[WHO 1999 classification of lung cancers: a guided tour].
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Adenocarcinoma↗
Biomedical subjects
E Brambilla
Publications and source records attributed to E Brambilla.
Antitumor activity of bax and p53 naked gene transfer in lung cancer: in vitro and in vivo analysis.
In vitro and in vivo data have demonstrated that virus-mediated p53 gene transfer can induce active cell death and lung tumor regression. In contrast, the therapeutic potential of bax, another apoptosis-inducing gene, has not been described. We compared p53 and bax cytotoxic effects by transient transfection of an average of 25 +/- 5% of the H-322 and H-358 bronchioloalveolar carcinoma cell lines in vitro. Under these conditions, bax expression killed 70 to 90% of the transfected cells whereas p53 killed only 40% of them. The killing activity of both genes involved apoptosis, as shown by TUNEL staining. Surprisingly, BrdU incorporation indicated that the cells that did resist Bax toxicity were blocked in the pre-S phase of the cell cycle, a result expected for p53 only. In vivo, repeated injections of naked DNA encoding Bax or p53 inhibited the growth of 4-mm preestablished H-322 tumors in nude mice. Growth retardation only, and not inhibition, was observed in H-358, a poorly transfectable and rapidly growing tumor. These results indicate that Bax and p53 share a similar, strong antitumor activity in vivo, even if the former is a more potent inducer of apoptosis in vitro.
Animals↗
The human p19ARF protein encoded by the beta transcript of the p16INK4a gene is frequently lost in small cell lung cancer.
The p16IN4/CDKN2/MTS1 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor called p16INK4a, which regulates retinoblastoma protein-dependent G1 arrest, and a cell cycle inhibitor designated p19ARF, which arrests cell growth in G1-S and also in G2-M. Whereas inactivation of p16INK4a has been described as a frequent event in lung cancer, the current function of p19ARF is still poorly understood. We have examined the expression of the human p19ARF (hp19ARF) protein in a large series of lung cancers using immunohistochemistry and showed that the protein was more frequently lost in high-grade neuroendocrine (NE) lung tumors (large cell NE carcinoma and small cell lung carcinoma; 51 of 78, 65%) than it was in non-small cell lung cancer (25 of 101, 25%). No deleterious mutation was found in exons 1beta and 2 of hp19ARF in those NE tumors with negative immunoreactivity, and a beta transcript was detected in the majority of them. Concomitant absence of hp19ARF and retinoblastoma proteins was frequently detected in high-grade NE lung tumors, whereas no relationship could be found between the status of hp19ARF and p53 proteins in those tumors. These results are consistent with an alternative growth suppressor function for hp19ARF in NE lung cancer that is distinct from that of p16INK4a. Moreover, the frequent uncoupling between the beta transcript and the hp19ARF protein suggests a novel mechanism of inactivation at the translational level.
Carcinoma, Small Cell↗
Serotonergic ergoline derivatives.
Novel classes of 13- and 14-tertbutyl-ergoline derivatives were prepared, and characterised in vitro for their affinity for adrenergic, dopaminergic and serotonergic binding sites. This study particularly examines the importance of the presence and the position of the tert-butyl group in conferring either significant 5-HT1A or 5-HT2 affinity and selectivity respectively.
Animals↗
Bidirectional modulation of insulin action by amino acids.
Amino acids have been shown to stimulate protein synthesis, inhibit proteolysis, and decrease whole-body and forearm glucose disposal. Using cultured hepatoma and myotube cells, we demonstrate that amino acids act as novel signaling elements in insulin target tissues. Exposure of cells to high physiologic concentrations of amino acids activates intermediates important in the initiation of protein synthesis, including p70 S6 kinase and PHAS-I, in synergy with insulin. This stimulatory effect is largely due to branched chain amino acids, particularly leucine, and can be reproduced by its transamination product, ketoisocaproic acid. Concurrently, amino acids inhibit early steps in insulin action critical for glucose transport and inhibition of gluconeogenesis, including decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2, decreased binding of grb 2 and the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2, and a marked inhibition of insulin-stimulated phosphatidylinositol 3-kinase. Taken together, these data support the hypothesis that amino acids act as specific positive signals for maintenance of protein stores, while inhibiting other actions of insulin at multiple levels. This bidirectional modulation of insulin action indicates crosstalk between hormonal and nutritional signals and demonstrates a novel mechanism by which nutritional factors contribute to insulin resistance.
Adaptor Proteins, Signal Transducing↗
Mechanisms of p16INK4A inactivation in non small-cell lung cancers.
The cyclin-dependent kinase inhibitor p16 (p16INK4A/CDKN2/MTS1) is a potent inhibitor of the cyclin D-dependent phosphorylation of the retinoblastoma gene (Rb) product, the inactivation of which induces loss of Rb-dependent G1 arrest through inappropriate phosphorylation of the Rb protein. To analyse the role of p16INK4A as a tumor suppressor in the genesis of non small cell lung cancers (NSCLC) and correlate loss of p16INK4A protein expression to genetic or epigenetic mechanisms, we have performed a comprehensive study of p16 status in a series of 43 NSCLC. To this end, we have investigated p16INK4A protein expression with immunohistochemistry, deletions of the gene by FISH, and determined the methylation status of exon 1alpha using a PCR-based methylation assay. Finally, possible mutations were studied by SSCP and subsequent sequencing. Twenty one of the 43 (49%) NSCLC studied exhibited an absence of p16INK4A nuclear staining. Of these, three (14%) had frameshift or missense mutations, seven (33%) displayed methylation of exon 1alpha and 10 (48%) displayed homozygous deletions. In total, 95% of the tumors with p16INK4A negative staining carried one of these three alternative genetic or epigenetic alterations. Furthermore, a high degree of chromosome 9 polysomy was found (58%) in those tumors with p16INK4A inactivation. Taken together these results suggest that deregulation of the p16 gene locus is a frequently occurring event in NSCLC through distinct mechanisms including rare point mutations, promotor methylation and frequent homozygous deletions. Furthermore, our data show that immunohistochemistry is a rapid and an accurate technique for screening of p16INK4A gene inactivation events that result in loss of protein expression.
Adenocarcinoma↗
IL-12 is involved in the induction of experimental autoimmune myasthenia gravis, an antibody-mediated disease.
IL-12 has been shown to be involved in the pathogenesis of Th1-mediated autoimmune diseases, but its role in antibody-mediated autoimmune pathologies is still unclear. We investigated the effects of exogenous and endogenous IL-12 in experimental autoimmune myasthenia gravis (EAMG). EAMG is an animal model for myasthenia gravis, a T cell-dependent, autoantibody-mediated disorder of neuromuscular transmission caused by antibodies to the muscle nicotinic acetylcholine receptor (AChR). Administration of IL-12 with Torpedo AChR (ToAChR) to C57BL/6 (B6) mice resulted in increased ToAChR-specific IFN-gamma production and increased anti-ToAChR IgG2a serum antibodies compared with B6 mice primed with ToAChR alone. These changes were associated with earlier and greater neurophysiological evidence of EAMG in the IL-12-treated mice, and reduced numbers of AChR. By contrast, when IL-12-deficient mice were immunized with ToAChR, ToAChR-specific Th1 cells and anti-ToAChR IgG2a serum antibodies were reduced compared to ToAChR-primed normal B6 mice, and the IL-12-deficient mice showed almost no neurophysiological evidence of EAMG and less reduction in AChR. These results indicate an important role of IL-12 in the induction of an antibody-mediated autoimmune disease, suggest that Th1-dependent complement-fixing IgG2a anti-AChR antibodies are involved in the pathogenesis of EAMG, and help to account for the lack of correlation between anti-AChR levels and clinical disease seen in many earlier studies.
Animals↗
Proinflammatory cytokines regulate antigen-independent T-cell activation by two separate calcium-signaling pathways in multiple sclerosis patients.
Central nervous system (CNS) lesions typical of multiple sclerosis (MS) are characterized by demyelinating inflammatory infiltrates that contain few CNS antigen-specific autoreactive T cells and a multitude of pathogenic non-antigen-specific mononuclear cells. Here, we report that in patients with MS the combined action of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-2, and IL-6 leads to the activation of most peripheral T cells (mainly CD4 memory) by promoting a persistent intracellular calcium increase via two independent signaling pathways. The activation of these pathways, one activated by IFNgamma and the other by the combination TNFalpha/IL-2/IL-6, is independent from myelin antigens and precedes by 2 weeks phases of disease activity (eg, clinical relapses and/or appearance of gadolinium-enhancing lesions on brain magnetic resonance imaging scans during 1 year of follow-up). Our results indicate that an appropriate combination of the four cytokines, three with a proinflammatory profile and one necessary for T-cell growth and differentiation, can activate in an antigen-independent fashion most peripheral T cells from MS patients. This mechanism is likely to contribute to the recruitment of nonspecific lymphocytes into the cellular activation processes leading to CNS demyelination and may represent a major target for immune intervention in MS.
Antigens↗
A simplified micromethod for fluoride analysis.
A simplified method for the determination ionic fluoride (F-) concentrations in microvolume samples using a F(-)-selective electrode is described. Portions (20 microl) of a series of F- standard solutions (1000-0.4 parts/10(9) F-) were placed between the surface of the F- electrode and the surface of a reference electrode mounted end to end with the aid of a steel holder. In this position the two surfaces were used as an analysis chamber, retaining the sample by surface tension without the use of sleeves or other devices. The precision of the results and the absolute detection limit of the modified method were determined. The analytical performance of this set-up was tested by comparing the results with those obtained from the conventional method. The regression between the modified-method data and the conventional-technique data showed a strong correlation (R2 = 0.993) between the results obtained using the two techniques. In the range of F- concentrations from 1000 and 0.48 parts/10(9), the precision of the results (coefficient of variation) was of 5-6%, improving to 0.9% at 0.9 parts/10(9) level.
Fluorides↗
Synthesis and in vitro and in vivo evaluation of dopaminergic ergoline derivatives.
A series of ergoline-amides was synthesised in the discovery of new dopaminomimetic agents. Several compounds exhibited in vivo high prolactin lowering activity (indirectly measured by the nidation test) in rats. For the most active, the potential anti-Parkinson activity was evaluated by observation of the contralateral turning behaviour in 6-OH-DA lesioned rats. The acute toxicity by oral route in mice was also studied.
Animals↗
Synthesis and antihypertensive activity of 2,4-dioxoimidazolidin-1-yl and perhydro-2,4-dioxopyrimidin-1-yl ergoline derivatives.
The synthesis and antihypertensive activity of a series of 2,4-dioxoimidazolidin-1-yl and perhydro-2,4-dioxopyrimidin-1-yl ergoline derivatives are reported. The oral antihypertensive activity was studied in spontaneously hypertensive rats (SHRs) by measuring systolic blood pressure by an indirect tail-cuff method at different times after treatment. The prolactin lowering activity (indirectly measured by the nidation test) in rats and the oral acute toxicity in mice were also studied. The results of this study revealed potent antihypertensive ergoline derivatives devoid of side-effects related to the dopaminergic stimulation and the importance of the delta 9,10 double bond for conferring high potency within these compounds.
Animals↗
Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity.
In the absence of a universal specific molecular tracer of apoptosis, structural DNA alterations provide the basis of labeling systems: double-strand fragmentation for TUNEL (terminal transferase-mediated dUTP nick end-labeling), denaturation for poly (A) in situ hybridization, immunogenicity of single strand DNA, all methods which imply limited specificity due to the unavoidable presence of DNA breaks in virtually all cells. Thus, TUNEL application has been restrained to a narrow spectrum of sample conditions which has limited, in particular, retrospective surveys and apoptotic nuclei-protein double labelings. In the apoptotic nucleus two main obstacles intervene between TUNEL reagents and their targets: DNA hypercondensation and proteins around DNA. The former increases in the course of apoptosis and both are worsened by crosslinking and precipitating fixatives. This point out that TUNEL is an ambitious approach whose target, apoptotic DNA breaks, is less accessible than breaks occurring in non-apoptotic less compacted DNA. However, TUNEL has an advantage: the far greater degree of apoptotic DNA fragmentation. How to obtain a frank differential staining between apoptotic and non-apoptotic DNA? It appears that the answer relies on the pretreatment step and not in modifying the TUNEL staining protocol, which is optimal. Adapted pretreatments are able to circumvent accessibility obstacles and to extend TUNEL applicability to the most demanding conditions, those of archived tissue samples and of TUNEL--protein double labelings.
Apoptosis↗
Solid alveolar rhabdomyosarcoma of the thorax in a child.
AIMS: This case illustrates the difficulties and pitfalls of diagnosis of alveolar rhabdomyosarcoma in its solid variant and in an unusual primary location, the mediastinum. CASE DETAILS: A 9-year-old boy presented with a primary thoracic tumour associated with metastasis in the left sacroiliac joint. Bronchial and mediastinal biopsies showed a malignant neoplasm with a solid sheet-like pattern of small round cells with a high nuclear to cytoplasmic ratio associated with little or no fibrosis usually evocative of a peripheral neuroectodermal tumour (PNET) at this age. Immunohistochemical positive staining with vimentin (80% of tumour cells), desmin (20%) and titin (30%) antibodies was suggestive of a rhabdomyosarcoma. In addition, all neural cell adhesion molecule (NCAM) markers tested were positive as well as MIC2, a marker for the Ewing family of sarcomas. There was no rhabdomyoid differentiation at ultrastructural examination. Molecular analysis with RT-PCR amplification of RNA isolated from the tumour demonstrated the presence of a PAX3/FKHR fusion transcript, product of a t(2;13) reciprocal translocation, a genetic marker specific for alveolar rhabdomyosarcoma. CONCLUSIONS: The diagnostic methodology of a small round cell tumour of the child must now include immunohistochemical study and molecular biology to confirm the diagnosis of alveolar rhabdomyosarcoma, in a solid and undifferentiated variant.
Bronchial Neoplasms↗
Neural cell adhesion molecules (NCAM) and NCAM-PSA expression in neuroendocrine lung tumors.
Neural cell adhesion molecules (NCAM) represent specific markers of neuroendocrine (NE) differentiation in lung cancer. Because the polysialic acid form (NCAM-PSA) has reduced adhesion properties, we hypothesized that NCAM-PSA expression could favor metastatic spread. Immunostaining of NCAM and NCAM-PSA were therefore compared in 120 NE lung tumors, including 17 typical carcinoids, 3 atypical carcinoids, 30 large cell NE carcinomas and 70 small cell lung carcinomas, as compared with 25 adenocarcinomas and 25 squamous cell carcinomas. Neural cell adhesion molecules were negative in adenocarcinomas and squamous cell carcinomas but were constantly expressed in all NE tumors from typical carcinoids to small cell lung carcinomas. NCAM-PSA expression was significantly more frequent in high-grade tumors, with 24 of 30 positive cases in large cell NE carcinomas and 65 of 70 positive cases in small cell lung carcinoma, than in carcinoids with 10 of 17 and 2 of 3 positive cases in typical carcinoids and atypical carcinoids, respectively. The neural cell adhesion molecule-polysialic acid form scores of staining were significantly higher in high-grade as compared with low-grade tumors (p = 0.002), and were correlated with nodal spread (p = 0.04) and metastasis (p = 0.016) across histologic classes but not in individual tumor type. We conclude that NCAM-PSA connotes poor differentiation and aggressive clinical behavior in the spectrum of NE lung tumors, but cannot be regarded as a prognostic factor in individual tumor classes.
Adenocarcinoma↗
Erdheim-Chester disease: a primary macrophage cell disorder.
Erdheim-Chester disease (ECD) is a rare focal or systemic infiltrative disorder resulting from xanthogranulomatous tissue deposition. Usually, bone marrow involvement affects long bone metaphyses symmetrically, but it spares the epiphyses. Retroperitoneal space, periaortic area, skin, and brain involvement have been described. Pulmonary involvement is frequent, occurring in 20% of cases. Reported histologic features in the lung include an infiltration of so-called lipid-laden macrophages and granulomatous lesions with fibrosis. Lung function outcome is unpredictable, but terminal respiratory failure is the most frequent cause of death. No effective treatment strategies have been described. We report a new case with lung and bone involvement occurring in a symptomatic woman. Histologic and electron microscopic analysis of the pulmonary infiltrate showed abnormal macrophages devoid of lipids forming nodular granulomas and rendering the previous hypothesis of this disease as a primary lipid storage disorder unlikely. These findings suggest that ECD histogenesis is instead based on a primary macrophage cell disease.
Adult↗
Mechanism of retinoblastoma gene inactivation in the spectrum of neuroendocrine lung tumors.
The retinoblastoma (RB) gene plays a key role in cell cycle control by regulation of G1 growth arrest. This gene is inactivated in some human cancers and in most small-cell lung carcinoma (SCLC) cell lines. The aim of this study was to analyze the mechanisms of RB silencing in freshly excised neuroendocrine (NE) tumors embracing the entire spectrum of NE lung neoplasms (typical and atypical carcinoids, large-cell neuroendocrine carcinomas [LCNECs], and SCLCs). To study the role and mechanism of RB inactivation in tumor differentiation and malignant potential, the status of the Rb protein was analyzed in 37 NE lung tumors, using immunohistochemistry with five Rb antibodies. Loss or altered expression of Rb protein was more frequently observed in high-grade NE lung carcinoma (23 of 28, 82%) than in typical and atypical carcinoids (1 of 9, 11%) (P < 0.001). Of 24 tumors with abnormal Rb staining, Southern blotting showed 1 to have undergone rearrangement, SSCP (single-strand conformation polymorphism) and sequencing showed that 6 (25%) exhibited mutations in exons 13-18 or 20-24 of the RB gene, and RT-PCR (reverse transcriptase-polymerase chain reaction) revealed that 14 (58%) showed a low level of or entirely absent RB mRNA (messenger RNA) expression, whereas hypermethylation of the CpG-rich island at the 5' end of the RB gene was not observed. Abnormal Rb protein expression was always associated with one of these three alternative mechanisms in the SCLCs analyzed, but in only 50% of LCNECs. These results indicate that inactivation of the RB gene is highly frequent in freshly excised high-grade NE lung tumors through distinct mechanisms including point mutations and frequent abnormal mRNA expression. Different modes of RB inactivation seem to be implicated along the spectrum of NE lung carcinomas, depending on differentiation state, phenotype, and malignancy grade.
Carcinoma↗
TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement.
TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70-80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens.
Apoptosis↗
Caries prevention during pregnancy: results of a 30-month study.
The purpose of this 30-month study was to explore the effectiveness of a caries-preventive regimen in lowering the salivary mutans streptococci level in pregnant women and, subsequently, in inhibiting the growth of these bacteria in their young children. Beginning at the end of the sixth month of pregnancy and continuing until delivery, subjects rinsed daily with 0.05 percent sodium fluoride and 0.12 percent chlorhexidine. The authors monitored the salivary mutans streptococci levels during the last six months of pregnancy and every six months thereafter for 24 months. They also measured bacterial levels in the children every six months until they reached age 24 months. The results show that treatment significantly reduced salivary mutans streptococci levels in mothers and delayed the colonization of bacteria in their children for about four months.
Analysis of Variance↗