiated carcinoma of the thyroid gland.

The expression of bcl-2 and p53 was investigated by immunocytochemistry in combination with that of conventional structural and differentiation antigens on the archival material of 22 cases of undifferentiated carcinoma (UC) and 19 of poorly differentiated carcinoma (PDC) of the thyroid gland. The restriction of bcl-2 expression to PDC in comparison to UC was 84.2% versus 13.6% of cases, respectively, in contrast to an almost equal percentage of p53 expression in the two histologic types, that is, 52.6% and 54.5% of cases of PDC and UC, respectively. However, the pattern of distribution of p53-immunoreactive cells was definitely different, being restricted to areas showing active infiltrating growth in PDC and involving almost all tumor cells in UC. Furthermore, in the subset of cases of UC showing the residual presence of a differentiated component, a distinctive mutual exclusion of bcl-2 and p53 immunoreactivity was observed in the two components. The results suggest that the evaluation of bcl-2 expression may be usefully applied to the differentiation of PDC from UC, whereas all morphologic findings related to p53 expression are in keeping with a significant role of the deregulation of this gene in the mechanism of dedifferentiation and progression of the disease.

Adult↗
PubMed1994

Assessment of cell proliferation in paraffin sections of normal bone marrow by the monoclonal antibodies Ki-67 and PCNA.

Assessment of the proliferative index of human bone marrow using immunohistology in tissue sections offers practical advantages over other techniques. It is simpler to perform and can be applied to archival material. Progress in this area has been hampered by technical problems. Ki-67 studies, limited to fresh or frozen tissues, have been contradictory, and the labeling indices have been criticized for being unrealistically low. Proliferating cell nuclear antigen (PCNA) studies have also been considered unreliable because labeling indices include nondividing cells. We stained 82 routinely processed nonneoplastic bone marrow biopsies for Ki-67 equivalent MIB 1 antibody and PCNA/PC10 using a microwave oven antigen retrieval technique. 44.7% +/- 12.0 SD of the bone marrow cells stained with Ki-67/MIB 1. PCNA/PC10 staining gave similar results (50.7% +/- 12.3 SD). Pro- and basophilic erythroblasts, myeloblasts, promyelocytes, myelocytes, and megakaryocytes stained with both monoclonal antibodies in a manner consistent with known morphologic and cytokinetic data. Patients in different clinical groups showed similar mean values, the only exception being a group of cytokine-treated patients, which showed higher values than the other cases. Immunoperoxidase staining of bone marrow sections with Ki-67/MIB 1 and PCNA/PC10 monoclonal antibodies is a reliable method for assessing proliferation of hematopoietic cells.

Adult↗
PubMed1994

Terminal deoxynucleotidyl transferase staining of malignant lymphomas in paraffin sections.

Terminal deoxynucleotidyl transferase (TdT) represents a useful marker for the diagnosis of acute lymphoblastic leukemia and lymphoblastic lymphoma. It is usually assayed in fresh tissue or cell suspensions by enzyme analysis, immunofluorescence, or immunoperoxidase. We have obtained satisfactory staining for TdT in routinely processed paraffin sections using a recently described antigen retrieval pretreatment based on microwave oven heating of the sections and a polyclonal rabbit anti-TdT serum. With this technique, we assayed paraffin sections from lymph nodes of 91 patients with a variety of malignant lymphomas, including 35 cases of lymphoblastic lymphoma. The specificity of this immunoperoxidase method of TdT analysis was confirmed by comparing the results obtained with conventional TdT analysis by indirect immunofluorescence. Neoplastic cells from 33 of the 35 (94%) patients with lymphoblastic lymphoma were TdT positive with both techniques. All remaining 50 cases of other subtypes of malignant lymphoma were TdT negative. Specificity was further confirmed by demonstrating TdT-positive blasts in paraffin sections of bone marrow biopsies from 44 patients with TdT-positive (by indirect immunofluorescence) acute leukemias and in paraffin sections of extramedullary leukemic infiltrates from eight patients with relapsed acute lymphoblastic leukemia. Other control material studied included eight lymph nodes with Hodgkin's disease, four normal spleen, seven reactive lymph nodes, and 25 nonneoplastic bone marrow samples, all of which gave the expected results, i.e., rare scattered TdT-positive cells in bone marrow and lymph nodes, absence of staining in Hodgkin's disease malignant cells and spleen. Our results confirm that TdT is a specific marker for the diagnosis and classification of lymphoblastic lymphoma.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Nucleotidylexotransferase↗
PubMed1993

Subtypes of Epstein-Barr virus in HIV-1-associated and HIV-1-unrelated Hodgkin's disease cases.

Epstein-Barr virus (EBV) type 2 is considered to be a much less potent transformer of lymphocytes than type 1. However, type-2 EBV may be involved in the pathogenesis of non-Hodgkin's lymphomas (NHLs) arising in immunocompromised patients, i.e., subjects with malaria or HIV-1 infection. To determine whether type-2 EBV may also play a role in Hodgkin's disease (HD) developing in immunocompromised patients, we characterized EBV subtypes in EBV-positive HD samples from 10 HIV-1-positive patients as well as from a control population of 24 HIV-1-negative patients. Type-2 EBV was detected in 5/10 HD samples from the HIV-1-positive group (1 case showed concomitant type-1 EBV positivity), but only in 1/20 HD samples from the HIV-1-negative group, indicating that, during HIV-1-induced immunodepression, type-2 EBV may be pathogenetically involved also in HD, as previously reported for HIV-associated NHLs.

Base Sequence↗
PubMed1993

Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro.

Two anti-nerve growth factor receptor (LNGFR or p75NGFR) antibodies, Me20.4 and Me8211, label stromal cells with dendritic features in fresh smears and in formalin-fixed, paraffin-embedded human bone marrow (BM). The LNGFR+ cells have an oval nucleus, a scanty cytoplasm with long dendrites that intermingle with the hematopoietic cells, line the abluminal side of sinus endothelial cells, and provide the scaffold for the hematopoietic marrow. At the electron microscopy level, the immunogold tag labels the body and the long branching dendrites of fibroblast-like cells with scanty cytoplasm containing mitochondria, endoplasmic reticulum, and dense bodies. The LNGFR+ cells are positive for alkaline phosphatase, reticulin, collagen III, vimentin, TE-7, and CD13 but negative for endothelial (vWF, CD34, Pal-E), neural (CD56, neurofilament) and leukocyte markers (CD45, CD68). The LNGFR+ stromal cells appear in the fetal BM before the hematopoietic activity begins, originate from the vessel adventitia, and radiate in the Bm cavity. Long-term BM culture (LTBMC) in vitro contain LNGFR+ stromal cells. We document the presence of RNA message for the low- (LNGFR) and the high-affinity NGF receptor (NTRK1) by using RT-PCR on fresh BM aspirate and on LTBMC. BM biopsies from patients with hematologic fibrogenic diseases and in cytokine-treated cancer patients are evaluated for LNGFR+ cells: the amount of stained cells is correlated with the traditional reticulin stain in cases of myelofibrosis, therapy-related myelodysplasia, leukemia, and detected an increase of stromal cells in cytokine-treated patients. The anti-LNGFR antibodies represent a specific membrane marker for the adventitial reticular cells (ARC) of the human marrow and allow precise evaluation and quantitation of this important BM microenvironment component in vivo and in vitro.

Adult↗
PubMed1993

Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections.

Enzymatic and non-enzymatic treatments for antigen unmasking on formalin-fixed, paraffin-embedded, dewaxed sections were optimized and compared by the use of a panel of antibodies of diagnostic relevance (anti-cytokeratins, vimentin, S-100, T- and B-cell receptors, Ki-67/MIB 1, muscle actin). Non-enzymatic unmasking was obtained by boiling the slides in a microwave oven in 0.01 M salt solution (pH 6) or in 6 M urea. Trypsin or pronase digestion was used for comparison and found to be necessary for some of the reagents. The investigation was then extended to 256 antibodies; the epitopic amino acid sequence was known for 48 of them. We found that enzymatic and non-enzymatic antigen unmasking are not dependent on the epitope sequence, but some antigens benefit selectively from one treatment but not from the other. Denaturation of proteins is the likely mechanism which leads to immunodetection on microwave oven-boiled slides; this suggestion is supported by the use of denaturating solutions and by the observation that endogenous enzymes were inactivated and a few antigens were no longer immunodetectable after boiling. Non-enzymatic methods for antigen unmasking are a powerful new tool for broadening the use of antibodies for immunostaining formalin-fixed, paraffin-embedded sections and should be used in parallel with the traditional enzymatic methods.

Antibodies↗
PubMed1993

Proliferating normal bone marrow cells do stain for Ki-67 antigen.

The normal human bone marrow has been investigated in the past for the presence of the proliferation associated antigen detected by the Ki-67 antibody and aberrantly low scores for this antigen have been reported. We used a new Ki-67 equivalent antibody (MIB 1) and a formalin fixation of smears and trephine biopsies, and we report 52.8% +/- 9.2 SD of the normal BM cells to stain for MIB 1. Late forms of the maturing blood cells compartment such as metamyelocytes and megakaryocytes were stained in cytokine-treated marrows.

Bone Marrow↗
PubMed1993

B and T lymphocyte subsets in fetal and cord blood: age-related modulation of CD1c expression.

Peripheral blood lymphocytes from fetal, term and adult subjects were analyzed with a panel of B- and T-lymphoid differentiation markers. CD1c +ve and CD5 +ve B cell (IgM +ve) percentage in fetal, cord and adult blood decreased significantly. Fetal and cord blood contained less CD2+ and CD3+ T lymphocytes and a higher percentage of CD38 +ve cells than adult blood. The phenotypic changes occurring during perinatal age in humans relate to the changes in the functional activities of the B and T lymphoid subsets from fetal to postnatal age.

Adult↗