[Linear growth of the chick embryo tibia cultured in presence of TAE (thymus gland aqueous extracts)].
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For theoretical and practical reasons, it is important to find out whether the differentiation of T cell precursors to the functional lymphocytes can be induced under in vitro conditions. Using the local GVHR assay (based on the enlargement of the popliteal lymph node), the inducibility of the precursors of reactive cells was studied with bone marrow, thymus, spleen, and lymph node cell suspensions submitted to short-term incubation with cell-free extracts from calf thymus, spleen or brain. GVHR-precursors from bone marrow were inducible not only specifically (i.e., with thymus extract) but also--and even to a higher degree--with spleen or brain extract. Thymus and spleen cell suspensions (the latter also depleted of the reactive subpopulation by treatment with anti-Thy 1.2 serum and complement) were, on the other hand, inducible mainly specifically, whereas lymph node cells were refractory to induction. The inductive action of tissue extracts obviously depends on the tissue origin of T cell precursors; their effects on pre- and postthymic differentiation of T lymphocytes are discussed.
Treatment of adult AKR mice with tissue-specific mitotic inhibitor mol. wt. 1,000--20,000 extracted from the AKR thymus significantly delayed the onset of spontaneous thymus leukaemia, whereas extracts of other organs were without effect. A slight prolongation of survival time was found with spleen extract when administered to BALB/c mice infected with Rauscher virus, which causes leukaemia starting in the spleen. Thymus extracts of MW 100,000--300,000 caused myocardial degeneration in some leukaemic and nonleukaemic mice.
Children suffering from JCA showed an elevated percentage of OKT4+ T lymphocytes and an increased ratio of OKT4+ to OKT8+ lymphocytes. Several children showing a significant aggravation of the disease, were selected for a 3-week treatment with TFX. In 7 out of 10 patients a pronounced clinical and laboratory improvement was registered, accompanied by a normalization of the OKT4+ to OKT8+ lymphocyte ratio.
The topical anti-inflammatory activity of four extracts from Thymus broussonetii Boiss (Labiatae) leaves, a herbal drug used in Moroccan traditional medicine, has been studied using the croton oil ear test in mice. A bioassay-oriented fractionation revealed that the pharmacological activity is mainly in the chloroform extract. Fractionation and analysis of this extract allowed the identification of ursolic acid and oleanolic acid as the main anti-inflammatory principles. Some flavonoids (luteolin, eriodictyol, thymonin) and glycosides (luteolin-7-O-glucoside, luteolin-3'-O-glucuronide, eriodictyol-7-O-glucoside) were also isolated from the methanol extract.
A cellfree extract prepared from the spleen cells of C3H mice is capable of suppressing antibody responses to SRBC when extract material is exposed to alloantigens. The observed immunosuppression was attributed to a soluble factor in the extract. This allogeneic suppressive factor (ASF) was detected in extracts prepared from the spleen cells of unirradiated mice as well as those of irradiated mice repopulated with thymocytes, provided that mice were previously immunized with SRBC. Donors of actively suppressive ASF preparations did not need to be previously exposed to alloantigens. Extracts from thymus and marrow cells of unirradiated mice and the spleen cells of irradiated mice repopulated with marrow cells (or no cells) did not contain ASF. C3H thymocytes stimulated with SRBC generated more ASF activity in spleens of C3BF1 hosts than in those of C3H hosts, indicating that alloantigenic stimulation enhances the production or activity of ASF. Once produced, C3H ASF was able to suppress antibody responses in cell transfer experiments only if exposed to C3BF alloantigens of either donor lymphoid cells or irradiated hosts. Once exposed to alloantigens, ASF appears to be capable of suppressing antibody responses of syngeneic C3H or semi-allogeneic C3BF cells. When both donor lymphoid cells and hosts were syngeneic with the donor of the ASF, there was enhancement of antibody formation in cell transfer experiments. C3H ASF did not interfere with education of C3BF thymocytes to SRBC or with the generation of precursors of anti-SRBC antibody-forming cells by C3BF1 marrow cells. ASF may interfere with cellular cooperative events necessary for humoral immune responses or with terminal differentiation of B cells. Production of ASF could partially account for the suppression of antibody responses observed during graft-vs-host reactions.
Endogenous factors inhibiting the proliferation of T-lymphocytes were investigated which may function as modulators of T-lymphocyte production within the thymus. An extract from calf thymus (T4) enriched in lymphocyte chalone arrests rat thymocytes at the G1 leads to S boundary and in the S phase of the cell cycle in short-term cultures. It also inhibits the proliferative response of human peripheral blood lymphocytes to PHA-P in a time-dependent manner, as well as the spontaneous proliferation of in vitro cultured human chronic leukaemic lymphoblasts. This crude extract contains two active moities which can be isolated by molecular filtration on Sephadex G-75 column. A species non-specific, cell line selectivity inhibitory effect is characteristic of the high molecular weight fraction (mol. wt. greater than 40,000). This activity is resistant to moderate heat treatment and trypsin but is sensitive to mild alkaline hydrolysis and to RNase A digestion. About ten protein components and a toluidine blue positive substance can be detected by analytical polyacrylamide gel electrophoresis. The active inhibitor, a proposed protein-RNA complex, might be identical with the chalone. The low molecular weight, non-dialysable factor (T4-4) inhibits [3H]thymidine incorporation into acid insoluble DNA in a cell non-specific manner. A possible relationship between the two activities is discussed.
Partially purified thymus products were used to evaluate the maturation of T lymphocytes in immunodeficient mice. Three different bovine thymic extracts, designated thymic extracts A, B, and C, were successful in increasing the longevity of conventionally raised nude mice. Daily injection of bovine thymic extracts A, B, and C and mouse thymus extracts failed to mature a population of T lymphocytes and restore the capacity to reject heart allografts. Preincubation of normal syngeneic bone marrow with thymic extract B in vitro before injection into nude mice also failed to reconstitute the host's ability to reject heart grafts. The number of antibody plaque-forming cells of sheep red blood cells in lethally irradiated, bone marrow-reconstituted mice could be increased by preincubating the bone marrow cells with bovine thymic extract fraction B before injection followed by daily injections. A similar but less marked increase in plaque-forming cells was obtained by the daily injection of bovine thymic extract fraction C. Complete functional maturation of T lymphocytes in immunodeficient animals appears to require more than thymic extract stimulation of bone marrow cells or pre-T lymphocytes.
A factor that decreases rosette formation between guinea pig T-cells and rabbit red blood cells (RRBC) was extracted from the thymus of the guinea pig. The active factor could be extracted from the spleen as well as the thymus, but not from the liver or kidney. The active factor of the thymic extract was found in the precipitates produced by 80% saturated ammonium sulfate and it was separated from the water-soluble fraction of the precipitates. The molecular weight of the partially purified substance was estimated to range between 10,000 and 30,000 by filtration through a diaflow membrane. From the studies on physicochemical characterization, it might be a heat-resistant basic peptide probably bound to a ribonucleotide moiety. This factor reduced rosette formation between RRBC and guinea pig T-cells, but did not reduce erythrocyte-antibody-complement rosette formation. This factor also inhibited mitogen (concanavalin A, phytohemagglutinin-P)- induced DNA synthesis of guinea pig lymphocytes and antigen-induced DNA synthesis of sensitized guinea pig lymphocytes.
A partially purified extract from thymus tissue termed thymosin Fraction 5 has been shown to reconstitute immunological deficiencies resulting from the lack of thymic function in several animal models, as well as humans with primary and secondary immunodeficiency diseases. Thymosin Fraction 5 consists of a family of polypeptides with molecular weights ranging from 1,000 to 15,000. Several of these polypeptides contribute individually to the biological activity of the parent compound. Two polypeptide components of thymosin Fraction 5, termed thymosin alpha1 and polypeptide beta1, have been characterized chemically and biologically. Thymosin alpha1 is a highly acidic molecule composed of 28 amino acid residues. This polypeptide has potent biological activity and has been found to be 10 to 1,000 times as active as thymosin Fraction 5 in one in vivo and several in vitro bioassay systems designed to measure differentiation and function of thymus-dependent lymphocytes (T cells). Polypeptide beta1, in contrast, is inactive in our bioassay systems, suggesting that it is not involved in thymic hormone action. Sequence analysis and homology studies have indicated that polypeptide beta1, although present in Fraction 5, does not contribute to the biological activity of thymosin Fraction 5.
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Polypeptide extracts from the pig spleen, liver, kidney, thymus, pancreas and periodont were obtained by the acid extraction in presence of Zn and MG cations. This extracts were analyzed by HPLC with determining amino acid sequence using Edman's method. According to the analysis results several fragments of haemoglobin are present in the group of parenchymal organs (epsylon-chain pig haemoglobin 56-66 and 55-66, alpha-chain 48-61), the main mass of substances is localized in more hydrophilic part of chromatogram with 20 aminoacid N-terminal ubiquitin fragments; hydrophylic polypeptides ubiquitin and 2 hydrophobic polypeptides were also found in the group, but haemoglobin peaks were absent. The main mass of polypeptide substances in periodont extract is localized in the hydrophobic region, the peaks of haemoglobin and ubiquitin are absent. We suppose that each group of similar morpho-functional tissues has similar chromatographic picture of polypeptide extracts. It can be connected with the processing of protein structures specific for each organ or tissue. This characteristic for each tissue peptide can take part in the signal transduction at the level of single population cells or between physiologically correlated organs.
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The soluble ribonucleoprotein nuclear antigen (RNP), associated with the Sm antigen and present in extracts of rabbit thymus acetone powder, was purified by ion-exchange chromatography. Analytical isoelectric focussing (IEF) showed two bands at pH 5.2 and two bands at pH 5.4. After treatment by RNAse these 4 bands disappeared, and a new band with a pI of 6.1, representing the intact protein moiety, appeared. During preparative IEF in granulated gels the antigenicity of RNP was lost. Only the Sm antigen could be detected by counter-immunoelectrophoresis. Fractions with Sm antigenicity refocussed as two intense bands (pI 6.8 and 7.2) and one or two fainter bands (pI 6.1). On reducing SDS-PAGE the SM antigen revealed two bands of apparent molecular weights of about 110,000 and 28,500, indicating a total molecular weight of 138,500.
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In this article we describe the chromatographic separation of acid nuclear protein fractions which have previously been shown to be active in DNA transfection experiments. By combining anionic and cationic ion exchangers, we were able to separate and identify some of the active proteins. In addition to HMG1, already known for its transfection activity, we have identified histone H1 and HMG17 as further transfection-active proteins. The highest transfection activity was associated with H1 and another nonidentified protein showing a somewhat higher electrophoretic mobility than H1. We have also found that the presence of CaCl2 in a low concentration in the cell culture medium is an important requirement for transfection.
Thymostimulin is a partially purified extract of calf thymus, consisting of a group of polypeptides with a total molecular weight of approximately 12,000 D. It is used as an immunologic response modifier in primary and secondary immunodeficiencies. We report a patient who had a severe anaphylactic reaction with the first injection of thymostimulin. Type I hypersensitivity to thymostimulin was suggested by an immediate skin test response, specific IgE determination by indirect ELISA and a positive histamine release test. This patient also reacted to bovine serum albumin (BSA), fetal calf serum (FCS) and cow hair and dander. The controls did not react to any of these tests. Clinicians need to be aware that the use of medical products of xenogeneic origin may lead to severe allergic reactions.