Effect of tailored practice and patient care plans on secondary prevention of heart disease in general practice: cluster randomised controlled trial.

OBJECTIVE: To test the effectiveness of a complex intervention designed, within a theoretical framework, to improve outcomes for patients with coronary heart disease. DESIGN: Cluster randomised controlled multicentre trial. SETTING: General practices in Northern Ireland and the Republic of Ireland, regions with different healthcare systems. PARTICIPANTS: 903 patients with established coronary heart disease registered with one of 48 practices. INTERVENTION: Tailored care plans for practices (practice based training in prescribing and behaviour change, administrative support, quarterly newsletter), and tailored care plans for patients (motivational interviewing, goal identification, and target setting for lifestyle change) with reviews every four months at the practices. Control practices provided usual care. MAIN OUTCOME MEASURES: The proportion of patients at 18 month follow-up above target levels for blood pressure and total cholesterol concentration, and those admitted to hospital, and changes in physical and mental health status (SF-12). RESULTS: At baseline the numbers (proportions) of patients above the recommended limits were: systolic blood pressure greater than 140 mm Hg (305/899; 33.9%, 95% confidence interval 30.8% to 33.9%), diastolic blood pressure greater than 90 mm Hg (111/901; 12.3%, 10.2% to 14.5%), and total cholesterol concentration greater than 5 mmol/l (188/860; 20.8%, 19.1% to 24.6%). At the 18 month follow-up there were no significant differences between intervention and control groups in the numbers (proportions) of patients above the recommended limits: systolic blood pressure, intervention 98/360 (27.2%) v control, 133/405 (32.8%), odds ratio 1.51 (95% confidence interval 0.99 to 2.30; P=0.06); diastolic blood pressure, intervention 32/360 (8.9%) v control, 40/405 (9.9%), 1.40 (0.75 to 2.64; P=0.29); and total cholesterol concentration, intervention 52/342 (15.2%) v control, 64/391 (16.4%), 1.13 (0.63 to 2.03; P=0.65). The number of patients admitted to hospital over the 18 month study period significantly decreased in the intervention group compared with the control group: 107/415 (25.8%) v 148/435 (34.0%), 1.56 (1.53 to 2.60; P=0.03). CONCLUSIONS: Admissions to hospital were significantly reduced after an intensive 18 month intervention to improve outcomes for patients with coronary heart disease, but no other clinical benefits were shown, possibly because of a ceiling effect related to improved management of the disease. TRIAL REGISTRATION: Current Controlled Trials ISRCTN24081411.

Are we ignoring the problem of sleep disorder in children with intellectual disabilities?

BACKGROUND: Sleep problems are more common amongst children with intellectual disability than other children. The implications for families, teachers and classmates, as well as the children themselves, are profound. AIM: A cross-sectional survey was undertaken with parents of children with intellectual disabilities in county Donegal. The aim was to examine the prevalence and types of sleep disorder in this group and to explore associated child and family characteristics. RESULTS: The response rate was 82.6%, with 96.8% meeting the criteria for a sleep disorder. This was higher than in other studies of children with and without intellectual disability or a diagnosed sleep disorder. CONCLUSION: Our findings underscore the need for an increased awareness amongst health and social care professionals of the extent and nature of the problem, as well as intervention options. A co-ordinated approach to detection and intervention is discussed and recommendations are made for future research in this area.

Secondary prevention of cardiovascular disease in different primary healthcare systems with and without pay-for-performance.

OBJECTIVE: To compare baseline cardiovascular risk management between people from two different healthcare systems recruited to a research trial of an intervention to optimise secondary prevention. DESIGN: Cross-sectional study. SETTING: 16 randomly selected general practices in Northern Ireland (NI) (UK NHS, strong infrastructure, pay-for-performance) and 32 in the Republic of Ireland (RoI) (mixed healthcare economy, less infrastructure, no pay-for-performance). PATIENTS: 903 (mean age 67.5 years; 69.9% male) randomly selected patients with known coronary heart disease. MAIN OUTCOME MEASURES: Blood pressure (BP), cholesterol, medications; validated questionnaires for diet (DINE), exercise (Godin) and quality of life (SF-12); healthcare usage. RESULTS: More RoI than NI participants had systolic BP >140 mm Hg (37% vs 28%, p = 0.01) and cholesterol >5 mmol/l (24% vs 17%, p = 0.02). RoI mean systolic BP was higher (139 vs 132 mm Hg). More RoI participants reported a high fibre intake (35% vs 23%), higher levels of physical activity (62% vs 44%) and better physical and mental health (SF-12); they also had more GP (5.6 vs 4.4) and fewer nurse visits (1.6 vs 2.1) in the previous year. Fewer participants in the RoI (55% vs 70%) were prescribed beta blockers. ACE inhibitor prescribing was similar for both groups (41%; 48%); high proportions were prescribed statins (84%; 85%) and aspirin (83%; 77%). CONCLUSIONS: BP and cholesterol are better controlled among patients in a primary healthcare system with a strong infrastructure supporting computerisation and rewarding measured performance, but this is not associated with healthier lifestyle or better quality of life. Further exploration of differences in professionals' and patients' engagement in secondary prevention in different healthcare systems is needed.

Gene expression in NKT cells: defining a functionally distinct CD1d-restricted T cell subset.

Since their discovery as cells bearing both TCRs and NK cell receptors, NKT cells have been intensively studied as a possible bridge between innate and adaptive immunity. Although their involvement in a wide variety of immune responses and in disease states have been well documented, molecular details of this functionality have been lacking. Recently, transcriptional profiling using microarrays has been applied to these cells, pinpointing gene-expression differences between this regulatory T cell subset and conventional T cells, and providing a framework for subset-specific therapeutic intervention in clinical settings.

Oligonucleotide arrays for expression monitoring.

This unit provides protocols for the amplification and labeling of mRNA (and the necessary controls) for hybridization to oligonucleotide arrays. It also describes methods for processing and normalizing the raw gene expression data in preparation for clustering and further analysis.

Preparation of mRNA for expression monitoring.

The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A(+)) versus total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.

Preparation of mRNA for expression monitoring.

The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A(+)) vs. total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.

Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology.

Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. Bone morphogenetic protein 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID-1, ID-2, ID-3, and ID-4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1, STAT1, and JunB. The changes in these nuclear binding factor mRNA levels were confirmed by real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). A further three transcription factors, core binding factor beta (CBFbeta), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment. In summary, we have used a gene chip microarray to identify a number of BMP-2 responsive genes in hMS cells. Thus, these studies provide potential candidate genes that may induce osteoblastic differentiation or, in the case of the ID proteins, block differentiation along alternate pathways.

Microbial lipopeptides induce the production of IL-17 in Th cells.

Naive Th cells can be directed in vitro to develop into Th1 or Th2 cells by IL-12 or IL-4, respectively. In vivo, chronic immune reactions lead to polarized Th cytokine patterns. We found earlier that Borrelia burgdorferi, the spirochaete that causes Lyme disease, induces Th1 development in alpha beta TCR-transgenic Th cells. Here, we used TCR-transgenic Th cells and oligonucleotide arrays to analyze the differences between Th1 cells induced by IL-12 vs those induced by B. burgdorferi. Transgenic Th cells primed with peptide in the presence of B. burgdorferi expressed several mRNAs, including the mRNA encoding IL-17, at significantly higher levels than Th cells primed with peptide and IL-12. Cytometric single-cell analysis of Th cell cytokine production revealed that IL-17 cannot be categorized as either Th1 or Th2 cytokine. Instead, almost all IL-17-producing Th cells simultaneously produced TNF-alpha and most IL-17(+) Th cells also produced GM-CSF. This pattern was also observed in humans. Th cells from synovial fluid of patients with Lyme arthritis coexpressed IL-17 and TNF-alpha upon polyclonal stimulation. The induction of IL-17 production in Th cells is not restricted to B. burgdorferi. Priming of TCR-transgenic Th cells in the presence of mycobacterial lysates also induced IL-17/TNF-alpha coproduction. The physiological stimulus for IL-17 production was hitherto unknown. We show here for the first time that microbial stimuli induce the expression of IL-17 together with TNF-alpha in both murine and human T cells. Chronic IL-17 expression induced by microbes could be an important mediator of infection-induced immunopathology.

CD1d on myeloid dendritic cells stimulates cytokine secretion from and cytolytic activity of V alpha 24J alpha Q T cells: a feedback mechanism for immune regulation.

The precise immunologic functions of CD1d-restricted, CD161+ AV24AJ18 (Valpha24JalphaQ) T cells are not well defined, although production of IL-4 has been suggested as important for priming Th2 responses. However, activation of human Valpha24JalphaQ T cell clones by anti-CD3 resulted in the secretion of multiple cytokines notably important for the recruitment and differentiation of myeloid dendritic cells. Specific activation of Valpha24JalphaQ T cells was CD1d restricted. Expression of CD1d was found on monocyte-derived dendritic cells in vitro, and immunohistochemical staining directly revealed CD1d preferentially expressed on dendritic cells in the paracortical T cell zones of lymph nodes. Moreover, myeloid dendritic cells both activated Valpha24JalphaQ T cells and were susceptible to lysis by these same regulatory T cells. Because myeloid dendritic cells are a major source of IL-12 and control Th1 cell differentiation, their elimination by lysis is a mechanism for limiting the generation of Th1 cells and thus regulating Th1/Th2 responses.

Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

Multiple differences in gene expression in regulatory Valpha 24Jalpha Q T cells from identical twins discordant for type I diabetes.

Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Valpha24JalphaQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Valpha24JalphaQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.

An essential role in liver development for transcription factor XBP-1.

XBP-1 is a CREB/ATF family transcription factor highly expressed in hepatocellular carcinomas. Here we report that XBP-1 is essential for liver growth. Mice lacking XBP-1 displayed hypoplastic fetal livers, whose reduced hematopoiesis resulted in death from anemia. Nevertheless, XBP-1-deficient hematopoietic progenitors had no cell-autonomous defect in differentiation. Rather, hepatocyte development itself was severely impaired by two measures: diminished growth rate and prominent apoptosis. Specific target genes of XBP-1 in the liver were identified as alphaFP, which may be a regulator of hepatocyte growth, and three acute phase protein family members. Therefore, XBP-1 is a transcription factor essential for hepatocyte growth.

Using DNA chips to unravel the genetics of type 1 diabetes.

Thousands of genes are currently being discovered by sequencing the human genome. Of these, hundreds if not thousands fall into regions of the genome identified by genetic studies as linked to the development of type 1 diabetes. Inheritance patterns for these regions suggest that diabetes results from the combinatorial interaction of susceptibility loci. The study of such complex events will require technologies that can simultaneously evaluate expression profiles and allelic differences for all these genes in order to dissect the mechanisms responsible for the development of disease. We will argue that DNA microarrays are the natural vehicle for the exploration of diabetes-related gene clusters, and the application of these arrays to understanding diabetes is discussed.

Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis.

The role of FSH in gonadal tumorigenesis and, in particular, in human ovarian cancer has been debated. It is also unclear what role the elevated FSH levels in the inhibin-deficient mouse play in the gonadal tumorigenesis. To directly assess the role of FSH in gonadal growth, differentiation, and gonadal tumorigenesis, we have generated both gain-of-function and loss-of-function transgenic mutant mice. In the gain-of-function model, we have generated transgenic mice that ectopically overexpress human FSH from multiple tissues using a mouse metallothionein-1 promoter, achieving levels far exceeding those seen in postmenopausal women. Male transgenic mice are infertile despite normal testicular development and demonstrate enlarged seminal vesicles secondary to elevated serum testosterone levels. Female transgenic mice develop highly hemorrhagic and cystic ovaries, have elevated serum estradiol and progesterone levels, and are infertile, mimicking the features of human ovarian hyperstimulation and polycystic ovarian syndromes. Furthermore, the female transgenic mice develop enlarged and cystic kidneys and die between 6-13 weeks as a result of urinary bladder obstruction. In a complementary loss-of-function approach, we have generated double-homozygous mutant mice that lack both inhibin and FSH by a genetic intercross. In contrast to male mice lacking inhibin alone, 95% of which die of a cancer cachexia-like syndrome by 12 weeks of age, only 30% of the double-mutant male mice lacking both FSH and inhibin die by 1 yr of age. The remaining double-mutant male mice develop slow-growing and less hemorrhagic testicular tumors, which are noted after 12 weeks of age, and have minimal cachexia. Similarly, the double-mutant female mice develop slow-growing, less hemorrhagic ovarian tumors, and 70% of these mice live beyond 17 weeks. The double-mutant mice demonstrate minimal cachexia in contrast to female mice lacking only inhibin, which develop highly hemorrhagic ovarian tumors, leading to cachexia and death by 17 weeks of age in 95% of the cases. The milder cachexia-like symptoms of the inhibin and FSH double-mutant mice are correlated with low levels of serum estradiol and activin A and reduced levels of aromatase mRNA in the gonadal tumors. Based on these and our previous genetic analyses, we conclude that elevated FSH levels do not directly cause gonadal tumors. However, these results suggest FSH is an important trophic modifier factor for gonadal tumorigenesis in inhibin-deficient mice.

Dissecting the marrow microenvironment.

Cloned human stromal cell lines representing functionally distinct cellular components of the marrow microenvironment were generated to serve as tools for identifying gene products that regulate hematopoiesis. Oligonucleotide arrays, or "gene chips" were used to provide a comprehensive comparison of gene expression among the cell lines. One line, designated HS-5, was found to secrete large amounts of cytokines, and conditioned media from this line was found to support the ex vivo expansion of both immature and mature progenitors. In contrast, a second line, designated HS-27a, does not secrete known cytokines but does support cobblestone area formation by CD34+/38lo cells. HS-27a, but not HS-5, was also found to express hJagged1, a ligand for Notch1, which may function to influence cell fate decisions of hematopoietic precursors. Both cell lines are currently being used to identify other gene products that regulate hematopoiesis and to generate reagents that will allow more formal evaluation of the putative role of hJagged1 in hematopoietic cell fate decisions.

Expression monitoring by hybridization to high-density oligonucleotide arrays.

The human genome encodes approximately 100,000 different genes, and at least partial sequence information for nearly all will be available soon. Sequence information alone, however, is insufficient for a full understanding of gene function, expression, regulation, and splice-site variation. Because cellular processes are governed by the repertoire of expressed genes, and the levels and timing of expression, it is important to have experimental tools for the direct monitoring of large numbers of mRNAs in parallel. We have developed an approach that is based on hybridization to small, high-density arrays containing tens of thousands of synthetic oligonucleotides. The arrays are designed based on sequence information alone and are synthesized in situ using a combination of photolithography and oligonucleotide chemistry. RNAs present at a frequency of 1:300,000 are unambiguously detected, and detection is quantitative over more than three orders of magnitude. This approach provides a way to use directly the growing body of sequence information for highly parallel experimental investigations. Because of the combinatorial nature of the chemistry and the ability to synthesize small arrays containing hundreds of thousands of specifically chosen oligonucleotides, the method is readily scalable to the simultaneous monitoring of tens of thousands of genes.

Transfection of TCR alpha-chains into suppressor and T helper cell hybridomas. Production of suppressor factors with predicted antigen specificity.

Conditioned medium from Ag-specific suppressor T cell hybridomas contains soluble factors (TsF) that modulate immune responses in an Ag-specific manner. We previously generated a series of TCR-alpha- and TCR-beta- expression variants from a 4-hydroxy-3-nitrophenyl acetyl (NP)-specific inducer suppressor T cell hybridoma and demonstrated that loss of TCR alpha-chain mRNA, but not TCR-beta chain mRNA, was accompanied by concomitant loss of suppressor bioactivity. Suppressor factor bioactivity was restored by expression of TCR alpha-chain cDNA, suggesting that the TCR alpha-chain plays a critical role in Ag-specific suppressor cell function. We have now transfected TCR alpha-chain from a Th cell clone specific for arsanylated peptides plus I-Ad into a TCR-alpha- derivative of an NP-specific inducer suppressor T cell hybridoma. The transfectants expressed a new hybrid TCR-alpha beta complex and produced soluble factors that suppressed azobenzenearsonate hapten (ABA) but not NP delayed-type hypersensitivity responses. These supernatants mediated suppression of the induction, but not the effector phase of the delayed-type hypersensitivity reaction. In reciprocal experiments we transfected a TCR alpha-chain from an NP-specific suppressor T cell hybridoma into a TCR-alpha- hybridoma derived from the ABA-specific Th cell hybridoma. The NP-specific TCR alpha-chain was expressed in the Th cell hybridoma, but the supernatant from this transfectant did not suppress DTH responses to either NP or ABA. However, the latter supernatants, when combined with cell lysates derived from a TCR-alpha- Ts hybridoma, specifically suppress NP DTH responses. These data are consistent with the interpretation that TCR alpha-chain imparts Ag specificity to the suppressor molecule and a second, yet undefined, component produced by the Ts hybridoma controls the immunoregulatory bioactivity.

T cell receptor alpha-chain defines the antigen specificity of antigen-specific suppressor factor but does not impart genetic restriction.

Previous studies utilizing NP (4-hydroxy, 3-nitrophenyl acetyl hapten)-specific, T suppressor hybridomas have indicated that expression of TCR-alpha, but not TCR-beta, mRNA is required for expression of Ag-specific suppressor factor bioactivity. Suppressor-effector factor has been shown to be Ag specific and I-J restricted. Although the expression of TCR-alpha mRNA was necessary for suppressor activity, the role of TCR-alpha, as it pertained to the functional properties of T cell suppressor factor (TsF), was not established. To determine which properties of TsF could be accounted for by TCR-alpha expression, TCR-alpha cDNA, derived from NP-specific, suppressor T cell (Ts) hybridomas, was transfected into recipient Ts hybridomas of a second Ag specificity. The resulting heterologous transfectants displayed NP-specific, genetically restricted TsF activity. The Ag specificity corresponded to that of the TCR-alpha donor; however, the genetic restriction was influenced by the recipient cell, implying that TCR-alpha did not control genetic restriction of the TsF. Examination of TCR-beta expression in one of the MHC-restricted transfectants indicated that the genetic restriction of TsF could not be accounted for by TCR-beta gene products. The data support the conclusion that TCR-alpha expression is not only obligate for TsF bioactivity, but that the Ag specificity of the TCR-alpha dictates the Ag specificity of the resulting suppressor factor.