Luc Thiberville

Human in vivo fluorescence microimaging of the alveolar ducts and sacs during bronchoscopy

The aim of the present study was to assess fibred confocal fluorescence microscopy (FCFM) as a tool for imaging the alveolar respiratory system in vivo during bronchoscopy. A 488-nm excitation wavelength FCFM device was used in 41 healthy subjects including 17 active smokers. After topical anaesthesia, the 1.4-mm miniprobe was introduced into the bronchoscope working channel and advanced distally to the alveoli. Morphometric and cellular analyses were performed on selected frames harbouring a minimal compression effect. In vivo acinar microimaging was obtained from each lung segment except for the apical and posterior segments of both upper lobes. Reproducible patterns, corresponding to the elastic framework of the axial and peripheral interstitial systems, were recorded from 192 separate acini. The mean±sd thickness of the acinar elastic fibres was 10±2.7 μm. Alveolar mouth diameters (mean±sd 278±53 μm) were normally distributed but appeared smaller in the right upper lobe and right medial basal segment. Lobular microvessels (median diameter 90 μm) were equally distributed throughout the lungs. Alveolar macrophages were not detectable in nonsmokers, whereas a specific tobacco-tar-induced fluorescence was observed in smoking subjects, providing fine details of the alveolar walls and macrophages. A strong correlation was found between the number of cigarettes smoked per day and the amount of large and mobile macrophages observed in vivo, as well as with the intensity of the macrophage alveolitis. Fibred confocal fluorescence microscopy enables accurate exploration of the peripheral lung in vivo in both smokers and nonsmokers.

Confocal Fluorescence Endomicroscopy of the Human Airways

Confocal endomicroscopes aim at providing to the clinician microscopic imaging of a living tissue. The currently available microendoscopic devices use the principle of confocal fluorescent microscopy, in which the objective is replaced by an optical fiber and a miniaturized scanhead at the distal end of the endoscope or by a retractable bundle of optical fibers. Such systems have recently been applied to the explorations of several organs, including the gastrointestinal tract, and more recently to the proximal and distal airways in vivo. Respiratory fluorescence microendoscopes use 488 nm or 660 nm excitation laser light and thin flexible miniprobes that are introduced into the working channel of the bronchoscope. The devices have a lateral resolution of 3 microm, a field of view of 600 microm, and produce real-time imaging at 9 frames per second. For in vivo imaging, the miniprobe is applied onto the bronchial wall surface or advanced into a distal bronchiole down to the acinus. In nonsmokers, the 488-nm excitation device images the autofluorescence of the elastin that is contained in the basement membrane of the proximal airways and that participates to the axial backbone of the peripheral interstitial respiratory system. In smokers, a specific tobacco tar-induced fluorescence allows in vivo macrophage and alveolar wall imaging. Using 660 nm excitation and topical methylene blue, the technique enables cellular imaging of both bronchial epithelial layer and peripheral lung nodules. This article reviews the capabilities and possible limitations of confocal microendoscopy for in vivo proximal and distal lung explorations.

Acquired homozygosity (isodisomy) of chromosome 3 during clonal evolution of a uveal melanoma: association with morphologic heterogeneity.

Cytogenetic investigation of untreated uveal melanoma has shown that the most frequent abnormality is monosomy 3, which occurs in approximately 60% of cases. One of our cases showed distinct pigmented and nonpigmented areas at gross dissection; representative tissue was collected separately from each area, cultured, and harvested using standard cytogenetic techniques. On histologic examination, the pigmented area was found to be composed of small epithelioid cells, whereas the nonpigmented area contained large, pleomorphic epithelioid cells. The karyotype of the pigmented tumor revealed monosomy 3, whereas the nonpigmented tumor showed two apparently normal chromosomes 3. Our purpose in the present study was to investigate the two tumor areas by molecular techniques to determine whether the karyotype of the nonpigmented tumor evolved directly from the pigmented tumor with duplication of the remaining chromosome 3 or whether the two sublines evolved in a divergent fashion from a common precursor stemline. DNA was extracted from normal lymphocytes and separately from both areas of the tumor. The DNA was analyzed using the polymerase chain reaction for polymorphic dinucleotide and tetranucleotide repeat sequences on chromosome 3. Both pigmented and nonpigmented areas of the tumor showed loss of heterozygosity at all informative loci on chromosome 3 that were tested. These results support our hypothesis that an abnormality on chromosome 3 plays a central role in the molecular pathogenesis of uveal melanoma and that some melanomas develop acquired homozygosity (isodisomy) by loss and duplication of the remaining, presumably abnormal, chromosome 3. Genes Chromosom Cancer 15:138–143 (1996).

Inhibition of tumor growth and metastatic spreading by overexpression of inter-alpha-trypsin inhibitor family chains.

The inter‐α trypsin inhibitor (ITI) family is a group of proteins built up from different combinations of 1 light chain (ITI‐L) and 3 highly homologous heavy chains (ITI‐H1, ‐H2 and ‐H3). To investigate a potential role of the ITI family chains in cancer and metastasis spreading, we engineered human H460M cell lines expressing both the green fluorescent protein (GFP) and one of these chains. These clones were subcutaneously injected in athymic nude mice, and lung metastasis number and primary tumor weight were determined after 28 days. Expression of the ITI‐L chain considerably decreased tumor weight and fluorescent lung metastasis number. ITI‐H1 and ITI‐H3 chain expression induced a significant decrease of metastasis number, whereas no decrease of tumor weight could be detected. In vitro, ITI‐L expression significantly decreased chemotaxis and ITI‐H1 and ITI‐H3 expression increased cell attachment. These results argue for the antitumoral or antimetastatic properties of ITI‐L, ‐H1 and ‐H3 chains.

In vivo molecular microimaging of pulmonary aspergillosis

The early diagnosis of invasive pulmonary aspergillosis (IPA) is challenging. Fibered confocal fluorescence microscopy (FCFM) is a new technique that allows in vivo imaging of the lung microstructure during bronchoscopy. In this study, we investigated the ability of FCFM to detect a fluorescent peptide-tracer bound to Aspergillus fumigatus in experimental IPA in 13 immunosuppressed, non-neutropenic rats. Subpleural IPA microabscesses were imaged through a transthoracic window using FCFM in vivo after i.v. injection of the c(CGGRLGPFC)-NH2([FITC]) peptide (n = 7) or saline. Results were compared to 10 immunosuppressed, non-infected rats and to six immunosuppressed Geosmithia argillacea-infected rats with and without i.v. injection of the peptide. The peptide in vitro specifically labeled A. fumigatus grown under biofilm growth conditions but not G. argillacea. In vivo, FCFM showed a local infiltration of fluorescent host cells in both Aspergillus and Geosmithia infections. Lung/inner thoracic wall fluorescence intensity ratio (FI) did not differ before and after peptide administration on healthy lung areas, on non-specific inflammatory areas, or on Geosmithia micro-abscesses. In contrast, FI increased from 1.05 without peptide to 1.83 after peptide injection on Aspergillus micro-abscesses (p < 0.0001). In peptide-injected rats, FI from IPA foci was higher than from non-specific inflammation or from Geosmithia abscesses (p ≤ 0.002). Using c(CGGRLFPC)-NH2([FITC]) peptide, FCFM allows the in vivo specific imaging of pulmonary aspergillosis. These data provide the basis for the in vivo diagnosis of human pulmonary aspergillosis using alveolar confocal endomicroscopy.

A model of spontaneous lung metastases visualised in fresh host tissue by green fluorescent protein expression

The authors describe a model of spontaneous lung metastases in nude mice using green fluorescent protein (GFP) expression as a marker. The human lung cell line H460M was transfected with the humanised GFP-S65T cDNA and a stable fluorescent cell line termed H460MGFP was obtained. The latter kept in vitro biological features when compared to the parental H460M cell line, which suggests that GFP-expression does not influence H460MGFP cell line behaviour. In order to evaluate their metastatic potential and to determine the number of spontaneous metastases, H460MGFP cells were subcutaneously inoculated into nude mice. Animals were sacrificed at time intervals and tissues (lung, liver, spleen, node, and kidney) were analysed under fluorescence microscopy. These experiments demonstrated that 2 weeks after subcutaneous inoculation, 75% of animals exhibited fluorescent spontaneous lung micrometastases. From the third week, 100% of animals exhibited an increasing number of metastases (10–16) which were only localised in the lungs. At the end of the study, the number of lung metastases had dramatically increased (42–400 at 7 weeks). Although these metastases were mainly localised in lung, a few mice had an invasion of neighbouring lymph nodes. The H460MGFP cell line allowed to follow the seeding and development of spontaneous lung metastases and may be considered a simple and powerful tool to study each step of the metastasis to screen new anticancer drugs.

Molecular follow-up of a preinvasive bronchial lesion treated by 13-CIS-retinoic acid

We report the result of the follow-up molecular analysis of a bronchial carcinoma in situ treated by 13-cis-retinoic acid, which relapsed 9 months after cessation of drug therapy. Loss of heterozygosity at 3p21 and 9p22 genomic sequences were assessed by polymerase chain reaction (PCR) after microdissection of the dysplastic epithelia. Despite a transient regression of the lesion to a lower grade with treatment, molecular analysis showed the persistence of a 3p and 9p deletion in all the bronchial biopsies taken in the same area during a 1 year follow-up, preceding by 9 months the recurrence of the carcinoma in situ. Our findings suggest that molecular follow-up analysis can help to assess the persistence of a malignant clone within a bronchial epithelium that displays a more benign phenotype under retinoid treatment on follow-up. Molecular analysis may be of great importance to evaluate the effects of chemoprevention and to determine the duration of such intervention in responder patients.

Molecular analysis of peripheral non-squamous non-small cell lung cancer sampled by radial EBUS.

Treatment optimization of non‐squamous non‐small‐cell lung cancers (nonSq‐NSCLC) relies on the molecular analysis of the tumour. We aimed to assess the predictive factors of molecular analysis feasibility (MAF) from samples of peripheral nonSq‐NSCLC obtained by radial endobronchial ultrasound bronchoscopy (r‐EBUS) and 1.5 mm microbiopsy forceps.

Detection by the polymerase chain reaction of two polymorphisms in exon 14 of the human inter-α-trypsin inhibitor heavy chain H1 gene

Two polymorphisms were detected within exon 14 of the inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) gene. The polymorphisms are detected by digesting the same 202-bp polymerase reaction product with the PstI and HphI restriction endonucleases. These gene polymorphisms lead to the change of two amino acids in the mature protein. The polymorphisms can be used for the analysis of 3p21 deletion in human carcinomas as well as to develop a better understanding of the protein polymorphism already described.

Photodynamic therapy using methylene blue in lung adenocarcinoma xenograft and hamster cheek pouch induced squamous cell carcinoma

BACKGROUND Photodynamic therapy (PDT) is used to treat early proximal bronchial cancer during a flexible bronchoscopy. The technique relies on the excitation of a photosensitizer by an appropriate wavelength, which is delivered into the bronchus in close contact with the tumor. OBJECTIVE To assess methylene blue (MB) as a PDT agent for the treatment of respiratory tract cancer in animal models. METHODS MB-induced PDT was performed on 7 subcutaneous NCI-H460 lung adenocarcinoma xenografts in nude mice and 9 induced squamous cell cancer in the hamster cheek pouch model. In mice, PDT was carried out on right-sided tumors after intratumoral injection of methylene blue 1% (w/v) and illumination at 630nm at 200J/cm (Diomed PDT 630), with the left tumor used as control (illumination alone or MB alone). The tumoral volume was assessed before and 15 days after PDT. RESULTS Fourteen xenografts were treated in mice, including seven treated with MB-PDT, producing a 52% mean tumor volume regression (1568mm(3)vs. 544mm(3)) compared to seven control cases in which tumor volume increased (p=0.007; Mann-Whitney test). Nine cheek pouch induced carcinomas were treated in the hamster group, with a mean volume decrease of 85.8% (from 44.8% to 100%) (initial mean volume=210mm(3)vs. post PDT mean volume=97mm(3)). Histology analysis showed 4/9 complete responses. CONCLUSION Intratumoral MB appears efficient as PDT agent for cancer treatment in animal models. Further studies are needed to assess the safety and efficacy of MB-associated PDT for the treatment of lung cancer in humans.