Daily injections of anakinra markedly improved clinical and laboratory manifestations in patients with neonatal-onset multisystem inflammatory disease, with or without CIAS1 mutations. (ClinicalTrials.gov number, NCT00069329 [ClinicalTrials.gov].).

This study represents the largest collection of patients with JLS ever reported. The insidious onset of the disease, the delay in diagnosis, the recognition of mixed subtype and the better definition of the other subtypes should influence our efforts in educating trainees and practitioners and help in developing a comprehensive classification system for this syndrome.

Objective. Neonatal-onset multisystem inflammatory disease (NOMID; also known as chronic infantile neurologic, cutaneous, articular [CINCA] syndrome) is characterized by fever, chronic meningitis, uveitis, sensorineural hearing loss, urticarial skin rash, and a characteristic deforming arthropathy. We investigated whether patients with this disorder have mutations in CIAS1, the gene which causes Muckle-Wells syndrome and familial cold autoinflammatory syndrome, two dominantly inherited disorders with some similarities to NOMID/CINCA syndrome.Methods. Genomic DNA from 13 patients with classic manifestations of NOMID/CINCA syndrome and their available parents was screened for CIAS1 mutations by automated DNA sequencing. Cytokine messenger RNA (mRNA) levels were assessed by real-time polymerase chain reaction on peripheral blood leukocyte mRNA, and serum cytokine levels were assayed by enzyme-linked immunosorbent assay. Protein expression was assessed by Western blotting of lysates from plastic-adherent peripheral blood mononuclear cells.Results. In 6 of the 13 patients, we found 6 heterozygous missense substitutions in CIAS1. Five of the 6 mutations are novel. None of these sequence changes was observed in a panel of >900 chromosomes from healthy controls. Two distinct nucleotide changes in a single codon in unrelated patients resulted in the same amino acid change. In 4 mutation-positive children whose parental DNA was available, no mutation was found in the parental DNA, supporting the conclusion that the mutations arose de novo. Consistent with

Glycoproteins located on the luminal surface of the plasma membrane of tick gut epithelial cells, when used to vaccinate cattle, are capable of stimulating an immune response that protects cattle against subsequent tick infestation. One such tick gut glycoprotein, designated Bm86, has been purified to homogeneity and the amino acid sequences of peptide fragments generated by endoproteinase Lys-C digestion have been determined. We report here the isolation and characterization of a cDNA that encodes Bm86. The nucleotide sequence of the cDNA contains a 1982-base-pair open reading frame and predicts that Bm86 contains 650 amino acids including a 19-amino acid signal sequence and a 23-amino acid hydrophobic region adjacent to the carboxyl terminus. The main feature of the deduced protein sequence is the repeated pattern of 6 cysteine residues, suggesting the presence ofseveral epidermal growth factor-like domains. A fusion protein consisting of 599 amino acids of Bm86 and 651 amino acids of /3-galactosidase was expressed in Escherichia coli as inclusion bodies. Ticks engorging on cattle vaccinated with these inclusion bodies were significantly damaged as a result of the immune response against the cloned antigen.The tick Boophilus microplus is a major ectoparasite of cattle in many parts of the world. A single female cattle tick takes up as much as 1.5 ml of bovine blood, increasing its body weight to =250 mg. It has been estimated that cattle in tropical areas of Australia may become infested with 1000 tick larvae per day, resulting in greatly reduced productivity. In addition, B. microplus is the vector of hematoprotozoal parasites such as Babesia bovis. Chemicals have been used extensively to control ticks and have been partially successful, but this approach suffers from certain drawbacks such as environmental and residue problems, the high incidence of acaricide resistance that has developed in tick populations in the field, the need for frequent administration, and high cost.Recently it was shown that cattle immunized against a Construction and Screening of cDNA Library. RNA was extracted (3) from adult B. microplus (picked from cattle 15 days after infestation), cDNA was synthesized from 4 ,ug of poly(A)+ RNA (4), and cDNA fragments larger than 800 base pairs (bp) were ligated to Agtll (5) to generate a library of 8 x 105 recombinant clones. Oligodeoxynucleotide probes (Table 1) were based on the sequences derived from peptides generated by endoproteinase Lys-C digestion of Bm86 (1).Three nitrocellulose filters were prepared from five 150-mm plates each containing 105 plaques. After prehybridization in 0.6 M sodium pyrophosphate/0.005% heparin (Sigma) at 40°C for 4 hr, hybridization was carried out for 16 hr at 40°C in the same solution with the radioactive oligonucleotide probe. Two of the filters were hybridized with the 63-mer probe while the third was hybridized with a mixture of the 50-, 51-, and 72-mer probes. The filters were washed in 0.3 M NaCl/0.03 M sodium citrate, pH 7.5/0.1% SDS at 45°C a...

NF‐Y binds a CCAAT motif found in many eukaryotic polymerase II‐dependent promoters. In the HLA‐DRA promoter it has been demonstrated that stereo‐specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild‐type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF‐Y binding. Interaction between NF‐Y and X box binding proteins was demonstrated by reciprocal co‐immunoprecipitation in the absence of DNA and co‐affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein‐DNA interactions and suggest that NF‐Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element.