Enhancement of the immune system of chemotherapy-treated cancer patients by simultaneous treatment with thymic extract,TP-1

Summary Thirty patients with incurable gastrointestinal cancer were treated in a randomized clinical trial, either with combination chemotherapy (5-fluorouracil, vincristine, and methyl-CCNU) or with the same chemotherapy and the thymic extract TP-1. T-lymphocyte counts slowly and steadily, increased in the TP-1-treated patients over a period of 4–5 months, in contrast to decreasing counts in the patients treated by chemotherapy only. The changes in both groups were significantly different from pretreatment counts (P<0.01). Skin tests for primary sensitization with DNCB were converted to positive or improved after TP-1 treatment (P<0.05 against control patients). In conclusion, TP-1 had a progressively accumulating effect on T-cell lymphopoiesis and activity (skin tests), which was strong enough to overcome chemotherapy-induced immunosuppression without any appreciable side effects.     

Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. II. Cytokine activities in murine thymic epithelial and mesenchymal cell culture supernatants

Two morphologically distinct primary cultures of murine thymic stroma were established and found to be of epithelial (MTEC) and mesenchymal (MTMC) origin. These cultures were generated by selective conditions of tissue disruption and were maintained on extracellular matrix in defined medium. Culture supernatants (CS) from these cultures (EC-CS and MC-CS respectively), were tested for cytokine production and for effects on thymocyte maturation. Both supernatants displayed the activities of IL-3 and of granulocyte/macrophage-CSF and not of IL-1, -2, -4, or IFN. In addition they were found to be mitogenic to murine thymocytes in a "spontaneous" [3H]TdR incorporation assay. The two supernatants differed, however, in their effect on Con A stimulation. EC-CS had a strong enhancing effect, both when used for preincubation (18 h) before Con A stimulation or when present simultaneously with it. MC-CS had a small inconsistent effect under these conditions. Also EC-CS enhanced IL-2 and IL-3 production by thymocytes. The responsive thymocyte subpopulation was the one that does not bind peanut agglutinin. CS of an established thymic epithelial cell line displayed only part of these activities at a considerably lower level. CS from primary kidney cell culture was completely devoid of activity. The results suggest that primary thymic stromal cell cultures, cultivated under the defined conditions described here, may better preserve physiologic secretory activities, and probably also other cell functions, compared with established cell lines. Furthermore, the results are compatible with the hypothesis that the soluble factors, secreted by thymic stromal cells, are active on either very early or late stages of thymic differentiation, whereas the main intrathymic stages of differentiation are conceivable dependent primarily on direct contact with stromal cells.     

Enhancement of natural killer cell activity in mice by treatment with a thymic factor

Summary The administration of a thymic factor, thymostimulin (TP-1), to mice resulted in considerable augmentation of natural killer (NK) cell activity as measured in a short-term assay against ⁵¹Cr-labeled YAC-1 target cells. Conditions suitable for detection of the thymostimulin-induced boosting of NK included multiple daily exposures to TP-1 (50 g/kg), and peak levels of reactivity were observed at 2–4 days after discontinuation of treatment. A strict age-dependency of the effect was also observed, with optimal augmentation of NK-cell activity when TP-1 was administered to mice at 4–6 weeks of age. The effect was not limited to TP-1 treatment but was also observed on administration of another thymic factor (thymosin ₁). The activated cells responsible for the increased natural cell-mediated cytotoxicity appeared to be typical murine NK cells, judging by both functional and antigenic criteria.     

Selective restoration of immunosuppressive effect of cytotoxic agents by thymopoietin fragments

Summary Swiss male mice were immunosuppressed by cyclophosphamide, cisplatinum, vincristine and methotrexate. The ability of the thymopoietin (TP) fragments TP-3, TP-4 and TP-5 to restore antibody production and phagocytosis was studied. Impaired antibody production after vincristine treatment was partially or totally restored by TP-3, TP-4 or TP-5. Only TP-3 or TP-5 interfered with the antibody-production-damaging effect of cisplatinum. The same effect of methotrexate could not be influenced by any of the TP fragments. The phagocytic capacity of peritoneal macrophages was reduced by vincristine, methotrexate and cyclophosphamide treatment. In this respect, TP-3 protected the function of macrophages against vincristine and cyclophosphamide treatment. TP-4 was active in the case of damage caused by vincristine and methotrexate, and TP-5 interfered with the phagocytosis-inhibiting effect of methotrexate. Each TP fragment seems to have a specific target orientation within the immune system. This also means that the proper TP fragment should always be chosen for combination therapy with various types of cytotoxic drugs.     

Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.     

Immunorestoration of old mice by injection of thymus extract: Enhancement of T cell-T cell cooperation in the in vitro antibody response

The effect of injecting a thymus extract (TP-1 or Thy-5) into immunodeficient old mice on the in vitro antibody response of their spleen cells was investigated by techniques suitable for dissecting out T- and B-cell reactivities. The anti-TNP antibody response of HRBC-primed spleen cells from old mice uninjected or injected with TP-1 or Thy-5 was elicited in vitro by TNP-HRBC or TNP-Ficoll. Treatment with TP-1 or Thy-5 was found to induce only a slight increase in the anti-TNP antibody response to both immunogens. The helper activity of HRBC-primed spleen cells from untreated or treated old mice was titrated by adding graded numbers of these primed cells to cultures containing a constant number of normal spleen cells from young mice and the immunogen TNP-HRBC. Under these conditions it was found that both thymus extracts are very effective in restoring T cell-T cell cooperation in the generation of helper cell activity.     

Neuroendocrine control of thymic hormonal production. II. Stimulatory effects of endogenous opioids on thymulin production by cultured human and murine thymic epithelial cells

Data have now accumulated to strongly demonstrate that several neuropeptides, including endogenous opioids, can have immunomodulatory functions. Most of the studies have so far focused on the direct action of these substances on lymphocytes. We decided to investigate whether thymic epithelial cells (TEC) - the major component of the thymic microenvironment - could also be modulated by endogenous opioids. Primary cultures of human and murine TEC were subjected to several opioids (alpha-beta- or gamma-endorphins, as well as met- or leuenkephalins) applied in concentrations ranging from 10(-6) to 10(-9) M. On the following days we measured the levels of thymulin (a chemically-defined thymic hormone known to stimulate some steps of T-cell differentiation) in the culture supernatants, as well as the numbers of thymulin containing cells, evaluated by immunofluorescence with an anti-thymulin monoclonal antibody. After treatment of TEC cultures with beta-endorphin or leu-enkephalin a significant increase in the levels of thymulin in the culture media was observed, paralleled by a rise in the percentage of thymulin containing cells. In addition, this stimulatory effect was dose-dependent. Preincubation of the opioids with the specific antibodies abrogated the opioid-induced stimulatory effect on TEC. Moreover, naloxone, an opioid receptor antagonist, blocked the effect of beta-endorphin on thymulin production, suggesting that the effect of this neuropeptide on epithelial cells was mediated by an opioid receptor. Importantly, no effect on thymulin production was observed with the other opioids used, whatever the dose. These results suggest that, at least in vitro, beta-endorphin and leu-enkephalin stimulate the hormonal function of the thymic epithelium. These findings lead to the general concept that the modulatory role of endogenous opioids on the immune system is not restricted to lymphocytes but can also take place at the level of cells belonging to T-cell differentiating microenvironments.     

Competence-Inducing Factor of Bacillus stearothermophilus

An incompetent mutant (4S Com(-)) does not release competence-inducing factor (CF) into the culture medium and is not infected with TP-1C phage deoxyribonucleic acid (DNA) unless CF is added to the transfection assay. A TP-1C phage-resistant mutant (4S Ton-r) releases relatively large amounts of CF into the culture medium but is not infected with TP-1C phage DNA, even in the presence of CF. The production of CF by the wild type or Ton-r mutant and the ability of the wild type or Com(-) mutant to react with CF does not occur after these cultures have grown at 67 C for 1 hr or longer. A preliminary characterization of the CF is described. The autolytic enzyme or the temperate phage of the wild type and the Ton-r and Com(-) mutants do not have competence-inducing activity.     

Enhanced Production of Virus-Inhibiting Factor (Interferon) in Human Diploid Cells by Ultraviolet Irradiation and Temperature Shift-Down after Stimulation with Newcastle Disease Virus

The production of the virus-inhibiting factor or interferon (IF) was highest in cells incubated at 37 C after inoculation with Newcastle disease (ND) virus and decreased as the incubation temperature was lowered. Shift-down of incubation temperature to 32 C or 34 C after incubation at 37 C for 4–7 hr enhanced IF production in cell cultures stimulated with ND virus, as compared with cultures incubated continuously at 37 C. Shift-down to 32 C after incubation at 37 C for 6 hr. was optimal for this enhancement of IF yield. Enhanced IF production was also observed in cell cultures irradiated by ultraviolet light 4–7 hr after stimulation with ND virus.     

Generation of lipopolysaccharide-induced interferon in spleen cell cultures

Incubation of murine spleen-cell cultures with lipopolysaccharide (LPS) induces interferon (IF) production. Maximal IF levels are obtained after incubation with 100 g/ml for 10 h. Two inbred mouse strains differing in their ability to generate LPS-induced IF in spleen-cell cultures were used: C3H/eB, which generates high levels of IF (about 60 units/ml), and C3H/HeJ, which fails to generate detectable quantities of IF. In a genetic analysis these strains were hybridized and IF production was determined in spleen-cell cultures from F₁ and F₂ generations, and from backcrosses of F₁ hybrids to parent strains. The results indicate that, in parent strains, a single dominant autosomal gene is responsible for differences in IF production in spleen cultures. LPS-induced IF in spleen-cell cultures resists pH 2 for as long as 48 h, but is labile to heating at 56° C for 30 min. Both macrophages and lymphocytes must be present in cultures for generation of LPS-induced IF. By using mixed cultures of macrophages and lymphocytes from C3H/eB and C3H/HeJ mice, it was shown that macrophages have to interact directly with LPS to enable IF production in the cultures.     

Thymic hormone-containing cells. V. Immunohistological detection of metallothionein within the cells bearing thymulin (a zinc-containing hormone) in human and mouse thymuses

Using an immunofluorescence (IF) assay, the presence of metallothionein (MT) was investigated in sections of normal and pathologic human thymuses as well as in cultures of thymic epithelial cells. This protein, known to have a high binding affinity for class II B transitional metals, such as zinc, was detected in the epithelial component of the thymus. Moreover, double labeling experiments with the anti-MT and an anti-thymulin monoclonal antibody showed that all cells containing thymulin, a thymic hormone whose active structure is known to contain zinc, also exhibited large amounts of metallothionein. These results, together with the fact that zinc and thymulin have been detected in the same type of cell organelles, lead to the conclusion that the MT present in thymic epithelial cells might be involved in the mechanism of zinc storage in these cells, thus favoring the secretion of thymulin in its biologically active, zinc-containing form.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.     

Depressed monocyte polarization and clustering of dendritic cells in patients with head and neck cancer: In vitro restoration of this immunosuppression by thymic hormones

The in vitro restoring effects of a thymic hormone preparation, TP-1, on defective monocyte and dendritic cell function in patients with head and neck squamous cell carcinoma (HNSCC) have been examined. TheN-formylmethionyl-leucyl-phenylalanine(fMLF)-induced polarization of monocytes isolated from the peripheral blood was significantly lower (a mean of 19%) than the polarization of monocytes isolated from healthy controls (a mean of 33%). After the in vitro addition of TP-1 this defective polarization was improved to the normal value of 33% polarized monocytes. The capability of dendritic cells prepared from the blood to form cellular clusters with allogeneic cells was impaired in 26/44 patients. In vitro addition of TP-1 again had restoring effects. The original defective dendritic cell clustering of 97 clusters/six microscopic fields (mean) was improved to a value of 121 clusters. The defects in monocyte polarization and clustering of dendritic cells could be ascribed to the presence in serum of a tumor-derived low-molecularmass factor low-M _r factor; <25 kDa) sharing structural homology with p15E, the capsular protein of murine and feline leukemogenic retroviruses. The incubation of low-M _r factor from the serum of HNSCC patients with healthy donor monocytes resulted in a significantly higher inhibition of fMLF-induced monocyte polarization than did incubation with control low-M _r factor (a mean of 42 versus 16% inhibition). This suppressive effect of patient low-M _r factor was abrogated with a mixture of two monoclonal antibodies against p15E as well as with TP-1. The observations here reported on the in vitro effects of TP-1 on depressed monocyte and dendritic cell function in HNSCC have provided one of the rationales for a TP-1 therapeutic pilot trial recently started in HNSCC patients.     

Chromosomal mapping of Bacillus thuringiensis by transduction.

Three groups of linked markers were mapped in Bacillus thuringiensis 4042B by using two-, three-, and four-factor crosses mediated by the temperate bacteriophages TP-13 and TP-18. The order of markers was (trp-11, trp-2)-(leu-1, leu-2)-his-1-(lys-1, lys-2)-cys-1 in the first group; met-1-(argCl, argOl)-met-2-(pyr-1, pyrA2) in the second group; and met-3-pur-1-(nal-1, nal-2)-str-1-(pur-2, pur-4)-pur-3 in the third group. Electron microscopic measurements of head sizes suggested that the volume of the TP-13 phage head is seven times greater than that of the TP-18 phage head. The TP-18 genome was shown by DNA restriction analysis to have a molecular mass of 36 megadaltons. TP-13 was useful for scanning large segments of the B. thuringiensis chromosome, and TP-18 was effective for ordering markers too closely linked for simple resolution with TP-13.     

Production of prostaglandin E2 and prostacyclin by thymic nurse cells in culture

To better define the thymic microenvironment, we have examined a specific population of thymic stromal cells, thymic nurse cells (TNC) for production of eicosanoids. TNC were prepared from BALB/c mice, cultured in complete medium, and culture supernatants were analyzed for the presence of various metabolites of arachidonic acid. Freshly isolated TNC produced large quantities of PGE2 and 6-keto PGF1 alpha (a stable metabolite of prostacyclin, PGI2). Both of these eicosanoids were produced continuously in culture, after an initial lag period of approximately 2 h. No significant production of the eicosanoids PGD2, thromboxane B2, or leukotrienes B4, C4/D4/E4 was seen in these cultures. Production of PGE2 and PGI2 by TNC was not stimulated by treatment with the calcium ionophore A23187, or by cell-cell interactions resulting from coculture of the TNC with free thymocytes. Eicosanoid production in these cultures was not due to production of these substances by cells likely to be present as contaminants, such as T rosettes or free thymocytes. These findings raise the possibility that PGE2 and/or PGI2 may provide signals to thymocytes at a specific developmental stage.     

Thymic stroma-derived T cell growth factor (TSTGF). IV. Capacity of TSTGF to promote the growth of L3T4- Lyt-2- thymocytes by synergy with phorbol myristate acetate or various IL

The present study investigates the role of thymic stroma-derived T cell growth factor (TSTGF) in promoting the growth of L3T4- Lyt2- (double-negative) thymocytes. Partially purified TSTGF samples were prepared from the culture supernatant of a newly established thymic stromal cell line, MRL104.8a. The TSTGF alone induced only marginal proliferation of double-negative thymocytes, whereas this factor exerted a potent growth-promoting effect on these cells in combination with PMA. Because such an enhanced proliferation was not inhibited by anti-IL-4 or anti-IL-2R antibody, this was not due to the stimulation of an autocrine mechanism involving the production and utilization of IL-4 or IL-2. In scrutinizing PMA-equivalent physiologic substance(s), IL-1 was revealed to be capable of replacing the role of PMA in the above co-stimulation cultures and including enhanced proliferation of double-negative thymocytes in combination with TSTGF. Although TSTGF plus IL-2 or IL-4 also exhibited an appreciable or moderate synergistic effect on the growth of double-negative thymocytes, its magnitude was weaker compared with that obtained by TSTGF plus IL-1. More important, the strikingly enhanced proliferation was induced in the combinations of TSTGF, IL-1, and IL-2 or IL-4 under conditions in which the proliferation induced by IL-1 plus IL-4 or IL-1 plus IL-2 was marginal or slight. Furthermore, such strongly enhanced proliferation was also observed in the double-negative thymocyte population which was additionally depleted of T3+ cells (namely, the L3T4- Lyt-2- T3- or dull population). These results indicate the crucial role of TSTGF in the proliferation of immature thymocytes by synergy with various cytokines.     

TP-58, a novel thienopyridine derivative, protects mice from concanavalinA-induced hepatitis by suppressing inflammation

Hepatitis represents a ubiquitous human health problem but effective therapies with limited side effects are still lacking. In this study, we investigated the effect and mechanism of TP-58, a novel thienopyridine derivative, on a murine fulminant hepatitis model induced by concanavalin A (ConA). We found TP-58 markedly alleviated ConA-caused liver injury and increased survival ratio of mice injected with a lethal dose of ConA. Oral administration of TP-58 significantly alleviated ConA-caused liver injury in mice by the reduction of serum aminotransferases and liver necrosis.The analysis of proinflammatory cytokines showed that TP-58 decreased both hepatic mRNA expressions and serum protein levels of TNF-α and IL-6. And the result from LPS-stimulated RAW 264.7 cells showed TP-58 suppressed the production of TNF-α, IL-6, and Nitro Oxide (NO) in the supernatant of LPS-stimulated RAW 264.7 cells. The study of activation of nuclear factor-κB (NF-κB) by electrophoretic mobility shift assay (EMSA) showed that TP-58 inhibited the activation of NF-κB both in vivo and in vitro. The inhibitory effect was also accompanied by a parallel reduction of IκB phosphorylation. These results indicate that TP-58 protects against liver injury by inhibition of the NF-κB-mediated inflammation and suggest a potential role of TP-58 against acute liver injury and other inflammatory diseases.     

Requirement of newly synthesized protein for the priming activity of interferon.

Pretreatment of mouse L cells with interferon (IF) enhanced IF production in response to polyinosinic-polycytidylic acid (poly I-poly C). Post-treatment of cells with IF caused no significant enhancement of IF production. The enhancing effect of IF pretreatment (priming) reached a maximum after incubation with IF (10 or 100 units/ml) for 4-6 hr at 37 C, but this effect was absent when the incubation was done at 4 C. Cells which were incubated for additional several hours at 37 C after IF pretreatment at 4 C did not develop the primed state nor the antiviral state. The presence of protein synthesis inhibitors during the IF pretreatment depressed, though not completely, the development of the primed state. The residual priming effect was lost when the cells were incubated with the inhibitors at 37 C for 2 hr before they were exposed to poly I-poly C. There was no significant difference in the binding rate of poly I-poly C to cells between IF-treated and untreated cells. The degradation rate of cell-bound poly I-poly C and its sensitivity to exogenous pancreatic ribonuclease in the pretreated cells were also similar to those in the untreated cells.