Biomedical subjects

Claude Lambert

Publications and source records attributed to Claude Lambert.

At least 19 recordsLinked to original sources

PubMed2007

Thematic workshop on fluorescence compensation settings in multicolor flow cytometry.

In his program of thematic one-day workshops, the French Association of Cytometry had organized a workshop dedicated to the fluorescence compensation settings in multicolor flow cytometry. This special day was in honor of our past President Jean Luc D'Hautcourt who has been involved in the quality of the use of flow cytometry in its clinical and research purposes. Review on fluorescence phenomena, compensation rules, settings, and few observed confounding situations were presented.

CD4-Positive T-Lymphocytes↗

PubMed2006

Enumeration of peripheral lymphocyte subsets using 6 vs. 4 color staining: a clinical evaluation of a new flowcytometer.

UNLABELLED: Technological advances in instruments allow the evaluation of many lymphocyte subsets in one step. The aim of this study was to evaluate the new FACSCanto flowcytometer in routine conditions, using a 6 color combination, single platform, whole blood, lysis, no wash protocol. METHODS: Two systems were simultaneously compared on 67 blood samples and external quality controls, using CD3,CD4, CD8, CD19, CD16/56, and CD45 in one tube TRUCOUNT beads (BD Biosciences) or two tubes (TetraChrome and Flowcount, Beckman-Coulter and DakoCytomation). RESULTS: The day-to-day instrument detection but automatic compensations were stable. Manual compensation settings were satisfactory using available facilities. Commercial and UK NEQAS quality control results were acceptable. The intra-experiment reproducibility was good (coefficient of variation (CV)<3%) but highly operator-dependent (CD4+ T cell count CVs from 1.2 to 9.7, six operators). Storage of samples was acceptable, but storage of stained samples altered absolute count reliability. Serial dilutions show a good count accuracy. The FACScanto T subsets and B cell data were highly correlated with our reference values (r2>0.87) and absolutes values were very close (slopes>0.89). The gating strategy, fluorochrome choice, and compensation setting are discussed. A few improvements are expected (sample loader, data management, auto-gating, acquisition parameters, sample mixing, absolute values calculation, etc). In conclusions, despite its complexity, 6 color staining is a reliable, stable, and highly informative technique for lymphocyte subset monitoring but remains to be optimized.

Adolescent↗

PubMed2005

The use of MN/CA9 gene expression in identifying malignant solid renal tumors.

OBJECTIVE: Small solid renal tumors are increasingly encountered. It is important to determine the malignancy of solid renal tumors for the choice of treatment. MN/CA9 is expressed in malignant renal cells but absent in normal cells. MN/CA9 is one of the most powerful gene markers available for RCC. The objective of this pilot study is to utilize MN/CA9 gene expression in FNA biopsy to determine the malignancy of imaging-indeterminate solid renal tumors. METHODS: A total of 35 patients with an imaging-indeterminate solid renal mass entered into this study. The molecular protocol consisted of a rapid column extraction of RNA and one-step RT-PCR for the detection of MN/CA9 gene expression. The preoperative molecular diagnosis was compared with postoperative pathology. RESULTS: There were 28 RCCs (19 clear cell carcinomas, 7 papillary carcinomas and 2 chromophobe carcinomas) and 7 benign tumors proved by postoperative pathology. The overall sensitivity and specificity for MN/CA9 were respectively 68% and 100%. MN/CA9 was positive in 16/19 (84%) FNA biopsies of clear cell RCCs. No false positive appeared for MN/CA9 gene expression. Moreover, MN/CA9 gene expression was positive in 8/13 (62%) of false negative or suspect cytology. CONCLUSION: Detection of MN/CA9 gene expression in FNA biopsy is possible. Its detection can be helpful in identifying the malignancy among renal tumors.

Adenocarcinoma, Clear Cell↗

PubMed2005

Evaluation of a panel of molecular markers for the diagnosis of malignant serous effusions.

PURPOSE: Our main goal was to evaluate a panel of molecular markers for the detection of cancer cells in serous effusions and to determine their value as an adjunctive reverse transcription-PCR (RT-PCR) test to cytologic examination. EXPERIMENTAL DESIGN: One hundred fourteen serous effusions from 71 patients with tumors and 43 patients with benign diseases were subjected to RT-PCR for expression of carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (Ep-CAM), E-cadherin, mammaglobin, mucin 1 (MUC1) isoforms MUC1/REP, MUC1/Y, and MUC1/Z, calretinin, and Wilms' tumor 1 susceptibility gene. RESULTS: CEA, Ep-CAM, E-cadherin, and mammaglobin were specifically expressed in malignant effusions. The sensitivity of RT-PCR in cytologically negative malignant effusions was 63.1% combining CEA and Ep-CAM (with 100% specificity) and reached 78.9% adding MUC1/Y or MUC1/Z (with 93% specificity). In the whole population of effusions, the combination of cytology with RT-PCR of CEA and Ep-CAM yielded a 90.1% sensitivity, a specificity and a positive predictive value of 100%, and a 86% negative predictive value for malignancy. Adding MUC1/Y or MUC1/Z to the panel, the sensitivity was 94.5% with 93% specificity, 95.7% PPV, and 90.9% negative predictive value. Moreover, CEA and mammaglobin were specifically expressed in epithelial malignancies, and mammaglobin was mainly expressed in effusions from breast carcinoma (97.3% of specificity). CONCLUSIONS: A combination of cytology and RT-PCR analysis of CEA and Ep-CAM significantly improved the detection sensitivity of tumor cells in serous effusions. RT-PCR analysis of CEA, Ep-CAM, and mammaglobin in serous effusions could be a beneficial adjunct to cytology for the diagnosis of malignancy.

Aged↗

PubMed2005

Decrease in serum procalcitonin levels over time during treatment of acute bacterial meningitis.

INTRODUCTION: The aim of this study was to describe the change in serum procalcitonin levels during treatment for community-acquired acute bacterial meningitis. METHODS: Out of 50 consecutive patients presenting with bacterial meningitis and infection at no other site, and who had received no prior antibiotic treatment, 48 had a serum procalcitonin level above 0.5 ng/ml on admission and were enrolled in the study. RESULTS: The mean age of the patients was 55 years, and mean Glasgow Coma Scale score on admission was 13. The time from symptom onset to admission was less than 24 hours in 40% of the patients, 24-48 hours in 20%, and more than 48 hours in 40%. The median (interquartile) interval between admission and initial antibiotic treatment was 160 min (60-280 min). Bacterial infection was documented in 45 patients. Causative agents included Streptococcus pneumoniae (n = 21), Neisseria meningitidis (n = 9), Listeria monocytogenes (n = 6), other streptococci (n = 5), Haemophilus influenzae (n = 2) and other bacteria (n = 2). The initial antibiotic treatment was effective in all patients. A lumbar puncture performed 48-72 hours after admission in 34 patients showed sterilization of cerebrospinal fluid. Median (interquartile) serum procalcitonin levels on admission and at day 2 were 4.5 (2.8-10.8) mg/ml and 2 (0.9-5.0) mg/ml, respectively (P < 0.0001). The corresponding values for C-reactive protein were 120 (21-241) mg/ml and 156 (121-240) mg/ml, respectively. Five patients (10%) died from noninfectious causes during their hospitalization. CONCLUSIONS: Serum procalcitonin levels decrease rapidly with appropriate antibiotic treatment, diminishing the value of lumbar puncture performed 48-72 hours after admission to assess treatment efficacy.

Acute Disease↗

PubMed2005

Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction.

Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa. We demonstrate that normal human vaginal epithelial cells constitutively secrete the chemokine macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20), known to recruit LC precursors (LCps) selectively via its cognate CC chemokine receptor 6 (CCR6). This secretion is up-regulated by the proinflammatory cytokine interleukin-1beta through the nuclear factor-kappaB pathway. Similar results were obtained with the human vaginal epithelial cell line SiHa, which displays numerous homologies with normal vaginal cells. The chemotactic activity of the secreted CCL20 was demonstrated by its ability to attract LCp CCR6+. Moreover, the use of neutralizing polyclonal antibodies directed against the CCL20 molecule abolished this migration completely, suggesting that CCL20 is the main attracting factor for LCps, which is produced by the vaginal cells. These data indicate that vaginal epithelial cells play an important role in the immunological defense by attracting immune cells to the site of epithelial/pathogen contact.

Antibodies↗

PubMed2005

Persistent oligoclonal CD4dimCD8+T cells in peripheral blood.

BACKGROUND: Routine CD4/CD8 T-cell phenotyping may shows a small fraction of CD4dimCD8+ T cells with a homogeneous appearance as described for lymphoproliferative syndromes or chronic infections. The aim of this study was to elucidate the significance of CD4dimCD8+ T cells and their degree of diversity. METHODS: Phenotyping was performed in 272 samples from healthy donors, elderly patients, and immunocompromised (human immunodeficiency virus or renal transplantation) patients. RESULTS: The CD4dimCD8+ T cells had decreased fluorescence intensity for CD4 but not for CD8. The frequency of patients with CD4dimCD8+ T cells (>20 cells/microl; 10.3% of patients with human immunodeficiency virus and 7.7% with renal transplantation) was not significantly different when compared with healthy donors (9.7%). The CD4dimCD8+ T cells did not express the activation marker CD69. The CD8 of CD4dimCD8+ T cells expressed the heterodimeric (beta) isoform. In 13 of 26 samples, the apparently highly homogeneous CD4dimCD8+ T cells expressed one predominant T-cell receptor Vbeta clonotype. These predominant clonotypes were widely distributed among patients: Vbeta 5.2, 17, 2, 3, 5.1, 13.1, 14, and 20. CONCLUSIONS: Whether these findings demonstrate an oligoclonal reaction to chronic inflammation or an emerging lymphoproliferative disorder must be elucidated in a long-term longitudinal study. By analogy to monoclonal gammopathy, we propose to name this phenomenon "oligoclonal clonopathy of undetermined significance."

Adolescent↗

PubMed2005

Selective sequestration of X4 isolates by human genital epithelial cells: Implication for virus tropism selection process during sexual transmission of HIV.

X4 and R5 HIV strains are present in the semen of men infected with HIV but R5 isolates are transmitted preferentially. The role of human epithelial cells in this selection is addressed. Three human cervical cell lines-CaSki, SiHa, and HEC1A-and normal human vaginal cells from HIV-negative donors were characterized for HIV receptor expression and incubated with X4 and R5 laboratory-adapted strains or primary isolates. The infection was assessed by detection of intracellular HIV DNA. The three cell lines were shown to express on their surface the CXCR4 and GalCer molecules, but not the CD4 and CCR5 ones. The three cell lines and normal human vaginal cells were found to be selectively permissive to X4 HIV entry; the preincubation of the cell lines with rhSDF-1 inhibited this infection. The detection of the intracellular proviral DNA in the cell lines and in normal human vaginal cells demonstrated a selective integration of X4 strains. Additional experiments showed that no extracellular RNA was detected in the supernatants of HEC1A cells infected by X4 isolates either after 18 days of culture or after incubation with PHA-stimulated PBMCs and that no transmission occurred after co-culture between infected HEC1A cells and PHA-stimulated PBMCs. These results suggest specific sequestration of X4 strains by genital epithelial cells, which could explain, at least in part, the HIV tropism selection process during sexual intercourse.

Cell Line↗

PubMed2005

Significance of unconventional peripheral CD4+CD8dim T cell subsets.

Routine T cells phenotyping occasionally reveals a CD4+CD8dim T cell subset with an apparently homogeneous dot plot. The aim of this study was to elucidate their immunological significance from analysis of 31 healthy donors, 21 elderly and 220 immune deficient patients. CD4+CD8dim T cells expressed reduced levels of CD8 (11-17,000 compared to 96-128,000 mol/cell on CD8+ T Cells). CD4 was expressed at the same level as on CD4+ T cells. The occurrence of raised CD4+CD8dim T cells (> 20 cells/muL) was similar in kidney transplant recipients (28.4%) and healthy donors (26%). It was somewhat lower in HIV+ patients (19.7%) possibly due to virally induced CD4+ T lymphopenia. However, an age effect is possible because the occurrence was raised (33.3%) in 70 volunteers (chi2 test NS). On the other hand, the size of the CD4+CD8dim subset was not correlated with age. CD4+CD8dim T cells did not express the activation markers CD69 (n = 220) or CD25 (n = 10) and expressed the homodimeric (alphaalpha) isoform of CD8, suggesting they are related to mucosal immunity (MALT). We selected 29 patients with unambiguous dot plots. In 26 of them one predominant TCR Vbeta clonotype was expressed on 18 to 94% of CD4+CD8dim T cells and never on more than 10% of conventional T cells. The predominant clonotypes were Vbeta8 (n = 5), Vbeta2 (n = 4), Vbeta13.1 and Vbeta 21 (n = 3 each). Whether this reveals a chronic stimulation or an emerging lymphoproliferative disorder must be elucidated. We propose to name this entity: "Oligoclonal Clonopathy of Undetermined Significance (OCUS)."

Adult↗

PubMed2005

Different DNA ploidy patterns for the differentiation of common subtypes of renal tumors.

OBJECTIVES: The common subtypes of renal tumors are conventional or clear cell carcinoma, papillary carcinoma, chromophobe carcinoma and oncocytoma. Each subtype has its distinct histogenesis and clinical evolution. DNA ploidy is viewed as a marker of gross genomic aberrations. The aim of this study is to evaluate the DNA ploidy in the common subtypes of renal tumors to increase our understanding of renal tumor biology and to broaden clinical application of DNA ploidy. METHODS: 38 renal tumor samples (13 clear cell RCCs, 12 papillary RCCs, 7 chromophobe RCCs, and 6 oncocytomas) were studied. Five biopsies of different parts of each fresh tumor were subjected to a flow cytometric analysis of DNA ploidy. RESULTS: All tumors except one papillary RCC generated interpretable DNA histograms. Flow cytometric analysis of oncocytomas showed the diploid pattern (29/30 frequencies) while the chromophobe RCC never showed the diploid pattern (0/55 frequencies) (p<0.01). 3/7 chromopbobe RCCs possessed the hypodiploid stemline. The hypodiploid stemline appeared neither in conventional RCCs (0/63 frequencies) nor in papillary RCCs (0/50 frequencies). The diploid pattern was dominant in conventional and papillary RCCs. 10/13 (76.9%) of clear cell RCCs and 9/11 (81.8%) of papillary RCCs possessed a homogeneous DNA ploidy pattern while only 1/7 (14.3%) has a homogeneous DNA ploidy pattern. 6/7 chromophobe RCCs had multiple aneuploid stemlines. CONCLUSIONS: Flow cytometric analysis reveals that conventional and papillary RCCs are more homogeneous than chromophobe RCC. Each subtype of renal tumors possesses a specific DNA ploidy pattern. The analysis of DNA ploidy is useful for the differentiation of common subtypes of renal tumors in morphologically difficult cases.

Adenocarcinoma, Clear Cell↗

PubMed2004

Recruitment of STAT3 for production of IL-10 by colon carcinoma cells induced by macrophage-derived IL-6.

The immunosuppressive cytokine IL-10 is associated with poor prognosis in colon cancer. Although macrophages are involved in antitumor defenses, production of IL-10 by tumor cells may permit malignant cells escape to cell-mediated immune defenses. To investigate interactions between macrophages and tumor cells in humans, we cultured macrophages isolated from patients and tested the effect of these macrophages on the production of IL-10 by several tumor cell lines. Macrophages were isolated from pleural effusions of patients with malignancy and from noncancer control patients. We demonstrated that culture supernatants of macrophages from both sources strongly stimulated IL-10 production by the three different human colon adenocarcinoma cell lines, Colo 205, Colo 320, and HT29. Recombinant IL-6, but not IL-10, TNF-alpha, and IFN-alpha, stimulated the secretion of IL-10 by colon tumor cells. mAbs against IL-6 and IL-6R prevented the effect of macrophage culture supernatants and of rIL-6, respectively, on the production of IL-10 by the three cell lines. Cocultures of macrophages and colon cancer cells showed that these tumor cells first stimulated macrophages to produce IL-6, which was then followed by IL-6-induced IL-10 production by colon cancer cells. Finally, we showed that IL-10 gene regulation was mediated by STAT3, which was phosphorylated after the binding of IL-6 to IL-6R. This is the first demonstration that IL-6, secreted by macrophages, can induce a STAT3-mediated IL-10 production by colon tumor cells.

Cell Line, Tumor↗

PubMed2004

CD3 bright lymphocyte population reveal gammadelta T cells.

BACKGROUND: In routine CD3/CD4/CD8 T-cell analysis, a CD3 bright population of lymphocytes is frequently observed. The aim of the present study was to identify the immunological significance of such CD3 bright lymphocytes. METHODS: We analyzed samples from 31 healthy adult volunteers, 78 human immunodeficiency virus (HIV)-positive, and 78 renal transplanted patients. RESULTS: A clearly distinct CD3 bright (frequently CD4-/CD8-) T-cell fraction was observed in 84% of donors and was directly correlated with the fraction of gammadelta T cells (r2 = 0.64). CD3 overexpression on gammadelta T cells was confirmed by a combination of monoclonal antibody staining (CD3-ECD, gammadeltaTCR-FITC, and alphabetaTCR-PE-Cy5) or immunomagnetic purification of gammadelta T cells (i.e., MdFI 20 vs 8.86). The gammadelta T cells expressed CD8 polypeptide chains (alpha and beta) in all possible combinations. The largest proportion, surprisingly, were cells expressing CD8betabeta homodimers (43.8 +/- 16.5%). CD8alphaalpha homodimers were expressed on 14.2% (+/- 12.3) of total gammadelta T cells, whereas CD8alphabeta heterodimers were expressed on 12.2% (+/- 7.5). We also observed a bimodal distribution of the intensity of CD3 fluorescence of gammadelta T cells in immunocompromised patients with a threshold at 105 cell/microl. CD3 bright gammadelta T cells were more frequently observed in HIV patients (29%) compared with renal transplant patients (11%) and healthy donors (3%; chi2 test: P = 0.0007). CONCLUSIONS: The simple observation of a CD3 bright T-cell subset on CD3/CD4/CD8 routine analysis suggests a high gammadelta T-cell fraction and, in our opinion, should be followed by a complementary analysis to determine precisely the number of gammadelta T cells and to identify their CD8alpha/beta phenotype. When CD3 bright T cells/microl were more than 40%, high gammadelta T cells were detected in more than 87% of cases, with a specificity of 76%. Occasionally, the CD3 bright subset appeared to be strongly homogeneous, suggesting an oligoclonal proliferation that could possibly reveal a chronic localized stimulation or an early lymphoproliferative disorder. Because the gammadelta T cells have interesting immunological peculiarities, the clinical significance of their quantitative abnormality should be clarified in diseases such as HIV, organ transplantation, autoimmunity and lymphoma.

Adult↗

PubMed2004

Cytometric bead assay of cytokines in sepsis: a clinical evaluation.

BACKGROUND: The clinical relevancy of an attractive new multiparametric method, cytometric beads assay (CBA), was evaluated for the monitoring of cytokines in sepsis. METHODS: A total of 52 samples (26 patients) were simultaneously tested by CBA and chemiluminescence (IL-6, IL-8, IL-1beta, and TNFalpha) or ELISA (IL-10, IL-12 p40, IL-12 p70, and soluble TNFalpha-RI). RESULTS: CBA standard curves were linear from 20-5,000 ng/L except for IL-1beta (40-5,000 ng/L). IL-6 and IL-8 were detected in 41 and 48 samples (44-5,000 ng/L and 22-5,000 ng/L), respectively, and out of range in six samples. IL-10 and IL-1beta were detected in 14 and 15 samples (21-1,548 ng/L and 29-582 ng/L), respectively. TNFalpha was rarely detected, and IL-12 p70 was never detected. Accuracy and repeatability were good (CV for IL-6 was 2.1-8.7%; for IL-8 3.7-5.3%; for IL-10 2.5-9.1%; for IL-12 5.2-6.5%; for IL-1 beta 4-12%; and for TNFalpha 3-19.3%). Reproducibility of four standard curves and 20 samples (-1.1 to +1.03%) was excellent between tests done at two- to six-week intervals. CBA values were correlated (r2 > 0.89; P < 0.001) with our reference methods, but were lower on CBA (TNFalpha 45% +/- 8; IL-6 49% +/- 7), probably due to interactions with patients' serum, as confirmed by spiking diluted standards in one patient's serum with end stage renal failure and high levels of TNF soluble receptor (i.e., TNFalpha levels 34 and 21%). CONCLUSIONS: Finally, these results suggest that IL-6, IL-8, IL-1beta, and Il-10 are clinically promising for sepsis evaluation. However, sensitivity of TNFalpha has to be improved and IL-12 p70 should be replaced with more relevant parameters such as TNF-R, procalcitonin, or neopterin.

Adolescent↗

PubMed2003

Rapid and sensitive detection of messenger RNA expression for molecular differential diagnosis of renal cell carcinoma.

PURPOSE: The aim of this study was to develop a practical technique to detect mRNA expression and to validate a panel of mRNA markers for molecular differential diagnosis of renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: The renal cancer cell line SKRC-52 was used to set up the technique, which consisted of column extraction of RNA and one-step reverse transcription-PCR. We validated a panel of gene markers, including MN/CA9, cadherin-6, vimentin, mucin1, and parvalbumin, and studied 50 renal tumors (30 conventional, 9 papillary, and 5 chromophobe RCCs and 6 oncocytomas), 10 normal tissues, and 10 normal blood samples. We mimicked fine needle aspiration (FNA) biopsy in 10 kidneys with conventional RCC and applied this technique to 10 preoperative FNA samples from imaging-indeterminate renal tumors. RESULTS: The technique could detect as few as 10 SKRC-52 cells with MN/CA9 as mRNA marker and was less time consuming and labor intensive. MN/CA9 was a sensitive and rather specific gene marker for conventional RCC. Cadherin-6 gene expression was a sensitive marker for conventional and papillary RCC. Vimentin was highly specific for conventional RCC. Mucin1 mRNA was sensitive for papillary and chromophobe RCC and oncocytoma. Parvalbumin mRNA was a sensitive and highly specific marker for both chromophobe RCC and oncocytoma. Thus, these mRNA markers represent the biomarker genes for the subtypes of renal tumors. Finally, we successfully applied the technique to FNA specimens. Five preoperative FNA samples were MN/CA9 gene positive, suggesting a RCC, whereas the routine cytology was positive in only three cases. CONCLUSIONS: A rapid and sensitive assay of mRNA markers was developed for molecular differential diagnosis of RCC. This molecular assay can be used as a powerful ancillary to surgical pathological diagnosis and cytological diagnosis of RCC.

Adenoma, Oxyphilic↗

PubMed2003

Macrophages from cancer patients: analysis of TRAIL, TRAIL receptors, and colon tumor cell apoptosis.

BACKGROUND: Tumor-infiltrating macrophages secrete cytokines, including Fas ligand, tumor necrosis factor-alpha (TNF-alpha), and TNF-related apoptosis-inducing ligand (TRAIL). TRAIL induces apoptosis in tumor cells but not in normal cells; however, regulation of TRAIL and its receptors in cancer patients is relatively uncharacterized. We investigated whether macrophages from cancer patients produce TRAIL and whether apoptosis in cultured colon adenocarcinoma cells involves TRAIL and its receptors. METHODS: Macrophages isolated from pleural effusions of nine cancer patients and five control patients with congestive heart failure (whose effusions contained no tumor cells) were cultured. Levels of TRAIL, TNF-alpha, interferon alpha, and Fas ligand in conditioned medium were measured by enzyme-linked immunosorbent assays. Apoptosis of human colon adenocarcinoma cell lines, including Colo 205, was determined by the Annexin V method and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick-end labeling (TUNEL). Cell-surface TRAIL receptors were measured by flow cytometry. RESULTS: Conditioned culture medium from macrophages isolated from pleural effusions containing 1%-5% tumor cells (CM-A) contained TRAIL at 980-1300 pg/mL, whereas that from macrophages from pleural effusions containing more than 50% tumor cells or containing no tumor cells (CM-B) contained TRAIL at 0-50 pg/mL. When cultured with medium containing 50% CM-A, 40% (95% confidence interval [CI] = 30% to 50%) of Colo 205 cells underwent apoptosis; when cultured with 50% CM-B, 8% (95% CI = 3% to 13%) underwent apoptosis. When Colo 205 cells were cultured with 50% CM-A, cell-surface expression of TRAIL death receptors DR5 and DR4 increased 13-fold and sixfold, respectively, compared with that of untreated Colo 205 cells. Recombinant TRAIL induced 90% (95% CI = 85% to 95%) of Colo 205 cells to undergo apoptosis and acted synergistically with TNF-alpha to induce apoptosis. CONCLUSION: Macrophages from cancer patients appear to be activated by tumor cells to produce TRAIL and to increase the expression of TRAIL death receptors DR4 and DR5 on tumor cells.

Adenocarcinoma↗

PubMed2003

Technique for obtaining highly enriched, quiescent immature Langerhans cells suitable for ex vivo assays.

Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a. These cells can capture a pathogen and then migrate and differentiate to a more mature stage. During this maturation process, dentritic cells express surface markers differentially. In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells. For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery. This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage. After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process. Both systems were equally efficient in terms of purification and yield. By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a(+) LCs, contrary to the repeated Ficoll floatation. This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells. Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.

Antibodies, Monoclonal↗

PubMed2003

[Molecular and cytometric analysis of renal cell carcinoma cells. Concepts, techniques and prospects].

At the present time, there is no reliable laboratory marker for the diagnosis and prognosis of clear cell renal cell carcinoma (RCC), while about 20% of small tumours detected by modern imaging techniques are benign and the clinical course is difficult to predict with considerable differences for the same stage and same grade. The molecular identification of clear cell RCC cells could satisfy these new requirements in the context of diagnosis of atypical or small renal tumours, allowing a more refined prognostic assessment, which is currently uncertain. Some of the antigens used for molecular diagnosis of clear cell RCC, such as cadherin-6, are present in the normal kidney, while others are newly formed antigens (TuM2PK, MN/CA9, CA12, calpain) or ectopic (PSMA, PSA, KLKI, cytokeratin 7 vimentin) or induce abnormal glycosylation (sialyl Lewis'X, galectins) indicating the malignant nature of the cells. The tumour's capacity for progression is related to dysregulations of the cycle (ras, Pax2, Tiam 1, waf/p21), division (tetracyclines, MIB1, PCNA, Nor Ag), apoptosis (bcl2, p53, CD95/Apo1), and the capacities for tissue invasion (proteases), disorganization (cadherin, catenins) or nidation (ICAM-1, CD44). Finally, chromosomal anomalies (mutations, translocations) also occur. MN/CA9, cadherin-6, vimentin, mucin 1 and DNA content are particularly useful for the diagnosis and/or prognosis of clear cell RCC. These markers can be analysed by extremely sensitive cytometric (flow cytometry, plate cytometry) or molecular methods (RT-PCR, in situ hybridization). These techniques lower the limit of detection of tumour cells in biological products (aspiration cytology, microbiopsy) and eventually in circulating blood. Proteomic and genomic methods (biochips) should considerably accelerate research in this field leading to the development of routine clinical applications.

Antigens, Neoplasm↗

PubMedDate not supplied

The in vivo DNA aneuploidization during expansion of conventional renal cell carcinoma.

Although numerous studies on the prognostic value of DNA aneuploidy in RCC have been reported, the in vivo DNA aneuploidization during RCC expansion has not been revealed. The present study was undertaken to observe the DNA aneuploidization during RCC expansion. We studied prospectively 67 consecutive conventional RCCs. The ploidy status was determined by analyzing five fresh tumor tissues from different areas by flow cytometry. The diploid, heterogeneous aneuploid tumors and homogeneous aneuploid tumors could be detected, respectively, in 44.8%, 23.9% and 31.3% of cases. The diploid tumors decreased significantly and aneuploid tumors increased significantly as the tumor expanded. The similar DNA content distribution was found between the heterogeneous aneuploid tumors and homogeneous aneuploid tumors. The hypertriploid clone was the most frequent in aneuploid tumors. The tumors of multiple aneuploid clones (16.4%) were mainly found in large-sized tumors. These results suggested that some RCCs underwent DNA aneuploidization during the tumor expansion and that a major route of aneuploidiztion (hypertriploidization) and several pathways existed. Our results also supported the idea that the progressive chromosomal instability was associated with continued tumor growth of RCC. The molecular mechanism and the clinical significance of aneuploidy phenotypes need to be further investigated.

Aneuploidy↗