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Aim: To enhance the efficiency of treatment in patients with secondary osteoarthritis (ОА) in the presence of comorbid condition, by using an interleukin-1 inhibitor (IL-1i) and laser therapy (LT)., Subjects and Methods: A total of 248 patients aged 38 to 65 years with RA and secondary OA who had predominantly Stage II in accordance with the Disease Activity Score 28 (DAS28) were examined. According to the received therapy, the patients were divided into 4 groups: 1) IL-1i + LT + mrthotrexate (MT); 2) IL-1i + MT; 3) LT + MT; 4) MT. The efficiency of treatment was evaluated from changes in the KOOS (Knee injury and Osteoarthritis Outcome Score), DAS 28, IL-1, and cartilage oligomeric matrix protein (COMP) 6 months later., Results: There were statistically significant functional improvements in KOOS and DAS28 in Groups 1 and 2 patients with secondary OA in RA. Clinical efficacy was confirmed by positive changes in serum IL-1 and COMP levels., Conclusion: Incorporation of an IL-1i into a therapy regimen for secondary OA in RA patients during basic therapy could not only improve the functional status of patients, but also decrease activity of the underlying disease according to the DAS 28.

We estimated the effectiveness and safety of methotrexate monotherapy of sarcoidosis in 63 patients refractory to the previous treatment. Methotrexate was given weekly at a dose of 5-20 mg. 54% of the patients reported improvement of their condition. Parameters of spirogram and the ray picture improved in 63.2 and 54% of the cases respectively. Methotrexate proved safe and had to be discontinued only in 12.6% of the patients. It is concluded that methotrexate can be used at least in half of the cases as a safe alternative to systemic steroid therapy.

Unlabelled: DAS28 index calculated with regard for ESR, the number of swollen/painful joints and evaluation of the patient's condition by VAS is universally used to estimate activity of rheumatoid arthritis (RA). There is a variant of calculation using C-reactive protein (CRP) instead of ESR. Our experience indicates that ESR decreases more slowly than CRP during treatment and better reflects dynamics of patients' condition. From the practical standpoint it is important to estimate activity of RA because therapeutic modalities are chosen based on the DAS28 value., Aim: To study the influence of pharmaceutical form of methotrexate on the acute-phase response in rheumatoid arthritis., Materials and Methods: The study included 32 patients (24 women, 8 men) aged 19-76 (mean 47.5 +/- 28.5) yr with active RA (DAS28 > 3.2) 4-30 months (11.5 +/- 7.4, median 8) in duration. Diagnosis was made using AXR criteria (1987), none of the patients previously received methotrexate injections. Inclusion criteria: initially high ESR (Westegren, mm/hr) and/or CRP (mg/l measured by a highly sensitive method). All patients were given methotrexate subcutaneously for 12 weeks as monotherapy (initial dose 10 mg, maximum one 25 mg/week). The cumulative dose was 211.36 +/- 17.2 mg., Results: Side effects did not require withdrawal of methotrexate. CRP level decreased faster than ERS: a 70% decrease of CRP by week 12 was recorded more frequently than that of ESR. Slow dynamics of the number of swollen joints compared with CRP may be due to the low cumulative dose of methotrexate. Duration of the disease had no effect on dynamics of acute phase characteristics., Conclusion: Methotrexate injections resulted in markedly delayed development of clinical signs of improvement compared with laboratory values. CFP levels fell down much faster than ESR, Remission or low activity of RA (estimated from DAS28) occurred only in 38% of the cases after 3 month monotherapy by methotrexate injections. It is concluded that efficacy of this drug should be estimated no sooner than 4 months after the onset of the treatment.

The aim of the work was to assess the quality of life in patients with rheumatoid arthritis treated with rituximab in combination with methotrexate or methotrexate monotherapy. The statistically significant improvement of quality of life in both groups 12 months after onset of the treatment was roughly identical. Rutiximab was prescribed after ineffective treatment with TNF-α inhibitors.

We have experimentally investigated the detoxifying and modifying effects of remaxol in the framework of traditional and high-dose chemotherapy in mice with transplanted tumors. The influence of remaxol in comparison with heptral was studied on the toxic and therapeutic action of cytostatic drugs gemzar, lastet (etoposide), and methotrexate during their traditional and high-dose administration in mice with transplanted P388 lympholeukosis. Remaxol demonstrated a detoxifying action with respect to these cytostatic agents, which decreased in the following series: gemzar lastet methotrexate (according to the results of lethality evaluation). Remaxol significantly increased the therapeutic efficacy of gemzar used in both traditional and high-dose regimes. This was manifested by slowing down tumor growth and increasing animal lifetime during combined administration of the drugs. This may be due to the remaxol potentiation of the antitumor effect of gemzar. In combination with lastet and methotrexate, remaxol does not alter their therapeutic efficacy. The detoxifying action of remaxol with respect to these cytostatics is more pronounced than that of heptral.

In the current study a technique for microencapsulation of human breast adenocarcinoma cells MCF-7 in alginate-chitosan microcapsules is used. Microencapsulation is proposed to generate multicellular tumor spheroids (MTS) based on these cells and to test them further as an in vitro model for anti-tumor drug screening. Cytotoxicity of methotrexate (MTX) was studied on the obtained MTS. A set of MTS with mean size of 150, 200 and 300 m was prepared in function of a cultivation time. After incubation of MTS in cultivation medium containing MTX at concentrations of 1, 2, 10, 50 and 100 nM for 48 hs cell viability was evaluated. MTS were shown to be more resistant to MTX than the monolayer culture, and the resistance to MTX was increased with enhancing a spheroid size. At MTX concentration of 100 nM a number of viable cells in MTS with the size of 300 m was 2.5-fold bigger than that one in monolayer culture. It is suggested that the cells in microencapsulated MTS can better mimic cell behavior in a small size solid tumor than the cells in a monolayer culture. In future microencapsulated MTS can be proposed as a novel in vitro model for anticancer drug screening.

Aim: To assess the effects of cyclophosphamide (CP), methotrexate (MT), and prednisolone (PS) on the levels of staphylococci, enterococci, Escherichia coli (EC) and lactose-negative enterobacteria (LNEB) in feces of white mice as well as to determine adhesive properties of isolated microorganisms., Materials and Methods: Drugs were administered intragastrically during 6 weeks; CP in dose 35.4 mg/kg daily; MT - 2.5 mg/kg over week; PS - 2.5 mg/kg daily; sterile distilled water was administered to controls. Microflora was assessed by common methods; adhesion of microorganisms to group 0 (I) human erythrocytes was determined., Results: Administration of prednisolone leads to increase of isolation rate of staphylococci, CP increased levels of LNEB and EC with suppressed enzymatic activity (P < 0.05). MT decreased isolation rate of typical EC (P < 0.05). MT also decreased degree of adhesion of EC and LNEB, whereas PS enhanced adhesiveness of LNEB, and CP decreased adhesion of staphylococci (P < 0.05). Statistically significant correlation between number and adhesiveness of enterobacteria was established (r = 0.73, P < 0.01)., Conclusion: Immunodepressors from different groups have non-similar effects on quantitative characteristics of microflora and adhesiveness of isolated opportunistic microorganisms.

The efficiency of the chemotherapeutic agent methotrexate (MTX) in tumor cells is limited by the frequent development of the drug resistance of tumor cells. We had previously shown in vitro using human acute leukemia cells with various sensitivity to MTX (T-lymphoblastic CCRF-CEM line and resistant CEM/MTX subline) that MTX incorporation into liposomes as a lipophilic prodrug, diglyceride conjugate (MTX-DG), allows for the overcoming of cell resistance due to the impaired active transmembrane transport. In this work, we have studied the profile of binding with carbohydrates of the cell lines mentioned using carbohydrate fluorescent probes (poly(acryl amide) conjugates). Lipophilic conjugates of tetrasaccharide SiaLe(x), 6'-HSO3LacNAc, and also inactive pentaol for incorporation into liposomes, have been synthesized. The cytotoxicity of MTX-DG liposomes equipped with the SiaLe(x) ligand toward the sensitive CCRF-CEM cell culture was demonstrated to be 3.5 times higher than that of MTX-DG liposomes bearing the control inactive pentaol. The activity of MTX liposomes bearing 6'-HSO3LacNAc toward resistant CEM/MTX was 1.6-fold increased. The use of carbohydrate ligands as molecular addresses for drug-carrying liposomes as a potential method of treating heterogeneous tumor tissue is discussed.

We have recently synthesized a lipid conjugate of the anticancer agent methotrexate (MTX-DG) and showed that the conjugate is quantitatively included in the lipid bilayer of liposomes prepared by a standard extrusion technique from an 8 : 1 : 1 (mol) egg phosphatidylcholine-yeast phosphatidylinositol-MTX-DG mixture. Both the size of liposomes (126 +/- 30 nm) and the MTX-DG concentration (4.4 mM) are relevant for systemic injections in mammals. The liposomal formulation of MTX-DG was shown to overcome the resistance of tumor cells in vitro to methotrexate: the cytotoxic activities (IC50) of MTX in cultures of the human T-lymphoblastic leukemia cell line CEM-CCRF and the MTX-resistant subline CEM/MTX were 0.075 +/- 0.005 and 16.4 +/- 4.9 microM, respectively, while, in the case of liposomes loaded with MTX-DG, the IC50 values were much closer: 0.77 +/- 0.06 and 3.8 +/- 1.9 microM.

A lipophilic methotrexate prodrug capable of incorporation into membranes of carrier liposomes was synthesized. The conjugate consists of a lipophilic rac-1,2-dioleoylglycerol anchor connected to methotrexate through beta(Ala)-N-carbonylmethylene linker, which should be located in the polar region of the lipid bilayer. The ester bond between the hydrophilic linker and the antitumor agent can be hydrolyzed by intracellular esterases. The liposomal formulation of the prodrug exhibited a cytotoxic activity in vitro. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

The effect of inhibitors of DNA methylation on light-sensitive developmental stages of the filamentous fungus Neurospora crassa was studied. Under conditions of nitrogen starvation, when blue light induced protoperithecia development and inhibited conidia formation, 5-azacytidine (3-300 microM) inhibited protoperithecia formation and stimulated conidia formation (a 700-fold increase after light induction). After treatment of the mycelium with 5-azacytidine, the protoperithecia formation was accompanied by inversely proportional changes in the formation of conidia, both in the dark and after illumination. In the mycelium cultivated on the Vogel's medium, 5-azacytidine (up to 30 microM) and methotrexate (up to 3 microM) stimulated the light-induced carotenoid synthesis by 30%, whereas higher concentrations of these agents were toxic to carotenoid synthesis and growth.

Superoxide-removing activity, the number of sulfo-hydryl NH2-groups and groups, mean mass molecular concentration, free and antioxidant activity of albumin were determined in blood serum and its albumin fraction in 20 patients with breast cancer T3-4N1-2Mo, before and after incubation with cyclophosphamide, adriablastin, methotrexate and vincristine at concentrations used in clinic. Our data suggested that, following incubation with blood plasma, chemotherapeutic drugs bound to albumin fraction and protein fragmentation took place. Preincubation of blood plasma with the drugs and their immobilization on albumin molecules caused protein fragmentation to occur. Fragments having pro-oxidant properties might have been absorbed and fixed by both tumor tissues and any others thus creating a pool of fast-dividing cells and organs. Therefore, cytostatic drugs can exert enhanced antitumor action and, at the same time, a highly toxic effect when administered with autoplasma during chemotherapy.

The lipid spectrum of erythrocyte membranes was studied by thin-layer and gas-liquid chromatography in patients with lung cancer in the process of antineoplastic cytostatic chemotherapy. The control group of examined subjects was formed of healthy volunteers. During a course of antineoplastic polychemotherapy according to the CAM scheme (cyclophosphan, adriamycin, methotrexate) disorders of the erythrocyte membrane lipid spectrum in patients with II-IV stage lung cancer remained or became still more manifest.

The effect of the expression of the exogenous human mutant p53 (Arg-->His in codon 273) on the amplification rate of the gene dhfr in permissive Rat-1 and LIM1215 cells was studied. It was shown that injection of a retroviral construction with p53His273 resulted in the accumulation of methotrexate-resistant variants with an increased number of dhfr copies in populations of recipient cells. Luria-Delbruck fluctuation analysis revealed a four- to six-fold increase in the rate of appearance of new methotrexate-resistant cells. Chromosomal analysis demonstrated an extrachromosomal location of amplified DNA in cells containing p53His273, as was the case for control sublines. The data obtained indicate that modifications of p53 may induce gene amplification not only via removing the proliferation block of cells with amplified genes in selective medium, but also via some other mechanisms, that seem to increase the chromosomal recombination rate.

Methotrexate (N10-methyl-4-amino-4-deoxyfolic acid), administered to rats subcutaneously at a dose of 0.25 mg/kg within 14 days, was found to decrease content of microsomal protein in liver tissue and content of cytochromes P-450 and b5 as well as the drug decreased activity of amidopyrine- and ethylmorphine-N-demethylases, aniline-p-hydroxylase, UDP-glucuronosyltransferase and urinary excretion of glucuronides by 21%, 60%, 39%, 33%, 53%, 21%, 41% and 28%, respectively. Phenobarbital, administered subcutaneously at a dose of 60 mg/kg within 14 days, elevated the parameters by 28%, 187%, 63%, 163%, 162%, 100%, 95% and 60%, respectively. The effect of phenobarbital in combination with methotrexate was decreased by 77%, 49%, 55%, 52%, 43%, 42% and 22%, respectively.

The data are analyzed on the bilateral asymmetry of the number of dorsal and scutellar chaeta observed under the action of methotrexate in Drosophila of wild strain. It was shown that the incidence of asymmetric specimens induced by methotrexate was abruptly increased as compared to the control. The asymmetry was found to have a statistically significant trend to left side both of the control and the variants of treatment. There was no sex difference revealed between targets of the asymmetry.

The ultrastructure of cells from seven human lung cancers and from the colonies formed by these cells in soft agar was investigated. Tumor cells developed to display the morphofunctional potentials of the initial tumors. Cultured cells of squamous-cell carcinomas contained numerous tonofilaments, those of adenocarcinomas developed microvilli on their apical surfaces and intracellular lumens. On the other hand, cells of squamous-cell carcinomas showed features specific of adenoma epithelium, i.e. well developed microvilli and intracellular lumens. Besides, cells of adenocarcinoma often contained large quantities of tonofilaments considered to be characteristic of epidermoid epithelium. The results obtained suggest a possibility of metaplastic transformation of the lung epithelium.

Morphofunctional peculiarities of tumor cells from 15 endometrial adenocarcinomas and 2 ovarian tumors have been investigated at the ultrastructural level. These cells could develop two types of colonies in soft agar: those with histotypical differentiation (numerous microvilli, well developed tight junctions, desmosomes, secretory granules), and those without it (absence of epithelial features, ability of tumor cells to produce filamentous extracellular matrix and striated collagen fibrils which are characteristic of fibroblastic cells). The addition of progesterone and tamoxifen to cell cultures resulted in rising the level of cell differentiation in the colonies. The fact that endometrial and ovarian cancer cells can express the properties specific of connective tissue cells may suggest a multipotention of the Mullerian epithelium derivatives to shed light on the histogenesis of the mixed Mullerian tumors of uterus.

The effect of emboli, saturated with methotrexate on the vascular wall and neighbouring tissues of experimental animals is investigated. The creation perspective of the new type of the cytostatics directed transport, promoting the prolonged effect of the preparation is shown.

The time course of in vitro cellular immune reactions by using cyclophosphane, prospidin , and methotrexate was examined in 39 patients with rheumatoid arthritis to make predictions of the efficacy of its cytostatic therapy. The agents were evaluated for effects on the expression of Ia-like antigens on the peripheral lymphocytes and synovial fluid of the patients. The optimal doses of the agents for this testing had been chosen in the lymphoid cells from MRL31 lpr mice. The active dosage forms of the cytostatics for the experiment were obtained by administering native agents into the retrobulbar plexus of Balb/c mice, followed by blood isolation of active metabolites . The Ia-like antigen pre-expression index which is the ratio of the proportion of Ia-positive lymphocytes in control cultures to that of the cells in the culture after drug addition was used as a marker of the baseline cellular immunity in the patients. The patients having the index more than 1.5 were considered to be sensitive, those with less than 1.5 were insensitive to the drug. Thus, the Ia-like antigen pre-expression index may be used as a predictor in the assessment of the expediency of prospidine use.

The effect of methylation antagonists (DL-ethionine, S-adenosyl homocysteine and folic acid analogs-aminopterine, ametopterine and tomisine) on vitamin B12 and porphyrin biosynthesis of P. shermanii resting cells was studied. DL-ethionine proved the most active inhibitor. Inhibition of vitamin B12 biosynthesis induced by DL-ethionine was accompanied by increase in porphyrin formation. DL-methionine reversed the inhibitory effect of DL-ethionine on the vitamin synthesis. Other compounds tested showed a less marked effect.

Folic acid and its antivitamin methotrexate exert contrary actions on the activity of monooxygenases of the rat liver endoplasmatic membranes. Under the influence of folic acid (intra-abdominally, 100 mg/kg-6 days, 25 mg/k-14 days) there is a growth in the B5 and P-450 cytochrome content, NADP.H-cytochrome P-450 and NAD.H-cytochrome B5-reductase activity and in the oxidation rate of NADP.H, NAD.H and ethylmorphine N-demethylation. In similar experimental conditions, methotrexate (subcutaneously 0.25 and 1 mg/kg for 6 days, 0.25 mg/kg for 14 days) decreases the rate of NAD.H and NADP.H oxidation and NAD.H-cytochrome B5-reductase activity, the content of cytochromes B5 and P-450 and the binding degree of the latter with ethylmorphine, aniline and their metabolic rate. A decrease of the dose of the agents with a simultaneous increase of the time of administration enhances the vitamin action and reduces that of the antivitamin.

A mathematical model of the folic acid cycle and of thymidylate, purines, methionine and serine biosynthetic reactions in leukemia L-1210 and Ehrlich ascite tumour cells was developed. This model was used for an analysis of biochemical criteria of different sensitivity of these tumours to methotrexate, such as differences in the rates of methotrexate transport into the cells, levels of target enzyme, dihydrofolate reductase, its affinity for the inhibitor and the capacity of "salvage" pathways of tetrahydrofolate formation. It was shown that low sensitivity of the Ehrlich ascite tumour cells to methotrexate is due to mainly a high activity of methionine synthetase, which represents a "salvage" pathway of tetrahydrofolic acid regeneration in the presence of methotrexate. The results of this analysis were used to predict a combined utilization of methotrexate and methionine synthetase inhibitor. The pretreatment of the tumour cells of the methionine synthetase inhibitor enhances the effects of methotrexate on the thymidylate and purines syntheses, thus increasing their sensitivity to this drug.

The data of the study on the effect of a single intraperitoneal administration of methotrexate (0.5 mg/kg) on the early stages of immunogenesis of mice are presented. A single administration of the drug in different periods before immunization (1-10 days) was shown to induce no suppression of the immune response. On the contrary, at the administration of methotrexate 6-9 days before antigen administration a distinct stimulating effect was noted (320-350% of the control level). The administration of the drug daily for 6 days produced no, significant changes in the immune response formation and only 7-8-day administrations led to a significant decrease of the immune response (24-50% of the control level). The results obtained suggest that the time of renewal of the population of immunocompetent cells in the mouse spleen is 6-9 days.

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates has been studied. The inhibition is reversible and characterized by positive kinetic cooperativity (Hill coefficient exceeds 1). The values of pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: 3',5'-dichloromethotrexate, 3'-chloromethotrexate, 5-methyl-5,6,7,8-tetrahydrofolic acid (0.24, 0.40 and 1.87 mM, respectively). Comparison of "half-saturation" concentrations for the above compounds and for methotrexate and folinic acid shows that pterin affinity to glycogen phosphorylase b is affected by substituents both in pteridine and in p-aminobenzoic moieties of the pterin molecule. The antagonism between 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates, on the one hand, and AMP and FMN, on the other, is revealed for combined action of modifiers on glycogen phosphorylase b.

A plasmid containing the bacterial gene of methotrexate-resistant dihydrofolate reductase (dhfr), under the control of early SV40 promoter, was introduced into murine teratocarcinoma CBA9H6 cells. From the whole pool of teratocarcinoma cells, which survived after transient methotrexate selection in vivo, the individual cells were isolated to give rise to 15 clones of tumors. Six of the 15 clones displayed nucleotide sequences of the original vector containing pBR322 sequences and the early SV40 promoter region; however, the bacterial dhfr gene was absent from the transformant clones. Possible causes of the loss of introduced dhfr gene from teratocarcinoma cells under non-selective conditions are discussed.

The content of direct plaque-forming cells (PFC) in the spleen of sheep erythrocytes immunized mice after a single administration to them of 2.5 mg/kg methotrexate on the 48th or 72nd hour of the immune response was measured. The PFC count was determined every 4 hours for 68 hours after introduction of methotrexate. In either of the cases the curve reflecting changes of the PFC count in the spleen was of an undulating nature. The alternating rises and falls of the PFC number proceeded at a high speed, viz. the time of doubling and half-periods of drop-off on individual lengths of the obtained curves was of the order of 2--4 hours. It is suggested that the PFC population accretion is ensured by a synchronous recruitment from the cells-precursors, antigen activated upon introduction of methotrexate. The decline in the number of PFC is, apparently, dependent upon their destruction due to an "unbalanced growth".

As evidenced from experiments conducted on rats immunodepressants-antimetabolites 6-mercaptopurine, methotrexate and 5-fluoruracil exert an antiexudative action and inhibit inflammation in its proliferative phase, as well as significantly change the count of leucocytes and their formula in the peripheral blood. It was established that the anti-exudate properties of these compounds may not be related to their immunodepressive action, while inhibition of proliferation is occasioned by their specific cytostatic effect.

Inhibition of serine-hydroxymethyl transferase activity by dimer of cycloserine--diketopiperazin (DP) was studied in vivo in mice liver tissue. DP (2500 mg and 4000 mg per kg of body weight) caused a decrease in the enzyme activity by 14% and 30%, respectively, within 2 hrs after intraperitoneal administration into the animals. Complete reactivation of the enzyme was observed in presence of pyridoxal phosphate added into the experimental samples. DP inhibited the rate of mice liver regeneration after partial hepatectomy, if it was administered separately or in combination with methotrexate within 8 days before and 4 days after the operation. The decreased rate of the tissue regeneration occurred simultaneously with a distinct decrease in the serine-hydroxymethyl transferase activity in vivo. Methotrexate did not affect the liver tissue regeneration after hepatectomy. A method is described for estimation of the serine-hydroxymethyl transferase activity in tissue extracts in presence of partially purified preparation of 5,10-methylene-H4-folate-dehydrogenase.

Different methods for obtaining early mitotic chromosomes have been comparatively analyzed. Lymphocyte cultures have been treated with methotrexate, actinomycin-D and with a combination of both agents. It is shown that a high rate of chromosomes in prophase, prometaphase and mid-metaphase can be achieved by the combined methotrexate and actinomycin-D treatment of the lymphocyte culture. Methotrexate treatment of cultures is preferable to obtain the great number of early metaphase chromosomes.

Administration to intact male rats of folic acid (25 mg/kg intragastrically for 14 days) was shown to increase and that of methotrexate (0.5 mg/kg subcutaneously) for 14 days) to decrease the activities of the rat liver uridine-diphosphate-glucuronosyl- and glutathione-S-transferases.

Tests staged with mice and rats demonstrated 5-fluorouracil, fluorafur, methotrexate, cyclophosphane and vinblastine capable of inhibiting to a different degree the primary immune response and the factors of nonspecific immunity. Methotrexate displays the highest activity and specificity of the immunodepressive effect. By using different doses of the drug in immunizing the animals with sheep erythrocytes and vi-antigens the factors of both specific and non-specific immunity were inhibited in all cases. With their single introduction 5-fluorouracil and fluorofur can stimulate the synthesis of humoral antibodies and have no effect on the immune response. With their multiple administration the immunological reactivity of the organism becomes depressed. The extent of changes involving non-specific factors doses not depend upon the pattern and schedule of the drug administration. Cyclophosphane and vinblastin inhibit the immune response when they are introduced 24 hours after immunization. Vinblastin had the faculty of bringing down the activity of the nonspecific immunity, whereas under the effect of cyclophosphane it did not change, except for the complement.

In cells of L1210 ascite leukemia cells, methotrexate inhibited H3-thymidine incorporation, blocked shortly (during 4 hours) the G1 leads to S transition, and did not affect cells in G2-phase or in the late S phase. Almost half a cell population was degenerated and cells in S- and G1-phases were affected in equal proportion. This may suggest that methotrexate is not S-phase specific for cells of leukemia L1210. A simultaneous administration of vinblastine increases the antitumour effect of methotrexate. Cells in G2-phase constitute, presumably, a significant proportion of cells recovered after methotrexate administration. A comparison of the data obtained with literature evidence shows that in the sensitive (leukemia L1210) and resistant (acute mieloid leukemia of man) forms of leukemia, methotrexate affects cells that are in S-phase, whereas cells being in G1-phase are affected only when the sensitive tumours are treated.

The injection of 5 X 10(7) sheep red blood cells (SRBC) to CBA and (CBA X C57BL/6)F1 mice was accompanied by the formation of antigen-induced B-suppressors (AIBs) in the spleen of the animals on day 7. When transferred to syngeneic intact recipients, AIBs inhibited their response to SRBC (control). Methotrexate in a dose of 2.5 mg/kg, cytozar in a dose of 1000 mg/kg and 5-fluorouracyl in a dose of 100 mg/kg, injected to donors simultaneously with SRBC (group A), had no influence on the number of IgM-forming cells and, when introduced on day 5 after the injection of SRBC, decreased the number of such cells in donors (group B) as determined by testing the donors' response on day 7. The suppressor activity of AIBs obtained from the donors of group A was equal to that of the control, while AIBs obtained from the donors of group B had lower suppressor activity in comparison with the control. These results suggest that the proliferation of the precursors of AIBs occurs between days 2 and 5 of immune response and the formation of AIBs themselves, between days 5 and 7.

The presence of the relationship between the content of formaldehyde and folic acid in solution and tissue has been studied. It has been demonstrated that folic acid and its amino derivatives reduce the content of formaldehyde in solution by over 50%. During the build up of the increased content of folic acids and its analogs, there takes place diminution of the content of exogenous formaldehyde in all the test organs. This was interpreted as evidence of a close interplay with folic acid metabolism as regards the mechanism of action of exogenous formaldehyde introduced with a purpose of organ conservation.

A mathematical simulation model for checking hypotheses concerning specific features of methotrexate action on the primary antibody response is described. In general consistent interpretation of 8 experimental curves (an intact immune response and one when methotrexate was injected 72 hr after immunization; the data taken from literature) was obtained: the number of IgM-AbFCs in the mouse spleen in the first 4.5 days of immunization and the AbFC's labeling indices for three different 3H-thymidine injection schedules. It is concluded that methotrexate effects include the drug-induced lethality of the B-cells in G1-phase (early) and S-phase, S-phase blockage of the cell transfer cell retention on the G1/S boundary and also a decrease of the number of non-dividing IgM-AbFCs due to RNA and protein synthesis impairment.

Spleen colonies in the irradiated mice are produced by both stem cells and by their more differentiated progeny. In the latter case the colonies are transitory, ceasing 10-11 days after cell injection. The transitory colonies may be the cause of systematic artifact during the determination of stem cell proliferation. It was shown in particular that the proliferation of stem cells after sublethal irradiation remains the same, while higher rates of suicide are determined by the death of the precursors of the transitory colonies. At the same time higher proliferation of stem cells is not artifact in lethally irradiated animals and is also detectable after exclusion of the effects of the precursors of the transient colonies.

Methotrexate administered to albino mice in a dose of 0.5 mg/kg for a period of 10 days caused significant (two period) changes in the central and peripheral immunocompetent tissue. During the first--destructive--period there occurred a marked reduction in the weight of the spleen and the thymus, a cut in the count of the nuclear cells in them, and also a significant depletion of the cellular composition of the lymphoid organs. Suspension of the cytostatic was followed by a gradual restoration of all the indices under study, slower in the thymus than in the spleen. In the animals immunized with the tetanus toxoid against the background of the methotrexate administration all the changes in the lymphoid organs developed under a preponderant influence of the cytostatic agent; the action of the antigen was depressed during the whole experiment. Specific antibodies appeared in the blood of these animals later and their titres were lower than in control; experimental mice were also less resistant to tetanus toxin.

Through an analysis of the immune response of mice to sheep's erythrocytes within a system of syngenic transfer the action of Methotrexate on the T- and B-lymphocytes was studied. Splenic cells of donors receiving methotrexate were introduced to lethally irradiated recipients together with the bone morrow or thymus cells. In the recipients' spleen the content of antibody-forming cells and hemolytic foci were determined. It was shown that in recipients receiving splenic cells alone the number of antibody-forming cells and of the foci sharply declined under the effect of methotrexate. Introduction of the bone marrow cells contributed to reconstitution of the response in recipients at the level of the antibody-forming cells, but not at that of the hemolytic foci. At the same time, thymocytes increased the content of the antibody-forming cells up to a level exceeding the control one, as well as the number of foci. The results proof methotrexate capable of inhibiting the proliferation of the T- and B-cells in the spleen of mice, induced by the antigen.

Cultivation of tumour L-cells in the presence of increasing folic acid concentrations led to the rise in the resistance of these cells population to metotrexate. With the subsequent cultivation, when the folic acid concentration was not increased the population of such cells became more sensitive to metotrexate even in comparison with the initial L-cells.

The effect of methotrexate given intraperitoneally to (CBA X C57BL/6j) F1 mice on the expression of alloantigens in the lymphatic node cell population was investigated. For this purpose cells from intact or methotrexate-treated F1 mice were injected into the foot-pad of CBA mice. The reaction was estimated as an increase of the regional popliteal lymphatic node in comparison with contralateral (intact) one. The injection of methotrexate in doses of 50 and 75 mg/kg significantly increased the alloantigenicity of the F1 mouse lymphatic node cell population.