[The immune cytochemical evaluation of reaction of phytohemagglutinin stimulation of lymphocytes with monoclonal antibodies Ki-67].

The article proposes new (immune cytochemical) mode of evaluation of reaction of blast-cell transformation of human lymphocytes under their stimulation with phytohemagglutinin instead of standard radiometric method using 3H-thymidine as a marker. The reaction was assessed by means of luminescent microscope under indirect immunofluorescence using nuclear antigen Ki-67 as a marker of proliferating cells. The Ki-67 was detected using monoclonal antibodies Ki-67. The functional capacity of lymphocytes was evaluated by amount of visualized (luminescent) Ki-67 positive cells appearing after three day stimulation of proliferation with phytohemagglutinin. The comparison was made between immune cytochemical (with Ki-67 marker) and radiometric (with marking by 3H-thymidine) methods of phytohemagglutinin stimulation of lymphocytes. The article presents substantiation of advantage of immune cytochemical method of evaluation of reaction of blast-cell transformation of human lymphocytes as compared with radiometric method concerning possibility of its broader implementation in laboratory practice.

[The value of monitoring of qualitative content of onco-antigens in serum in diagnostics of malignant tumors].

The results of monitoring of qualitative content of 8 onco-antigens (CK, CA-242, CA-19-9, CA-125, REA, AFP, SCC, NSE) in serum of 414 patients are presented. The examination of 62 patients with tumors of different localization was carried our in dynamics before and after treatment. The monitoring of concentration of onco-antigens in serum permitted to apply early diagnostics of malignant process to 9 out of 28 patients with unclear clinical symptomatic. The technique informativeness is demonstrated in pre-clinical diagnostic and prognosis of course of oncological diseases. The possibility of transitory increase of concentration of certain onco-antigens under non-oncologic diseases returning to normal values after application of corresponding pharmaceutical treatment is revealed. The broader implementation of this technique into practice of public health is recommended.

[The levels of key nucleolar proteins in the lymphoid cells of healthy individuals and patients with lymphoproliferative diseases].

Immunocytochemical staining with specific antibodies was used to study the expression of three nucleolar proteins (fibrillarin, B23/nucleofozmin, and SURF6), which were involved in pRNA maturation, in the lymphoid cells of healthy individuals and patients with lymphoproliferative diseases and to compare it with the expression of the known proliferation marker Ki-67 protein. The results indicated that fibrillarin was detectable at the comparable level in the lymphoid cells of the patients and in the peripheral blood lymphocytes of the healthy individuals. In one fourth of the patients, the proportion of cells containing B23/nucleofozmin was noticeably higher than that in the lymphocytes of donors; however, there was no great difference in patients with different types of the disease. The number of SURF6-positive cells was directly correlated with that of Ki-67-positive cells. The maximum level (47-67%) of SUR6-positive lymphoid cells was found in splenic lymphosarcomas and mantle cell lymphoma. The findings suggest that SURG6 protein may be of additional diagnostic and prognostic value.

Enhanced sensitivity of nucleoli in human proliferating cells to inhibition of protein synthesis with anisomycin.

We describe the reaction of nuclei in cultured human cells from different tissues to inhibition of total protein synthesis with anisomycin - ribotoxin, which is now considered as a potential antitumor drug. It was shown that nucleoli in sensitive cells demonstrate typical reaction: under the action of the inhibitor, labile nucleolar protein, a component of RNA polymerase I transcription complex (previously called A3 antigen), rapidly migrates from the nucleolus to numerous discrete foci in the nucleoplasm. These changes are specific for translation suppression and are not induced by other influences on the cells. Migration of A3 antigen into the nucleoplasm manifests primarily in cells at the stage of DNA replication and is absent in resting cells. These results suggest that localization of A3 antigen can be a marker of artificial suppression of translation in proliferating human cells in vitro.

[Antibodies to synthetic fragments of nucleophosmin for the specific detection of its monomeric and oligomeric forms].

Immunoactive fragments corresponding to the N-terminal (19-36) and C-terminal (283-294) regions of the NPM1.1 isoform of nucleophosmin and their shortened fragments were chosen and synthesized. Rabbits were immunized with free full-size peptides and their protein conjugates. Antibodies produced against the 19-36 and 283-294 peptides were purified by affinity chromatography on bromocyanogen-activated sepharose that was preliminary conjugated with the synthetic peptides. An analysis of immunoblots of lysates of the HeLa and Ramos cells demonstrated that the antibodies produced against the 19-36 peptide detected the monomeric form of nucleophosmin, whereas the antibodies against the 283-294 peptide predominantly revealed its oligomeric form. It was established by immunocytochemical analysis that the antibodies induced by the 19-36 peptide stained the nucleoplasm and perinuclear space of the cytoplasm of the HeLa and Ramos cells, but did not stain the nucleoli, while the antibodies against the 283-294 peptide stained only the nucleoli of the same cells. On the basis of these results, one could propose that the monomeric and oligomeric forms of nucleophosmin were located in the nucleoplasm and nucleoli of the examined cells, respectively. Thus, antibodies which can predominantly detect monomeric and oligomeric forms of nucleophosmin were produced for the first time. An analysis of the monomeric-oligomeric state and the location of the nucleophosmin in tumor cells could be performed using these antibodies.

[Isolation of the protein B23/nucleophosmin from HeLa cell nuclei].

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35-40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C- and N- terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.

[The cytological indicators of the overall suppression of protein synthesis as revealed by staining with a new monoclonal antibody].

In this work we describe how the nucleolus reacts to inhibition of protein synthesis as revealed by labeling with a new monoclonal antibody A3. In normal cells A3 antigen is observed as numerous foci within the nucleolus. During mitosis A3 antigen is located in a few foci on chromosomes. Regions of A3 localization are susceptible to pepsin treatment but are not susceptible to RNAse A treatment. This fact indicates that A3 antigen is of protein nature. On the ultra structural level, A3 antigen is localized primarily at the periphery of fibrillar centers. Taken together these properties of A3 antigen suggest that it's a component of the RNA polymerase I transcription machinery. A3 antigen has an intriguing property, namely, an ability to migrate from the nucleolus to the nucleoplasm upon inhibition of protein synthesis with anisomycin, puromycin or cycloheximide. The obtained results show that the localization of A3 antigen revealed by the new monoclonal antibody may serve as a cytological indicator of the overall level of protein synthesis in vitro.

Changes in the status of nucleolus during long-term culturing of human HeLa cells.

Changes in the immunocytochemical status of the nucleoli during long-term (6-8 months) in vitro culturing of HeLa (carcinoma of the cervix uteri) cells were described using new A3 monoclonal antibodies selectively reacting with human cell nucleoli. The appearance of cells with abnormal location of A3 antigen was paralleled by a significant increase of culture sensitivity to some external factors (protein synthesis inhibition and oxidative stress). The data indicate that location of one of the nucleolar antigens is an indicator of the qualitative status of HeLa cells in the culture.

[Possibilities of early diagnosis of tumors].

AIM: To study diagnostic significance of blood serum cancer antigens levels in patients who subsequently develop pulmonary cancer (PC) and gastrointestinal cancer (GIC). MATERIAL AND METHODS: ELISA was used to study expression of tumor antigens (CEA, NSE, CA19-9, CA242, AFP) in the blood serum of 27 PC and 31 GIC patients; 22 patients with lymphatic tumors and 32 patients with pulmonary and gastrointestinal inflammation served control. After removal of the tumor the same antigens including cytokeratines (CK) and differentiated leukocytic markers were studied immunocytochemically in the tumor cells with relevant monoclonal antibodies. RESULTS: Sera of patients with verified afterwards cancer contained elevated concentrations of the antigens: NSE and CEA in PC, CA19-9, CA242 in GIC. The expression of these antigens including CK was found also in tumor cells of these patients. Atypical cells of lymphatic tumors had hemopoietic markers in the absence of CK. In inflammation and in lymphatic tumors, tumor antigens levels remained normal. CONCLUSION: The test for tumor antigens levels in the serum may be used for early (preoperative) diagnosis of cancer, especially in tumors with difficult access or if they are asymptomatic.

[Factors of an unfavorable prognosis in patients with B-cell chronic lymphoid leukemia: a retrospective analysis of 206 cases]. / Faktory neblagopriiatnogo prognoza u bol'nykh B-kletochnym khronicheskim limfoleikozom: retrospektivnyi analiz 206 sluchaev.

AIM: To assess factors of an unfavourable prognosis in a group of intermediate risk of B-cell chronic lymphoid leukemia (BCCLL). MATERIAL AND METHODS: 206 BCCLL patients (mean age 55.5 years, male/female = 1.66) entered the study conducted by Hematological Research Center in 1992-2000. RESULTS: Nine patients under 35 years of age did not survive 5 years except one female who achieved a complete remission on fludarabin. The type of bone marrow infiltration (diffuse vs interstitial and nodular), the time of lymphocyte count doubling (under or over 12 months) discriminate the patients by prognosis in the group of intermediate risk: medians of overall survival 65 months vs 148 months and 72 vs 133 months, respectively (p < 0.005 for both curves, log-rank criterion). Survival medians in groups with low (< 50% cells) and high (> 50% cells) expression of CD38+ cells in the group of intermediate BCCLL risk comprise 55 and 106 months (p = 0.005). The type of bone marrow infiltration and time of doubling of lymphocyte count overlap: > 70% patients with a diffuse type of bone marrow infiltration have the time of doubling under 12 months and vice versa while expression of CD38 do not overlap with these values. Combination of two signs (type of bone marrow infiltration and CD38 expression or time og lymphocyte count doubling and CD38 expression) allows more precise identification of prognostically unfavourable groups. Medians of survival for combination of the first two signs (two positive against two negative) comprise 51 months vs 169 months (p < 0.0001), for combination of the latter two signs 55 months vs 106 months was not reached (p < 0.001). Although most patients with a tumor form of BCCLL are referred to stage II, the prognosis in this form is much worse than in stage II, survival medians are 44 and 69 months, respectively (p < 0.05). A mutation status of the genes of a variable region of immunoglobulins enable identification of the group of patients with a relatively benign course of BCCLL (survival medians 61 and 289 months, p < 0.0001). CONCLUSION: In patients under 35 years of age BCCLL runs unfavourably and seems to require intensive polychemotherapy. Usage of a combination of the signs (CD38, time of doubling of lymphocyte count and type of bone marrow infiltration) is a simple and reliable method of identification of prognostically different categories of patients in the group of an intermediate BCCLL risk. Prognosis in patients with a tumor form of BCCLL is unfavourable: medians of survival in patients with a tumor form and stage III-IV are comparable. Mutational status of the genes of immunoglobulin variable region may serve a marker of a long-term prognosis.

[Immunophenotyping in diagnosis of chronic lymphoproliferative diseases]. / Immunofenotipirovanie v diagnostike khronicheskikh limfoproliferativnykh zabolevanii.

With due respect to their many-year experience, the authors focused their attention on the peculiarities of implementation and registration aspects of the immunofluorescence method of immunotyping made in cell suspensions and in tissue imprints; additionally they made a system of a set of monoclonal antibodies (used for the purpose), which enable the differential diagnosis of reactive conditions related with various malignant lymphoproliferative diseases (LPD). It order to specify a nature of lymphoid cells it is suggested to undertake the immunophenotyping withing a gradually expanding set of monoclonal antibodies, which reflects different parameters of lymphoid cells like linear attributes, clonal characteristics, differential-diagnostic markers, functional status and proliferative activity. Typical marker phenotypes of lymphoid cells observed in the main B- and T-cell LPDs are described; a possibility is mentioned that there can be errors in interpreting the phenotyping results while diagnosing an LPD.

A major nucleolar protein B23 as a marker of proliferation activity of human peripheral lymphocytes.

A novel monoclonal antibody (Mab) (called 3C9) against a major nucleolar phosphoprotein B23 was used to study B23 qualitative and quantitative alterations in phytohemagglutinin (PHA) -stimulated human peripheral blood lymphocytes in indirect immunofluorescence and Western blots. It was shown that lymphocyte proliferation was accompanied by gradual augmentation of nucleoli and their accumulation of the protein B23 up to 2-fold by 16 h and 40-50 fold by 72 h, as compared with the non-stimulated cells. By parallel immunolabeling with the anti-Ki-67 antibody, it was shown that the early changes of B23 amount and localization occurred before an appearance of Ki-67 protein, a well-known marker of proliferating cells. Our results evidence that antibodies against B23 might be applied for recognition of human peripheral lymphocytes at early stages of their activation for proliferation, preceding the S-phase.

[Identification of nucleolar antigen in different types of cells using domestic monoclonal antibodies]. / Vyiavlenie iadryshkogo antigena v razlichnykh tipakh kletok s pomoshch'iu otechestvennykh monoklonal'nykh antitel.

Murine hybridoma 3C9 producing BCA-PC monoclonal antibodies and selectively staining the nucleoli in various cells, most intensely in actively proliferating cell cultures, was obtained by somatic hybridization of cells. BCA-PC MAb belong to IgM. As a rule, IgM antibodies are virtually unavailable for intracellular antigens, and therefore a special method has been developed for fixation and performance of indirect immunofluorescence with BCA-PC. By its molecular weight (37 and 43 kD) and distribution in the cellular nucleus, the antigen detected by BCA-PC resembles the nucleolar protein B23 located in the nucleolar granules. The content of B23 increases with malignant degeneration of cells and during cell proliferation. It is therefore believed that the new MAbs will help characterize the proliferative status of cells which determines the malignancy of a pathological process.

[Differential diagnosis of complicated cases of acute leukemia by morphocytochemical and immunological methods]. / Differentsial'naia diagnostika slozhnykh sluchaev ostrogo leikoza s pomoshch'iu kompleksa morfotsitokhimicheskikh i immunologicheskikh metodov.

Twelve intricate cases with different variants of acute leukemia were diagnosed by morphocytochemical and immunological methods. In six patients with initially diagnosed acute lymphoblastic leukemia (ALL) comprehensive studies revealed acute myelomonoblastic leukemia (AMML), in four ALL patients AML(M1,M0) and AMML were diagnosed, and in two patients with AML B-lymphoblastic lymphoma/leukemia was detected. These data indicate that immunophenotyping methods in complex with morphological and cytochemical studies helped correctly diagnose the disease variants, particularly in cases difficult to diagnose.

[MTT-colorimetric method for detection the cytotoxic activity of human natural killer cells]. / MTT-kalorimetricheskii metod opredeleniia tsitotoksicheskoi aktivnosti estestvennykh killernykh kletok.

A test system has been adapted for evaluating the cytotoxicity of human natural killer cells (NK) towards tumor targets K-562 in vitro. The method is based on the capacity of mitochondrial enzymes of viable cells to reduce the yellow soluble salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a purple-blue insoluble formasane precipitate which is quantified spectrophotometrically after dissolution in an organic solvent. The amount of MTT-formasane production is directly proportionate to the count of viable cells. The optimal conditions for the cytotoxicity assay were determined: cell count 10 x 10(3) or 20 x 10(3) cells/well for K-562 as targets and 100 or 200 x 10(3) cells/well for peripheral blood mononuclears as effectors at effector/target cell ratios 20:1, 10:1, and 5:1. This colorimetric method has several advantages over radioactive methods: multiwell scanning spectrophotometers permit the processing of numerous samples and are safe. The results of MTT assay and morphological assay for measuring the NK cytotoxicity were in good correlation and were similarly sensitive.

[Significance and features of MLC reaction in histocompatibility determination of donor and recipient in allogenic bone marrow transplantation to patients with hematological malignancies]. / Znachenie i osobennosti reaktsii smeshannoi kul'tury limfotsitov v opredelenii gistosovmestimosti donora i retsipienta pri transplantatsii allogennogo kostnogo mozga u bol'nykh gemoblastozami.

AIM: To characterize mixed lymphocyte culture (MLC) reaction used for determination of donor-recipient compatibility before bone marrow transplantation to patients with hematological malignancies and to assess the reaction significance. MATERIAL AND METHODS: The analysis was made of compatibility testing in MLC standard reaction performed in 134 patients with hematological malignancies with HLA-A, -B identical donors-sibs and in 5 patients with haploid identical donors. RESULTS: Out of 134 patients, 22(91%) appeared compatible to donor sibs in MLC reaction, 12(9%) patients were incompatible. Mean RR for MLC-compatible couples made up: in RvD direction 77 +/- 0.17%, DvR 2.61 +/- 0.32%. 93% of RR values ranged from +15 to -15%, the rest--from +25% to -25%. Bone marrow transplantation was made in 83 patients. Graft retention was observed in 77(93%) patients. Acute and chronic graft versus host reaction developed in 15 and 17 patients, respectively. CONCLUSION: An optimal protocol is proposed for examination of compatibility donor-recipient in MLC reaction in patients with hematological malignancies. It is intended for allogenic bone marrow transplantation in hematological departments.

[Analysis of the proliferative activity of a cell using new monoclonal antibodies to nucleolar protein B23/nucleophosmin]. / Analiz proliferativnoi aktivnosti kletok s pomoshch'iu novykh monoklonal';nykh antitel k iadryshkovomu belku B23/nukleofozminu.

Nowadays, antinucleolar antibodies are widely used for exploration of the nucleolar organization and molecular mechanisms of ribosome production. Here we have described a new monoclonal antibody against the major nucleolar phosphoprotein B23/nucleophosmin (3C9) that is involved in the terminal stages of ribosome production. It is used to examine immunocytochemical peculiarities of the nucleolus in terms of the cell proliferative status and also during mitosis. In human peripheral blood lymphocytes, activated for proliferation with phytohaemagglutinin (PHA), PHA stimulation of lymphocytes was shown to result in accumulation of protein B23 in augmentative nucleoli. A comparative study of 3C9 and two other anti-B23 antibodies 20B2 and anti-B23 by Western blots and indirect immunofluorescence favored the idea that 3C9 cross-reacted with the major isoform of B23, B23.1, that have an apparent molecular weight of 40 kDa.

[The immunodiagnosis of breast cancer metastases to the bone marrow]. / Immunodiagnostika metastazov raka molochnoi zhelezy v kostnyi mozg.

Thirty-eight patients with stages II-III breast cancer after mastectomy, two patients with other tumors, and six patients with lymphosarcoma were examined. For detecting cancer metastases to the bone marrow, morphological analysis of puncture biopsy specimens was carried out in all patients with breast cancer; in patients with other diseases, puncture biopsy specimens of the tumor or lymph node were examined. Immunodiagnosis was carried out by the immunocytochemical method with detection of tumor antigens--monoclonal antibodies to cytokeratins, to breast cancer (ICO-25 and ICO-103), and to hemopoietic cells. All patients with breast cancer were divided into two groups: 1) 9 patients with cancer metastases to the bone marrow, confirmed by x-ray, histological, and morphological methods and 2) 29 patients without documented metastases to the bone marrow. Immunocytochemical methods detect isolated tumor cells in patients with suspected metastases to the bone marrow, lymph nodes, and other organs in cases when these cells are undetectable by routine morphological and histological methods.

[Priorities in research of hemoblastosis]. / Prioritetnye napravleniia issledovanii po probleme gemoblastozov.

Evidence is provided for that it is urgent to elaborate a problem of hemoblastosis and hemopoietic depressions within the framework of a special federal research and technological programme. Priorities of research lines in this areas, trends of their development till 2005 are presented.