Evaluation of cellular tumour rejection mechanisms in the peritumoral bladder wall after bacillus Calmette-Guérin treatment.

OBJECTIVE: To compare the immunological status of normal and peritumoral bladder walls, and to characterize immunocompetent cells before and during intravesical instillations of bacillus Calmette-Guérin (BCG). PATIENTS AND METHODS: Twenty-three patients with superficial urothelial bladder carcinoma (stages pTa to pT1, grades 1-3) were treated with six weekly instillations of 150 mg of BCG (Pasteur strain). Biopsies of cystoscopically normal bladder wall were taken before, 3 weeks and 3 months after BCG instillation. The controls comprised bladder biopsy specimens from 13 brain-dead ventilated kidney donors. Local infiltrating cell types, i.e. lymphocyte infiltrates (CD4, CD8, CD20, CD3, interleukin-2-receptor-positive, natural killer, gammadelta), macrophages and dendritic cells, adhesion and costimulatory molecules (ICAM-1 and B7-BB1) and major histocompatibility complex (MHC) class I and class II antigens were assessed using semi-quantitative immunohistochemical analysis. RESULTS: Before BCG the peritumoral bladder wall had fewer macrophages than control bladder wall. BCG treatment restored normal numbers of macrophages and enhanced T helper lymphocytes, B lymphocytes, natural killer cells, activated lymphocytes, dendritic cells, normal MHC class I, adhesion (ICAM-1) and costimulatory (B7-BB1) expression. The enhancement of these immunological variables was transient, with a return to baseline 3 months after BCG instillation. CONCLUSIONS: These results support the concept that there is a host-immune escape associated with bladder cancer. BCG therapy may temporarily restore impaired tumour rejection mechanisms in the peritumoral bladder wall, suggesting a need for maintenance therapy after the first course of BCG.

[Detection of circulating prostatic cells with RT-PCR PSA in prostatic cancer]. / Détection des cellules circulantes prostatiques par RT-PCR PSA dans le cancer de prostate.

OBJECTIVES: The detection of circulating prostatic cells by molecular biology techniques (RT-PCR) can be useful in the staging of localized prostate cancer prior to radical prostatectomy in some institutions. After describing their technique, the authors report their results. PATIENTS: 80 RT-PCR were performed: 32 in a control group (including 11 women free of any neoplastic disease, 11 healthy men, and 10 men with benign prostatic hyperplasia before resection), and 48 in patients with prostate cancer (43 with clinically localized cancer and 5 with metastatic cancer). RESULTS: In the control group, none of 11 women had a positive RT-PCR, 1 of the healthy men was positive (orchidopexy) and 3 of the 11 patients with benign prostatic hyperplasia were positive, but none of them had tumour on the resection chips. None of the 5 metastatic patients were positive. In the patients treated by radical prostatectomy, no correlation was observed between RT-PCR results, pathological stage, positive resection margin status and laboratory progression after radical prostatectomy. CONCLUSION: This PSA RT-PCR technique developed in this institution does not appear to be useful for the molecular staging of prostate cancer. This study demonstrates the difficulty of standardization of this technique which limits its routine use.

[In vitro detection of prostate cancer circulating cells by immunocytochemistry, flow cytometry and RT-PCR PSA]. / Détection in vitro des cellules circulantes dans le cancer de la prostate par immunocytochimie, cytométrie en flux et RT-PCR PSA.

OBJECTIVE: To evaluate 3 in vitro methods detection (immunocytochemistry, flow cytometry and RT-PCR PSA) of circulating prostate cancer cells from a model of uncap dilution in immortalised lymphocytes. METHODS: In vitro comparison of 3 techniques (immunocytochemistry, flow cytometry, RT-PCR PSA) was performed from a range of dilutions of LbCap cells in immortalised human lymphocytes (concentration range: 1 LnCap cell per 100 lymphocytes to 1 LnCap cell per 100 million lymphocytes). Cells were detected by anti-PSA (prostate specific antigen) and PAP (prostatic acid phosphatase) antibody by immunochemistry, by fluorescent linked antipancytokeratin antibody by flow cytometry and RT-PCR PSA. RESULTS: The limit of detection was 1 LnCap cell per 200,000 lymphocytes (1/2.10(5)) for immunochemistry, 1 LnCap cell per 1,000 lymphocytes (1/1.10(3)) for flow cytometry and 1 LnCap cell per 10 million lymphocytes (1/10(7)) for RT-PCR PSA. CONCLUSION: RT-PCR, due to its most perceptible limit of detection, appears to be the method of choice for the detection of prostatic epithelial cells. Immunocytochemistry has the advantage of providing a quantitative approach. Flow cytometry is limited by the limit of detection of the apparatus used. The prognostic significance of detection of circulating prostate cancer cells remains to be clarified, but the detection of these cells and their correlation with the primary tumour will provide a better understanding of metastatic phenomena.

[Detection of circulating epithelial cells in prostate cancer]. / Détection des cellules épithéliales circulantes dans le cancer de la prostate.

INTRODUCTION: The prostate cancer, which is the second cause of cancer, is in constant increase. This is linked to the ageing of population which is in fact a medical and socioeconomic problem. The curative therapeutic attitude of the localized prostate cancer are either wath-full waiting, radiotherapy or radical prostatectomy. With the actual clinical and radiological techniques of stadification, 40% of the localized prostate cancer are extraglandular on the prostatectomy specimen. Recently, the RT-PCR seems to be a more specific and sensitive technique to detect circulating epithelial cells compared to others techniques (immunocytochemistry or flow cytometry). The detection of these circulating cells, would be correlated to the anatomopathologic stage. The use of RT-PCR as a tool of molecular stadification should be considered. METHODS: This study evaluates and compares with in vitro pattern, the different techniques of separation (ficoll, gradient, osmotic shock) and detection (immunocytochemistry, flow cytometry and RT-PCR). RESULTS: The ficoll gradient is the best technique of cellular separation preserving cellular morphology and RNA. The detection level are 1 cell LnCap per 40,000 lymphocytes cell by immunocytochemistry, 1/1000 by flow cytometry, 1/10,000 to 1/1,000,000 by RT-PCR. CONCLUSION: The RT-PCR with its low detection level and its high specificity, is the best technique of detection of epithelial circulating cells in localized stage. The advantage of immunocytochemistry is its simplicity. Its use for circulating cells caracterization can be considered for metastatic stage.

[The PSA gene: value in detection of circulating prostate cancer cells]. / Le gène du PSA: intérêt pour la détection des cellules circulantes dans le cancer de la prostate.

There has been a renewed interest in detection of circulating cancer cells due to the simplicity, specificity and sensitivity of a molecular biology technique: RT-PCR. This detection is based on the concept of specificity of an RNA sequence expressed by a cancer cell. PSA appears to be the ideal marker for circulating prostate cancer cells due to its specificity. The authors review the genes of the kallikrein family and discuss the advantages and difficulties involved in the use of PSA messenger RNA as a marker for circulating prostate cancer cells.

Developmental changes of acidic fibroblast growth factor (aFGF) transcription and expression in mouse brain.

In order to increase our knowledge of the in vivo role of acidic fibroblast growth factor (aFGF) in the central nervous system, we have examined aFGF levels during mouse brain development. Using a specific polyclonal antibody raised against aFGF, we measured levels of aFGF-immunoreactive material (IRMaFGF) in extract of total mouse brain taken at different days of development. We found that the level of measurable IRMaFGF remained low and without significant variation during fetal brain development (0.2 ng/mg of extracted proteins). During the first 11 days postnatal (P0 to P11), IRMaFGF increased from 0.5 to 1.5 ng/mg. Between P11 and P14 IRMaFGF levels went up more rapidly, reaching 5 ng/mg. From P30 to adulthood a constant value of 2.5 ng/mg was measured, aFGF content in the different brain extracts was further characterized by its affinity for heparin-Sepharose, its elution at 1 M NaCl from this column and its capacity to induce thymidine incorporation in quiescent fibroblasts. These results were confirmed at the mRNA level. Northern blot analyses of poly A+ mRNA from brains with a specific riboprobe for bovine aFGF, revealed a major 4.5-Kb transcript and a minor 2.7-Kb transcript detectable only in postnatal brains. A similar pattern to that observed for IRMaFGF was seen with these mRNA transcripts, indicating that these aFGFmRNA are translated in the mouse brain. Our results suggest that aFGF may act in the postnatal phases of brain maturation.

Proliferating satellite cells express acidic fibroblast growth factor during in vitro myogenesis.

Recent in vitro studies have indicated that the proliferation of satellite cells, which are involved in muscular regeneration in vivo, is stimulated by exogenous addition of fibroblast growth factor (FGF). We present evidence that satellite cell cultures produce acidic, but not basic FGF. Acidic or basic FGF content was measured by enzyme immunoassay on cellular extracts after partial purification by heparin-Sepharose chromatography. During maximal cell proliferation, the level of acidic fibroblast growth factor (aFGF) was increased over fivefold from the values obtained before plating. aFGF content drastically dropped at the postmitotic stage to almost the threshold of detection, and remained weak as differentiation was completed. The immunolocalization of aFGF using highly purified anti-aFGF antibodies confirmed these results and indicated that aFGF was cytoplasma- or membrane-associated. Our work suggests that an endogenous production of aFGF by satellite cells may trigger cell proliferation by an intra- or autocrine mechanism, and therefore play an important role in muscular regeneration.

Immunological study of acidic fibroblast growth factor (aFGF) distribution in the eye.

During the last ten years, several groups, including the present authors, have detected growth factor activities in various ocular tissues, and the presence of a ubiquitous Eye-Derived Growth Factor (EDGF) has been described. More recently, isolation and characterization of this growth factor activity from the retina led to the identification of two molecules. These molecules were shown to be identical to other growth factors isolated from neuronal and non-neuronal tissues and are now designated as acidic and basic fibroblast growth factor (aFGF, bFGF). The biological function and the reason for the ubiquitous distribution of these factors remain unclear. Understanding may be improved by quantification of this distribution in various tissues during development. In the present study, specific polyclonal antibodies were raised against acidic FGF, aFGF was determined in various ocular tissues by enzyme immunoassay, and the localization of immunoreactive aFGF by immunohistological staining with fluorescent antibodies or with enzyme- or gold-labeled antibodies was studied. In almost all tissues tested aFGF was found; but the retina, cornea, and vitreous body contained the highest levels of aFGF per gram of tissue. In the retina, aFGF was associated primarily with the nerve fiber layer and the inner and outer segments of the photoreceptors, whereas corneal aFGF was detected in the cytoplasma of the basal layer of epithelial cells.

Development and testing of radio and enzyme immunoassays for acidic fibroblast growth factor (aFGF).

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor from bovine brain stimulate growth in a variety of tissues in several species. Despite the 55% amino acid sequence homology of the two forms of FGF, a specific immunoassay of aFGF has been developed using a polyclonal antibody raised in a rabbit. Two immunoassays were compared: a radioimmunoassay (RIA) using 125I aFGF and an enzyme immunoassay (EIA) using aFGF coupled to the tetrameric form of acetylcholinesterase (aFGF-AchE) as tracer. With EIA, the detection limit was 1.5 ng/ml, versus 2.2 ng/ml with RIA, while the dose at 50% was 5.9 ng/ml for EIA and 9.6 ng/ml for RIA. Using a modified EIA procedure where aFGF-AchE was added 2 h after the other reagents, the dose at 50% binding was 1.5 ng/ml. Examples of the performance of both immunoassays are presented for various brain extracts of different species including human. The aFGF content obtained by these methods correlates (CR = 0.987) with the values obtained by biological assay.