Development of an electronic emergency department-based geo-information injury surveillance system in Hong Kong.

OBJECTIVES: To describe the experience in the development of an electronic emergency department (ED)-based injury surveillance (IS) system in Hong Kong using data-mining and geo-spatial information technology (IT) for a Safe Community setup. METHODS: This paper described the phased development of an emergency department-based IS system based on World Health Organization (WHO) injury surveillance Guideline to support safety promotion and injury prevention in a Safe Community in Hong Kong starting 2002. RESULTS: The initial ED data-based only collected data on name, sex, age, address, eight general categories of injury types (traffic, domestic, common assault, indecent assault, batter, industrial, self-harm and sports) and disposal from ED. Phase 1--manual data collection on International Classification of External Causes of Injury pre-event data; Phase 2--manual form was converted to electronic format using web-based data mining technology with built in data quality monitoring mechanism; Phase 3--integration of injury surveillance-data with in-patient hospital information; and Phase 4--geo-spatial information and body mapping were introduced to geo-code exact place of injury in an electronic map and site of injury on body map. CONCLUSION: It was feasible to develop a geo-spatial IS system at busy ED to collect valuable information for safety promotion and injury prevention at Safe Community setting. The keys for successful development and implementation involves engagement of all stakeholders at design and implementation of the system with injury prevention as ultimate goal, detail workflow planning at front end, support from the management, building on exiting system and appropriate utilisation of modern technology.

Survey on medical records and EHR in Asia-Pacific region: languages, purposes, IDs and regulations.

OBJECTIVES: To clarify health record background information in the Asia-Pacific region, for planning and evaluation of medical information systems. METHODS: The survey was carried out in the summer of 2009. Of the 14 APAMI (Asia-Pacific Association for Medical Informatics) delegates 12 responded which were Australia, China, Hong Kong, India, Indonesia, Japan, Korea, New Zealand, the Philippines, Singapore, Thailand, and Taiwan. RESULTS: English is used for records and education in Australia, Hong Kong, India, New Zealand, the Philippines, Singapore and Taiwan. Most of the countries/regions are British Commonwealth. Nine out of 12 delegates responded that the second purpose of medical records was for the billing of medical services. Seven out of nine responders to this question answered that the second purpose of EHR (Electronic Health Records) was healthcare cost cutting. In Singapore, a versatile resident ID is used which can be applied to a variety of uses. Seven other regions have resident IDs which are used for a varying range of purposes. Regarding healthcare ID, resident ID is simply used as healthcare ID in Hong Kong, Singapore and Thailand. In most cases, disclosure of medical data with patient's name identified is allowed only for the purpose of disease control within a legal framework and for disclosure to the patient and referred doctors. Secondary use of medical information with the patient's identification anonymized is usually allowed in particular cases for specific purposes. CONCLUSION: This survey on the health record background information has yielded the above mentioned results. This information contributes to the planning and evaluation of medical information systems in the Asia-Pacific region.

Arrhythmia and sudden death associated with elevated cardiac chloride channel activity.

The identification and analysis of several cationic ion channels and their associated genes have greatly improved our understanding of the molecular and cellular mechanisms of cardiac arrhythmia. Our objective in this study was to examine the involvement of anionic ion channels in cardiac arrhythmia. We used a transgenic mouse model to overexpress the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-regulated chloride channel. We used RNase protection and in situ hybridization assays to determine the level of CFTR expression, and radiotelemetry and in vivo electrophysiological study in combination with pharmacological intervention to analyse the cardiac function. Cardiac CFTR overexpression leads to stress-related sudden death in this model. In vivo intracardiac electrophysiological studies performed in anaesthetized mice showed no significant differences in baseline conduction parameters including atrial-His bundle (AH) or His bundle-ventricular (HV) conduction intervals, atrioventricular (AV) Wenckebach or 2:1 AV block cycle length and AV nodal functional refractory period. However, following isoproterenol administration, there was marked slowing of conduction parameters, including high-grade AV block in transgenic mice, with non-sustained ventricular tachycardia easily inducible using programmed stimulation or burst pacing. Our sudden death mouse model can be a valuable tool for investigation of the role of chloride channels in arrhythmogenesis and, potentially, for future evaluation of novel anti-arrhythmic therapeutic strategies and pharmacological agents.

The -844C/T polymorphism in the Fas ligand promoter associates with Taiwanese SLE.

FasL expression is critical in T-cell activation-induced apoptosis, which is involved in lupus pathogenesis. This study identified two SNPs in the FasL promoter regions from -1145 to -45 by genomic DNA sequencing. The -844C/T polymorphism was previously described by its location in and affect on the CCAAT/enhancer-binding protein beta (C/EBPB beta)-binding site and the other (-1094A/C, a novel polymorphism) was located at the NF-kappaB transcription-binding site. FasL gene promoter polymorphisms were genotyped in 260 systemic lupus erythematosus (SLE) patients and 280 healthy controls using MassArray matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The distribution of FasL promoter -844C/C genotype, predominant in Taiwanese, was skewed in Taiwanese SLE patients (odds ratio: 1.53; P-value=0.014). FasL promoter -844C/T polymorphism genotype distributions of Taiwanese, African Americans, and Caucasians differed. Moreover, no particular clinical association of -844C/T and -1094A/C polymorphisms with SLE was found in patients in Taiwan. This study confirmed that -844C/C genotype is associated with lupus susceptibility. The -1094A/C polymorphism is not significantly associated with lupus disease susceptibility, albeit the role of NF-kappaB pathway in FasL promoter activation remains unclear. Fas/FasL pathway may contribute to SLE polygenic disease entity.

Fcgamma receptor IIa, IIIa, and IIIb polymorphisms of systemic lupus erythematosus in Taiwan.

OBJECTIVE: To determine whether the distribution of Fcgamma receptor IIa, IIIa, and IIIb polymorphisms confers a risk factor for disease susceptibility, and correlates with the clinical characteristics and serological parameters of patients with SLE in Taiwan. METHODS: Genotyping of Fcgamma receptors IIa H/R131, IIIa F/V158, and IIIb NA1/NA2 was performed in 302 patients with SLE and 311 healthy blood donor controls. The distribution of Fcgamma receptor IIa, IIIa, and IIIb genotypes in patients and controls was analysed. Frequencies of three Fcgamma receptor polymorphisms were also compared between lupus patients with and without different clinical manifestations and autoantibodies. RESULTS: No significant skewing in the distribution of Fcgamma RIIa H/R131, Fcgamma RIIIa F/V158, and Fcgamma RIIIb NA1/NA2 was found between patients and controls in Taiwan. The following clinical associations were found: Fcgamma RIIIb NA1/NA1 protected against neuropsychiatric lupus (p = 0.028) but conferred susceptibility to discoid rash (p<0.005); increased Fcgamma RIIIa V/V158 was associated with infections (p = 0.039); increased Fcgamma RIIa H/H131 was associated with earlier onset of lupus (p = 0.01). CONCLUSION: Fcgamma receptor IIa, IIIa, and IIIb polymorphisms may be responsible for the development of distinct manifestations of lupus patients in Taiwan, but there is no significantly skewed distribution in the susceptibility to lupus as a whole.

Prostate-specific targeting using PSA promoter-based lentiviral vectors.

The prostate-specific antigen (PSA) promoter is known to be highly tissue specific. Although its tissue specificity has been confirmed, its efficiency of gene transcription is significantly lower compared to known nonspecific viral promoters. These lower levels of promoter activity therefore pose a problem when developing an efficacious gene vector for prostate cancer gene therapy. Thus, selecting an appropriate therapeutic gene and vector system to carry the gene driven by the PSA promoter (PSAP) is important. In the studies described here, a human immunodeficiency virus (HIV)-1-based lentiviral vector carrying either the enhanced green fluorescent protein (EGFP) reporter or the diphtheria toxin A (DTA) gene was constructed. The results demonstrate that the PSA promoter in a lentiviral vector drives genes in prostate cells with satisfactory efficacy and specificity. The tissue-specific expression of the DTA protein efficiently eradicates LNCaP prostate cells in culture. We also infected prostate cancer cells and control cells carried by nude mice with the EGFP lentiviral vector. Significant numbers of EGFP-positive LNCaP cells were detected in all the mice bearing these tumors, but no EGFP-positive control cells were detected in any other mouse tissue. The high levels of expression in prostate cells, compared with the low levels of background expression in other cells, show that the PSAP-lentiviral vector could be a potential useful tool for gene therapy of metastatic prostate cancer.

Regulated expression of the human CFTR gene in epithelial cells.

We developed an epithelium-specific, inducible cystic fibrosis transmembrane conductance regulator (CFTR) expression system. In this system we used a human cytokeratin 18 expression cassette to drive epithelium-specific expression of the reverse tetracycline transactivator (rtTA), which turns on CFTR expression from a Tet-inducible promoter in the presence of doxycycline. CFTR expression was monitored by reverse-transcription polymerase chain reaction, immunostaining, and Western blotting. We confirmed that protein expression was dose-dependent in double stable transfected cell lines, with no detectable protein in the absence of doxycycline. However, low levels of CFTR mRNA could be detected in the uninduced state. When clones capable of inducing high levels of CFTR expression were analyzed, we observed a decrease in cell proliferation, consistent with reports in other cell lines (NIH3T3 and BTS). We generated transgenic mice expressing rtTA from the K18 expression cassette and demonstrated that the system retained its tissue specificity for lacZ reporter expression in vivo. When mice were induced with doxycycline, high levels of expression were found in the trachea, upper bronchi, and submucosal glands. Therefore, this inducible system can improve our understanding of the role of CFTR in the lung and should help in the design of safe and effective CF therapies.

Human immunodeficiency virus Env-independent infection of human CD4(-) cells.

CD4(-) epithelial cells covering mucosal surfaces serve as the primary barrier to prevent human immunodeficiency virus type 1 (HIV-1) infection. We used HIV-1 vectors carrying the enhanced green fluorescent protein gene as a reporter gene to demonstrate that HIV-1 can infect some CD4(-) human epithelial cell lines with low but significant efficiencies. Importantly, HIV-1 infection of these cell lines is independent of HIV-1 envelope proteins. The Env-independent infection of CD4(-) cells by HIV-1 suggests an alternative pathway for HIV-1 transmission. Even on virions bearing Env, a neutralizing antibody directed against gp120 is incapable of neutralizing the infection of these cells, thus raising potential implications for HIV-1 vaccine development.

Targeting transgene expression to airway epithelia and submucosal glands, prominent sites of human CFTR expression.

Targeting therapeutic gene expression to disease-affected tissues is an essential component of effective and safe gene therapy. After birth, CFTR gene expression in human lungs is localized predominantly in the epithelial cells lining the upper airways, especially in the ducts and serous tubules of the submucosal glands. We have developed a K18 expression cassette, based on the DNA control elements of the human cytokeratin 18 gene. Temporal and spatial analyses of transgenic mice demonstrated that this expression cassette targets transgene expression to almost all cell types in which CFTR is expressed. Airway epithelium expression started as early as 11.5 days of gestational age and continued into the adulthood of the transgenic mice. In these adult mice, the pattern of the reporter expression strikingly matched that of the human cytokeratin 18 and human CFTR genes. The transgene expression was epithelium-specific and undetectable in connective tissue, muscle, bone, cartilage, blood, and endothelial cells. Significantly, high levels of expression were detected in tracheal submucosal glands. Together, these results suggest that our K18 expression cassette has a high potential for clinical application in gene therapy for patients with cystic fibrosis.

A human epithelium-specific vector optimized in rat pneumocytes for lung gene therapy.

Gene therapy vectors based on mammalian promoters offer the potential for increased cell specificity and may be less susceptible than viral promoters to transcription attenuation by host cytokines. The human cytokeratin 18 (K18) gene is naturally expressed in the lung epithelia, a target site for gene therapies to treat certain genetic pediatric lung diseases. Our original vector based on the promoter and 5' control elements of K18 offered excellent epithelial cell specificity but relatively low expression levels compared with viral promoters. In the present study, we found that adding a stronger SV40 poly(A) signal boosted primary rat lung epithelial cell expression but greatly reduced cell specificity. Addition of a 3' portion of the K18 gene to our vector as a 3' untranslated region (UTR) improved epithelial cell-specific expression by reducing expression in lung fibroblasts. The effect of the 3' UTR was not related to gross differences in cell-specific splicing. A deletion variant of this UTR further increased lung epithelial cell expression while retaining some cell specificity. These data illustrate the possibilities for using 3' UTR to regulate cell-specific transgene expression. Our improved K18 vector should prove useful for pediatric lung gene therapy applications.

Screening for hypercholesterolaemia in patients with other risk factors for coronary heart disease.

OBJECTIVE: To determine the prevalence of hypercholesterolaemia in individuals with other major risk factors of coronary heart disease and markers of hypercholesterolaemia. METHODS: A community-based cross-sectional study on a random sample of 261 persons aged between 35 and 69 years residing in the flatted housing estate of West Coast, Singapore, was conducted in 1997. A questionnaire, together with a medical examination and investigation involving the use of Reflotron machine, were used to collect the information required. RESULTS: 43.2% of the population had at least 1 major risk factor, with 18.1% being smokers, 18.1% with hypertension, 6.5% with diabetes mellitus, 5.1% being obese, and 5.0% with a family history of coronary heart disease. Higher percentages of individuals with hypercholesterolaemia were found when each risk factor was present. 9.5% had 2 or more risk factors, of which, 21.1% had high cholesterol levels. A high prevalence of hypercholesterolaemia that was statistically significant was found amongst subjects with corneal arcus below the age of 60. CONCLUSIONS: The risk factors of coronary heart disease remain prevalent in our population. We recommend screening for serum cholesterol only in those with at least 2 major risk factors of coronary heart disease in the general population between 35 and 69 years of age.

Hormonal regulation and genomic organization of the human amiloride-sensitive epithelial sodium channel alpha subunit gene.

To investigate the regulation of the amiloride-sensitive epithelial sodium channel (ENaC) expression, we have characterized the genomic structure and performed promoter analyses of the alpha subunit of the human (h) ENaC gene. Genomic clones containing the alphahENaC gene were isolated and subjected to restriction-mapping analysis. The alphahENaC gene was shown to be composed of 13 exons and 12 introns. Primer extension analysis confirmed that transcription initiation occurred at the beginning of the reported alphahENaC cDNA, but also indicated potential heterogenous initiation sites. Examination of a 3.1 kb 5' flanking sequence revealed a notable absence of CCAAT or TATA-like elements but suggested three GC boxes and several putative transcription factor binding sites, including a glucocorticoid response element (GRE) consensus. A 250 bp minimal promoter was capable of directing expression of a secreted alkaline phosphatase reporter. This promoter activity was enhanced 2.5- and 4-fold by upstream flanking sequences. Dexamethasone treatment induced levels of expression from the longer, GRE-containing promoter fragments from 8- to 20-fold, but not from the minimal promoter. Precise deletion of the 15-bp, dyad GRE sequence completely abolishes the response of reporter expression to dexamethasone induction. These experiments indicate that glucocorticoid augmentation of lung epithelial Na+ transport occurs, at least in part, by direct stimulation of transcription of the ENaC genes.

Previous cesarean section and abortion as risk factors for developing placenta previa.

OBJECTIVE: To determine the risk of subsequent occurrence of placenta previa in women with a history of previous cesarean sections and/or spontaneous and induced abortions. METHODS: A retrospective analysis of all single gestation deliveries at National University Hospital of Singapore from 1993-1997 was done. Women with placenta previa were identified by clinical or ultrasonographic diagnosis. RESULTS: Of the 16,169 singleton deliveries, 164 women (1.0%) had placenta previa. Women with placenta previa had a significantly higher incidence of previous cesarean sections (p < 0.001). Among the 164 women with placenta previa, women with 1, 2, and 3 previous cesarean sections had 2.2 (95% CI 1.4, 3.4), 4.1 (95% CI 1.9, 8.8) and 22.4 (95% CI 6.4, 78.3) times increased risk of developing placenta previa respectively. Similarly, women with 2 or more previous abortions had a 2.1 (95% CI 1.2, 3.5) times increased risk of subsequently developing placenta previa. CONCLUSION: There is a strong association between previous cesarean section and risk of subsequent development of placenta previa. This risk increased with the number of previous cesarean sections. Increasing frequency of abortions was also found to predispose a woman to placenta previa.

Development of Th1 and Th2 populations and the nature of immune responses to hepatitis B virus DNA vaccines can be modulated by codelivery of various cytokine genes.

In this study, we provide direct evidence that the magnitude and nature of the immune response to a DNA vaccine can be differentially regulated by codelivery of various mouse cytokine genes. Mice immunized with a hepatitis B virus (HBV) DNA vaccine and the IL-12 or IFN-gamma gene exhibited a significant enhancement of Th1 cells and increased production of anti-HBV surface IgG2a Ab, as well as a marked inhibition of Th2 cells and decreased production of IgG1 Ab. In contrast, coinjection of the IL-4 gene significantly enhanced the development of specific Th2 cells and increased production of IgG1 Ab, whereas Th1 differentiation and IgG2a production were suppressed. Coinjection of the IL-2 or the granulocyte-macrophage-CSF gene enhanced the development of Th1 cells, while the development of Th2 cells was not affected, and the production of IgG1 and IgG2a Ab were both increased. The CTL activity induced by HBV DNA vaccination was most significantly enhanced by codelivery of the IL-12 or IFN-gamma gene, followed by the IL-2 or granulocyte-macrophage-CSF gene, whereas codelivery of the IL-4 gene suppressed the activity. When challenged with HBV surface Ag (HBsAg)-expressing syngeneic tumors, significant reduction of tumor growth was observed in mice that were coadministered the IL-12 gene but not the IL-4 gene. Taken together, these results demonstrate that application of a cytokine gene in a DNA vaccine formulation can influence the differentiation of Th cells as well as the nature of an immune response and may thus provide a strategy to improve its prophylactic and therapeutic efficacy.

Construction of vectors expressing bioactive heterodimeric and single-chain murine interleukin-12 for gene therapy.

It has been well demonstrated that interleukin-12 (IL-12) could be useful to defend against a variety of pathogens, to suppress tumor growth and metastasis, and even to be employed as an adjuvant of vaccines to enhance beneficial type 1 T helper (Th1) cell response over detrimental type 2 T helper (Th2) cell responses. To apply IL-12 genes in gene therapy such as a DNA vaccine, a pIL-12 vector was constructed that contained two cytomegalovirus (CMV) promoters to drive the expression of p35 and p40 subunits, respectively. In addition, a pscIL-12 vector was designed with a linker to fuse p35 cDNA with p40 cDNA to produce a single-chain IL-12 protein, ensuring not only that the expression of p35 and p40 subunits was equally expressed, but also that no free p40 subunits interfered with IL-12 activity. The data suggested pIL-12 could produce a rather high level of biologically active IL-12 after transfection of COS cell lines as well as C2C12 muscle cell lines, as measured by both concanavalin A blast proliferation assay and enzyme-linked immunosorbent assay. Interestingly, the pscIL-12 vector could also express a bioactive murine IL-12 fusion protein in vitro. Furthermore, in vivo functional studies also demonstrated that mice co-immunized with a pS vector expressing the major envelope protein of hepatitis B virus (HBV) and IL-12 vectors encoding native IL-12 or single-chain IL-12 fusion protein elicited higher levels of IgG2a anti-HBs antibody and of Th1-related cytokine. Because p35 and p40 genes can be expressed in a vector by using a single promoter, pscIL-12 should be useful in future applications for nucleic acid vaccination or for gene therapy against diseases.

Identification and characterization of human genes encoding Hprp3p and Hprp4p, interacting components of the spliceosome.

Nuclear RNA splicing occurs in an RNA-protein complex, termed the spliceosome. U4/U6 snRNP is one of four essential small nuclear ribonucleoprotein (snRNP) particles (U1, U2, U5 and U4/U6) present in the spliceosome. U4/U6 snRNP contains two snRNAs (U4 and U6) and a number of proteins. We report here the identification and characterization of two human genes encoding U4/U6-associated splicing factors, Hprp3p and Hprp4p, respectively. Hprp3p is a 77 kDa protein, which is homologous to the Saccharomyces cerevisiae splicing factor Prp3p. Amino acid sequence analysis revealed two putative homologues in Caenorhabditis elegans and Schizosaccharomyces pombe. Polyclonal antibodies against Hprp3p were generated with His-tagged Hprp3p over-produced in Escherichia coli . This splicing factor can co-immunoprecipitate with U4, U6 and U5 snRNAs, suggesting that it is present in the U4/U6.U5 tri-snRNP. Hprp4p is a 58 kDa protein homologous to yeast splicing factor Prp4p. Like yeast Prp4p, the human homologue contains repeats homologous to the beta-subunit of G-proteins. These repeats are called WD repeats because there is a highly conserved dipeptide of tryptophan and aspartic acid present at the end of each repeat. The primary amino acid sequence homology between human Hprp4p and yeast Prp4p led to the discovery of two additional WD repeats in yeast Prp4p. Structural homology between these human and yeast splicing factors and the beta-subunit of G-proteins has been identified by sequence-similarity comparison and analysis of the protein folding by threading. Structural models of Hprp4p and Prp4p with a seven-blade beta-propeller topology have been generated based on the structure of beta-transducin. Hprp3p and Hprp4p have been shown to interact with each other and the first 100 amino acids of Hprp3p are not essential for this interaction. These experiments suggest that both Hprp3p and Hprp4p are components of human spliceosomes.

The hsp90-binding antibiotic geldanamycin decreases Raf levels and epidermal growth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase.

We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras.Raf, Ras.Src, Raf.Mek, and Raf.Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf.hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.

Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2.

DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In this study, we show that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of interleukin-2 (IL-2). Plasmid vectors encoding the major (S) or middle (pre-S2 plus S) envelope proteins of hepatitis B virus (HBV) were constructed and compared for their potential to induce hepatitis B surface antigen (HBsAg)-specific immune responses with a vector encoding the middle envelope and IL-2 fusion protein or with a bicistronic vector separately encoding the middle envelope protein and IL-2. Following transfection of cells in culture with these HBV plasmid vectors, we found that the encoded major protein was secreted while the middle protein and the fusion protein were retained on the cell membrane. Despite differences in localization of the encoded antigens, plasmids encoding the major or middle proteins gave similar antibody and T-cell proliferative responses in the vaccinated animals. The use of plasmids coexpressing IL-2 and the envelope protein in the fusion or nonfusion context resulted in enhanced humoral and cellular immune responses. In addition, the vaccine efficacy in terms of dosage used in immunization was increased at least 100-fold by coexpression of IL-2. We also found that DNA vaccines coexpressing IL-2 help overcome major histocompatibility complex-linked nonresponsiveness to HBsAg vaccination. The immune responses elicited by HBV DNA vaccines were also modulated by coexpression of IL-2. When restimulated with antigen in vitro, splenocytes from mice that received plasmids coexpressing IL-2 and the envelope protein produced much stronger T helper 1 (Th1)-like responses than did those from mice that had been given injections of plasmids encoding the envelope protein alone. Coexpression of IL-2 also increased the Th2-like responses, although the increment was much less significant.

Development of an epithelium-specific expression cassette with human DNA regulatory elements for transgene expression in lung airways.

The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. Utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5' genomic sequences and one of its introns, we have developed a novel expression cassette that can efficiently express reporter genes, as well as the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, in cultured lung epithelial cells. CFTR transcripts expressed from the native K18 enhancer/promoter include two alternative splicing products, due to the activation of two cryptic splice sites in the CFTR coding region. Modification of the K18 intron and CFTR cDNA sequences eliminated the cryptic splice sites without changing the CFTR amino acid sequence, and led to enhanced CFTR mRNA and protein expression as well as biological function. Transgenic expression analysis in mice showed that the modified expression cassette can direct efficient and epithelium-specific expression of the Escherichia coli LacZ gene in the airways of fetal lungs, with no detectable expression in lung fibroblasts or endothelial cells. This is the first expression cassette which selectively directs lung transgene expression for CFTR gene therapy to airway epithelia.

Functional mapping of the N-terminal regulatory domain in the human Raf-1 protein kinase.

Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways from the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain. Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain. Effects of the deletion mutations on kinase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conserved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase activity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is transforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal half containing the kinase domain of Raf-1 is required for activation by Src. These findings demonstrate that N-terminal deletions in Raf-1 do not necessarily result in constitutively elevated basal kinase activity and that the N-terminal regulatory domain is completely dispensable for Raf-1 activation by Src.