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The cardinal sign of acute stress is thymic involution, which subsequently attenuates the activity of immunocompetent cells, notably T-lymphocytes, macrophages and NK cells. Sodium diethyldithiocarbamate (DTC), a low molecular weight sulphur compound, may function as a thymic hormone to induce precursor cells to become functionally mature T-lymphocytes. The studies were conducted on Balb/c mice exposed to restraint stress twice for 12 h at 24 h intervals. DTC at a dose of 20 mg/kg or calf thymus extract (TFX) at a dose of 10 mg/kg were injected i.p. four times at 24 h intervals prior to the exposure. It has been found that restraint stress markedly reduces the number of thymocytes which is concomitant with reduction in the weight of the thymus. In our study the changes sustained for 10 days of the observation. Besides, alterations in proliferative response of the thymocytes stimulated in vitro with concanavalin A (Con A) and phytohemagglutinin (PHA) were observed. The proliferative response of thymocytes to Con A was reduced 24 h after the exposure to restraint stress, but between days 4 and 7 it was found at increased levels, which decreased again on day 10. In contrast, the proliferative response of thymocytes to PHA was depressed for the entire 10 day period of the observation. It has been found that DTC and TFX administered to mice prior to restraint stress successfully counteract stress-induced immunosuppression, albeit TFX exerts stronger protective and regenerating impact on the thymus than DTC. TFX totally inhibits the suppressive effect of stress on proliferative activity of the thymocytes stimulated in vitro with Con A and PHA, stimulates restoration of thymic cells and increases the weight of the thymus. In contrast, DTC is not able to counteract the decrease in proliferative response of thymocytes to PHA.

Sera from anti-Ro(SSA) positive and negative patients with rheumatoid arthritis (RA) were compared with those from patients with primary Sjogren's syndrome (pSS) and systemic lupus erythematosus (SLE) with regard to anti-La(SSB) antibodies. Several assays for anti-La(SSB) (RNA precipitation, counterimmunoelectrophoresis, and immunoblotting) demonstrated the presence of such antibodies in selected anti-Ro(SSA) positive (10/19 in RA and 18/37 in pSS and SLE) but not in anti-Ro(SSA) negative sera. In agreement with previous reports, anti-La(SSB) antibodies from pSS and SLE patients uniformly reacted with various cellular extracts used as sources of La(SSB) antigen, including extracts from human cultured cells (HeLa), calf thymus and rabbit thymus. In contrast, while all 10 anti-La(SSB) sera from RA patients reacted with the human HeLa cell extracts, only three of them also reacted with calf and rabbit thymus extracts. These results were further supported by the analysis of a cohort of 70 consecutively selected sera (displaying monospecific anti-Ro(SSA) reactivity against calf thymus extract) from patients with various rheumatic disorders, where human-specific anti-La(SSB) were detected in two out of the five of RA patients studied.

The purpose of this study was to determine whether specific autoantibodies could be identified that are associated with eosinophilia-myalgia syndrome (EMS). Sera from 44 patients with EMS were tested by indirect immunofluorescence, immunodiffusion against calf thymus extract, and immunoprecipitation from HeLa cell extract. Antinuclear antibodies were detected in the sera of 24/39 patients with EMS (61.5%) by indirect immunofluorescence against HEp-2 cells. Seven patients (16%) were demonstrated to have specific autoantibodies by immunoprecipitation in which at least two shared patterns were noted. In three sera immunoprecipitation identified a similar 63-kDa band (Ab-1). An additional four sera shared a pattern of bands consisting of a strong 110-kDa protein and a weak 95-kDa protein (Ab-2). Absorption of HeLa extract with a strongly positive Ab-2 serum confirmed that the four patients shared the same antibody. In conclusion, the detection of these autoantibodies provides evidence of autoimmunity in EMS, and may distinguish this syndrome from clinically related conditions.

The human promyelocytic cell line HL-60 can be differentiated with retinoic acid (RA) along the granulocytic pathway. Numerous studies have identified many synergistic combinations of RA with cytostatics, cytokines and other inducers. A combination of RA with the crude thymus extract Thymex-L increased differentiation of HL-60 cells as confirmed by two functional assays and morphology, whereas the extract itself did not show any effect. The functional markers phagocytosis-associated chemiluminescence and nitroblue tetrazolium reduction were more enhanced (up to 4-fold with 1000 micrograms/ml Thymex-L) than morphology. The effect was found over a wide RA concentration range (10(-11) - 10(-6) M) and was dependent on extract concentration. The half-maximal induction of both functional markers was reached at 400 micrograms/ml. To achieve the same effect with the combination in comparison with RA alone, an RA dose reduction of about 100-fold was estimated. The effect was also seen when the cells were pretreated with the thymus extract for two days. The enhancement of RA action by Thymex-L was not correlated with an increase of extracellular or intracellular RA concentration. The active compound in Thymex-L is heat stable and bigger than 5 kDa as confirmed by gelfiltration. The defined thymus peptides thymosin alpha 1, prothymosin alpha 1 and thymopentin were unable to synergistically enhance HL-60 differentiation. These data suggest that the treatment with a thymus extract can increase the sensitivity of HL-60 cells for RA. This may have clinical implications.

Poly(ADP-ribose) polymerase responds to DNA strand breaks in nuclei by producing ADP-ribose polymers covalently attached to proteins. Here we report two fast protein liquid chromatographic applications to aid investigations on poly(ADP-ribosyl)ation. The first rapidly purifies poly(ADP-ribose) polymerase from crude calf thymus extract. The purification protocol, involving successive fractionations over four columns, reduces the time for polymerase purification from four days to 14 h resulting in a > 50% increase in enzyme-specific activity. The second application employs a complex salt gradient to reproducibly separate ADP-ribose polymers into individual size classes.

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.

The aim of this study was to examine the utility of diagnostic assays based on recombinant SS.B/La (rSS.B). Using this antigen, we have developed an ELISA and an immunoblot and compared these recombinant antigen-based assays with traditional thymus extract-based counterimmunoelectrophoresis (CIEP). Using the recombinant ELISA, the incidence of anti-SS.B in 184 normal blood donors was 2.2% (four sera). These four sera were all low titre, i.e., 3-5 SD above the mean. Of 38 sera positive for anti-SS.B by CIEP, 37 were positive in both recombinant assays (97.4% concordance). Anti-SS.B titre in CIEP correlated strongly with results of the rSS.B-based ELISA, but the ELISA was 3,000-fold more sensitive. In an analysis of 152 autoimmune sera containing anti-DNA, anti-RNP, anti-centromere, anti-SS.A/Ro or anti-cardiolipin, all of which were negative for anti-SS.B/La by CIEP, the recombinant assays detected 17 new anti-SS.B positives. These positive results were found only in sera which had previously been characterised by CIEP as anti-SS.A/Ro positive. Anti-SS.B/La antibodies detected by recombinant SS.B assays were found to be highly predictive of primary Sjögren's syndrome. Our results show that rSS.B can have an important role in the design of sensitive and specific assays for anti-SS.B. The diagnostic significance of anti-SS.B/La as a guide to primary Sjögren's syndrome is not diminished by the increased sensitivity of recombinant SS.B assays.

Antibodies to uridylic acid rich small nuclear ribonucleoprotein particles (UsnRNP) are mainly detected in patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Particularly those directed against epitopes of the 70K protein of U1snRNP serve as important markers for the diagnosis of MCTD. To establish an ELISA for determination of anti-70K protein antibodies in patients' sera a 1239 bp long cDNA insert coding for the epitopes of the 70K protein was ligated into a fusion expression vector. The bacterially expressed fusion protein was purified by chromatography on DEAE cellulose. Microtiter plates were coated with the fusion protein as well as with partially purified calf thymus extract (CTE) containing all natural UsnRNP antigens and RNase digested calf thymus extract (CTERNase) in which the natural 70K antigen was destroyed by the nuclease treatment. 10,888 sera of patients with suspected or overt rheumatic disease were analyzed for antibodies against these antigens simultaneously. Antibodies against CTE or CTERNase were not detected in 9123 sera, none of these showed reactivity with the 70K protein indicating a high degree of specificity of the assay. Positive results in each the 70K protein, CTE as well as the CTERNase ELISAs were obtained with 474 sera. 319 sera were only positive with CTE and 70K protein. Of these 793 anti-70K protein ELISA positive sera, 79% could be confirmed by immunoblot. Of 967 sera reacting with CTE and CTERNase but not with the recombinant 70K protein, 31% contained antibodies against various other UsnRNP proteins as shown by immunoblotting. 2.4% of these sera revealed also antibodies against the 70K protein. The use of the recombinant 70K protein as antigen meets the criterion for a simple and specific assay to detect anti-U1snRNP antibodies. Nevertheless, the sole use of this recombinant protein for anti-U1snRNP antibody screening may not be appropriate, because antibodies against other frequently occurring U1snRNP proteins (A, C) cannot be detected with this test. Therefore it should be used together with a natural UsnRNP antigen until further studies in patients with well established diagnoses will show whether natural antigens may be omitted.

A method enabling the direct assay in a cell culture system of material separated by thinlayer chromatography (TLC) without prior elution of active material from the TLC sheet is described. After chromatographic development on a plastic cellulose sheet, small disks were punched out from the sheet and transferred to microwells used for cell culture. Lymphoid cells in culture medium were incubated with the disks and the DNA synthesis was measured by [3H]thymidine incorporation. The reliability of the method was tested with l -alanine and thymidine, which are known to enhance the DNA synthesis of lymphoid cells and to interfere with the uptake of labeled thymidine, respectively. The incorporation of [3H]thymidine was stimulated with disks containing l -alanine and inhibited with disks containing unlabeled thymidine, completely in agreement with the expected results. Elution experiments showed that more than 75% of l -alanine and thymidine on the TLC disks was eluted into the culture medium within 1 h. Elution of material of higher molecular weight was also demonstrated. The method was used for the separation and assay of a thymocyte-specific growth factor isolated from calf thymus and greatly facilitated the detection of the active material after TLC.

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 ( p I = 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. Mn2+ was essential for the reaction and could not be replaced by Mg2+. 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.

Well known thymic-dependent properties of T-lymphocytes, E-rosetting, and phytomitogenic responsiveness, were observed to be lowered in some patients with chronic virus-B hepatitis. On the assumption that these deficiencies may be due to lack of thymic influence, we decided to attempt their repair by administration of the thymus extract “TFX-Polfa” to the patients. The earliest effect of treatment observed was an increase in percentage of E-rosetting cells to a value within the normal range. Prolonged treatment resulted in the increase of lymphocyte response to stimulation with concanavalin A, and thereafter in significant increase in reactivity to phytohemagglutinin. It is suggested that these quantitative and qualitative improvements elicited by TFX in the peripheral blood lymphocyte pictures of the patients arise out of its ability to induce in vivo sequential processes of differentiation of thymus-dependent lymphocytes normally accomplished by the thymus.

Three subspecies of RNA polymerase II, designated IIO, IIA, and IIB, have been described in a variety of eukaryotic cells and shown to differ in the molecular weight of their largest subunit, designated IIo, IIa, and IIb, respectively. The objectives of this study were to establish the in vivo molecular structure of RNA polymerase II in mammalian cells and to examine conditions that influence the stability of RNA polymerase II subspecies. Subunit affinity-purified antibodies were used to determine the relative concentration of subunits IIo, IIa, and IIb in crude extracts of calf thymus tissue, cultured bovine kidney cells, and HeLa cells. HeLa cells contain exclusively RNA polymerase IIO whereas both cultured bovine kidney cells and calf thymus tissue contain RNA polymerases IIO and IIA. RNA polymerase IIB was not detected at significant levels in any of the cell extracts examined. Cell extracts were aged at either 4 degrees or 37 degrees C and the stability of RNA polymerases IIO and IIA determined by protein blotting. In the presence of buffer normally used for RNA polymerase purification, subunit IIo disappears from calf thymus extracts within 24 h at 4 degrees C or within 5 min at 37 degrees C. RNA polymerase IIO is partially stabilized by the inclusion of protease inhibitors and further stabilized by the presence of relatively high concentration of EDTA and EGTA. The prior fractionation of nuclei does not have an appreciable effect on RNA polymerase II stability. An increase in the amount of reducing agent causes a dramatic reduction in the stability of subunit IIo. The following manuscript (Bartholomew, B., Dahmus, M. E., and Meares, C. F. (1986) J. Biol. Chem. 14226-14231) examines the transcriptional activity of RNA polymerases IIO and IIA in reactions dependent on the major late promoter of adenovirus-2. Photoaffinity labeling of subunits IIo and IIa, relative to their concentration in the transcription extract, indicates that the transcriptional activity of RNA polymerase IIO is greater than 10 times that of IIA.

The major NaCl-stimulated protein-tyrosine kinase activity found in soluble thymus extracts, as measured by the phosphorylation of angiotensin I, is a 40-kDa enzyme known as p40 (Zioncheck, T. F., Harrison, M. L., and Geahlen, R. L. (1986) J. Biol. Chem. 261, 15637-15643). Antibodies prepared against p40 cross-react with a 72-kDa protein-tyrosine kinase (p72) from spleen or thymus that is closely related to p40 by peptide-mapping experiments. The recovery of p40 from spleen homogenates is reduced while the recovery of p72 is enhanced by the addition of high concentrations of leupeptin or soybean trypsin inhibitor to the homogenization media. The generation of p40 in spleen homogenates occurs with a concomitant increase in protein-tyrosine kinase activity. Activated catalytic fragments of 38-43 kDa can be generated by the treatment of partially purified p72 with trypsin or papain. The p72 protein-tyrosine kinase is found at the highest levels in spleen, thymus, and lung, tissues that also have high protein-tyrosine kinase activity and generate high levels of p40 following homogenization. p72 is also found in certain T and B cell-derived cell lines and in NIH3T3 cells.

Antibodies to the RNase-sensitive RNP and to the RNase-resistant Sm nuclear antigens were used to affinity purify these antigens from a saline extract of rabbit thymus acetone powder. Determination of the protein subunits recovered by either glycine-HCl, pH 2.8, or 2.5 M MgCl2 elution on gradient sodium dodecyl sulfate-polyacrylamide electrophoresis containing mercaptoethanol revealed that RNP was composed of five proteins with mol. wts from 10,000 to 15,000 whereas Sm contained the same or similar five chains plus six additional subunits with mol. wts from 21,000 to 42,000. RNase treatment of the thymus extract increased the recovery in Sm of the same bands compared to untreated extract. Thus, RNP and Sm appear to have different numbers of protein components and RNP may be a subset of Sm. Sucrose gradient centrifugation of the 125I-labeled, pH 2.8 eluted antigens gave peaks of 3 and 6S for RNP and Sm, respectively. Sucrose gradient centrifugation of the crude untreated thymus extract followed by quantitative single radial immunodiffusion analysis of each fraction produced a broad peak from 16S to the top of the gradient while pretreatment of the extract with RNase resulted in a discrete 6S peak. These results indicate that in rabbit thymus acetone powder native RNP and Sm exist as larger polydisperse complexes with additional material including RNA and that after acid elution or RNase treatment the antigens are found in a smaller monodisperse form.

The increased detection of anti-Jo-1 antibody afforded by the use of the purified antigen, histidyl-tRNA synthetase, in counterimmunoelectrophoresis is demonstrated. Using purified antigen, anti-Jo-1 antibody was detected in the sera of 16 33 (48.5%) patients with confirmed myositis and in 20 45 (44.5%) patients with confirmed or possible myositis. This rate is approximately double that obtained with commercial thymus extracts both in this study and seven others reported in the literature. The presence of antibody shows marked correlation with the activity of myositis at the time of serum sampling and with the presence of interstitial lung disease. Detection rates are similar in patients with polymyositis and dermatomyositis both with and without additional connective tissue diseases.

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, Zn2+, 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.

A tRNA pseudouridine synthase has been extensively purified from steer thymus extracts, using undermodified tRNA from hisT- mutants of Salmonella typhimurium as a substrate. The enzyme synthesizes a group of psi residues in the anticodon region of various hisT- isoacceptors and behaves like a eukaryotic homologue of Salmonella tRNA psi synthase I. The thymus enzyme requires a thiol and a monovalent cation (NH4+ or K+) for optimum activity; no energy sources or cofactors are required. The activity is inhibited by single tRNAs or bulk tRNA from all sources tested, and by ribosomal RNAs, various polyribonucleotides, and DNA. The enzyme modifies the two hisT- tRNAPhe isoacceptors, both tRNATyr acceptors and at least five of the tRNALeu isoacceptors to products that coelute with the respective wild type species on reverse-phase columns. With pure hisT- tRNA2Phe as substrate, the enzyme specifically converts residue U39 to psi. Interestingly, a psi residue is still present at position 32, in the anticodon loop of hisT- tRNA2Phe, indicating the existence of other uncharacterized pseudouridylation enzymes in S. typhimurium. These composite results show that the thymus enzyme can form psi at residues 38, 39, and 40 in the anticodon region of appropriate hisT- isoacceptors. During the enzyme purification, a second activity is partially resolved, which releases 3H from wild type S. typhimurium [pyrimidine-5-3H]tRNA. This activity may be associated with an enzyme that pseudouridylates sites that are uniquely modified in eukaryotic tRNAs, but not in Salmonella tRNAs. Our observations support the view that the psi residues in tRNA are synthesized by a family of enzymes, whose members act on uridine residues in specific regions of the molecule.

Twenty-one immunodeficient patients with recurrent herpes simplex labialis (HSL) were randomly allocated to either the saline or the bovine thymus extract Thymostimulin (TS) group and treated for a period of 6 months. A total of 17 recurrences with a mean severity index of 266.2 +/- 192 in the TS-treated group versus a total of 62 recurrences in the placebo group (score 458.8 +/- 299.6) were observed after 12 months of follow-up. A significant increase of total WBC (P less than 0.05 versus control group), lymphocyte count, and T-cell number (P less than 0.001) were detected in the TS group after 6 months as well as after 12 months. In vitro lymphoproliferative responses to herpes simplex virus antigen and natural killer cell activity were also significantly higher (P less than 0.05) in the TS group, whereas no significant difference in antibody titers to herpes simplex could be detected between the two groups. TS may be potentially useful in preventing viral reactivation in immunocompromised hosts by potentiating cell-mediated immune responses. Further studies evaluating TS for prophylaxis of infection in patients at risk should be considered.

Advantages in the use of indirect immunofluorescence for the identification of terminal transferase (TdT) in single cells may be offset by lack of specificity, as compared to the biochemical assay of the enzyme, especially in analyses of lymphocyte populations. False positive results were obtained in 15 15 tonsillectomy samples and in 9 27 specimens from children with acute lymphoblastic leukemia in remission, perhaps due to antihuman lymphocyte activity in the rabbit heteroantisera which are used in the indirect immunofluorescence technique. This phenomenon may be more pronounced in ‘activated’ normal lymphocytes. Such reactions are not due to antibodies directed against adenovirus, papovavirus or EB virus antigens, although these are common constitutents of human tonsillar cells. Additional problems with TdT heteroantisera may result from immunization with non-TdT determinants in calf thymus extracts, as was manifest in human non-lymphoid (KB) cells cultured in fetal bovine serum. These difficulties will be overcome only by production of a monoclonal antibody using human TdT as the antigen.

The nuclear autoantigen SS-B (Sjögren's syndrome B antigen) was purified from rabbit thymus extract by immunoaffinity chromatography with human autoantibodies, and used to immunise BALB/c mice for production of monoclonal antibodies. Fusion of spleen cells from an immunised mouse with NS-1 myeloma cells resulted in the isolation of 3 clones secreting anti-SS-B antibody. Subclasses were shown to be IgG2b by immunodiffusion. Specificity of the monoclonal antibodies (MCA) was determined by ELISA and indirect immunofluorescence. By immunoblotting all 3 MCA identified a single 45 K immunoreactive polypeptide in rabbit thymus, identical with the major polypeptide recognised by human sera containing anti-SS-B. Affinity columns prepared from the 3 MCA all bound SS-B from rabbit thymus extract, without binding other nuclear antigens. Immunofluorescence studies on standard substrates showed that SS-B was located predominantly in the nucleoplasm but in cells transformed by EBV or phytohaemagglutinin more prominent nucleolar and cytoplasmic staining was seen.

DNA ligase II has been purified about 4,000-fold to apparent homogeneity from a calf thymus extract. The ligase consists of a single polypeptide with a molecular weight of 68,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On fluorography after electrophoresis, a DNA ligase-[3H]AMP complex gave a single band corresponding to a molecular weight of 68,000. The Km values of the ligase for ATP and nicked DNA (5'-phosphoryl ends) were obtained to be 40 and 0.04 microM, respectively. Antibody against calf thymus DNA ligase II was prepared by injecting the purified enzyme into a rabbit. The antibody cross-reacted with DNA ligase II but not with calf thymus DNA ligase I. DNA ligase II was not affected by antibody against calf thymus DNA ligase I with a molecular weight of 130,000 (Teraoka, H. and Tsukada, K. (1982) J. Biol. Chem. 257, 4758-4763). These results indicate that DNA ligase II (Mr = 68,000) is immunologically distinct from DNA ligase I (Mr = 130,000).

Numerous polymyositis sera were found to give complement fixation reactions with an antigen in calf thymus extract. Because of the weak and inconstant precipitin reactions characteristic of this system, the specificity of the reaction could not be classified by the Ouchterlony method of double diffusion in agar gel. To identify the specificity of this reaction, the following method was developed. Papain digestion of any of the polymyositis sera converted the complement fixing capacities of the sera to potent and specific inhibitors of the reaction characteristic of these polymyositis sera. Papain digestion of sera from patients with a variety of connective tissue diseases containing a multiplicity of antibodies to antigens in calf thymus extract failed to inhibit the complement fixation reaction characteristic of the polymyositis sera. Thus far, therefore, antibodies to this antigen in calf thymus extract appear most specific for patients with polymyositis syndromes, particularly those with dermatomyositis.

The graft-vs.-host (GVH) reaction, induced by the injection of parental strain lymphoid cells into F 1 mice, suppressed the immune response to sheep blood cells (SRBC). The degree of immunosuppression was related to the time post GVH induction when F 1 mice were treated with SRBC and to the dose of parental cells used to induce the GVH reaction. Attempts were made to restore the PFC response in animals immunosuppressed by the GVH reaction. It was demonstrated that, when animals experiencing a GVH reaction were treated with F 1 thymus cells or thymus extract on days 6 and 7 post-GVH induction, the PFC response to SRBC was partially restored—up to 50% of the normal control value. Spleen and bone marrow cells failed to restore the PFC response of animals experiencing a GVH reaction. F 1 thymus cells were more effective than parental thymus cells. Using an anti-theta C3H serum, it was demonstrated that thymus-derived cells of F 1 origin were not diminished in the thymus, spleen, and lymph nodes of animals experiencing a GVH reaction. It is suggested that GVH-induced immunosuppression is caused by depletion of a thymic mediator, which plays an essential role at some critical stage following antigenic stimulation.

Hypothermia tolerance was studied in guinea pigs with experimental myasthenia gravis induced by immunization with homologous thymus extract and Freund's complete adjuvant. Untreated guinea pigs and guinea pigs immunized with liver extract and Freund's complete adjuvant served as controls. Thymus-immunized guinea pigs survived to a significantly lower temperature at asystole than did the liver-immunized controls. This finding is consistent with a slight diminution in temperature maintenance mechanisms in guinea pigs with experimental autoimmune thymitis. The observed increase in hypothermia tolerance may be due to impaired shivering as a consequence of the partial neuromuscular block demonstrable in the myasthenia gravislike state.

Anti-theta serum inhibits a portion of spleen spontaneous rosette-forming cells (sRFC) and all of thymus sRFC (as seen after in vivo hydrocortisone treatment). Bone marrow sRFC are also partially sensitive to anti-theta serum but at much higher concentrations. The rosette inhibiting activity of anti-theta serum is absorbed by thymus cells and brain; it is also absorbed by bone marrow cells but to a much lesser degree. Theta-positive sRFC are inhibited by low and noncytotoxic concentrations of azathioprine (10−6 M) and ALS ( 1 2000 – 1 16,000 ) in the presence of complement. Bone marrow sRFC are much less sensitive to azathioprine and ALS than spleen and thymus sRFC. Spleen sRFC from neonatally thymectomized and Nude mice lose their susceptibility to anti-theta serum, azathioprine and ALS. Conversely, thymosin (a cell free thymus extract) confers on normal bone marrow sRFC a sensitivity identical to that of spleen sRFC.

An enzyme activity which catalyzed the deacetylation of histone was found in the calf thymus extract. 14 C-Acetyl-histone prepared by incubating calf thymus nuclei with sodium 14 C-acetate served as the substate and treatment of the histone with pronase destroyed most of the susceptibility to the enzymatic deacetylation. 14 C-Acetyl-histone prepared by chemical acetylation with 14 C-acetic anhydride served as the substrate to a less extent.

Fifty patients received 0.5 c.c. of thymus extract, this being the only maximum dose with which we had any experience. Only 1 patient was 3 fingers dilated. The others ranged from no dilatation to 2½ fingers. Nine occipitoposteriors, 1 face, and 1 set of twins were encountered. The frequency of the pains was only slightly increased. The duration of the pains was seldom affected. A definite rhythm was established. The intensity was distinctly increased. The patients stated that they experienced somewhat stronger pains after the administration of thymus. All of which resulted in earlier, full dilatation and delivery, not, however, as rapidly nor as forcefully as with thymophysin. The blood pressures were not affected.

This paper contains evidence of the presence in calf thymus extracts of an enzymatic factor able to produce hyperchromicity in native DNA in vitro without causing fragmentation of the individual strands.

54 CROSS-REACTING ANTIBODY (AB) TO ALFALFA SEED (AS) AND DEOXYRIBONUCLEIC ACID (DNA) IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). E. J. Bardana, M.D., M. R. Malinow, M.D., S. Craig, and P. McLaughlin Portland, Oregon. Dietary AS reduces plasma cholesterol in rabbits and monkeys. Ingestion by a man lowered cholesterol, but caused an SLE-like illness (Lancet 1:615, 1981). Three of 5 female monkeys fed 45% ground AS in semi-purified diet (SPD) developed an SLE-like illness as opposed to none on SPD alone. Hemolytic anemia, antinuclear AB, hypocomplementemia and anti-native DNA were noted (Am. J. Kidney Dis. 1:345, 1982). Further studies incriminated germinated AS (sprouts) and suggested L-canavanine as the responsible component (Science 216:415, 1982). To explore these observations in humans, sera from 35 patients with SLE, 28 with non-SLE rheumatic disorders and 39 normals were studied by immunodiffusion for AB to: AS extract (50 mg/ml and 100 mg/ml); sonified native DNA (1 mg/ml and 0.5 mg/ml); denatured DNA (0.5 mg/ml); and rabbit thymus extract (RTE) (20 mg/ml). Eleven of 35 SLE sera (31%) and 1 of 39 normals (3%) showed precipitins to AS extract. None were seen in patients with other rheumatic disorders (p

Summary The effect of a purified bovine thymus gland extract (Dr. Kurt Mulli, GmbH, Hamburg, West Germany) was studied in 12-week old male Wistar rats on the following: thyroid weights and morphology, T3-T4 serum levels, thyroid lactic dehydrogenase, ATP-ase, acid phosphatase, and non-specific esterase activities. Thymus extract was administered intramuscularly daily for 21 days at doses of 0.5, 1.0 and 2.0 ml/kg. Mesurements were made on day 3, 7, 14 and 21 of treatment. Thyroid histology and enzyme activity were studied only on 21-day specimens. Thymus extract significantly decreased average thyroid gland weights in a dose-dependent manner irrespective of treatment duration. T3 serum levels were consistently lower in thymus-treated rats irrespective of treatment dose or duration. Changes from control levels were not statistically significant due to large standard deviations. T4 serum levels were significantly lower than control levels in rats treated with thymus extract for 14 and 21 days. Histology of thyroids from 21-day treated animals revealed a marked reduction in both thyroid follicle size and colloid content with an increase in connective tissue, resorption vacuoles and hyperemia. Histochemical study of thyroid enzyme activity showed lactic dehydrogenase increase in follicle epithelial cells, acid phosphatase increase in follicle epithelial cells and decrease in interstitium, ATP-ase increase in granular storage area and non-specific esterase increase in follicle epithelial cell. The data suggest the presence of an unidentified specific anti-thyroid factor(s) in the thymus gland extract.