Ig heavy chain variable region gene complex of lupus mice exhibits normal restriction fragment length polymorphism.

B cell hyperactivity, hypergammaglobulinemia, and autoantibody expression, the hallmarks of systemic lupus erythematosus, might be associated with structural abnormalities within the Ig heavy chain variable region (Igh-V) gene complex. The Igh-V loci from several lupus-prone mouse strains, their ancestors, and other nonautoimmune mice were therefore analyzed by restriction fragment length polymorphisms with DNA probes corresponding to seven VH gene families. These seven families comprise the majority of the known polymorphic murine VH gene repertoire, including some involved in autoantibody generation. Our study showed that the Igh-V loci from lupus and haplotype-matched nonlupus mice resulted in essentially identical restriction fragment patterns, a finding which suggests that the Igh-V gene complex does not carry a primary defect responsible for autoimmune disease.

Genetic elements used for a murine lupus anti-DNA autoantibody are closely related to those for antibodies to exogenous antigens.

The mRNAs encoding heavy and light chains of a hybridoma-derived monoclonal IgM kappa anti-DNA autoantibody from lupus-prone MRL/Mp-lpr/lpr mice (Ighj) have been transcribed into cDNA copies and molecularly cloned, and their complete nucleotide sequences have been determined. The mRNA for the heavy chain variable region, including leader peptide and 5' untranslated region, is transcribed from a heavy chain variable region (VH) gene closely related (and possibly allelic) to VH genes of the C57BL/6 (Ighb) nitrophenyl antibody family. The deduced amino acid sequence corresponding to the light chain variable region of this autoantibody shows extensive similarities with non-autoantibody molecules of the V kappa 1 group, suggesting a common variable gene origin. The joining segments, constant regions, and 3' untranslated regions of both the heavy and light chain mRNAs are nearly identical to corresponding sequences of non-autoantibodies from normal mice. Our findings suggest that this anti-DNA autoantibody originated from the same germline repertoire as antibodies to exogenous antigens.

Delineation of a defect in T cell receptor beta genes of NZW mice predisposed to autoimmunity.

In an attempt to determine whether genes involved in T cell antigen recognition are structurally abnormal and thereby promote murine systemic lupus, we analyzed the structural integrity of the D, J, and C region elements of the T cell receptor alpha and beta chain genes in all major lupus strains and several normal strains. Within the limits of restriction fragment length polymorphism analysis, all strains had an identical genomic organization, except the NZW mice, in which a deletion of the C beta 1-D beta 2-J beta 2 elements was found. Sequence analysis of NZW genomic elements containing this deletion placed its probable origin within the first exon of C beta 1, and extending to a complementary region within the first exon of C beta 2. The significance of this abnormality in the pathogenesis of systemic autoimmune disease remains to be determined.

Immunoglobulin kappa light chain variable region gene complex organization and immunoglobulin genes encoding anti-DNA autoantibodies in lupus mice.

We have investigated the genetic origin of autoantibody production in several strains of mice that spontaneously develop a systemic lupus erythematosus-like disease. Restriction fragment length polymorphism analyses of gene loci encoding kappa light chain variable regions (Igk-V) demonstrated, as shown previously for the Ig heavy chain locus, that autoantibody production and disease occur in different Igk-V haplotypes. Moreover, autoimmune mice with known genetic derivation inherited their Igk-V loci essentially unaltered from their nonautoimmune ancestors. New Zealand black lupus mice, with unknown genetic derivation, had a possibly recombinant Igk-V haplotype, composed of V kappa loci that were primarily indistinguishable from those of nonautoimmune strains from either of the two potential donor haplotypes. The heavy and light chain gene segments (variable, diversity, joining) encoding anti-DNA antibodies were diverse and often closely related, or even identical, to those found in antibodies to foreign antigens in normal mice. Only 1 of 11 sequenced variable region genes could not be assigned to existing variable region gene families; however, corresponding germline genes were present in the genome of normal mice as well. These data argue against abnormalities in the genes and mechanisms generating antibody diversity in lupus mice and suggest a remarkable genetic and structural diversity in the generation of anti-DNA binding sites.

Structural characterization of a heterogeneous family of rat brain mRNAs.

A large heterogeneous family of RNAs derived from a single rat gene contains members that differ from each other at one or more of three positions. Their 5' ends are nested and transcription can begin at 22 or more sites covering 265 nucleotides. Many of the 5' ends are detectable only in brain RNAs, and even 5' ends common with other tissues appear with different absolute and relative abundances in brain RNA. The central portions of the RNAs are of two forms, differing only by the presence or absence of 17 nucleotides; these forms are probably produced by alternative splicing. Polyadenylation occurs at either of two sites. This complicated family of 88 RNAs encodes two novel putative proteins that differ at their C termini.

Common and idiosyncratic patterns of cytokine gene expression by Epstein-Barr virus transformed human B cell lines.

Epstein-Barr virus (EBV) transformed human B cells proliferate indefinitely in vitro, and it has been proposed that cytokine-mediated autocrine loops contribute to the maintenance of the lymphoblastoid phenotype. We used a novel multiprobe RNase protection assay to quantify cytokine mRNA species expressed by EBV-transformed lymphoblastoid cell lines (LCL), derived either by the transformation of B cells with B95-8 or wild-type EBV or by the in vitro outgrowth of EBV-associated B cell lymphomas to identify cytokines that are commonly expressed in all LCL and thus more likely to be essential for immortalization of B cells. All 16 LCL expressed high levels of tumor necrosis factor (TNF)alpha, TNFbeta, and transforming growth factor (TGF)beta1 mRNA, while interleukin (IL)-10 transcripts were detected in most LCL but at a lower level. Expression of IL-1alpha, IL-1beta, IL-6, IL-12p35, IL-12p40, IL-13 and IFNgamma mRNA was variable among the LCL tested. Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-4, and IL-5 mRNA were undetectable in all LCL. Furthermore, we found that IL-10, TNFalpha, and TNFbeta mRNA were induced in EBV-negative B cell lines after infection with EBV. These data define common versus idiosyncratic patterns of cytokine expression by LCL and, in the former case, such cytokines as TNFalpha, TNFbeta, and IL-10 emerge as strong candidates that are essential for the autocrine regulation of EBV-immortalized B cells.

Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H(2) synthases by ibuprofen, naproxen, and indomethacin.

A series of nonsteroidal anti-inflammatory drugs (NSAIDs) [S(+)-naproxen, ibuprofen isomers, and indomethacin] were evaluated for their activation of peroxisome proliferator-activated receptor (PPAR) alpha and gamma isoforms in CV-1 cells co-transfected with rat PPAR alpha and gamma, and peroxisome proliferator response element (PPRE)-luciferase reporter gene plasmids, for stimulation of peroxisomal fatty acyl CoA beta-oxidase activity in H4IIEC3 cells, and for comparative inhibition of ovine prostaglandin endoperoxide H synthase (PGHS)-1 and PGHS-2 and arachidonic acid-induced human platelet activation. Each drug produced a concentration-dependent activation of the PPAR isoforms and fatty acid beta-oxidase activity, inhibition of human arachidonic acid-induced platelet aggregation and serotonin secretion, and inhibition of PGHS-1 and PGHS-2 activities. For PPARalpha activation in CV-1 and H4IIEC3 cells, and the stimulation of fatty acyl oxidase activity in H4IIEC3 cells, the rank order of stereoselectivity was S(+)- ibuprofen > R(-)-ibuprofen; S(+)-ibuprofen was more potent than indomethacin and naproxen on these parameters. On PPARgamma, the rank order was S(+)-naproxen > indomethacin > S(+)-ibuprofen > R(-)-ibuprofen. Each drug inhibited PGHS-1 activity and platelet aggregation with the same rank order of indomethacin > S(+)-ibuprofen > S(+)-naproxen > R(-)-ibuprofen. Notably, the S(+)-isomer of ibuprofen was 32-, 41-, and 96-fold more potent than the R(-)-isomer for the inhibition of PGHS-1 activity, human platelet aggregation, and serotonin secretion, respectively. On PGHS-2, the ibuprofen isomers showed no selectivity, and indomethacin, S(+)-ibuprofen, and S(+)-naproxen were 6-, 27-, and 5-fold more potent as inhibitors of PGHS-1 than PGHS-2 activity. These results demonstrate that the mechanisms of action of NSAIDs on these cell systems are different, and we propose that the pharmacological effects of NSAIDs may be related to both their profile of inhibition of PGHS enzymes and the activation of PPARalpha and/or PPARgamma isoforms.

Genomic organization and expression of B and T cell antigen receptor genes in murine lupus.

Studies with genomic DNAs of lupus mice and monoclonal autoantibodies suggest that autoantibody expression does not result from defects in immunoglobulin germline genes nor in mechanisms generating antibody repertoires. Genomic and expression abnormalities of T cell antigen receptor genes have been identified in lupus mice, but their possible contribution to disease manifestations remains to be established.

Advances in analytical techniques for neutron capture therapy: thin layer chromatography matrix and track etch thin layer chromatography methods for boron-10 analysis.

A new track etch autoradiographic technique for quantitating boron-10 containing compounds used for neutron capture therapy is described. Instead of applying solutions of Cs2B12H11SH and its oxidation products directly to solid-state nuclear track detectors, diethylaminoethyl cellulose thin layer chromatography (TLC) plates are utilized as sample matrices. The plates are juxtaposed with Lexan polycarbonate detectors and irradiated in a beam of thermal neutrons. The detectors are then chemically etched, and the resultant tracks counted with an optoelectronic image analyzer. Sensitivity to boron-10 in solution reaches the 1 pg/microliter level, or 1 ppb. In heparinized blood samples, 100 pg boron-10/microliter are detected. This TLC matrix method has the advantage that sample plates can be reanalyzed under different reactor conditions to optimize detector response to the boron-10 carrier material. Track etch/TLC allows quantitation of the purity of boron neutron capture therapy compounds by utilizing the above method with TLC plates developed in solvent systems that resolve Cs2B12H11SH and its oxidative analogs. Detectors irradiated in juxtaposition to the thin layer chromatograms are chemically etched, and the tracks are counted in the sample lane from the origin of the plate to the solvent front. A graphic depiction of the number of tracks per field yields a quantitative analysis of compound purity.

A prototype epithermal neutron beam for boron neutron capture therapy.

An epithermal neutron beam has been designed and tested at the Georgia Institute of Technology's 5-MW Research Reactor. The prototype facility consists of aluminum and sulfur disks in a tangential beam port for fast neutron filtration. A cadmium sheet at the port exit removes the thermal neutrons from the transmitted beam, leaving an intensely epithermal neutron beam spanning five energy decades, each contributing to the flux demanded by boron neutron capture therapy. The thermal neutron flux generated by the incident epithermal neutrons in a polyethylene head phantom peaks at a depth of 3 cm and remains above the incident thermal flux to a 7-cm depth. The beam thus provides the penetration required for treating deep-seated gliomas. Photon contamination in the prototype facility is high, and a number of basic modifications are proposed for reducing it to safer levels.

The tuberous sclerosis 2 gene product can localize to nuclei in a phosphorylation-dependent manner.

The tuberous sclerosis 2 (TSC2) gene has been genetically mapped to a disease characterized by abnormal cell proliferation that results in the production of tumorous lesions in a variety of tissues. The molecular mechanism for TSC2 mediation of tuberous sclerosis is unclear but it appears to be related to its ability to cytoplasmically interact with a second gene, TSC1, mapping to the disease. These proteins are linked to constraints on cell cycle signaling pathways and therefore envisioned to function as tumor suppressor genes. In previous studies we have demonstrated TSC2 associations with steroid receptor family members and modulation of their gene expression capabilities. Here we provide evidence for TSC2 translocation to the nucleus and a possible role for phosphorylation in both TSC2 translocation and TSC2 modulation of steroid receptor-mediated transcription.

Complete cDNA sequence of the fourth component of murine complement.

We have used the method of Okayama and Berg to construct cDNA clones that span the entire length of the fourth component of murine complement (C4) from mouse strain B10.WR. The cDNA sequence spans 5372 nucleotides and encodes a pre-pro-C4 molecule 1738 amino acids in length; it includes 56 and 99 untranslated bases at the 5' and 3' ends, respectively, of the C4 mRNA. The deduced pre-pro-C4 molecule includes a 19 amino acid signal peptide and two highly basic interchain regions that presumably are excised during maturation of the protein. The nucleotide sequence shows 79% identity with the human C4 cDNA sequence over the length of the protein-coding region. A comparison of the B10.WR C4 sequence with those of C4 and sex-limited protein (Slp) from mouse strain FM (i) suggests that the difference in hemolytic activity between the C4 proteins from B10.WR and FM is due to structural changes distant from both the C1 cleavage site and the internal thioester site and (ii) raises the possibility that the C4 gene from B10.WR has undergone genetic exchange with its adjoining Slp genes.

Correlations of autoimmunity with H-2 and T cell receptor beta chain genotypes in (NZB X NZW) F2 mice.

Accelerated autoimmunity as expressed by the classical autoimmune strain mouse (NZB x NZW)F1 is thought to be the result of major histocompatibility complex (MHC)-associated NZW genes acting on a genetic predisposition for autoimmunity as expressed by the NZB mouse. To evaluate more accurately both H-2 and T cell receptor (TcR) beta chain involvement in F1 disease, we studied the segregation of NZB (H-2d, TcRB) and NZW (H-2z, TcRW) haplotypes of these genetic elements and the development of autoimmunity in (NZB x NZW)F2 generation mice. F2 mice with the H-2d/z genotype lived shorter average life-spans and expressed elevated levels of antibodies to gp70, ssDNA and dsDNA, while those with the TcRW/W genotype (homozgous for the NZW TcR deletion) expressed elevated levels of autoantibodies but had relatively long life-spans. On the other hand, mice with the TcRB/B genotype (homozygous for the NZB TcR) produced consistently low levels of autoantibodies but died at an early age. The most severely affected F2 population were the mice carrying both the TcRB/B and H-2d/z alleles. These mice died on an average within the first 5 months of life, but produced the lowest levels of antibodies to gp70, single-stranded DNA and double-stranded DNA. These data confirm the contribution of NZW H-2-linked genes to accelerated autoimmunity in the F1 hybrid and, furthermore, define NZB TcR-linked components as primary developers of this phenomenon. They also suggest a limited, if any, contribution of both the NZW TcR deletion and traditional autoantibodies to F1 accelerated autoimmunity.

Catabolites of cholesterol synthesis pathways and forskolin as activators of the farnesoid X-activated nuclear receptor.

The nuclear receptors are a family of transcriptional mediators that, upon activation, bind DNA and regulate gene transcription. Among these receptors, the farnesoid X-activated receptor (FXR) has recently been identified as one activated by bile acids and farnesol. To investigate the potential of other sterols to activate FXR, as well as to examine relevant relationships among identified activators of FXR, the current study used a mammalian cell transcription assay to quantify and compare activation potential. In addition to the classical bile acids deoxycholate (DCA) and chenodeoxycholate (CDCA), FXR was shown to be transcriptionally active in the presence of the androgen catabolites 5alpha-androstan-3alpha-ol-17-one (androsterone) and 5beta-androstan-3alpha-ol-17-one (etiocholanolone), as well as the sterol bronchodilatory drug forskolin. Conversely, cholesterol and several other key precursors to the androgens and bile acids were either not active or only slightly active. Furthermore, it was observed that the bile acid ursodeoxycholate (UDCA) could inhibit DCA and CDCA activation of FXR in a manner parallel to its ability to antagonize DCA and CDCA induction of apoptosis. By far, the most efficacious activator of FXR was forskolin. Interestingly, although it is classically viewed as an initiator of the adenylate cyclase/protein kinase A (PKA) pathway, PKA inhibition did not inhibit forskolin's activation of FXR nor was cyclic AMP (cAMP) able to stimulate FXR-mediated transcription. These data would suggest that forskolin acts as a ligand for FXR rather than as a secondary activator of FXR and could have important implications with respect to its potential toxicity and pharmacological use.

Induction of IL-1 secretion from human monocytes by Fc region subfragments of human IgG1.

Peripheral blood-derived human monocytes and the murine P388D1-monocytes-like cell line are induced to secrete IL-1 when stimulated with Fc region but not F(ab) region subfragments obtained from the cleavage of human IgG1 with papain or pepsin. The portion of the Fc region of IgG1 responsible for stimulation of IL-1 secretion appears to be located within the C gamma 3 domain of the molecule. This hypothesis is supported by the observation that the biologically active pepsin-derived pFc' subfragment is located within the C gamma 3 domain and the long-term papain digests containing predominately Fc' are also active. In contrast, short term papain digests containing mostly intact Fc fragments were found to be unable to induce IL-1 secretion.

Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors.

Peroxisomes are cytoplasmic organelles which are important in mammals in modulation of lipid homeostasis, including the metabolism of long-chain fatty acids and conversion of cholesterol to bile salts (reviewed in refs 1 and 2). Amphipathic carboxylates such as clofibric acid have been used in man as hypolipidaemic agents and in rodents they stimulate the proliferation of peroxisomes. These agents, termed peroxisome proliferators, and all-trans retinoic acid activate genes involved in peroxisomal-mediated beta-oxidation of fatty acids. Here we show that the receptor activated by peroxisome proliferators and the retinoid X receptor-alpha (ref. 6) form a heterodimer that activates acyl-CoA oxidase gene expression in response to either clofibric acid or the retinoid X receptor-alpha ligand, 9-cis retinoic acid, an all-trans retinoic acid metabolite; simultaneous exposure to both activators results in a synergistic induction of gene expression. These data demonstrate the coupling of the peroxisome proliferator and retinoid signalling pathways and provide evidence for a physiological role for 9-cis retinoic acid in modulating lipid metabolism.

Mechanisms of peroxisome proliferation by perfluorooctanoic acid and endogenous fatty acids.

1. The effects of endogenous fatty acids and perfluorooctanoic acid (PFOA) and its analogs on peroxisomal acyl CoA oxidase (ACO) and microsomal laurate hydroxylase (LH) activities were evaluated in primary cultures of rat hepatocytes and activation of peroxisome proliferator-activated receptor alpha (PPARalpha) in CV-1 cells. The rank order for the stimulation of ACO activity in hepatocytes for selected compounds was PFOA >> octanoic acid>octanedioic acid, perfluorooctanol (inactive). Increases in ACO activity by PFOA, like those of ciprofibrate, were associated with a marked increase in peroxisome number and cytosolic occupancy volume. Maximal effects of ciprofibrate and PFOA on the stimulation of ACO activity were not additive, suggesting that these two compounds share a common pathway of peroxisome proliferation. 2. Saturated monocarboxylic acids of C4 to C18 chain length were inactive, and, among dicarboxylic acids, only small elevations (40-45%) in ACO activity were observed with the long-chain C12 and C16 dioic acids. Of the C18 fatty acids tested, only oleic and linoleic acids, at 1 mM, produced a two- to three-fold elevation in ACO and LH activities. In comparison with endogenous fatty acids, PFOA was more potent and exhibited a different time course and greater magnitude of stimulation of ACO and LH activities in cultured hepatocytes. 3. Addition of mitochondrial beta-oxidation inhibitors (3-mercaptopropionic and 2-bromooctanoic acids) did not alter ACO activity in the presence of octanoic acid or octanedioic acid; nor did they modify the stimulation of ACO activity by PFOA. The carnitine palmitoyltransferase I inhibitor 2-bromopalmitic acid produced a 2.5-fold increase in ACO stimulatory activity and reduced both ciprofibrate- and PFOA-mediated stimulations of ACO activity. 4. Cycloheximide treatment reduced PFOA- and ciprofibrate-induced ACO activities; however, the response to oleic acid was not blocked and increased slightly. 5. In rat and human PPARalpha transactivation assays, the rank order of activation was ciprofibrate > PFOA > oleic acid > or = octanoic acid > octanedioic acid or perfluorooctanol (inactive). PFOA, ciprofibrate and oleic acid were activators of rPPARalpha at concentrations that correlated favorably with the changes in ACO activity in cell culture. Octanoic acid did not increase ACO activity and was a weak activator of PPARalpha. 6. Our findings suggest that fatty acids such as oleic acid (endogenous fatty acids) and PFOA (a stable fatty acid) act through more than one pathway to increase ACO activity in rat hepatocytes. We conclude that the potent effects of PFOA are primarily mediated by a mechanism that includes the activation of liver PPARalpha.