The present study describes a technique for quantitation of mRNA in a few isotypic cells obtained from an intact organ structure by combining laser-assisted cell picking and real-time PCR. The microscopically controlled lasering of selected cells in stained tissue sections was applied to lung alveolar macrophages, which are unique in that they can alternatively be gathered as a pure cell population from intact lungs by bronchoalveolar lavage as a reference technique. TNF-alpha was chosen as the transcriptionally inducible target gene to be quantified in alveolar macrophages of control rat lung, as well as low- and high-challenge lungs stimulated by endotoxin and IFN-gamma nebulization. Online fluorescence detection for quantitation of the number of amplified copies was based on 5' nuclease activity of Taq polymerase cleaving a sequence-specific dual-labeled fluorogenic hybridization probe. A pseudogene-free sequence of PBGD served as an internal calibrator for comparative quantitation of target. A quick procedure and minimized loss of template were achieved by avoiding RNA extraction, DNase digestion and nested-PCR. Using this approach, we demonstrated dose-dependent manifold upregulation of the ratio of TNF-alpha mRNA copies per one copy of PBGD mRNA in alveolar macrophages of the challenged lungs. The quantitative data obtained from laser-picked alveolar macrophages were well matched with those of lavaged alveolar macrophages carried out in parallel. We suggest that this new combination of laser-assisted cell picking and real-time PCR has great promise for quantifying mRNA expression in a few single cells or oligocellular clusters in intact organs, allowing assessment of transcriptional regulation in defined cell populations.

Tetra-amelia is a rare human genetic disorder characterized by complete absence of all four limbs and other anomalies. We studied a consanguineous family with four affected fetuses displaying autosomal recessive tetra-amelia and craniofacial and urogenital defects. By homozygosity mapping, the disease locus was assigned to chromosome 17q21, with a maximum multipoint LOD score of 2.9 at markers D17S931, D17S1785, D17SS1827, and D17S1868. Further fine mapping defined a critical interval of approximately 8.9 Mb between D17S1299 and D17S797. We identified a homozygous nonsense mutation (Q83X) in the WNT3 gene in affected fetuses of the family. WNT3, a human homologue of the Drosophila wingless gene, encodes a member of the WNT family known to play key roles in embryonic development. The Q83X mutation truncates WNT3 at its amino terminus, suggesting that loss of function is the most likely cause of the disorder. Our findings contrast with the observation of early lethality in mice homozygous for null alleles of Wnt3. To our knowledge, this is the first report of a mutation in a WNT gene associated with a Mendelian disorder. The identification of a WNT3 mutation in tetra-amelia indicates that WNT3 is required at the earliest stages of human limb formation and for craniofacial and urogenital development.

PIK3CA, encoding the catalytic subunit p110alpha of phosphatidylinositol 3-kinase (PI3K), is activated in malignant diseases. However, the role of the PIK3CA gene aberrations for tumourigenesis of head and neck squamous cell carcinoma (HNSCC) is to date unclear. The present study was designed to determine the genomic aberration of PIK3CA in invasive HNSCC and dysplastic precursor lesions by fluorescence in situ hybridization (FISH) with a YAC probe, containing the PIK3CA gene, on isolated interphase nuclei from histomorphologically well-defined regions of formalin-fixed tissue sections and to compare these data with protein and mRNA expression of p110alpha. The mRNA and protein levels of p110alpha were assessed, respectively, by in situ hybridization and immunohistochemistry on consecutive tissue sections. Copy number gains at 3q26 were observed in one of six low-to-moderate dysplasias (17%) and in seven of nine high-grade dysplasias (78%), as well as in 11 carcinomas (100%). In addition, one of seven high-grade dysplasias (14%) and 6 of 11 carcinomas (55%) had amplifications of 3q26. The majority of cases with copy number gain in more than 50% of the cells and/or amplification in more than 10% of cells showed increased p110alpha mRNA and protein expression, whereas only two cases (18%) (one high-grade dysplasia and one carcinoma) with no gain or low-level gain displayed increased p110alpha protein expression. These data suggest that 3q26 copy number gain and amplification represent early genomic aberrations in HNSCC carcinogenesis. In addition, p110alpha mRNA and protein expression in HNSCC may be regulated by these genomic aberrations as well as by epigenetic events.

We report here on the characterization and immunohistochemical localization in human tissues of calcitonin receptor-like receptor (CRLR) which was recently found to mediate the effects of both calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM). Western blot analysis using antibodies raised against the first extracellular loop and the carboxy-terminal part of hCRLR, respectively, detected two major bands corresponding to about 70 and 60 kDa in membrane preparations of cultured endothelial cells and numerous organs including lung, heart ventricle and kidney. Immunohistochemical analysis of the cardiovascular system revealed CRLR-like immunoreactivity (CRLR-LI) in the endothelium of all blood vessels including large and small arteries, veins and capillaries, and in heart muscle cells and endocardium. The lung showed intense staining over the alveolar capillaries. Within the digestive tract, staining was observed over the cells lining the excretory ducts of the parotid gland, over the epithelium of the fundic glands of stomach, endocrine cells of the duodenum and ileum and some myenteric ganglia. The kidney presented staining of the juxtaglomerular arteries, the glomerular capillaries and chief cells of the collecting duct. Within the endocrine organs, a strong CRLR-LI signal was observed over the Langerhans islets, and weak immunoreactivity in the Leydig cells of testis. Spleen showed intense staining in trabecular veins and sinuses. Macrophages displayed a variable immunoreactivity. Our data demonstrate a wide distribution of CRLR throughout the human body and suggest CRLR to be involved in the mediation of a variety of actions in addition to vascular control.

Cardiac allograft vasculopathy is a major cause of morbidity and mortality of cardiac transplant recipients. The underlying cause of this disease remains unclear. Histological studies have implicated accelerated hemostasis and intravascular fibrin deposition in its pathogenesis. In the present study a defined model of this disease in the rat was used to elucidate the implication of tissue factor in the production of the hypercoagulable state observed in cardiac allograft vessels. Tissue factor protein and mRNA expression were studied in rat heart allografts developing allograft vasculopathy resembling human disease. Immunohistochemistry demonstrated tissue-factor-positive cells present in the allograft coronary intima and adventitia. Significant staining for tissue factor was detected in the endothelium lining coronary lesions in cardiac allografts and in interstitial mononuclear cells, respectively. Both transplant coronary endothelial cells and mononuclear cells contained tissue factor mRNA as indicated by oligo-cell reverse transcription polymerase chain reaction after laser-assisted cell picking. In contrast, tissue factor mRNA and protein were not or negligibly detectable within the coronary intima of nontransplanted control hearts. Thus, the present study clearly demonstrates that aberrant tissue factor expression occurs within the coronary intima after cardiac transplantation. Tissue factor, activating downstream coagulation mechanisms, may account for the intravascular clotting abnormalities observed in cardiac allografts and may represent a key factor in transplant atherogenesis.