The function of E- and Id proteins in lymphocyte development.

Helix-loop-helix proteins are essential factors for lymphocyte development and function. In particular, E-proteins are crucial for commitment of lymphoid progenitors to the B- and T-cell lineages. E-proteins are negatively regulated by the Id class of helix-loop-helix proteins. The Id proteins function as dominant-negative inhibitors of E-proteins by inhibiting their ability to bind DNA. Here, we review the role of E-proteins and their Id protein antagonists in lymphocyte proliferation and developmental progression. In addition, we discuss how E-protein activity and Id gene expression are regulated by T-cell receptor (TCR) and pre-TCR-mediated signalling.

Early thymocyte development is regulated by modulation of E2A protein activity.

The E2A gene encodes the E47 and E12 basic helix-loop-helix (bHLH) transcription factors. T cell development in E2A-deficient mice is partially arrested before lineage commitment. Here we demonstrate that E47 expression becomes uniformly high at the point at which thymocytes begin to commit towards the T cell lineage. E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage. However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase-dependent pathway. Consistent with these observations, a deficiency in E2A proteins completely abrogates the developmental block observed in mice with defects in TCR rearrangement. Thus E2A proteins are necessary for both initiating T cell differentiation and inhibiting development in the absence of pre-TCR expression. Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.

High-efficiency expression and solubilization of functional T cell antigen receptor heterodimers.

The T cell receptor (TCR) zeta chain was attached to the TCR alpha and beta extracellular domains to induce efficient expression of alpha beta heterodimers that can recognize complexes of antigen with major histocompatibility complex (MHC) molecules. Chimeric constructs expressed in RBL-2H3 cells were efficiently transported to the cell surface uniquely as disulfide-linked heterodimers. Transfectants were activated by specific antigen-MHC complexes, which demonstrated that the expressed alpha beta was functional and that CD3 was not required for antigen-MHC binding. Constructs with thrombin cleavage sites were efficiently cleaved to soluble disulfide-linked heterodimers. Thus, attachment of TCR zeta domains and protease cleavage sites to TCR alpha and beta induces expression of demonstrably functional heterodimers that can be solubilized.

Selection of amino acid sequences in the beta chain of the T cell antigen receptor.

The induction of an immune response in mammals is initiated by specifically reactive T lymphocytes. The specificity of the reaction is mediated by a complex receptor, part of which is highly variable in sequence and analogous to immunoglobulin heavy- and light-chain variable domains. The functional specificity of the T cell antigen receptor is, however, markedly different from immunoglobulins in that it mediates cell-cell interactions via the simultaneous recognition of foreign antigens and major histocompatibility complex-encoded molecules expressed on the surface of various lymphoid and nonlymphoid cells. The relation between the structure of the receptor and its functional specificity was investigated by analyzing the primary sequences of the receptors expressed by a series of T lymphocyte clones specific for a model antigen, pigeon cytochrome c. Within this set of T lymphocyte clones there was a striking selection for amino acid sequences in the receptor beta-chain in the region analogous to the third complementarity-determining region of immunoglobulins. Thus, despite the functional differences between T cell antigen receptors and immunoglobulin molecules, analogous regions appear to be important in determining ligand specificity.

Transcription factors in hematopoiesis.

The advent of gene targeting in the mouse has led to rapid advances in the identification of factors controlling gene expression that are essential for normal hematopoietic development. Recent work has also uncovered roles for some of these factors in leukemogenesis and in the global regulation of chromatin structure.

E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas.

The E2A gene products, E12 and E47, are critical for proper early B-cell development and commitment to the B-cell lineage. Here we reveal a new role for E2A in T-lymphocyte development. Loss of E2A activity results in a partial block at the earliest stage of T-lineage development. This early T-cell phenotype precedes the development of a T-cell lymphoma which occurs between 3 and 9 months of age. The thymomas are monoclonal and highly malignant and display a cell surface phenotype similar to that of immature thymocytes. In addition, the thymomas generally express high levels of c-myc. As assayed by comparative genomic hybridization, each of the tumor populations analyzed showed a nonrandom gain of chromosome 15, which contains the c-myc gene. Taken together, the data suggest that the E2A gene products play a role early in thymocyte development that is similar to their function in B-lineage determination. Furthermore, the lack of E2A results in development of T-cell malignancies, and we propose that E2A inactivation is a common feature of a wide variety of human T-cell proliferative disorders, including those involving the E2A heterodimeric partners tal-1 and lyl-1.

Integrated Data Repository Toolkit (IDRT). A Suite of Programs to Facilitate Health Analytics on Heterogeneous Medical Data.

BACKGROUND: In recent years, research data warehouses moved increasingly into the focus of interest of medical research. Nevertheless, there are only a few center-independent infrastructure solutions available. They aim to provide a consolidated view on medical data from various sources such as clinical trials, electronic health records, epidemiological registries or longitudinal cohorts. The i2b2 framework is a well-established solution for such repositories, but it lacks support for importing and integrating clinical data and metadata. OBJECTIVES: The goal of this project was to develop a platform for easy integration and administration of data from heterogeneous sources, to provide capabilities for linking them to medical terminologies and to allow for transforming and mapping of data streams for user-specific views. METHODS: A suite of three tools has been developed: the i2b2 Wizard for simplifying administration of i2b2, the IDRT Import and Mapping Tool for loading clinical data from various formats like CSV, SQL, CDISC ODM or biobanks and the IDRT i2b2 Web Client Plugin for advanced export options. The Import and Mapping Tool also includes an ontology editor for rearranging and mapping patient data and structures as well as annotating clinical data with medical terminologies, primarily those used in Germany (ICD-10-GM, OPS, ICD-O, etc.). RESULTS: With the three tools functional, new i2b2-based research projects can be created, populated and customized to researcher's needs in a few hours. Amalgamating data and metadata from different databases can be managed easily. With regards to data privacy a pseudonymization service can be plugged in. Using common ontologies and reference terminologies rather than project-specific ones leads to a consistent understanding of the data semantics. CONCLUSIONS: i2b2's promise is to enable clinical researchers to devise and test new hypothesis even without a deep knowledge in statistical programing. The approach presented here has been tested in a number of scenarios with millions of observations and tens of thousands of patients. Initially mostly observant, trained researchers were able to construct new analyses on their own. Early feedback indicates that timely and extensive access to their "own" data is appreciated most, but it is also lowering the barrier for other tasks, for instance checking data quality and completeness (missing data, wrong coding).

Secure Secondary Use of Clinical Data with Cloud-based NLP Services. Towards a Highly Scalable Research Infrastructure.

OBJECTIVES: The secondary use of clinical data provides large opportunities for clinical and translational research as well as quality assurance projects. For such purposes, it is necessary to provide a flexible and scalable infrastructure that is compliant with privacy requirements. The major goals of the cloud4health project are to define such an architecture, to implement a technical prototype that fulfills these requirements and to evaluate it with three use cases. METHODS: The architecture provides components for multiple data provider sites such as hospitals to extract free text as well as structured data from local sources and de-identify such data for further anonymous or pseudonymous processing. Free text documentation is analyzed and transformed into structured information by text-mining services, which are provided within a cloud-computing environment. Thus, newly gained annotations can be integrated along with the already available structured data items and the resulting data sets can be uploaded to a central study portal for further analysis. RESULTS: Based on the architecture design, a prototype has been implemented and is under evaluation in three clinical use cases. Data from several hundred patients provided by a University Hospital and a private hospital chain have already been processed. CONCLUSIONS: Cloud4health has shown how existing components for secondary use of structured data can be complemented with text-mining in a privacy compliant manner. The cloud-computing paradigm allows a flexible and dynamically adaptable service provision that facilitates the adoption of services by data providers without own investments in respective hardware resources and software tools.

Late-onset adrenal steroid 3 beta-hydroxysteroid dehydrogenase deficiency. I. A cause of hirsutism in pubertal and postpubertal women.

To investigate the adrenal cause of hyperandrogenism in peri- and postpubertal hirsute women, baseline and ACTH-stimulated serum concentrations of delta 5-17-hydroxypregnenolone (delta 5-17P), dehydroepiandrosterone (DHEA) and its sulfate, 17-hydroxyprogesterone (17-OHP), cortisol, delta 4-androstenedione, and testosterone were determined in 116 women with hirsutism or acne of peri- and postpubertal onset with or without menstrual abnormalities. The results were compared with the same steroid concentrations in 30 normal age-matched women. Sixteen of the 116 women with hirsutism whose ACTH-stimulated 17-OHP levels (mean +/- SD, 5404 +/- 3234 ng/dl; normal, 334 +/- 194) were markedly elevated while their ratios of delta 5-17P to 17-OHP (0.4 +/- 0.2; normal, 3.4 +/- 1.5) were low were diagnosed as having nonclassical symptomatic 21-hydroxylase deficiency. Seventeen other hirsute women, including 3 siblings, had very high responses of delta 5-17P (2276 +/- 669 ng/dl; normal, 985 +/- 327) and DHEA (2787 +/- 386 ng/dl; normal, 1050 +/- 384) to ACTH stimulation, with significantly elevated ratios of delta 5-17P to 17-OHP (11 +/- 2.0; normal, 3.4 +/- 1.5) and DHEA to delta 4-androstenedione (7.5 +/- 2.3; normal, 4.6 +/- 1.5). In these hirsute women, the morning serum delta 5-17P and DHEA concentrations were elevated, had a diurnal variation, and were suppressed with dexamethasone administration. We propose that partial adrenal 3 beta-hydroxysteroid dehydrogenase deficiency is the cause of hirsutism in these women. This may represent an allelic variant at the genetic locus for 3 beta-hydroxysteroid dehydrogenase deficiency similar to that reported for symptomatic nonclassical 21-hydroxylase deficiency producing peripubertal excess androgen syndrome.

The influence of MHC gene products on the generation of an antigen-specific T-cell repertoire.

We have previously described the B10.A pigeon cytochrome c-specific response in terms of clonal phenotypes and T-cell receptor (TcR) gene usage. All B10.A T-cell clones studied respond to antigen in association with syngeneic B10.A APCs and cross-react to antigen in association with one or two allogeneic variants of the I-E-encoded MHC molecules. In congenic strains of mice expressing these allogeneic MHC alleles [B10.A(5R) and B10.S(9R)], pigeon cytochrome c-specific T cells exhibit very similar MHC cross-reactivities. Our goal was to determine whether the same MHC cross-reactive T-cell clones were expressed in each appropriate strain, or whether each T-cell repertoire was unique. The results indicate that identical V alpha-J alpha and V beta-J beta combinations were expressed by the major pigeon cytochrome c-specific response phenotype in B10.A and B10.A(5R) mice. Previous functional data supports this overlap in expressed T-cell clones. B10.A and B10.S(9R) mice exhibit similar response phenotypes to pigeon cytochrome c but express distinctly different TcR genes. The results of these studies support the existence of at least two different mechanisms in determining MHC-linked immune response polymorphisms.

Serum thyroid hormone abnormalities in psychiatric disease.

Psychiatric illness is a cause of "euthyroid sick syndrome" (ESS), defined as abnormal concentrations of circulating iodothyronines in euthyroid subjects with nonthyroidal illness (NTI). We describe a prospective study of 150 consecutive psychiatric admissions studied by endocrine and psychologic techniques. Based on 150 admission blood samples, we found a 7% incidence of ESS and with serial samples (74 patients) the incidence was 27%, demonstrating that ESS can develop after hospital admission. Of the 20 patients with ESS, 11 had elevation of both serum total T4 concentrations (T4) and free thyroxine index (FTI) while their serum total T3 concentrations (T3) remained normal; 5 had elevation of FTI without elevation of T4 or T3; and 4 had low T4 and low FTI and normal TSH. In 2 of the 4 patients in the last category, the T3 was also low. The free T3 index (FT3I) was normal in all but 1 patient who had low FT3I and FTI, low T4 and T3, and normal TSH. The serum thyroid hormone abnormalities were transient in the ESS patients during the 10 day period with 2 exceptions; 1 patient had persistently elevated T4 and FTI with normal T3 and FT3I values while another patient had persistently depressed T4 and FTI without abnormality of FT3I or TSH. Multivariate statistical analysis demonstrated a difference (P less than .06) in the psychologic attributes of somatic and autonomic symptoms in ESS patients compared to controls. We conclude that ESS is as common amongst psychiatric admissions as in general hospital patients previously studied and that blood thyroid function tests should be interpreted cautiously in all hospitalized patients.

Analysis of human primary bone cells by fluorescence activated cell scanning: methodological problems and preliminary results.

We describe the development of flowcytometrical methods to analyse human primary osteoblast-like cultures obtained from trabecular bone explants in comparison to the human osteosarcoma cell line HOS 58. Two antigens typical of osteoblasts were studied: bone alkaline phosphatase and collagen/procollagen I; the non-specific attachment protein fibronectin served as control. The morphology of all different antigens is shown by immunocytochemistry before flowcytometrical analysis. The establishment of flowcytometry is described in detail. While all antigens tested were nearly 100% positive in the HOS 58 cells in immunocytochemistry and flowcytometry, in primary osteoblast-like cells results varied widely between both methods. Cell permeabilisation before flowcytometry improved the homogeneity of results, probably by increasing the accessibility of the specific antibody to intracellular compartments. Though up to 80% of cells were lost during preparation the ratio of positive versus negative cells in specific experiment was not dependent on the cell recovery. Therefore, the cells finally analysed seemed to be representative of the total population.

Intraoperative localization of subcortical brain tumors: further experience with B-mode real-time sector scanning.

Intraoperative ultrasonography is a potentially useful tool for the neurosurgeon faced with the task of finding and removing small subcortical brain tumors. With the B-mode real-time sector scan equipment now available, satisfactory images of the intracranial contents can be obtained. Others have reported obtaining images by applying the transducer directly to the dura mater or cortex. This carries the risk of pressure damage to the brain. Furthermore, the presence of acoustical noise in the region close to the transducer results in poor image resolution in the superficial region of the cortex. To circumvent these two problems, we have used a saline-filled cylinder placed over the craniotomy site to achieve acoustical coupling. This technique also increases the area of cortex visualized by the pie-shaped beam of the sector scan by separating the transducer from the brain surface.

Neurosurgical intra-operative ultrasound: tumor localization and characterization.

Intra-operative real-time ultrasound provides useful information for the neurosurgeon faced with the task of localization and extirpation of small subcortical brain tumors.

Human osteoblast-like cells phagocytose metal particles and express the macrophage marker CD68 in vitro.

Periprosthetic osteolysis is a major cause of aseptic loosening in artificial joint replacement. It is assumed to occur in conjunction with the activation of macrophages. We have shown in vitro that human osteoblast-like cells, isolated from bone specimens obtained from patients undergoing hip replacement, phagocytose fine particles of titanium alloy (TiAlV). The human osteoblast-like cells were identified immunocytochemically by the presence of bone-specific alkaline phosphatase (BAP). With increasing duration of culture, a variable number of the osteoblastic cells became positive for the macrophage marker CD68, independent of the phagocytosis of particles, with a fine granular cytoplasmic staining which was coexpressed with BAP as revealed by immunodoublestaining. The metal particles were not toxic to the osteoblastic cells since even in culture for up to four weeks massively laden cells were vital and had a characteristic morphology. Cells of the human osteosarcoma cell line (HOS 58) were also able to phagocytose metal particles but had only a low expression of the CD68 antigen. Fluorescence-activated cell scanning confirmed our immunocytochemical results. Additionally, the cells were found to be negative for the major histocompatibility complex-II (MHC-II) which is a marker for macrophages and other antigen-presenting cells. Negative results of histochemical tests for tartrate-resistant acid phosphatase excluded the contamination by osteoclasts or macrophages in culture. Our observations suggest that the osteoblast can either change to a phagocytosing cell or that the phagocytosis is an underestimated property of the osteoblast. The detection of the CD68 antigen is insufficient to prove the monocytic lineage. In order to discriminate between macrophages and osteoblasts additional markers should be used. To our knowledge, this is the first demonstration of cells of an osteoblastic origin which have acquired a mixed phenotype of both osteoblasts and macrophages.

Parental values, reinforcement, and young children's prosocial behavior: a longitudinal study.

In this study, the authors assessed the relation of parental reinforcement and parental values to young children's prosocial behaviors. Parents' dyadic interactions with their 1- to 2-year-old children were videotaped in the home on two occasions approximately 6 months apart. The children also were videotaped playing with a peer at 3 1/2 to 4 1/2 years of age. Parental reinforcement of the children's prosocial behaviors was coded, as were the children's prosocial behaviors with the peer. The frequency of girls' spontaneous prosocial behaviors decreased in the early years; modest consistency was observed for boys (but not girls) across the two parental sessions. No relation existed between the frequency of children's prosocial behaviors with their parents and their behaviors with peers. Both maternal and paternal valuing of compliance were negatively related to the mothers' use of reinforcement for children's spontaneous prosocial behaviors. Parental reinforcement of compliant prosocial behaviors was negatively related to children's compliance with a peer's request for prosocial behavior and positively related to defensive behavior with the peer. Fathers' valuing of prosocial behavior was associated with children's compliance with the peer's requests for prosocial action. Parents who valued compliance had children who exhibited low levels of compliant prosocial behaviors with the peer, possibly because of the depressed level of peer interaction.

Ectopic expression of E47 or E12 promotes the death of E2A-deficient lymphomas.

Mice with null mutations in the E2A gene are highly susceptible to the spontaneous development of thymic lymphomas. To understand better how E2A deficiency may contribute to lymphomagenesis, we have observed the consequences of enforced expression of the E2A gene products E12 and E47 in cell lines derived from lymphomas that arose spontaneously in E2A-deficient mice. E2A-expressing cells are steadily eliminated from lymphoma cultures into which E47 or E12 was introduced. The mechanism underlying the loss of E2A-expressing cells does not involve an arrest in cell-cycle progression. Rather, the E2A proteins activate a programmed cell death pathway in these lymphomas. This E2A-mediated cell death appears to be preceded by a loss of mitochondrial transmembrane potential. These data provide direct evidence that E2A gene products can act as tumor suppressors.