[Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots].

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.

[Microtubules suppress blebbing and stimulate lamellae extension in spreading fibroblasts].

We compared spreading of Vero fibroblasts when microtubules were depolymerized or stabilized. After initial attachment cells start blebbing that continues for different time and abruptly transfers into spreading. After spreading initiation, most cells spread in an anisotropic manner through stochastic formation of lamellipodia. A second mode was rapid, isotropic spreading via formation of circular lamellum that occurs in 15% of cells. The rate of spreading was maximal at the beginning and decreased during the first hour according to logarithmic law. After 60 min many cells formed stable efges and started migrating on the substrate. However, cell area slowly continued to increase. Actin bundles are formed 20 min after cell attachment and they first run along cell boundary. This system disassembles within 20-40 min and is substituted with stress fibers crossing the cell. In the isotropically spread cells no actin bunbles are seen. Microtubules in the spreading cells enter into large blebs and all nascent lamella and later form radial array. When MTs has been depolymerized or stabilized blebbing started before cells attached to the substrate and continue much longer than in control cells. In both cases the initial rate of spreading decrease several fold, and remains constant for many hours. After 24 h the mean area occupied by cells with altered MT system was the same as in control. Alteration of MT system had moderate effect on actin system--formation of actin cables started at the same time as in control (within 20 min upon cell attachment), however, they grew even in cells undergoing prolonged blebbing. Actin cables running along cell margin were similar to tat in control cells, but they did not disappear up to 1 h. When stabilized, microtubules form chaotic array: they do not enter blebs and in spread cells run parallel to the cell margin at a distance of 3-5 microm. We conclude that dynamic microtubules speed up completion of blebbing and promote early stages of fibroblasts spreading.

[Expression of early hematopoietic markers in cord blood and mobilized blood].

G-CSF mobilized peripheral blood and cord blood are major sources of hematopoietic progenitor cells. These cells are characterized by the expression of "early" antigens. We have evaluated the coexpression of hematopoietic cell markers CD34, CD133, CD90, CDCP1, CD117 and activation antigen CD38 using multicolor flow cytometry. We show that (1) cells being positive for every single antigen form a separate population. (2) Percentage of cells expressing each "early" antigen are twice more in the cord blood than in the mobilized blood. The content of cells with complex progenitor phenotype (CD34+/CD38-/CD117, CD133+/CD34+/CD38-, CDCP1+/CD34+/CD38- etc.) is equal in mobilized and cord blood. (3) There are strong positive correlations between the expression of CD34, CD133, CD117 and CDCP1 in both groups. Positive correlation exists for CD90 with CD34, CD133, CDCP1 and CD117 only in cord blood and is not significant in mobilized blood. The analyses of early antigens coexpression with activation marker CD38 revealed that hypothesis on sequential activation and loss of expression of the aforementioned antigens is not confirmed. We assume that there is global regulation of the expression of CD34, CD133, CDCP1 and CD117. Yet expression of CD38 could be reversibly abolished during maturation of the hemapoetic cells and CD117 could be expressed not only on myeloid cells.

[Nine-year experience in the treatment of patients with diffuse large B-cell lymphosarcoma].

AIM: To ascertain indications to standard (CHOP-21/R-CHOP-21) and intensive (mNHL-BFM-90) treatment in patients with diffuse large B-cell lymphosarcoma (DLBCL) with involvement of lymphoid organs. MATERIAL AND METHODS: The trial, performed from January 2002 to December 2010, enrolled 139 DLBCL patients with affected lymph nodes (LN), tonsils, spleen, bone marrow (BM). The diagnosis was made according to WHO criteria. The patients were examined according to the protocol of lymphoproliferative diseases. Biopsy material from all 139 patients was studied immunohistochemically on paraffin blocks (LN, tonsils, spleen, BM) using a wide panel of antibodies. The same examinations of BM were made in all 18 cases of BM involvement. Cytogenetic examination was performed in 106 patients: 48 standard cytogenetic tests, 139 - FISH for t (14;18) as well as rearrangement of locus 3q27. Patients with a poor prognosis (n = 86, 61.8%) received intensive therapy according to mNHL-BFM-90 program. The signs of a poor prognosis were the following: massive tumor (tumor size more than 7.5 cm), invasion into the adjacent organs or tissues, stage III-IV disease by Enn-Erbor, high concentration of LDG. Patients without a poor prognosis (n = 53, 38.2%) received standard treatment CHOP-21 (n = 28) or R-CHOP-21 (n = 25). RESULTS: A complete remission without recurrences was achieved in all 53 patients without signs of unfavourable prognosis (100%). Overall 5-year survival was 96%, 2 patients died in remission of other causes. Of 86 patients with a poor prognosis a complete remission was achieved in 64 (74.4%) patients. Overall and recurrence-free 5-year survival was 65 and 86%, respectively. CONCLUSION: Standard treatment provided long-term complete remission in all the patients without poor prognosis. Intensive (mNHL-BFM-90) treatment produced the best results in generalized lesion without BM involvement. Overall 5-year survival was 84% in these patients and 12% in patients with BM involvement.

[Applications of fluorescent semiconductor nanocrystals in microscopy and cytometry].

Quantum dots (QD) nanocrystals consisting of CdSe core with ZnS shell are a novel class of fluorophores with tremendous potential in microscopy and cytometry techniques. The unique optical features of Qdots, namely, high photostability and extinction coefficient, wide absorption and narrow emission spectra, and large Stokes shift make them desirable fluorescent tags for diverse biomedical applications. Applications of this novel technology in microscopy and cytometry produce reliable multicolor specimens due to increased photostability, ability for multiplexing and narrow emission spectra of nanocrystals. QD conjugates are available on the market and could be prepared in the laboratory. This paper describes the application of QD-conjugates for immunophenotyping and FISH assessment of cells and tissues, and the requirements for microscope and flow cytometer reengineering for successful use of QD in multiplex fluorescent format. Despite the considerable progress, important methodological issues still need to be solved in terms of QD nanocrystals' size, heterogeneity, functionalization and stability of their conjugates. We discuss practical approaches and challenges that need to be addressed to make QD immunostaining a standard method in biology.

[Diffuse large B-cell lymphoma with primary involvement of mediastinal lymph nodes: diagnosis and treatment].

AIM: To diagnose diffuse large B-cell lymphosarcoma (DLBCLS) with primary involvement of the mediastinal lymph nodes (LN) and to evaluate the efficiency of aggressive polychemotherapy (PCT). SUBJECTS AND METHODS: The study included 15 patients (6 men and 9 women aged 18 to 70 years; median 38 years) followed up at the Hematology Research Center, Russian Academy of Medical Sciences, in 2004 to 2009. Three and 12 patients had Stages II and IE DLBCLS, respectively. B symptoms were found in 14 (93.4%) patients. Increased lactate dehydrogenase (LDH) concentrations were detectable in 14 (93.4%) patients; tumors of 10 cm or more (bulky disease) were seen in 11 (73.3%). Enlarged cervical, supraclavicular, and axillary lymph nodes were found in 9 (60%) patients; lung involvement via extension in 9 (60%), and invasion into the pericardium in 5 (33.3%) and soft tissues of the anterior thoracic wall in (13.3%). There were no signs of involvement of extranodal organs at the moment of diagnosis. All the 15 patients received PCT according to the modified NHL-BFM-90 program: 4 to 6 courses depending on the response to the therapy; 10 (66.6%) and 5 (33.3%) patients had 4 and 6 courses, respectively; for consolidating purpose, 11 (78.5%) patients were prescribed radiotherapy applied to the mediastinum in a cumulative dose of 36 Gy due to the fact that they had a residual mass. RESULTS: Thirteen (86.6%) patients achieved a complete remission (CR). Primary PCT resistance was confirmed in one case. Another patient was stated to have near-complete remission. No recurrences were notified during the follow-up. The mean CR duration was 24.5 (range 2-49) months. CONCLUSION: DLBCLS with primary LN involvement is an individual nosological entity to be differentiated from primary mediastinal large B-cell lymphosarcoma. In most cases, DLBCLS shows signs of a poor prognosis, which makes it necessary to perform aggressive PCT.

[Radial-organized microtubules provide maintenance of the cell shape and more effective intercellular transport than in the case of free microtubules].

Microtubules take part in very different cell processes including cell polarization and migration, intercellular transport and some others. Therefore the microtubules spatial organization is crucial for normal cell behaviour. Fibroblasts have radial microtubule array consisting of microtubules running from the centrosome. This microtubule array includes two components: (1) centrosomal microtubules with their minus ends attached to the centrosome and with their plus ends radiating to the cell periphery and (2) free microtubules with the ends non-attached to the centrosome. Distinction in the dynamic properties, intercellular organization and structure of centrosome-attached and free microtubules allow us to assume that their functions in the cell are also different. In order to investigate centrosome-attached and free microtubules functions we used the cytoplasts--experimentally denucleated cellular fragments and under certain condition lacking of the centrosome as well--which contain only free microtubules. Centrosome-containing cytoplasts do not differ significantly in the form, general morphology and the size from the intact cells. At the same time centrosome-lacking cytoplasts keep extremely thinned out network of microtubules located in the central area of the cytoplast. These cytoplasts lose the original cell shape usual for fibroblasts and get rough, with protrusions, lamella; the internal architecture of the cytoplasm and organoids arrangement is also broken. Saltatory movements in the centrosome-containing cytoplasts are similar to those in the intact cells, and saltatory movements in centrosome-lacking cytoplasts show half the speed and smaller distances compared with intact cells. Besides, the saltatory movements of granules in the centrosome-lacking cytoplasts occur mainly in the central regions of the cytoplasts and they are less ordered than in the intact cells and in the cytoplasts kept the centrosome. We believe that radial organization of the microtubules provide effective transport and dynamical interactions of microtubules plus ends with cortical structures of the cell, which are sufficient for maintenance of typical fibroblast-like shape, whereas disorganized free microtubules by themselves cannot keep up the shape and intercellular organization characteristic of fibroblasts.

[A morphometric analysis of the nuclei and nucleoli in tumor cells in lymphogranulomatosis, diffuse large B-cell lymphoma and anaplastic large cell lymphoma].

AIM: To make a comparative morphometric analysis of the nuclei and nucleoli of tumor cells in lymphogranulomatosis (LGM), diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL) for differential diagnosis of these lymphomas. MATERIAL AND METHODS: Biopsy material (lymph node biopsies) was frozen in hexane, fixed and stained, then microscopic pictures were made. RESULTS: Mean area of tumor cell nuclei in LGM was 97.25 +/- 68.77 mcm2, in DLBCL and ALCL--55.89 +/- 20.13 mcm2 and 70.31 +/- 34.64 mcm2, respectively. The area differences were significant (p < 0.001). Hodgkin's and Berezovsky-Rid-Sternberg cell bucleoli area was the largest (11.44 +/- 7.83 mcm2). The nucleoli of the former are larger than those of the latter. Mean area of the nucleoli in DLBCL was 3.05 +/- 1.58, in ALCL--5.53 +/- 4.94 mcm2. The differences are significant (p < 0.001). CONCLUSION: Nucleoli in Hodgkin 's cells are significantly larger than those in the tumor cells in ALCL and DLBCL and the nucleoli with the area more than 12 mcm2 can be used in differential diagnosis between LGM and DLBCL but not between LGM and ALCL.

[Features of immune status among native population of Yamal in the conditions of Europeanization of traditional food].

The destruction of nutrition traditional structure and imbalance of food allowance by the basic kinds of plastic and energy materials impose a negative mark on efficiency of immune protection of north Aboriginals.

[Correlation of lipid spectrum with red blood cells izoantigene polymorphism and nutritional status of isetski believers].

In genetically homogeneous groups food habits associate with a number of food substances which act with food stuffs, and render essential influence on a lipid spectrum wheys of blood. Thus can influence character of current not only not infectious, but also on infectious inflammatory diseases which associate with secondary immune.

[High-dose therapy of Burkitt's lymphoma in patients over 40 years of age].

AIM: To analyse efficacy and tolerance of high-dose polychemotherapy (PCT) of Berkitt's lymphoma (BL) in patients aged over 40 years. MATERIAL AND METHODS: High-dose PCT was given to 6 BL patients aged 41-56 years (median 48.1 years). RESULTS: Complete clinicohematological remissions were achieved in 4 patients. In two of them the treatment was discontinued after three blocks of PCT because of severe infectious complications. According to 4-12 month follow-up, remission continues. Remission was not achieved in two patients: one patient had primary resistance, the other died of sepsis after the second PCT course before remission. The time to remission did not correlate with age. Duration of myelotoxic agranulocytosis varied from 2 to 24 days. Duration of agranulocytosis did not correlate with age. Infections complicated 19 of 20 PCT blocks. Severity of complications caused withdrawal of three patients. CONCLUSION: BL is biologically heterogenous as it demonstrates different responses to BL-M-04 program. Causes of slow regression of tumor mass in some patients need further investigations. In spite of a great number of infectious complications high-dose therapy has no alternative.

[Efficacy of therapy of different variants of anaplastic large T-cell lymphomas].

AIM: To compare efficacy of NHL-BFM-90 and CHOP-like courses in the treatment of anaplastic large cell lymphoma (ALCL). MATERIAL AND METHODS: Twenty-two patients with ALCL participated in the study. The diagnosis was made basing on the findings of clinical, device, morphological, immunohistochemical and molecular-genetic examinations with application of a panel of monoclonal antibodies to CD30, ALK, CD3, CD4, CDS, CD7, CD34, CD15, CD68, CD20, CD45RO, CD45RA, Ki-67. 14 cases of 22 were negative by kinase of anaplastic lymphocytes (ALK-) and 8 were positive (ALK+). Mean age of ALK-ALCL patients was 39.6 +/- 4.1 years, of ALK+ALCL patients - 23.4 +/- 2.6 years. 14 patients were treated by the protocol NHL-BFM-90, 8 were initially treated with other schemes (CHOP, MACOP-B, BEACOPP and others). All 14 patients treated according to NHL-BFM-90 had ALCL stages III-IV with B-symptoms. 12 patients who completed treatment by the above protocol achieved complete remission after the forth course, 2 patients failed the treatment. Of 8 ALCL patients treated initially according to other schemes, a complete remission was achieved in 4 patients (2 had stage II). One of 4 patients with remission had recurrence. Four patients who had failed to achieve complete remission died of the disease progression. CONCLUSION: ALCL occurs more frequently in young and middle-aged patients. The disease has an aggressive course with rapid generalization. For such processes it is more preferable to use a modified protocol NHL-BFM-90.

[Treatment of life-threatening airway obstruction in patients with malignant lymphomas].

Case histories of patients with hemoblastosis admitted to a hospital for acute respiratory failure due to tumor-induced obstruction were retrospectively analyzed. The causes of obstruction, antitumor therapy, methods for provision of airway patency, and major critical syndromes were analyzed. Ten patients with life-threatening tumor-induced airway obstruction were hospitalized from 1995 to 2007. The patients suffered from malignant lymphomas in all cases. The causes of impaired airway patency were the compression of the trachea and large bronchi with a tumor, lymph nodes, affected thyroid, as well as the superior vena cava syndrome, and tumor-induced lesion of soft tissues of the neck and chest. Airway patency was effected with tracheal intubation in 9 cases, it was maintained by noninvasive mask ventilation in 1 patient. Translaryngeal tracheal intubation and tracheostomy were used for artificial ventilation (AV) in 6 and 3 patients, respectively. Multidrug therapy (MDT) was performed in all the patients. Airway patency restored in 9 patients after MDT initiation. The duration of AV was 5.8 +/- l.7 days. The length of stay in an intensive care unit was 90%; intrahospital survival was 70%; 28-day and 3-year survivals were 90 and 24%, respectively. Two of the 3 patients who had undergone were observed to have serious complications (cicatrical stenosis, tracheoesophageal fistula). Hemoblastosis patients with tumor-induced obstruction need for provision of airway patency and for immediate initiation of MDT and/or radiotherapy. Orothracheal or nasotracheal intubation of the trachea is the methods of choice in providing airway patency. Tracheostomy is not indicated in most cases. Short-term prognosis is good in this cohort of patients. Long-term prognosis is determined by a lot of factors of which tumor sensitivity to cytostatics and radiation is most important.

[On the relation of the microtubule length and dynamics: boundary effect and properties of an extended radial array].

In interphase cells, microtubules (MT) are long and form extended radial array. The length of individual MTs in living cells exhibits substantial stochastic fluctuations while the average length distribution in a cell remains nearly constant. We present a quantitative model that describes relation of the MT length and dynamics in the steady state in the cell using the minimal set of parameters (cell radius, tubulin concentration, critical concentration for plus end elongation, and the number of nucleation sites). The MT array is approximated as a radial system, where MT minus ends are associated with the nucleation sites on the centrosome, while plus ends grow and shorten. Dynamic instability of MT plus ends is approximated as a random walk process with boundary conditions and the behavior of MT array is quantified using diffusion and drift coefficients (Vorobjev et al., 1997, 1999). We show that establishment of the extended steady-state array could be accomplished solely by the limitation of the MT growth by the cell margin. We determined for the cell radius, tubulin concentration, critical concentration for plus end elongation, and number of nucleation sites the reference point in the parameter space where plus ends of individual MT on average neither elongate nor shorten. In this case average length of MT is equal to the half of cell radius. When any parameter is shifted from its reference value MTs become longer or shorter and consequently acquire positive or negative drift of their ends. In the vicinity of reference point, change in any parameter has major effect on the MT length and rather small effect on the drift. When mean length of the MTs is close to the cell radius the drift of the free plus ends becomes substantial, resulting in processive growth of individual MTs in the internal cytoplasm accompanied by apparent stabilization of the plus ends at the cell margin. Under these conditions small changes in parameters have significant impact on the magnitude of drift. Experimental analysis of the MT plus ends dynamics in different cultured cells shows that in most cases plus ends display positive drift, which, in the framework of the presented model, is in agreement with the simultaneous presence of long MTs.

[The spatial organization of centrosome-attached and free microtubules in 3T3 fibroblasts].

Microtubules spatial organization is essential for different cellular processes to proceed normally. It is supposed traditionally, that the fibroblasts have radial microtubule array consisting of long microtubules running from the centrosome. However, the detailed analysis of the microtubule array in the internal cytoplasm has never been performed. In the current study we used laser photobleaching for the analysis of the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, and then in the bleached zone growth of microtubules in the centrosome region and in the peripheral parts of cytoplasm was analyzed. In most cases microtubules growth in the bleached zone occurred rectilinearly, on the distance up to 5 microm they seldom bend more than 10-15 degrees. We considered a growing fragment of the microtubule as a vector with the beginning in the point of occurrence and with the end in a point where growth terminated (or the end point after 30 s if microtubule's persistent growth proceeded longer). We defined the direction of microtubules growth in different parts of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the centrosome we directed the vector of comparison inside of the bleached zone from the centrosome to the beginning of the growing microtubule segment; in fibroblast lamella and in fibroblast trailing part we used, the vector of comparison was directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing immediately from the centrosome grew along the cell radius. However at a distance of 10 microm from the centrosome radially growing microtubules gave 40% from the overall number, and at a distance of 20 microm--only 25%. The rest of microtubules grew in different directions, with the preferred angle between their growth direction and cell radius around 90 degrees. Fibroblast lamella and trailing part 80% of all microtubules grew along the cell long axis or at the angle no more than 20 degrees, and 10-15% of microtubules grew along cell axis but towards the centrosome. Thus, in 3T3 fibroblasts the radial system of microtubules is perturbed starting from the distance of several microns from the centrosome. In the internal cytoplasm the microtubule system is completely disordered, and in the stretched parts of the polarized cell (lamella, trailing edge) the microtubule system again becomes well organized--microtubules are preferentially oriented along the long cell axis. From the results obtained we conclude that orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from the centrosome, but their orientation is preset by local factors.

[Primary diffuse large B-cell lymphosarcoma of the spleen].

AIM: To investigate characteristics of the course and efficacy of treatment of diffuse large B-cell lymphosarcoma (DLBL) with primary lesion of the spleen. MATERIAL AND METHODS: From 1998 to 2006, primary splenic lesion was registered in 15 of 120 patients with DLBL and affected lymph nodes (LN), spleen and Waldeyer's ring. The diagnosis was made according to WHO criteria. Of them 14 patients had splenectomy as the first stage of therapy. The operation was followed with 6 to 8 courses of CHOP-21 (8 patients), 4 courses of R-CHOP-21 and radiotherapy (one patient). One patient received 7 courses of CHOP-21 followed by splenectomy. Because of the presence of several signs of unfavourable prognosis 5 patients under 60 years were given intensive therapy: 4-6 courses of the modified program NHL-BFM-90, 2 of 5 patients received radiotherapy. RESULTS: All the patients with primary DLBL of the spleen had two and more signs of unfavourable prognosis: elevated concentration of serum LDG, size of the tumor more than 10 cm, high proliferative activity of tumor cells, B-symptoms, severe condition. Seven patients had centroblastic, 8--anaplastic variants of DLBL. Tumor cells in primary DLBL of the spleen had no specific immunophenotype. Complete remission of the disease was achieved in 9 (90%) of 10 patients treated on programs CHOP-21, R-CHOP-21, in 4 of 4 patients on the modified program NHL-BFM-90. Mean follow-up was 39.3 months (from 7 to 103 months). CONCLUSION: For primary DLBL of the spleen characteristic are long-term remissions on first line therapy according to CHOP-21 program irrespective of morphology and immunophenotype.

[Free and centrosome-attached microtubules: quantitative analysis and the modeling of two-component system].

In the internal cytoplasm of interphase cells the density of microtubules is the highest in the centrosome area and decreases to the cell periphery. As a rule, the quantity of fluorescent microtubules cannot be counted up in the internal cytoplasm, but it is possible to estimate microtubules quantity using measuring of their optical density. In living 3T3 and CHO cells the microtubules optical density decreased according to different mathematical dependences that apparently reflected the differences of their microtubule system organization. To determine appropriateness that circumscribe the reduction of microtubules optical density from the centrosome region to the direction of cell margin, we modeled cell contours with the certain ratio and interposition of centrosome-attached and free microtubules in vector schedules CorelDraw program. The decrease of optical density was analyzed in MetaMorph program as it was described earlier (Smurova et al., 2002). It was shown that fluorescent microtubules optical density decreased exponentially (y = ae(-bx)) if the system joined only microtubules growing from the centrosome up to the cell margin. The curve became smoother in the case of not all radial centrosome-attached microtubules reached the margin, and adding of free microtubules into the system led to the sharp fall in optical density in the centrosome area and to its gradual decrease at the cell periphery. The increase in free microtubules quantity changed the character of the curve describing the reduction of optical density microtubule system which included free and centrosome-attached microtubules in proportions of 5 : 1 was described by the equation of linear regression (f= k . x + b). Thus, the mathematical dependence describing the microtubules distribution from the centrosome to the cell periphery, depends on the ratio of microtubules and their relative positioning in the cell volume. The data obtained using model systems have coincided with the results of experiments. The graphs which described the increase in microtubules optical density during microtubule repolymerization after nocodazole treatment, corresponded to the graphs for model cells. Thus, the method we used allows to analyze the microtubule system in the cases when the direct observation of individual microtubules is difficult.