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The objective: to study a response of the spleen extracellular matrix (ECM) to the liposome-encapsulated dextrazide (LEDZ) administration in the stabilization period of BCG-induced granulomatosis.Subjects and Methods. A model of generalized tuberculosis inflammation was reproduced by injecting mice with mycobacteria from BCG vaccine (BCG-infected mice). Intact mice served as controls. During inflammation stabilization, the infected mice were dosed with LEDZ by inhalation and intraperitoneal injection, and main ECM components were assessed.Results. The BCG-infected mice showed a decrease in the structural components of proteoglycans (PGs) but demonstrated elevated profibrotic fractions of hydroxyproline (Hyp), enhanced activity of matrix metalloproteinases (MMPs) and no differences in the content of their tissue inhibitors (TIMPs), that indicated the fibrosis of the organ. Despite the way of LEDZ administration, the level of uronic acids in PGs and activity of hyaluronidases increased but the content of protein-bound Hyp was reduced with no differences in the content of free Hyp versus infected untreated mice. The administration of LEDZ by inhalation reduced content of profibrotic fractions of Hyp but increased galactose in PGs increased to the level of control mice. The intraperitoneal LEDZ administration decreased the protein content in PGs.

Using specific antibodies we studied the content of nucleolar SURF-6 protein, which participates in rRNA processing, in mouser spleen lymphocytes activated for proliferation with concanavalin A and compared it with the content of nucleolar nucleophosmin/B23 protein and DNA replication factor PCNA, well-known markers of proliferating cells. Using immunocytochemistry and immunoblotting methods we demonstrate that the concentration of all these proteins increases simultaneously with increasing the proportion of proliferating cells. Unlike nucleophosmin/B23, SURF-6 protein was not revealed in quiescent lymphocyte nucleoli, while the increase of its level in activated lymphocytes preceded elevation of PCNA level. These observations suggest that nucleolar protein SURF-6 can act as a marker of early T lymphocyte activation for proliferation and that it could participate in cell cycle regulation in mammals.

Using comet assay, a statistically significant increase (p < 0.05) in the level of DNA breaks in spleen cells was revealed in male CBA/lac mice exposed to gamma-radiation (1.7 cGy/day) or 90Sr (150-250 Bq/day) for 210 days. The level of DNA breaks also increased under combined exposure to both gamma-radiation and 90Sr (p < 0.05), but to a lesser degree than under exposure to each of these factors alone. Upon additional in vitro treatment of spleen cells with hydrogen peroxide, the relative increase in the level of DNA breaks was smaller in cells of irradiated mice than in the control. The ratio of the level of DNA breaks after hydrogen peroxide treatment to that before this treatment in control mice was 4.2 +/- 0.9, compared to 1.4 +/- 0.6 in gamma-irradiated mice, 1.9 +/- 0.8 in 90Sr-irradiated mice, and 2.3 +/- 0.8 in mice exposed to both gamma- and 90Sr-irradiation.

Cell hybrids obtained by fusing mouse PCC4azal teratocarcinoma cells and spleen cells induced to proliferation and treated with the demethylating agent 5-azacytidine prior to fusion are described. The obtained hybrids demonstrated no expression of T lymphocyte marker genes CD11 and CD45, which indicates possible somatic nucleus reprogramming by factors present in teratocarcinoma cells. Irrespective of culture conditions, cell hybrids demonstrated a relatively stable chromosome number: they lost on average no more than four chromosomes after 30 passages. Culturing in medium containing hypoxanthine, aminopterin, and thymidine (selective conditions) decreased the differentiation capacity of cell hybrids compared to nonselective conditions, which is likely due to the inhibition of their metabolism. For the first time, teratocarcinoma cell hybrid differentiation into cardiomyocytes under the influence of DMSO has been demonstrated in vitro.

The content of total T- and B-lymphocytes, T-rosette-forming at 37 degrees, T-autorosette-forming (ARF), FcIgM+, FcIgG+ and TDT+ lymphocytes is determined in mice after unilateral nephrectomy. The number of ARF, FcIgM+ and TDT+ lymphocytes increased 17 and 48 hours after the nephrectomy. The coincidence in time of the lymphocyte population increase with acquisition of morphogenetic activity by the spleen lymphoid cells allows one to assume their participation in the regenerative proliferation of renal epithelium. The alterations in the content of other lymphocyte populations were in the limit of random deviations.

CBA mice were given a single Desferal dose (50 mg/kg). The mice received 3H-Thymidine 1 hour before they were killed. They were killed 1, 2, 6, or 9 hours after Desferal. Radiometry, and autoradiography were used to determine the level of DNA synthesis. Desferal was found to stimulate splenic 3H-incorporation 3 hours after drug injection and to increase the DNA-synthesizing cell fraction. In contrast, the agent inhibited 3H-incorporation in the bone marrow and thymus 9 hours after injection. This suppressive affect was accompanied by a decrease in the DNA-synthesizing cell fraction in these organs. In the thymus, there was a significant increase in the DNA-synthesizing cell fraction among lymphoblasts 1 and 3 hours following the administration of Desferal.

Mammalian brain-derived "tetrodotoxin-sensitive protein" has been shown to share an epitope with as yet unidentified structure of the human and rodent lymphocyte surface. Previously obtained observations that a monoclonal antibody to this epitope induces a proliferative response of murine splenic mononuclear cells are confirmed. However, this antibody fails to modify the phytohemagglutinin-induced response. Moreover, lectin with submitogenic concentration inhibited the antibody-induced response provided that monocytes were present in the culture. The antibody-induced proliferation appeared to be less monocyte-dependent than the lectin-induced one. Taken together, these findings argue against hypothesis that a lymphocyte structure with epitope of the "tetrodotoxin-sensitive protein" is associated with either T cell receptor for antigen or interleukin-2 receptor.

Proteins released by mouse spleen nuclei under conditions of limited hydrolysis with DNase I and micrococcal nuclease were compared. Analysis of these proteins by polyacrylamide gel electrophoresis in the presence of SDS revealed the essential similarity in the qualitative composition of released proteins. Characteristics of the most prominent component were studied in both cases and it was shown that this component is identical. It has a molecular weight of 25 500 according to electrophoresis data and 2300 as determined by equilibrium sedimentation. Amino acids composition and N-terminal amino acid were studied. It was shown that its N-amino acid is arginine.

The activity of natural killers from the mouse spleen was determined by 51Cr clearance from labeled target cells (YAC-1) at different times after resection of the two-thirds of the spleen. In the early periods after operation, there was a decrease in the natural cytotoxic activity of splenocytes followed by a relatively rapid rise for 8 days until the maximum exceeding the cytotoxicity of intact spleen cells was reached. However, the total lytic potential of the regenerating spleen did not return to normal during 28 days.

The content of thymic serum factor in mice during the early postnatal ontogenesis is at the level characteristic of immunologically mature animals. Their spleen rosette-forming cells are, however, much less sensitive to antilymphocytic serum than those of adult animals. The sensitivity to the latter did not increase after incubation of splenocytes with thymosin or upon its multiple introduction to the neonatal mice. The results obtained suggest the refractoriness of the spleen rosette-forming cells to the thymic hormones during early postnatal ontogenesis.

Cytostatic and cytotoxic activity of mouse spleen cells against normal and tumour target cells has been studied. The comparative analysis of mouse spleen cell cytostatic and cytotoxic activity after exposure to cyclophosphamide has shown that the effectors of natural cytotoxic activity are highly sensitive to cyclophosphamide, while cytostatic effectors are heterogeneous in their sensitivity to cyclophosphamide. Pretreatment of spleen cells with alpha-interferon produced an increase in cytotoxic and cytostatic activity against tumour target cells. The cells of lymphoid organs (spleen, bone marrow, thymus) had greater distinctions in cytotoxic than in cytostatic activity against tumour target cells.

Two-step treatment of mouse spleen nuclei with staphylococcal nuclease was used to isolate residual nuclear structures lacking a considerable part of chromation. Partial disruption of the nuclear envelope after the first step of digestion was shown to be essential for obtaining residual nuclear structures. Isolated residual nuclear structures contained condensed chromatin (residual chromatin) which was not solubilized upon additional staphylococcal nuclease treatment and amounted to approximately 20% of total nuclear chromatin. Residual chromatin was almost deprived of nonhistone chromosomal proteins. It contained a full complement of histones and consisted of nucleosomal chains having different lengths--from one to 50-60 nucleosomes. Some of the condensed chromatin chains were anchored to the nuclear matrix.

The restriction analysis of BALB/c and AKR mice genome DNA was used to show the translocation and amplification of gene which is expressed with the Rauscher erythroleukemia as the 35S nuclear RNA. The homology of this RNA to 10 defined oncogenes was studied. It was found that the cDNA 35S RNA is efficiently hybridized only with v-sis oncogene. The clones homologous to the 35S RNA, v-sis oncogene and 3'-area of the Rauscher leukemia virus genome are isolated from the genes' library of mice erythroleukemic cells.

After a single intraperitoneal inoculation of C57BL/6 mice with enterotoxin A of Staphylococcus aureus (SEA) the activity of natural killers (NK) increases and phase change of interferon production by spleen cells upon reinduction in vitro occurs. Multiple daily inoculations of mice with SEA maintain the activity of splenic NK at a similar high level. With an adequate control (multiple administration of the medium) NK activity was maintained at the same high level. Interferon production by spleen cells of mice which were multiply inoculated with the medium upon reinduction in vitro was the same as in the control animals, whereas after multiple inoculations of mice with SEA, spleen cells in vitro produced lower amounts of interferon.

The effect of the in vivo treatment with synthetic interferon inducer B-58 on natural killer (NK) and cytostatic cell activity was studied in CBA and A/Sn mice. A marked increase in NK cell activity against target cells YAC-1 was observed in the spleen of CBA mice within 4 days after treatment. On the other hand, NK activity in A/Sn mice was not affected by B-58. However, B-58 was shown to enhance cytostatic cell activity both in CBA and A/Sn mice, when tested against target cells P 815.

In CBA mice the absolute and relative (per 10(6) spleen cells) number of antibody-forming cells (AFC) in the spleen was cut by half on the 1st, 4th, and 7th days after acute hypoxia (12 hours, 6700 m), and on the 1st and 4th days after cessation of chronic hypoxia (16 days, 16 hours, 6700 m). The number of AFC in the spleen returned to the normal level on the 7th day after cessation of chronic hypoxia. Single or double erythropoietin injections caused approximately a 1.15--2-fold decrease in spleen AFC number in posthypoxic mice in comparison with control animals.

Manganese, calcium and mercury ions, as well as p-chloromercury benzoate and dithiothreitol are studied for their effect on the activity of inorganic pyrophosphatase (EC 3.6.1.1) of mice spleen. It is shown that Ca2+ and Mn2+ are inhibitors of this enzyme, but Mn2+ in low concentrations may replace Mg2+ in the pyrophosphatase reaction. Hg2+ and p-chloromercury benzoate inhibit the pyrophosphatase activity essentially but not completely. Mice spleen pyrophosphatase is very labile: its preincubation without the substrate for 30 min at 37 degrees C leads to a complete loss of the activity. Neither glycerol, nor glutathione and cysteine but magnesium ions, dithiothreitol and 2-mercaptoethanol protect the enzyme from inactivation. The enzyme is purified by the sulphate ammonium salting-out, gel filtration on Sephadex G-100 as well as by isoelectrofocusing in 5% PAAG. Then pyrophosphatase is eluted from gel and subjected to electrophoresis in the plane layer of the linear gradient of 5-15% PAAG with SDS or 5-25% PAAG without denaturing conditions. One zone corresponding to the molecular mass of 70 kDalton is obtained. It is splitted into two zones in electrophoresis with SDS and 2-mercaptoethanol.

The effect of the thymus preparation thymalin (1000 micrograms/mouse, i.p.) on the system of cyclic nucleotides in the spleen has been studied. The preparation produced two-phasic changes in the content of cAMP and cGMP, as well as in the activity of cAMP phosphodiesterase. The changes in the spleen cAMP content are consequent on those in the activity of phosphodiesterase.

Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) stimulated murine splenocytes in vitro to produce cytotoxic factors (CF) that killed target cells L-929. This effect was synergic at LPS dose of 10 ng/ml and MDP dose of 10 micrograms/ml. CF production started 2 hours after spleen cell activation and was maximum in 6 hours. CF were produced by macrophages as well as by lymphocytes stimulated by LPS, MDP or their combination. However, synergic effect of immunomodulators was registered only if nonfractionated spleen cells were stimulated during 24 hours. Lymphocytes depleted on T cells did not lack the ability to generate CF upon activation. In addition, LPS and MDP activated synergically the production of interleukin-I by spleen cells in vitro.

The content (per 10 nucleated cells) and avidity curves of IgM positive B-lymphocytes forming rosettes with trinitrophenyl-sheep erythrocytes (TNP-RFC) were compared in two mouse lines (BALB/c C57BL/6), F1 hybrids (BALB /c X C57BL/6) and backcross hybrids (F1 X BALB/c, F1 X C57BL/6). Trinitrophenyl-bovine serum albumin (TNP24BSA) and dinitrophenyl-bonvine serum albumin (DNP23BSA) and sulfanil-BSA (Sulf17-BSA) in different dilutions were used as inhibitors. BALB/c spleen contained by 60% more TNP-RFC than C576/6 spleen. F1 hybrids (F1 X BALB/c) contained on the average by 35% more TNP-RFC than (F1 X C57BL/6). Inhibition curves (avidity curves) were essentially different in the two mouse lines and F1 hybrids. A conclusion was drawn that the TNP-REC content and avidity were under a strong genetic control. Together with the assumption of random expression of V-genes (idiotype genes?) in the lymphocytes the above data suggest that the simplest mechanism of the genetic control could be a definitive ratio between the corresponding groups of V-genes.

Properties of condensed residual chromatin of mouse spleen, a component of residual nuclear structures, were studied. Extraction of the structures with buffers of different NaCl concentrations showed that the condensed chromatin consists of condensed nucleosomal chains. On increasing the ionic strength the complexes gradually fell apart into separate nucleosomal chains. DNA of condensed chromatin was accessible to staphylococcal nuclease and DNAase I, but digestion of this DNA was not accompanied by solubilization of the residual chromatin. Besides the essentially decreased total content of nonhistone chromosomal proteins the condensed chromatin practically did not contain HMG proteins. The nucleosome repeat length of this chromatin was shorter than that of chromatin solubilized by staphylococcal nuclease.

The mitogen activity of a low molecular lymphocytosis stimulating substance isolated from the thymus (LSS) has been investigated in cultured mouse CBA spleen cells. LSS caused transformation of lymphocytes to the blast cells capable of mitotic division. The initial signs of transformation were observed after 24 hours of incubation, reaching the maximum in 48 hour old cultures. The stimulating action of LSS held true for about 7 days. The intensity of blastogenesis depended on the dose of LSS. A high concentration LSS inhibited the blastogenic response. B-lymphocytes are not sensitive to LSS. It is concluded that the low molecular humoral factor of the thymus activated specifically the thymus-depended lymphocytes.

Some properties of mouse spleen cytosol inorgainc pyrophosphatase (PPi-ase) (E. C. 3.6.1.1) as well as the effect of methylene diphosphonic acid (PCP) on the PPi-ase activity were studied. Specific staining for the enzyme PAAG disc-electrophoresis was developed; it was shown that the PPi-ase formed only one band in 7.5% PAAG. The enzyme pH optimum being 8.0, the optimal [Mg++]/[PPi] ratio was about 2; Km =7.7x10(-4) M, Vmax=0.77 mkM. min-1. mg protein-1. PCP was shown to competitively inhibit the pyrophosphatase reaction, Ki=2.5x10(-4) M +/- 0.2x10(-4) M.

Accumulation of erythropoietic, granulopoietic cell elements, a change in the ration and absolute count of the T, B cells, and "null" lymphocytes, as well as a sharp inhibition of primary immune response were found in the spleen of CBA mice treated with antierythrocyte sera. The inductive phase of immunogenesis proved to be sensitive to the action of antierythrocyte sera. The data obtained apparently demonstrated the existence of a functional relationship between the immunocompetent spleen cells and their non-lymphoid hemopoietic microenvironment.

Synthetic polyanion pyran (copolimer of divinyl ether and maleic anhydride) injected to mice increases the titre of antibody to sheep red blood cells as well as the activity of serum DNAase I and DNAase I splenic inhibitor. Simultaneously a growth of the spleen weight takes place. A possible role of the DNAase I inhibitor system in the mechanism of the adjuvant action of synthetic polyanion is discussed.

The migration activity of the spleen cells from intact mice is inhibited by the stem cell inhibitory factor (SCIF) released by lymphocytes treated with antilymphocytic globulin. The degree of the migration inhibition is proportional to the activity of SCIF in the colony-formation inhibition. The macrophage-migration inhibitory factor (MIF), obtained in the H-2 system exhibited a stimulating effect on the colony formation in mice when used in vitro for the treatment of bone marrow transplants. This activity of MIF corresponds to its migration-inhibitory effect on the spleen cells. Incubation of the bone marrow cells with MIF for 30 minutes is more effective than the 2-hour treatment. The observed effects are interpreted as an indication of non-identity of SCIF and MIF.

Non-histone protein selectively released from mouse spleen nuclei by mild hydrolysis with micrococcal nuclease or DNase I was introduced into L cells by the microinjection method. It is shown that this protein selectively accumulates in cell nuclei. Analysis of the mitotic cells after microinjection of 125I labeled protein demonstrated its location in chromosomes of L cells.

The spleen (2/3) was removed in CBA male mice (the 1st group); in the 2nd group the bone marrow from the right posterior shin was removed. The hemopoietic splenic colonies were counted on the 8th day after the lethal irradiation and injection of 1 X 10(-6) nucleated cells of the intact spleen. A significant increase of the number of colonies in comparison with their number in control intact mice was observed. The authors suppose that this increase could also be caused by the local influence of the regenerating stroma of the spleen and by some stimulating factor discharge by the regenerating hemopoietic tissue.

Splenocytes obtained from mice on the 5th day after the injection of sheep red blood cells (SRBC) lost irreversibly approximately 50% of their surface immunoglobulins in the course of four-hour in vitro incubation. On the contrary, there were no changes in the total amount of surface immunoglobulins following in vitro incubation of splenocytes obtained on the 9th day after the antigen injection. Metabolism of antigen-binding receptors of immune splenocytes was studied by the method of rosette-formation. Incubation for 20 hours of splenocytes obtained on the 5th day after the SRBC injection demonstrated a loss of 70% of rosette-forming (RFC). The remaining RFC belonged to the theta-positive lymphocytes with the half-life of their antigen-binding receptors of approximately 4 hrs. Similar half-life was found for the RFC receptors of mouse spleen on the 9th day after the antigenic stimulation. No reduction of the RFC count occurred during their in vitro incubation for 20 hrs. It is supposed that a reduction in the number of the RFC obtained at the peak of the primary immune response could result from the inability of immune lymphocytes to synthesize new receptors or from blocking the newly-formed receptors by a soluble factor produced by immune lymphocytes following their in vitro incubation.

A study was made of He-Ne laser radiation (lambda = 632.8 nm) on spontaneous chemiluminescence of mouse splenic cells and that stimulated by addition of Candida albicans. Irradiation with low-intensity red light was shown to stimulate cell chemiluminescence and to intensify that stimulated by C. albicans within the dose range from 100 to 300 J/m2 with a maximum at about 200 J/m2.

Non-lethal and small lethal doses of short-wave (254 nm) and long-wave UV light (365 nm), the SUV and LUV rays, when acting upon the murine spleen cells, induce an increase in expression of the membrane markers of T-lymphocytes [theta-(Thy-I)-antigens] and B-lymphocytes (MBLA-antigens and EAC receptors). The sum of cells with T- and B-lymphocyte markers in an irradiated suspension is even more than the total amount of cells in a non-irradiated suspension (100%), which indicates the possibility of expression of those antigens and receptors of the plasmic membrane which are not revealed in non-irradiated lymphocytes. In the range of isolethal dosages (LD15-LD28) LUV irradiation suppresses, while that of SUV induces further increase of the expression of theta- and MBLA-antigens. From among the membrane markers of B-lymphocytes, the MBLA-antigens are more resistant against LUV and SUV irradiation than EAC-receptors.

Significant suppression of the entry of leukemic spleen cells in mitosis was found to be produced by the administration of colchamin (5 mg/kg) to mice with transplanted leukemia La. These data allow a conclusion that the duration of mitosis and the rate of mitotic activity in tissues cannot be determined by the colchamin method.

Early alterations in the enzyme activities controlling cyclic adenosine-3',5'-monophosphate (cAMP) metabolism after the irradiation (800 rad) of mice were found in the lymphoid cells of the spleen and thymus. Postradiation disturbances in these activities' ration induced alterations in cAMP steady-state concentrations in the cell. It was also demonstrated that irradiation reduced lymphoid cell ability to accumulate cAMP in response to isoproterenol administration.

The effect of distribution of injected colony-forming units (CFU) in the recipient's organism was studied. The distribution was assessed by the "sedimentation factor" (f) criterion. This factor was found by the number of colonies revealed on histological sections of the spleen and femoral bone marrow in comparison with the number of colonies visible on the surface of the spleen. Macroscopic count of the colonies on the spleen permits, to assess the f value both in the whole spleen volume and in the femoral bone marrow. In this case f value was independent of the character of CFU differentiation. f value could change depending on the physiological condition of CFU population. In irradiation of the bone marrow CFU in a dose of 200, 400 and 600 R f value in the spleen decreased in comparison with control, but remained unchanged in the bone marrow.

The inorganic pyrophosphatase activity was determined in different tissues of mice. The immunization of mice by sheep erythrocytes increased the inorganic pyrophosphatase activity of the spleen. The in vivo administration of bisphosphonates (40 mg per 1 g of mass), which are structural analogs of inorganic pyrophosphate (methylene bisphosphonic acid--MBPA, hydroxyethylidene bisphosphonic acid--HEBPA and aminomethylene bisphosphonic acid--AMBPA), inhibited the inorganic pyrophosphatase activity only by MBPA in the thymus and spleen but not in liver. The addition of MBPA, HEBPA as well as of phosphonoacetic acid, imidobisphosphate, bis(phosphonomethyl)-phosphonic acid, MBPA and phosphoric acid monoanhydride to cytosol from the mouse spleen led to the competitive (relative to the [Mg (PPi)2-] complex) inhibition of the inorganic pyrophosphatase activity. AMBPA didn't possess the analogous effect.