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Mouse spleen cells sequentially agglutinated by soybean agglutinin (SBA) and peanut agglutinin (PNA) previously had been shown to be sufficiently depleted of graft-versus-host activity to allow reconstitution of lethally irradiated allogeneic recipient mice. We have now tested the extent of T-cell depletion in this cell fraction by various in vitro assays, including cytotoxicity testing with anti-Thy-1 antiserum, mitogenic response to the T-cell mitogens concanavalin A and phytohemagglutinin and allogeneic responsiveness in the mixed lymphocytes culture assay. By these criteria the SBA+, PNA+ spleen fraction, used previously in the in vivo experiments, was found to possess about 1% T-cell contamination. The slight contamination with T cells previously found in the singly agglutinated SBA fraction can be removed by a second fractionation with SBA, thus eliminating the possibility that a minor T-cell subpopulation bears receptors for SBA. Finally, we demonstrated that the twice-agglutinated fraction, by SBA and PNA or by SBA alone, contains a significant number of prothymocytes, thereby indicating that mouse prothymocytes bear receptors for both SBA and PNA. The implication of these findings to bone marrow transplantation in humans is discussed.
Tumor cell fractions isolated from tumor lines SH-3 (breast carcinoma) and RPMI-7932 (malignant melanoma) by differential centrifugations were capable of transforming lymphocytes into cytotoxic effector cells. Lymphocytes cultured alone in human AB plasma did not become cytotoxic to tumor cells. However, when cultured with tumor cell fractions sedimented at 1000 X g(R1), 20,000 X g(R2), and 100,000 X g(R3), these lymphocytes became markedly cytotoxic to specific tumor targets in a 3.5-hr (51)Cr release assay. R2 fractions were significantly more immunogenic than were R3 fractions (p less than 0.05). Although lymphocytes sensitized with SH-3 tumor cell fractions were cytotoxic to SH-3 tumor cells, they were also cytotoxic to cells from RPMI-7932 and RPMI-8322 (malignant melanoma) tumor lines and vice versa. Cells from tumor lines HT-29 (colon carcinoma) and COLO 110 (ovary carcinoma) were significantly less susceptible to lysis by effector cells generated against SH-3. These immune cells, although capable of killing cells from tumor lines, were not able to lyse cells from autochthonous normal lymphoid lines or normal lymphocytes that have been transformed by phytohemagglutinin. Tumor cell fractions were not immunogenic at low (5- to 20-mul/0.75 ml) concentrations; an increase of 4- to 10- fold in their concentrations was usually followed by a decrease in immunization.
The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.
Undifferentiated human lymphoblasts (culture LS-2) were separated according to cell size during their exponential growth phase by way of centrifugal elutriation. The cell fractions thus obtained were characterized in terms of different cell cycle stages by flow cytometric measurement of their deoxyribonucleic acid (DNA histogram), the [3H]thymidine labeling index, and by determining the rate of [3H]thymidine incorporation. In these cell fractions the activities of thymidine kinase, thymidylate synthase, DNA polymerase, dihydrofolate reductase, methionine synthase, and hexokinase were determined. The results showed that all the enzymes investigated exhibited activities in all cell fractions. With the exception of DNA polymerase, all of the enzymes exhibited the lowest level of activity in the fraction containing the highest proportion of G0 + G1 phase cells (fraction 2); the activity of thymidine kinase was particularly low. This would suggest that thymidine kinase is not active in G0 + G1 phase cells and that the activity measured in fraction 2 is perhaps attributable to contamination of this fraction by S and G2 + M phase cells.
BACKGROUND: The thymidine labeling index (T-LI) evaluated with the autoradiographic technique on fresh specimens is the most objective and specific marker of tumor proliferative cell fraction. In this study we investigated T-LI and PCNA expression in 68 cases of transitional cell bladder carcinomas in order to evaluate whether PCNA immunostaining in paraffin sections could be a valid alternative to autoradiographic methods for cell kinetic detection. Immunohistochemistry was applied after different fixatives: usual formalin, neutral 4% buffered formalin and Bouin. The results revealed that only in buffered formalin-fixed tumor specimens, considering the most darkly stained cells, the PCNA-LI in all cases overlapped with T-LI. Higher values than those observed with autoradiography were detected in PCNA values obtained after usual formalin fixation whereas no or faint PCNA staining was seen in material fixed in Bouin. Thus, only cells with strong PCNA staining can be considered as significant in evaluating the proliferative activity in transitional cell bladder carcinoma. By using immunohistochemical methods it should thus be possible to overcome many of the inherent difficulties of autoradiography and likewise to obtain accurate and significant information on the biological aggressiveness of bladder tumors and consequently of patient prognosis.
A procedure was developed for the isolation from human bone marrow of a cell fraction enriched for haematopoietic precursors and depleted of T lymphocytes. T cells are eliminated from bone marrow by rosetting with sheep red blood cells, followed by differential agglutination of residual T lymphocytes in the non-rosetting population by the lectin, soybean agglutinin. The fraction unagglutinated by the lectin contains a high proportion of colony-forming cells and non detectable T cell alloreactivity in vitro. Similar results were obtained with monkey bone-marrow cells, suggesting that monkeys can be used for evaluation of this fractionation technique for bone-marrow transplantation across histocompatibility barriers.
Monodispersed cells from anterior pituitaries of male rats were prepared by Pronasedissociation and incubated with [3H]progesterone or [3H]testosterone. The cells were then separated into enriched fractions consisting of gonadotropic, somatotropic or chromophobic cells by velocity sedimentation at unit gravity for 4 h. The uptake of [3H]progesterone and [3H]testosterone by the gonadotropic enriched cell fraction was 1.8 to 3.2 times greater than the somatotropic and chromophobic enriched cell fractions. The gonadotropic and chromophobic enriched cell fractions metabolized [3H]progesterone and [3H]testosterone appreciably. The principal metabolites were identified and quantitated by reverse isotopic dilution. After incubation with [3H]progesterone, the principal metabolite was [3H]5alpha-dihydroprogesterone which ranged from 11.5% for the gonadotropic cells to 7.6% for the chromophobic cells. Smaller amounts of 3alpha-hydroxy-5alpha-pregnan-20-one (3.7 to 4.8%) and 20alpha-dihydroprogesterone (2.1 to 4.3%) were also identified. After incubation with [3H]testosterone, the principal metabolite was 5alpha-dihydrotestosterone which ranged from 12.6% for the gonadotropic cells to 10.3% for the chromophobic cells. Smaller amounts of 5alpha-androstane-3alpha, 17beta-diol (4.1 to 5.5%) and androstenedione (1.8 to 3.0%) were identified. After incubation with [3H]progesterone or [3H]testosterone the same metabolites were also identified in the somatotropic cell fraction but were thought to be present because of contamination with gonadotropic cells. Dissociated pituitary cells from orchidectomized rats had a 2-fold increase in the uptake of [3H]testosterone and a greater metabolism of [3H]testosterone to 5alpha-dihydrotestosterone as compared to pituitary cells from intact rats (12.6 vs 25.7%).
A method has been devised to fractionate cells of Tetrahymena pyriformis, yielding pure or highly enriched preparations of cilia, cilia-associated soluble material, pellicles, mitochondria, microsomes, and postmicrosomal supernatant. The method prevents the destructive action of lipolytic enzymes commonly associated with this organism. Analysis of the membrane lipids of these fractions reveals significant differences in lipid composition. Most noteworthy are the high concentrations of phosphonolipid and tetrahymanol in the surface membranes.
In the present study we have isolated and purified fractions of nonparenchymal liver cells were isolated by collagenase-pronase digestion of the biliary and connective hepatic tissue, which remained undissociated after collagenase perfusion of the liver. Fractionation of the nonparenchymal fractions was then achieved by centrifugal elutriation. Both normal rats and rats with proliferated bile duct-like structures, which were induced either by a 14-day bile duct ligation or by feeding 0.1% alpha-naphthylisothiocyanate for 28 days, were used in these studies. Using a normal rat liver, the fraction richest in biliary epithelial cells was that obtained at a pump flow rate of 36-40 ml/min. In this fraction 1.8-3.8 x 10(6) cells per liver were recovered and up to 55% of them were positive for gamma-glutamyl transpeptidase and cytokeratins 7 and 19, all of which were histochemically or immunohistochemically detected solely in the biliary structures in the intact rat liver. When the nonparenchymal cells were isolated from hyperplastic livers, the number of cells recovered in such a fraction ranged from 12 to 19 x 10(6) per liver, and as many as 60%-85% of the cells expressed phenotypes of biliary epithelial cells. These results indicate that (a) by centrifugal elutriation a fraction of nonparenchymal cells enriched in cells with biliary epithelial phenotypes can be obtained from rat liver and (b) the hepatic hyperplasia induced by biliary obstruction or alpha- naphthylisothiocyanate feeding is a useful and valid strategy for improving both the yield and the purity of the isolated biliary epithelial cells.
High-dose chemotherapy with peripheral blood stem cell (PBSC) transplantation in advanced germ cell tumour (GCT) patients is widely applied. The aims of this study were: (1) To examine the presence of alphafetoprotein (AFP) bearing tumour cells in PBSC harvests from advanced GCT patients obtained after multiple cycles of induction chemotherapy. (2) To determine whether induction chemotherapy contributed to in vivo purging of the tumour. We evaluated cryopreserved PBSC samples from 5 patients with advanced stage II/III AFP producing GCT. PBSC were separated after the first, second and third cycles of induction chemotherapy. Those samples were analysed using the nested reverse transcription polymerase chain reaction (RT-PCR) method to detect AFP mRNA. Although, in all patients, AFP mRNA was detected in PBSC samples after the first or second cycle of induction chemotherapy, but was not detected in 3 of 4 samples after the third cycle of chemotherapy. Although it is not clear whether tumour cells contaminating PBSC fraction contribute to disease relapse, PBSC harvested after at least 3 cycles of induction chemotherapy might be recommended to avoid such a possibility.
Tumor clonogenic cell content is believed to play an important role in the outcome of radiotherapy. However, there is no proven method to assess the number of clonogens in human tumors accurately. All currently available assays employ in vitro plating efficiency or in vivo TD50 (the average number of cells needed to induce tumors in 50% of injected mice) to estimate the tumor clonogenic ability. In this study, a monolayer mass primary culture system was used to estimate the clonogenic cell fraction in human tumors. For this purpose, 25 growth curves were performed for 25 tumor specimens derived from 21 head and neck and 4 cervical squamous cell carcinomas. The exponential portion of each growth curve was extrapolated through the ordinate (day 0) to estimate the clonogenic cell fraction; this method is only an estimate because it assumes no lag phase before exponential growth of clonogenic cells. The mean clonogenic cell fraction, expressed as clonogens/tumor cells inoculated, was relatively low (mean: 0.71%, range: 0.11-9.28), and the variation was wide (coefficient of variation = 148%). On the other hand, the doubling time of the growing population was 1.46 days and exhibited a very narrow range (0.98-2.24, coefficient of variation = 24%). The mean and range of clonogenic cell fraction were found to be in agreement with published values of soft agar colony forming efficiencies in both murine and human tumors. However, further investigation is necessary to determine how accurately this method measures the relative clonogenic cell content in human tumors. Clinical correlations between clonogenic cell fraction values and the response to radiotherapy are still too early to determine.
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Despite a focused effort within the myogenic cell transplantation community, little progress has been made toward the reliable identification and isolation of progenitors that are capable of tolerating the initial posttransplantation environment and effectively regenerating clinically relevant quantities of muscle. The future success of myogenic-based treatment modalities requires an enhanced understanding of the highly heterogeneous nature of the myogenic progenitor cell pool, which has been previously documented by numerous researchers. Further, for translation of experimental animal results to clinical application, reliable in vitro selection criteria must be established and must be translatable across species. While research into the utility of surface markers is ongoing, as an alternative we have investigated in vitro cell behavioral characteristics under imposed conditions which challenge the propensity of myogenic progenitors to choose between various cell fates (i.e., proliferation, quiescence, or differentiation). Previous observations in the mouse suggest an enhanced in vivo regenerative capacity of myogenic populations with respect to their in vitro ability to maintain a proliferative and undifferentiated state [J. Cell Sci. 115 (2002) 4361]. From these observations it is thus proposed that such behavior may represent an a priori indicator of regenerative capacity following transplantation. To challenge this proposition, a rat cell isolation and transplantation model was evaluated in an identical manner. In agreement with the results obtained from the mouse, a significant correlation between regenerative capacity and induction of differentiation was observed. These results contribute to the growing body of scientific evidence documenting the underlying behavioral differences that exist between various myogenic progenitors while also, importantly, providing evidence that such differences may significantly impact the functional capabilities of these cells posttransplantation. This information further implies that from a therapeutic standpoint isolation strategies aimed toward obtaining efficient myogenic progenitors should, in the absence of a reliable surface marker(s), focus on identifying populations displaying desirable in vitro behavior (i.e., high proliferative capacity and low induced differentiation). Incorporating such criteria into cell isolation and/or purification schemes may yield significant returns in the clinical myogenic transplantation setting.
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Bronchial epithelial cells isolated by protease digestion can be cultured in vitro for the study of proliferation and differentiation. However, these cells represent a heterogenous population, the components of which likely interact with one another. We attempted to utilize density gradient centrifugation as a method to prepare subpopulations of these bronchial epithelial cells. The suspension of the cells obtained by protease digestion of the bovine bronchi was mixed with an equal volume of colloidal silica reagent, Sepracell-MN, and centrifuged to form a continuous density gradient. Two distinct cell layers were identified in addition to a cell pellet at the bottom. Cells from fraction A (top layer) were more than 95% ciliated cells by morphologic examination. These ciliated cells were recovered intact as assessed by trypan blue dye exclusion and by watching beating of their cilia. The cells from fraction C (bottom layer) were 89.9 +/- 3.88% nonciliated, small round cells with a densely staining nucleus and scant cytoplasm. Comparison of cell morphology of these cells with basal cells in vivo and electron microscopic examinations suggested that these cells were basal cells. These basal cells showed an exponential cell proliferation until confluence in Ham's F12 with supplements, LHC9, and a 1:1 mixture of Medium-199 and modified Eagle's medium with 2% fetal calf serum. In contrast, the cells from fraction A grew minimally in all conditions tested. This difference was also shown in the study of DNA synthesis by [3H]thymidine uptake. Enzyme-linked immunosorbent assay for release of bovine fibronectin into cultured media indicated that fraction C cells secreted much more fibronectin (532 +/- 5.28 ng/10(6) cells/h) than fraction A cells (73.4 +/- 1.00). We also used Percoll as a density-gradient reagent and showed potential usefulness in the preparation of cell fractions of bronchial epithelial cells. In conclusion, it was possible to separate ciliated and nonciliated, presumably basal, cells of bovine bronchial epithelial cells. These differed in growth and fibronectin secretion. Studies of airway cell biology may be aided by the availability of more homogenous cell populations.
BACKGROUND: The molecular mechanics of inclusion body formation is still far from being completely understood, specially regarding the occurrence of properly folded, protein species that exhibit natural biological activities. We have here comparatively explored thermally promoted, in vivo protein aggregation and the formation of bacterial inclusion bodies, from both structural and functional sides. Also, the status of the soluble and insoluble protein versions in both aggregation systems have been examined as well as the role of the main molecular chaperones GroEL and DnaK in the conformational quality of the target polypeptide. RESULTS: While thermal denaturation results in the formation of heterogeneous aggregates that are rather stable in composition, protein deposition as inclusion bodies renders homogenous but strongly evolving structures, which are progressively enriched in the main protein species while gaining native-like structure. Although both type of aggregates display common features, inclusion body formation but not thermal-induced aggregation involves deposition of functional polypeptides that confer biological activity to such particles, at expenses of the average conformational quality of the protein population remaining in the soluble cell fraction. In absence of DnaK, however, the activity and conformational nativeness of inclusion body proteins are dramatically impaired while the soluble protein version gains specific activity. CONCLUSION: The chaperone DnaK controls the fractioning of active protein between soluble and insoluble cell fractions in inclusion body-forming cells but not during thermally-driven protein aggregation. This cell protein, probably through diverse activities, is responsible for the occurrence and enrichment in inclusion bodies of native-like, functional polypeptides, that are much less represented in other kind of protein aggregates.