Thymus Extract Research Articles

10-S DNA polymerase from calf thymus which copies both poly(rA) · oligo(dT) and activated DNA

A novel DNA polymerase, which could use both poly(rA) · oligo(dT) and activated calf thymus DNA efficiently as template-primers, was purified 20 000-fold from calf thymus extract. These activities were co-purified throughout successive column chromatographies and banded at the same position in either electrofocusing ( pI = 6.5–7.0 ) or sucrose rate-zonal centrifugation (10–10.5 S). The most purified fraction (DNA-cellulose fraction) possessed specific activities of 3900 units/mg of protein with poly(rA) · oligo(dT) and 32 000 units/mg of protein with activated DNA. The poly(rA) · oligo(dT)-dependent activity differed from the previously described DNA polymerase γ from other sources in the following ways: 1. The activity was inhibited by 100–300 mM KCl and 80 mM potassium phosphate buffer. 2. The activity was 4-fold higher at 26°C than at 37°C. 3. The K m value for dTTP was 2.6–3.0 · 10 −4 M, which is several hundred-fold greater than that of DNA polymerase γ. 4. Mn 2+ was essential for the reaction and could not be replaced by Mg 2+. The activated DNA-dependent activity shared many properties with DNA polymerase α, except that it was less sensitive to N- ethylmaleimide and anti-α polymerase immunoglobulin G. The 10-S DNA polymerase was dissociated into 8.5-S and 3.3-S by treatment with Triton X-100.

Increased number of precursors of rosette-forming cells sensitive to thymus hormone in the spleen of mice vaccinated with smallpox vaccine

Injection of smallpox vaccine into female C57BL/6j mice aged 2 months led on the first few days to a sharp increase in the number of precursor cells of rosetteforming lymphocytes sensitive to the differentiating effect of thymus extract. By the tenth day after vaccination the normal number of these cells was restored.

Bovine thymus poly(adenosine diphosphate ribose) polymerase.

About 1,300-fold purification of poly(adenosine diphosphate ribose) polymerase has been achieved from the extract of bovine thymus with a recovery of 10 to 20%. The final preparation has a purity of 99%, and the enzyme is composed of a single peptide with a molecular weight of 130,000. The purified enzyme required NAD+, Mg2+, a thiol compound, DNA, and histones for full activity. Whereas DNA is essential for activation of the enzyme, histones are not. The observed stimulation of the reaction by histones is shown to be due to masking of the inhibitory effect of contaminating denartured DNA in native DNA preparation. The concentration of DNA required for half-maximal enzyme activity (apparent Km for DNA) is proportional to the concentration of enzyme in the reaction mixture. The minimum estimation of the number of nucleotide pairs of DNA required for half-maximal activation of one enzyme molecule is 220 to 240 for bulk of calf thymus DNA, while the value is 10 for a calf thymus DNA fraction, "active DNA," which was separated from the enzyme fraction in a stage of the purification. These results suggest that the enzyme is activated by binding to a specific site on calf thymus DNA. The apparent Km for NAD+ and the maximum velocity of the enzyme are estimated to be 60 micrometer and 0.91 mumolper min per mg, respectively.

Effect of tissue specific mitotic inhibitors on survival time of spontaneous and virus-induced murine leukaemia and influence on myocardial degeneration.

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.

Enrichment of in vitro and in vivo immunologic activity of purified fractions of calf thymic hormone.

Thymus humoral factor (THF), a thymus hormone which participates in the processes leading to acquisition of immunocompetence of lymphoid cells has been isolated in our laboratory by a stepwise gel filtration through various Sephadex columns. THF so isolated appears to be a polypeptide of 3,000 mol wt which contains approximately 30 amino acid residues. Here we have tested the biological activity of THF fractions of successive degrees of purity upon lymphoid cells from both intact and neonatally thymectomized mice. The lymphoid cell populations were treated with the various THF fractions by in vitro incubation for a short time and by repeated injection in vivo. The treated cells evidenced increased ability to react in the graft-versus-host assay in vivo and in mixed lymphocyte cultures in vitro concomitantly with the rise of intracellular cAMP. On the other hand no activity whatsoever was shown by any of the control materials tested. These bioassays permitted isolation of fractions progressively more active than the original crude dialyzate of thymus extract tested. Thus the active peptide component of THF eluted from DEAE Sephadex A-25 column was estimated to be 2 X 10(4)-fold more active than the crude dialyzate of thymus extract which served as a starting material.

681 THYMOSIN TREATMENT OF DI GEORGE SYNDROME

A 2½-month-old infant with complete DiGeorge syndrome was treated with bovine thymosin fraction V. Initial evaluation revealed normal numbers of blood lymphocytes, 29% EAC rosettes, 41% sIg lymphocytes and no T-rosettes. T-rosettes rose to 15% upon in vitro incubation with thymosin. There was no in vitro response to PHA, PWM, SLO, candida or allogeneic cells, and delayed skin reactivity to SKSD, candida and DNCB was absent. IgG, A, M and E were 175 mg%, 15 mg%, 40 mg% and 10 Iu/ml respectively. He received 10 mg/day of thymosin for 21 days, followed by 20 mg/day for an additional 17 days, and weekly injections of the same dose thereafter. During therapy the lymphocyte count did not change, but T-rosettes rose to 26% while EAC rosettes and sIg lymphocytes decreased to 14% and 10% respectively. Lymphocytes remained unreactive in vitro to PHA, PWM, candida and allogeneic cells, but reacted to SLO (S.I.=42). Delayed skin reactions remained negative. IgG, A and M were 120 mg%, 20 mg% and 62 mg% at 4 months of age, while IgE rose to 255 Iu/ml. No specific antibodies were detected. He died of sepsis at 4½ months of age. In the postmortem no thymus or parathyroids could be found, and the lymphoid tissue revealed depleted T-dependent areas with poorly developed germinal centers. Similar partial reconstitution of T-cell functions, presumably resulting from the maturation of null-cells, have been observed in other immunodeficiencies treated with thymic extracts.

Effect of thymosin and lipopolysaccharide on murine lymphocyte cyclic AMP

The effects of thymosin and lipopolysaccharide (LPS) on cyclic AMP levels in lymphocytes were evaluated using three independent assays which included adenine prelabeling, protein kinase binding, and radioimmunoassay. All three assays proved to be both sensitive and accurate in assessing relative changes in lymphocytes after incubation in vitro with various agents. The assays confirmed that basal and stimulated levels of cyclic AMP depended on the origin of the lymphocyte population. Each of the three techniques demonstrated that pyrogen-free bovine thymosin fraction 5 did not elevate thymocyte cAMP levels. In contrast, it was found that lipopolysaccharide (LPS) significantly elevated cAMP levels in both spleen and thymus lymphocytes. These studies indicate that assays for measuring the activity of thymic extracts in which the intracellular levels of cyclic nucleotides are a criterion for activity are only valid if the preparations are not contaminated with endotoxins.

723 STEM CELL DEFECTS IN SEVERE COMBINED IMMUNODEFICIENCY (SCID)

Fractionated bone marrow cells from normal volunteers can regularly be induced to bear the human T-lymphocyte antigen (HTLA) marker to form E-rosettes and respond to concanavalin A (Con A) following coculture with normal thymic epithelial mono-layers (TEM) or supernatants (SUP). T-cell differentiation done in this way on marrow cells of 5 patients with SCID revealed the following results: Previously, Touraine et al found no induction of HTLA marker in the marrow of a patient with SCID after incubation with thymic extract suggesting absence of stem cells but Incefy et al showed that some patients may have stem cells inducible to HTLA but not to E-rosettes or functional markers. We conclude that in variants of SCID precursor cells are present but are arrested at different steps along the T-cell line. In T-cell differentiation HTLA marker probably appears before E-rosettes, followed by a state of responsiveness to mitogens. (Supported by grants CA-17404, CA-19267 and Judith Harris Selig Foundation)

Characterization of a Soluble Factor that Specifically Suppresses the in Vitro Generation of Cells Cytotoxic for Syngeneic Tumor Cells in Mice

Abstract A tumor-specific soluble factor found in extracts of thymocytes from mice bearing small P815 tumors has been demonstrated. This factor is capable of significantly suppressing the in vitro generation of syngeneic cells cytotoxic for P815 targets if it is added to culture vessels within the first 30 hr of culture. The suppressive factor eluted from Sephadex G-100 after hemoglobin and was estimated to have a m.w. in the range of 40 to 60,000. Preparative isoelectric focusing of thymic extracts established that the suppressive material has an isoelectric point in the range of pH 4.6 to 4.9. The suppressive activity of extracts could be removed by passage of the material through immunoadsorbent columns prepared from membrane proteins of P815 cells but not by analogous columns prepared by using L1210 membrane proteins. The suppressive material was not removed by its passage through immunoadsorbent columns containing anti-mouse immunoglobulin.

In vitro allogeneic response of human lymphocytes dependent upon dialyzable plasma components and a thymic hormone, THF

The in vitro response to allogeneic stimulation of human lymphocytes obtained from umbilical cord blood and from adult peripheral blood is dependent upon the presence of dialyzable plasma components. We observed here a reduced allogeneic response of human lymphocytes in the presence of a dialyzed human plasma as compared to their reactivity in the presence of whole human plasma. This impaired mixed lymphocyte culture response was restored by the addition of thymus humoral factor (THF), a calf thymus extract. It is suggested that dialysis depletes the plasma of its natural thymic hormone(s) content, this being replaced by the addition of THF. Restoration of the in vitro allogeneic response of human lymphocytes by THF was shown to be specific, since such effect was not observed when calf spleen extract or bovine serum albumin were tested.

The biochemistry of lymphocyte-derived mediators of immunological inflammation.

Evidence is presented to indicate that there exists in lymphoid tissue, as a result of transforming lymphocytes, a new lymphokine which is chemotactically specific for lymphocytes, called 'lymphotactin'. Lymphotactin has been purified to electrophoretic homogeneity; has a molecular weight of 10,500 D and an isoelectric point of 5.9. Its role in amplifying the immune defense system by recruitment of naive lymphocytes into propinquity with the challenging antigens is suggested. Purification of macrophage migration inhibitory factor from thymus extracts to electrophoretic homogeneity leads to a compound of molecular weight of 36,500 D and an IEP of 6.9. Chemically it contains sialic acid and o-methyl glucopyranoside as its only carbohydrates. Purified MIF activates the macrophage phagocytically. Skin reactive factor and lymph node permeability factor have been isolated and purified and are found to be inhibited by pepstatin and antihistamine and to have an isoelectric point of pH 4.2 and a molecular weight of 50,000--100,000 D. It is believed that this anionic permeability increasing agent actually arises from the lysosomes of macrophages and lymphoblasts (the normal small lymphocyte having essentially no lysosomal organelles). The mononuclear cell infiltration characteristic of crude SRF and LNPF may proceed from their being contaminated with lymphotactin.

The in vitro effect of a thymic epithelial culture supernatant on mixed lymphocyte reactivity and intracellular cAMP levels of thymocytes and on antibody production to SRBC by nu/nu spleen cells

Addition of supernatants from rat thymic epithelial cultures (TES) to rat thymocytes stimulated with T-cell-mitogens or allogeneic cells leads to an increase in 14C-TdR incorporation. Furthermore, in the presence of TES, spleen cells from athymic nu/nu mice exhibit an enhanced in vitro antibody production to SRBC, whereas TES has no such effect on spleen cells from T-cell-deprived mice. If TES is added together with thymocytes to T-cell-deprived spleen cell cultures, the number of plaque-forming cells to SRBC is enhanced, suggesting that TES induces a helper cell function in thymocytes which, if added alone, have no effect. TES also increases intracellular levels of cAMP in thymocytes in vitro and appears to act on a membrane site distinct from the β-adrenergic receptor. TES fails to affect mitogen responses, MLR and cAMP levels of lymphocytes from other lymphoid organs. The biological activity of TES as compared to that of thymic extracts is discussed.

Effect of thymus extract and of late thymectomy on interferon production

Mice aged 10 days do not produce interferon in response to injection of Newcastle disease virus (NDV). A single injection of a fraction of calf thymus extract, obtained by the method of Gyulling et al., into newborn mice conferred the ability to produce interferon on mice at the age of 10 days. In response to injection of NDV, adult rats produce interferon in high titers. The intensity of interferon synthesis was unchanged 2–3 months after thymectomy.

Immunotherapy of cancer with thymosin.

AbstractBovine thymosin fraction 5, a partially purified thymic extract, has been employed in phase I and phase II clinical studies in immunodeficient cancer patients. Some improvement in cell‐mediated immune functional parameters has been seen, notably an increase in percentages of peripheral blood T lymphocytes as detected by rosette formation with sheep erythrocytes. Thymosin‐induced rosette‐forming cell increases demonstrated in vitro prior to the institution of thymosin treatment generally were predictive of those patients who showed the in vivo rise in circulating T cells following thymosin administration. Although some patients exhibited clinical improvement, it is still too early to infer from these open studies whether or not these improvements can be attributed to thymosin treatment. Well‐controlled randomized trials are in progress to objectively examine the clinical efficacy of thymosin fraction 5 therapy in cancer patients.Thymosin fraction 5 contains several thymus‐specific polypeptides which appear to act on lymphoid cells at a number of stages during their differentiation. Several of these individual polypeptides have been isolated, and one has been sequenced (thymosinα1) and found to have immunoenhancing activity. Cancer patients may be viewed in many cases as exhibiting an “acquired” disease‐related or treatment‐related deficiency in cell‐mediated immunity. This may render immunotherapeutic manipulations nonbeneficial and may in fact contribute to tumor progression and/or metastasis. By treating such patients with thymosin fraction 5 or the specific polypeptide(s) within fraction 5 required to allow the appearance of immunocompetent T lymphocytes, these patients may become amenable to immunotherapy, more resistant to infection with opportunistic organisms, and perhaps may develop endogenous antitumor defenses.

Rationale for combined use of fetal liver and thymus for immunological reconstitution in patients with variants of severe combined immunodeficiency

Bone marrow cells from a patient with severe combined immunodeficiency were studied in vitro for thymus-dependent lymphocyte (T cell) differentiation by using, at varying times, thymic epithelial monolayers and culture supernatants, thymopoietin, ubiquitin, and thymic extract as inducing agents. On initial evaluation, with thymopoietin or human thymic extract, only a partial differentiation of marrow cells was achieved into cells bearing the human T cell antigenicity without the capacity to form rosettes with sheep erythrocytes, suggesting that the stem cells were defective. Two fetal liver transplantations aimed at reconstitution were unsuccessful, despite evidence of chimerism. Induction studies at that time demonstrated rosetting capacity (with sheep erythrocytes) of the patient's bone marrow cells after coculture with thymic epithelial monolayers but not with their supernatants. An 18-week fetal thymus (irradiated) was then transplanted, but the transplantation was unsuccessful and no clear evidence of chimerism was demonstrated. Subsequently, transplantation of another fetal liver resulted in chimerism and immunologic reconstitution. Serum thymic factor activity rose from 1:2 before transplantation to 1:16 after reconstitution. The combined use of fetal thymus and liver may provide effective immunological reconstitution in some variants of severe combined immunodeficiency.

A low-molecular-weight homogeneous fraction of the thymus stimulating immunogenesis

The effect of a purified acetic acid extract of thymus (“thymarin”) and of three of its fractions obtained by ion-exchange chromatography, and consisting of individual substances of polypeptide nature, on the immune response was studied in mice immunized with sheep's erythrocytes. Thymarin and one of its fractions with a molecular weight of about 5000 were shown to have a marked stimulating action on the thymus-dependent immune response (the formation of cells producing IgM and IgG antibodies and the circulating antibody level).

Biochemical characterization of alkaline phosphatase in guinea pig thymus

1. 1. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) in guinea pig thymus was extracted optimally in 10 mM Tris · HCl buffer at pH 8.0 containing 5 g/l Triton X-100. 2. 2. α-Glycerophosphate, β-glycerophosphate and phenolphthalein monophosphate were hydrolyzed by thymus extract with a pH optimum at 9.8–10.0, whereas p- nitrophenylphosphate and ga-naphthylphosphate were hydrolyzed with pH optima at 10.7—10.8 and β-naphthylphosphate at pH 11.2. p- Nitrophenylphosphate and phenolphthalein monophosphate proved to be the most suitable substrates. 3. 3. Alkaline phosphatase was effectively inhibited by EDTA, Zn 2+, histidine and urea therefore resembling the inhibition characteristics of alkaline phosphatase in the placenta and kidney, but not that in the liver and intestine, which differed markedly. 4. 4. DEAE-cellulose chromatography and polyacrylamide disc electrophoresis revealed three enzyme peaks which did not differ in their substrate specificities and modifier characteristics. 5. 5. Polyacrylamide disc electrophoresis of thymus, serum, placenta, kidney, liver, bone and intestine revealed no alkaline phosphatase bands definitely unique to thymus.

Identification of a thymic inhibitor ('chalone') of lymphocyte transformation as a spermine complex.

CHALONES are defined as endogenous, tissue-specific but species nonspecific inhibitors of cell proliferation. Their potential use in the control of neoplasia is well recognised1–3. Circumstantial evidence for the existence of a lymphocyte chalone has been produced4–8, but neither its chemical nature nor its molecular weight have been satisfactorily ascertained. During our search for a lymphocyte chalone, an inhibitor of lymphocyte transformation has been reproducibly isolated from porcine thymic tissue. We report here that though the inhibitor was non-dialysable, its active moiety was identified biologically and chemically as spermine, molecular weight only 202. This accords with the formation in thymic extracts of a tightly bound spermine complex. As the complex displayed some tissue specificity it is possible that an unidentified tissue-specific material acts as a carrier for spermine. In relatively crude extracts of tissues it is probable that spermine-elicited inhibition swamps any authentic chalone activity.

Immunochemical studies on acetylcholine receptor from Torpedo californica

Acetylcholine receptor (AChR) was purified from electric organ tissue of Torpedo california by solubilization of membrane fragments with 1% Triton X-100 followed by affinity chromatography on a Naja naja siamensis neurotoxin-Sepharose resi. The molecular and pharmacological properties of the purified recerptor were determined, and it was shown to have a specific activity of 8–12 nmoles toxin bound per mg protein, corresponding to 80,000–125,000 daltons per toxin-binding site. Experimental autoimmune myasthenia gravis was consistently induced in rabbits following a single injection of purified AChR. The cellular and humoral immune responses to the purifed AChR were analyzed in the injected animals. By lymphocyte transformation in vitro it was shown that such animals responded not only to the homologous immunogen— Torpedo AChR—but also to extracts of rabbit skeletal muscle or thymus. Humoral rabbit anti-AChR antibodies gave one precipitation line with AChR in immunodiffusion and immunoelectrophoresis. The antigenic determinants against which antibodies were produced are different from the toxin-binding sites of the AChR. However, anti-AChR antibodies inhibit toxin-binding, probably due to steric hindrance. A possible molecular model is presented for explaining the relationship between the physiological active site, the ‘myasthenic determinant’ and other antigenic determinants on the AChR molecule.

Thymosin reconstitution of T cell deficitsin vitro in cancer patients

Thymosin, a soluble extract of fetal calf thymus, has increased cellular immunity in children with thymic deficiency. Prior to therapy, an increase in thymus-dependent lymphocyte (T cell) levels in vitro after incubation with thymosin correlated with a rise in peripheral blood T cell levels and improvement in other parameters of cellular immunity. These correlations constituted the basis for a study of the effects of thymosin on T cell levels in vitro in cancer patients. Groups studied were 350 untreated patients with local-regional solid malignancies, 157 patients cured of these tumors, 340 patients studied at 523 intervals during radiation therapy, 80 patients receiving chemotherapy for disseminated solid malignancies, and 427 normal volunteers. Although there were significant differences among the groups in mean leukocyte, lymphocyte and T cell levels, among those with low T cell levels in each group there was a significant inverse relation between T cell levels after incubation with thymosin in vitro and initial T cell levels, with the exception of patients receiving chemotherapy. In patients receiving chemotherapy, T cell levels increased independently of initial T cell levels. These in vitro observations are consistent with evidence that a major effect of thymosin is maturation of T cell precursors; however, the effect is that of reconstitution at low T cell levels, and not of elevation to levels significantly above normal. The results provide a rationale for clinical trials with thymosin to maintain immune competence during radiation therapy and chemotherapy, and for a two-phase approach to immunotherapy of cancer utilizing thymosin for reconstitution of cellular defects followed by administration of agents that potentiate cellular immunity.