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.     

Prospects of AIDS therapy by thymic humoral factor, a thymic hormone

Research performed in our laboratory has established that thymic humoral factor (THF), a peptide hormone isolated from calf thymus, leads to clonal expansion, differentiation and maturation of T-cell lymphocytes. THF augments the response to T-cell lectins, mixed lymphocyte reactions, graft-versus-host reactivity, antibody response to SRBC, cytotoxic responses and production of interleukin-2. Clinical data based on the use of THF of natural sources in about 200 patients indicate that it regulates differentiation of T-cell precursors, leading to normalization of the ratio between helper and suppressor/cytotoxic subsets. THF reconstitutes the defective cell-mediated immunity in patients affected by various types of neoplasms and suffering from secondary immune deficiencies, as a result of chemo and/or radiotherapy. THF-gamma 2 was purified and synthesized and found to be an octapeptide which retains all the biological activity of the thymus extracts. It is presently available for extensive clinical use.     

Characterization of a heat stable, 12,000 Da, glucagon-like immunoreactive (GLI) peptide from porcine ileum that stimulates hepatic glucose production in vivo and in vitro

Glucagon-like immunoreactivity (GLI) was extracted from porcine ileal mucosa with boiling 2 M HAc followed by elution on DEAE A-50 and fractionation on Sephadex G-50 F. Intact GLI was isolated from mesenteric blood by fractionation of several plasma samples from a mesenteric vein of a dog on Sephadex G-50 M followed by refractionation of the pooled GLI from these columns on G-50 F. Analysis of the semipurified mucosal and plasma GLI on Sephadex G-50 SF, eluted with 0.1 M Tris/HCl + 8 M urea, pH 7.5, demonstrated a single, sharp peak of GLI with a relative molecular mass (Mr) between 12,000 and 13,000. Electrophoresis on PAGE gels at acid pH with 2 M urea demonstrated a single charged GLI species in both the plasma and mucosal fractions. Stimulation of the release of GLI from a loop of ileum isolated in situ in an anesthetized dog followed removal of the known sources of glucagon (stomach, pancreas, and duodenum) resulted in an immediate and sustained increase in hepatic glucose production. The isolated GLI from ileal mucosa (5 X 10(-8) M) stimulated gluconeogenesis from 10 mM [14C]alanine in hepatocytes isolated from fed rats. The stimulation was equal to 25% of the maximal stimulation observed with 10(-8) M glucagon. These experiments demonstrate that the ileum synthesizes and secretes a GLI peptide with a Mr of approximately 12,000 that stimulates hepatic glucose production in vivo and in vitro.     

NH2-terminal big gastrin immunoreactivity in human urine

The concentrations and molecular forms of urinary and plasma gastrin from normal subjects were studied by radioimmunoassays using two region-specific antisera. Urinary concentration of NH2-terminal big gastrin (G-34) immunoreactivity was several hundred times as great as that of COOH-terminal gastrin immunoreactivity. Fractionation of urine extract showed a broad giant peak of NH2-terminal G-34 immunoreactivity (gastrin fragments "U") eluting in a later position than G-34(1-17) by Sephadex G-50 column chromatography. HPLC revealed that urinary NH2-terminal G-34 immunoreactivity was composed of four fragments including G-34(1-8), G-34(1-9), and G-34(1-10). Sephadex G-50 column chromatography of plasma extract revealed two or three peaks of NH2-terminal G-34 immunoreactivity, and a major peak eluted in the same position as urinary gastrin fragments "U". These results and data on renal clearances suggest that most of all gastrin fragments "U" in plasma are excreted in urine without renal reabsorption, whereas almost all of plasma COOH-terminal gastrin peptides including G-34 and little gastrin (G-17) are removed and metabolized in the kidney.     

Simultaneous microdetermination of homovanillic acid and 5-hydroxyindoleacetic acid in brain tissue using Sephadex G-10 and QAE-Sephadex A-25

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in animal brains were simultaneously purified by two steps of column chromatography on Sephadex G-10 and QAE-Sephadex A-25. Perchloric acid extracts of brain tissue were directly passed through a column of Sephadex G-10. The gel retained both HVA and 5-HIAA, thereby separating them from Cl0^?₄ which interferes with subsequent purification process and from endogenous substances which give blank fluorescence. HVA was loosely adsorbed on the gel and was easily desorbed with dilute acetic acid. This effluent was successively passed onto a column of QAE-Sephadex A-25 placed beneath the G-10 column and the adsorbed HVA was eluted with 0.1 M Na₂HPO₄. The 5-HIAA remaining on the Sephadex G-10 without being desorbed by acetic acid was eluted with dilute ammonia. The recovery of both acid metabolites by this column procedure was more than 90%. Thus, it is possible to determine the levels of HVA and 5-HIAA in single brains of small rodents.     

Role of L-alanine in the response of human lymphocytes to PHA and Con A

The response of umbilical cord blood lymphocytes (UCBL) to PHA and Con A is strongly dependent upon the presence of dialyzable plasma components. The impaired response of human cells in the presence of dialyzed human plasma was restored by calf thymus extract. In the present experiments we have further purified calf thymus extract by means of paper electrophoresis and chromatography on thin-layer cellulose plates. We reached the conclusion that the active material in this model is a single amino acid, alanine. When the different isomeric forms of alanine were tested, we found that L-alanine is the only biologically active material. In addition, we have observed here that the allogeneic response of UCBL is also dependent upon the presence of dialyzable plasma components. This impaired allogeneic response of UCBL in the presence of DHP was restored by addition of L-alanine. The present findings suggest that, under in vitro conditions, for human cells to respond to mitogenic or allogeneic stimulation L-alanine is essential.     

Glycoprotein biosynthesis by organ cultures of hamster trachea

Conditions have been developed for maintaining hamster tracheas in organ culture for at least 10 days. Secreted glycoproteins labelled with [¹⁴C]glucosamine and [³H]fucose were isolated from the spent medium and digested with papain, and the digest was fractionated on DEAE-Sephadex by stepwise elution with NaCl. The fractions eluted by 0.1 and 0.2 M NaCl and some of the products eluted with 0.4 M NaCl were shown to be derived from epithelial glycoproteins. Glycosaminoglycans were eluted by 0.4 M and by 1.25 M NaCl. Glycopeptides isolated from the epithelium by homogenization, ethanol precipitation and papain digestion, and defined as “intracellular”, gave a very similar profile on DEAE-Sephadex. The 0.1 M glycopeptide peak was the major fraction of epithelial origin from both secreted and “intracellular” material; it labelled extensively with both glucosamine and fucose and had a molecular weight of approx. 5000 (as judged by its elution from Sephadex G-75). This fraction was purified further by chromatography on Sephadex G-75 and DEAE-Sephadex; its amino acid and carbohydrate compositions were determined.     

Identification of a protease inhibitor from rat peritoneal macrophages as poly(ADP-ribose)

Rat peritoneal macrophages contain a chymotrypsin-like protease and its specific inhibitor, both being associated with chromatin of the cells. The inhibitor was separated from the protease by gel filtration through a Sephadex G-75 column, further treated with trypsin, DNase and RNase, and then purified successively on Sephadex G-75, Sephadex G-25, and dihydroxyboryl Bio-Gel P-60 columns. The purified inhibitor had a molecular weight in the range from 2,000 to 3,500 and an absorption maximum at 260 nm at pH 7.0. When the inhibitor was digested by snake venom phosphodiesterase, the inhibitory potency was lost, yielding 5'-AMP and 2'-(5'-phosphoribosyl)-5'-AMP as the digestion products which were identified by high pressure liquid chromatography. The inhibitory potency was neutralized specifically by anti-poly(ADP-ribose) antiserum. The profile of inhibition by the isolated inhibitor was nearly identical with that produced by authentic poly(ADP-ribose). It was therefore concluded that the inhibitor isolated was identical with poly(ADP-ribose), whose chain length ranged from 4 to 7 ADP-ribosyl units. This is the first demonstration that a intracellular protease inhibitor can be endogenous poly(ADP-ribose).     

Response of lymphoid cells to thymic hormonal factors isolated from mouse thymus

Thymic hormonal factors were isolated from mouse thymus by two methods. (1) Thymic cytosols in phosphate buffer saline were filtered through Sephadex G100 with 0.1 M NH4HCO3 (pH 8.0) as buffer and the protein peaks were collected. (2) Protein having thymosin activity (F5) was isolated from thymic cytosols after heat inactivation, salt fractionation and desalting on Sephadex G25. Molecular weights of all the proteins were determined on SDS-PAGE. Biological activity of thymic proteins was studied by in vitro and in vivo assays, using synthetic thymosin alpha 1 as the standard. Thymocytes treated with different thymic proteins showed maximum stimulation at 16 h of incubation period. Preincubation of the thymocytes with the thymic proteins and subsequent incubation with Con-A decreased the stimulation index. Incubation of spleen lymphocytes with thymic proteins increased the percentage of Tdt+ cells. The antitumor effects of thymic proteins carried out on animals having leukemia, showed statistically significant results. Clinically however, the antitumor effects of the thymic proteins alone and in combination chemotherapy were negligible at 1 mg/kg body weight dose level.     

A putative 12 transmembrane domain cotransporter expressed in thymic cortical epithelial cells

We have isolated a full-length cDNA clone (thymic stromal origin (TSO)-1C12) from a SCID thymus library using a probe from a PCR-based subtractive library enriched for sequences from fetal thymic stromal cells. TSO-1C12 mRNA is expressed mainly in the thymic cortex and is highly enriched in SCID thymus. Expression per cell is highest during fetal thymus development and decreases after day 16. Antipeptide Abs immunoprecipitated a hydrophobic, plasma membrane glycoprotein (thymic stromal cotransporter, TSCOT) whose translated sequence has weak homology to bacterial antiporters and mammalian cation cotransporters with 12 transmembrane domains. TSCOT represents a new member of this superfamily that is highly expressed in thymic cortical epithelial cells.     

The serum thymic factor (FTS)

Summary The thymus produces a nonapeptide capable of inducing T cell surface markers and T cell functions in immature lymphoid cells. This peptide is found partly bound to a carrier protein in the circulation from where it has been isolated (hence its name of serum thymic factor (FTS)). Immunofluorescence studies using an antibody raised against synthetic FTS has shown that it is produced by the thymic epithelium. Its mode of action at the cellular level involves the binding to specific high affinity receptors.     

Neuropeptide Y- and somatostatin-like immunoreactivities in ganglioneuroma, ganglioneuroblastoma and neuroblastoma

Neuropeptide Y (NPY)- and somatostatin (SS)-like immunoreactivities (LI) were investigated in tumor tissues of one ganglioneuroma (GN), 3 ganglioneuroblastomas (GNB) and one neuroblastoma (NB) by radioimmunoassay. NPY-LI was detected from all 5 tumor tissues (16.4-1247 pmol/g wet tissue). Sephadex G-50 column chromatography and reverse phase high performance liquid chromatography (HPLC) revealed that most of the NPY-LI in tumor extracts was eluted in an identical position to synthetic human NPY except one GNB (case 2). In this case, most of the NPY-LI was eluted in a higher molecular weight region than synthetic human NPY in Sephadex G-50 column chromatography and in a more hydrophobic position in HPLC. SS-LI was detected from 4 tumor extracts except one GNB (case 2) (21.3-787 pmol/g wet tissue). Sephadex G-25 column chromatography and reverse phase HPLC revealed that SS-LI in tumor extracts was eluted just after the void volume and then in the same positions as SS-28 and SS-14. These results suggest that NPY, SS-14 and SS-28 exist in tumor tissues of GN, GNB and NB, and most of the NPY-LI in one GNB was a higher molecular and more hydrophobic form of NPY-LI.     

Mechanism of thymic involution (analysis of intrinsic and extrinsic aspects)

The mechanisms of thymic involution with age are analyzed in terms of both intrinsic and extrinsic aspects. The intrinsic factors include: decrease in the number of thymocyte-precursors (pro-T cells) in the bone marrow, in emigration of pro-T cells into the thymus, in proliferation of thymic lymphocytes and decrease in extrathymic factors promoting proliferation of thymic lymphocytes. The extrinsic factors capable to exert modulating effects on the thymic function are humoral factors of the hypothalamic-pituitary origin. It seems much easier to manipulate the extrinsic factors by controlling neuro-endocrine stimulation, and to restore the thymic involution. This is quite encouraging, because it permits restoring the impaired immune functions of the aged people.     

Insulin-like substance and insulin-degrading complex in hemolysates of human erythrocytes

Human erythrocyte lysate was fractionated on various gel filtration media and immunoreactive insulin, insulinase and the influence of individual fractions of the insulin-degrading activity were determined. The hemolysate was shown to contain a complex of substances including an insulin-like substance, insulinase, protease inhibitor and insulinase activator. The insulin-like substance eluted from a Sephadex G-50 column in the same manner as native insulin, and its concentration exceeded the plasma level. Insulinase (Mr 100,000) degraded insulin to the trichloroacetic acid soluble fragments but did not degrade protein or glycoprotein hormones from human pituitaries. Insulinase was inhibited by low temperature, aprotinin and by a newly discovered protease inhibitor from erythrocytes which also inhibits serine proteases--trypsin and chymotrypsin. Another newly discovered substance eluted from a Sephadex G-100 column in the region of low molecular weight substances and showed an insulinase activating activity. The elution patterns of the protease inhibitor and insulinase activator suggest the possibility of the presence of more than one inhibiting and activating factor. The experimental results suggest that the insulin-degrading complex plays a role of a regulator of plasma insulin level. The nonpancreatic origin of the insulin-like substance is also possible.     

Conversion of high molecular weight CRF (corticotropin-releasing factor) or PRF (prolactin-releasing factor) into lower molecular weight forms by boiling at low pH.

Extracts (0.1 N HCl) of bovine hypophyseal stalk had 2 CRF peaks, one (CRF-A) in the void volume with Sephadex G-100 chromatography, the other more retarded (CRF-B). PRF activity of the same extracts eluted in 2 peaks from G-100, one in the void volume (PRF-A) and the other (PRF-B) between A and B CRF peaks. On rechromatography, isolated CRF-A and PRF-A remained in the void volume. However, heating to 100 C at pH 1–2 for 15 min converted CRF-A to CRF-B and PRF-A to PRF-C, which eluted after PRF-B on G-100. We conclude that CRF or PRF can be converted from high to low molecular weight forms with full retention of biological activity.     

Endogenous thymic factors regulating cell proliferation and analysis of their mechanism of action.

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.     

Allergenic activity of some kinds of plant pollen

Allergenic active fractions of ragweed, wormwood, goosefoot and sunflower pollen with a molecular weight of 37 000, 19 000, 35 000 and 14 000, respectively, were isolated by chromatography on Sephadex. All studied allergens had similar properties: affinity to Sephadex which was more pronounced to Sephadex G-75 than to G-100; absorbing components had no activity; allergenic and antigenic activities were determined only in fractions eluted in the bed volume of the column. Affinity of the allergans to Sephadex did not depend on the eluent type, but decreased in potentiation of the buffer ionic strength.     

The role of C1 esterase inhibitor in the activation of C1r, a subcomponent of the first component of complement from human plasma.

Clr was isolated from human serum by DEAE-cellulose column chromatography in the presence of EDTA. The isolated Clr did not hydrolyze N(alpha)-acetyl-L-arginine methyl ester, unless activated by brief treatment with trypsin [EC 3.4.21.4]. On thecolumn, the C1 esterase inhibitor activity was found to coincide with Clr but not C1s (another subcomponent of the first component) C1r was isolated from the euglobulin fraction of human serum by DEAE-cellulose column chromatograph. On Sephadex G-200 column chromatography, Clr was eluted in the void volume, whereas Clr was eluted in a position corresponding to a molecular weight of 140,000-160,000. The results indicated that, on activation, Clr was converted to an enzyme of lower molecular weight...     

In vivo effects of catecholamines and glucocorticoids on mouse thymic cAMP content and thymolysis

In vivo administration of the beta-adrenergic agonist, isoproterenol, induced a rapid, dose-dependent increase in the mouse thymic cyclic AMP (cAMP) content. Hydrocortisone (1 mg/animal), at a concentration which by itself did not alter the cAMP content of the thymus, markedly potentiated the effect of isoproterenol (5 micrograms/animal). Isoproterenol or hydrocortisone treatment led to a significant decrease in thymic weight and an even greater decrease in thymocyte number. In addition, the simultaneous administration of both agents produced additive effects on thymic atrophy. It appears from these results that glucocorticoids and catecholamines exert a negative control on the thymic size by increasing the programmed cell death of some cell subpopulations. Thus, glucocorticoids and catecholamines, either alone or in association, may influence the immune system under physiological or pathophysiological conditions.