Hemophagocytic lymphohistiocytosis in acute African swine fever clinic.

INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) usually has been defined as the combination of a proliferation of cytologically benign, actively phagocytic macrophages in bone marrow, spleen, lymph nodes, etc. in association with fever, cytopenia, splenomegaly, and hypertriglyceridemia. HLH is often triggered by viral infection. The aim of this study was to ascertain the features of HLH involvement in African swine fever virus (ASFV) (genotype II) pathogenesis. METHODS: The serum levels of macrophage colony-stimulating factor (MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF), as well as the histological constitution (for hemophagocytic macrophages detection) of various organs of pigs infected with ASFV genotype II were investigated. The diagnosis of HLH was made according to universally accepted human criteria. RESULTS: The association of fever, cytopenias, splenomegaly, and hemophagocytosis was present in 87.5% of the infected pigs (absence of hyperthermia in one of eight pigs). Marked hypertriglyceridemia was observed at 3-4days post infection. Previously it was shown that ASFV induced a significant decrease in the level of fibrinogen from day 5 till the end of experiment. Progression of the HLH coincided with a temporary increase in the serum levels of MCSF levels (early stage of disease) and GMCSF levels (2-3 pays post infection). CONCLUSIONS: Hemophagocytic syndrome should be suspected in ASFV (genotypeII) infected pigs.

[Influence of ionizing radiation on enzymatic activity and state of nucleus-nucleolar apparatus in rat hepatocytes].

The effects of a single exposure of rats to the whole-body roentgen irradiation at the doses of 3.5 Gy and 4.5 Gy on the activity of creatine kinase, purine nucleoside phosphorylase, alanine aminotransferase, aspartate aminotransferase, as well as on the state of the nuclear-nucleolar apparatus in rat hepatocytes on the 6th and 13th days after radiation exposure have been studied. Irradiation at the above doses induced changes in the levels of enzymatic activity of different values and different directions within the same time periods, as well as oscillating changes in this type of enzymatic activity over time. This demonstrates various radiosensitivity and adaptation abilities of these enzymatic activities. The changes in the enzymatic activity significantly correspond to the changes in the morphometric indices of nuclear-nucleolar apparatus of hepatocytes, as well as the distribution of hepatocytes within the ploidy classes: in particular, stabilization of the enzymatic activity on the 13th day after irradiation correlates with the increased transcriptional activity, which is detectable through the increased number of nucleoli per nucleus and the expanded space of a hepatocyte nucleus. The compensation mechanisms are likely to be targeted at the changes in the functional activity of surviving hepatocytes, rather than at the replacement of the damaged cells by the new ones.

[The reaction of the neuroblastoma cells in the culture on the influence of tretionine and neurotoxine].

Effect of the tretionine (retinoid) and aluminum chloride (neurotoxin) on the growth and differentiation of neuroblastoma cells in culture after their introduction into the medium separately and in combination was studied. The introduction of these substances creates a new information field in the medium, which becomes apparent by the reactions of neuroblastoma found on the populational and cellular levels of its organization. The presence of tretionine stimulates proliferation and induces differentiation of the cells into astrocytes. Aluminum chloride inhibits cell proliferation and enhances the process of their destruction in the monolayer. The variety of the reactions of neuroblastoma cells to the presence of these substances in the medium indicates the existence and functioning of a mechanism that selects from the information introduced only the portion which may contribute to adaptation of neuroblastoma cells to the changed culture conditions.

[Pathology of lymphoid tissue cells infected by African swine fever virus in vitro].

The authors studied the pathology of bone marrow (BM) lymphoid cell from pigs infected by African swine fever virus (ASFV) in vitro. Monocytes were shown to be primarily afflicted in unstimulated BM culture. These cells disappeared completely 72 hours after infection. Just 24 hours following ASFV infection, there were atypical lymphocytes amounting to 12% of the general lymphoid population at hour 72 after inoculation.The area and perimeter of minor, middle, and large lymphocytes tended to reduce during both BM cell cultivation and inoculation. Lymphoblasts and monocytes were generally triploid in both the control and test groups, but among them there were diploid, triploid, and tetraploid cells. Cytophotometric assay revealed that the amount of nuclear DNA significantly increased in BM lymphoblasts and monocytes in the early stages of ASFV infection (within 24 hours). This effect was also rather pronounced in the lymphoblasts in the later stages (at hour 72).

Properties of cells with increased resistance to some picornaviruses.

The resistance to picornaviral infection cells of susceptible lines has similar changes in the phenotype. They have decreased number of nucleoli and increased percentage of euploidy. Also the percentage of euploid cells those were resistant to the picornaviral infection increased in all highly transformed cultures. In resistant cells of all cultures has been found reduction of DNA. RNA amount also decreased both in nucleus and in cytoplasm. All these data correlated with the increased euploidy of the resistant population. The resistant cells had a less transformed phenotype, and decreased proliferative activity. Decreased nucleolar status became apparent by reduction of absolute and relative nucleolar indices. Consequently the reduction of viral titer (viral titters reduction) in resistant cells could be the direct result of diminished activity of the RNA synthesis machinery. It is important to note that the cells lose resistance while another type of virus, even from the same family, infects the culture once.

Phenotypic and cytologic studies of lymphoid cells and monocytes in primary culture of porcine bone marrow during infection of African swine fever virus.

We have modeled in vitro infection of African swine fever virus (ASFV) in primary unstimulated cells of the porcine bone marrow and have studied the phenotypical changes in the population of porcine lymphoid cells by cytophotometry. Monocytes and large-sized lymphocytes completely vanished in 72 h of infection which is result of high sensitivity of those cells to ASFV. We describe DNA synthesis in monocytes at 24 h post infection. Cytophotometry of the uninfected cells revealed the few number of atypical lymphocytes and lymphoblasts after 72 h of cultivation; whereas in viral infected cultures, atypical cells appeared in large quantity (about 14%) with 24 h. Most of atypical lymphocytes and lymphoblasts had altered nucleus, and only a small number of atypical cells had additional nucleus. The cytophotometry of main and additional nuclei showed that DNA content didn't exceed diploid standard which indicates that the additional nuclei were consequence of fragmentation of nuclei in lymphocytes.

[The influence of solcoseryl on population and cellular parameters of HeLa and RD cultures].

Changes of population and cellular parameters of HeLa and RD cultures after introducing of solcoseryl in culture medium were studied by methods of scanning cytophotometry and cytomorphometry. Monolayer density, proliferation activity, the number of dead cells in a monolayer, the number of nucleoli in nuclei and distribution of cells in the populations by this parameter, RNA and DNA masses in nuclei and nucleoli, total volumes and surface areas of the nuclei and nucleoli were determined. It has been shown that solcoseryl differently affects the cultures both on population and on cellular levels of their organization. The results of multi-parametric analysis of the influence of solseryl on the cultures allow considering it as a biologically active compound with the features typical for cell and cell population growth regulating factors.

[Multiparametric study and cluster-analysis of the transformed cultures of human cells].

The cells of nepatocarcinoma (HEp-G2), adenocarcinoma of large intestine (Caco-2), embryonal kidney (HEK-293), neuroblastoma (SH-SY5Y), rabdomyosarcoma (RD), and larynx cancer (Hep-2) were studied by the methods of scanning cytophotometry, cytochemistry and cytomorphometry during 96 h of cultivation. The density of monolayers, proliferation activity, the number of dead cells, DNA content in the nuclei and distribution of the cells in the population by this parameter, total DNA content in the nucleoli (circumnucleolar chromatin), the number of nucleoli in the nuclei, distribution of cells by their number, the volume and area of the nucleus surface, total volume and area of the nucleoli surface were determined. The data obtained were used in the treelike cluster analysis of the cultures by Pierson correlation. As a result, the SH-SY5Y culture was put in a separate cluster, while Caco-2, HEp-G2, HEK-293, Hep-2 and RD cultures were placed in the tree of another cluster. The least transformed culture of neuroblastoma (SH-SY5Y) had no relationship with other cultures, which showed various rate of similarity. The cultured HEK-293, Hep-2 and RD appeared to be close to each other by all parameters. The parameters studied are of different significance for the formation of general pattern of the cell cultures. The greatest "weight" is carried by the parameters, which characterize the population as whole: the density of the monolayer, mitotic coefficient and the number of dead cells. They are followed by the content of DNA in the nuclei, the total area of the nucleoli surface, and ratios of DNA content in the nucleoli to DNA content in the nucleus and of total surface of the nucleoli to the surface of the nuclei. Other parameters are not so significant.

Behavior of lymphoid cell population, cell nuclei and nucleoli in periodic disease and leukemia.

Similar behavior of lymphoid cells, their nuclei and nucleoli in periodic disease and leukemia attest to nonspecific reaction of the immune system to these diseases, but the intensity of this reaction and mechanisms of the population recovery are different. DNA hyperreplication plays an important role in this process: in periodic disease it is realized via gene amplification, which manifests by the formation of H2c nuclei and increase in the number of nucleoli, while in leukemia bone marrow lymphoblasts double the DNA content during S phase, maturate during G2 phase, and then divide. We called this mechanism "reserve lymphopoiesis" by analogy with reserve erythropoiesis discovered previously by us.

[Human large intestine adenocarcinoma cells (CaCo-2) in the process of cultivation].

Using cytomorphometry and cytophotometry cells of human large intestine adenocarcinoma (CaCo-2) were studied under condition of a 10 day cultivation. A reverse dependence was established between proliferative activity and monolayer density. The increase of the latter inhibits proliferation and promotes the formation of islets of polymorph cells. 2c-cells could be seen only at the beginning of culture growth; a larger part of cells polyploidized by cell blocking in G2-phase. These cells do not divide, which is testified by the absence of 2c-cells, but some part of 4c-cells start the next cycle, accumulates 8c-DNA and then divides, replenishing the 4c-cells population. In the process of cultivation, we observed an increase in the number and total volume of nucleoli in the nuclei, and a rise in DNA amount in the peri-nucleolar chromatin. The formation of numerous 4c-cells with multi-nucleolar nuclei may define an increase of functional activity of CaCo-2 culture as the whole, whereas the formation of separated groups of such cells in the monolayer may denote a possible initiation of their differentiation.

Comparison of impact of EMCV replication on the nuclear apparatus of NIH 3T3 and HEp-2 cells.

We have investigated differences between the actions of encephalomyocarditis virus (EMCV) on cytometric indices in cultured NIH 3T3 and HEp-2 cells, which are characterized by different levels of transformation. HEp-2 cells surviving 48 h after EMCV infection showed lower nuclear ploidy, reduced nuclear area, fewer nucleoli and a higher percentage of euploid cells. There was a significant increase of nucleolar/nuclear DNA 6-24 h after EMCV infection. However, EMCV had markedly different effects on NIH 3T3 cells: there was a consistent increase in population ploidy, but the average number of nucleoli and the number of euploid cells in the population remained constant. The nucleolar/nuclear DNA ratio was almost unchanged. These different viral effects might be explained by the contrasting levels of differentiation of the cultured cell lines. The number of nucleoli does not depend on the amount of nuclear DNA in either viral-infected or intact cells but on the euploidy-to-aneuploidy ratio. The ratio of the sums of the nucleolar perimeters to the nuclear perimeter increases linearly with the number of nucleoli per nucleus in both intact and virus-infected cells. In both cell lines, the amount of DNA per nucleolus decreases as the number of nucleoli increases.

[Nuclei and nucleolar organizing regions in chromosomes of lymphocytes on different stages of periodic disease]. / Povedenie iader i iadryshkoobrazuiushchikh raionov khromosom limfotsitov na raznykh stadiiakh razvitiia periodicheskoi bolezni.

By scanning cytomorphometry a cytological study was first performed on the behavior of nuclei and nucleolar organizing regions (NOR) in chromosomes of peripheral blood lymphocytes of healthy men and of patients with periodic disease (familial Mediterranean fever, FMF) on different stages of development, including its complication with amyloidosis. The volume and total surface of nuclei, the sum total volume and sum total surface of NOR, the mean number of NOR for one nucleus and distribution of nuclei according to NOR number were measured. It is shown that the parameters of nuclei and NOR for patients with FMF on all stages clearly and trustworthy differ from those for healthy men. They are sufficiently informative, can be successfully used in clinical practice and even serve as an early diagnostic test for amyloidosis complication.

Comparative analysis of effects of various types of double-stranded RNA on cultured human laryngeal cancer cells.

We compared the effects of Na+ and Ca2+ double-stranded RNA on cultured human laryngeal cancer cells by cytomorphometry and cytophotometry. Both agents inhibited proliferation and other cell functions, but to a different extent: Ca2+ double-stranded RNA was more active than Na+ double-stranded RNA.

[Behavior of HEp-2 cell line cells, their nuclei and nucleoli during cultivation and under the effect of Na-ds-RNA]. / Povedenie kletok perevivaemoi kul'tury HEp-2, ikh iader i iadryshek v protsesse kul'tivirovaniia i pod deistviem Na-ds-RNK.

The behavior of human larynx cancer cells (HEp-2) and of their nuclei and nucleoli during the cultivation without the influence of Na-ds-RNA and after its introduction into the medium was investigated by methods of cytomorphometry and cytophotometry. The density of monolayer (the number of cells on the area unit), percentage of two-nuclear cells, the number of nucleoli in the nuclei, mitotic coefficient, volume and total surface of nuclei and nucleoli have been measured. In addition, the mass of DNA in the nuclei and that of the total RNA and DNA in the nuclei and in each nucleolus was measured. Cells in the culture, not subjected to the influence of Na-ds-RNA, were weakly differentiated, kept active proliferation, and their population contained a small number of two-nuclear elements and a high share of multi-nuclear cell. During cultivation, these indices became even more pronounced, which is typical for the increase in cell malignancy. Under the influence of Na-ds-RNA, the proliferate activity decreases, the number of double-nuclear cells increases, while that of multi-nucleolar cell decreases; also, the share of cells with one- and two-nucleolar nuclei increases. The authors conclude that Na-ds-RNA may have antineoplastic activities, clearly evidenced from its influence on the culture of transformed HEp-2 cells.

[The effect of calcium precipitate of double-stranded RNA on erythropoiesis in rats with acute anemia]. / Vliianie kal'tsievogo pretsipitata dvuspiral'noi RNK na éritropoéz u krys pri ostroi anemii.

On the model of experimental acute anaemia of rats, induced by injection of phenilhydrazine, the influence of calcium precipitate of double-stranded RNA (Ca-ds-RNA), introduced during the crise of anaemia, on the process of erythron restoration, was studied. In the presence of Ca-ds-RNA the number of pro- and erythroblasts in fission increases by 1.5 times, compared with "pure" anaemia situation, and accordingly there is a marked decrease in the share of microcytes, which play an important role in the restoration of cell number, and in the erythron recovery after the crise of anaemia. Less pronounced is the influence of Ca-ds-RNA on the macrocytosis, which keeps its value in spite of the increase in the speed of development and of the number of normocytes after Ca-ds-RNA introduction. The mechanism of Ca-ds-RNA inclusion in the system, which controls the process of erythropoiesis during anaemia, is discussed. A conclusion is drawn that Ca-ds-RNA may directly affect the inductive stage of erythropoiesis, stimulating the formation of competent erythroid cells in population of stem haemopoiethic cells and their proliferation. Further development of bone marrow cells takes place according to the known programme of erythropoiesis whose variations are stimulated by the current conditions of its realization, but not by the presence of Ca-ds-RNA.

[Changes in the kinetics of the hemoglobin, total protein and DNA content in rat erythroid cells in experimental anemia. II. The bone marrow]. / Izmeneniia v kinetike soderzhaniia gemoglobina, summarnogo belka i DNK v éritroidnykh kletkakh krys pri éksperimental'noi anemii. II. Kostnyi mozg.

Cytophotometry and population analysis of rat bone marrow erythroid cells, during anaemia induced by phenylhydrazin (4-8 days after the beginning of infections), revealed that in the period of restoration of erythron after the rats acute anaemia in the bone marrow blood formation the reciprocally complementary processes of microcytosis, macrocytosis and normal erythropoiesis occur. These processes are based on the normally functioning regulation mechanisms and on the reserve mechanisms, including DNA hyper-replication, which are turned on in the extreme conditions. The various paths of the bone marrow erythroid cells development during anaemia, the means and rates of the micro- and macrocytes formation and part of each line of the erythrocyte development in the restoration of blood cells quantity and haemoglobin homeostasis are analysed. It is suggested that mechanisms of the reserve erythropoiesis, activated during the acute anaemia of adult animals, could function also during the process of normal primary erythropoiesis of embryos.

[Immunocytophotometric and cytogenetic analyses of T-lymphoblastoid cell lines in experimental HIV Infection]. / Immunotsitofotometricheskii i tsitogeneticheskii analiz kletok T-limfoblastoidnykh linii pri éksperimental'noi VICh-infektsii.

The accumulation of viral proteins p24 and gp41 in MT-4 cells was shown to occur asynchronously. No increase in the number of polydiploid cells, no polyploid mitoses or chromosome defects were found indicating the antiproliferative effect of HIV on MT-4 cells. A cyclic pattern of the infection course was observed in infected Jurkat-tat cells. The accumulation of viral proteins was concerted, their maximum amounts preceding the cell death. HIV-1 did not inhibit cell division and caused strong disorders in the chromosome structure.

[Changes in the population composition and the rates of proliferation and specialization of erythroid cells in rats with experimental anemia]. / Izmeneniia sostava populiatsii, tempov proliferatsii i spetsializatsii kletok éritroidnogo riada krys pri éksperimental'noi anemii.

Cytomorphologic, morphometric and population analyses have revealed that during acute anemia, induced by phenylhydrazin, the proliferative activity of erythroblasts increases, an abrupt change in the structures of erythroid populations of both bone marrow and peripheral blood is observed, together with the increase in the rates of their specialization. Also, the ratio of the stem cells which differentiate by the erythroid pathway is increased, and the macro- and microcytosis mechanisms, which are essentially salient in normal conditions, are switched on. The roles of macro- and microcytes in the restoration of the normal amount of erythroid cells and haemoglobin contents in the blood are discussed.

[Biological effect of human erythropoietin in transgenic mice]. / Biologicheskii éffekt éritropoétina cheloveka u transgennykh myshei.

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).

[DNA synthesis and content in the macrophage nuclei of normal mice, during carcinogenesis and during the administration of retinoids to the animals]. / Sintez i soderzhanie DNK v iadrakh makrofagov myshei v norme, pri kantserogeneze i pri vvedenii zhivotnym retinoidov.

It has been found that nearly 50% of the lymph node and spleen macrophages (MP) of the CBA line mice contain DNA at levels superior to the diploid value (H2c--H4c in mononuclear MP, and up to H16c among polynuclear ones, the latter comprising 2.5-9.0% of the whole MP population). No DNA synthesis and mitosis were detected by autoradiography, cytophotometry, and cytomorphological analysis. During carcinogenesis the proportion of MP with elevated DNA amounts ("activated MPs") decreases due to their migration to tumours. Also immature MPs (1.6%) appear in the population, which synthesize DNA, but do not divide. Injection of retinoids restores the percentage of MPs with elevated DNA amounts to the levels characteristic of the intact animals, the fraction of DNA-synthetizing cells increasing up to 2.8%. It is proposed that retinoids may accelerate the processes of MP maturation, activation and renewing. A mechanism of cooperative action of MPs and retinoids is discussed in addition to the role of DNA hyper-replication.