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Biomedical subjects

F Grignani

Publications and source records attributed to F Grignani.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cellular stress and glucocorticoid hormones protect L929 mouse fibroblasts from tumor necrosis factor alpha cytotoxicity.

Adaptive responses to the environment depend on the induction of the "stress response" in less differentiated organisms and cultured cells and the activation of the hypothalamic-pituitary-adrenal axis in animals and humans. This indicates that adrenal steroids and stress proteins play an important role in regulating cell survival in response to noxious stimuli. In an in vitro model, we analyzed the effects of either dexamethasone (DEX) treatment or environmental changes which can elicit a stress response, on the survival of cultured L-929 mouse fibroblasts exposed to the cytotoxic cytokine tumor necrosis factor alpha (TNF-alpha). DEX treatment produced a significant reduction in the apoptotic death of L-929 cells produced by TNF-alpha. Abrogation of the protective effect of DEX by actinomycin D and cycloheximide demonstrated that protection against TNF-alpha requires de novo synthesis of mRNA and proteins. The results were similar when L-929 cells were exposed to metabolic (serum starvation) or thermal (heat shock) stresses before TNF-alpha treatment. In both cases the stress process afforded significant protection against TNF-alpha cytotoxicity. Inhibition of mRNA and protein synthesis abrogated the protection exerted by stress (serum starvation) or produced massive death during the stress event (heat shock). The similarities in the protective activities of DEX and stress response and the reported interactions between heat shock proteins and glucocorticoid hormones suggest that stress proteins and glucocorticoids both belong to an ancient evolutionary pathway which controls cell survival.

Animals↗
PubMed1993

Major pulmonary resections: pneumonectomies and lobectomies.

We report on our experience in 20 patients who underwent major thoracoscopic pulmonary resections between October 1991 and November 1992. These consist of 2 left pneumonectomies, 17 lobectomies, and 1 segmentectomy. The indications were strictly limited to benign pulmonary diseases and stage I (TNM) primary lung cancer. A hilar lymphadenectomy was performed in all cases of malignancy. Our surgical technique is described. Our findings demonstrate the feasibility of performing major video-assisted thoracoscopic pulmonary resections, even though the definite role of this procedure in the management of lung cancer must still be defined.

Female↗
PubMed1993

Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: a further pitfall in the use of the MTT assay for evaluating cell growth and survival.

The natural isoflavone genistein inhibits the growth of a number of tumour cell lines in vitro. During investigations on the antiproliferative effects of genistein we observed that, with respect to direct cell counting, a tetrazolium (MTT) colorimetric assay consistently underestimated the growth inhibitory activity of the substance. Cell proliferation was markedly inhibited by genistein in three tumour cell lines (MCF-7, human breast tumour; Jurkat cells, human T-cell leukaemia; L-929, mouse transformed fibroblasts) when cell number was evaluated by direct counting, whereas a 72-h MTT assay failed to reveal any growth-inhibitory effect. Cell cycle analysis by propidium iodide staining and flow-cytometry revealed a G2/M cell cycle arrest after genistein treatment. Genistein-treated cells displayed an increase in cell volume and in mitochondrial number and/or activity, as revealed by enhanced formazan generation and increased uptake of the vital mitochondrial dye rhodamine 123. These results suggest that alterations in cell cycle phase redistribution of tumour cells by genistein may significantly influence mitochondrial number and/or function and, consequently, MTT reduction to formazan. This may constitute an important bias in analysing the effects of genistein, and possibly other drugs that block the G2/M transition, on growth and viability of cancer cells in vitro by MTT assay.

Animals↗
PubMed1993

The molecular genetics of acute promyelocytic leukemia.

The chromosome breakpoints of the acute promyelocytic leukemia (APL)-specific 15;17 translocation have recently been isolated. They are localized on a previously unknown gene, PML, on chromosome 15 and in the gene that encodes the alpha retinoic acid receptor (RAR alpha) on 17. The translocation, which is balanced and reciprocal, leads to the formation of two fusion genes, PML/RAR alpha and RAR alpha/PML. Both are expressed in APL. The PML/RAR alpha gene codes for two abnormal proteins: the PML/RAR alpha fusion protein and an abnormal PML protein, the RAR alpha/PML gene encodes the RAR alpha/PML fusion protein. Experiments to investigate the biological activity of the abnormal translocation products are in progress. Preliminary results suggest that the PML/RAR alpha fusion protein is responsible for two important properties of the APL phenotype: the differentiation block characteristic of the leukemic blasts and the high sensitivity of the blasts to the differentiative action of retinoic acid (RA) both in vivo and in vitro. The mechanism through which PML/RAR alpha exerts its biological function remains unknown. However, there is accumulating evidence that it acts by interfering with normal endogenous pathways of both RAR alpha and PML. The RAR alpha receptor is implicated in regulating the myeloid differentiation induced by RA. Although the physiological function of PML is not known, it is probably a transcription factor. Definition of the molecular architecture of the t(15;17) has furnished further tools for: (1) molecular diagnosis of APL and (2) highly sensitive evaluation of the neoplastic clone during antileukaemic therapy. The molecular identification of residual APL disease after anti-leukaemia therapy allows patients at risk of relapse to be identified.

Carrier Proteins↗
PubMed1993

A prospective analysis of chemotherapy following surgical resection of clinical stage I-II small-cell lung cancer.

Thirty-seven (37) consecutive patients with clinical Stage I (T1-2NO, Mo), and Stage II (T1-2N1 Mo) central small-cell lung cancer (SCLC) underwent complete surgical resection of the primary tumor. Ten patients were subsequently pathologically Stage I, 14 patients were Stage II, and 8 were Stage III (T3;N2). The pathologically Stage I, II, and III patients were then treated with chemotherapy consisting of cyclophosphamide (1 g/m2), doxorubicin (50 mg/m2), and vincristine 2 mg (CAV) every 3 weeks for six courses followed by prophylactic cranial irradiation (2000 cGy in 10 fractions). Median survival in Stage I patients is 162 weeks and calculated 5-year survival is 50%; for Stage II patients, median survival is 86 weeks and calculated 5-year survival is 35%. T3;N2 patients have a median survival of 63 weeks; calculated 5-year survival is 21%. Our data suggest surgery plus adjuvant chemotherapy and cranial irradiation results in long-term survival in early central SCLC. These data support the need for randomized surgical trials in Stage I, II, and III central SCLC.

Adult↗
PubMed1993

Frequency of RAS and p53 mutations in acute promyelocytic leukemias.

The frequency of RAS and p53 mutations was investigated in 30 acute promyelocytic leukemias by single strand conformation polymorphism analysis and direct sequencing of genomic DNA. Only two cases bore N-RAS codon 12 mutations and none had p53 mutations responsible for aminoacid substitutions. It would, therefore, seem that neither RAS nor p53 are involved in acute promyelocytic leukemogenesis.

Base Sequence↗
PubMed1993

Videothoracoscopic excision of a mediastinal thymoma.

The authors have applied the advances in optics, endotelevision monitoring, and instrumentation that have led to the development of videoendoscopic abdominal surgery to performing major excisional procedures in the thorax. A videothoracoscopic technique for the removal of a mediastinal thymoma is described. The procedure was done on a 24-year-old woman suffering from a benign lymphoepithelial thymoma of the mediastinum. The mediastinal mass was mobilized under videolaparoscopic guidance and extracted from the thorax through a small (4 cm) inframammary incision. The postoperative course was uneventful, and the patient experienced minimal postoperative pain. She was discharged on the 3rd postoperative day with excellent functional and cosmetic results and resumed normal activity 4 days later.

Adult↗
PubMed1993

Cytostatic and cytotoxic effects of tumor necrosis factor alpha on MCF-7 human breast tumor cells are differently inhibited by glucocorticoid hormones.

To investigate the mechanisms of growth inhibition exerted by TNF-alpha on tumor cells in vitro, we analyzed the cytokine effects on growth and cell-cycle parameters of cultured MCF-7 human breast cancer cells. TNF-alpha exerted a dose-dependent inhibition of MCF-7 cell growth, which reached its maximum at 1000 U/ml TNF-alpha concentrations. Flow-cytometric analysis of cell nuclei revealed two main components in TNF-alpha activity: an earlier cytostatic effect (G1/S block), was followed by nuclear shrinkage and cytolysis. The 55-60-kDa TNF-alpha receptor is involved in the growth inhibitory activity of the cytokine, since the H398 anti-55-kDa receptor antibody significantly counteracted the cytostatic and cytotoxic effects of TNF-alpha while an antibody (htr-9) with agonistic activity on the same receptor produced both cytostasis and cytolysis. Culture conditions strongly influenced the MCF-7 cell response to TNF-alpha. Serum deprivation of log-growing (i.e., high S phase percentage) cultures potentiated the cytotoxic effect, while reduction in S phase cell percentage by preculture in serum-free medium resulted in a significant inhibition of TNF-alpha action. Mitogenic hormones, such as insulin and 17 beta-estradiol+insulin, restored the sensitivity of MCF-7 cells precultured in serum-free medium to both the cytostatic and cytolytic effects of TNF-alpha. The synthetic glucocorticoid hormone dexamethasone, at micromolar concentrations, counteracted the TNF-alpha effect on MCF-7 cell growth. Flow-cytometric analysis showed that dexamethasone did not antagonize the cytostatic activity of either TNF-alpha or htr-9 agonistic antibody, but only the subsequent cytolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1992

Human peritoneal mesothelial cells produce many cytokines (granulocyte colony-stimulating factor [CSF], granulocyte-monocyte-CSF, macrophage-CSF, interleukin-1 [IL-1], and IL-6) and are activated and stimulated to grow by IL-1.

To investigate the role of peritoneal mesothelial cells in regulating hematopoiesis, as well as inflammation, healing, and tissue regeneration processes, long-term cultures of peritoneal mesothelial cells from human endocavitarian fluids were established. The purity of the cell population was assessed by morphologic and immunocytochemical criteria. Five peritoneal mesothelial cell cultures were analyzed for cytokine expression. Macrophage colony-stimulating factor (M-CSF), granulocyte-CSF (G-CSF), interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6 transcripts were constantly but variably detected throughout the culture period, while granulocyte-monocyte-CSF (GM-CSF) expression started as the cell culture aged. No IL-2, IL-3, IL-4, IL-5, or IL-7 transcripts were detected in the same samples. Corresponding cytokine activities were detected in the supernatants of the cultures. Peritoneal mesothelial cells proliferated after the addition of exogenous IL-1 beta or IL-1 alpha, whereas the addition of recombinant GM-CSF, G-CSF, M-CSF, or IL-6 failed to trigger proliferation. IL-1 receptor type I transcripts were detected in peritoneal mesothelial cells. Moreover, IL-1 was able to upregulate the expression of the genes that code for G-CSF, GM-CSF, IL-1 alpha, and IL-1 beta in these cells. These data indicate that peritoneal mesothelial cells produce many cytokines and suggest that IL-1 is a regulatory molecule for peritoneal mesothelial cells.

Antigens, CD↗
PubMed1992

A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.

A new SH2-containing sequence, SHC, was isolated by screening cDNA libraries with SH2 representative DNA probes. The SHC cDNA is predicted to encode overlapping proteins of 46.8 and 51.7 kd that contain a single C-terminal SH2 domain, and an adjacent glycine/proline-rich motif with regions of homology with the alpha 1 chain of collagen, but no identifiable catalytic domain. Anti-SHC antibodies recognized three proteins of 46, 52, and 66 kd in a wide range of mammalian cell lines. These SHC proteins complexed with and were phosphorylated by activated epidermal growth factor receptor. The physical association of SHC proteins with activated receptors was recreated in vitro by using a bacterially expressed SHC SH2 domain. NIH 3T3 mouse fibroblasts that constitutively overexpressed SHC acquired a transformed phenotype in culture and formed tumors in nude mice. These results suggest that the SHC gene products couple activated growth factor receptors to a signaling pathway that regulates the proliferation of mammalian cells.

3T3 Cells↗
PubMed1992

Characterization of the PML-RAR alpha chimeric product of the acute promyelocytic leukemia-specific t(15;17) translocation.

The acute promyelocytic leukemia 15;17 chromosomal translocation fuses the PML gene to the RAR alpha locus. The resulting chimeric gene encodes for a putative PML-RAR alpha fusion protein. PML is a putative transcriptional factor and RAR alpha is one of the nuclear retinoic acid receptors through which retinoic acid regulates gene expression. In this study, we investigated the retinoid binding and biochemical properties of the PML-RAR alpha protein by size exclusion high-performance liquid chromatography and immunoblot analysis and compared them with those of normal RAR alpha. The introduction of the expression vector PSG5/PML-RAR alpha into COS-1 cells led to high levels of expression of the PML-RAR alpha fusion protein. This protein was primarily localized in the nucleus and bound retinoids with the same affinity and specificity as the wild type RAR alpha receptor. The PML-RAR alpha fusion protein, but not the RAR alpha, was found in high molecular weight complexes with either itself or other nuclear factors. In the acute promyelocytic leukemia-derived cell line NB4, which contains the t(15;17) chromosomal marker, the PML-RAR alpha product was also found as a high molecular complex. The interaction of the PML-RAR alpha with itself or with other nuclear proteins may be important in understanding the role of the PML-RAR alpha fusion protein in promyelocytic leukemogenesis.

Carrier Proteins↗
PubMed1992

Identification of DNA rearrangements at the retinoic acid receptor-alpha (RAR-alpha) locus in all patients with acute promyelocytic leukemia (APL) and mapping of APL breakpoints within the RAR-alpha second intron. Italian Cooperative Study Group "GIMEMA".

Seventy patients with acute promyelocytic leukemia (APL) were characterized at the DNA level using genomic retinoic acid receptor-alpha (RAR-alpha) probes on Southern blot experiments. Sixty-two cases were defined as M3 according to the French-American-British (FAB) criteria, and eight had a diagnosis of microgranular or variant (M3v) APL. The use of two restriction enzymes and three probes exploring the second intron of the RAR-alpha gene allowed us to detect specific abnormal DNA fragments in every case, with clustering of rearrangements within the 20-kb intronic region between RAR-alpha exons II and III. A more detailed mapping of APL breakpoints was performed in 52 cases in which three EcoRI subregions of the RAR-alpha second intron were analyzed with corresponding probes. Comparison of clinical and hematological features in the three subgroups of patients with distinct RAR-alpha breakpoints did not show significant differences regarding age, peripheral blood (PB) counts, presence of coagulopathy, or FAB classification (M3 v M3v). Interestingly, a significant difference was observed in the M/F ratio of the three subgroups, with a higher incidence of rearrangements at the 5' end of the RAR-alpha second intron in female patients, and more frequent 3' breakpoints in males. The results of this study indicate that a unique genomic alteration consistently occurs on the 17q- derivative of the APL specific t(15;17) aberration. Moreover, the clinical relevance of RAR-alpha gene analysis both at diagnosis and in follow-up studies is further emphasized.

Adolescent↗
PubMed1992

Expression pattern of the RAR alpha-PML fusion gene in acute promyelocytic leukemia.

Two chimeric genes, PML-RAR alpha and RAR alpha-PML, are formed as a consequence of the acute promyelocytic leukemia (APL)-specific reciprocal translocation of chromosomes 15 and 17 [t(15;17)]. PML-RAR alpha is expressed as a fusion protein. We investigated the organization and expression pattern of the RAR alpha-PML gene in a series of APL patients representative of the molecular heterogeneity of the t(15;17) and found (i) two types of RAR alpha-PML mRNA junctions (RAR alpha exon 2/PML exon 4 or RAR alpha exon 2/PML exon 7) that maintain the RAR alpha and PML longest open reading frames aligned and are the result of chromosome 15 breaking at two different sites; and (ii) 10 different RAR alpha-PML fusion transcripts that differ for the assembly of their PML coding exons. A RAR alpha-PML transcript was present in most, but not all, APL patients.

Base Sequence↗
PubMed1992

Genomic variability and alternative splicing generate multiple PML/RAR alpha transcripts that encode aberrant PML proteins and PML/RAR alpha isoforms in acute promyelocytic leukaemia.

The acute promyelocytic leukaemia (APL) 15;17 translocation generates a PML/RAR alpha chimeric gene which is transcribed as a fusion PML/RAR alpha mRNA. Molecular studies on a large series of APLs revealed great heterogeneity of the PML/RAR alpha transcripts due to: (i) variable breaking of chromosome 15 within three PML breakpoint cluster regions (bcr1, bcr2 and bcr3), (ii) alternative splicings of the PML portion and (iii) alternative usage of two RAR alpha polyadenylation sites. Nucleotide sequence analysis predicted two types of proteins: multiple PML/RAR alpha and aberrant PML. The PML/RAR alpha proteins varied among bcr1, 2 and 3 APL cases and within single cases. The fusion proteins contained variable portions of the PML N terminus joined to the B-F RAR alpha domains; the only PML region retained was the putative DNA binding domain. The aberrant PML proteins lacked the C terminus, which had been replaced by from two to ten amino acid residues from the RAR alpha sequence. Multiple PML/RAR alpha isoforms and aberrant PML proteins were found to coexist in all APLs. These findings indicate that two potential oncogenic proteins are generated by the t(15;17) and suggest that the PML activation pathway is altered in APLs.

Amino Acid Sequence↗
PubMed1992

Human T-helper clones induce IgG production in a subclass-specific fashion.

The mechanisms governing the induction of IgG subclasses by T-helper cells in humans were investigated. As preliminary bulk-culture experiments had indicated that a direct B cell contact with viable T cells was an essential requirement for optimal IgG subclass production, 256 CD4+ human T cell clones were preactivated with PHA and cultured in direct contact with autologous B cells. These clones induced IgG production in a strikingly subclass-specific fashion. Moreover, the distribution of subclass-specific helper clones was very similar to the IgG subclass profile observed in serum and peripheral lymphoid tissue plasma cells (IgG1 approximately 60%, IgG2 approximately 30%, IgG3 approximately 5-10%, IgG4 less than or equal to 5%) and unlike that observed in resting B cells (which is IgG1 approximately 40% and IgG2 approximately 50%). It would, therefore, seem that a predominance of T cells capable of delivering IgG1-specific, as opposed to IgG2-specific, help is an essential factor for the preferential induction of IgG1 antibodies during B cell proliferation and differentiation. There was no relationship between IL2, IL4, IL6, and IFN-gamma secreted by the T-helper clones and their IgG subclass induction patterns. In addition, only a few supernatants were able to reproduce the helper effects of the clones themselves. Therefore, direct contact of B cells with helper clones is crucial for IgG-subclass production in humans.

B-Lymphocytes↗
PubMed1992

Double-blind randomized phase I study on the clinical tolerance and biological effects of natural and recombinant interferon-beta.

The clinical tolerance of and the effects recombinant human interferon-beta (rHuIFN-beta) obtained from mammalian cells (Chinese hamster ovary cells) exerts on 2',5'-oligoadenyl (2-5A) synthetase activity, human-Mx protein, neopterin, beta 2-microglobulin, interleukin-1 (IL-1) alpha and beta synthesis were compared to those of natural IFN-beta in 12 healthy volunteers. Each subject received a single i.m. injection of 6 x 10(6) IU rHuIFN-beta and natural IFN-beta according to a randomized double-blind cross-over study design. Both were well tolerated and provoked similar changes in clinical indices. Moreover, rHuIFN-beta and natural IFN-beta induced significant and similar increases in 2'-5' adenylates, human Mx protein, and neopterin levels, but neither modulated beta 2-microglobulin, IL-1 alpha or beta synthesis. The sum of these findings indicates that rHuIFN-beta and natural IFN-beta are biologically equivalent. In view of these results, we are of the opinion that these two types of IFN are probably also therapeutically equivalent and, in consequence, that trials to evaluate the response of viral and neoplastic disease patients to rHuIFN-beta are fully justified.

2',5'-Oligoadenylate Synthetase↗