Therapeutic antibodies elicited by immunization against TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in the pathogenesis of several chronic inflammatory diseases. Monoclonal antibodies against TNF-alpha are currently used for the treatment of rheumatoid arthritis and Crohn's disease. This report describes a simple and effective method for active immunization against self TNF-alpha. This vaccination approach leads to a T-cell-dependent polyclonal and sustainable anti-TNF-alpha autoantibody response that declines upon discontinuation of booster injections. The autoantibodies are elicited by injecting modified recombinant TNF-alpha molecules containing foreign immunodominant T-helper epitopes. In mice immunized with such molecules, the symptoms of experimental cachexia and type II collagen-induced arthritis are ameliorated. These results suggest that vaccination against TNF-alpha may be a useful approach for the treatment of rheumatoid arthritis and other chronic inflammatory diseases.     

Interstrain crosses enhance excision of Tc1 transposable elements in Caenorhabditis elegans

Summary We have studied spontaneous and UV mutagenesis of the glyU gene in Escherichia coli trpA461 (GAG) strains carrying the pIP11 plasmid, in which the dnaQ gene encoding the 3–5 exonuclease subunit (epsilon) of DNA polymerase III is fused to the tac(trp-lac) promoter. We have used a pair of M13glyU phage in which the gene encoding the glycyl-tRNA is cloned in opposite orientations, consequently the phage present either GGG or CCC anticodon triplets for mutagenesis. The presence of IPTG, the inducer of the tac-dnaQ fusion, results in about 100-fold decrease in frequency of spontaneous Su⁺ (GAG) mutations arising in the CCC phage. The enhanced expression of tac-dnaQ reduces 10-fold the frequency of UV-induced Su⁺ (GAG) mutations in the CCC phage and nearly completely prevents generation by UV of Su⁺ (GAG) mutations in the GGG phage, in which UV-induced pyrimidine photoproducts can be formed only in the vicinity of the target triplet. These results suggest that both locally and regionally targeted mutagenesis is affected by overproduction of the epsilon subunit. By delayed photoreversal mutagenesis we have shown that UV-induced chromosomal mutagenesis of the umuC36 trpA461 strain harboring pIP11 is completely abolished in the presence of IPTG. This result seems to indicate that the misinocorporation step of DNA translesion synthesis is affected by excess of the epsilon subunit. Finally, we have introduced the pIP13 plasmid carrying the dnaQ gene into the recA1207 strain, which is deficient in the recombinase activity of RecA but constitutive in the protease activity. We demonstrate that the transformant shows much higher UV sensitivity than recA1207 carrying the vector plasmid pBR325, indicating that translesion synthesis significantly contributes to DNA repair capacity of cells deficient in recombination.     

Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans

We have sequenced 11 representative mutations of the unc-54 myosin heavy chain gene of Caenorhabditis elegans that affect the synthesis, assembly or enzymatic activity of the encoded myosin heavy chain. Six of the sequenced unc-54 mutations cause premature termination of protein synthesis. Four mutations (e1092, e1115, e1213, e1328) were ochre mutations, one mutation (e903) was a frameshift, which caused premature termination at a nearby UGA terminator, and one mutation (e190) was a deletion that altered the reading frame and caused termination at an ochre codon. Two mutations (e675 and s291) were inphase deletions, which resulted in a shortened myosin rod segment. These aberrant myosins fail to assemble into normal thick filaments. The sequence alterations of the missense mutations (e1152, s74, s95) indicated amino acid residues that are critical for myosin function. The mutation e1152 causes the production of a myosin heavy chain that fails to assemble into thick filaments. It had two adjacent amino acid substitutions at the extreme amino terminus of the rod, indicating a role for subfragment-2 in thick filament assembly. Mutants homozygous for s74 or s95 are very slow-moving, although they make myosin heavy chains that assemble normally. The encoded amino acid substitutions of s95 and s74 are in the 23 X 10(3) Mr and 50 X 10(3) Mr domains of the myosin head, flanking the ATP binding site. The sequenced mutations are distributed throughout the gene in the order predicted from genetic fine-structure mapping experiments. Seven of eight point mutations isolated following ethylmethane sulphonate mutagenesis were G X C to A X T transitions. A single X-ray-induced allele proved to be a deletion of two adjacent thymidine residues. The three deletion mutations were found in a region of the myosin rod with numerous direct and inverted nucleotide sequence repeats, but their origin cannot be accounted for by homologous recombination. Instead, a comparison of the deletion junctions suggests that the deletions arose by a site-specific mechanism.     

Paramyosin of Caenorhabditis elegans

Paramyosin has been isolated from the nematode, Caenorhabditis elegans. Its identity has been established by a variety of criteria, including purification, molecular weight, immunological cross reactivity with known paramyosin and formation of characteristic paracrystals. The presence of paramyosin in both pharyngeal and body-wall musculature was shown by a technique that allows analysis by sodium dodecyl sulphate gels of the protein in a single worm. The possibility of defining the role of paramyosin in the structure and function of the invertebrate muscle through the isolation of mutants in this protein is discussed.     

A mutant affecting the heavy chain of myosin in Caenorhabditis elegans

A set of non-complementing, closely linked, ethyl methanesulphonate-induced mutations in Caenorhabditis elegans specifically affects the structure and function of body-wall muscle cells but not the pharyngeal musculature. One of these mutations, e675, is semidominant and results in the production of a new protein of about 203,000 molecular weight in addition to normal myosin at about 210,000 M_r. The abnormal polypeptide chain is structurally very similar to normal myosin heavy chain when maps of iodinated peptides are compared.The E675 mutant shows a clear relation between defective movement, disruption of the body-wall muscle structure, and the molecular defect in the myosin heavy chains. The altered chain is synthesized in heterozygotes, suggesting that the e675 mutation is either in a structural gene for the heavy chain or in a cis acting control element. The hypothesis that there are two classes of myosin heavy chain within the same cells is discussed.     

Mutants with altered muscle structure in Caenorhabditis elegans

In the small nematode, Caenorhabditis elegans, mutants with a disorganized myofilament lattice structure have been identified by polarized light and electron microscopy. Genetic analysis places the mutations in 12 complementation groups which are distributed over the six linkage groups of C. elegans. The phenotypes are described for the mutants from the 9 complementation groups not previously reported on in detail. Most are paralyzed, but some exhibit essentially normal movement; mutants of two loci show changes only in later larval stages and adulthood. Morphological studies show that, in general, all the members of a complementation group show similar changes in muscle structure and that these changes are distinctive for that group. In mutants of several genes, disorganization of the myofilament lattice is general with no one component of the lattice more obviously altered than others. In mutants of other genes specific structures are prominently altered. In one of the instances where thick filaments appear to be abnormal, double mutants combining mutations in this gene (unc-82 IV) with mutations in the gene for a myosin heavy chain (MacLeod et al., 1977a,b) or paramyosin (Waterston et al., 1977) were used to show that the unc-82 gene product probably affects thick filament assembly through its actions on paramyosin. Some possible implications of the morphological features of the mutants as well as the conclusions derived from the genetic studies are discussed.     

Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans.

Caenorhabditis elegans has 12 tRNA(UGGTrp) genes as defined by Southern analysis. In order to evaluate the function of the individual members of this multigene family, we sought to recover amber (UAG)-suppressing mutations from reversion experiments with animals carrying amber mutations in a nervous system-affecting gene (unc-13) or a sex-determining gene (tra-3). Revertants were analyzed by Southern blot, exploiting the fact that the CCA to CTA change at the anticodon creates a new XbaI site. Five different members of the tRNATrp gene family were identified as suppressors: sup-7 X, sup-5 III, sup-24 IV, sup-28 X, and sup-29 IV. All five suppressor genes were sequenced and found to encode identical tRNA(UAGTrp) molecules with a single base change (CCA to CTA) at the anticodon compared with their wild-type counterparts. The flanking sequences had only limited homology. The relative expression of these five genes was determined by measuring the efficiencies of suppressers against amber mutations in genes affecting the nervous system, hypodermis, muscle, and sex determination. The results of these cross-suppression tests showed that the five members of the tRNA(Trp) gene family were differentially regulated in a tissue- or development stage-specific manner.     

Screening children from overseas for infections: is it justified?

OBJECTIVES--To investigate current practice of screening children from abroad for infections after coming to the United Kingdom, and to make recommendations for future practice. DESIGN--A review of literature and a questionnaire sent to all health authorities and boards in the United Kingdom. SETTING--All health authorities and boards in the United Kingdom. SUBJECTS--167 Health authorities or boards that completed questionnaires (response rate 80%), 59 of which used a screening programme. MAIN OUTCOME MEASURE--Response to questionnaire on policies for screening children for infections on their return from overseas. RESULTS--12 Of the 59 authorities screened all children and one screened only those from the West Indian subcontinent. 13 Authorities excluded children from school while awaiting results; 58 screened for tuberculosis and four for diphtheria. CONCLUSIONS--There is a wide variation in screening policies around the country with no national consensus. Screening for diphtheria, typhoid, and salmonellosis is hard to justify and is probably not effective. Screening for tuberculosis, however, is supported by many authorities, is widely practised, and probably is effective. There is a strong case for rationalisation of screening.