MAC (morphology-antibody-chromosome). Un nuevo método para el estudio de las neoplasias hematológicas / MAC (morphology-antibody-chromosome). A new method from study of the hematology neoplasic

Los estudios realizados en el campo de la citogenética del cáncer han permitido determinar que las alteraciones cariotípicas de las células neoplásicas se encuentran distribuidas en el genoma en forma específica, encontrándose cromosomas particularmente asociados a diferentes tipos de neoplasias (1)(2). En lo que respecta a las neoplasias hematológicas, el análisis citogenético ha demostrado ser de gran importancia para la evaluación del diagnóstico, pronóstico y tratamiento de las mismas (3)(4). Los avances logrados en la citogenética de neoplasias han sido posibles debido al desarrollo y mejoramiento de distintas técnicas, que permiten la obtención de extendidos cromosómicos de alta calidad, donde es posible detectar alteraciones muy pequeñas del material genético. Sin embargo, las técnicas tradicionales de citogenética llevan a la destrucción de la membrana plasmática y del contenido citoplasmático. Si bien esto permite una mejor extensión y dispersión de los cromosomas, impide, por otro lado, la identificación de las células cuyos cariotipos son analizados. Esto último es de particular importancia en el caso de la médula ósea, donde existen numerosas progenies celulares capaces de originar mitosis para el análisis cromosómico. siendo imposible a través de las técnicas convencionales de citogenética, poder determinar si las anomalías cromosómicas observadas se encuentran o no restringidas a aquellas que muestran clonalidad. Actualmente es posible estudiar poblaciones celulares presentes en sangre periférica y médula ósea, a través de diferentes técnicas de inmunohistoquímica que, en los últimos años se han perfeccionado notablemente con el desarrollo de los anticuerpos monoclonales y los métodos enzimáticos de inmunomarcación. Los estudios previos realizados para determinar el origen de las células hematopoyéticas cariotípicamente anormales, se basaron en análisis morfológicos y citogenéticos separados de la misma muestra, o en la creación de poblaciones hemogéneas, a partir de células aisladas o cultivos de células progenitoras. Los primeros intentos de identificación directa de células mitóticas fueron hechos por Rastrick et al (5), usando el cromosoma Philadelphia (PH1) como un marcador de células leucémicas y la incorporación de hierro radiactivo como un indicador de precursores eritroides, en un paciente con leucemia mieloide crónica (LMC)> Blocstock y Garson (6) usaron la misma técnica en células de médula ósea, de pacientes con leucemia mieloide

MAC (morphology-antibody-chromosome). Un nuevo método para el estudio de las neoplasias hematológicas / MAC (morphology-antibody-chromosome). A new method from study of the hematology neoplasic

Los estudios realizados en el campo de la citogenética del cáncer han permitido determinar que las alteraciones cariotípicas de las células neoplásicas se encuentran distribuidas en el genoma en forma específica, encontrándose cromosomas particularmente asociados a diferentes tipos de neoplasias (1)(2). En lo que respecta a las neoplasias hematológicas, el análisis citogenético ha demostrado ser de gran importancia para la evaluación del diagnóstico, pronóstico y tratamiento de las mismas (3)(4). Los avances logrados en la citogenética de neoplasias han sido posibles debido al desarrollo y mejoramiento de distintas técnicas, que permiten la obtención de extendidos cromosómicos de alta calidad, donde es posible detectar alteraciones muy pequeñas del material genético. Sin embargo, las técnicas tradicionales de citogenética llevan a la destrucción de la membrana plasmática y del contenido citoplasmático. Si bien esto permite una mejor extensión y dispersión de los cromosomas, impide, por otro lado, la identificación de las células cuyos cariotipos son analizados. Esto último es de particular importancia en el caso de la médula ósea, donde existen numerosas progenies celulares capaces de originar mitosis para el análisis cromosómico. siendo imposible a través de las técnicas convencionales de citogenética, poder determinar si las anomalías cromosómicas observadas se encuentran o no restringidas a aquellas que muestran clonalidad. Actualmente es posible estudiar poblaciones celulares presentes en sangre periférica y médula ósea, a través de diferentes técnicas de inmunohistoquímica que, en los últimos años se han perfeccionado notablemente con el desarrollo de los anticuerpos monoclonales y los métodos enzimáticos de inmunomarcación. Los estudios previos realizados para determinar el origen de las células hematopoyéticas cariotípicamente anormales, se basaron en análisis morfológicos y citogenéticos separados de la misma muestra, o en la creación de poblaciones hemogéneas, a partir de células aisladas o cultivos de células progenitoras. Los primeros intentos de identificación directa de células mitóticas fueron hechos por Rastrick et al (5), usando el cromosoma Philadelphia (PH1) como un marcador de células leucémicas y la incorporación de hierro radiactivo como un indicador de precursores eritroides, en un paciente con leucemia mieloide crónica (LMC)> Blocstock y Garson (6) usaron la misma técnica en células de médula ósea, de pacientes con leucemia mieloide

Characterization, in some human breast cancer cell lines, of gastrin-releasing peptide-like receptors which are absent in normal breast epithelial cells.

The binding of 125I-Tyr4 bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinity, single-site class of receptors in 3 cell lines (KD of 0.75 x 10(-9) and 10(-9) M, Bmax of 0.75 x 10(-13) and 9.7 x 10(-13) M/mg protein in MDA-MB231 and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analogues (natural or synthetic) inhibited the binding of 125I-Tyr4 bombesin in the following order of potency: gastrin-releasing peptide (GRP, EC50 = 1.7 x 10(-10) M) greater than BIM 26159 greater than bombesin, Tyr4 bombesin greater than BIM 26147 greater than litorin greater than neuromedin C. In contrast, 125I-Tyr4 bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex 125I-Tyr4 bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role.

[The recording of human fibroblast metabolism in vitro by the chromatographic-mass spectrometric determination of the metabolites excreted]. / Registratsiia metabolizma fibroblastov cheloveka in vitro putem khromato-mass-spektrometricheskogo opredeleniia ékskretiruemykh imi metabolitov.

Metabolites excreted into culture medium by human skin fibroblasts have been studied by high resolution gas chromatography and mass spectrometry. Parameters for 29 metabolites have been obtained and 11 of them have been identified. Excreted metabolites reflect activity of certain metabolic processes in fibroblasts. Comparison of chromatographic and mass spectrometric parameters of cellular metabolites with the metabolites excreted with urine revealed that most metabolites excreted from fibroblasts differ from urine metabolites. The possibility for secondary transformation of cell metabolites in organism and specificity of metabolism in different tissues has been discussed.

T-cell activation in Crohn's disease. Increased levels of soluble interleukin-2 receptor in serum and in supernatants of stimulated peripheral blood mononuclear cells.

Serum levels of soluble interleukin-2 receptor were determined in 29 patients with active and quiescent Crohn's disease. In addition, the ability of peripheral blood mononuclear cells of 23 of these patients to generate soluble interleukin-2 receptor following mitogenic stimulation was studied in vitro. Serum soluble interleukin-2 receptor concentrations of patients with active Crohn's disease (n = 19) were significantly elevated (757 +/- 438 U/ml) compared with levels in patients with inactive disease (n = 10; 412 +/- 120 U/ml) and healthy control individuals (n = 40; 375 +/- 102 U/ml; p less than 0.003 and p less than 0.0005, respectively). Serial determinations of serum soluble interleukin-2 receptor concentration in a follow-up of 11 hospitalized patients treated for highly active disease showed a decrease from 1252 +/- 494 U/ml to 527 +/- 193 U/ml (p less than 0.004) that corresponded to clinical improvement, as assessed by Crohn's disease activity index and a reduction of inflammatory parameters. In vitro phytohemagglutinin stimulation of peripheral blood mononuclear cells derived from patients with Crohn's disease resulted in elevated soluble interleukin-2 receptor production not only in patients with active disease (3987 +/- 2439 U/ml), but also in patients with inactive disease (3297 +/- 2282 U/ml), compared with the amount of soluble interleukin-2 receptor produced by mononuclear cells of healthy individuals (1523 +/- 1152 U/ml; p less than 0.005 and p less than 0.02, respectively). In addition, cultivation of mononuclear cells without mitogen resulted in higher soluble interleukin-2 receptor production in patients with active disease than in patients with inactive disease (p less than 0.02). However, patients suffering from active ulcerative colitis also had significantly increased serum levels of soluble interleukin-2 receptor (1080 +/- 400 U/ml) compared with the levels in patients with chronic disease (455 +/- 140 U/ml; p less than 0.0025). In addition, peripheral blood mononuclear cells derived from patients with ulcerative colitis produced significantly more soluble interleukin-2 receptor upon mitogenic stimulation with phytohemagglutinin (2314 +/- 936 U/ml), than cells from healthy controls (1523 +/- 1152 U/ml; p less than 0.05). The finding of elevated soluble interleukin-2 receptor serum levels in patients with active Crohn's disease and its increased production by mononuclear cells of patients with both active and inactive disease is a further example of an alteration of the immune system in this condition; however, this alteration can also be found in other inflammatory bowel diseases.

A reversible multi-well chamber for incubation of cultured cells with small volumes: application to screening of hybridoma fusions using immunofluorescence microscopy.

Immunofluorescence microscopy is a powerful technique for detecting the location of surface and intracellular antigens in individual cells. However, using standard methods, processing large numbers of samples for immunofluorescence is cumbersome and difficult. To simplify greatly this process, we have developed a chamber that reversibly creates multiple small wells in a large (150 mm) tissue culture dish. This device allows the rapid and convenient processing of hundreds of samples each of 100 microliters volume. Each sample is examined using a short working distance, high numerical aperture immersion objective for maximum sensitivity and resolution. This apparatus makes immunofluorescence a practical method for the primary screening of hybridoma clones.

The effects of thrombin on bovine aortic endothelial and smooth muscle cells.

Recent evidence suggests that thrombin interacts with various cell types, stimulating cellular proliferation and protein and prostanoid production. To further delineate its role in vascular healing, we have studied the effects of thrombin on proliferation and matrix production by the cells of the vessel wall. The addition of thrombin (1 unit/ml) to cultures of bovine aortic smooth muscle cells resulted in an increase in cell proliferation (p less than 0.01) and number (p less than 0.03), whereas in cultures of bovine aortic endothelial cells thrombin produced a decrease in cell proliferation (p less than 0.01) and number (p less than 0.02). Thrombin also altered matrix composition in cultures of these cells. In both bovine aortic endothelial cells and bovine aortic smooth muscle cell cultures grown in the presence of thrombin, total protein content was significantly increased when compared to controls (p less than 0.03). In bovine aortic endothelial cell cultures the addition of thrombin resulted in a decrease in collagen content (p less than 0.01) and an increase in sulfated glycosaminoglycan content (p less than 0.02). In contrast, in bovine aortic smooth muscle cell cultures thrombin resulted in an increase in collagen content (p less than 0.03), whereas glycosaminoglycan content was unaffected. These findings suggest that thrombin may significantly influence vascular healing and function by altering cell number and matrix composition.

[The effect of inhibitors of ADP ribosylation on the restoration of the mitotic cycle and on DNA repair after the action of ionizing radiation in Chinese hamster cells]. / Vliianie ingibitorov ADF-ribozilirovaniia na vosstanovlenie mitoticheskogo tsikla i na reparatsiiu DNK posle deistviia ioniziruiushchei radiatsii v kletkakh kitaiskogo khomiachka.

Specific inhibitors of poly(ADP-ribose)polymerase-3-aminobenzamide and 3-metoxybenzamide (6, 12 mM) have been shown to: 1) reduce survival of X-irradiated CHO K1 cells to a slight degree; 2) increase S- and particularly G2-delays in X-irradiated cells, while progressing through the cell cycle as analysed by the DNA flow cytofluorimetry; 3) reduce effectiveness of DNA single-strand breaks repair. The above data suggest a definite role of ADP ribosylation in the cell repair activity.

Enzymatic quantification of sphingosine in the picomole range in cultured cells.

An enzymatic method to quantify the mass levels of free sphingosine in cellular lipid extracts was developed. The assay is based upon the observation that ceramide is phosphorylated by Escherichia coli diacylglycerol kinase. Although sphingosine is not recognized by the enzyme, it can be converted to a substrate by acylation with hexanoic anhydride. Using a mixed micellar assay, previously reported for the mass quantification of diacylglycerol, the short-chain ceramide (N-C6-sphingosine), generated by acylation, is quantitatively phosphorylated to N-C6-[32P]sphingosine phosphate. This assay allows quantification of sphingosine over a broad range from 25 to 5000 pmol. When this assay was applied to standard compounds, reverse-phase thin-layer chromatography of the reaction products was adequate to separate the phosphorylated derivatives of long-chain ceramide and N-C6-sphingosine. However, the presence of other lipids in extracts from biological samples (mainly monoalkylglycerols which are also a substrate for the diacylglycerol kinase) interfered and necessitated an additional purification step. The most efficient purification step devised was a combination of anion- and cation-exchange chromatography. The mass levels of free sphingoid bases in different cultured cells were quantified using this assay. Levels varied between 8 to 20 pmol/10(6) cells. When normalized to phospholipids, sphingosine levels varied between 0.01 and 0.04 mol%. The lowest levels were found in L929 cells, while Schwann cells derived from Twitcher mice contained the highest levels. These levels were significantly higher than those of Schwann cells derived from normal mice.

Incorporation of contrast media in cultured cells.

Monolayer cultures of human prostatic (PC-3) and cervical (NHIK 3025) carcinoma cells were grown on formvar film and exposed to moderate concentrations of contrast agents for 30 minutes to 4 hours. After the exposure period, the monolayers were quickly frozen, and cryosections were examined by electron microscopy and X-ray microanalysis. Iodine was not detected in control cells, but was found in the cells that had been exposed to iodine-containing contrast media. The amount of intracellular iodine increased with increasing exposure dose and time. Because the cells mostly presented no sign of membrane damage, our findings support the view that contrast media have the ability to enter intact cells.

Desmin distinguishes cultured fat-storing cells from myofibroblasts, smooth muscle cells and fibroblasts in the rat.

To differentiate cultured rat liver myofibroblasts, fat-storing cells, aortic smooth muscle cells and skin fibroblasts from each other, desmin and vimentin stainings were undertaken by indirect immunofluorescence using monoclonal antibodies. In myofibroblasts, the reaction with antibodies to vimentin was positive but that with antibodies to desmin was virtually negative. In primary cultures as well as subsequent passage of fat-storing cells, reactions with antibodies to both desmin and vimentin were positive. In primary culture of smooth muscle cells, both reactions were positive, but in the first passage, smooth muscle cells lost the reactivity with antibodies to desmin. Fibroblasts showed a positive reaction with antibodies to vimentin and a negative one with antibodies to desmin. Thus, immunohistochemistry of intermediate filaments allows for the differentiation between fat-storing cells, which are desmin- and vimentin-positive, and myofibroblasts or fibroblasts, which are desmin-negative but vimentin-positive. Smooth muscle cells are also vimentin-positive and become desmin-negative after the first passage.

Purification of proteins from cell culture supernatants.

Desired proteins excreted by animal cells usually reach rather low concentrations in the culture supernatant and have to be purified from an excess of serum proteins which are added to the animal cell culture to maintain its viability and/or productivity. Defined media offer among others the advantage of an easier purification procedure. In addition lysis of cells may contribute also to the complexity of the protein mixture encountered. If fetal calf serum or new born calf serum is used in cultivation, bovine serum albumin and globulins will be the most abundant protein in the supernatant. The interaction of albumin especially with hydrophobic proteins represents significant problems for the effective purification of minor constituent. Isolation of a desired protein follows the general scheme: concentration: by precipitation, ultrafiltration, batch adsorption or partition in aqueous phase system; enrichment: by chromatography or partition; high resolution purification: by chromatography and/or immuno adsorption; final concentration and finishing: pyrogen removal, sterilization, formulation. Biochemical engineering aspects of proteins purification are discussed using human interferon-beta produced in a recombinant mouse cell line as an example. The process developed encloses extraction of interferon-beta from the production medium in aqueous two-phase systems and subsequent recovery of interferon-beta from the top phase by high pressure liquid chromatography.

A convenient system for the serial quantitation of viable cells measuring luminescence of ATP.

Measurement of intracellular ATP using luminescence offers an alternative method for the routine determination of viable animal cells in a large number of cultures. Optimal conditions of the new technology are described as is its application in various test systems.

A rapid and sensitive method for the detection of mycoplasmas in infected cell cultures using 6-methyl purine deoxyriboside.

6MPDR causes the death of cells lightly infected with mycoplasmas. This is brought about by the action of the mycoplasmal enzyme adenosine phosphorylase which converts 6MPDR to highly toxic metabolites. If the medium conditions are adjusted to favour the growth of mycoplasmas by the addition of pig serum to the medium, infections as low as 1 mycoplasma per 200,000 cells can be detected within 7 days. This finding enables mycoplasma tests to be carried out rapidly and reliably by untrained personnel.

Double-beam autocompensation for fluorescence polarization measurements in flow cytometry.

The degree of depolarization of fluorescent light emitted from an organic dye, which is used as molecular probe, is a powerful tool in probing the microenvironment. By fluorescence depolarization the macromolecular structure can be investigated as well as the the mobility of the marker molecule itself or of the complex formed by the probe. Additional information such as energy transfer rates, donor-acceptor distances, and orientations are also measurable. These data are of particular interest if they can be measured from whole cells. Using flow cytometry, we can analyze a large number of cells with high statistical significance in a short period of time. We describe a newly developed double-beam epi-illumination arrangement for fluorescence polarization measurements that uses an autocompensation technique. This new technique permits the various depolarizing effects within the optical as well as the electronic components of the system to be continually compensated for on a cell by cell basis. Simultaneous measurements of other cell parameters for cell cycle analysis by total fluorescence intensity remains possible. The sensitivity of the system to measure polarization was determined as +/- 0.006 p (0 less than or equal to p less than or equal to 0.5 in isotropic media), which amounts to +/- 1.2% of the maximum p value. Polarization data for latex microspheres plotted in the histogram mode were measured with a standard deviation of 0.006, which proved the high resolution and the high performance of the system.