Phenotypic protein profiling of different B cell sub-populations using antibody CD-microarrays.

Antibody microarrays enable extensive protein expression profiling, and provide a valuable complement to DNA microarray-based gene expression profiling. In this study, we used DotScan antibody microarrays that contain antibodies against 82 different cell surface antigens, to determine phenotypic protein expression profiles for human B cell sub-populations. We then demonstrated that the B cell protein profile can be used to delineate the relationship between normal B cells and malignant counterparts. Principle component analysis showed that the lymphomas did not cluster with the normal memory B cells or germinal centre B cells, but they did cluster with germinal centre founder cells and naïve B cells.

[Cellulose decomposition under nitrogen deficiency by bacteria isolated from the intestines of phytophagous vertebrates]. / Rasshcheplenie tsellulozy pri defitsite azota bakteriiami, vydelennymi iz kishechnika rastitel'noiadnykh pozvonochnykh.

A nitrogen-fixing strain identified as Klebsiella pneumonia 402-2 and two endoglucanase-synthesizing Bacillus strains were isolated from the intestines of phytophagous animals. One of the Bacillus strains was identified as Bacillus subtilis GL. Klebsiella pneumoniae 402-2 increased the endoglucanase activity of both Bacillus strains in mixed cultures. The data on the taxonomic position of strains 402-2 and GL and on the nitrogen-fixing capacity of strain 402-2 were confirmed by sequencing and analyzing their 16S rRNA genes and by amplifying the nitrogenase gene nifH.

[Specific features of nitrogen transformation in the soddy-podzolic soil in the areas of habitat of the common vole Microtus arvalis]. / Osobennosti transformatsii azota v dernovo-podzolistoi pochve na uchastkakh, zaselennykh obyknovennoi polevkoi Microtus arvalis.

The influence of common vole Microtus arvalis on processes of nitrogen fixation and denitrification in the soddy-podsolic soil was studied. In the common vole colonies, the level of nitrogen fixation was reliably lower and that of denitrification higher, than in the control soil outside these colonies. Nitrogen-containing excretory products of voles accumulating in the soil are among the main factors that determine the activity of these processes.

Immunophenotyping of leukemias using a cluster of differentiation antibody microarray.

Different leukemias express on their plasma membranes particular subsets of the 247 defined cluster of differentiation (CD) antigens, which may resemble those of precursor cells along the lineages of differentiation to mature myeloid and lymphoid leukocytes. The extent of use of CD antigen expression (immunophenotyping) for identification of leukemias has been constrained by the technique used, flow cytometry, which commonly specifies only three CD antigens in any one assay. Currently, leukemias and lymphomas are diagnosed using a combination of morphology, immunophenotype, cytochemistry, and karyotype. We have developed a rapid, simple procedure, which enables concurrent determination of 50 or more CD antigens on leukocytes or leukemia cells in a single analysis using a microarray of antibodies. A suspension of cells is applied to the array, and cells only bind to antibody dots for which they express the corresponding CD antigen. For patients with significantly raised leukocyte counts, the resulting dot pattern then represents the immunophenotype of those cells. For patients at earlier stages of disease, the diagnosis depends on recognition of dot patterns distinct from the background of normal leukocytes. Distinctive and reproducible dot patterns have been obtained for normal peripheral blood leukocytes, chronic lymphocytic leukemia (CLL), hairy cell leukemia, mantle cell lymphoma, acute myeloid leukemia, and T-cell acute lymphoblastic leukemia. The consensus pattern for CD antigen expression found on CLL cells taken from 20 patients in descending order of cells bound was CD44, HLA-DR, CD37, CD19, CD20, CD5, CD52, CD45RA, CD22, CD24, CD45, CD23, CD21, CD71, CD11c, and CD9. The antigens that provided the best discrimination between CLL and normal peripheral blood leukocytes were CD19, CD20, CD21, CD22, CD23, CD24, CD25, and CD37. Results obtained for the expression of 48 CD antigens from the microarray compared well with flow cytometry. The microarray enables extensive immunophenotyping, and the intact cells captured on antibody dots can be further characterized using soluble, fluorescently labeled antibodies.

Lipid peroxidation during experimental cholera intoxication.

Cholera intoxication in albino mice was induced by intraperitoneal injection of endotoxin in doses of LD(16), LD(25), and LD(50) and combination of endo- and enterotoxin in doses equivalent to LD(25). Dose-dependent activation of superoxide dismutase, phasic changes in the contents of MDA and conjugated trienes and dienes, and modulatory influence of enterotoxin on catalase activity in the blood were observed during intoxication.

Western blot analysis of bile or intestinal fluid from patients with septic shock or systemic inflammatory response syndrome, using antibodies to TNF-alpha, IL-1 alpha and IL-1 beta.

Septic shock or systemic inflammatory response syndrome (SIRS) often develops in patients following burns, traumatic injury, surgery or biliary obstruction. Although the inflammatory cytokines TNF-alpha and IL-1 have been strongly implicated in the development of these syndromes, treatment of patients by the systemic administration of inhibitors of TNF-alpha or IL-1 has shown limited effectiveness. Recent reports suggest that septic shock may be perpetuated by inflammatory cytokines secreted by the liver in response to bacterial translocation resulting from cytokine-induced gastrointestinal damage. The present study sought to demonstrate the presence of high levels of inflammatory cytokines in the bile or small intestine of patients suffering from septic shock or SIRS, with a view to the development of strategies for the reduction of gastrointestinal damage through intraduodenal administration of cytokine inhibitors. Western blot analysis of human bile or intestinal fluid using anti-TNF-alpha antibodies resulted in the detection of a number of bands in samples from patients with septic shock or SIRS. However, these proteins differed antigenically from human recombinant TNF-alpha (rTNF-alpha) and showed no activity in a biological assay for TNF-alpha. Antibodies to IL-1 alpha and IL-1 beta detected several strong bands, some of which appeared to be identical to recombinant IL-1 alpha and IL-1 beta. It is concluded that proteins resembling several known inflammatory cytokines are present in the bile and intestine of septic shock patients, but it is suggested that further work is required to determine the nature and function of these molecules.

Drug resistance mechanisms and MRP expression in response to epirubicin treatment in a human leukaemia cell line.

A drug resistant series of sublines were developed by treating the human leukaemia CCRF-CEM cell line with 16-1000 ng/ml of the anthracycline, epirubicin. The sublines developed resistance in two stages, neither involving detectable levels of P-glycoprotein. Treatment with up to 50 ng/ml epirubicin produced sublines with cross resistance limited to the anthracyclines and etoposide. Treatment with 100-1000 ng/ml epirubicin produced sublines with increased expression of the mrp gene, increased resistance to the anthracyclines and etoposide, additional cross resistance to vincristine and colchicine, decreased drug accumulation and reversal of resistance by verapamil and by buthionine sulphoximine (BSO; an inhibitor of glutathione synthesis). Our results indicate an interaction between MRP and glutathione metabolism as a mechanism for multidrug resistance.

Identification of Yersinia pestis with varied plasmid composition using monoclonal and polyclonal fluorescent immunoglobulins.

The efficiency of serological identification of Yersinia pestis strains which contain different plasmids was assessed with polyclonal and monoclonal immunoglobulin preparations in the direct fluorescent antibody method. Plague polyclonal luminescent immunoglobulins recognize only those Y. pestis strains which contain pPst, pFra plasmids or both. Anticapsular plague monoclonal antibodies interact only with capsule-forming plague agent strains (pFra+) grown at 37 degrees C. With plague monoclonal lipopolysaccharide antibodies one can identify all Y. pestis strains irrespective of their plasmid content and cultivation temperature. However, these antibodies cross-react with Yersinia pseudotuberculosis bacteria in 60% of cases. The problem of laboratory diagnosis of the plague organism, whatever its plasmid profile, can be solved through the development of a test kit involving two preparations such as plague lipopolysaccharide monoclonal luminescent antibodies and pseudotuberculosis specific luminescent adsorbed immunoglobulins.

The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells.

The MTT cell viability assay is widely used in determining drug sensitivity profiles for patients with hematological malignancies and in primary screening of potential chemotherapeutic drugs. Because the multidrug resistance (MDR) phenotype is associated with these malignancies, and since many vital dyes are effluxed from MDR expressing cells, we have investigated whether the MDR phenotype interferes with the MTT assay. In CCRF-CEM and K562 human leukemic cell lines and drug-resistant sub-lines developed from them, comparison of the MTT assay with other cell viability assays showed significant variation in IC50 concentrations, although the resistance relative to the sensitive parent cell was correlated. Inclusion of verapamil, an inhibitor of drug efflux activity, had no effect on the MTT assay.

[A micromethod of determining the activity of factor XIII in whole blood]. / Mikrometod opredeleniia aktivnosti faktora XIII v tsel'noi krovi.

A simple, available, and sensitive micromodification of Sigg and Baluda's methods for measurements of Factor XIII activity is suggested with registration of the blood clot hemolysis degree in the urea. The method was tried in rabbits and guinea pigs during immunization against cholera. The results of Factor XIII activity measurements were found to be unrelated to blood fibrinogen level but they are in slight correlation with the blood fibrinolytic activity.

Clonal B-cell proliferation in an infant with congenital HIV infection and immune thrombocytopenia.

An infant with congenital human immunodeficiency virus (HIV) infection had immune thrombocytopenic purpura (ITP) develop at four months of age. A bone marrow aspirate had normal results in morphologic characteristics and cellularity. Flow cytometry analysis of the marrow cells showed that the predominant cell in the "lymphocyte" cluster was of B-lineage and common acute lymphocytic leukemia antigen (CALLA) positive. Southern blot analysis of marrow DNA demonstrated gene rearrangements in both the immunoglobulin (Ig) heavy chain and kappa light chain loci, confirming the presence of a clonal B-cell lymphoid proliferation. At one year of age the patient is clinically well without evidence of malignant lympho-proliferative disease. This case exemplifies a limited clonal B-cell expansion in the bone marrow of a patient with HIV infection and a benign hematologic condition.

[Interserovar antitoxic immunity to Vibrio cholerae toxins]. / Mezhserovarnyi antitoksicheskii immunitet k toksinam kholernykh vibrionov.

The immunochemical affinity of V. cholerae enterotoxins, serovars Inaba and Ogawa, has been shown in animal experiments on cross antitoxic immunity in the small intestine, the passive hemagglutination test and the toxin neutralization test. However, antitoxic interaction with both enterotoxins is characteristic only for antibodies to V. cholerae of serovar Inaba, while in animals immune to Ogawa toxin the choleragenic effect of enterotoxins produced by V. cholerae of both serovars in retained. The possible mechanisms of one-sided cross interserovar antitoxic immunity in cholera are discussed.

Virus-specific polypeptides of caprine arthritis-encephalitis virus recognized by monoclonal antibodies to virion proteins p24 and p14.

The proteins of an isolate of caprine arthritis-encephalitis virus (CAEV) were analysed by SDS-PAGE and Western blotting. Monoclonal antibodies (MAbs) produced to the main core protein p24 and the small structural protein p14 also recognized two major polypeptides of Mr 41K and 55K in infected cell material, consistent with a precursor role for these gag polypeptides. In addition, the p24 MAbs detected a 33K polypeptide in extracellular virus preparations, while the p14 MAbs reacted strongly with a polypeptide of 18K (corresponding to structural protein p18) and weakly with another of 21K. The use of these MAbs in an indirect fluorescent antibody method revealed an intracytoplasmic location of these viral antigens in both mononucleated and multinucleated (syncytial) infected cells. Cross-reactivity with several other isolates indicated that these MAbs may be useful for diagnosis of CAEV infection.

[Interrelation between the blood neutrophil damage test and the passive hemagglutination reaction in detecting antibodies]. / Vzaimosviaz' mezhdu testom povrezhdeniia neitrofilov krovi i reaktsiei passivnoi gemaggliutinatsii v obnaruzhenii antitel.

Statistical analysis of the results of examinations of vaccinees against plague has revealed that the values of the neutrophil damage index (NDI) and antibody titers as determined in the passive hemagglutination (PHA) test qualitatively (but not quantitatively) correlate. The statistical series for the PHA test and NDI belong to different variations, i. e. they describe different functions of antibodies with respect to the same antigen. Besides, the determination of NDI permits the detection of antibodies in 95% of cases with probability equal to 0.06, while the PHA test determines antibodies in 67% of the vaccinees with probability equal to 0.30. The determination of NDI, used as an alternative qualitative method for antibody detection, is particularly effective in the evaluation of faintly immunogenic antigens, as well as at the early and late periods of immune state.

[Modeling of myocardial pathology in mice with the surgical methods]. / Modelirovanie patologii miokarda u myshei operativnymi metodami.

A description of three operations on the heart in mice is presented: a supravalvular narrowing of the ascending aorta, banding of the apex of the heart, and ligation of the coronaries. These surgical interventions permit to obtain in mice a functional overloading and hypertrophy of the myocardium, a cardiac aneurysm and occlusive myocardial infarction.

[Quantitative characteristics of the multiplication of muscle cells in the course of postnatal cardiogenesis in mice]. / Kolichestvennaia kharakteristika razmnozheniia myshechnykh kletok v techenie postnatal'nogo kardiogeneza u myshei.

Changes in the absolute number of muscle cells and nuclei in the ventricle during the 1st year of postnatal life were followed in males of the CC57BR mice. The counts were carried out in the cell suspension obtained from the fixed cardiac muscle by means of alkaline dissociation. The number of muscle cells and nuclei reliably increased during the 1st month of life and ceased to increase when the maturity was attained (2-3 months). The number of muscle cells increased twice and that of muscle nuclei almost 4 times. A comparative estimate was made of the role of proliferation and growth of muscle cells in the increase of ventricle weight with the age. The importance of individual variation of the number of muscle cells and nuclei during early postnatal ontogenesis for the study of the myocardium adaptive growth is discussed.

[Determination of the quantity of cells in different organs and tissues following alkaline dissociation]. / Opredelenie kolichestva kletok v razlichnykh organakh i tkaniiakh posle shchelochnoi dissotsiatsii

A tissue following fixation for 10-15 days with formalin was treated with 50% KOH aqueous solution during 18 hours, which led to complete dissociation of the tissue into cells. Suspension of cells was placed into the counter of Fuchs-Rosenthal and numbers of nuclei in cells of definite tissues were counted. On preparations of the same suspension in 1000 cells distribution of the latter according to the number of nuclei was determined. Then a number of cells in 1 mg of tissue and in the organ were estimated. The count of muscular cells in the heart of a mouse showed that the relative error of the determination of the number of cells did not exceed 2.0%.