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PubMed2000

Immunohistochemical study of tyrosine phosphorylation signaling in the involuted thymus.

Thymic involution has been reported to be an important parameter of the degree and duration of child abuse. In the present study, we assessed the status of tyrosine phosphorylation signaling, which is known to play a key role in the physiological function of the thymus, in involuted thymuses of abused children through immunohistological studies performed with anti-phosphotyrosine antibodies. We found that tyrosine-phosphorylated proteins were present in high amounts in Hassall's corpuscles (HC) in the medulla of control thymuses. In involuted thymuses of abused children, expression of tyrosine-phosphorylated proteins was reduced with accompanying morphological changes of HC, such as reduction in size or calcification. These findings lead us to the suggestion that tyrosine phosphorylation signaling is reduced in involuted thymuses of abused children and that reduction of the signaling may be associated with morphological changes of HC as observed in involuted thymuses of abused children. In order to certify the suggestion, we investigated expression of tyrosine-phosphorylated proteins in involuted thymuses of stressed rats as well as in control thymuses. Immunohistochemistry revealed that tyrosine-phosphorylated proteins were expressed in control thymuses, more abundantly in the medulla, and reduced remarkably in involuted thymuses of stressed rats. Further, immunoblot analysis also showed that expression of phosphotyrosine-containing proteins was reduced in thymus extracts of involuted thymuses of stressed rats, thus supporting the suggestion. Our results also raise the possibility that components of tyrosine phosphorylation signaling could be a molecular marker for thymic involution.

Animals↗
PubMed1985

The arrest of T cell maturation in spontaneously hypertensive rats with a deficient production of thymic factors.

Spontaneously hypertensive rats (SHR) which regularly develop hypertension and periarteritis nodosa showed a progressive loss of T cell numbers and functions early in life. When six months old and compared with W rats, the original strain of SHR, they were found to possess a reduced number of thymocytes that formed rosettes with guinea pig erythrocytes and that reacted to anti-Thy1. 1 and anti-T (W3/13) sera. The number of spleen and lymph node cells which reacted to anti-T (W3/13) and anti-helper T (W3/25) sera also decreased. The antibody response of 3-month-old SHR spleen cells to SRBC was about one-fifth that of Wistar rats and progressively declined with age. Subcutaneous grafting of 3-month-old SHR thymus tissues failed to promote differentiation and functions of T cells in the spleen of nude mice, whereas 3-month-old W thymus tissues showed significant recovery of T cell generation and functions. The T cell functions of old SHR was completely restored by grafting adult W thymus tissues but was not restored by grafting adult SHR thymus tissues. Similar recovery was also obtained by the injection of thymus extracts from W thymus tissues. These results suggest that thymic epithelial cell products regulating T cell maturation may decrease in SHR with increasing age.

Animals↗
PubMedDate not supplied

[Detection of autoantibodies to extractable nuclear antigens in autoimmune diseases of rheumatic origin using immunoblotting--comparison with counter-electrophoresis].

Sera from 82 patients with rheumatic autoimmune disease were tested for anti-ENA antibodies by immunoblotting and counterimmunoelectrophoresis (CIE), using HeLa cell extract and rabbit thymus extract, respectively, as antigens. Anti-ENA antibodies were more frequently detected and could be better differentiated by immunoblotting rather than by CIE. There was especially an increase in anti-Sm antibodies (23, in contrast to four positive results), which was only detectable in serum samples for the 59 SLE patients. The sera of the six patients with MCTD all reacted with the 68 U1-RNP antigen. The sera of the five patients with Sjögren's syndrome only recognized the 50k La antigen, while other anti-ENA antibodies were not observed. In SLE anti-La antibodies were often associated with other anti-ENA antibodies. Seven out of eight SLE patients showing a combined detection of antibodies against Sm, U1-RNP and La by immunoblotting demonstrated severe organ involvements, especially lupus nephritis. Therefore, the characterization of anti-ENA antibodies by immunoblotting may contribute to improve the differentiation of connective tissue diseases.

Antibodies, Antinuclear↗
PubMed1987

[Detection and differentiation of autoantibodies to extractable nuclear antigens with immunoenzyme tests].

Nuclear antigens were extracted from calf thymus with phosphate-buffered saline The 60-80% ammonium sulfate fraction (ASF) of thymus extract contained SS-B-, the 30-60% ASF Sm- and U1-RNP-, and the chromatographically purified 30-60% ASF only Sm antigen. With these fractions enzyme immunoassays were developed for quantitative determination of Sm-, U1-RNP-, and SS-B autoantibodies. Out of 144 sera with antinuclear antibodies 85% were positive in the enzyme immunoassays, 41% in immunofluorescence tests on liver sections (12% speckled, 29% homogeneous immunofluorescence pattern), and 10% in Ouchterlony tests. 26% of the sera were positive in enzyme immunoassays and immunofluorescence tests. All antibody specificities detected by immunodiffusion could be confirmed by enzyme immunoassay. The enzyme immunoassay is far more sensitive then immunodiffusion and in contrast to immunofluorescence allows antibody specificities to be determined. Enzyme immunoassays are recommended for the diagnosis of rheumatic diseases.

Antibodies, Antinuclear↗
PubMed1988

[Identification of pro-opiomelanocortin and its C-terminal fragments in the mammalian thymus].

Immunoreactive alpha-, beta-, gamma-endorphins and beta-lipotropin were detected in perfused calf thymus extracts at the following concentrations (fmol/mg) tissue, M +/- m): 1.32 +/- 0.08, 1.53 +/- 0.45, 0.0186 +/- 0.0022 and 0.741 +/- 0.157, respectively. It was demonstrated for all ligands tested that the synthetic peptide and increasing amounts of the extract cause a similar displacement of the corresponding 125I-peptide from its complex with specific antiserum. Using the immunoblotting technique with a highly specific antiserum to bovine beta-lipotropin, the extracts of calf thymus, rat thymocytes and bovine hypophysis were found to contain two polypeptides with Mr of 32 and 14 kD, whose mobility corresponds to that of proopiomelanocortin and beta-lipotropin.

Animals↗
PubMed1984

Modulation of Schistosoma mansoni egg-induced granuloma formation. III. Evidence for an anti-idiotypic, I-J-positive, I-J-restricted, soluble T suppressor factor.

Modulation of pathogenic egg-induced hepatic granuloma formation in chronically Schistosoma mansoni-infected mice is an immunoregulatory process. Adoptive transfer and in vitro studies have demonstrated that this suppression involves various T lymphocyte circuitries, and the participation of soluble suppressor factors has recently been noted in these systems. The present study has partially characterized a soluble suppressive activity extracted from the thymus glands of chronically infected mice (SmTsF) that modulates granuloma formation in acutely infected mice. The suppressive effect of SmTsF could be administered by multiple i.v. injections or by slow release from osmotic minipumps implanted i.p. Homologous and reciprocal transfers of SmTsF prepared from B10.A(3R) and B10.A(5R) donors indicated that SmTsF-induced suppression required homology between the donor and recipient at the I-J subregion of the major histocompatibility complex. Furthermore, the use of immunoabsorbents prepared with anti-I-Jk and anti-I-Jb sera demonstrated that CBA/J (H-2k) SmTsF was retained by, and could be recovered from, anti-I-Jk insoluble columns, but was unaffected by parallel treatment with anti-I-Jb sera. Subsequent immunoabsorbent studies showed that SmTsF did not bind to soluble egg antigenic (SEA) columns, and thus demonstrated a lack of idiotype, anti-antigen activity. However, columns prepared by using anti-SEA IgG from chronically infected syngeneic mice retained SmTsF suppressive activity, and it could be recovered by alkaline elution. These data are compatible with an interpretation that the suppressive activity expressed anti-idiotypic reactivity. Thus a thymus extract obtained from chronic, modulated, S. mansoni-infected mice can induce granuloma suppression in acutely infected mice. This activity is associated with an I-J determinant-bearing, possibly anti-idiotypic moiety or moieties. These observations further implicate some of the Ts cascades reported in other systems in the regulation of cell-mediated pathogenesis in chronic experimental schistosomiasis.

Animals↗
PubMed1984

T-subset abnormalities in thalassaemia intermedia: possible evidence for a thymus functional deficiency.

Peripheral blood T-lymphocyte subsets, evaluated by means of a series of monoclonal antibodies, were assessed in 14 patients affected by thalassaemia intermedia, 7 of them previously splenectomized. A significant reduction of T+4 cells ('helper' T cells) was found in almost all patients, whereas T+8 cells ('suppressor/cytotoxic') showed a marked increase only in splenectomized subjects. Together with these quantitative T-subset abnormalities, which seemed to be partly affected by either splenectomy or high serum iron levels, an unusual circulating T-cell subpopulation labelled by T6 monoclonal antibody was detected in all patients. Complete disappearance of T+6 cells ('thymocyte-like' T lymphocytes) and normalization of the T4/T8 ratio was observed after 'in vitro' incubation of patient's lymphocytes with a crude thymus extract (Thymostimulin). This would suggest the presence of a so far unreported thymus-dependent defect of T-lymphocyte phenotypic maturation occurring in thalassaemia intermedia.

Adolescent↗
PubMed1970

Studies on some physicochemical properties of a thymus humoral factor conferring immunocompetence on lymphoid cells.

By means of an assay of graft-versus-host activity some properties of the thymic humoral factor which confers immunocompetence upon lymphoid cells in vitro have been studied. Allogeneic and xenogeneic thymic preparations were found to activate lymphoid cells from neonatally thymectomized mice, enabling initiation of a graft-versus-host response. Thus, this thymus factor is apparently neither strain nor species specific. The active principle of calf thymus extracts was found to be in the supernatant after prolonged ultracentrifugation. When exhaustive dialysis and ultrafiltration through Diaflo membranes were performed, the active thymus agent was found to pass through both the dialysis sac and Diaflo UM-2 membranes. The molecule which confers immunocompetence upon lymphoid cells thus seems to be of molecular weight of an order of magnitude of 1000 or less. Dialyzed thymus preparations injected into neonatally thymectomized mice also restored the capacity of spleen cells of these mice to induce graft-versus-host activity. When injected into intact mice, thymus extract also increased the proportion of competent cells in the spleens of these animals, probably by activation of target cells originating outside the spleen.

Animals↗
PubMed1971

Contribution of a thymic humoral factor to the development of an immunologically competent population from cells of mouse bone marrow.

The hypothesis that cells located in mouse bone marrow can acquire immunological competence by a process that involves interaction with a noncellular component of the thymus was tested using an in vitro assay of graft-versus-host reactivity as a criterion of cell competence. When suspensions of C57BL bone marrow cells were incubated in thymus extract and injected into mice incapable of inducing a response in the graft-versus-host assay as a result of neonatal thymectomy, or adult thymectomy plus irradiation, or because of genetic similarity with the (C3H x C57BL)F(1) tissue used for challenge in the assay, competent cells were recovered from the spleens of the injected mice. The reactive cells were shown to be of bone marrow origin since immune reactivity was related to the genetic makeup of the bone marrow cells rather than that of the intermediate recipients. A thymic factor was involved in the process leading to immune reactivity by these cells, as bone marrow cells incubated in xenogeneic or syngeneic thymic extracts induced a graft-versus-host response after passage through nonresponsive mice, whereas incubation of bone marrow cells in xenogeneic lymph node or spleen extracts or in culture medium only did not lead to subsequent reactivity. Participation of peripheral lymphoid tissue seemed essential in this process since bone marrow cells tested directly after exposure to thymic extract failed to induce a graft-versus-host response. C57BL bone marrow cells exposed to thymus extract and cultured together with fragments of (C3H x C57BL)F(1) spleen tissue in vitro were competent to induce a graft-versus-host response; thus, these components would seem to be sufficient as well as necessary for the immunodifferentiation process leading to graft-versus-host activity. It is concluded that one step in the process by which bone marrow cells acquire competence vis-a-vis the graft-versus-host response depends upon a thymic agent that is noncellular and extractable, and that another stage in this process is under the influence of components found within the peripheral lymphoid tissue environment. It is suggested that differentiation of precursor cells to competence could occur by progressive development of the cells in separate compartments of the lymphoid system.

Animals↗
PubMed1975

Cytotoxic effects on splenic ultrafiltrates upon leukaemic lymphocytes.

Ultrafiltrates from spleen inhibited both DNA synthesis and the proliferation of normal lymphocytes stimulated inculture from both mouse and man without apparent cytotoxicity. However, the same doses of this spleen ultrafiltrate will kill up to two-thirds of the leukaemic lymphoblasts from both mouse and man after 24 h incubation. This unique lymphocytotoxic effect could also be demonstrated on fresh primary cultures of leukaemic lymphocytes and was highly effective on slowly growing established cell lines under crowd culture conditions. Furthermore. ultrafiltrated thymus extract did not affect the DNA synthesis rates of the viability of NC-37 lymphoblasts, which have B cell characteristic. Thymus extract was cytotoxic to Molt cells, which have T cell characteristics.

Animals↗
PubMed1996

Purification of a novel MHC class I element binding activity from thymus nuclear extracts reveals that thymic RBP-Jkappa/CBF1 binds to NF-kappaB-like elements.

We purified a DNA binding protein that recognizes a portion of the MHC class I regulatory element region 1/NF-kappaB binding site whose expression correlates with the expression of a MHC class I transgene in the thymus. The N-terminal amino acid sequence and the molecular size matched the RBP-Jkappa protein, also known as the EBV C-promoter binding factor, CBF1. Anti-peptide sera reactive with RBP-Jkappa/CBF1 also reacted with this protein in gel mobility shift assays. Although RBP-Jkappa/CBF1 is ubiquitously expressed, binding to the MHC class Ia NF-kappaB site was limited to the thymus. Comparison of the DNA binding specificities of RBP-Jkappa/CBF1 in thymic and splenic nuclear extracts revealed strong binding from both extracts to an IFN-beta kappaB site containing the RBP-Jkappa/CBF1 consensus sequence (CGTGGGAA). In contrast, only the thymic nuclear extract showed strong DNA binding activity with probes containing the NF-kappaB recognition sequences present in the MHC class Ia, IL-2Ralpha, and granulocyte-macrophage CSF promoters. Thus, RBP-Jkappa/CBF1 in thymic extracts demonstrates a clearly distinguishable DNA binding specificity that correlates with tissue-specific expression of a class I transgene. This, coupled with the fact that our previous study showed enhanced expression of the transgene in CD4+CD8+ thymocytes, suggests that RBP-Jkappa/CBF1 may play a role in the development of the immune system.

Amino Acid Sequence↗
PubMed1988

Thymic humoral factor gamma 2: purification and amino acid sequence of an immunoregulatory peptide from calf thymus.

Thymic humoral factor gamma 2 (THF-gamma 2), an octapeptide essential for immune regulation, was purified from calf thymus. The purification of THF-gamma 2, monitored in vitro and in vivo in mouse splenocyte proliferation assays, was achieved by gel filtration of low molecular weight thymus extracts followed by ion-exchange chromatography and sequential reversed-phase high-performance liquid chromatography. The process yielded 5 micrograms of THF-gamma 2/1000 kg of thymus tissue. The concentration of THF-gamma 2 required for augmentation of lymphocyte proliferation and interleukin 2 production was 5 ng/mL in vitro and 10 ng/kg per mouse in vivo. THF-gamma 2 has the amino acid sequence Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. The proposed structure has been confirmed because a peptide was synthesized on the basis of this sequence that showed activity identical with that of the biological molecule. It shows no homology to the amino acid sequence of other thymic hormones nor is it part of any peptide or protein of known sequence. THF-gamma 2 retains essentially all of the biological activity of the thymus extract from which it is derived.

Amino Acid Sequence↗