Lymphocyte transformation in human plasma without addition of synthetic medium. A study of immune function in patients with chronic uraemia or diabetes mellitus.

The in vitro response of lymphocytes obtained from normal subjects, uraemic patients on haemodialysis and diabetic patients was studied using cultures containing either medium plus plasma (medium cultures) or plasma alone (plasma cultures). The study demonstrated that plasma alone can adequately support lymphocyte transformation induced by nonspecific mitogens (PHA and PWM) and allogeneic lymphocytes in mixed lymphocyte culture (MLC) reaction. This investigation further confirms our previously reported findings that uraemic patients undergoing haemodialysis have a normal lymphocyte response in MLC and to PHA and PWM. Plasma cultures give results similar to conventional medium cultures in subjects where lymphocyte transformation is normal. The lymphocyte hyporactivity observed in diabetics is, however, better shown in the plasma cultures. The suppressed response of the diabetic patient's lymphocytes to PHA and PWM both in the presence of autologous and normal AB plasma suggests intrinsic lymphocyte dysfunction as the explanation for impaired immune function. Plasma cultures may provide a better in vitro system for the evaluation of immune function in certain groups of patients where it is desirable to distinguish between intrinsic abnormalities of lymphocyte function and the effect of humoral immunosuppressive factors.     

Thymus lymphocytes in uraemic rats and the effect of thymosin fraction 5 in vitro.

Cellular immunity of thymus lymphocytes in uraemic rats was studied. Severe and moderate uraemia was induced in rats, and sham-operated and normal rats were used as the controls. As a result, the response of thymus lymphocytes to concanavalin A (Con A) significantly decreased in severely uraemic rats, but did not change in moderately uraemic rats. However, when the thymus lymphocytes were pretreated with thymosin fraction 5, the response to Con A was ameliorated in severely uraemic rats. There was a significant correlation between the effect of thymosin fraction 5 on Con A response and Con A response of thymus lymphocytes. In addition, serum from severe uraemic rats suppressed the response of normal thymus lymphocytes to Con A. These results indicate that severe uraemia may cause an impairment in maturation of thymus lymphocytes, which can be improved by thymosin fraction 5 in vitro.     

Tumor Associated Immunosuppression in Mice Bearing Anaplastic Carcinoma

The effect of anaplastic carcinoma (15091A) and fibroblast cell culture supernates was examined in conjunction with Concanavalin A (Con A) on the splenic lymphocyte transformation of syngeneic A/J mice. Though both the tumor and normal cell culture supernates caused a dose-dependent suppression of the in vitro DNA synthesis of lymphocytes, larger depressive effects were observed with the former. Similar results were observed with supernates collected from tumor cell and fibroblast cultures in 10% heat inactivated fetal calf serum or serum-free media. The results of these experiments indicate that the depressed immunological reactivity observed in animals and humans with cancer may be attributed to enhanced release of immunosuppressive factor(s) by malignant tissue into the body fluids of hosts.     

Immunosuppressive factors in uraemic sera are composed of both dialysable and non-dialysable components

The immunosuppressive factors in uraemic sera were studied. Sera were obtained from 46 uraemic patients with a mean serum creatinine of 1.24 mM/1 +/- 0.36 (s.d.) prior to being introduced to dialysis therapy. Normal peripheral blood mononuclear cells, which were incubated in vitro with pokeweed mitogen (PWM) in the presence of 10% uraemic sera plus 10% pooled normal human serum sustaining the culture, showed decreased 3H-thymidine uptake compared to the control culture with normal sera. The immunosuppressive activity of uraemic sera was partially lost by in vitro dialysis, although a moderate degree of suppressive potency to the normal cells cultured with PWM remained in the dialysed uraemic sera. The remaining inhibitory activity in the dialysed sera was found to be unrelated to anti-lymphocyte antibodies.     

Tumor Associated Immunosuppression in Mice Bearing Anaplastic Carcinoma

The effect of anaplastic carcinoma (15091A) and fibroblast cell culture supernates was examined in conjunction with Concanavalin A (Con A) on the splenic lymphocyte transformation of syngeneic A/J mice. Though both the tumor and normal cell culture supernates caused a dose-dependent suppression of the in vitro DNA synthesis of lymphocytes, larger depressive effects were observed with the former. Similar results were observed with supernates collected from tumor cell and fibroblast cultures in 10% heat inactivated fetal calf serum or serum-free media. The results of these experiments indicate that the depressed immunological reactivity observed in animals and humans with cancer may be attributed to enhanced release of immunosuppressive factor(s) by malignant tissue into the body fluids of hosts.     

Cellular immunity in renal failure: depression of lymphocyte transformation by uraemia and methylprednisolone. Intra-individual consistency of lymphocyte responses to the in vitro suppressive effect of steroid

In vitro parameters of the cell-mediated immunity were assessed in patients with chronic renal failure on haemodialysis (HD). The in vitro mitogen responses of uraemic lymphocytes are depressed compared to control lymphocyte cultures. Prolonged or shortened incubation of uraemic cultures does not normalize the mitogen responses, and the difference is reflected in both DNA, RNA and protein synthesis of lymphocytes. Lymphocyte responses of control cultures incubated with uraemic plasma are similar to those of cultures with saline, and significantly stronger (p less than 0.05) than those of the uraemic lymphocyte cultures. The relative in vitro immunosuppressive effect of steroid is stronger in uraemic cultures. Thus the depressed uraemic lymphocyte responses may be associated with steroid-sensitive cellular interactions independent of incubation periods. However, both control and uraemic lymphocyte cultures have a reproducible individual in vitro lymphocyte response to the immunosuppressive effect of steroid.     

血吸虫感染宿主免疫抑制现象的研究 Ⅱ.日本血吸虫感染鼠血清的免疫抑制作用

在正常鼠脾淋巴细胞和刀豆素A(ConA)的培养物中,加入感染日本血吸虫的同系鼠血清,发现血清浓度在2.5,5和10％时,对ConA诱导的增生应答均有抑制作用,进一步实验是在脾淋巴细胞和ConA的培养物中加入10％的感染后1～10周的鼠血清,以观察对脾淋巴细胞增生应答的抑制作用有无显著变化。结果表明,正常鼠脾淋巴细胞对ConA的应答,加入感染后第5周血清时,其抑制作用明显减弱。然而,感染鼠脾淋巴细胞对ConA的增生应答,加入感染后第5周血清时,其抑制作用的减弱不明显。提示感染血清对ConA增生应答的免疫抑制作用可能受双重因素影响,包括感染后某时期血清本身抑制作用的强弱,以及这一时期的淋巴细胞对ConA应答能力大小。     

In vitro reactivity of lymphocytes obtained from uraemic patients maintained by heamodialysis.

Lymphocytes obtained from uraemic patients maintained on intermittent haemodialysis had a normal ability to respond to and stimulate allogeneic lymphocytes obtained from normal subjects in the mixed lymphocyte culture (MLC) reaction. The response of these uraemic lymphocytes to PHA and pokeweed mitogen (PWN) was also normal. Uraemic plasma from eight out of twenty-six patients studied, however, possessed blocking factor activity which suppressed the MLC reactivity of normal random donors and also the mitogenic response of allogeneic lymphocytes but not of autologous uraemic lymphocytes. The blocking factor activity was attributed to a non-dialysable factor present in the plasma of the patients investigated.     

Serum effects of mitogenic reactivity in subjects with systemic lupus erythematosus, rheumatoid arthritis and scleroderma. Technical considerations and lack of correlation with anti-lymphocyte antibodies.

Methodological problems which affect the assessment of humoral effects on mitogenic reactivity include: (1) the source and concentration of serum used to support cell cultures; (2) the day to-day variability of inhibitory effects and (3) the specific activity of [3H]thymidine added to the culture. These problems were alleviated by addition of half concentration (7-5%) of pooled normal human serum to all cultures, the intoruction of anti-lymphocyte serum as a suitable internal control for monitoring the suppressability of lymphocytes and a reduction of specific activity of the [3H]thymidine to 1-3 C2/mM. Inhibitory factors were loosely bound to the lymphocyte surface and eluted off after incubation at 37 degrees C for 1 hr. Cells from twenty-five subjects and paired controls were cultured simultaneously in medium containing either 15% normal human serum (NHS) or 7-5% patient and 7-5% NHS. The cells were stimulated with various dilutions of phytohaemagglutinin, Con A or pokeweed mitogen. Lupus serums suppressed the reactivity of autologous lymphocytes to PHA and pokeweed mitogen. Serums from subjects with RA and scleroderma did not significantly inhibit blastogenesis of autologous lymphocytes. One-half of the lupus serums significantly inhibited the reactivity of homologous lymphocytes to two of three mitogens. Only one of eight scleroderma serums and none of twelve RA serums and none of twelve RA serums had this effect. All patients serums were examined for antilymphocyte antibodies by microcytotoxicity and immunofluorescent techniques. These antibodies were usually found in SLE, and were often observed in subjects with rheumatoid arthritis but not scleroderma. A firm relationship between serum suppressors of lymphocyte blastogenesis and anti-lymphocyte antibodies was not found.     

Glucocorticoid inhibitory action on the response to PHA of residual circulatory lymphocytes in renal transplant patients following immunosuppressive therapy

The inhibitory effect of glucocorticoids on the in vitro response to phytohaemagglutinin of the residual circulating T lymphocytes in renal transplant patients during maintenance immunosuppressive therapy with glucocorticosteroids and azathioprine has been investigated. As in normal subjects, the steroid-induced inhibition of transplanted patients' lymphocyte response was inversely correlated with the mitogen concentration used. On the other hand, the response of the various lymphocyte preparations from transplanted patients appeared less inhibited by steroids than the corresponding preparations from normal subjects. The addition to the culture of the adherent cell product interleukin 1 was effective in removing to a similar extent the steroid inhibitory effect on lymphocytes from normal and transplanted subjects. Thus, the lesser inhibitory effect of glucocorticoids on transplanted patient lymphocytes could be explained by the higher percentages of monocytes present in all peripheral blood mononuclear cell preparations. These results suggest that during immunosuppressive therapy with glucocorticoids and azathioprine the residual circulating lymphocytes have a responsiveness to in vitro dexamethasone suppression similar to that of normal peripheral blood lymphocytes.     

Influence of oxygen tension, sulfhydryl compounds, and serum on the motility and virulence of Treponema pallidum (Nichols strain) in a cell-free system.

The motility and virulence of Treponema pallidum (Nichols strain) were monitored during incubation in a modified tissue culture medium to study the effects of oxygen tension and medium composition on survival of the organism. A basal medium of Eagle minimal essential medium with 50% fresh, heat-inactivated normal rabbit serum was used inasmuch as better survival occurred with 50% normal rabbit serum than with lower concentrations. Addition of 0.5 to 2.0 mM dithiothreitol or 2.0 mM dithioerythritol to the basal medium led to significantly longer retention of T. pallidum viability in the presence of 3% oxygen than under aerobic or anaerobic conditions. The results of this investigation lend support to the classification of T. pallidum as a microaerophilic organism and provide direction for the design of potentially successful culture systems, with or without tissue culture cells.     

Placental isoferritin as a physiological downregulator of cellular immunoreactivity during pregnancy.

CM-H-9 monoclonal antibody specific for placental isoferritin (PLF) was used to quantify PLF in the serum and on peripheral blood lymphocytes derived from term delivery women and healthy controls. It was found that the mean level of PLF in maternal sera was 50.4 +/- 50.1 U/ml, whereas in normal adults the mean serum PLF level was very low (4.5 +/- 7.7 U/ml). Furthermore, term mothers exhibited a sub-population of peripheral blood lymphocytes (PBL) which stained positively with CM-H-9 MoAb (mean 11.6 +/- 7.8%). Such lymphocytes were scarce in normal non-pregnant women (mean 0.8 +/- 1.2%). The effect of PLF on lymphocyte transformation in MLC was studied in mixed lymphocyte cultures of maternal-newborn and normal non-related PBL controls. It was found that placental isoferritin was immunosuppressive in comparison to normal adult ferritin. The suppressive effect was significantly higher in maternal-newborn MLC compared to normal adult controls. Furthermore, it was found that PLF which is present in maternal serum has an immunosuppressive activity since, its removal on CM-H-9 MoAb affinity column abrogated this effect. The results of this study suggest that both PLF and PLF binding lymphocytes play a role in the development of immunosuppression during pregnancy. Therefore the lack of one of the factors may result in diminished immunosuppression or in fetal rejection.     

In vitro reactivity of human lymphocytes in chronic uraemia: analysis and interpretation

Altered cellular immunity complicating chronic uraemia includes lymphocytopenia, thymic atrophy, impaired allograft rejection and delayed hypersensitivity in skin tests, diminished appearances of lymphocytes on skin windows, and shortened in vitro survival of uraemic lymphocytes. Studies were undertaken to further characterize these lymphocyte defects.

Lymphocytes separated from peripheral blood of twenty-four uraemic patients were compared with those from fifty-one normal persons in their rates of RNA and DNA synthesis in both PHA-stimulated and unstimulated cultures, as determined by incorporation of radiolabelled precursors. Synthesis, expressed as absolute incorporation/10⁶ viable lymphocytes, was accelerated in the majority of both stimulated and unstimulated uraemic cultures. Serial studies over several months of twenty-five uraemic patients (fifteen maintained by haemodialysis, ten by renal allotransplantation), compared with nineteen normal controls, showed these differences to be consistent and persistent. While normal lymphocytes exhibited stability, uraemic cells fluctuated widely in their serial synthesis rates. In 25% of such cultures, unstimulated synthesis exceeded that induced by PHA. Increased synthesis without PHA, reflecting enhanced spontaneous blastogenesis, is compatible with decreased survival, numbers, and functional capacity of uraemic lymphocytes.

Reports by others of diminished PHA-responsiveness of uraemic lymphocytes are based upon whole-culture incorporation and/or expressed as ratios between stimulated and unstimulated cultures. Both shortened survival and accelerated spontaneous nucleic acid synthesis by uraemic lymphocytes cause such ratios to be misleadingly low. Such factors as cell numbers and viability at harvest, counting efficiency, and culture sterility are essential to avoid misinterpretations of such data based on incorporation of radiolabelled precursors to the nucleic acids.

     

Human lymphocyte transformation induced by mitogens and antigens in a serum-free tissue culture system

The use of serum to supplement lymphocyte tissue culture media introduces uncontrolled variables; different serum sources, lots and concentrations can produce variability in experimental results, serum can stimulate or inhibit lymphocytes, and components of serum can react with substances whose effects on lymphocytes are being studied. To avoid these problems, we studied the ability of human peripheral mononuclear cells to survive and to respond to stimulation in an entirely synthetic medium, RPMI-1640 supplemented with L-glutamine, gentamicin, HEPES buffer and magnesium. Optical cell concentration in this serum-free RPMI-1640 was 2.5 × 10⁶ cells/ml, whereas optimal cell concentration in serum containing RPMI-1640 was 1 × 10⁶ cells/ml. In this serum-free RPMI-1640, 50% of the cellular input was recovered as viable cells after 7 days of culture, which was similar to results in serum containing RPMI-1640. Mononuclear cell transformation was induced by phytohemagglutinin, concanavalin A, pokeweed mitogen, streptolysin O and candida. Optimal doses of stimulants and the kinetics of the responses were similar in serum-free and serum containing RPMI-1640. This system can be used to avoid the problems inherent in systems which supplement tissue culture media with serum.     

Further studies on replication of virulent Treponema pallidum in tissue cultures of Sf1Ep cells.

A number of parameters aimed at optimizing culture conditions for both Sf1Ep cells and Treponema pallidum have been investigated. Optimum temperature for replication of T. pallidum ranged between 33 and 35 degrees C. At 33 degrees C, replication occurred in the presence of atmospheric oxygen concentrations of less than 0.3 to 10%, the optimum range being 1.5 to 5%. No replication occurred in the presence of 12.5% oxygen. When both temperature and oxygen concentrations were varied between 33 and 35 degrees C and 1.5 to 5%, respectively, little differences in replication were noted. Although variation in the oxygen concentration within each temperature group had little or no effect on replication, it did have an effect on motility, which remained greater in the 5% oxygen concentration after 9 to 12 days of cultivation. Optimum concentration of fetal bovine serum in the culture medium was 20%, although replication occurred in concentrations ranging from 5 to 30%. If carefully screened, calf serum could be substituted for fetal bovine serum. Testis extract was an essential component of the culture medium. Although extract obtained from an adult rabbit--either normal or T. pallidum infected--was slightly superior, replication of T. pallidum occurred when rat or hamster testis extract was substituted.     

Effect of iron deficiency on the response of mouse lymphocytes to concanavalin A: the importance of transferrin-bound iron.

The in vitro response to Con A of lymphocytes from iron-deficient and normal mice in media containing either 10% fetal calf serum, apotransferrin or 20% iron-saturated transferrin was similar for the iron-deficient and control groups. However, the degree of proliferation in serum-free medium containing apotransferrin was significantly lower in all groups, compared to the responses in media containing either 20% iron-saturated transferrin or 10% fetal calf serum. Proliferation of lymphocytes from normal, iron-deficient or iron-repleted mice was lower in media supplemented with serum from iron-deficient mice than when serum from normal or iron-repleted mice was used. Addition of sufficient iron to bring the iron level of the deficient serum to that of normal serum significantly improved its ability to promote proliferation, while in vivo repletion of iron-deficient mice resulted in a restoration of normal lymphocyte responses to Con A. The proportion of cells positive for Thy 1.2, Ly 1 and Ly 2 antigens did not differ significantly between any groups of mice. Protein synthesis by cells proliferating in serum-free medium containing apotransferrin or 20% iron-saturated transferrin was the same in all groups of mice. These results indicate that decreased lymphocyte proliferative responses in iron deficiency may be due to inadequate levels of circulating transferrin-bound iron, rather than to intrinsic defects in the cells themselves or changes in the proportions of different T-cell subsets, and that iron availability does not affect protein synthesis by proliferating lymphocytes.     

Lack of immunosuppression by ketoconazole and itraconazole.

The antifungal drugs ketoconazole and itraconazole were evaluated for their effects in the following test systems: in vitro, phytohaemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells and IL-2-driven proliferation of CTLL-2 cells; in vivo, antibody response to sheep red blood cells (SRBC) and delayed-type hypersensitivity (DTH) reaction to oxazolone. At a concentration of 10 microM, ketoconazole moderately and itraconazole strongly inhibited thymidine (Thd) incorporation in human peripheral blood mononuclear cells cultured in medium supplemented with 5% human serum. Increasing the serum concentration from 5 to 20% almost completely reversed these inhibitory effects. Also, cell viability, found to be less than 15% in cultures containing 10 microM itraconazole was restored by increasing the serum concentrations in the culture medium. Similar observations were made in experiments using IL-2-stimulated CTLL-2 cells: the growth inhibition in the presence of 10 microM ketoconazole or 1 microM itraconazole could be counteracted by increased serum supplementation. In vivo, subchronic intraperitoneal dosing with 40 mg/kg ketoconazole or itraconazole to mice had no effect on the antibody response to SRBC as measured by the number of splenic IgM and IgG plaque-forming cells and did not significantly affect the DTH response to oxazolone. These data indicate that neither ketoconazole nor itraconazole exert immunosuppressive properties in vivo. Their in vitro inhibitory effects on PHA-induced lymphocyte proliferation and IL-2-dependent CTLL-2 growth are reversed by the serum supplementation to the culture medium and these activities should therefore be considered as in vitro artefacts.     

Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells in Cultures with Different O<Subscript>2</Subscript> Content in the Medium

We studied the effects of multipotent mesenchymal stromal cells isolated from the adipose tissue on proliferation and viability of immunocompetent cells at different concentration of O₂ (5 and 20%) in culture medium. It was shown that co-culturing with multipotent mesenchymal stromal cells 3-fold reduced proliferative index of phytohemagglutinin-activated lymphocytes, while their viability remained unchanged and did not depend on partial oxygen pressure in the medium. These findings suggest that low O₂ concentration in tissues will not affect immunosuppressive properties of multipotent mesenchymal stromal cells, which is very important for their application in regenerative medicine.     

L-alanine--an essential amino acid for growth of lymphocytes in vitro.

A factor an ultrafiltrated (UM 10) extracts of calf thymus, liver and spleen stimulated the uptake of 3H-thymidine in DNA of cultured lymphocytes. The factor was purified by ion-exchange chromatography and gel chromatography and identified as alanine, which is lacking in the culture medium, RPMI 1640. An optimal amount of L-alanine increased the uptake of 3H-thymidine in DNA of lymphocytes from different species by about 100%. L-alanine should be regarded as a growth factor for lymphocytes in vitro and should be added to RPMI 1640, when this tissue culture medium is used for lymphocytes.     

Alterations in mononuclear cell tumour necrosis factor-alpha (TNF-alpha) response in patients on long term cuprophane haemodialysis.

We have investigated TNF-alpha secretory response of peripheral blood mononuclear cells (PBMC) from 13 uraemic patients undergoing regular haemodialysis with cuprophane membrane (CM). Sixteen healthy subjects and five uraemic patients under conservative therapy were also studied as controls. Cells of haemodialysis patients exhibited increased TNF-alpha release in vitro in the absence of activating stimuli other than culture conditions, as compared with normal and uraemic controls. In contrast to normal cells, this spontaneous secretion of TNF-alpha from dialysis PBMC could not be significantly reduced by addition of polymyxin B to culture medium, thus indicating its independence of trace amount of lipopolysaccharide (LPS) present in the medium as contaminant. Furthermore, predialysis PBMC were considerably more sensitive to stimulation with 10(7) pg/ml of LPS under in vitro culture conditions than normal and uraemic controls. To elucidate a role of direct contact with CM in stimulation of TNF-alpha release from monocytes, PBMC were cultured on CM in vitro. Contact with CM stimulated TNF-alpha secretion from PBMC above the level of cells cultured on tissue culture plastic. This response persisted with time in culture in contrast to a transient LPS-induced TNF-alpha release. Furthermore, PBMC stimulated by contact with CM for 2 days did not lose the capacity to secrete TNF-alpha in response to a subsequent LPS stimulation, while a 2-day treatment of cells with LPS was followed by LPS refractory state. Therefore, direct contact with CM induces in PBMC a long-lasting TNF-alpha response which is not down-regulated by the acquisition of refractoriness in a manner similar to that which occurs in the case of LPS stimulation. These in vitro findings provide a possible explanation of the observation that predialysis PBMC exhibit elevated TNF-alpha secretory capacity.