An economic evaluation of screening 60- to 70-year-old adults for hearing loss.

BACKGROUND: Hearing loss is common among older adults and has consequences for sufferers, families and society, but there is substantial unmet need for intervention. Screening could expedite intervention and improve outcomes. METHODS: We use Markov models to estimate the incremental cost-effectiveness ratio (ICER) of potential screening programmes compared with current provision (GP-referral), from a health service perspective. Alternative options are investigated through scenario analysis. One-way and probabilistic sensitivity analyses are undertaken. RESULTS: All modelled screens are cost-effective and reduce unmet need for hearing aids. The most cost-effective option identified is a one-stage audiometric screen for bilateral hearing loss ≥30 dB hearing level (HL) at age 60, repeated at ages 65 and 70. This option has an ICER of £1461 compared to GP-referral and would mean an additional 15 437 adults benefiting from hearing intervention per 100 000 population aged 60. The cost-effectiveness acceptability curve shows that screening is more cost-effective than GP-referral provided a Quality Adjusted Life Year is valued at £2000 or more. CONCLUSIONS: Adult hearing screening would provide a cost-effective way to improve quality of life for older adults. We recommend piloting an audiometric screen offered to all adults age 60, 65 and 70 years to identify bilateral hearing loss of at least 30 dB HL.

Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer: evidence of a reversible acid-denatured molten globule.

The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation. A reversible acid-denatured molten globule state was observed from circular dichroism and fluorescence spectroscopy data. The molten globule state was characterized by a loss of tertiary structure with associated retention of secondary structure near pH 3. Once returned to pH 7, the acid-denatured state refolded over the course of 7 days resulting in approximately 90% recovery of the native structure. The molten globule state was characterized by a broadening of structural features in the second-derivative spectra of Fourier transform infrared spectroscopy. A component band at 1650 cm(-1) was shown to be alpha-helix and originate from amide carbonyl vibrations of the isoleucine zipper. Differential scanning calorimetry measurements characterized the pH-sensitive molten globule state at pH 3.3 as one lacking a well-defined unfolding transition with an accompanying baseline shift at 58 degrees C (a consequence of increased heat capacity). The tendency to form molten globules during acid denaturation stress permits an opportunity to study the process of partial protein unfolding with implications concerning stability. Although reversible molten globules can be formed, it is important to recognize the unusual nature since the molten globule state is formed exclusively within the beta-sheet receptor-binding region.

Effects of insulin and LongR(3) on serum-free Chinese hamster ovary cell cultures expressing two recombinant proteins.

Insulin is the most commonly used growth factor for sustaining cell growth and viability in serum-free Chinese hamster ovary (CHO) cell cultures. In the present study insulin and IGF-1 analogue (LongR(3)) were compared for their ability to support growth, viability, and production of two serum-free CHO cell lines expressing recombinant protein. The first cell line, VA12, expresses protein B, and the second cell line, CL23, expresses protein C. Both molecules are recombinant cytokine receptors. VA12 will grow in serum-free media lacking growth factor, while CL23 requires either insulin or LongR(3) for cell growth. Both cell lines, however, require a growth factor for optimal performance under production conditions. In this study, LongR(3) was better able to sustain the viability of both cell lines under production conditions than insulin. These data indicate that while insulin and LongR(3) can both serve as growth and viability factors for CHO cells, LongR(3) is the preferred growth factor for cell lines VA12 and CL23.

Note: evaluation of selective media for the enumeration of Bifidobacterium sp. in milk.

Pure cultures of three species of bifidobacteria (Bifidobacterium longum, Bif. adolescentis and Bif. bifidum), Lactobacillus acidophilus and a mixed culture of Lact. delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus were each enumerated on two differential media and six selective media for the enumeration of bifidobacteria. The appearance of the colonies on the differential media was as expected but when mixed cultures were present, it proved extremely difficult to distinguish one species from another. Of the selective media, AMC, RMS, NPNL and BL-OG performed well in that they gave good recoveries of bifidobacteria and were inhibitory to the growth of Lact. delbrueckii subsp. bulgaricus, Strep. salivarius subsp. thermophilus and Lact. acidophilus. However, of these four media, AMC was most convenient as it is based on a commercially available medium, whereas the others must be made up from individual constituents. The AMC agar is thus a good choice for the routine enumeration of bifidobacteria from mixed cultures.

Incorporation of an isoleucine zipper motif enhances the biological activity of soluble CD40L (CD154).

Recent progress in the understanding of immune function indicates that the interaction of CD40L with its receptor, CD40, plays a pivotal role in both humoral immunity and cell-mediated defense against pathogens. Functional studies of this interaction on both dendritic cells and malignant cells have demonstrated that CD40L also plays an important role in immune surveillance and anti-tumor immunity. CD40L exists in nature predominantly as a membrane-anchored molecule. To develop CD40L as a potential therapeutic, it is important to optimize soluble forms of this molecule that could be used in a clinical setting. Several reports have shown that soluble forms of CD40L, like CD40 antibodies, are biologically active. In the present report we demonstrate that the incorporation of an isoleucine zipper trimerization motif significantly enhances the biological activity of soluble CD40L.

The production and quality of tomato concentrates.

The standards and specifications for the quality and composition of tomato concentrates are reviewed. The main quality parameters of tomato puree and paste are color, consistency and flavor. Overall, there is an absence of standardization of methods and instruments to define quality. While color can now be measured objectively, there are currently no standard color requirements for tomato concentrates. Rheological measurements on both tomato juice and concentrates are reviewed; the power law finds wide applicability, although other rheological characteristics, particularly time dependency, have received far less attention and there has been little effort to relate rheological understanding to the commonly used empirical tests such as consistency measurements. The volatiles responsible for flavor and odor have been identified to the point where the natural odor of tomato paste can be imitated. Attempts to develop objective methods as a substitute for sensory assessment are reviewed.

Improved bicistronic mammalian expression vectors using expression augmenting sequence element (EASE).

We have recently reported on the isolation of a 5.7 kb segment of Chinese hamster ovary cell genomic DNA, Expression Augmenting Sequence Element (EASE), which when used in bicistronic expression vectors allows the development of stable Chinese hamster ovary cell pools in a five to seven week time period that express high levels of recombinant protein (6-25 mug 10-6 cells/day depending on the protein). In the present study, we have mapped the activity of the EASE to a 2.1 kb region using colony forming assays and developed bicistronic expression vectors with the smaller EASE or control lambda DNA. The recovery of pools expressing the hematopoietic growth factor, FLT3 Ligand, in methotrexate-containing media took 1 to 4 weeks less when using EASE expression vectors compared with control vectors. The cell pools developed with the EASE and control vectors had similar final protein expression levels. Southern blot analysis suggested the expression cassette from the EASE containing vectors integrated in tandem arrays arranged in either head to head or head to tail fashion. By contrast, control vectors appeared to integrate with multiple interruptions to the expression vector. Thus, the EASE, within a bicistronic expression vector, appeared to facilitate tandem vector integration and reduce the time required to develop cell pools for protein expression.

Hematologic effects of flt3 ligand in vivo in mice.

We have investigated the effects of in vivo treatment with flt3 ligand (FL) on murine hematopoiesis, including mobilization of progenitors into the peripheral blood (PB). Mice were injected once daily with 10 micrograms recombinant human FL for 15 days. On days 3, 5, 8, 10, 15, and 22, mice were killed and analyzed for the number of leukocytes and colony-forming units (CFU) in bone marrow (BM), spleen, and PB. Splenic and PB cellularity increased with time in FL-treated mice. In the spleen, there was an increase in B cells, myeloid cells, and nucleated erythroid cells; in the PB, there was an increase in lymphocytes, granulocytes, and monocytic cells. The maximal number of CFU in the BM was observed after 3 days of FL treatment, giving 3.7- and 7.3-fold increases in CFU-granulocyte-macrophage (CFU-GM) and CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), respectively, compared with mouse serum albumin (MSA)-treated controls. After 8 days of FL treatment, there was a maximal 123- and 108-fold increase in splenic CFU-GM and CFU-GEMM, respectively. The maximal number CFU-GM and CFU-GEMM were seen in PB on day 10, with 537- and 585-fold increases, respectively. Burst-forming units-erythroid (BFU-E) increased in the same time frame as those of CFU-GM and CFU-GEMM in BM, spleen, and PB, although the magnitude was not as great. Primitive day-13 CFU-spleen (CFU-S) and phenotypically defined stem cells were also mobilized into the PB of FL-treated mice with similar kinetics and magnitude to that of CFU-GM and CFU-GEMM. We conclude from these studies that FL, when administered as a single agent, is a potent mobilizer of hematopoietic progenitors into the PB.

An alternatively spliced Pit-1 isoform altered in its ability to trans-activate.

Although alternative splicing has been shown to give rise to isoforms of a number of transcription factors, such isoforms have not previously been detected for the POU homeodomain protein Pit-1. Screening of a rat pituitary GH3 cell cDNA expression library yielded a clone, termed pCMVPit-1a, encoding a 35.8 kD protein (Pit-1a) containing a 26 amino acid insert in the Pit-1 trans-activation domain. The position of the insert, plus Southern blot analysis, implied that Pit-1a mRNA arises by alternative splicing of the Pit-1 gene transcript. Pit-1a mRNA was detected in GH3 rat pituitary tumor cells at levels about 1/7 that of Pit-1 mRNA. Pit-1a mRNA-specific sequences were also detected in rat and mouse pituitary, and in mouse thyrotropic tumor TtT cells. DNA mobility shift assays showed that Pit-1a binds specifically to Pit-1 binding sites in the proximal prolactin promoter, but produces DNA-protein complexes of markedly different mobilities than Pit-1. In stably transfected CHO cells which accumulated approximately equal levels of either of the two proteins, Pit-1 trans-activated a prolactin promoter-driven CAT construct, while Pit-1a yielded no detectable transactivation, implying a trans-activation ratio for Pit-1a/Pit-1 of less than 0.05. Thus, the insertion of 26 amino acids of similar composition into the activation domain of Pit-1 has at once affected both the mode of binding of this protein and its ability to function as a trans-activator.

Point mutational analysis of the hamster dihydrofolate reductase minimum promoter.

We have shown previously that 48 base pairs (bp) of 5'-flanking sequence are necessary for correct initiation at the major transcriptional start site of the Chinese hamster dihydrofolate reductase (dhfr) gene (Ciudad et al., 1988). As an upstream element, this sequence alone confers 25% of maximum promoter activity. The 5' half of this sequence is particularly well conserved among mammalian species; it contains one Sp1 binding site (GC box) and one CAA element. In the present work, we have analyzed the role of this region by extensive point mutational analysis. Twenty-three dhfr minigene constructs containing 1- or 2-base substitutions in this region of the promoter were tested by measuring their ability to transfect DHFR-deficient Chinese hamster ovary cells to a DHFR+ growth phenotype. Eight mutants, all in or near the GC box, exhibited reduced transfection efficiency. Promoter disfunction in these mutants was confirmed by RNase protection analysis of stable transfectants. Gel retardation experiments showed that mutants affected in the consensus sequence for Sp1 binding were deficient in binding a protein found in nuclear extracts of Chinese hamster ovary cells. Purified human transcription factor Sp1 was also unable to bind a promoter sequence bearing one of these single base substitutions, suggesting that Sp1 itself is involved in dhfr transcription in vivo. We conclude that most single base mutations in the GC box severely cripple or eliminate promoter function by inhibiting binding of transcription factors to this regulatory sequence and that Sp1 is likely to be involved in dhfr transcription in vivo. We also found that the well conserved CAA element is not absolutely necessary for transcription.

Use of a selectable reporter for the isolation of mammalian regulatory genes.

A general method is described for isolating the genes encoding differentiation-specific activators of transcription using genetic selection. Employing regulation of the prolactin encoding gene (PRL) as a model, we have shown that the hamster dihydrofolate reductase-encoding gene (dhfr) is an effective dominant selectable reporter in this methodology. The dhfr coding region was ligated to the rat PRL promoter, and the resultant construct was stably transfected into DHFR- Chinese hamster ovary (CHO) cells, where it had little or no activity. Transfection of these cells with plasmid DNA, containing the coding region of a pituitary-specific transcription factor (Pit-1/GHF-1) in a eukaryotic expression vector, resulted in transfectants in which activation of the chimeric construct, pPRLdhfr, had occurred, enabling these cells to be selected on the basis of their DHFR+ phenotype. Our results suggest that this strategy could be used to isolate unknown genes that regulate a variety of differentiated functions.

Isolation of novel cDNA transformation markers from SV40-transformed human keratinocytes.

Differential screening of a cDNA library was used to isolate probes for mRNAs that are induced in simian virus 40 (SV40)-transformed human keratinocytes. Several of these cDNAs hybrid select mRNAs which encode transformation-induced proteins found in the cytoskeletal component of SV40-transformed keratinocytes. One of these cDNAs was used to study the phenotype of normal and transformed cell lines derived from various tissues. We found that mRNA encoding the novel transformation-induced proteins is expressed in two squamous carcinoma cell lines derived from the oral epithelium, four SV40-transformed keratinocyte cell lines, and two SV40-transformed fibroblasts. Normal or transformed lymphoid cells or cell lines derived from carcinoma of the cervix do not express mRNAs which hybridize to these probes. The results from this study suggest that these probes may be used to detect markers of transformation in certain cell types.