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PubMed2001

Reactivity of anti-proliferating cell nuclear antigen (PCNA) murine monoclonal antibodies and human autoantibodies to the PCNA multiprotein complexes involved in cell proliferation.

Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.

Animals↗
PubMed1996

Prevalence and clinical significance of antibodies to ribonucleoproteins in systemic lupus erythematosus in Malaysia.

One hundred and seventy patients with systemic lupus erythematosus (SLE) were studied for the prevalence of antibodies to the small RNA-associated proteins Ro/SSA, La/SSB, Sm, U1RNP and Sm. The relationship of these autoantibodies to different races, sexes and clinical manifestations of SLE was evaluated. Passive immunodiffusion was employed using human spleen extract as antigen source for Ro and rabbit thymus extract for La, Sm and U1RNP. We found the prevalence of antibodies to be as follows: anti-Ro/SSA, 36%; anti-La/SSB, 8%; anti-Sm, 15% ; anti-U1RNP, 21%. Except for a low prevalence of anti-La, the prevalence of these antibodies was similar to that in Western studies, The prevalence of anti-Ro/SSA is similar to that reported in the Western studies, but lower than that reported in the Oriental patients from Singapore and Hong Kong. Linkages of anti-Ro with anti-La antibodies were usual; however, although anti-Sm antibodies were usually associated with anti-U1RNP, they were more frequently associated with anti-Ro antibodies. The Malay patients had a high prevalence of anti U1RNP compared to other races. No gender difference was detected. Anti-Sm antibody was associated with serositis and anti-U1RNP antibodies with Raynaud's phenomenon. No association was found between the presence of skin renal or cerebral manifestations and any specific antibodies or combination of antibodies.

Adolescent↗
PubMed1986

Murine lupus monoclonal antibodies define five epitopes on two different Sm polypeptides.

Monoclonal anti-Sm (Smith) antibodies derived from the mouse strain MRL/lpr were isolated and characterized by binding to purified antigen, immunoprecipitating characteristic uridine-rich RNAs from Hela cell extracts, and by Western blot analysis using rabbit thymus extract. Five different Sm epitopes were demonstrated by epitope blockade and probing Western blots with the monoclonal antibodies. Human anti-Sm serum inhibited each monoclonal antibody from binding to antigen, indicating that both human and mouse antibodies bind to the same Sm epitopes. Human anti-Sm antibodies bound to 28,000 and 16,000 MW polypeptides, a small number also binding to a 14,000 MW polypeptide. The monoclonal antibodies also bound to the 28,000 and/or the 16,000 polypeptide, and provided evidence to suggest that these two Sm polypeptides bear some structural similarities, but are distinct molecules.

Animals↗
PubMed1976

[The influence of low-molecular extracts from heterologous thymus, epiphysis and hypothalamus on the immune response in mice].

Extracts from bovine tissue were prepared by the acetic acid treatement and purified by gel filtration. All the preparations were injected into CBA mice for 10 days. Extracts from the thymus and the epiphysis proved to increase the number of antibody-forming cells generated in the spleen and the level of serum hemagglutinins in response to the immunization with sheep red cells. As to the hypothalamic extract, it depressed the immune response.

Animals↗
PubMed1983

Separation of two molecular species of the Sm antigen by affinity chromatography with murine monoclonal and human anti-nuclear autoantibodies.

Nuclear ribonuclear protein (nRNP) and Sm were extracted from a 30-60% ammonium sulphate fraction of rabbit thymus extract by affinity chromatography. Immunoadsorbent columns were prepared from IgG extracted from an anti-nRNP serum, an anti-Sm serum and two monoclonal autoantibodies derived from a MRL/lpr mouse. All four immunoadsorbents isolated both nRNP and Sm antigens indicating that they exist as the 'nRNP/Sm complex' A species of Sm which did not bind to the anti-nRNP column and which was subsequently purified with the anti-Sm column was termed 'free Sm'. The Sm in the complex and free Sm were immunologically identical on immunodiffusion and gave similar polypeptide bands on polyacrylamide gel electrophoresis. However, they differed in that complexed Sm was sensitive to heat and RNAase. These studies provide direct evidence of a physical association between the nRNP and Sm antigens and indicate an additional molecular species of Sm whose resistance to RNAase may be due to different associated RNA species.

Animals↗
PubMed2001

In vivo and in vitro apoptosis of human thymocytes are associated with nitrotyrosine formation.

Most thymocytes are deleted by thymic selection. The mechanisms of cell death are far from being clear. Peroxynitrite is a powerful oxidant produced in vivo by the reaction of superoxide (O2*-) with nitric oxide (NO*) and is able to mediate apoptosis. The aim of this study was to analyze whether NO and peroxynitrite could play a role in human thymocyte apoptosis. The results indicate that 3-(4-morpholinyl)-sydnonimine (SIN-1, an O2*- and NO* donor) and chemically synthesized peroxynitrite, but not S-nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO* donor), have a strong apoptotic effect on human thymocytes (annexin V staining and TUNEL reaction). This effect was inhibited by exogenous superoxide dismutase (SOD), which interacts with O2*- and inhibits the formation of peroxynitrite. Because peroxynitrite formation requires NO*, thymic stromal cells were investigated to determine if they produced NO*. Inducible NOS was synthesized in cultured thymic epithelial cells in certain conditions of cytokine stimulation, as shown by messenger RNA levels, protein analysis, and nitrite production in the supernatants. SIN-1-treated thymocytes had high levels of tyrosine nitration, abolished by the addition of exogenous SOD. Tyrosine nitration was also detected in thymus extracts and sections, suggesting the presence of peroxynitrite in situ. In thymus sections, clusters of nitrotyrosine-positive cells were found in the cortex and corticomedullary areas colocalized with cells positive in the TUNEL reaction. These data indicate an association between human thymocyte apoptosis and nitrotyrosine formation. Thus, the results support the notion of a physiologic role for peroxynitrite in human thymocyte apoptosis. (Blood. 2001;97:3521-3530)

Annexin A5↗
PubMed1968

Cell to cell interaction in the immune response. I. Hemolysin-forming cells in neonatally thymectomized mice reconstituted with thymus or thoracic duct lymphocytes.

An injection of viable thymus or thoracic duct lymphocytes was absolutely essential to enable a normal or near-normal 19S liemolysin-forming cell response in the spleens of neonatally thymectomized mice challenged with sheep erythrocytes. Syngeneic thymus lymphocytes were as effective as thoracic duct lymphocytes in this system and allogeneic or semiallogeneic cells could also reconstitute their hosts. No significant elevation of the response was achieved by giving either bone marrow cells, irradiated thymus or thoracic duct cells, thymus extracts or yeast. Spleen cells from reconstituted mice were exposed to anti-H2 sera directed against either the donor of the thymus or thoracic duct cells, or against the neonatally thymectomized host. Only isoantisera directed against the host could significantly reduce the number of hemolysin-forming cells present in the spleen cell suspensions. It is concluded that these antibody-forming cells are derived, not from the inoculated thymus or thoracic duct lymphocytes, but from the host. Thoracic duct cells from donors specifically immunologically tolerant of sheep erythrocytes had a markedly reduced restorative capacity in neonatally thymectomized recipients challenged with sheep erythrocytes. These results have suggested that there are cell types, in thymus or thoracic duct lymph, with capacities to react specifically with antigen and to induce the differentiation, to antibody-forming cells, of hemolysin-forming cell precursors derived from a separate cell line present in the neonatally thymectomized hosts.

Animals↗
PubMed1982

Protein antigens of the RNA-protein complexes detected by anti-SM and anti-RNP antibodies found in serum of patients with systemic lupus erythematosus and related disorders.

Evidence has been obtained previously indicating that the antigens reacting with the anti-Sm and anti-RNP sera are present as a large complex, and similar protein bands are obtained with both types of sera. Inthe present study, it proved possible to break up this complex using SDS treatment before immunoprecipitation. After such treatment, different protein bands were immunoprecipitated by the two antisera; Sm determinants resided, at least partially, in a 19-kd protein. Sequential immunoprecipitation with and without prior SDS treatment provided further evidence for these specificities and suggested that two classes of particles exist in different tissues, one containing proteins immunoreactive with the Sn and RNP antisera and the other containing proteins immunoreactive only with the Sm antisera. The latter particle contained all the bands seen with the first type except for the absence of the 19-kd band. Nitrocellulose blot analyses confirmed the assignment of the 25- and 16-kd polypeptides to Sm antigenic determinants; analyses for RNP proved les informative by this technique. Some differences in the banding patterns were obtained using cells from different species: the 25-kd Sm band was usually double in human cells and single in rat and rabbit tissue. Methods of extraction also caused some differences which was especially true for the rabbit thymus extract widely used for Sm and RNP studies. Additional immunoreactive bands at 68 and 70 kd also were detected when the Sm and RNP antisera were used in nitrocellulose blot analyses. Furthermore, evidence was obtained for a number of other antibodies in lupus sera which have not as yet been detected by serological methods.

Antibody Specificity↗
PubMed1983

Isolation of intact Sm/RNP antigens from rabbit thymus.

A comparison of the immunologically reactive components of the highly conserved Sm and RNP autoantigens from various mammalian tissue sources suggested the complete absence of a major 26K to 27K Sm-specific polypeptide in rabbit thymus extracts prepared by conventional procedures. A simple modification of the solubilization protocol, achieved by sonicating a suspension of commercial rabbit thymus acetone powder in 0.35 M NaCI, gave an extract containing the full complement of immunologically reactive Sm and RNP proteins detectable in other mammalian species. Without further manipulation, extracts were immediately passed through an immunoaffinity column constructed from human SLE IgG with both anti-Sm and anti-RNP reactivities. The proteins of the purified Sm/RNP were recovered in sufficient quantities for direct analysis by protein staining or immunoblot assays. The antigenic polypeptides were recovered intact and consisted of a single 73K RNP-specific species together with Sm-specific proteins of 26K to 27K (a doublet) and 13K. These proteins were easily visible by protein stain as were nonantigenic components of 35K, 32K, 11K, and less than 10K. The same polypeptides were present in affinity-purified Sm/RNP from HeLa cells, although the RNP protein was slightly smaller. The resolution and integrity of the complexes isolated by this simple two-step procedure, requiring less than 4 hr for completion, is remarkable, and the protein composition of the product compares quite favorably with antigens isolated from other sources by considerably more lengthy and laborious procedures.

Animals↗
PubMed1988

An extract of bovine thymus stimulates human keratinocyte growth in vitro.

An extract prepared from newborn calf thymus stimulated proliferation of human keratinocytes cultured from newborn foreskins and from skin biopsies of 26 adult volunteers aged 19 to 70 years. Growth over the 7-day assay period in the basal medium was age-dependent, with newborn cultures achieving a 10-fold increase in cell number over seeding density, old adult cultures barely maintaining their seeding density and young adult cultures intermediate in proliferative capacity. Maximally stimulatory extract concentration was 5-fold higher for newborn than for adult keratinocytes, with adult cultures experiencing toxicity at doses still growth-promoting for newborn cultures. At optimal extract concentration the maximal average increase in cell yield (66.3% for newborn, 53.6% for young adult, and 18.1% for old) indicated decreased mitogen responsiveness or increased inhibitor sensitivity with increasing donor age. Stimulation of cholera toxin-treated cultures was equally high, ranging from 39.4% to 145.9%, suggesting that the extract acts through a cyclic AMP-independent pathway. Thymic extract did not increase colony forming efficiency. Our findings provide further support for the concept of functional interactions between the skin and the immune system, in addition to the recognized morphologic similarities between thymic cells and keratinocytes. Furthermore, these data confirm earlier findings of an inverse relationship between mitogen responsiveness and donor age for cultured cells.

Adult↗
PubMed1975

[Study of the thymus as a source of humoral factors stimulating antibody formation].

A fraction stimulating antibody formation in neonatal animals was obtained in separation of the calf thymus extract (1A) prepared by the method of Goldstein et al. on a column with Sephadex G-25 (fine). Testing of the activity of analogous fractions of the extracts of calf tonsils, spleen, lymph nodes and the liver led to a supposition that the thymus was the only source of specific humoral factor stimulating antibody formation, which could accumulate in the peripheral lymphoid formations. Low activity of tonsillar extracts failed to confirm the hypothesis on the role played by the tonsils as the central immunity organ similar to the bursa Fabricii.

Animals↗
PubMed1985

Thymic epithelial cells of severely undernourished mice: accumulation of cholesteryl esters and absence of cytoplasmic vacuoles.

The thymic epithelium was compared in weanling male and female CBA/J mice when fed ad libitum and when subjected to severe food intake restriction for 14 days. The restriction protocol elicited predominantly a metabolic response to caloric deficit rather than to protein deficiency. Electron microscopy revealed intracytoplasmic accumulations of large, circular, homogeneously electron-dense profiles (with no limiting membrane) in a high proportion of cortical and medullary epithelial cells of thymuses from restricted mice, but not from controls. The electron-dense material was not preserved in the absence of osmium. Thin-layer chromatography (TLC) indicated elevated levels of free and esterified cholesterol, particularly the latter, in whole thymus extracts of restricted mice. Measurements of total cholesterol levels in the thymic extracts were consistent with the results obtained by TLC. In addition, cryostat sections of thymuses from restricted mice, but not from controls, exhibited numerous stained foci throughout the cortex and medulla when treated with oil red O (a general neutral lipid stain) or by the Schultz procedure which is specific for cholesterol. Collectively the results suggest accumulations of cholesteryl esters, together with some free cholesterol, as non-membrane-bound droplets in the cytoplasm of thymic epithelial cells of undernourished mice. It is also of interest that the lipid-laden epithelial cells exhibited none of the cytoplasmic vacuoles observed in controls and believed to be important in thymic hormone secretion. This work provides the first direct evidence of thymus epithelial abnormalities in severe protein-energy malnutrition.

Animals↗
PubMed2006

Study on the antinociceptive effects of Thymus broussonetii Boiss extracts in mice and rats.

In Morocco, Thymus broussonetii is widely used in folk medicine for the treatment of a variety of diseases including gastroenteric and bronchopulmonary disorders and to relieve dolorous process. The antinociceptive effect of the aqueous, butanolic and ethyl acetate extracts of this species was examined in rats and mice using chemical and thermal models. The results obtained showed that aqueous and butanolic extracts exerted an antinociceptive activity in the two phases of formalin (50-300 mg/kg), tail immersion and writhing tests. Whereas, the ethyl acetate extract reduced the nociceptive response only in the second phase of formalin (100-300 mg/kg) and writhing tests. The aqueous extract, which is the most effective, contains active analgesic principles acting both centrally and peripherally. Furthermore, this antinociceptive effect has been avoided by naloxone at a dose of 1mg/kg in the first phase of formalin and hot plate tests indicating that this extract acts partly through an opioid-mediated mechanism. The present results demonstrated that Thymus broussonetii contains active constituents which possess antinociceptive activity justifying its popular use to relieve some pains.

Acetic Acid↗
PubMed1993

C-reactive protein (CRP) binding to the Sm-D protein of snRNPS. Identification of a short polypeptide binding region.

C-reactive protein (CRP) binds to chromatin, histones, and small nuclear ribonucleoproteins (snRNPs) through a phosphocholine (PC)-inhibitable, calcium-dependent binding site. snRNPs process pre-mRNA to mature mRNA and are composed of small uridine-rich RNAs (designated U1, U2, U5 and U4/U6) and associated proteins. We have shown that CRP binds to snRNPs in intact cells and to the U1 snRNP-specific 70 K protein in cell extracts. To determine whether CRP bound to other snRNP proteins, snRNPs were purified from rabbit thymus extract and CRP binding was assessed by blotting. CRP bound to a protein with the same mobility as Sm-D as well as to the 70 K protein. CRP specifically bound to and precipitated a fusion protein containing full-length Sm-D, confirming the binding of CRP to Sm-D. Binding was inhibited by PC and by EDTA. Binding studies using deletion mutants of the Sm-D fusion protein revealed that CRP binding was mediated by the C-terminal region of Sm-D, a region which binds autoantibodies and is proposed to bind to RNA. A comparison of the peptide regions on different autoantigens suggests that there is a shared motif to which CRP binds.

Amino Acid Sequence↗
PubMed1981

Identification of the immunogenically active components of the Sm and RNP antigens.

The spectrum of cellular targets in the autoimmune diseases is both large and varied and includes among the nuclear components the so-called Sm and RNP antigens associated with systemic lupus erythematosus. The use of immunoaffinity chromatography with dual specificity for the Sm and RNP antigens has allowed for their substantial purification from rabbit thymus in parallel and in quantity. In lieu of a functional assay, the use of a counterimmunoelectrophoresis assay provided a sensitive and rapid means of monitoring the distribution of the two antigens during purification and ensured the isolation of complexes containing the components required for antigenicity. The resulting purified complex consisted of nine polypeptides having molecular weights of approximately 9000 to 44,000 and two small RNAs of similar size. However, limited proteolysis of the isolated complex suggested that most of these polypeptides were not actually required for antigenic activity. Unlike Sm in crude thymus extracts, purified Sm was RNase sensitive. Thus, one of the major diagnostic criteria used to distinguish Sm and RNP antigens in crude extracts was shown to be invalid for purified material, suggesting that both antigens from rabbit thymus are actually ribonucleoprotein complexes.

Animals↗
PubMed1983

Generation of anti-NZB red blood cell antibody-forming plasma cells from bone marrow cultures of syngeneic and allogeneic mice: functional modulation of helper T-cell subsets in autosensitization.

An in vitro anti-NZB red blood cell (RBC) autoantibody-forming system was developed by culturing young (antiglobulin negative) or old (antiglobulin positive) NZB mouse bone marrow cells in the presence of young or old NZB thymocyte homogenates (or RNA thymus extracts) and young NZB-RBC. Using young NZB bone marrow cells, the greatest number of autoantibody forming cells were seen following stimulation with a mixture of young and old thymocyte homogenates (TH). A higher response was seen with old NZB bone marrow cells stimulated by old NZB thymocyte homogenates and RBC. Phenol-extracted thymic RNA retained activity when added to bone marrow culture containing NZB-RBC. Thymus RNA from 9 to 10 days old NZB mice had no activity although their bone marrow cells responded well to stimulation by young and old thymic RNA together with RBC. By analysing results obtained using T-cells enriched for helper and suppressor activities, we have concluded that abrogation of self-tolerance in mice is due to the appearance of functionally modulated helper T-cell subsets and to imbalance between helper and suppressor T cells.

Aging↗