Isolation and characterization of Thy 1 homologue from human thymus

A 40 000 M.W. glycoprotein was isolated from human thymus. This molecule binds lentil lectin, reacts with an antiserum made against the p25 antigen (the human Thy 1 homologue) and possesses almost identical amino acid composition as the p25 antigen and its 40 000 M.W. dimer.     

The effect of a calf thymus acid lysate on bone marrow cell growth in vitro

Granulocyte-macrophage colony forming units (CFU-GM) were studied in cultures of bone marrow from 16 apparently healthy normal controls, 9 patients with the myelodysplastic syndrome, 5 patients with myeloproliferative disease and 2 with myeloma. Supernatants from non-stimulated 72 hr cultures of nonadherent mononuclear blood cells ("lymphocytes") stimulated the forming of an average of 38.4 colonies per 100,000 cells from normal marrow. The addition of GIBCO's commercial conditioned medium or of a medium produced by lymphocytes stimulated with different concentrations (5, 10 and 20 mcg/ml) of an acid lysate of thymus (thymomoduline), increased growth to 65.2 - 55.4 colonies (p less than 0.001 to 0.05). Similarly, a significant increase (p less than 0.05) was found in the number of clusters and colonies formed in cultures of marrow from patients with the myelodysplastic syndrome. In contrast, no growth was found when the thymus acid lysate was added directly to the bone marrow cultures, suggesting that the lysate induces the production of colony stimulating activity by lymphocytes, but does not contain it. Similarly no significant increase was found as regards the initially high number of colonies from the five patients with myeloproliferative disease, or as regards the initially low number in the two myeloma patients.     

Comparison of mutagenicity and calf thymus DNA adducts formed by the particulate and semivolatile fractions of vehicle exhausts

In this study we compared the ability of extractable organic material from particulate and semivolatile fractions of gasoline emission to induce mutations in bacteria and form adducts with calf thymus (CT) DNA with corresponding data obtained from diesel exhaust. Exhaust particles from gasoline-powered passenger cars were collected on filters and semivolatile compounds were collected on polyurethane foam (PUF). The mutagenicity of the soluble organic fraction (SOF) was determined in Salmonella typhimurium strain TA98 and the DNA binding of aromatic compounds in the extracts was assessed by in vitro incubations with CT DNA and rat liver S9 (oxidative activation) or xanthine oxidase (reductive activation) followed by butanol-enhanced (32)P-postlabeling analysis. Semivolatile fractions of gasoline emission collected on PUF formed more CT DNA adducts than filter extracts under all reaction conditions, but showed a lower mutagenic potential than the corresponding particulate samples. This suggests that the capacity of PUF to collect exhaust particle-derived compounds and/or the efficiency of xanthine oxidase and enzymes in the rat liver S9 to activate these compounds to DNA binding metabolites was higher than expected. Gasoline extracts, benzo[a]pyrene and diesel particulate matter (SRM 1650) formed more S9-mediated DNA adducts as their dose increased, although a linear dose-response was not observed for the gasoline exhausts. Lower concentrations of gasoline and diesel extracts bound to DNA with greater efficiency than did 8-fold higher doses, suggesting complex interactions and/or an inhibition of S9 enzyme activities by the high doses. Diesel extracts formed higher levels of adducts than gasoline extracts, especially with the reductive activation system, suggesting that diesel extracts contain high levels of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs). The higher direct-acting Salmonella mutagenicity in diesel extracts in comparison with gasoline extracts is consistent with diesel extracts containing higher concentrations of nitro-PAHs. The results of this study indicate that diesel extracts are more mutagenic and form more DNA adducts than gasoline extracts and that the effects of extract dose on DNA adduct formation are complex.     

Viscosity and conformation of calf thymus histones

The gross conformation of the four main histone fractions was determined from viscosity measurements in aqueous solutions and in solutions containing inorganic electrolytes. In distilled water the histones behaved like flexible linear polyelectrolytes, i.e. the reduced specific viscosity increased with dilution exponentially. In the presence of acid or salts, the viscosity decreased and the normal linear dependence of the reduced specific viscosity on concentration was observed. The viscosity of the very lysine-rich Fraction 1 (which is also relatively rich in proline) was much higher than that of the other histone fractions. Phosphate and sulfate ions were more effective in diminishing the viscosity of aqueous solutions of the Fraction 1 than chloride or acetate ions.     

Experimental myasthenia gravis, myositis and myocarditis in guinea-pigs immunized with subcellular fractions of calf thymus or calf skeletal muscles in freund''s complete adjuvant

Experimental thymitis and a partial neuromuscular block was produced in guinea-pigs by immunization with the subcellular fractions of calf thymus and skeletal muscle homogenates. A significant incidence of thymitis was observed in guinea-pigs immunized with the soluble or with the microsomal fractions of thymus or of skeletal muscle. The mitochondrial fraction of thymus or skeletal muscle was less effective in producing thymitis. In guinea-pigs immunized with the subcellular fractions of skeletal muscle additional evidence of experimental myositis (60%) and myocarditis (40%) was shown. All three skeletal muscle fractions were equally effective in the production of experimental muscle lesions.

A close correlation was found between the presence of thymitis and the evidence of a partial neuromuscular block. In contrast, a partial neuromuscular block was not observed in animals with experimental myositis but without thymitis. This finding gives further support to the hypothesis that the inflamed thymus is a source of a factor which blocks neuromuscular transmission in experimental myasthenia gravis.

     

Characterization of Thy 1.2 positive cells in mouse bone marrow by sedimentation and mitogen stimulation.

By the use of unit gravity velocity sedimentation it was found that the majority of Thy 1.2 positive cells in the bone marrow of BALB/c mice sedimented in the same fractions as small lymphocytes of the marrow. This was shown both in normal, neonatally thymectomized and congenitally athymic mice. In all three groups of mice, two populations of Thy 1.2 positive cells were found. This indicates that these cells are cycling in the bone marrow. Long-lived T cells of normal bone marrow were included in the slowly sedimenting Thy 1.2 positive population (peak at 3 mm/h). Results after stimulation of bone-marrow cells with phytohaemagglutinin or concanavalin A indicated that the majority of Thy 1.2 positive cells in the bone marrow of thymus-deprived mice are effete end-products.     

Extraction and differentiation of the Su autoantigen from calf thymus nuclei

Antibodies to Su antigen have been reported previously as a distinct antigen-antibody system associated with connective tissue diseases; most specifically systemic lupus erythematosus and undifferentiated connective tissue disease. The Su antigen was first identified by double immunodiffusion using calf thymus nuclear extract (CTNE) as a source for Su antigen. In this report, enhanced extraction of Su antigen was achieved using deoxyribonuclease I (DNase) for preparation of CTNE. Only the Sm antigen was found in comparable quantities in the DNase CTNE. Western immunoblotting and immunoprecipitation employing DNase CTNE and extracts of [35S]methionine-labeled HeLa cells respectively were used for further characterization and differentiation of the Su antigen. Sera from patients positive for Su antibodies by double immunodiffusion were found to react most specifically with antigen components in a molecular weight range of approximately 50-55 kDa. These methods should assist in further understanding the biochemical properties of the Su antigen.     

Rational design of a meningococcal antigen inducing broad protective immunity

The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.     

Isolation and purification of a small peptide with activity of increasing e-receptor expression from calf thymus

A small peptide of molecular mass lower than 1,000 daltons was isolated and purified from a crude extract of Calf thymus. It has been demonstrated to have the activity of increasing E-rosette formation and E-receptor expression of human and porcine T-lymphocytes with three different in vitro assays. Amino acid composition analysis showed that this peptide consists of glutamic acid, aspartic acid and glycine residues at a molecular ratio of 3:3:2. A hypothesis of a multi-factoral and multistaged mechanism of regulation of thymocyte differentiation and maturation in the thymus is proposed.     

Neuraminidase in juvenile calf thymus: determination and characterization by a continuous fluorometric assay procedure

A continuous fluorometric neuraminidase assay has been developed. Within the pH range optimal for neuraminidases (from 3 to 6) the fluorescence intensity of 4-methylumbelliferone exceeds that of the glycoside 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid about 50 times (at lambda Ex = 335 nm and lambda Em = 445 nm), allowing the precise, simple and time-saving continuous fluorometric registration of enzymatically released 4-methylumbelliferone. In juvenile calf thymus a neuraminidase consisting of two components differing in pH optima and resistence to freezing and to detergents could be found. A beta-galactosidase activity in juvenile calf thymus could be proved by the same assay procedure using the synthetic substrate 4-methylumbelliferyl-beta-D-galactopyranoside.     

Steroid economising effects of a calf thymus extract in three patients with juvenile chronic arthritis

Summary Recent therapeutic trials in rheumatology using different immunomodulating agents have given encouraging results. In this study an aqueous calf thymus extract (CTE) was administered to three patients, two with systemic juvenile chronic arthritis (JCA), Still's disease, who could not be weaned from steroids during 2 years of conventional therapy, and one girl with a chronic juvenile monarthritis who had responded unsatisfactorily to nonsteroidal antirheumatics for 19 months. A striking clinical improvement was observed in all three patients. Prednisone (PRED) was discontinued in one case with systemic (JCA) and 0.25 mg/kg body weight/day is presently being given to the other patient. The girl is doing well on 4 mg chloroquin kg body weight/day; indomethacin (IND) was discontinued. Laboratory data including cellular immunoreactivity normalized in all three patients.     

Induction of a T-cell specific antigen on bone marrow lymphocytes with thymus RNA.

Expression of a rabbit T-cell specific antigen can be induced on bone marrow lymphocytes following exposure to an RNA extract obtained from the thymuses of young rabbits. The presence of the antigen was demonstrated using goat anti-rabbit T-cell serum in a complement-dependent cytotoxicity assay. The T-cell antigen first appeared 3 h after addition of the thymus RNA to bone marrow cell cultures and the maximum number of cells expressing the T-cell antigen was observed within 24 h. RNA obtained from a source other than the thymus was found to be ineffective in inducing expression of the T-cell antigen. The induction of the antigen appears to be dependent on the presence of intact thymus RNA, as RNase treatment but not trypsin treatment, destroyed the ability of the RNA to induce the T-cell antigen.     

Characterization of antigenic polypeptides of the RNP, Sm and SS-B nuclear antigens from calf thymus

Antinuclear autoantibodies are a hallmark of autoimmune diseases. The RNP, Sm and SS-B nuclear antigens from calf thymus in whole tissue, nuclear extracts and fractions have been studied by using different techniques including immunodiffusion, counterimmunoelectrophoresis and protein blotting. Such studies were done in order to obtain a precise characterization of the polypeptide components of those antigens. From our results it can be established that: one 69.8 Kd polypeptide (for whole tissue and nuclei) and a number of well-defined 32-38-Kd polypeptides (for nuclear extracts and ammonium sulfate fractions) show an antigenic character against anti-RNP sera; anti-Sm sera from different patients show in all cases a variable component of antigenic polypeptides, including one 28.8, 29.7 Kd doublet and two singlets of 14.8 and 11.0 Kd; and a 52.0-Kd SS-B antigenic polypeptide is found for whole tissue, which is gradually degraded in nuclei and nuclear extracts to a more stable 47.1-Kd polypeptide.     

HIV-1 pathogenesis and therapeutic intervention in the SCID-hu Thy/Liv mouse: a model for primary HIV-1 infection in the human thymus

The SCID-hu Thy/Liv mouse is a model for the analysis of human thymopoiesis. It has been constructed by engrafting fragments of human fetal liver and thymus into the immunodeficient C.B-17 scid/scid (SCID) mouse. The resulting ‘Thy/Liv’ organ promotes long-term differentiation of human T cells. Given the apparently normal physiology of the SCID-hu Thy/Liv organ, it has been used to explore the pathophysiologic mechanisms of HIV-1 infection in vivo, and to test therapeutic modalities such as anti-HIV-1 drugs and haematopoietic stem cell (HSC)-based gene therapy. In this review, I will summarise what we have learned from the SCID-hu Thy/Liv model, with a focus on recent findings in HIV-1 replication and therapy. Unique HIV-1 determinants have been identified which are required for replication in the Thy/Liv organ but not for replication in PBMC or in T cell lines in vitro. The mechanism of HIV-1 induced thymus depletion is not clear. It is correlated with high levels of HIV-1 replication. Both direct and indirect mechanisms may be involved. In addition to preclinical evaluation of anti- HIV-1 drugs, the SCID-hu Thy/Liv mouse has also been successfully used to test the feasibility of HSC-based gene therapy. A number of improved SCID-hu models have been constructed to meet different requirements. Using these SCID-hu Thy/Liv models, current/future efforts will provide insightful information for understanding pathogenesis and designing therapeutic interventions against HIV-1 infection in humans, especially in paediatric patients. © 1997 John Wiley & Sons, Ltd.     

Antigens in thymus and muscle effective in inducing experimental autoimmune thymitis and the release of thymin

Hartley guinea-pigs immunized with homologous thymus or muscle in Freund's complete adjuvant (FCA) developed experimental autoimmune thymitis, with lymphocytic accumulations in the thymic medulla and electromyographic evidence of a block in neuromuscular transmission similar to that of humans with myasthenia gravis. These animals immunized with homologous tissues had a higher incidence of thymitis than animals similarly immunized with bovine tissues; yet, unlike the latter, no serum autoantibodies could be demonstrated by immunofluorescence.

Muscle was considered to be thymitogenic because there are common antigens in striated muscle and myoid cells of the thymus. Muscle was thymitogenic in doses down to 50 μg while thymus was thymitogenic in doses down to 5 μg. Since muscle contains far more muscle anitgen than thymus, these results were interpreted as showing that thymus probably contains thymitogen(s) which are distinct from, and even more potent than, muscle thymitogen.

Animals with thymitis also had electromyographic evidence of neuromuscular block to single stimuli, increasing with repetitive stimulation. The incidence of neuromuscular block closely paralleled the incidence of thymitis, whether the thymitis was induced by thymus or muscle. Thus the release of thymin, the thymic substance causing neuromuscular block, appeared to be a general consequence of inflammation of the thymus and was not related to the type of antigen initiating thymitis.