A normative study of Nelson's (1976) modified version of the Wisconsin Card Sorting Test in healthy older adults.

A modified version of the Wisconsin Card Sorting Test (mWCST) proposed by Nelson (1976) was administered to 229 healthy community-dwelling older adults, composed of 97 men and 132 women, ages 45 to 91. Investigating the impact of demographic factors such as age, education, and gender on mWCST performance, results indicated that mWCST performance was significantly affected by both age and education. Unlike the standard WCST, however, gender did not significantly influence mWCST performance. Because demographic factors appear to exert a significant influence on mWCST performance of normal older adults, demographically-corrected norms were calculated according to the procedure described by Heaton, Grant, and Matthews (1991). In addition, longitudinal analysis of mWCST performance revealed that significantly fewer nonperseverative errors were committed at retest approximately one year later. Number of categories completed and perseverative errors did not appear to demonstrate significant practice effects in this sample. Accounting for demographic influences and the inspection of practice effects on serial administration of the mWCST may improve upon its sensitivity and specificity for use in the clinical assessment of executive function in older adults.

Interleukin-10 is an autocrine growth factor for acquired immunodeficiency syndrome-related B-cell lymphoma.

Interleukin-10 (IL-10) is an acid-sensitive protein of 35 kD that has pleiotropic effects including inhibition of cytotoxic T-cell response, induction of major histocompatibility complex type II in B lymphocytes, induction of B-cell growth and differentiation, and autocrine growth factor activity in monocytes. We and others have shown that IL-10 is produced spontaneously by blood mononuclear cells from human immunodeficiency virus-seropositive patients. In an attempt to ascertain the potential role of IL-10 in acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma, we evaluated the expression of human IL-10 in both tumor-derived B-cell lines and primary tumor cells. Expression of human IL-10 (hIL-10) mRNA and protein was detected in four of five cell lines examined. An IL-10 antisense oligonucleotide inhibited IL-10 mRNA expression and IL-10 protein production. The proliferation of all B-cell lines was inhibited by an antisense oligonucleotide in a dose-dependent manner that was abrogated by the addition of recombinant hIL-10 protein. No effect of antisense oligonucleotide was observed in the B-cell line not producing hIL-10. Evaluation of primary tumor cells from patients with AIDS-lymphoma cells showed similar production and response to IL-10. These data suggest an autocrine growth mechanism for IL-10 in AIDS-related lymphoma cells and that IL-10 may be important in its pathogenesis.

Interleukin 10: a novel stimulatory factor for mast cells and their progenitors.

We have characterized the mast cell stimulating activity of murine cytokine synthesis inhibitory factor, referred to as interleukin 10 (IL-10). It was found that IL-10 alone failed to support the growth of mast cell lines and mast cell progenitors. Nevertheless, it dramatically enhanced their growth when combined with IL-3 or IL-4. Moreover, IL-4 plus IL-10 supported the proliferation of mast cells as well as IL-3, suggesting that these two factors may provide a pathway for their development independent of IL-3. However, optimal mast cell growth was stimulated by the combination of IL-10, IL-4, and IL-3. This particular set of cytokines are coordinately produced by activated T cells and may constitute an effective network regulating early and late stages of mast cell development during certain immune responses.

Isolation of monoclonal antibodies specific for IL-4, IL-5, IL-6, and a new Th2-specific cytokine (IL-10), cytokine synthesis inhibitory factor, by using a solid phase radioimmunoadsorbent assay.

Hybridomas were produced from a rat that was immunized with partially purified proteins from supernatants of induced Th2 cells. These preparations were enriched for cytokine synthesis inhibitory factor (CSIF, IL-10). The mAb in the supernatants were screened by a solid phase radioimmunoadsorbent assay using 35S-methionine-labeled secreted proteins from a lectin-stimulated Th2 clone. A total of 18 anticytokine mAb were isolated, comprising 6 anti-CSIF, 1 anti-IL-4, 1 anti-IL-5, and 10 anti-IL-6 mAb. The anti-CSIF mAb were separable into three groups. mAb in groups A and B neutralized and depleted bioactivity, and bound to overlapping but nonidentical subpopulations of CSIF molecules. The 2 mAb in group C did not neutralize CSIF activity, and bound to CSIF molecules not recognized by mAb from groups A or B. A two-site sandwich ELISA for CSIF could be established with the group A antibody, SXC1, combined with any of the three group B antibodies. The sensitivity of this assay was equivalent to that of the CSIF bioassay. These antibodies have been used to show that CSIF is responsible for most or all of the ability of Th2 supernatants to inhibit cytokine synthesis by Th1 cells. In addition, the ELISA has been used to confirm that CSIF is produced by Th2 but not Th1 clones.

Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas.

We found a unique thymocyte growth-promoting activity in supernatants (SN) from subclones of the B cell lymphoma CH12.LX. We have tentatively named this activity B-TCGF (for B cell-derived T cell growth factor) and characterized the activity produced by the CH12.LX.4866 subclone. This SN did not induce thymocyte proliferation alone, however, it enhanced both adult and fetal (Day 15 of gestation) murine thymocyte proliferation in the presence of IL-2, IL-4, or IL-7. Other known cytokines were screened for a B-TCGF-like activity using both adult and fetal thymocytes. IL-6 was found to be active only on adult thymocytes, while TNF alpha and GM-CSF were found to be active only on fetal thymocytes. However, neutralizing antibodies against these cytokines did not block the B-TCGF activity present in CH12.LX.4866 using either adult or fetal thymocytes. These observations suggest that the B-TCGF activity is mediated by a novel factor(s). The apparent molecular weight of this novel molecule(s) was 27-50 kDa determined by sizing HPLC.

Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones.

A cytokine synthesis inhibitory factor (CSIF) is secreted by Th2 clones in response to Con A or antigen stimulation, but is absent in supernatants from Con A-induced Th1 clones. CSIF can inhibit the production of IL-2, IL-3, lymphotoxin (LT)/TNF, IFN-gamma, and granulocyte-macrophage CSF (GM-CSF) by Th1 cells responding to antigen and APC, but Th2 cytokine synthesis is not significantly affected. Transforming growth factor beta (TGF-beta) also inhibits IFN-gamma production, although less effectively than CSIF, whereas IL-2 and IL-4 partially antagonize the activity of CSIF. CSIF inhibition of cytokine synthesis is not complete, since early cytokine synthesis (before 8 h) is not significantly affected, whereas later synthesis is strongly inhibited. In the presence of CSIF, IFN-gamma mRNA levels are reduced slightly at 8, and strongly at 12 h after stimulation. Inhibition of cytokine expression by CSIF is not due to a general reduction in Th1 cell viability, since actin mRNA levels were not reduced, and proliferation of antigen-stimulated cells in response to IL-2, was unaffected. Biochemical characterization, mAbs, and recombinant or purified cytokines showed that CSIF is distinct from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IFN-gamma, GM-CSF, TGF-beta, TNF, LT, and P40. The potential role of CSIF in crossregulation of Th1 and Th2 responses is discussed.

The characterization of four monoclonal antibodies specific for mouse IL-5 and development of mouse and human IL-5 enzyme-linked immunosorbent.

Four rat mAb directed against mouse IL-5 have been characterized by their ability to remove and neutralize mouse IL-5 activity in various bioassays. All four mAb absorbed IL-5 activity from solution. Although all were able to neutralize mouse IL-5 bioactivity, two were significantly more effective. These two were also able to neutralize the activity of mouse IL-5 delivered to B cells during a cognate-linked interaction with a Th cell clone. A two-site sandwich ELISA specific for mouse IL-5 was developed by using pairs of mAb. The mouse IL-5 ELISA is capable of detecting natural or mouse rIL-5 in supernatants, crude bacterial lysates, and high concentrations of mouse serum, and has a detection limit of less than 20 pg. Two of these antibodies cross-reacted with and neutralized human rIL-5, and one of these was used for development of an ELISA for human IL-5.

Increased autoantibody production by NZB/NZW B cells in response to IL-5.

We previously demonstrated that B cells from NZB/NZW but not nonautoimmune mice secrete high levels of autoantibodies in response to factor(s) derived from type 2 Th cell (Th2) clones. Supernatants from type 1 Th cell clones, which contain a different set of lymphokines, were not stimulatory. In the present experiments, we attempted to define the active Th2 factor(s) and to better understand the cellular basis for the hyperresponsiveness. In response to optimal concentrations of supernatant (Th2-Sup), B cells from 3-mo-old NZB/NZW mice produced up to 40-fold greater amounts of IgM anti-DNA compared with unstimulated B cells, whereas BALB/c B cells produced levels only slightly above background. Although Th2-Sup contained large amounts of IL-4, comparable concentrations of rIL-4 alone did not stimulate NZB/NZW B cells. Furthermore, a blocking anti-IL-4 mAb did not prevent Th2-Sup-stimulated autoantibody production. Th2-Sup was fractionated by HPLC, and the stimulatory factor(s) was found in fractions known to contain IL-5 (also known as B cell growth factor II). Indeed, a highly purified preparation of IL-5 reproduced the effects of Th2-Sup by stimulating NZB/NZW B cells to produce high levels of IgM anti-DNA antibodies while enhancing production by nonautoimmune cells only slightly. In limiting dilution studies, NZB/NZW compared with BALB/c spleens contained a three- to four-fold greater frequency of DNA-specific B cells that were responsive to IL-5. Together, the results suggest a potential role for IL-5 in the pathogenesis of NZB/NZW autoimmune disease.

Identification of IL-6 as a T cell-derived factor that enhances the proliferative response of thymocytes to IL-4 and phorbol myristate acetate.

Thymocytes undergo a vigorous proliferative response when stimulated with a combination of IL-4 and PMA. We have found that conA-induced supernatants from a number of Th cell clones could enhance the level of IL-4/PMA-induced proliferation of unseparated thymocytes 0.5- to 2-fold and of peanut agglutinin-positive thymocytes 2- to 10-fold. These supernatants did not contain IL-2 or IFN-gamma, and the enhancing activity could be chromatographically separated from IL-3, -4, -5, and granulocyte/macrophage CSF. The possibility that the thymocyte enhancement factor contained in these supernatants was IL-6 was suggested when murine rIL-6 was found to have similar activity. Further evidence for the identity of these two factors was obtained when an IL-6 assay, based on plasmacytoma growth, was used to test column fractions showing thymocyte enhancement. All fractions active in the thymocyte enhancement assay also had activity in the plasmacytoma growth assay. These observations suggest that the thymocyte-stimulating activity present in the T cell supernatants was due to IL-6.

Expression, renaturation and purification of recombinant human interleukin 4 from Escherichia coli.

The lymphokine human interleukin 4 (IL-4) has been expressed from a plasmid in the cytoplasm of Escherichia coli. Advantage has been taken of insolubility of the human IL-4 in E. coli for rapid purification of this protein in only a few steps. We describe extraction and renaturation procedures which solubilize human IL-4 yielding biologically active protein. The protein was purified to homogeneity by one passage over a gel-filtration column. The refolded human IL-4 was characterized by N-terminal sequence analysis, amino acid analysis and bioassays. The refolded E. coli-derived human IL-4 has biological activity on T and B cells and binds to the human IL-4 receptor, comparable to mammalian expressed human IL-4, indicating that the protein is folded correctly.

A mouse T cell product that preferentially enhances IgA production. I. Biologic characterization.

Supernatants from a subset of helper T cell clones can enhance IgA, IgE, and IgG1 production in cultures of lipopolysaccharide-stimulated, T cell-depleted spleen cells. The lymphokine interleukin (IL)-4 has been shown to cause the IgE and IgG1 enhancement produced by these supernatants. IgA enhancement, however, is mediated by a factor distinct from IL-4, although IL-4 can potentiate the effect of the IgA-enhancing factor. IgA-enhancing factor is also distinct from IL-1, IL-2, IL-3, granulocyte-macrophage colony-stimulating factor, and interferon-gamma and acts directly on B cells. Purified IgA-enhancing factor enhances IgA production three- to sixfold yet causes less than a twofold increase in other isotypes. The IgA enhancing activity is not inhibited by concentrations of interferon-gamma that inhibit IL-4 activities. In the accompanying article, we show that this IgA enhancing activity is a novel property of the lymphokine IL-5.

A mouse T cell product that preferentially enhances IgA production. II. Physicochemical characterization.

Certain subsets of helper T cells, following stimulation with concanavalin A, secrete factors that specifically enhance the production of IgG1, IgE, and IgA by lipopolysaccharide-stimulated B cells. In the previous report, we describe a factor from the helper T cell line MB2-1 which enhances IgA production. IgA-enhancing factor has been purified from serum-free supernatants of this cell line. The purified lymphokine is a family of microheterogeneous polypeptides presumably modified post-translationally. IgA-enhancing factor has a native m.w. of 45,000 to 60,000 with subunits of between 24,000 and 28,000 under reducing conditions. Upon Edman degradation, a single amino-terminal sequence is detected which is identical to that of the lymphokine interleukin 5. IgA-enhancing factor activity is thus mediated by the same polypeptide that has been characterized as type II B cell growth factor, T cell-replacing factor, and eosinophil-differentiation factor.

Contribution of tryptophan residues to the combining site of a monoclonal anti dinitrophenyl spin-label antibody.

Two Fab fragments of the monoclonal anti dinitrophenyl (DNP) spin-label antibody AN02 were prepared by recombination of specifically deuterated heavy and light chains. In the recombinant H(I)L(II) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the heavy chain. In the recombinant H(II)L(I) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the light chain. Saturation of three resonances of H(I)L(II), assigned to tryptophan protons of the light chain, resulted in magnetization transfer to the aromatic proton at position 6 of the DNP ring and to the CH2 protons of the glycines linked to the DNP in a diamagnetic hapten (DNP-DG). Saturation of three resonances of H(II)L(I) assigned to tryptophan protons of the heavy chain resulted in magnetization transfer to the CH2 protons of the glycines in DNP-DG. From the dependence of the magnetization transfer on the irradiation time, the cross relaxation rates between the involved protons were estimated. The inferred distances between these protons of the hapten and certain tryptophan protons are 3-4 A. It is concluded that in the combining site of AN02 there is one tryptophan from the light chain and one tryptophan from the heavy chain that are very near the hapten. When all tyrosines and phenylalanines were perdeuterated and all tryptophan aromatic protons were deuterated except for the protons at positions 2 and 5, titration of the Fab fragments with variable amounts of paramagnetic hapten showed that one proton from the light chain tryptophan is near (less than 7 A) the unpaired electron and that three other protons are significantly closer than 15 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of Escherichia coli dnaC. Identification of a cysteine residue possibly involved in association with dnaB protein.

The nucleotide sequence of the Escherichia coli dnaC gene and the primary structure of the dnaC protein were determined. The NH2-terminal amino acid sequence of the dnaC protein matched that predicted from the nucleotide sequence of the 735-base pair coding region. The dnaC gene lacks characteristic promoter structures; neither the "Pribnow box" nor the "-35 sequence" was detected within 222 base pairs upstream from the initiator ATG codon. There is, however, a typical Shine-Dalgarno sequence 7-10 base pairs before the ATG codon. An upstream open reading frame, separated by just 2 base pairs from the coding region of dnaC, encodes the COOH-terminal half of the dnaT product (protein i; Masai, H., Bond, M. W., and Arai, K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1256-1260). The dnaC protein contains 245 amino acids with a calculated molecular weight of 27,894 consistent with the observed value (29,000). Similar to dnaG and dnaT, dnaC uses several minor codons; the significance of these minor codons to the low level expression of the protein product in E. coli cells remains to be determined. The in vitro site-directed mutagenesis method was employed to determine the functional region involved in interaction with dnaB protein. The first cysteine residue located in the NH2-terminal region of the dnaC protein (Cys69) was shown to be important for this activity. Overall sequence homology between dnaC protein and lambda P protein, functionally analogous to the dnaC protein in the lambda phage DNA replication, is not extensive. There are, however, several short stretches of homologous regions including the NH2-terminal eight amino acids and the Cys78 region of dnaC protein.

An RNA polymerase II transcription system from rat liver. Purification of an essential component.

An enzyme system capable of supporting accurate initiation of transcription by RNA polymerase II has been prepared from rat liver. Transcription depends upon at least three activities in addition to RNA polymerase II. One activity, designated alpha, has been purified 30,000-fold to near homogeneity. From all promoters tested, the synthesis of full-length runoff transcripts by RNA polymerase II depends upon alpha. alpha appears to consist of a single 35,000-dalton polypeptide and is inactivated by N-ethylmaleimide and heat. Similarities between alpha and transcription activities in fractions TFIIB (Matsui, T., Segall, J., Weil, P. A., and Roeder, R. G. (1980) J. Biol. Chem. 255, 11992-11996) and [CB] (Samuels, M., Fire, A., and Sharp, P. A. (1982) J. Biol. Chem. 257, 14419-14427) from human cells are discussed.

T-cell and mast cell lines respond to B-cell stimulatory factor 1.

The murine lymphokine B-cell stimulatory factor 1 (BSF-1) has been described previously in terms of its action on B lymphocytes. We now provide evidence that BSF-1 is also responsible for two additional biological activities. The first of these is the stimulation or maintenance of a state of activation in mouse T-cell lines. The second activity is the increase in the proliferative rate of certain mast cell lines costimulated with interleukin 3. The T-cell and mast cell activities are mediated by purified BSF-1 and copurify with BSF-1 from supernatants of certain T-cell lines. Each of these activities is inhibited by monoclonal anti-BSF-1 but not by monoclonal anti-interleukin 2 antibody. The antibody inhibition results also indicate that BSF-1 is the major or only source of these two activities in the activated T-cell supernatants that we have tested.

B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide-activated B cells.

Supernatants from some mouse helper T cell (TH) lines contain an activity that can enhance IgE production by lipopolysaccharide (LPS)-stimulated B cells by at least two orders of magnitude. During purification, this activity could not be resolved from B cell stimulatory factor-1 (BSF-1). Highly purified BSF-1 from a different source, the T lymphoma cell line EL-4, enhanced IgE production to the same extent as TH supernatants, which suggests that BSF-1 is responsible for this increase in IgE production. Monoclonal antibody to BSF-1 totally inhibits the IgE-enhancing activity of a TH supernatant, lending further support to this conclusion. The effects of BSF-1 on LPS-stimulated B cells are specific for IgE and, as previously reported, IgG1 and IgG3, because the levels of IgM, IgG2a, IgG2b, and IgA in the cultures change relatively little when BSF-1 is added.

A single gene from yeast for both nuclear and cytoplasmic polyadenylate-binding proteins: domain structure and expression.

Nuclear and cytoplasmic poly(A)-binding proteins have been purified from Saccharomyces cerevisiae, and antisera have been used to isolate a gene that encodes them. The gene occurs in a single copy on chromosome 5 and gives rise to a unique, unspliced 2.1 kb transcript. The nuclear protein appears to be derived from the cytoplasmic one by proteolytic cleavage into 53 and 17 kd polypeptides that remain associated during isolation. DNA sequence determination reveals four tandemly arrayed 90 amino acid regions of homology that probably represent poly(A)-binding domains. A 55 residue A-rich region upstream of the initiator methionine codon in the mRNA shows an affinity for poly(A)-binding protein comparable to that of poly(A)180-220, raising the possibility of feedback regulation of translation.