Biomedical subjects
J Shoham
Publications and source records attributed to J Shoham.
Molecular cloning of the bovine thymopoietin gene and its expression in different calf tissues: evidence for a predominant expression in thymocytes.
Thymopoietin (TP), a 49 amino acid peptide, is regarded as a thymic hormone, secreted specifically by some epithelial cells in the thymic stroma and exerting a multitude of effects on maturation and function of T lineage cells. As part of our study on the molecular biology of TP, we isolated cDNA clone coding for a bovine TP precursor and used it as a probe to analyze the presence of mRNA coding for TP in different tissues. The cDNA clone reported here is 1.1 kb long and contains an open reading frame (ORF) of 741 bp which corresponds to 247 amino acids. The 147 bp coding for the entire bovine TP are at the 5' end of the ORF. A DNA fragment coding for amino acids 1-42 of bovine TP was used as a probe to look for hybridizable RNA sequences, extracted from various calf tissues, by the S1 nuclease protection method. Our results indicate that the TP gene is expressed predominantly in lymphatic tissues. Lymphatic tissues with the highest levels observed were thymocytes and not thymic stroma. Lower, but still significant, amounts were present in tonsils, neck lymph nodes, and small intestine (probably because of its lymphatic part--the Peyer's patches), whereas cultured thymic stromal cells, spleen tissue and peripheral blood mononuclear cells displayed a low level of TP mRNA. The TP gene expression in all other (non-lymphatic) tissues tested, was weak, barely detectable or virtually absent. However, the cerebellum could be singled out with relatively strong expression of TP mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. IV. Cytokines secreted by human thymic epithelial cells in culture and their activities on murine thymocytes and bone marrow cells.
In previous reports we described our approach to the cultivation of murine and human thymic epithelial cells in primary cultures, using defined, serum-free growth factor-supplemented medium and extracellular matrix-coated culture plates. The cells in these cultures displayed high metabolic activity and their supernatant was highly active on thymocytes. In the study reported here we analysed cytokine activities in the supernatant of human thymic epithelial cell cultures (HTES), by using the respective cytokine-dependent cell lines and by neutralization with specific monoclonal antibodies. Three cytokine activities were detected--interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) and macrophage (M)-CSF. Other cytokine activities tested for [IL-1, IL-2, IL-7, interferon (IFN) and tumour necrosis factor (TNF)] were negative. The effect of HTES on concanavalin A (Con A)-induced proliferation of murine thymocytes could be completely abolished by anti-IL-6 antibodies, but not by antibodies to CSF, whereas enhancement of bone marrow cell proliferation by HTES was partially inhibited by either anti-G-CSF or anti-M-CSF antibodies and completely inhibited by both antibodies, but not at all by anti-IL-6. We can thus distinguish between thymocyte-related cytokines (IL-6) and bone marrow (myeloid/monocyte) related ones (G-CSF, M-CSF) in HTES.
Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. III. Growth conditions of human thymic epithelial cells and immunomodulatory activities in their culture supernatant.
We report here on a new approach to the cultivation of human thymic epithelial (HTE) cells, which apparently allows more faithful preservation of cell function. This approach, previously developed by us for mouse thymic epithelial (MTE) cells, is based on the use of culture plates coated with extracellular matrix (ECM), and on the use of serum-free, growth factor-supplemented medium. The nutritional requirements of HTE and MTE are somewhat different. Although both are critically dependent on ECM and insulin, they differ in their dependency on other growth factors: selenium and transferrin are much more important for HTE cells, whereas epidermal growth factor and hydrocortisone play a more essential role in MTE cultures. The epithelial nature of the cultured cells is indicated by positive staining with anti-keratin antibodies and by the presence of desmosomes and tonofilaments. The ultrastructural appearance of the cells further suggests high metabolic and secretory activities, not usually found in corresponding cell lines. The culture supernatant (CS) of HTE cells exhibited a strong enhancing effect on thymocyte response to Con A stimulation, as measured by cell proliferation and lymphokine production. The effect was observed on both human and mouse thymocytes, but was much stronger in the homologous combination. Thymic factors tested in parallel did not have such a differential effect. The dose-effect relationships were in the form of a bell-shaped curve, with fivefold enhancement of response at the peak and a measurable effect even with 1:1000 dilution, when human thymocytes were used. The responding thymocytes were those which do not bind peanut agglutinin and are resistant to hydrocortisone. The culture system described here may have advantages for the in vitro study of thymic stromal cell function.
Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. I. Growth conditions and morphology of murine thymic epithelial and mesenchymal cells.
We report here the successful selective cultivation of murine thymic mesenchymal reticular cells (MTMC) and murine thymic epithelial cells (MTEC) grown on extracellular matrix in the presence of defined medium. The selective growth of these two cell types was based on 1) conditions of tissue disruption and 2) differential growth requirements. Both cell types were dependent on transferrin, high density lipoproteins, insulin, hydrocortisone, and epidermal growth factor, whereas MTMC was dependent also on selenium and 3,5,3'-triiodothyronine. The elimination of single factors or extracellular matrix resulted in specific and different changes in the growth pattern of each cell subpopulation. Cells of both types exhibited the ultrastructural features of high metabolic activity. The epithelial nature of MTEC cultures was defined by bundles of tonofilaments and desmosomes and by positive staining to keratins and negative to vimentin. In addition MTEC were positively stained with mAb to thymic medullary epithelial cells and by Ulex europeus agglutinin, and were able to form Hassall's corpuscles, suggesting their medullary origin. MTEC were also H-2 and Ia positive. In contrast MTMC were positive for vimentin and periodic acid-Schiff, low positive for H-2, and negative for keratin and Ia. Both cells did not contain nonspecific esterase, nor did they phagocytize latex beads. With the use of all these criteria we classified MTEC as epithelial cells from the medullary compartment of the thymus and MTMC as reticular cells of mesenchymal origin.
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.
Augmentation of tumor cell immunogenicity by viruses--an approach to specific immunotherapy of cancer.
Several viruses have been evaluated as potential agents for cancer treatment using either their oncolytic properties, in order to lyse cancer cells, or their potential augmenting effects on the immune response to tumors. However, the direct oncolytic effect was found to be limited in time, in scope and in specificity, whereas the use of viral oncolysates to augment antitumor immunity was shown to be better than tumor cell homogenates or extracts but inferior to noninfected intact tumor cells, attesting for the importance of membrane architecture in preserving immunogenicity of tumor specific surface antigens. In order to get the maximum benefit from this approach we selected a nonlytic virus-tumor cell combination, using Newcastle disease virus as a nonpathogenic virus, to treat the experimental tumor model, Lewis lung carcinoma (3LL) in mice. The virus effectively infected 3LL cells without any cytopathic effect. The infected cells induced strong antitumor immunity, as judged by the appearance of immune cells in the spleen (Winn test and lymphocytotoxicity) and by the resistance to challenge with the 3LL cells after immunization. The antitumor immunity was superior to that obtained with intact noninfected tumor cells. We also designed a treatment protocol using the same virus-tumor cell preparation to treat mice after tumor inoculation. This treatment resulted in cure of 40% of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Enhancement of human monocyte cytotoxicity by both interferon-gamma and -beta and comparison to other stimuli.
Recombinant interferon preparations caused a dose-dependent increase of human monocyte cytotoxicity to the K562 and Daudi cell lines. Both rIFN-gamma and rIFN-beta enhanced this function to a similar extent, while rIFN-alpha c had less effect when compared on the basis of their anti-viral effects. Endotoxin and concanavalin A increased basal monocyte cytotoxicity while phagocytosis of latex particles had no effect. The increased monocyte cytotoxic effect of rIFN-beta was completely abrogated by monoclonal antibody to IFN-beta, while monoclonal antibody to IFN-gamma had no effect. However, monoclonal antibody to IFN-gamma only reduced the increased cytotoxic effect caused by rIFN-gamma by 25%. Catalase inhibited both basal monocyte cytotoxicity and the increase in cytotoxicity following addition of rIFN-gamma only slightly, suggesting that mechanisms other than the oxidative burst were active and could be induced by rIFN-gamma.
Contact-mediated maturational effects of thymic stromal cells on murine thymocytes in culture.
The effect of direct contact between thymic stromal cells and thymocytes on differentiation markers and functions of the latter was studied. The thymic stromal cells included two epithelial and one fibroblast cell lines, previously described. Murine thymocytes were incubated with confluent monolayers of these cells or their supernatants for 24 hr, using monolayers of non-thymic cells and their supernatants as controls. Then, the thymocytes were tested for changes in expression of several surface antigens [Thy-1, Lyt-1, Lyt-2, L3T4, IL-2-receptor (IL-2R)], spontaneous [3H]thymidine incorporation (STI), lectin-induced proliferative response (PR) and lymphokine (IL-2 and IL-3) production. All three thymic stromal cell lines reduced the expression of Thy-1, Lyt-1 and Lyt-2 significantly. The expression of L3T4 was also reduced in some of the experiments, while IL-2R was not expressed by the thymocytes, neither before nor after the co-culture. The thymic stromal cell lines also increased the spontaneous [3H]thymidine incorporation and lymphokine production by the thymocytes and inhibited their proliferative response to lectins. Under the same experimental conditions, the culture supernatants of the thymic stromal cells and the non-thymic cells did not have any effect on the thymocytes, either when collected and used separately or when used in a co-culture system which allowed thymocyte contact with the medium but not with the stromal cells (Transwell system). These results suggest a specific effect of thymic stromal cells, epithelial as well as fibroblasts, on thymocyte maturation. The effect is mediated by direct cell contact and not by secreted factors.
Placebo-controlled trial of topical interferon in labial and genital herpes.
The efficacy of topical interferon-beta (IFN-beta) treatment was assessed in 25 patients with herpes of the lips or genitals who completed a 2-year follow-up in a double-blind placebo-controlled trial. IFN-beta gel (10(5) U/g) 4 times daily (about 2 x 10(4) U) applied locally during eruptions (about 10 days) reduced the mean number of recurrences (p less than 0.007) and the duration of eruptions (p less than 0.007): in the placebo group these indices did not change significantly. Reduction of symptoms and severity was noted in 11 of 12 patients on IFN-beta and in only 1 on placebo. No important side-effects were recorded. Topical IFN-beta may therefore be advantageous as a time-limited local treatment of recurrent herpes simplex virus infections of the genitals and lips.
Antigen-stimulated lymphokines from patients with cutaneous leishmaniasis induce monocyte killing of Leishmania major intracellular amastigotes.
The specific immune response of patients with cutaneous leishmaniasis including the ability of their lymphokines to enhance the monocytes' leishmaniacidal activity was studied. In 16 patients with cutaneous leishmaniasis, their concanavalin A-induced lymphocyte proliferative responses, interferon-gamma and interleukin 2 activities and the ability of their concanavalin A-induced lymphokines to kill monocyte intracellular amastigotes were not different from normal controls. Antigen-stimulated lymphocyte cultures showed that 13 of 13 patients had an increased lymphocyte proliferative response; 11 of 16 produced interleukin 2 and 12 of 13 produced interferon-gamma; in addition, 10 of 11 of these antigen-induced supernatants increased the monocytes' killing of Leishmania major amastigotes. Antibody levels to parasite membrane antigens determined by radioimmunoassay showed that 8 of 13 titers were greater than 10 and 4 of 13 titers were 2 to 10 times higher than control. Our findings demonstrate that patients with cutaneous leishmaniasis elicit a specific immune response to L. major antigens and part of this response is the production of lymphokine capable of promoting monocyte killing of intracellular amastigotes.
Modulation of immune response and tumor development in tumor-bearing mice treated by the thymic factor thymostimulin.
Thymostimulin (TS), a partially purified thymic factor, has a significant impact on tumor development in C57B1/6 mice inoculated with Lewis lung carcinoma (3LL) cells, as judged by its effect on time of tumor appearance after tumor cell transplantation. In a previous study, we determined the conditions under which survival rate of the tumor-bearing mice can be significantly increased by TS treatment. In the study communicated here we analyzed host defense mechanisms that are modified by TS treatment in the tumor-bearing mice. In general, immune parameters that were increased or stimulated by the presence of the tumor were further increased in the TS-treated animals (number of lymphoid spleen cells, their response in mixed lymphocyte tumor cultures, their natural killer cell activity, and their ability to produce colony-stimulating factor), or reached earlier maximum levels (spontaneous [3H]thymidine incorporation, a reflection of in vivo spleen cell activation). Responses which reflect tumor-induced immunosuppression (proliferative response induced by phytohemagglutinin or concanavalin A stimulation) were restored to normal level by TS. Specific tumor-related reactions (specific cell-mediated cytotoxicity) were preserved in the TS-treated animals. The wide spectrum of TS effects had, nevertheless, certain elements of selectivity; e.g. colony-stimulating factor, but no interferon production is enhanced by TS in the tumor-bearing mice in diametric contrast to TS effect in Mengo virus-infected mice. The spectrum of TS effects was also dependent on the type of tumor cell used. The results indicate that the significant effect of TS on 3LL tumor development in mice is associated with a strong, multifaceted effect of TS on the immune system.
Solid-phase radioimmunoassay for the measurement of surface antigens expressed on intact lymphocytes.
A solid-phase radioimmunoassay was developed for the measurement of lymphocyte surface antigens. The assay was performed in microplates, using cells that were initially fixed to the wells by air drying. The method was used for the measurement of Thy-1, Lyt-1,2,3, IL-2-R, H-2Kb and DR antigens on the surface of mouse thymus, spleen and bone marrow cells, mouse cell lines CTLL, EL-4 and DA-1 and human thymocytes and consisted of sequential incubations with rat or mouse monoclonal antibodies directed against the above antigens, rabbit anti-rat or goat anti-mouse IgG and 125I-protein A. The assay permits the processing of large numbers of samples, is easy to perform, reliable and highly specific.
Topical application of human fibroblast interferon (IFN) in cutaneous leishmaniasis.
Fifteen patients with single or multiple lesions of cutaneous leishmaniasis were randomly assigned to apply either IFN-beta-containing cream or a placebo cream base. Despite 8 weeks of therapy no significant improvement of the lesions was achieved. Although topical IFN-beta was not effective in this study, several laboratory studies suggest that IFN, particularly IFN-gamma, may be of therapeutic value in cutaneous leishmaniasis.
The enhancing effect of interferon-beta and -gamma on the killing of Leishmania tropica major in human mononuclear phagocytes in vitro.
Both native human IFN-beta or -gamma added to human monocytes in culture increased their leishmaniacidal effect on intracellular Leishmania tropica major (L. major) amastigotes. This effect was dose-dependent, and was apparent if the IFN was added either before or after infection of the monocyte cultures with the promastigote form of the parasite. Compared on the basis of antiviral activity, IFN-gamma was shown to have a leishmaniacidal effect approximately three times greater than IFN-beta. Recombinant IFN preparations showed similar effects. In addition, IFN-gamma increased H2O2 production from human monocytes in culture in a dose-dependent manner. Monoclonal antibody to IFN-gamma abrogated both its effect on the leishmaniacidal capacity and on H2O2 production by the monocytes. These results suggest that IFN-gamma may be of therapeutic value in cutaneous leishmaniasis.
The effects of Con A-induced lymphokines from the T-lymphocyte subpopulations on human monocyte leishmanicidal capacity and H2O2 production.
The effects of concanavilin A (Con A)-induced lymphokines from human peripheral blood mononuclear cells, enriched helper and suppressor lymphocyte populations and recombinant interferon-gamma (IFN-gamma) on the ability of monocytes to promote killing of intracellular Leishmania major (L. major) amastigotes and to induce the production of hydrogen peroxide (H2O2) was examined. All these mitogen-induced supernatants contained active IFN-gamma. There were no differences in the ability of these supernatants or IFN-gamma to promote the monocyte killing of the L. major amastigotes or H2O2 production. An increase in H2O2 production by monocyte monolayers was observed following the addition of either of these supernatants or IFN-gamma alone. In addition, a marked increase in the production of H2O2 was observed following priming with either of these mitogen-induced supernatants or IFN-gamma and the addition of a second stimulus, phorbol myristate acetate (PMA). Monoclonal antibody to IFN-gamma abrogated the increase in production of H2O2 by all these mitogen-induced supernatants; however, this antibody only resulted in partial inhibition of the leishmaniacidal effect of these lymphokines on human monocytes. These results would suggest that IFN-gamma is the component of the lymphokine that is largely or exclusively responsible for H2O2 production, while other factors in addition to IFN-gamma are important in promoting oxygen-independent mechanisms for the killing of intracellular L. major amastigotes.
A newly designed whole microplate automatic harvester for lymphocyte stimulation assays.
A unique automated sampling manifold designed to recover cells grown in standard 96 well microplates from their culture medium is described. Cells are recovered and washed on fiber glass filter discs. Incorporation of radioisotopes into cells, can then be measured by appropriate counting of the filter discs. Typical applications include termination of mixed lymphocyte cultures, assays of mitogen stimulation of lymphocytes and antigen-specific lymphocyte transformation and assays of interferon activity. The harvester can also be used in other biological systems where collection and washing of precipitates is desired.
Antibody-dependent cellular cytotoxicity to human colon-tumour cells. I. Lack of tumour specificity in a population study.
The humoral and cellular components of the antibody-dependent cellular cytotoxicity (ADCC) against allogeneic human colonic tumour cell lines were evaluated. The 2 colon cell lines used in this study (HT-29 and ACC-20) were found by immunofluorescence to have carcinoembryonic antigen (CEA) on their surface, and to become sensitive to the lytic effect of unstimulated lymphocytes after coating with heterologous anti-CEA. This reaction was used to evaluate the ADCC activity of mononuclear cells from the peripheral blood of patients with gastrointestinal cancer (mostly local extensive colo-rectal). Remarkable variability was found in the lytic capability (2-50% specific lysis) of both cancer and non-cancer mononuclear cells, with no significant difference between them. Sera from 127 cancer patients and 91 non-cancer patients were tested, using the reaction with heterologous anti-CEA as positive control and as a reference point. In 46 cases (21%) the sera were reactive in this system, and 43 of them were of Blood Group O. However, there was no difference between the cancer patients and the normal controls. The antigenic determinant involved in this reaction is not the Blood Group A specificity but, most probably, a polypeptide common to CEA and A (as shown in the following publication). In addition, trials for the elimination of the non-tumour-specific reaction, by absorption or inhibition, failed to disclose a tumour-specific one. The value of the ADCC assay in monitoring human tumour immunity, and possible ways of eliminating reactivity to normal antigens in this system, are discussed in the light of these findings.