Centrifugal separation of carcinoma or atypical cells in voided urine

A simple density gradient method was used to separate atypical and cancer cells from non-cancer cells in voided urine from patients with transitional cell atypia (moderate and grave atypia) and bladder cancer (squamous cell carcinoma and transitional cell carcinoma). Prior to cell separation, the Saccomanno preserved cells were dispersed by homogenization. After cell separation (5 min x 1400 rpm), atypical and cancer cells were enriched up to 20-fold. Also, most of the leucocytes (68-98%) and squamous cells (47-82%) were absent from density gradient specimen fractions containing the largest percentages of atypical and cancer cells. Peak purity ranges of atypical or cancer cells from different sample classes showed a large degree of overlap. This permitted the pooling of density gradient fractions enriched for atypical or cancer cells, thus increasing the efficiency of the method. Also, following centrifugation, the Papanicolaou-stained specimen fractions showed less background staining than the unprocessed controls, and the cells retained diagnostic morphologic features. We infer that this method may be a useful, low-cost approach for the morphologic study of developing cancers, not only from the urinary bladder, but also from the respiratory tract.     

Prostatic carcinoma cells in urine specimens

Between January 1985 and March 1990, the Cytology Laboratory of Hanover General Hospital examined cytologic preparations from four patients which revealed cells consistent with prostatic adenocarcinoma. In three of these cases, malignant cells were positive for prostatic-specific antigen. A fifth patient's specimen contained prostatic-specific antigen positive cells compatible with prostatic origin, but without overtly malignant features. All five patients had high grade prostatic adenocarcinoma (Gleason's Grade 8–10), and urinary tract symptoms. the most useful cytologic features indicating prostatic origin were large and often multiple nucleoli.     

Inflammatory response in induced sputum mononuclear cells from patients with acute exacerbation of asthma

Examination of sputum provides a direct method to investigate airway inflammation non-invasively in particular Th1 (IL-2, IFN-gamma) and Th2 (IL-4, IL-10) cytokine production. IL-2, IL-4, IL-10 and IFN-gamma cytokine were studied in induced sputum mononuclear cells of asthmatic patients. Sputum induction was performed on 10 patients and 10 normal controls. Basal and mitogen-stimulated cytokine production was determined in induced sputum T-cell culture. Supernatants were collected and assayed not only with specific ELISA but also with polymerase chain reaction (PCR) techniques. Data showed a significantly higher production of IL-10 by both the ELISA and the RT-PCR techniques in asthmatic patients compared with sputum mononuclear cells from healthy controls. IL-4 production was detected at a low level using the ELISA method in asthmatic patients. The RT-PCR analysis detected a significantly IL-4-mRNA expression in all asthmatic patients, compared with controls. Results of IL-10 and IL-4 mRNA expression were reproducible. We did not find any alteration in the expression of the type 1 derived cytokines (IL-2 and IFN-gamma) in asthmatic patients or in healthy controls. Our study showed a tendency of induced sputum mononuclear cells to express a Th2-like cytokine pattern in acute exacerbation of asthmatic patients, where IL-10 and IL-4 are synthesized in larger amounts. The combination of sputum induction as a non-invasive tool to explore the lung and the identification of disease-associated cytokine expression and of specific cytokine mRNA should help elucidate mechanisms of the immunologically mediated inflammatory responses in asthma.     

Cytodiagnosis of Hodgkin's disease in sputum specimens

In patients with histologically proven Hodgkin's disease, knowledge of the extent of involvement of lymph nodes and other organs has proven valuable in the determination of treatment and prognosis. One of the most common sites of involvement outside the hematopoietic system has been shown to be the lung parenchyma; in this study, six patients with a tissue-proven diagnosis of Hogdkin's disease and positive cytologic findings in the sputum were reviewed. Three cell types not found in normal sputum specimens were identified in these patients and were correlated with the histologic patterns of the tumors as seen in lung biopsies. Our results suggest the usefulness of sputum examination as an adjunctive or possibly a substitute diagnostic procedure in the evaluation of patients with Hodgkin's disease and possible lung involvement. They also suggest that in some cases the cytologic diagnosis can be quite specific in the identification of neoplastic cells as consistent with a diagnosis of Hodgkin's disease.     

Cell-cycle characteristics of undifferentiated and differentiating embryonal carcinoma cells

The cell-cycle parameters of undifferentiated and differentiating embryonal carcinoma cells were determined. Undifferentiated cultures of an F9 subclone, OTF9-63, had a 12-hr generation time, 10-hr S, and 2-hr G2 + M. G1 was less than 0.5 hr. In contrast, OTF9-63 cells induced to differentiate by treatment with retinoic acid had a 16.8-hr generation time, 12.5-hr S, 2-hr G2 + M, and 2.3-hr G1. Similar results were obtained with undifferentiated cultures and aggregation-induced differentiating cultures of PSA-1 cells. These data demonstrate that the undifferentiated stem cells have little or no G1, and that both G1 and S lengthen during differentiation.     

New microbiota found in sputum from patients with community-acquired pneumonia

Community-acquired pneumonia （CAP） is a major concern in hospitals and the bacterial community of which has not been systemically discussed yet. Sputum from patients in the acute stages is a kind of accessible sample reflecting its fea- tures. In our study, we analyzed 45 sputum samples from 45 patients with CAP. Eighteen sputum samples from healthy people were chosen as the controls. Pyrosequencing of the 16s rDNA V3 hypervariable regions of aH the bacteria con- tained in the sputum was used as a culture-independent method to disclose the community constitution. Also, our published data for hospital-acquired pneumonia （HAP） in sputum was used for comparison. By pyrosequencing, 〉90,000 DNA reads were detected. After being analyzed by tools in the Ribosomal Database Project, the reads were clas- sified into five main phyla and 〉100 genera. At the phyla level, the reads＇ distribution of CAP is similar to that of healthy people and at genera level, the occurrence of each genus possesses their feature in three categories. Genera such as Streptococcus and Neisseria showed stability in their percentages, indicating that such genera are rarely affected by exogenous bacteria or antibiotics. The role of other genera such as Moraxella and Rothia in CAP should be emphasized. According to our analysis, the bacterial communities of CAP are with slight change when compared with those of healthy people, but have a large gap between HAP. Meanwhile, Rothia might be an important endogenous pneumonia-causing factor.     

Genetically tagged putative differentiation intermediates derived from undifferentiated HT29 human colon carcinoma cells.

The HT29 colonic carcinoma cell line has proven to be a very practical tool for modelling aspects of colonic cell differentiation and toxification by chemotherapeutic agents. As an approach to subclone and clarify molecular events involved in sublineage maturation, non-differentiated HT29 cells were electroporated with a dominant marker gene (NeoR) to convey aminoglycoside resistance (G418R). Transfectants surviving passage in glucose-G418 medium were >200 times the abundance of transient G418R cells of controls. Genomic analysis showed that each clonal type was unique in NeoR integration pattern while mitochondrial DNA copy was relatively unchanged. All of the randomly generated NeoR clones resembled the parental phenotype, but some over-produced the mucin, secretory cell type or the cell death phenotype after culturing in 2 mM sodium butyrate medium. Re-exposure to glucose medium restored the parental-like phenotype.     

Glycopeptide in sputum from allergic asthma patients

The glycopeptide and glycosaminoglycan content of sputa from allergic asthma, bronchiectasis, and common cold patients was assayed. The glycopeptide content was higher in sputum from allergic asthma patients than that in bronchiectasis and common cold patients, while no significant difference in the glycosaminoglycan content was detected among these materials. Fractionation of the glycopeptide by DEAE-cellulose column chromatography yielded four glycopeptide fractions at concentrations of 0.05 to 0.3 M NaCl from the allergic asthma samples, whereas it yielded three fractions at concentrations of 0.05 to 0.2 M NaCl from the bronchiectasis and common cold samples. They were characterized by increases in sialic acid and sulfate as the molarity of NaCl increased. Hexose was the main component and hexosamine was the next in each fraction from all materials. The increase in sputum glycopeptide in the allergic asthma samples was due to a large increase in sialic acid- and sulfate-rich glycopeptide.     

"Tween egg medium" for isolating mycobacteria from sputum specimens.

'Tween egg medium,' in which Tween 80 was used as a carbon source, showed a higher rate of positive cultures than Ogawa egg medium in isolation of mycobacteria from sputum specimens.     

Interferon-gamma secretion and perforin expression are impaired in CD8+ T lymphocytes from patients with undifferentiated carcinoma of nasopharyngeal type

An efficent antitumor and antiviral cellular immune response requires optimal interferon-gamma (IFN-gamma) secretion and perforin expression in CD8(+) T cells. The aim of this study was to define whether CD4(+) and CD8(+) T cells from patients with undifferentiated carcinoma of nasopharyngeal type (UCNT), a tumor regularly associated with the Epstein-Barr virus (EBV), have abnormal phenotype profiles, cytokine production, perforin and CD3-zeta expressions. Our data showed that CD4 and CD8 subset distribution was not grossly altered in the peripheral blood of UCNT patients, while tumor biopsies contained an increased proportion of CD8(+) T cells. The analysis of the CD4(+) subset showed a defect in interleukin-2 (IL-2) production and a moderate increase of IL-10 production, a situation consistent with a Th1/Th2 imbalance. We have also demonstrated that CD8(+) lymphocytes from UCNT patients had a marked impairment of IFN-gamma secretion and perforin expression. This impairment was not related to the presence of detectable EBV DNA in the plasma. In UCNT patients, the blockade of the perforin pathway and of IFN-gamma production may constitute important mechanisms for immune escape by the tumor and for impaired control of EBV replication.     

Expression of ADAMs and their inhibitors in sputum from patients with asthma

ADAMs (a disintegrin and metalloprotease) constitute a family of cell surface proteins containing disintegrin and metalloprotease domains which associate features of adhesion molecules and proteases. ADAMTSs (a disintegrin and metalloprotease with thrombospondin motifs) bear thrombospondin type I motifs in C-terminal extremity, and most of them are secreted proteins. Because genetic studies have shown that ADAM-33 gene polymorphisms are associated with asthma, we designed this study to assess mRNA expression profile of several ADAM and ADAMTS proteases in sputum from patients with asthma and to investigate the relationship between expression of these proteases and asthma-associated inflammation and airway obstruction. mRNA expression profile of selected ADAM and ADAMTS proteinases (ADAM-8, -9, -10, -12, -15, -17, and -33; ADAMTS-1, -2, -15, -16, -17, -18, and -19), their physiological inhibitors TIMP-1 and TIMP-3, and RECK, a membrane-anchored MMP activity regulator, was obtained by RT-PCR analysis performed on cells collected by sputum induction from 21 patients with mild to moderate asthma and 17 healthy individuals. mRNA levels of ADAM-8, ADAM-9, ADAM-12, TIMP-1, and TIMP-3 were significantly increased, whereas mRNA levels coding for ADAMTS-1, ADAMTS-15, and RECK were significantly decreased in patients with asthma compared with control patients. ADAM-8 expression was negatively correlated with the forced expiratory volume at the first second (FEV(1)) (r = -0.57, P < 0.01), whereas ADAMTS-1 and RECK expressions were positively correlated to FEV(1) (r = 0.45, P < 0.05, and r = 0.55, P = 0.01, respectively). We conclude that expression of ADAMs and ADAMTSs and their inhibitors is modulated in airways from patients with asthma and that these molecules may play a role in the pathogenesis of asthma.     

MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation

Background

Irreversible airflow obstruction in Chronic Obstructive Pulmonary Disease (COPD) is thought to result from airway remodelling associated with aberrant inflammation. Patients who experience frequent episodes of acute deterioration in symptoms and lung function, termed exacerbations, experience a faster decline in their lung function, and thus over time greater disease severity However the mechanisms by which these episodes may contribute to decreased lung function are poorly understood.This study has prospectively examined changes in sputum levels of inflammatory cells, MMP-9 and TIMP-1 during exacerbations comparing with paired samples taken prior to exacerbation.

Methods

Nineteen COPD patients ((median, [IQR]) age 69 [63 to 74], forced expiratory volume in one second (FEV1) 1.0 [0.9 to1.2], FEV1% predicted 37.6 [27.3 to 46.2]) provided sputa at exacerbation. Of these, 12 were paired with a samples collected when the patient was stable, a median 4 months [2 to 8 months] beforehand.

Results

MMP-9 levels increased from 10.5 μg/g [1.2 to 21.1] prior to exacerbation to 17.1 μg/g [9.3 to 48.7] during exacerbation (P < 0.01). TIMP-1 levels decreased from 3.5 μg/g [0.6 to 7.8] to 1.5 μg/g [0.3 to 4.9] (P = 0.16). MMP-9/TIMP-1 Molar ratio significantly increased from 0.6 [0.2 to 1.1] to 3.6 [2.0 to 25.3] (P < 0.05). Neutrophil, eosinophil and lymphocyte counts all showed significant increase during exacerbation compared to before (P < 0.05). Macrophage numbers remained level. MMP-9 levels during exacerbation showed highly significant correlation with both neutrophil and lymphocyte counts (Rho = 0.7, P < 0.01).

Conclusion

During exacerbation, increased inflammatory burden coincides with an imbalance of the proteinase MMP-9 and its cognate inhibitor TIMP-1. This may suggest a pathway connecting frequent exacerbations with lung function decline.     

Serum CYFRA21-1 as a prognostic marker for patients with undifferentiated nasopharyngeal carcinoma

The purpose of this study was to evaluate the potential of serum CYFRA21-1 and carcinoembryonic antigen (CEA) as prognostic markers in patients with undifferentiated nasopharyngeal carcinoma. Sixty-one patients who received definitive radiotherapy/chemoradiotherapy were analysed retrospectively. We investigated the association of the follow-up results with pretreatment level, post-treatment level and change of serum CYFRA21-1 and CEA, respectively. Patients with low pretreatment CYFRA21-1 had a significantly better overall survival. There were no significant associations among the remaining serum markers, and the survival and recurrence rates on multivariate analysis. The present study shows that pretreatment CYFRA21-1 level is a potential factor for predicting long-term survival.     

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.