Investigation was carried out on the epithelial cells obtained from 32 patients with benign and malignant tumors of large intestine aged from 42 to 80 years. Ratio of single-stranded and double-stranded DNAs in the epithelial cells of unchanged colorectal mucosa (12 patterns), adenomatous polyps (4) and adenocarcinomas (29) was studied using fluorimetric analysis. Increased instability of DNA secondary structure was revealed in the tumor cells comparing to the cells of unchanged colorectal epithelium. Relative accumulation of single-stranded DNA reflects structural and functional changes in gene apparatus of cells under malignization.
It is not clear whether screening for breast cancer works as public health policy and whether early indicators of effect predict an ultimate reduction in mortality. The malignant potentials of 248 breast cancers detected by the screening service in Finland were compared with those of 490 control cancers diagnosed before the screening service was established. Aggressiveness was assessed by DNA flow cytometry and clinical status by cancer size and node involvement. After the first screening round, the results of DNA flow cytometry were the same in cancers diagnosed by screening and in controls; these findings are consistent with the hypothesis that the biological aggressiveness of breast cancer remains constant as the cancer progresses. The proportion of patients with node-negative and small T1 cancers after the first screening was higher among the screened population than among controls, indicating earliness of diagnosis among those screened. Cancers diagnosed in the first round had a low malignant potential, as indicated by the DNA flow-cytometry and by clinical stage. Lower aggressiveness of cancers found by screening than of control cancers would indicate overdiagnosis or length-biased sampling, but not earliness of diagnosis. Screening with mammography is practised as a public-health policy in Finland. The results predict that the mortality reduction found in randomised trials can be repeated with a screening service.
PURPOSE. To investigate antigen (Ag) specificity, activation, and effector function of the Ag-specific T cells involved in the development of experimental immune-mediated blepharoconjunctivitis (EC), an experimental conjunctivitis. METHODS. EC was induced in Brown Norway rats by injection of ovalbumin (OVA)-specific T cells followed by OVA challenge with eye drops. Eyes, including the conjunctivas, were harvested at different time points after challenge. The dependence of EC onset on the challenging Ag was assessed by challenge with an irrelevant Ag or stimulatory OVA peptides. To show the infiltration of transferred T cells into the conjunctiva, T cells were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) before transfer. The activation of T cells in the conjunctiva was assessed by measuring phosphorylation of Lck-associated molecules by Western blot analysis. Conjunctivas were also examined by immunohistochemistry and used for reverse transcription-polymerase chain reaction to determine the phenotype of the infiltrating cells and cytokine, chemokine, and chemokine receptor expression. To investigate infiltration of non Ag-specific T cells into the conjunctiva, ragweed (RW)-primed lymphocytes were transferred into OVA-specific T-cell receptor transgenic (DO11.10) mice. The mice were then challenged with RW and the conjunctivas were harvested for immunohistochemistry to detect T cells derived from DO11.10 mice. RESULTS. EC was induced only when challenged with OVA protein or stimulatory OVA peptides, and CFSE-labeled transferred cells were found in the conjunctiva. Phosphorylation of Lck and an 85-kDa Lck-associated molecule were observed in the conjunctiva 6 hours after challenge. Many cytokines and chemokines began to be expressed at 6 hours, and individual expression patterns over time correlated well with the infiltration patterns of different inflammatory cells. In DO11.10 mice that received RW-primed lymphocytes, T cells derived from the recipient mice infiltrated the conjunctiva after RW challenge. CONCLUSIONS. Ag-specific T cells initiate EC by first infiltrating the conjunctiva, where they become activated by the specific Ag in the conjunctiva.
Ameloblastoma is the most common clinically significant odontogenic tumor. It is considered benign but locally invasive and associated with variable clinico-pathological behavior. Ameloblastic carcinoma is a malignant tumor having features of ameloblastoma in addition to cytologic atypia with or without metastasis. It is aggressive and associated with poor prognosis. The aim of this study was to examine which epithelial and stromal markers are predictive of histologically diagnosed ameloblastic carcinoma and can sufficiently differentiate it from solid/multicystic ameloblastoma (SA). We examined immunohistochemically Ki-67, epithelial membrane antigen (EMA), alpha-smooth muscle actin (alpha-SMA), calponin, p63 and DNA content using image (ICM) and flow cytometry (FCM) in three ameloblastic carcinomas and up to 18 SAs. The important findings were that Ki-67 labeling index was significantly higher in ameloblastic carcinoma than SA while EMA, calponin, p63, ICM and FCM did not sufficiently differentiate the two groups of lesions. Expression of alpha-SMA was consistently obtained within the epithelial island cells of ameloblastic carcinoma and not in SA, although the marker was well expressed in the stroma of both lesions. We therefore conclude that the presence of alpha-SMA within the epithelial islands is highly predictive of ameloblastic carcinoma.
The utilization of the intracellular and extracellular sources of carbon and energy during the mitotic cycle of yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida boidinii, Candida tropicalis has been studied. Increase in the consumption rate of carbon and energy sources and in the exogenous respiration rate at G1- and G2-phases of the mitotic cycle is shown. The rate of the endogenous respiration of the cells at these phases decreased. The hypothesis has been proposed that during the mitotic cycle of the yeast cell the regular alternation of exotrophy (the utilization of the extracellular carbon and energy sources by a cell) and endotrophy (the process of the utilization of the intracellular carbon and energy sources by a cell) occurs. It is possible to reveal the exotrophic cells by the cytological method which is based on the calculation of dead cells after incubation of the yeast suspension in amyl alcohol solution. This method has revealed that exotrophic and endotrophic processes do not predominate one over another but alternate at the mitotic cycle. Exotrophy and endotrophy are phase-specific processes. The G1- and G2-phases are exotrophic processes, phases S and M are endotrophic ones.
HIV-1 viral load quantitation is now recognized as a useful tool to monitor the efficiency of antiviral treatment and a powerful predictor of disease outcome. Three HIV-1 viral load quantitation methods have been currently available as commercial kits in Canada since 1996.
To evaluate the ability to quantify HIV-1 RNA in plasma of the Amplicor HIV Monitor Test, the NASBA HIV-1 RNA QT Assay and the Quantiplex HIV RNA Assay, version 2.0, at comparable lower detection limits.
Blood was collected from 50 HIV-1-infected patients at various stages of infection and therapy. CD4+ cell count were estimated by flow cytometry. Plasma was isolated and tested in duplicate on four occasions using viral load kits from a single lot. HIV RNA data, performance, sensitivity and intra- and inter-assay variability were compared.
RNA could be quantified in 33 patients by each technique. An inverse correlation was observed between viral load level and CD4+ cell counts in patients with counts below 200. Monitor could detect RNA in 94% of patients, but it showed the greatest variability and failure rate. Quantiplex 2.0 could detect HIV-1 RNA in 78%, and NASBA in 88% of the patients at theoretically equivalent lower detection limits, suggesting that the detection limit of Quantiplex 2.0 may be higher than 500 HIV-1 RNA copies per ml. NASBA had the fewest invalid tests and good reproducibility, comparable to that of Quantiplex 2.0. The mean values from NASBA and Monitor were the most similar but the best correlation was observed between Monitor and Quantiplex 2.0 results.
Monitor, NASBA and Quantiplex results were comparable, although those obtained by Quantiplex were significantly lower. Performing this study at comparable detection limits showed that the detection limit of Quantiplex 2.0 may be higher than stated by the manufacturer.
PURPOSE: The aim of this study was to investigate the phenotypes of antigen (Ag) presenting cells (APCs) in the conjunctiva during the development of experimental immune-mediated blepharoconjunctivitis (EC), which serves as a model for investigation of severe types of human allergic conjunctivitis. METHODS: Brown Norway rats treated by ovalbumin (OVA) were used in this study. To confirm the restriction of MHC class II by OVA-specific T cells, monoclonal Abs against MHC class II were added to the conventional proliferation assay. To evaluate the MHC class II expression in the conjunctiva during the development of EC, an immunohistochemical analysis, either as the single or double staining, was performed. Conjunctival fibroblast cell lines were established from naive rats and the MHC class II expression was evaluated by flow cytometric analysis. To examine the roles of costimulatory molecules, OVA-specific T cells were stimulated with anti-TcR Ab and anti-CD28 Ab and then subjected for Western blotting to evaluate the ERK phosphorylation. Finally, in vivo expression of B7 molecules was examined immunohistochemically. RESULTS: OVA-specific T cells recognized OVA in the context of MHC class II. MHC class II was expressed in conjunctival macrophages but not in fibroblasts. EC induction was accompanied by abundant infiltration of macrophages positive for MHC class II. MHC class II was also expressed in conjunctival epithelial cells by EC induction. Stimulation from CD28 was necessary for ERK phosphorylation. B7-2, but not B7-1, was expressed in the conjunctiva. CONCLUSION: Conjunctival macrophages may represent a major source of APCs for the induction of EC in the conjunctiva.
Thyroid carcinoma is rare and constitutes about 1% of all malignant tumours. For some time it has been known that age, sex, histology, and tumour grade are factors of importance for prognosis. In Stockholm we have analyzed the amount of nuclear DNA in thyroid tumours for the last 10 years. In our studies and those of other nuclear DNA-analysis has given important prognostic information for differentiated thyroid tumours in addition to that provided by histopathology, and may be of value preoperatively for treatment planning. DNA-analysis is well established in our clinical work and is one of the factors considered in a prospective randomized study concerning patients with papillary carcinoma in the Stockholm-Uppsala area. Tumours with diploid nuclear DNA pattern are randomized between lobectomy and total thyroidectomy in this study.
Flow cytometric analysis of leukocyte surface antigens has been used to characterize infectious and septic processes in patients. We wanted to investigate how the sampling and processing temperature, the anticoagulant used, and the storage of the sample influence leukocyte immunophenotyping. Four blood samples, two using acid citrate dextrose and two using heparin as an anticoagulant, were taken from five intensive-care unit patients with severe sepsis and five healthy volunteers. The samples were collected, stored, and processed either at +4?C or at room temperature (RT). The samples were processed for flow cytometric analysis within 1 ?hr of collection or after 6 or 24? hr storage. The surface antigens of interest were neutrophilic CD11b and CD64, monocytic CD11b, CD14, CD40, CD64, CD80 and HLA-DR, and lymphocytic CD69 (separately in CD4+ and CD8+ T cells, B cells, and natural killer cells). The fluorescence intensities were higher at RT than at +4?C. During storage the intensities increased at RT, but at +4?C there were only minor changes. The effects were similar with both anticoagulants studied. According to our results, flow cytometric analysis of leukocyte surface antigen expressions should be performed using +4?C temperature throughout the process and within 6? hr.
The occurrence of abnormal nuclear DNA content in major salivary gland adenomas is not well known and its correlation with tumor recurrence has not been documented previously. From 1987 to 1991, 119 consecutive major salivary gland adenomas were operated on at Turku University Central Hospital. These tumors were analyzed by flow cytometry and 100 (84%) were found to be diploid, 12 (10%) near-diploid and 7 (6%) aneuploid with DNA indexes > 1.15. The mean proliferation rate measured as a percentage of cells in the S-phase fraction was 2.5 +/- 1.6%. The histological slides were then blindly reclassified according to current World Health Organization classification. As a result histological classification was changed in 3 tumors: malignant cells were found in 2 aneuploid tumors and 1 diploid neoplasm. Preoperative cytological fine-needle aspiration biopsy had been considered as possibly malignant in 2 of these cases. Among all case material 10 specimens were recurrent tumors; although the tendency to recur depended on the extent and adequacy of the surgery performed, multiple recurrences were associated with non-diploid tumors.