Development and validation of the Axillary Sweating Daily Diary: a patient-reported outcome measure to assess axillary sweating severity.

BACKGROUND: Hyperhidrosis is estimated to affect ~ 4.8% of the US population, and most patients experience a negative psychological impact. Here, we describe development and psychometric evaluation of a patient-reported outcome (PRO) measure to assess severity of axillary hyperhidrosis in clinical trials that meets current U.S. regulatory standards to support product approvals. METHODS: Three rounds of hybrid concept-elicitation/cognitive-debriefing qualitative interviews were conducted in adults with clinician-diagnosed primary axillary hyperhidrosis, followed by similar interviews in children/adolescents. The draft measure included diary items for presence, severity, impact and bothersomeness (basis of the Axillary Sweating Daily Diary [ASDD]), exploratory weekly impact items, and a single-item Patient Global Impression of Change (PGIC). Phase 2 (adults only) and phase 3 (adults and children ≥9 years) clinical trial data were utilized to evaluate measurement properties of the resulting draft measure: floor/ceiling effects, nonresponse bias, test-retest reliability, construct validity, and responsiveness were assessed. The primary concept of interest was axillary sweating severity (ASDD Item 2); however, additional supportive concepts were explored to allow for development of a comprehensive hyperhidrosis measure. RESULTS: Twenty-nine patient interviews were conducted (N = 21 adult and N = 8 children/adolescents), resulting in the ASDD (4 items, patients ≥16y) and child-specific ASDD-C (2 items ≥9y to <16y), as well as 6 Weekly Impact items and the PGIC (patients ≥16y). No floor/ceiling effects or response biases were identified. Consistency between hypothesized and observed correlation patterns between ASDD/ASDD-C items and other efficacy measures supported construct validity. Intraclass correlation coefficients supported test-retest reliability (0.91-0.93; Item 2). Large effect sizes (- 2.2 to - 2.4) demonstrated that the ASDD/ASDD-C Item 2 could detect changes in hyperhidrosis severity, supporting the measure's responsiveness. Patients perceiving a moderate improvement in symptoms on the PGIC experienced an average 3.8-point improvement on ASDD axillary sweating severity (Item 2); thus, a 4-point responder threshold was defined as a clinically meaningful change. CONCLUSIONS: Qualitative and quantitative evidence support the reliability and validity of the ASDD/ASDD-C and its use in the clinical evaluation of axillary hyperhidrosis treatments. Further evaluation of this measure in future research studies is warranted to demonstrate consistent performance across different axillary hyperhidrosis populations and in different study contexts.

Mouse hematopoietic stem cells and the interaction of c-kit receptor and steel factor.

Hematopoietic stem cells (HSCs) are distinguished from other hematopoietic progenitors in bone marrow by their unique ability to undergo multilineage differentiation and self-renewal. Two mouse mutations, dominant spotting (W) and steel (Sl), have pleiotropic effects on hematopoiesis, gametogenesis, and melanoblast development. These two mutations have been shown to be intrinsic (W) and microenvironmental (Sl) defects. Recently, molecular studies revealed that the W and Sl loci encode the c-kit receptor and steel factor (SLF), respectively. The c-kit receptor is expressed on HSCs and hematopoietic progenitors, while SLF is produced by stromal cells. SLF acts on hematopoietic progenitors synergistically with other growth factors. Here we review the effect of these mutations on mouse hematopoiesis, and show that SLF acts on HSCs and other myeloerythroid progenitors, but that it, in our hands, does not play a critical role in HSC generation or self-renewal. Rather, SLF is the most potent co-mitogen (with IL-1, IL-3, IL-6, G-CSF, GM-CSF, or M-CSF) found that acts on these cells, but the effect of such treatments is the rather specific and massive expansion of myeloerythropoiesis, not lymphopoiesis, and perhaps at the expense of HSC self-renewal.

Structural and functional analysis of the murine adenosine deaminase gene.

We describe the structural and functional analysis of cosmid clones that span the entire murine adenosine deaminase gene. Functional analysis indicated that these clones are capable of encoding murine adenosine deaminase activity when introduced into human cell lines. Structural analysis revealed that the gene consists of 12 exons distributed over approximately 25 kb. The exact size of each exon and the sequence of each exon/intron junction were determined. The results show that the 1056-nucleotide open reading frame for adenosine deaminase extends from exon 1 to exon 11, and that exon 12 contains untranslated sequences only. During the course of these investigations, we discovered that a gene encoding an abundant 1.3-kb polyadenylated transcript overlaps the 3' end of the murine adenosine deaminase gene and is transcribed from the opposite strand.

GC-rich murine adenosine deaminase gene promoter supports diverse tissue-specific gene expression.

The murine adenosine deaminase (ADA) gene has a GC-rich promoter that is structurally typical of many mammalian "housekeeping" gene promoters. The ability of the ADA gene promoter to support diverse tissue-specific gene expression was investigated. Endogenous ADA gene expression in different mouse tissues was found to vary over a greater than 3000-fold range in a highly complex pattern. This range of expression was also observed in cultured human cell lines derived from different tissues. The ADA levels in all tissues and cell lines examined correlated closely with steady-state ADA mRNA levels. Several of the mouse tissues examined also showed stage-specific variation during postnatal development. In order to determine whether tissue-specific ADA expression was controlled by cis-acting sequences upstream of the coding region, constructs containing a reporter gene regulated by the ADA gene's 5' flanking sequences were used to generate transgenic mice. All transgene-expressing mice obtained showed diverse reporter gene expression in the tissues analyzed. Our results demonstrate that both in vivo and in the context of an integrated transgene this GC-rich promoter can support highly diverse gene expression in all tissues of the animal.

Developmental expression of adenosine deaminase in the upper alimentary tract of mice.

The distribution and localization of adenosine deaminase (ADA) was studied during postnatal development of the alimentary tract in mice. There was detectable enzyme activity in all organs examined, but a range of more than 10,000 fold in the relative levels of specific activity was observed among adult tissues. A comprehensive survey of multiple adult tissues revealed that the highest levels of ADA occur in the upper alimentary tract (tongue, esophagus, forestomach, proximal small intestine). Immunohistochemical analysis revealed that ADA was predominantly localized to the epithelial lining of the alimentary mucosa: the keratinized squamous epithelium that lines the forestomach, esophagus, and surface of the tongue; and the simple columnar epithelium of the proximal small intestine (duodenum, proximal jejunum). Biochemical analysis revealed that ADA was one of the most abundant proteins of these mucosal tissue layers, accounting for 5%-20% of the total soluble protein. Tissue-specific differences in ADA activity correlated both with levels of immunoreactive protein and RNA abundance. The level of ADA activity in the upper alimentary tissues was subject to pronounced developmental control, being low at birth and achieving very high levels within the first few weeks of postnatal life. The appearance in development of ADA-immunoreactivity coincided with maturation of the mucosal epithelium. These results suggest that ADA is subject to strong cell-specific developmental regulation during functional differentiation of certain foregut derivatives in mice.

Molecular cloning of the murine adenosine deaminase gene from a genetically enriched source: identification and characterization of the promoter region.

A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.

Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells.

We demonstrate that an adenosine deaminase (ADA) cDNA gene can function as a dominant selectable and amplifiable marker for gene transfer experiments in mammalian cells. Cells that incorporate the gene can be selected by growth in the presence of low concentrations of the ADA inhibitor 2'-deoxycoformycin with cytotoxic concentrations of adenosine or its analogue 9-beta-D-xylofuranosyl adenine. The DNA copy number of the transfected ADA minigene in the isolated transformants of Chinese hamster ovary cells can be amplified greater than 100-fold by growth in ADA selection media and increasing concentrations of 2'-deoxycoformycin. This selection scheme may allow for the introduction and subsequent amplification of heterologous DNA in a variety of mammalian cells.

Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line.

Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein.

Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli.

Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.

Amplification and molecular cloning of murine adenosine deaminase gene sequences.

A previously isolated mouse Cl-1D derived cell line (B-1/25) overproduces adenosine deaminase (EC 3.5.4.4) by 3200-fold. The present studies were undertaken to determine the molecular basis of this phenomenon. Rabbit reticulocyte lysate and Xenopus oocyte translation studies indicated that the B-1/25 cells also overproduced adenosine deaminase mRNA. Total poly(A+) RNA derived from B-1/25 was used to construct a cDNA library. After prehybridization with excess parental Cl-1D RNA to selectively prehybridize nonamplified sequences, 32P-labeled cDNA probe synthesized from B-1/25 total poly(A+) RNA was used to identify recombinant colonies containing amplified mRNA sequences. Positive clones containing adenosine deaminase gene sequences were identified by blot hybridization analysis and hybridization-selected translation in both rabbit reticulocyte lysate and Xenopus oocyte translation systems. Adenosine deaminase cDNA clones hybridized with three poly(A+) RNA species of 1.5, 1.7, and 5.2 kilobases in length, all of which were overproduced in the B-1/25 cell line. Dot blot hybridization analysis using an adenosine deaminase cDNA clone showed that the elevated adenosine deaminase level in the B-1/25 cells was fully accounted for by an increase in adenosine deaminase gene copy number. The adenosine deaminase cDNA probes and the cell lines with amplified adenosine deaminase genes should prove extremely useful in studying the structure and regulation of the adenosine deaminase gene.

Selective overproduction of adenosine deaminase in cultured mouse cells.

The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the protein and nucleic acid probes required to study adenosine deaminase gene structure and regulation. Since adenosine deaminase expression is not required for growth of cells in culture, the first step necessary to isolate adenosine deaminase gene amplification mutants was to devise selective conditions in which adenosine deaminase activity was required for survival. This was accomplished by developing a new selection system, termed 11AAU, which selected simultaneously for adenosine deaminase and adenosine kinase. The 11AAU selection medium consists of alanosine (0.05 mM) to block de novo AMP biosynthesis, adenosine (1.1 mM) to provide a salvage route for AMP biosynthesis via the adenosine kinase reaction, and uridine (1.0 mM) to alleviate the block in UMP biosynthesis caused by adenosine at the concentration employed. Because adenosine is highly cytotoxic at 1.1 mM, adenosine deaminase expression is required to detoxify excess adenosine by converting it to inosine. We used 11AAU selection in conjunction with stepwise selection for increasing resistance to deoxycoformycin, an adenosine deaminase inhibitor, to obtain highly drug-resistant cells with a 6000-fold increase in adenosine deaminase activity. Adenosine deaminase accounted for approximately 50% of the soluble protein in highly drug-resistant lines and was indistinguishable from that in the parent as judged by isoelectric focusing, electrophoretic mobility on starch gels, and by deoxycoformycin binding studies. Increased adenosine deaminase was also correlated with the presence of numerous double-minutes, cytogenetic structures indicating the presence of amplified DNA. Growth in the absence of selection was accompanied with the loss of double-minutes and a ten-fold decline in adenosine deaminase levels. Based on the stepwise selection protocol employed, the instability of the phenotype, and the presence of double-minutes, we believe that the increased adenosine deaminase is most likely the result of amplification of adenosine deaminase genes.