Efficacy and tolerability of 16% subcutaneous immunoglobulin compared with 20% subcutaneous immunoglobulin in primary antibody deficiency.

Multiple subcutaneous immunoglobulin (SCIG) products are available to treat primary antibody deficiency (PAD). The efficacy and tolerability of 16% SCIG (Vivaglobin(®) ) was compared with 20% SCIG (Hizentra(®) ) in PAD subjects. The study was a prospective, single-centre, open-label study of PAD subjects transitioning Vivaglobin to equivalent Hizentra doses, rounded to the nearest vial size. Comparisons included immunoglobulin (Ig)G levels; tetanus, varicella and Streptococcus pneumoniae titres; adverse events (AEs), annual infection rate and quality of life during 8 weeks of Vivaglobin and 24 weeks of Hizentra. Thirty-two subjects (aged 2-75 years) participated. Rounding to the nearest Hizentra vial size resulted in a 12·8% (± 2·9%) increase in SCIG dose. Median immunoglobulin (Ig)G level following 8 weeks of Vivaglobin was similar to 24 weeks of Hizentra (1050 versus 1035 mg/dl, respectively; P = 0·77). Both products had similar protective titres to tetanus, varicella and serotypes of S. pneumoniae, which were variable but well above protective levels. After 12 weeks of Hizentra, subjects reported fewer local site reactions compared with Vivaglobin. Switching products resulted in increased systemic AEs in some subjects but, overall, not significantly higher than during Vivaglobin treatment. Average infusion time decreased from 104·7 min (3·3 sites) with Vivaglobin to 70·7 min (2·2 sites) with Hizentra (P = 0·0005). Acute serious bacterial infections were similar. Treatment satisfaction was superior with Hizentra. Hizentra and Vivaglobin have similar pharmacokinetics and efficacy. Although transition to a different SCIG product initially increased AEs, Hizentra is well tolerated and can be infused more rapidly and with fewer sites compared to Vivaglobin.

Intensive educational course in allergy and immunology.

A one-day intensive educational course on allergy and immunology theory and diagnostic procedure significantly increased the competency of allergy and immunology fellows-in-training.

Formation of circular amplifications in Saccharomyces cerevisiae by a breakage-fusion-bridge mechanism.

Primary gene amplification, the mutation from one gene copy per genome to two or more copies per genome, is a major mechanism of oncogene overexpression in human cancers. Analysis of the structures of amplifications can provide important evidence about the mechanism of amplification formation. We report here the analysis of the structures of four independent spontaneous circular amplifications of ADH4:CUP1 in the yeast Saccharomyces cerevisiae. The structures of all four amplifications are consistent with their formation by a breakage-fusion-bridge (BFB) mechanism. All four of these amplifications include a centromere as predicted by the BFB model. All four of the amplifications have a novel joint located between the amplified DNA and the telomere, which results in a dicentric chromosome, and is adjacent to all the copies of the amplified DNA as predicted by the BFB model. In addition we demonstrated that two of the amplifications contain most of chromosome VII in an unrearranged form in a 1:1 ratio with the normal copy of chromosome VII, again consistent with the predictions of the BFB model. Finally, all four amplifications are circular, one stable endpoint for molecules after breakage- fusion-bridge.

Intramolecular interaction of yeast TFIIB in transcription control.

The general transcription factor TFIIB is a key component in the eukaryotic RNA polymerase II (RNAPII) transcriptional machinery. We have previously shown that a yeast TFIIB mutant (called YR1m4) with four amino acid residues in a species-specific region changed to corresponding human residues affects the expression of genes activated by different activators in vivo. We report here that YR1m4 can interact with several affected activators in vitro. In addition, YR1m4 and other mutants with amino acid alterations within the same region can interact with TATA-binding protein (TBP) and RNAPII normally. However, YR1m4 is defective in supporting activator-independent transcription in assays con-ducted both in vitro and in vivo. We further demonstrate that the interaction between the C-terminal core domain and the N-terminal region is weakened in YR1m4 and other related TFIIB mutants. These results suggest that the intramolecular interaction property of yeast TFIIB plays an important role in transcription regulation in cells.

Mutational studies of yeast transcription factor IIB in vivo reveal a functional surface important for gene activation.

Recent experiments in yeast (Saccharomyces cerevisiae) cells have identified a species-specific region of yeast transcription factor IIB (TFIIB) located at residues 144-157. According to the human TFIIB structure, this region is part of a solvent-exposed helix in the first repeat of the carboxyl-terminal core domain. In this report, we systematically analyze four positions in this region (Lys-147, Cys-149, Lys-151, and Glu-152) that together have been shown previously to be important for yeast TFIIB's function in vivo. Our experiments suggest that all of these four positions, and in particular positions 151, 149, and 152, are critical for yeast TFIIB's ability to support cell growth. In addition, we describe an intragenic suppressor screening experiment to identify mutations that reverse, or partially reverse, the temperature-sensitive phenotype of a yeast TFIIB derivative bearing amino acid changes at these four positions to human residues. The suppressor mutations reveal changes at positions 115, 117, and 182 that are located outside the species-specific region of yeast TFIIB, suggesting an extended surface available to interact with other proteins. Finally, we demonstrate that the suppressor mutations restore gene activation in vivo, further supporting the idea that one important function of yeast TFIIB in living cells is to mediate gene activation.

Identifying a species-specific region of yeast TF11B in vivo.

The general transcription factor IIB (TFIIB) is required for RNA polymerase II transcription in eukaryotes. It provides a physical link between the TATA-binding protein (TBP) and the RNA polymerase and is a component previously suggested to respond to transcriptional activators in vitro. In this report, we compare the yeast (Saccharomyces cerevisiae) and human forms of the protein in yeast cells to study their functional differences. We demonstrate that human TFIIB fails to functionally replace yeast TFIIB in yeast cells. By analyzing various human-yeast hybrid TFIIB molecules, we show that a 14-amino-acid region at the amino terminus of the first repeat of yeast TFIIB plays an important role in determining species specificity in vivo. In addition, we identify four amino acids in this region that are critical for an amphipathic helix unique to yeast TFIIB. By site-directed mutagenesis analyses we demonstrate that these four amino acids are important for yeast TFIIB's activity in vivo. Finally, we show that mutations in the species-specific region of yeast TFIIB can differentially affect the expression of genes activated by different activators in vivo. These results provide strong evidence suggesting that yeast TFIIB is involved in the process of transcriptional activation in living cells.

B-ATF: a novel human bZIP protein that associates with members of the AP-1 transcription factor family.

A new member of the ATF/CREB family of transcription factors, called B-ATF, has been isolated from a cDNA library prepared from Epstein-Barr virus stimulated human B cells. B-ATF is a 125 amino acid nuclear protein possessing a basic leucine zipper domain that is most similar to the basic leucine zipper of ATF-3. Northern blot analysis of polyadenylated mRNA isolated from a variety of human tissues and established cell lines indicates that the 1.0 kilobase B-ATF mRNA is expressed differentially, with the strongest hybridization detected in lung and in Raji Burkitt's lymphoma. Efficient homodimerization of the B-ATF protein cannot be detected using the yeast two hybrid system or using in vitro binding assays with glutathione-s-transferase-B-ATF and maltose binding protein-B-ATF fusion proteins produced in E. coli. However, a yeast two hybrid library screen has identified the human oncoprotein JunB as a specific binding partner for B-ATF. Glutathione-s-transferase-B-ATF heterodimerizes efficiently with in vitro translated JunB, c-Jun, and JunD, but only weakly associates with c-Fos. In addition, electrophoretic mobility shift assays demonstrate that a B-ATF/c-Jun protein complex can interact with DNA containing a consensus binding site for AP-1, suggesting that B-ATF functions as a tissue-specific modulator of the AP-1 transcription complex in human cells.

Phenotypic identification of amplifications of the ADH4 and CUP1 genes of Saccharomyces cerevisiae.

Primary gene amplification, i.e., mutation from one gene copy to multiple gene copies per genome, is important in genomic evolution, as a means of producing anti-cancer drug resistance, and is associated with the progression of tumor malignancy. Primary amplification has not been studied in normal eukaryotic cells because amplifications are extremely rare in these cells. A system has been developed to phenotypically identify co-amplifications of the ADH4 and CUP1 genes of Saccharomyces cerevisiae and 21 independent spontaneous amplifications have been isolated.

Sensitivity of a disposable end-tidal carbon dioxide detector.

A small disposable carbon dioxide detector that can be used to provide evidence of correct endotracheal tube placement is now commercially available (FEF). The device contains an indicator that changes color when exposed to carbon dioxide. This study measured the lowest concentration of carbon dioxide causing a perceivable color change in the device. Ten volunteers were blinded to the concentrations of carbon dioxide in an airway circuit/lung model, and the minimal concentration of carbon dioxide that caused a perceivable color change was recorded. The mean minimum concentration required for detection of a color change was 0.54% (4.1 mm Hg) and ranged from 0.25 to 0.60% (1.9 to 4.6 mm Hg). We conclude that this device should produce a detectable color change even in patients with low end-tidal carbon dioxide, as might be observed during cardiopulmonary resuscitation.