Depression of lymphocyte reactivity to phytohemagglutinin by serum from patients with liver disease

The influence of serum from patients with a variety of liver abnormalities on the in vitro response of normal human peripheral blood lymphocytes to stimulation with phytohemagglutinin was studied. These experiments demonstrate that serum from patients with acute viral hepatitis, chronic Australia antigenemia, common bile duct obstruction, primary biliary cirrhosis, hepatic necrosis secondary to halothane, and alcoholic cirrhosis suppresses DNA synthesis by stimulated normal lymphocytes. Sera obtained from two patients 1 week after surgical correction of their common duct obstruction no longer demonstrated lymphocyte suppression. Dilution of normal serum with serum from either of three patients resulted in more rapid decrease of thymidine uptake by stimulated lymphocytes than when normal serum was diluted with culture medium. This indicates the presence of an inhibitory factor (s) in the patients' sera. No correlation was shown between the extent of thymidine uptake by stimulated lymphocytes and the bilirubin, transaminase, or alkaline phosphatase levels in the serum in which they were cultured. The addition of bile salts to stimulated lymphocyte cultures did result in suppression of the response but only at concentrations much higher than would be expected in serum of the patients studied. Cell death after 72-hr incubation was 42% in normal serum and only 50% in serum from a patient who had demonstrated prominent suppression of DNA synthesis.     

Ecteinascidia turbinata extracts inhibit DNA synthesis in lymphocytes after mitogenic stimulation by lectins.

Aqueous ethanol extract of a tunicate which was previously found to exert antitumor and immunosuppressive activities in vivo was tested for its effect on normal human lymphocytes in vitro. The extract suppressed the uptake of tritiated thymidine by lymphocytes stimulated with mitogen. This suppressive effect did not require continuous presence of the extract. Treatment of lymphocytes prior to mitogenic stimulation resulted in suppressive effect. The fact that suppression by the extract could also be achieved 24 hr after exposure to mitogen, an interval which was found to suffice for the attainment of maximal commitment for blastogenic transformation indicates that Ete can act at a stage subsequent to the binding of the lectin and elicitation of a mitogenic signal(s).     

The influence of in vitro and in vivo exposure to antibiotics on mitogen-induced proliferation of lymphoid cells in rainbow trout (Oncorhynchus mykiss)

The influence of five different antimicrobial drugs on mitogen-induced lymphoid cell proliferation in vitro and after oral administration of the drugs was studied in rainbow trout (Oncorhynchus mykiss). The drugs tested were: oxolinic acid, oxytetracycline, florfenicol and trimethoprim in combination with sulfadiazine in ratio 1:5. In the in vitro tests, trimethoprim:sulfadiazine increased the 3H-thymidine incorporation into the DNA of phytohaemagglutinin and lipopolysaccharide-stimulated lymphoid cells while all the other drugs tested interfered negatively with the incorporation in a dose dependent manner. In the experiment with oral drug administration, the fish were fed 10 days with a therapeutic dose of the drugs. After the drug treatment, lymphoid cells were isolated from the head kidney of the fish and tested for proliferating capacity. All drugs except trimethoprim+sulfadiazine, suppressed the mitogenic response of the head kidney cells. The suppression of the response was more severe in phytohaemagglutinin-stimulated than in lipopolysaccharide-stimulated cells indicating that the T-cells were more vulnerable to the toxic effects of the drugs than B-cells.     

Lymphocyte transformation in vitro : V. Thymidine incorporation into unstimulated lymphocytes

In vitro conditions and kinetics of ¹⁴C-thymidine incorporation into unstimulated lymphocytes were studied. Lymphocytes cultured during 3 to 9 days displayed a progressive increase of thymidine uptake with time. The addition of varying numbers of autologous mitomycin-treated lymphocytes to cultures containing a fixed number of untreated lymphocytes markedly enhanced thymidine uptake per non-mitomycin-treated lymphocytes. In the latter cultures a sharp rise of thymidine uptake between the 7th day of culture was seen. Supernatants of non-stimulated lymphocytes, whether mitomycin-treated or not, showed no stimulating effect on autologous normal cells in these experiments. Although the mechanism of the enhancing effect of mitomycin-treated autologous lymphocytes remains unclear, it obviously may interfere in culture experiments in which the antigen specific stimulatory effect of mitomycin-treated cells is measured, which can only be detected by including the appropriate controls.     

Study of trimethoprim sensitivity in strains of Yersinia pestis isolated from various natural foci of plague

The effect of exogenic addition of purines and pyrimidines on trimethoprim sensitivity was studied in 134 vaccinal and virulent strains of the plague causative agent isolated at various periods in 12 natural plague foci. 74 strains proved to be sensitive to low concentrations of trimethoprim in the cultivation media irrespective of the presence or absence of thymine or thymidine in them. In this respect the strains differed from many other bacterial species which in the presence of thymidine or thymine were resistant to high concentrations of trimethoprim. 60 natural arginine deficient strains of the plague causative agent from Transcaucasia and Mongolia showed high levels of resistance to trimethoprim on media with thymine or thymidine. The possible mechanism of the plague microbe sensitivity to trimethoprim in the presence of thymine or thymidine is discussed with an account of the literature and original data.     

The determination of individual sensitivity to 5-fluorouracil in patients with malignant tumors in different locations]

Possible determination of individual chemosensitivity of tumors to 5-fluorouracil was investigated using new original data and suggestions that lymphocytes of the tumor carrier have an ability to assimilate chemical drugs. Such an ability is likely due to the fact that 5-fluorouracil as an antimetabolite converted its action by inclusion of lymphocytes to the nucleic acid metabolism. The relation between the phenomenon and the chemotherapy efficacy is possibly realized through two mechanisms. One of them is the direct cytotoxic action of chemical drugs on lymphocyte tumor-associated clones resulting in impairment of the tumor growth control. The other is possible participation of lymphocytes in transport of chemical drugs to the tumor tissue. The in vitro model for estimation of tumor carrier chemosensitivity by the level of the nucleic acid metabolism reflects the impact of the immune system on realization of the antitumor effect of chemical drugs.     

Inhibition by somatostatin of the proliferation of T-lymphocytes and Molt-4 lymphoblasts

The proliferation of Molt-4 lymphoblasts and phytohemagglutinin-stimulated human T lymphocytes in vitro was inhibited significantly by 10(-12) to 10(-10) M to 10(-13) to 10(-9) M somatostatin, as assessed by the uptake of [3H]thymidine and [3H]leucine, respectively. The inhibitory effect of somatostatin was not attributable to cytotoxicity and was associated with a mean degradation of 52 and over 95% of immunoreactive somatostatin, respectively, after 3 and 24 hr of incubation at 37 degrees C. The specific suppression of Molt-4 lymphoblasts and T lymphocytes by somatostatin represents a distinct mechanism for the specific regulation of immunological responses by neuropeptides of the peripheral nervous system and gastrointestinal tract.     

Chromosomal studies in vivo and in vitro of trimethoprim and sulphamethoxazole (co-trimoxazole)

Because both trimethoprim (TMP) and sulphamethoxazole (SMX) could interfere with folate metabolism and the former is a pyrimidine analogue it seemed advisable to try to ascertain whether these drugs alone, or in combination, determine damage in human chromosomes.In nine patients who had been taking a standard daily dosage of co-trimoxazole for periods varying from 3 to 112 weeks no damage in chromosomes of lymphocytes was found. In vitro experiments were cultured in concentrations of TMP and SMX considerably higher than the steady state plasma levels of patients being treated with the combination of drugs, also showed no chromosomal damage.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

Effect of x-rays on the intracellular transport of thymidine in human lymphocytes stimulated by phytohemagglutinins

The effect of X-irradiation on thymidine transport in human lymphocytes PHA stimulated was investigated. Mediated transport sistem, the predominant mechanism at low extracellular concentration of thymidine (less than 10(-7) M) in the medium is highly radiosensitive. The transport sistem was damaged considerably by high doses of X-rays (at least until 10 Krad); the decrease of thymidine uptake was a function of the time of incubation after irradiation. It suggest that repair mechanisms are not involved at high doses of X-rays within 120 minutes of incubation.     

Thymine and Thymidine Uptake by Haemophilus influenzae and the Labeling of Deoxyribonucleic Acid

The influence of ribo- and deoxyribonucleosides and ribo- and deoxyribonucleotides on the uptake of radiolabeled thymidine and thymine by Haemophilus influenzae during growth was investigated. A nucleoside-degrading enzyme similar to that reported in Escherichia coli was found to break down thymidine unless other nucleosides were present to divert its action. The presence of other nucleosides permitted a nearly quantitative uptake of even low levels of thymidine. This quantitative uptake of thymidine in the presence of an excess of other nucleosides suggests that the uptake mechanism for thymidine is specific in this organism. Under optimal conditions, as much as 50% of the deoxyribonucleic acid (DNA) thymine was derived from exogenous thymidine. Thymine was not taken up by H. influenzae but, in the presence of purine deoxynucleosides, labeled thymine entered the cells, presumably as thymidine. Ribonucleosides or ribonucleotides inhibited thymine conversion to thymidine, but, as noted above, they were degraded by a cellular enzyme. Thus, unless the ribonucleoside level was excessively high, a cell level of growth was reached at which the inhibiting ribonucleoside was broken down and labeled thymine appeared in the cells at an increasing rate. Twenty-five per cent of the DNA thymine of this organism was labeled with exogenous thymine. The information noted above serves as the basis for isotopically labeling the DNA.     

Inhibition of DNA synthesis in varicella-zoster virus-infected cells by BV-araU.

The inhibitory effect of BV-araU on DNA synthesis in human embryonic lung cells infected with varicella-zoster virus (VZV) or herpes simplex virus type 1 (HSV-1) was compared with that of acyclovir. Cellular uptake of [3H]thymidine and its incorporation into DNA was markedly stimulated by the infection with VZV or HSV-1, suggesting that the incorporation was mainly due to viral DNA synthesis. DNA synthesis in VZV-infected cells was dose-dependently suppressed by BV-araU and acyclovir, although cellular uptake of [3H]thymidine decreased in cells treated with a high concentration of drugs for an extended time. DNA synthesis in HSV-1-infected cells was also markedly inhibited by both drugs in a dose-dependent manner, without affecting cellular uptake of [3H]thymidine. The concentration of drugs inhibiting DNA synthesis was well correlated to their in vitro anti-VZV and anti-HSV-1 activities. The inhibitory concentration of BV-araU for DNA synthesis in VZV-infected cells was one-thousandth of that of acyclovir. Our results suggest that the antiviral action of BV-araU against VZV and HSV-1 is based on the inhibition of DNA synthesis in herpesvirus-infected cells.     

Iron and transferrin uptake by phytohemagglutinin-stimulated human peripheral blood lymphocytes

Iron (Fe) and transferrin (TF) uptake by human peripheral blood lymphocytes stimulated in vitro with phytohemagglutinin was measured. Pulses of 59FeTF or 125I-TF were added to the cultures either at time 0 or 8 hr before the end of a 72-hr incubation. In time-course experiments, peak iron and transferrin uptake coincided with the peak of tritiated thymidine uptake taken as a measure of cellular activation. Iron, but not transferrin, was accumulated by the cells. Non-linear relationships existed between both iron and transferrin uptake and the degree of activation. Both rose markedly above basal levels only at a level of activation at least 50% of the maximum observed. The results suggest that although iron utilization is related to cellular activity, the uptake mechanism is only activated when an increased iron metabolism has exhausted internal stores.     

Immunological effect of co-trimoxazole on platelets.

Diminished survival of transfused platelets occurred in two patients given co-trimoxazole, and a third patient taking this drug developed thrombocytopenia. By means of an indirect immunofluorescence assay antibodies against donor platelets coated with co-trimoxazole were found in the sera in all cases. These antibodies were directed against the trimethoprim component of co-trimoxazole and not against sulphamethoxazole. Co-trimoxazole is a potent antimicrobial agent and is advocated for treatment and prophylaxis in leukaemia. Hence its adverse effect on platelets is of great importance.     

Isolation and Characterization of Thymineless Mutants from Ultraviolet-Sensitive Bacillus subtilis Strains

Thymineless mutants of Bacillus subtilis 168 ind, hcr-9 were isolated by using trimethoprim. These and other Thy⁻ strains differed drastically from Thy⁺ ones in their patterns of [³H]thymidine uptake and growth in trimethoprim-containing medium. Transformation was negligible between most mutants derived from the ultraviolet-sensitive strain 168 ind, hcr-9 but significant between 168 ind, thy and these mutants. The latter and these new mutants all grow in the presence of trimethoprim plus thymidine or thymine and fail to grow if thymine or thymidine is omitted.     

Inhibition of mitogen-induced lymphocyte transformation by local anesthetics.

Chlorpromazine (CPZ) and lidocaine were added to cultures of mouse spleen cells stimulated by concanvalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and lipopolysaccharide (LPS). Concentrations of CPZ greater than 5 x 10(-6)M and concentrations of lidocaine greater than 2 x 10(-3)M totally inhibited the mitogenic responses to all four mitogens. Minimal inhibitory concentrations of neither drug interferred with cell viability as determined by trypan blue uptake or 51Cr release. The effects were totally reversed by the removal of the drugs from the culture. Addition of the drug at intervals after mitogen exposure demonstrated that the inhibited event occurred relatively soon after exposure to the mitogen. For example, the addition of lidocaine or CPZ more than 24 hr after Con A stimulation had no effect on tritiated thymidine incorporation. Elevated concentrations of cyclic AMP, cyclic GMP (or their derivatives) or calciunown membrane active actions of these drugs and the rapid reversibility of the effect strongly support the idea that the local anesthetics act on the surface membrane of lymphocytes. Binding of radiolabeled Con A or LPS to lymphocyte membranes in the presence of lidocaine or CPZ was not inhibited. The possibility exists that CPZ and lidocaine disorganized cell membranes so as to interfere with the surface membrane elaboration or action of a second messenger, or interfere with cell-cell interactions.     

pH-sensitive non-phospholipid vesicle and macrophage-like cells: binding, uptake and endocytotic pathway

Phospholipid and non-phospholipid vesicles are extensively studied as drug delivery systems to modify pharmacokinetics of drugs and to improve their action in target cells. It is believed that the major barrier to efficient drug delivery is entrapment of drugs in the endosomal compartment, since this eventually leads to its degradation in lysosomes. For these reasons, the knowledge of internalization pathway plays a fundamental role in optimizing drug targeting. The aim of this work is to characterize pH-sensitive Tween 20 vesicles, their interaction with macrophage-like cells and their comparison with pH-sensitive liposomes. The effect of different amounts of cholesteryl hemissucinate on surfactant vesicle formation and pH-sensitivity was studied. To evaluate the initial mode of internalization in Raw 264.7 and the intracellular fate of neutral and pH-sensitive formulations, flow cytometry in presence and in absence of selected inhibitors and fluorescence microscopy in absence and presence of specific fluorescent endocytotic markers were used. The obtained results showed that the surfactant vesicle pH-sensitivity was about two or three fold higher than that obtained with pH-sensitive liposomes in the presence of serum in vitro. The uptake mechanism of surfactant vesicles, after incubation with macrophage-like cells, is comparable to that of liposomes (clathrin-mediated endocytosis).     

Suppression of B cell function by methotrexate and trimetrexate. Evidence for inhibition of purine biosynthesis as a major mechanism of action

Methotrexate (MTX) is a widely used drug in the treatment of a variety of human neoplasms. Trimetrexate (TMQ) is a lipid-soluble quinazoline derivate of MTX that, unlike MTX, is not dependent upon membrane folate transport for cellular entry. A number of studies have demonstrated that MTX and, more recently, TMQ possess potent immunosuppressive properties. To examine the cellular events associated with the immunomodulatory effects of anti-folates on humoral immunity, a murine B cell maturation model was used. In vitro, MTX and TMQ reduced the number of antibody-forming cells to SRBC, as well as IgM production. B cells stimulated with anti-Ig demonstrated a dose-related suppression in [3H]UdR incorporation after addition of either drug, suggestive of a decrease in de novo DNA synthesis. B cell activation events preceding S phase were also suppressed by both anti-folates, as evidenced by inhibition of RNA synthesis. However, neither drug affected surface expression of Ia Ag nor inositol phosphate accumulation. Addition of TdR caused a slight non-significant increase in the antibody-forming cell response in the presence of 10(-7) M MTX. However, addition of hypoxanthine or adenine, but not guanine, resulted in complete restoration. Timed addition revealed that the ability of MTX to suppress antibody responses was diminished if added after 48 h of culture, similar to the reversal of this suppression mediated by hypoxanthine. Cell cycle analysis of LPS-stimulated B lymphocytes demonstrated that both drugs modulated events preceding, as well as during, the S phase. The present studies suggest that although drug-induced impairments in dTMP biosynthesis may be responsible for deficient lymphoid proliferation, anti-folate-induced impairment in purine biosynthesis is a major mechanism in anti-folate-induced suppression of humoral immunity.     

The effect of diphenylhydantoin and cortisol on the cell cycle

Normal human lymphocytes cultured in the presence of phytohemagglutinin were blocked in G0G1 when diphenylhydantoin (DPH) or cortisol 3.6 X 10(-4) M was added at the beginning of culture. The suppression of culture growth was analyzed by flow cytometry and confirmed by [3H]thymidine incorporation and mitotic rate analysis. The correlation of these measurements with flow cytometry was good for DNA synthesis and excellent for mitosis. There was an additive effect on the G0G1 retention of cells when both drugs were present in the culture. These data may partially explain the suppression of cell-mediated immunity which occurs in DPH-treated patients.     

Deficient natural killer function in patients receiving immunosuppressive drugs: analysis at the cellular level

Renal transplant recipients receiving low maintenance immunosuppression (azathioprine, 2 mg/kg/day, and prednisolone, 10 mg/day), tolerating their transplants well, and without viral infection disclose a profound depression of NK activity as assessed by 51Cr-release assay. By combining the analysis of the different steps of cytolysis with the agarose single-effector assays and the estimation of circulating large granular lymphocytes (LGL), the defect is shown to be due to a significant decrease of the number of NK cells capable of binding (% target-binding cells 2.0 +/- 0.3 versus 5.7 +/- 0.7 in normals, P less than 0.001) and killing (% cytotoxic target-binding cells 12.4 +/- 1.9 versus 22.0 +/- 0.5 in normals, P less than 0.001) of targets. There is also a significant reduction (P less than 0.001) of both percentages (1.0 +/- 0.2 versus 3.3 +/- 0.4 in normals) and absolute values (9.8 +/- 2.4 versus 62.3 +/- 8.0/microliters in normals) of LGL. These observations indicate that depressed NK activity is due mostly to depletion of NK cells. Functional impairment of NK cells can also be involved. Lack of direct in vitro effects of drugs (6-mercaptopurine, hydrocortisone, and methylprednisolone) at concentrations likely to be reached in vivo during treatments and relative resistance of NK activity after in vivo steroid administration suggest that immunosuppressive drugs act at the precursor cell level or on regulatory mechanisms. Despite functional integrity of two suppressor cell systems of allogeneic NK activity (suppression induced by preculture of lymphocytes with Con A and suppressor granulocytes) in immunosuppressed patients, tested on normal NK cells, NK cells of immunosuppressed patients did not disclose greater susceptibility to Con A-induced suppression. This analysis indicates that the depletion phenomenon is probably a major mechanism in NK depression of patients receiving immunosuppressive drugs.