CD40 expression in human thyroid tissue: evidence for involvement of multiple cell types in autoimmune and neoplastic diseases.

CD40, a member of the tumor necrosis factor-alpha (TNF-alpha) receptor family of surface molecules, is expressed by a variety of cell types. It is a crucial activational molecule displayed by lymphocytes and other bone marrow-derived cells and recently has also been found on nonlymphoid cells such as fibroblasts, endothelia, and epithelial cells in culture. While its role in lymphocyte signaling and activation has been examined in great detail, the function of CD40 expression on nonlymphoid cells, especially in vivo, is not yet understood. Most of the studies thus far have been conducted in cell culture. In this article, we report that several cell types resident in thyroid tissue in vivo can display CD40 under pathological conditions. Sections from a total of 46 different cases were examined immunohistochemically and included nodular hyperplasia, chronic lymphocytic thyroiditis, diffuse hyperplasia, follicular neoplasia, papillary carcinoma, and medullary carcinoma. Thyroid epithelial cells, lymphocytes, macrophages, endothelial cells, and spindle-shape fibroblast-like cells were found to stain positively in the context of inflammation. The staining pattern observed in all cell types was entirely membranous. In general, epithelial staining was limited to that adjacent to lymphocytic infiltration except in 5 of 17 cases of neoplasia and in diffuse hyperplasia. Moreover, we were able to detect CD40 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) in human thyroid tissue. These results constitute convincing evidence for expression of CD40 in nonlymphocytic elements of the human thyroid gland. Our findings suggest a potentially important pathway that might be of relevance to the pathogenesis of thyroid diseases. They imply the potential participation of the CD40/CD40 ligand bridge in the cross-talk between resident thyroid cells and bone marrow-derived cells recruited to the thyroid.

The sequence of the tms transcript 2 locus of the A. tumefaciens plasmid pTiA6 and characterization of the mutation in pTiA66 that is responsible for auxin attenuation.

The incorporation of Ti plasmid sequences, the T-DNA, into the genomes of dicotyledenous plants causes the formation of tumors. Here we report the nucleotide sequence of one of the T-DNA "oncogenes", the transcript 2 gene of pTiA6 and we further characterize the 2.7 Kb element that has spontaneously inserted into this gene in plasmid pTiA66. The results indicate that the transcript 2 portion of the T-DNA has an open reading frame that could encode a polypeptide of 49.8 Kd. The open reading frame is surrounded by sequences that typically have roles in eucaryotic gene expression. Nucleotide sequence and Southern blot analysis also indicates that the 2.7 Kb insert in the transcript 2 gene of pTiA66 is located within the coding sequence of the gene and suggests that the element is an insertion sequence. We designate this element, IS66.

Neoplastic progression in crown gall in tobacco without elevated auxin levels.

We have isolated two stable variants from a crown-gall teratoma tissue of tobacco (Nicotiana tabacum L.) transformed by Agrobacterium tumefaciens strain A66, a mutant of the virulent A6 strain containing an insertion sequence in the tumor-inducing (Ti) plasmid at the locus coding for auxin biosynthesis. Normally tobacco cells transformed by strain A66 spontaneously form shoots in culture and will not grow on hormone-free medium unless shoots develop. The variant tissue lines, isolated from the teratoma tissue after prolonged culture in the dark, grew as friable and unorganized tissues on hormone-free growth medium. Growth of the variants was more sensitive to auxin feeding than growth of the parental teratoma line, and the auxin dose-response curves of the variant lines were similar to those obtained with A6-transformed tobacco cells. Southern blot analysis of DNA from the parental teratoma line and one of the variants showed no differences in copy number or organization of the oncogenic DNA sequence (T-DNA) transferred from the bacterium, indicating that the variant phenotype did not result from reversion of the A66 mutation. Radio-immunoassay analysis showed similar levels of indole-3-acetic acid (IAA) in the variants and parental teratoma line (3-50 and 38-42 pmol·(gFW)(-1), respectively), whereas an A6-transformed cell line contained much higher IAA levels (150-1200 pmol·(g FW)(-1)). Low levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid in the variants and the parental teratoma line (<5 nmol·(g FW)(-1)) as compared with that found in the A6-transformed line (>100 nmol· (g FW)(-1)) provided additional, indirect evidence for low auxin levels in the variant lines. These results indicate that crown-gall teratoma tissues of tobacco may switch to the unorganized, auxin-sensitive phenotype without an increase in auxin content.

Genomic sequence of the murine guanylin gene.

Guanylin, a 15-amino-acid peptide, is an endogenous ligand of the intestinal receptor guanylate cyclase-C. After binding to this receptor, guanylin increases the intracellular concentration of cyclic GMP and induces chloride secretion. We have isolated a genomic clone containing the entire murine guanylin gene. The guanylin gene is composed of three exons that span 1700 bp. The first 133 nucleotides of upstream promoter sequence lack the canonical TATA, CAAT, and SP1 elements. Guanylin transcription is nearly exclusively limited to the intestine, and the presence of guanylin mRNA is greatest in the distal colon and ileum. Therefore, characterization of the guanylin promoter is likely to provide another paradigm for intestine-specific gene regulation.

Variation in hormone autonomy and regenerative potential of cells transformed by strain A66 of Agrobacterium tumefaciens.

Mutant Agrobacterium tumefaciens strain A66 is shown to differ from its wild-type progenitor (strain A6) by a spontaneous 2.7 kb DNA insert into the T-DNA region of its Ti plasmid. Tobacco stems transformed by A66 exhibit an attenuated response characterized by slow growth and shoot proliferation. Clonal analysis demonstrates that this response is due to an alteration in the growth and regenerative potential of transformed cells, rather than to variation in the frequency of fully autonomous cells within the primary tumor. Cloned A66 transformed tobacco cells exhibit an auxin requirement for growth that can be overcome by shoot proliferation. Other host species, however, may complement the A66 mutation yielding fully auxin-independent tumors when transformed by this bacterium.

Hepatocyte nuclear factor-1alpha regulates transcription of the guanylin gene.

To study the molecular mechanisms controlling guanylin expression, we have cloned the mouse guanylin gene, including 2.7 kb of upstream sequence. We show that the first 133 base pairs (bp) of the upstream guanylin promoter are sufficient to drive near maximal (6-fold over basal) luciferase reporter gene expression in Caco-2 intestinal cells; at least 300 bp of upstream promoter are required for reporter gene expression in HT-29 intestinal cell lines. Using electromobility shift assays, we demonstrate that nuclear proteins bind to the hepatocyte nuclear factor-1 (HNF-1) consensus sequence in the guanylin promoter. The HNF-1 consensus sequence, located in the immediate 5' flanking region, is required for transcriptional activation of the guanylin gene in both intestinal cell lines. Mutagenesis of the HNF-1 consensus sequence abolishes transcriptional activation of guanylin promoter-luciferase reporter gene constructs. Cotransfection of these constructs with HNF-1alpha augments transcriptional initiation of the reporter gene. In contrast, HNF-1beta has no significant effect on transcription of the reporter gene. These experiments demonstrate that HNF-1alpha is an important regulatory element in the transcriptional activation of guanylin.

Structure and synthesis of histopine, a histidine derivative produced by crown gall tumors.

Histopine, an unusual amino acid derivative of histidine isolated from crown gall tumors of sunflowers (Helianthus annus) inoculated with Agrobacterium tumefaciens strain B6, was previously assigned the gross structure N-(1-carboxyethyl) histidine (2). A diastereomeric mixture containing histopine (2a and 2b) was readily prepared by reductive alkylation of (S)-histidine (1) with pyruvic acid and sodium cyanoborohydride. The individual diastereomers were prepared by reaction of (S)-histidine with (R)- and (S)-2-bromopropionic acid. (R)-N-(1-Carboxyethyl)-(S)-histidine (2a) supports the growth of A. tumefaciens whereas (S)-N-(1-carboxyethyl)-(S)-histidine (2b) is inactive. Therefore, we assign structure 2a to histopine.

Computer programs for the assembly of DNA sequences.

A collection of user-interactive computer programs is described which aid in the assembly of DNA sequences. This is achieved by searching for the positions of overlapping common nucleotide sequences within the blocks of sequence obtained as primary data. Such overlapping segments are then melded into one continuous string of nucleotides. Strategies for determining the accuracy of the sequence being analyzed and reducing the error rate resulting from the manual manipulation of sequence data are discussed. Sequences mapping from 97.3 to 100% of the Ad2 virus genome were used to demonstrate the performance of these programs.

A specific endonuclease from Bacillus caldolyticus.

The purification and characterization of a new restriction endonuclease, BclI from the extreme thermophile Bacillus caldolyticus is reported. This enzyme recognizes the sequence : formula: (see text) and cleaves at the positions indicated by the arrows.

Octopine and nopaline metabolism in Agrobacterium tumefaciens and crown gall tumor cells: role of plasmid genes.

Crown gall tumors produced octopine or nopaline or neither compound, depending on the bacterial strain that incited the tumor. The genes specifying production of octopine or nopaline by the tumor were transferred to recipient bacterial strains when the large plasmid associated with virulence was transferred by either conjugation or deoxyribonucleic acid-mediated transformation. Our results, which confirm the work of others (Bomhoff et al., 1976; Goldman et al., 1968; Petit et al., 1970), indicate that, in general, the strains that utilize octopine induce tumors that synthesize octopine, and those that utilize nopaline induce tumors that synthesize nopaline. However, there were several notable exceptions. One class utilized both octopine and nopaline, but the tumors induced by these strains produced only nopaline. Another class utilized nopaline, but their tumors synthesized neither nopaline nor octopine. Mutants were isolated from a number of either octopine- or nopaline-utilizing strains that no longer could utilize the relevant guanido amino acid. These strains, which were mutant in the gene specifying octopine or nopaline oxidase, still retained the permease for these amino acids as well as virulence. Tumors induced by these mutants still synthesized approximately the same levels of octopine and nopaline as tumors induced by their parents. These results suggest that the plasmid gene that determines production of octopine or nopaline by the tumor is distinct from the plasmid gene that determines their catabolism by the bacteria.

Prostaglandin-endoperoxide H synthase-2 expression in human thyroid epithelium. Evidence for constitutive expression in vivo and in cultured KAT-50 cells.

Prostaglandin-endoperoxide H synthase (PGHS) (EC 1.14.99.1) expression was examined in human thyroid tissue and in KAT-50, a well differentiated human thyroid epithelial cell line. PGHS-1 is found constitutively expressed in most healthy tissues, whereas PGHS-2 is highly inducible and currently thought to be expressed, with few exceptions, only in diseased tissues. Surprisingly, PGHS-2 mRNA and protein were easily detected in normal thyroid tissue. KAT-50 cells express high levels of constitutive PGHS-2 mRNA and protein under basal culture conditions. Compounds usually associated with PGHS-2 induction, including interleukin-1beta (IL-1beta), phorbol 12-myristate 13-acetate, and serum transiently down-regulated PGHS-2 expression. Human PGHS-2 promoter constructs (-1840/+123 and -831/+123) fused to a luciferase reporter and transfected into untreated KAT-50 cells exhibited substantial activity. NS-398, a highly selective inhibitor of PGHS-2 could inhibit substantial basal prostaglandin E2 production. Exogenous IL-1 receptor antagonist or IL-1alpha neutralizing antibodies could attenuate constitutive PGHS-2 expression in KAT-50 cells, suggesting that endogenous IL-1alpha synthesis was driving PGHS-2 expression. Our findings suggest that normal thyroid epithelium expresses high constitutive levels of PGHS-2 in situ and in vitro and this enzyme is active in the generation of prostaglandin E2. Thus, unprovoked PGHS-2 expression might be considerably more widespread in healthy tissues than is currently believed.

Cultured human fibroblasts express constitutive IL-16 mRNA: cytokine induction of active IL-16 protein synthesis through a caspase-3-dependent mechanism.

Human fibroblasts can express numerous regulatory molecules that influence immune function. IL-16, a ligand for CD4, is a chemoattractant molecule expressed by lymphocytes, eosinophils, mast cells, and lung epithelium. It appears that the sole target for IL-16 is the CD4-bearing cell. Here we demonstrate that fibroblasts from several tissues can express IL-16 mRNA and protein as well as IL-16-dependent chemoattractant activity. The transcript is expressed abundantly under basal culture conditions as a 2.5-kb band on Northern analysis, similar to that observed in lymphocytes. IL-16 protein and activity are undetectable in fibroblast cultures under these same control conditions. However, when treated with proinflammatory cytokines such as IL-1beta, they express very high levels of IL-16 protein and chemoattractant activity, a substantial component of which can be blocked with IL-16-neutralizing Abs. The amount of IL-16 protein released into the medium is 3- to 4-fold greater, on a per cell basis, than that observed in lymphocytes. The induction of IL-16 protein by IL-1beta can be attenuated with specific inhibition of caspase-3, which could be detected in IL-1beta-treated fibroblasts. IL-1beta also induces RANTES mRNA, protein, and activity, and most of the chemoattractant activity released from fibroblasts not derived from IL-16 can be attributed to RANTES. Human fibroblasts appear to be an important source of IL-16 and through expression of this molecule may have key roles in the recruitment of CD4+ cells to sites of inflammation. IL-16 expression and the mechanism involved in its regulation appear to be cell type specific.

Cytokine-mediated PGE2 expression in human colonic fibroblasts.

We investigated prostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84, HT-29, and LS 174T) and the effect of PGE2 on fibroblast morphology. Cytokine-stimulated PGE2 production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) protein and mRNA expression were evaluated. Basal PGE2 levels were low in all cell types (0.15-6.47 ng/mg protein). Treatment for 24 h with interleukin-1beta (IL-1beta; 10 ng/ml) or tumor necrosis factor-alpha (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions of PGE2 synthesis in CCD-18Co cultures and similar results in primary fibroblast cultures; maximal inductions with IL-1beta in colonic epithelial cell lines were from zero to fivefold. Treatment of CCD-18Co fibroblasts with IL-1beta caused maximal 21- and 53-fold increases, respectively, in PGHS-2 protein and mRNA levels without altering PGHS-1 expression. PGE2 (0.1 micromol/l) elicited a dramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and target for colonic prostanoids in vivo.

Leukoregulin induction of prostaglandin-endoperoxide H synthase-2 in human orbital fibroblasts. An in vitro model for connective tissue inflammation.

Several proinflammatory cytokines can increase prostaglandin E2 (PGE2) synthesis in a variety of cell types, constituting an important component of the inflammatory response. We demonstrate here that leukoregulin, a 50-kDa product of activated T lymphocytes, dramatically increases PGE2 synthesis in cultured human orbital fibroblasts. This up-regulation is mediated through an induction of prostaglandin-endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Steady-state levels of PGHS-2 mRNA are increased within 1.5 h of leukoregulin addition and are near maximal by 6 h, when they are 50-fold or higher above basal levels. The increase in PGHS-2 mRNA levels is partially blocked by cycloheximide, suggesting de novo synthesis of an intermediate protein may be required for a maximal leukoregulin response. Nuclear run-on studies indicate PGHS-2 gene transcription is up-regulated by leukoregulin 2-fold after 2 and 6 h. PGHS-2 protein, as assessed by Western blotting and two-dimensional protein gel analysis, is increased dramatically in orbital fibroblasts. This lymphokine-dependent expression of PGHS-2 is blocked by dexamethasone, and the increase in PGE2 and cAMP levels following leukoregulin treatment is also blocked by indomethacin and by SC 58125, a newly developed PGHS-2-selective cyclooxygenase inhibitor. The dramatic increase in cAMP levels causes marked alteration in orbital fibroblast morphology. PGHS-2 expression in dermal fibroblasts is also increased by leukoregulin; however, the response is considerably less robust, and these cells do not undergo a change in morphology. Both orbital and dermal fibroblasts express high levels of PGHS-1 mRNA and protein, the other abundant form of cyclooxygenase. In contrast to its effects on PGHS-2 expression, leukoregulin fails to alter PGHS-1 levels in either orbital or dermal fibroblasts, suggesting that PGHS-1 is not involved in cytokine-dependent prostanoid production in human fibroblasts. The increased PGHS-2 expression elicited by leukoregulin in orbital fibroblasts may be a consequence of both transcriptional and post-transcriptional effects. These observations help clarify the pathogenic mechanism relevant to the intense inflammation associated with Graves' ophthalmopathy. Lymphocytes trafficked to orbital tissues have a putative role, through the cytokines they release, in the activation of fibroblasts in this autoimmune disease.

Nucleotide sequences from the adenovirus-2 genome.

The sequence of 15,441 nucleotides from the adenovirus-2 genome has been determined and includes the regions between coordinates 0-32% and 89-100%. These regions contain the early (E) transcription units E1A, E1B, E2B, and E4, the genes for polypeptides IVa2 and IX, the COOH terminus of fiber polypeptide, as well as the two virus-associated RNAs and the leader sequences for the major late mRNAs. Analysis of tryptic peptides from the terminal protein and its precursor (Smart, J. E., and Stillman, B. W. (1982) J. Biol. Chem. 257, 13499-13506) has allowed the gene for the precursor terminal protein to be positioned between coordinates 28.0 and 23.5 on the 1-strand. A minimum Mr = 74,500 is predicted. A second, longer open reading frame is also found on the 1-strand between coordinates 22.9 and 14.2 and predicts a polypeptide of at least Mr = 120,000. Many open reading frames longer than 10,000 exist within this sequence although less than half of them can be assigned to previously characterized polypeptides. As with other viral genomes, the available coding information is highly compressed. Intergenic distances are very short and examples are found of genes which overlap either on the same strand or the complementary strand.