Sub-projecto
Sub-project

Investigador responsável
Sub-project leader

Expression of C-CAM1 in lung

António José Garcia Segorbe Luis

Genes for metabolizing genotoxics and mutations of gene TP53 in neoplasms of the lung

Fernando Jesus Regateiro

Lung permeability

Luis Alberto Cardoso Oliveira

Ga2len

Luis Alberto Cardoso Oliveira

TOBACCO-FREE HEALTH SERVICES

Luis Alberto Cardoso Oliveira

Smoking-related Assault

Luis Alberto Cardoso Oliveira

Allergic inflammation

Luis Alberto Cardoso Oliveira

Multiple Drug Allergy Syndrome

Luis Alberto Cardoso Oliveira

Allergy in the elderly

Luis Alberto Cardoso Oliveira

Deep Lung and HIV+ therapy

Manuel Fontes Baganha

Bronchopulmonary carcinomas

Lina Maria Rodrigues Carvalho

Combustion Respiratory Impact

Carlos Manuel Silva Robalo Cordeiro

AIRWAYS INFLAMMATION

Carlos Manuel Silva Robalo Cordeiro

MDR-TB IMMUNOGENOMICS

António José Garcia Segorbe Luis

Sub-projecto
Sub-project

Analysis of C-CAM1 (CD66a) isoforms expression in lung carcinomas

Investigador responsável
Project leader

António José Garcia Segorbe Luis

Resumo da actividade desenvolvida

The adhesion molecule CEACAM-1 (CD66a) acts as a tumor suppressor in several experimental systems where loss of CEACAM1 expression results in enhanced tumor-cell growth and tumorigenicity. Immunohistochemistry of paraffin lung biopsies and preneoplastic lesions with specific monoclonal antibodies for C-CAM1, E-cadherin, integrins and other biological/ genetic markers such as HER2 and EGFR may play essentials roles in cell metastation and tissue morphogenesis.

Bronchial biopsy specimens with preneoplastic lesions were studied by immunohistochemistry using antibodies against LP34, CK7, Cromo A, p53, Ki67,c-erbB-2 and EGFR. HER2 and EGFR gene copy numbers per cell were evaluated by fluorescent in situ hybridization.
Results: Differentiation markers such as LP34, CK7 and Cromo A allowed us to accurately differentiate preneoplastic lesions of the bronchial epithelial. Expression of p53 was also found in preneoplasic lesions, especially in dysplasia and carcinoma in situ. Polysomy was found early in the hyperplasia-metaplasia-dysplasia sequence independently on the type of the lesion. Amplification of HER2/neu and EGFR occur more frequently in dysplasia and carcinoma in situ.
Conclusion: The expression of p53 occurs in all stages of preneoplasic lesions. Genetic and protein expression of EGFR and HER2/neu are detected in preneoplastic lesions with increased expression in pre-invasive lesions like dysplasia and carcinoma in situ.

Inflammatory myofibroblastic tumor (IMT) is a rare mesenchymal neoplastic proliferation, represented by myofibroblastic spindle cells, inflammatory cells, macrophages and collagen deposition. Its distinctive histological appearance has given this lesion a wide variety of names, including “plasma cell granuloma”, “inflammatory pseudotumor”, “fibrous histiocytoma”, “inflammatory fibrossarcoma” and “xanthogranuloma. Surgery with tumour free margins is the treatment of choice to achieve cure. However, rare cases curse with malignant progression, relapse and metastatic capability .
IMT can arise anywhere in the body, but occurs primarily in the lungs. Outside the lung, the most common sites are the mesentery, retroperitoneum, and pelvis. Pulmonary IMT may be asymptomatic or associated with chest pain and dyspnoea. Both etiology and reactive versus neoplastic pathogenesis of IMT remain uncertain and controversial. However, it has been identified a subset of IMT whose karyotypes contain clonal chromosomal rearrangements involving band 2p23. Such 2p23-rearrangments target the ALK locus and result in various fusion genes containing the catalytic domain of ALK. These clonal mutations support IMT as a neoplastic process and are associated with ALK expression in IMT myofibroblastic cells. So far, immunohistochemical studies have detected ALK expression in 40-60% of IMTs. By molecular analyses, several ALK fusion genes have been demonstrated in IMTs: TPM3 (at 1p23), TPM4 (at 19p13), RANBP2 (at 2q13), CLTC (at 17q23), ATIC (at 2q35), CARS (at 11p15) and SEC31L1 (at 4q21). These fusion genes are diagnostically useful molecular markers in IMTs and it is plausible that these specific genetic alterations could correlate with the clinical course and/or certain pathological features of IMT.
To contribute to a better understanding of the morphology and histogenesis of inflammatory myofibroblastic tumours of the lung, 19 cases of formalin-fixed paraffin-embedded tissue of pulmonary IMTs were analyzed to ascertain their clinical, histopathological and molecular features. They were evaluated not only by anti-ALK immunohistochemical studies, but also by polymerase-chain reaction (PCR) and fluorescence in situ hybridization (FISH) assays.

Sub-projecto
Sub-project

GENES FOR METABOLIZING GENOTOXICS AND MUTATIONS OF GENE TP53 IN NEOPLASMS OF THE LUNG

Investigador responsável
Project leader

Fernando Jesus Regateiro

Resumo da actividade desenvolvida

Genotoxic metabolizing enzymes and TP53 mutations in lung cancer
Summary

A total of 463 blood samples were already collected: 209 from patients with lung cancer, without any chemotherapy or only one cycle and 254 as controls, from patients without lung cancer attending the Clinic of Pneumology. For 55 patients with lung cancer, diagnosis was confirmed by pathology studies on biopsies obtained by bronchoscopy. These biopsies were paraffin embedded for later TP53 mutation analysis.
DNA from all 463 blood samples were extracted. Concerning DNA patients samples: 72 were analysed for fast metabolizer (wild type), slow metabolizer (mutant) or intermediate metabolizers (heterozygotes) for the CYP2D6/BstNI polymorphism; 152 were analysed for fast (wild type) and slow (mutant) alleles of the polymorphic NAT2 gene, by PCR amplification followed by restriction enzyme digestion; 197 were submitted to multiplex PCR amplification for GSTM1 and GSTT1 simultaneously evaluation, in a single PCR reaction.
Concerning control population, 146 were already analysed for CYP2D6/BSTNI polymorphism and 207 for NAT2 gene; 254 were amplified for GSTM1 and GSTT1 by multiplex PCR.
Information about age, sex, profession, family history, smoking and food habitudes, histological type of the tumor and related information about tumor evolution was collected for further epidemiological studies.
 

Table. CYP2D6, NAT2, GSTMi and GSTT1 genotypes distribution in controls and patients.        

  W=wild allele; M= mutated allele; N=gene deleted

Sub-projecto
Sub-project

Ga2len-the Global Allergy and Asthma European Network-spreading excellence

Investigador responsável
Project leader

Luis Alberto Cardoso Oliveira

Resumo da actividade desenvolvida

Ga2len Activities During 2006

Participation in Ga2len Meetings (Viena – Austria) 8-14th June 2006
. General Assembly
. T-reg. Meeting
. Severe Asthma
Participation in Gallen Assembly in Brussels, 29th November 2006
Participation in Gallen Meeting in Stockholm
Severe asthma
Organization and participation on Ga2len symposium during the HB2006- Health Building 2006 international Meeting –Lisbon, Jun 2006

Development of SARI project
.Samples of plasma, serum and whole blood was collected and stored
.Spirometery analysis using the NDD Easy One spirometer was done to some patients
.Skin prick test
.Interview for application questionnaires was done to the study population
.Data entry in Epidata program

Selection of asthmatic patients for T. - Reg. cells was done. Blood was collected and cell isolated and stored

Skin prick test and questionnaires to patients according to Ga2len recommended allergen panel.

Sub-projecto
Sub-project

Kinetic and dynamic evaluation of allergic inflammation

Investigador responsável
Project leader

Luis Alberto Cardoso Oliveira

Resumo da actividade desenvolvida

The in vivo dynamic of specific allergic response was studied on 14 allergic respiratory patients after challenge test simultaneously to the re-injection of leukocytes labelled with Tc99m-HMPAO. Static and dynamic acquisitions were performed in several regions of interest (ROI). For each study a qualitative analyses were performed and quantitative evaluation of inflammatory activity. We had select additional patients submitted to specific immunotherapy, on maintenance dose, to be study concerning immune inflammatory response after specific challenge test.
The qualitative analysis could evidence an earlier inflammatory response after allergic challenge test. An involvement of structures related to central immune areas (bone marrow and mediastinum) was showed, during allergic reaction and the inflammatory activity was time-related.
We are able to conclude this study during the beginning of 2007 and analyse all the groups in this in vivo methodology of allergic response.

Sub-projecto
Sub-project

Clinical and Immunological Characterization in Multiple Drug Allergy Syndrome Patients

Investigador responsável
Project leader

Luis Alberto Cardoso Oliveira

Resumo da actividade desenvolvida

Summary

In this year we studied:

1) The perioperative anaphylaxis in multiple drug allergy patients. The clinical characterization and skin prick tests to neuromuscular blocking agents and others general anesthetics were performed. This work obtained the first award SPAIC/ Shering Plough, for the best communication in Portuguese Immunoallergy Annual Meeting - XXVII SPAIC 2006. The results have also been presented in meeting and in Brasilien Allergy and Immunopatology Congress.

2) We also studied the immediate contrast medium hypersensitivity in this patients. Two of 8 patients included presented Multiple Drug Allergy Syndrome. Case history data, skin prick tests (SPT) and intradermal tests with iodixanol, iopromide, iobitridol and amitrozoate–Na+-meglumine were performed. All patients were submitted to SPT with common aeroallergens and latex. Patch tests with the former ICM and with povidone-iodine (2%) and potassium iodine were also done. The IgE mediated allergy is suggested by positive intradermal tests in 4 of the 8 patients (50%), 3 of them with previous severe reactions after ICM injection. This work is included in European Network Drug Allergy (ENDA) multicenter study - Contrast medium task force protocol performed by Drug Allergy Interest Group from the European Academy of Allergology and Clinical Immunology.

3) 23% of our Multiple Drug Allergy patients presented moderate or severe asthma. An article about options therapy in severe asthma was done.

Sub-projecto
Sub-project

Allergy in the elderly

Investigador responsável
Project leader

Luis Alberto Cardoso Oliveira

Resumo da actividade desenvolvida

Summary

We selected more patients to obtain a larger cohort of patients with long lasting asthma to repeat some important immunological and inflammatory markers. Lymphocytes phenotypes related to asthma pathogen and to ageing process (CD4, CD8), apoptosis, naïve and memory subtypes and activation markers were studied in the all sample.
The evaluation of inflammatory markers, such as neopterin, continued.
Respiratory functional changes of patients with long lasting asthma were also analyzed.
In some selected patients Treg cells were also studied.
We also determined eNO in a subgroup of individuals.
Immunoblotting to dermatophagoids pteronyssinus was done to some patients that were sensitized to this house dust mite.

Sub-projecto
Sub-project

Bronchopulmonary carcinomas: An immunohistochemical study of gene products, suppressor genes, proliferation and drug resistance markers.

Investigador responsável
Project leader

Lina Maria Rodrigues Carvalho

Resumo da actividade desenvolvida

Summary

Activities
Task 1 – Application of the markers in the selected cases.
Task 2 – Compilation of databases.
Task 3 – Statistical study of the results.

Description
Task 1 – The bronchopulmonary carcinomas have been selected retrospectively from archive cases. They were reevaluated and classified by OMS Lung Tumors Classification.
We have selectecd the tissue samples for the application of each immunohistochemical marker.
Task 2 – Data relative to age, sex, tobacco habits, local of the lesion, survival and other clinical important data will be compiled. The results of the immunohistochemistry markers will be compiled in the form of tables and graphics. The results are going to be classified as negative and positive with different grades of expression.
Task 3 – The results obtained will be subject of statistical study using the adequate methods trying to identify differences statistical significant between the established groups.

Task 1 is in course until June 2007.
Task 2 – between July and September 2007.
Task 3 - between October and December 2007.

This project is to be concluded in December 2007

Sub-projecto
Sub-project

OCCUPATIONAL AND ENVIRONMENTAL EFFECTS REGARDING THE ACUTE AND CHRONIC INHALATION OF COMBUSTION BY-PRODUCTS IN HIGH RISK PROFESSIONAL CLUSTERS

Investigador responsável
Project leader

Carlos Manuel Silva Robalo Cordeiro

Resumo da actividade desenvolvida

Currently undergoing the animal experimentation phase.

Phase II (with collection of Exhaled NO and exhaled breath) will be possible this year, because we could guarantee the collaboration of a firefighter corporation during the summer.

Sub-projecto
Sub-project

MARKERS OF INFLAMMATION IN AIRWAYS DISEASES

Investigador responsável
Project leader

Carlos Manuel Silva Robalo Cordeiro

Resumo da actividade desenvolvida

During 2006:


1 –Th1/Th2 differentiation studies in BAL and blood, in Interstitial Lung Diseases: samples recovered in more 30 patients

2 – Genetic expression of fibrosis mediators in BAL and blood, in Interstitial Lung Diseases: samples recovered in more 20 patients with ILD

3 – Development of the prospective study on mathematical analysis of clinical and laboratorial data to the therapeutic decision in Sarcoidosis

4 – Collection of Exhaled Breath Condensate samples from patients with Cystic Fibrosis and from a control group, including smokers and nonsmokers, to study Volatile organic compounds

5 – Implementation of a Cooperative Protocol with the University of Maastricht, on Antioxidation in Sarcoidosis