Patrícia Couceiro

Nitric oxide stimulates the proliferation of neural stem cells bypassing the epidermal growth factor receptor.

Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC‐18 (10 μM) increased cell proliferation, whereas higher concentrations (100 μM) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen‐activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin‐dependent kinase inhibitor 1, p27KIP1, allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS−/− mice) prevented the increase in cell proliferation observed following seizures in wild‐type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. STEM CELLS 2010;28:1219–1230

Promoter hypermethylation of DNA repair genes MLH1 and MSH2 in adenocarcinomas and squamous cell carcinomas of the lung

Five years survival of lung cancer is 16%, significantly lower than in prostate (99.9%), breast (88.5%) and colon (64.1%) carcinomas. When diagnosed in the surgical stage it increases to 50% but this group only comprises 14-16% of the cases. DNA methylation has emerged as a potential cancer-specific biomarker. Hypermethylation of CpG islands located in the promoter regions of tumour suppressor genes is now firmly established as an important mechanism for gene inactivation. This retrospective study included 40 squamous cell carcinomas and 40 adenocarcinomas in various surgical TNM stages to define methylation profile and possible silencing of DNA repair genes - MLH1 and MSH2 - using Methylation-Specific PCR and protein expression by immunohistochemistry in tumoural tissue, preneoplastic lesions and respiratory epithelium with normal histological features. The protein expression of MLH1 and MSH2 genes, in the available preneoplastic lesions and in normal cylindrical respiratory epithelium appeared reduced. The frequency of promoter hypermethylation found on these DNA repair genes was elevated, with a higher prevalence of methylation of MLH1 gene in 72% of squamous cell carcinoma. The differences are not so obvious for MSH2 promoter hypermethylation. No correlation was found among the status of methylation, the protein expression and the clinicopathological characteristics. With a larger study, a better characterization of the hypermethylation status of neoplastic and preneoplastic lesions in small biopsies would be achieved, inherent to tumour histology, heterogeneity and preservation, and finally differences in the study population to elucidate other possible mechanisms of altered expression of the hMLH1 and hMSH.

Projecto de estadiamento do cancro do pulmão pela IASLC: Estudo comparativo entre a 6.ª edição TNM em vigor e a 7.ª edição proposta

Resumo A futura 7.a edicao TNM da Classificacao de Tumores Malignos, a publicar em 2009, contempla varias recomendacoes estabelecidas pela IASLC para alteracoes nos componentes T, N e M, no que diz respeito ao estadiamento do cancro do pulmao. As alteracoes no estadiamento global dos doentes, que decorrem destas recomendacoes, incluem: a reclassificacao de tumores com mais de 7cm de T2 para T3; a mudanca de algumas categorias dadas a nodulos pulmonares tumorais adicionais em algumas localizacoes; reclassificar o derrame pleural como componente M; devendo ainda mover -se os casos T2b N0 M0 do estadio IB para o IIA, os casos T2a N1 M0 do estadio IIB para IIA e os casos T4 N0 -1 M0 do estadio IIIB para IIIA. Foram aplicadas as definicoes da nova proposta TNM – 7.a edicao a 203 casos de carcinoma broncopulmonar, correspondendo a carcinoma epidermoide (83) e adenocarcinoma (120) em estadios cirurgicos, da casuistica do Servico de Anatomia Patologica dos Hospitais da Universidade de Coimbra, tendo-se verificado as seguintes alteracoes: vinte casos no estadio IB movidos para o estadio IIA e tres casos movidos para IIB; dezoito casos no estadio IIB movidos para o estadio IIA e um caso movido para IIIA; dois casos no estadio IIIB movidos para o estadio IV; seis casos no estadio IV movidos para o estadio IIIA e um caso movido para o estadio IIIB. Conclui-se assim que ocorreram alteracoes de estadiamento global dos doentes em 51 dos 203 casos analisados (25,1%).

Regulation of striatal astrocytic receptor for advanced glycation end-products variants in an early stage of experimental Parkinson's disease.

Convincing evidence indicates that advanced glycation end‐products and danger‐associated protein S100B play a role in Parkinsons disease (PD). These agents operate through the receptor for advanced glycation end‐products (RAGE), which displays distinct isoforms playing protective/deleterious effects. However, the nature of RAGE variants has been overlooked in PD studies. Hence, we attempted to characterize RAGE regulation in early stages of PD striatal pathology. A neurotoxin‐based rodent model of PD was used in this study, through administration of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) to C57BL/6 mice. Animals were killed 6 h post‐MPTP to assess S100B/RAGE contents (RT‐qPCR, ELISA) and RAGE isoform density (WB) and cellular distribution (immunohistochemistry). Dopaminergic and gliotic status were also mapped (HPLC‐ED, WB, immunohistochemistry). At this preliminary stage of MPTP‐induced PD in mice, RAGE inhibitory isoforms were increased whereas full‐length RAGE was not affected. This putative cytoprotective RAGE phenotype paired an inflammatory and pro‐oxidant setting fueling DAergic denervation. Increased RAGE inhibitory variants occur in astrocytes showing higher S100B density but no overt signs of hypertrophy or NF‐κB activation, a canonical effector of RAGE. These findings expand our understanding of the toxic effect of MPTP on striatum and offer first in vivo evidence of RAGE being a responder in early stages of astrogliosis dynamics, supporting a protective rather tissue‐destructive phenotype of RAGE in the initial phase of PD degeneration. These data lay the groundwork for future studies on the relevance of astrocytic RAGE in DAergic neuroprotection strategies.

EGFR/erB-1, HER2/erB-2, CK7, LP34, Ki67 and P53 expression in preneoplastic lesions of bronchial epithelium: an immunohistochemical and genetic study

A prognostic interpretation of preneoplastic lesions would have impact in bronchial carcinoma early diagnosis and through the study of Erb-B family receptors as they have an important role in lung carcinogenesis. The existence of drugs as tyrosine kinase inhibitors stressed the importance of studying gene alterations for selected chemoprevention schemes and characterization of carcinogenesis. Bronchial preneoplastic lesions were characterized by immunohistochemistry using the antibodies LP34 (high weigh molecular cytokeratin), CK7, chromogranin A, Ki67, p53, C-erbB-2 and EGFR. HER2 and EGFR gene copy number was also evaluated by fluorescent in situ hybridization in those lesions. The expected results defined the origin cell for basal cell hyperplasia and squamous metaplasia as adaptative lesions and dysplasia. By known experiences and published data, beyond the stem cell, the spectral evolution of bronchial preneoplastic lesions was demonstrated by characterizing basal cells (LP34) and their neoplastic potentiality. Dysplasias showed a higher expression of EGFR, Ki67 and p53 with a stepwise increase with the gravity of the respective grading. C-erbB-2 immunohistochemical overexpression was a rare event in preneoplastic lesions. Polysomy was the main mechanism for EGFR and HER2/neu higher gene copy number and together with increased proliferation index (Ki67) will account to preview bronchial carcinogenesis.

The effects of physical exercise on nonmotor symptoms and on neuroimmune RAGE network in experimental parkinsonism

Parkinsons disease (PD) prodromal stages comprise neuropsychiatric perturbations that critically compromise a patients quality of life. These nonmotor symptoms (NMS) are associated with exacerbated innate immunity, a hallmark of overt PD. Physical exercise (PE) has the potential to improve neuropsychiatric deficits and to modulate immune network including receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs) in distinct pathological settings. Accordingly, the present study aimed to test the hypothesis that PE 1) alleviates PD NMS and 2) modulates neuroimmune RAGE network in experimental PD. Adult Wistar rats subjected to long-term mild treadmill were administered intranasally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probed for PD NMS before the onset of motor abnormalities. Twelve days after MPTP, neuroimmune RAGE network transcriptomics (real-time quantitative PCR) was analyzed in frontal cortex, hippocampus, and striatum. Untrained MPTP animals displayed habit-learning and motivational deficits without gross motor impairments (cued version of water-maze, splash, and open-field tests, respectively). A suppression of RAGE and neuroimmune-related genes was observed in frontal cortex on chemical and physical stressors (untrained MPTP: RAGE, TLR5 and -7, and p22 NADPH oxidase; saline-trained animals: RAGE, TLR1 and -5 to -11, TNF-α, IL-1β, and p22 NADPH oxidase), suggesting the recruitment of compensatory mechanisms to restrain innate inflammation. Notably, trained MPTP animals displayed normal cognitive/motivational performances. Additionally, these animals showed normal RAGE expression and neuroprotective PD-related DJ-1 gene upregulation in frontal cortex when compared with untrained MPTP animals. These findings corroborate PE efficacy in improving PD NMS and newly identify RAGE network as a neural substrate for exercise intervention. Additional research is warranted to unveil functional consequences of PE-induced modulation of RAGE/DJ-1 transcriptomics in PD premotor stages.NEW & NOTEWORTHY This study newly shows that physical exercise (PE) corrects nonmotor symptoms of the intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of experimental parkinsonism. Additionally, we show that suppression of neuroimmune receptor for advanced glycation end products (RAGE) network occurs in frontal cortex on chemical (MPTP) and physical (PE) interventions. Finally, PE normalizes frontal cortical RAGE transcriptomics and upregulates the neuroprotective DJ-1 gene in the intranasal MPTP model of experimental parkinsonism.

EGFR unusual mutation status in lung adenocarcinomas

Lung cancer is the most common cause of cancer deaths in both men and women. Adenocarcinoma represents about 28% of the NSCLC cases in men and 42% in women. EGFR is a member of the ERBB family of tyrosine kinases (TK). EGFR mutations are more frequently observed in female, non-smokers, East-Asian and in patients with adenocarcinomas, and predict response to TK Inhibitors (TKIs). Sections of adenocarcinomas of the lung, formalin-fixed paraffin-embedded tissues (FFPE), were selected to analyze mutations in EGFR exons 19 and 21 by DNA extraction for polymerase chain reaction (PCR). Exon 19 was studied by fragment analysis and exon 21 was studied by direct sequencing. The analysis of FISH results was done by Cappuzzo’s score to EGFR gene. Determination of EGFR protein expression was done by immunohistochemistry (IHC) (Zymed Laboratories). The author’s present two cases of lung adenocarcinoma that harbours coexisting EGFR exon 19 and 21 mutations and one case of EGFR multiple in frame-deletions. The patients were female (n = 3), with mixed type adenocarcinoma overexpressing EGFR by IHC. Most reports demonstrate one EGFR mutation per adenocarcinoma. We demonstrated that a single adenocarcinoma can harbour more than one EGFR activating mutations.

KRAS and EGFR mutations coexisting in lung adenocarcinoma

Lung adenocarcinoma represents about 42% and 28% of NSCLC in women and men. Adenocarcinomas incidence is still rising being the most frequent type of NSCLC diagnosed in USA. Both EGFR and KRAS gene mutations can contribute to the development of NSCLC, namely adenocarcinomas. EGFR and KRAS mutations are considered by some authors as mutually exclusive explained by the fact that KRAS-MAPK pathway is one of the downstream signalling pathways of EGFR. Lung cancers with KRAS mutations are resistant to EGFR tyrosine kinase inhibitors. Sections of the adenocarcinoma of the lung, formalin-fixed paraffin-embedded tissues (FFPE), were selected to analyze mutations in EGFR exons 19 and 21 and KRAS - codons 12 and 13 by DNA extraction for polymerase chain reaction (PCR). Exon 19 was studied by fragment analysis and exon 21, codons 12 and 13 were studied by direct sequencing. The analysis of FISH results was done by Cappuzzo’s score to EGFR gene. Determination of EGFR protein expression was done by immunohistochemistry (IHC) (Zymed Laboratories). The authors present a rare case with synchronous EGFR and KRAS mutations. The patient is a 77 years old, male with a central 3cm mixed adenocarcinoma. The tumor showed EGFR protein overexpression identified by IHC and chromosome 7 high polyssomy by FISH. The authors call attention to the fact that although EGFR and KRAS mutations are almost always mutually exclusive in some cases they may coexist in the same neoplasia.

EGFR/erb-1, HER2/erb-2, CK7, LP34, Ki67 and p53 expression in preneoplastic lesions of bronchial epithelium

A prognostic interpretation of preneoplastic lesions would have impact in bronchial carcinoma early diagnosis and through the study of Erb-B family receptors as they have an important role in lung carcinogenesis. The existence of drugs as tirosine kinase inhibitors (TKis) stressed the importance of studying gene alterations for selected chemoprevention schemes and characterization of carcinogenesis. Bronchial preneoplastic lesions were characterized by immunohistochemistry using the antibodies LP34 (high weigh molecular cytokeratin), CK7, Chromogranin A, Ki67, p53, C-erbB-2 and EGFR. HER2 and EGFR gene copy number was also evaluated by fluorescent in situ hybridization (FISH) in those lesions. The expected results defined the origin cell for basal cell hyperplasia and squamous metaplasia as adaptative lesions and dysplasia. By known experiences and published data, beyond the stem cell, the spectral evolution of bronchial preneoplastic lesions was demonstrated by characterizing basal cells (LP34) and their neoplastic potentiality. Dysplasias showed a higher expression of EGFR, Ki67 and p53 with a stepwise increase with the gravity of the respective grading. C-erbB-2 immunohistochemical overexpression was a rare event in preneoplastic lesions. Polysomy was the main mechanism for EGFR and HER2/neu higher gene copy number and together with increased proliferation index (Ki67) will account to preview bronchial carcinogenesis.