Biomedical subjects
Publications and source records attributed to C Monaco.
In this article, the clinical, angiographic, and postmortem features of unstable angina are reviewed and its pathogenesis is discussed. Coronary plaque inflammation may play a key role in the pathogenesis of unstable angina and the evidence for this assertion is examined. Finally, the therapeutic implications of the involvement of inflammation in acute coronary syndromes are outlined.
Inherited activating mutations of Ret, a receptor tyrosine kinase, predispose to multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma. To investigate the effects induced by acute stimulation of Ret, we transfected both PC12 and NIH 3T3 cells with a molecular construct in which the ligand-binding domain of the epidermal growth factor receptor was fused to the catalytic domain of Ret. Acute stimulation of the chimeric receptor induced PC12 cells to express a neuronal-like phenotype. Moreover, we introduced the dominant mutation, responsible for the multiple endocrine neoplasia type 2B, in the catalytic domain of the Ret chimera. Expression of the mutant chimera, in the absence of ligand stimulation, induces the PC12 cells to acquire a flat morphology with short neuritic processes and transforms the NIH 3T3 cells. Stimulation of the mutant chimera with epidermal growth factor causes a drastic overgrowth of long neuritic processes, with the induction of the suc1-associated protein tyrosine phosphorylation in PC12 cells and higher transforming efficiency in NIH 3T3 cells. These data indicate that the gain-of-function MEN2B mutation does not abrogate ligand responsiveness of Ret and suggest that the presence of Ret ligand could play a role in the pathogenesis of the MEN2B syndrome.
BACKGROUND: Elevated levels of C-reactive protein (CRP) are associated with an unfavorable clinical outcome in patients with unstable angina. To determine whether ischemia-reperfusion injury causes this acute-phase response, we studied the temporal relation between plasma levels of CRP and ischemic episodes in 48 patients with unstable angina and 20 control patients with active variant angina, in which severe myocardial ischemia is caused by occlusive coronary artery spasm. METHODS AND RESULTS: Blood samples were taken on admission and subsequently at 24, 48, 72, and 96 hours. All patients underwent Holter monitoring for the first 24 hours and remained in the coronary care unit under ECG monitoring until completion of the study. On admission, CRP was significantly higher in unstable angina than in variant angina patients (P < .001). In unstable angina, 70 ischemic episodes (1.5 +/- 2 per patient) and in variant angina 192 ischemic episodes (9.6 +/- 10.7 per patient) were observed during Holter monitoring (P < .001), for a total ischemic burden of 14.8 +/- 30.2 and 44.4 +/- 57.2 minutes per patient, respectively (P < .001). The plasma concentration of CRP did not increase in either group during the 96 hours of study, even in patients who had episodes of ischemia lasting > 10 minutes. CONCLUSIONS: The normal levels of CRP in variant angina, despite a significantly larger number of ischemic episodes and greater total ischemic burden, and the failure of CRP values to increase in unstable angina indicate that transient myocardial ischemia, within the range of duration observed, does not itself stimulate an appreciable acute-phase response.
BACKGROUND: Elevated plasma levels of C-reactive protein have been found in the majority of patients with unstable angina. The evidence of elevated levels of acute-phase proteins in unstable angina is in line with a growing body of evidence that suggests that inflammation plays a role in this syndrome and is an indirect sign of increased production of interleukin-6, which is the major determinant of acute-phase-protein production by the liver. However, in unstable angina, there is no direct proof of the role played by interleukin-6. METHODS AND RESULTS: We measured levels of interleukin-6 in 38 patients with unstable angina at the time of their admission to the coronary care unit and in 29 patients with stable angina. In the same groups of patients, we also measured C-reactive protein. Interleukin-6 (undetectable, ie, < 3 pg/mL, in healthy volunteers) was detectable in 23 (61%) of 38 patients with unstable angina but in only 6 (21%) of 29 with stable angina (P < .01). Median interleukin-6 levels were 5.25 pg/mL (range, 0 to 90 pg/mL) in patients with unstable angina but were below the detection limit of the assay in patients with stable angina (range, 0 to 7 pg/mL). A significant correlation was observed between interleukin-6 and C-reactive protein levels (r = .4, P = .013). CONCLUSIONS: Our study demonstrates that raised levels of interleukin-6 are common in unstable angina, correlate with C-reactive protein, and are associated with prognosis, thus confirming the importance of the cytokine pathway for the production by the liver of acute-phase proteins and strengthening the importance of inflammation in this syndrome. Further studies are required to elucidate better the role of interleukins in unstable angina.
Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P < 0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P < 0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.
Germ-line missense mutations of the receptor-like tyrosine kinase ret are the causative genetic event of the multiple endocrine neoplasia (MEN) type 2A and type 2B syndromes and of the familial medullary thyroid carcinoma. We have used the rat pheochromocytoma cell line, PC12, as a model system to investigate the mechanism or mechanisms by which expression of activated ret alleles contributes to the neoplastic phenotype in neuroendocrine cells. Here we show that stable expression of ret mutants (MEN2A and MEN2B alleles) in PC12 cells causes a dramatic conversion from a round to a flat morphology, accompanied by the induction of genes belonging to the early as well as the delayed response to nerve growth factor. However, in the transfected PC12 cells, the continuous expression of neuronal specific genes is not associated with the suppression of cell proliferation. Furthermore, expression of ret mutants renders PC12 cells unresponsive to nerve growth factor-induced inhibition of proliferation. These results suggest that induction of an aberrant pattern of differentiation, accompanied by unresponsiveness to growth-inhibitory physiological signals, may be part of the mechanism of action of activated ret alleles in the pathogenesis of neuroendocrine tumors associated with MEN2 syndromes.
Chromosome 3p deletions in breast cancer have been detected at 3p12-p21 by cytogenetic and loss of heterozygosity studies. Recently, we have cloned the FHIT (fragile histidine triad) gene, located at 3p14.2. Abnormalities of the FHIT locus were found in many established cancer cell lines, and the gene was abnormally transcribed in primary tumors of the digestive tract and lung. In this report, we describe the analysis of breast cancer, cell lines, and primary tumors for alterations in transcription of the FHIT gene; about 20% of the samples exhibited altered transcripts. In most of the cases, aberrant transcripts were missing exons. Lack of expression of FHIT mRNA was observed in another 10% of primary tumor samples. These results suggest that alterations in the FHIT gene may play an important role in breast cancer tumorigenesis and suggest that the MIT gene product functions in the control of the tumorigenic phenotype in a large variety of human neoplasms.
Heterozygosity for ataxia-telangiectasia (A-T), a cancer-prone recessive syndrome, has been associated with an increased risk of breast cancer. The gene for A-T (ATM) is located at chromosomal region 11q22-q23, a region of frequent loss of constitutional heterozygosity in breast and other tumors. Loss of constitutional heterozygosity at 1lq22-q23 was found in 47% of informative cases in the series of primary tumors analyzed in this study. To investigate the role of ATM in breast cancer, we have determined the complete genomic organization of the gene, developed an exon-scanning PCR single-strand conformation polymorphism (PCR-SSCP) assay for mutation detection of ATM, and screened 38 consecutive breast tumors for mutations using both genomic DNA- and cDNA-based assays. In addition to common ATM polymorphisms detected both in the coding sequence and in flanking introns, seven unique SSCP alleles were identified in six tumor DNAs. Sequence analysis of these alleles revealed rive nucleotide substitutions that were predicted to change the encoded amino acid. However, PCR-SSCP and nucleotide sequencing analysis of the paired blood samples and of an extended sample size of a total of 224 chromosomes indicated that these SSCP patterns represent constitutional rare polymorphisms with a frequency between 0.005 and 0.023. Because the majority of A-T mutations are null mutations and none of the ATM alleles found in breast cancer samples would lead to the truncation of the translation product, we conclude that, in this initial sample of sporadic breast cancer patients, there was no evidence for an increased number of A-T carriers. In addition, because no somatic mutations were found, our study rules out the ATM gene as the frequently altered tumor suppressor gene at 11q23.
OBJECTIVES: This study sought to assess neutrophil activation in acute coronary syndromes and its relation to ischemic episodes. BACKGROUND: Neutrophil activation has been reported in unstable angina and acute myocardial infarction; however, it is not clear whether it is related exclusively to ischemia-reperfusion injury. METHODS: We measured the index of intracellular myeloperoxidase in 1) patients with unstable angina, myocardial infarction, variant angina and chronic stable angina and in normal subjects (protocol A); and 2) in patients with unstable angina and acute myocardial infarction during the first 4 days of the hospital period (protocol B). To assess whether neutrophil activation was triggered by ischemia, the myeloperoxidase intracellular index was analyzed before and after spontaneous ischemic episodes and before and after ischemia induced by an exercise stress test in 10 patients with chronic stable angina. In 11 patients with unstable angina, we also compared values of the myeloperoxidase intracellular index at entry with those after waning of symptoms. RESULTS: In protocol A, the myeloperoxidase intracellular index was significantly reduced in patients with unstable angina and acute myocardial infarction compared with patients with stable and variant angina and normal subjects (p < 0.01). In protocol B, the myeloperoxidase intracellular index did not change over time in patients with unstable angina and myocardial infarction. However, in 11 patients with waning symptoms, the myeloperoxidase intracellular index was significantly higher afer symptoms had waned (p < 0.05). In patients with unstable angina, 23 ischemic episodes were studied; no changes in the myeloperoxidase intracellular index were observed. In 10 patients with chronic stable angina and positive exercise stress test results, no significant differences in the myeloperoxidase intracellular index were observed after stress-induced ischemia. CONCLUSIONS: Our study confirms that neutrophils are activated in acute coronary syndromes but suggests that their activation may not be only secondary to ischemia-reperfusion injury.
The results of our study suggest that the acute phase response may be partly related to a yet unknown primary inflammatory component in unstable angina. Further studies are needed to elucidate the actual role of inflammation in unstable angina and its relation to activation of the coagulation system.
OBJECTIVE: p53 is a well-known nuclear phosphoprotein encoded by a suppressor gene know to be mutated in various kinds of human tumours. A relationship between p53 gene mutation and tumour progression seems to be a common feature of several neoplasias. DESIGN: In order to investigate the role of p53 mutations in human thyroid tumours, DNA samples derived from fifty-six neoplastic tissues, ranging from benign adenomas to undifferentiated carcinomas, were examined for the presence of p53 gene mutations. METHODS: The analysis has been conducted using polymerase chain reaction (PCR) amplification of the exons 5-9 of the p53 gene followed by single strand conformation polymorphism (SSCP) and sequence analyses. RESULTS: One anaplastic carcinoma and one papillary carcinoma showed p53 gene mutations in exons 5 and 8, respectively. A cell line established from the papillary carcinoma showed the same mutation present in the original tumour. Both p53 mutations were heterozygous. The p53 positive samples were analysed for other genetic alterations frequently detected in human thyroid carcinomas (mutations of the RET, TRK, and ras oncogenes): both p53-mutated samples proved to be mutated at level of codon 13 of the c-Ki-ras gene. CONCLUSIONS: Our data confirm that p53 gene alterations are rare in well-differentiated thyroid tumours, that they are an important requirement for the establishment in culture of human thyroid carcinoma cell lines, and that they can be associated with other genetic alterations, namely ras mutations, in the malignant progression of thyroid tumours.
Previous research has yielded an inconclusive picture of the effects of music on motor performance. Using a 3-factor within-subjects design [mellow/4:4 time), frenetic/(2:8 time), and white noise music conditions], each of 19 schizophrenic inpatient volunteers performed a Purdue Pegboard and Finger Oscillation (Tapping) Test following 1-min. presentations of 3 types of music. Pegboard performance was higher after frenetic music but unaffected by mellow music; there was no effect on tapping.
We have previously generated transgenic mice bearing a molecular construct obtained by fusing the rat thyroglobulin promoter with the human Kirsten ras oncogene (KRAS). These mice showed thyroid abnormalities, although at very low incidence and after a long latency period. A six-month thyrotropin (TSH) stimulation of thyroid glands, followed by a two-month suspension, induced a significant increase in the number of lesions in transgenic mice as compared with a nontransgenic control group. Our goal was to follow the progression and the reversion of the tumorigenesis process in relationship with the levels of expression of the KRAS in this experimental model. In situ hybridization was used to detect expression of KRAS mRNA in sections of thyroids of the various groups of mice. A positive hybridization was observed in follicular cells of TSH-stimulated transgenic mice, whereas no expression could be appreciated in control nontransgenic mice. A positive signal was also observed in thyroid glands excised from transgenic mice after the 2-month suspension of treatment; however, the number of expressing cells was decreased compared with transgenic mice killed immediately after 6 months of a goitrogen regimen. Finally, every cell in the single thyroid carcinoma observed after the two-month suspension was positive for the transgene mRNA. This study further strengthens the role of the expression of mutated KRAS in the early stages of thyroid follicular cell transformation and indicates that when the expression of the mutated KRAS becomes independent of exogenous TSH stimulation, this event coincides with a further progression towards tumorigenesis.
We have earlier shown that expression of the human activated Ki-ras, directed by the rat thyroglobulin (TG) promoter in the thyroid gland of transgenic mice, is able to induce thyroid benign tumors, albeit at low incidence. A likely explantation of our results is that the low levels of exogenous Ki-ras transcripts are not sufficient to induce multifocal tumors in the thyroid gland. We have performed experiments to analyze the effects of a similar construct in vitro upon thyroid-cell proliferation and differentiation. Transfection of FRTL-5 rat thyroid cells with the human Ki-rasval12 fused to the rat TG promoter is rapidly followed by reduced expression of the differentiation markers thyroglobulin, thyroperoxydase and thyrotropin receptor, but not by fully malignant cell transformation. The data reported support the hypothesis that Ki-ras mRNA levels are critical to the process of complete neoplastic transformation of thyroid epithelial differentiated cells in vitro.
The expression of the receptor-like tyrosine kinase RET is associated with tumors, tissues or cell lines of neural crest origin. In addition RET products (Ret) are involved in determining cell fate during the differentiation of the enteric nervous system and during renal organogenesis. However, as yet, no direct evidence exists to indicate that the Ret kinase activity might interfere in a specific way with cellular differentiation, or proliferation, of a neural crest derived cell line. By using two constitutively activated forms of RET (RET/PTC1 and RET/PTC3) in transient transfection experiments, we have obtained evidence that active RET could reprogramme the gene expression pattern in the rat pheochromocytoma PC12 cell line. Transcription driven by gene promoters, such as NGFI-A and vgf, which belong, respectively, to primary and delayed response genes to nerve growth factor (NGF), and by the neuron-specific enolase (NSE) promoter, is rapidly induced by the expression of activated RET oncogenes. This induction is not elicited in other non neural derived cell types tested. We also demonstrate that endogenous ras activity is required for RET induction of these neural markers. Finally, in the RET/PTC transfected PC12 cells, NGF is unable to induce further their transcription. This suggests that RET/PTC could share an intracellular signalling pathway with the NGF-receptor.
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Transgenic mice have been generated bearing three fusion genes consisting of: (a) a 900-base pair rat thyroglobulin promoter followed by a gene coding for a chloramphenicol acetyl transferase activity; (b) the same promoter followed by the complementary DNA of the human activated Ki-ras oncogene; (c) a 2000-base pair rat thyroglobulin promoter followed by the complementary DNA of the human activated Ki-ras. We have shown that the 900-base pair rat thyroglobulin promoter is able to direct the expression of the reporter gene specifically in the thyroid gland of transgenic mice. The mice bearing the two Ki-ras constructs, which express the transgene in thyroid glands, show thyroid abnormalities, although at very low incidence. These lesions appear after a long latency and with a benign aspect, thus suggest that, in agreement with literature data on naturally occurring human thyroid tumors, the action of an activated ras gene is not sufficient to attain a complete malignant conversion of thyroid glands in vivo. However, ras expression in thyroid follicular cells represents a favorable ground for tumor development, as shown by the fact that goitrogen stimulation experiments increase the occurrence of tumors.
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