Small dense LDL cholesterol in human subjects with different chronic inflammatory diseases.

BACKGROUND AND AIMS: Chronic inflammatory diseases (CID) are associated with a profound increase in cardiovascular (CV) risk resulting in reduced life expectancy. However, LDL-cholesterol is reported to be low in CID patients which is referred to as the "LDL paradoxon". The aim of the present study was to investigate whether LDL-particles in CID exhibit an increased content of the highly atherogenic small-dense LDL subfraction (sdLDL). METHODS AND RESULTS: In this prospective, single center, observational study we enrolled 141 patients with CID (RA n = 59, inflammatory bowel disease (IBD) n = 35, ankylosing spondylitis (SpA) n = 25, Psoriasis n = 22) in 2011 through 2013 to evaluate sdLDL levels before as well as 6 and 26 weeks after initiation of different anti-cytokine therapies (anti-TNFα, anti-IL-6R antibodies). sdLDL levels were compared to 141 healthy individuals in a case control design. Compared to healthy controls, all CID patients displayed a significantly higher sdLDL content within the LDL cholesterol fraction: RA 35.0 ± 9.2% (p < 0.001), SpA 42.5 ± 10.5% (p < 0.001), IBD 37.5 ± 7.1% (p < 0.001), Psoriasis 33.6 ± 4.6% (p < 0.01). Furthermore, the sdLDL/LDL ratio was significantly higher in male compared to female RA subjects (p < 0.05). Neither anti-TNFα nor anti-IL6R medication altered sdLDL levels despite a significant improvement of disease activity. CONCLUSION: In several different chronic inflammatory disease entities, LDL-cholesterol is shifted toward a pro-atherogenic phenotype due to an increased sdLDL content which might in part explain the LDL paradoxon. Since premature CV disease is a major burden of affected patients, specifically targeting lipid metabolism should be considered routinely in clinical patient care. CLINICAL TRIALS: Registration at German Clinical Trial Register (DRKS): DRKS00005285.

Zebrafish screen identifies novel compound with selective toxicity against leukemia.

To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells.

Prepuberal intranasal dopamine treatment in an animal model of ADHD ameliorates deficient spatial attention, working memory, amino acid transmitters and synaptic markers in prefrontal cortex, ventral and dorsal striatum.

Intranasal application of dopamine (IN-DA) has been shown to increase motor activity and to release DA in the ventral (VS) and dorsal striatum (DS) of rats. The aim of the present study was to assess the effects of IN-DA treatment on parameters of DA and excitatory amino acid (EAA) function in prepuberal rats of the Naples high-excitability (NHE) line, an animal model for attention-deficit hyperactivity disorder (ADHD) and normal random bred (NRB) controls. NHE and NRB rats were daily administered IN-DA (0.075, 0.15, 0.30 mg/kg) or vehicle for 15 days from postnatal days 28-42 and subsequently tested in the Làt maze and in the Eight-arm radial Olton maze. Soluble and membrane-trapped L-glutamate (L-Glu) and L-aspartate (L-Asp) levels as well as NMDAR1 subunit protein levels were determined after sacrifice in IN-DA- and vehicle-treated NHE and NRB rats in prefrontal cortex (PFc), DS and VS. Moreover, DA transporter (DAT) protein and tyrosine hydroxylase (TH) levels were assessed in PFc, DS, VS and mesencephalon (MES) and in ventral tegmental area (VTA) and substantia nigra, respectively. In NHE rats, IN-DA (0.30 mg/kg) decreased horizontal activity and increased nonselective attention relative to vehicle, whereas the lower dose (0.15 mg/kg) increased selective spatial attention. In NHE rats, basal levels of soluble EAAs were reduced in PFc and DS relative to NRB controls, while membrane-trapped EAAs were elevated in VS. Moreover, basal NMDAR1 subunit protein levels were increased in PFc, DS and VS relative to NRB controls. In addition, DAT protein levels were elevated in PFc and VS relative to NRB controls. IN-DA led to a number of changes of EAA, NMDAR1 subunit protein, TH and DAT protein levels in PFc, DS, VS, MES and VTA, in both NHE and NRB rats with significant differences between lines. Our findings indicate that the NHE rat model of ADHD may be characterized by (1) prefrontal and striatal DAT hyperfunction, indicative of DA hyperactivty, and (2) prefrontal and striatal NMDA receptor hyperfunction indicative of net EAA hyperactivty. IN-DA had ameliorative effects on activity level, attention, and working memory, which are likely to be associated with DA action at inhibitory D2 autoreceptors, leading to a reduction in striatal DA hyperactivity and, possibly, DA action on striatal EAA levels, resulting in a decrease of striatal EAA hyperfunction (with persistence of prefrontal EAA hyperfunction). Previous studies on IN-DA treatment in rodents have indicated antidepressant, anxiolytic and anti-parkinsonian effects in relation to enhanced central DAergic activity. Our present results strengthen the prospects of potential therapeutic applications of intranasal  DA by indicating an enhancement of selective attention and working memory in a deficit model.

[Microbiome and nutrition. The way to a future therapy for chronic inflammatory bowel diseases?]. / Mikrobiom und Ernährung. Therapie der Zukunft für chronisch-entzündliche Darmerkrankungen?

The complex microbiome of the human gut contains an excessive amount of genetic information that is more than 100-fold larger than the human genome. In patients with inflammatory bowel disease diversity of the gut microbiome is significantly reduced and moreover specific phyla are overrepresented or underrepresented. However, the functional capacity of the microflora to generate certain metabolic products containing lipids or amino acids- and more complex regulatory substances is more important that the mere annotation of the microorganisms. Modern pharmacological approaches target the functional capacity and constitution of the microbiome. An important strategy is the development of controlled release formulations that deliver defined lipid, carbohydrate or amino acid products derived from nutritional components targeting gut areas distal to the absorption zones of the upper gastrointestinal tract.

Tbl3 regulates cell cycle length during zebrafish development.

The regulation of cell cycle rate is essential for the correct timing of proliferation and differentiation during development. Changes to cell cycle rate can have profound effects on the size, shape and cell types of a developing organ. We previously identified a zebrafish mutant ceylon (cey) that has a severe reduction in T cells and hematopoietic stem/progenitor cells (HSPCs). Here we find that the cey phenotype is due to absence of the gene transducin (beta)-like 3 (tbl3). The tbl3 homolog in yeast regulates the cell cycle by maintaining rRNA levels and preventing p53-induced cell death. Zebrafish tbl3 is maternally expressed, but later in development its expression is restricted to specific tissues. Tissues expressing tbl3 are severely reduced in cey mutants, including HSPCs, the retina, exocrine pancreas, intestine, and jaw cartilage. Specification of these tissues is normal, suggesting the reduced size is due to a reduced number of differentiated cells. Tbl3 MO injection into either wild-type or p53-/- mutant embryos phenocopies cey, indicating that loss of tbl3 causes specific defects in cey. Progression of both hematopoietic and retinal development is delayed beginning at 3 day post fertilization due to a slowing of the cell cycle. In contrast to yeast, reduction of Tbl3 causes a slowing of the cell cycle without a corresponding increase in p53 induced cell death. These data suggest that tbl3 plays a tissue-specific role regulating cell cycle rate during development.

Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency.

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.

Expert evaluations of fatigue questionnaires used in rheumatoid arthritis: a Delphi study among patients, nurses and rheumatologists in the Netherlands.

OBJECTIVES: Evaluating fatigue items from traditional questionnaires and a new scale (BRAF-MDQ) by experts in rheumatoid arthritis (RA). This evaluation was part of a study to select fatigue items to develop an item bank for a Dutch computer-adaptive test (CAT) for RA. Experts' opinions were incorporated since they are essential for content validity of measurement instruments. METHODS: The 60 items of the SF-36 subscale vitality, FACIT-F, POMS subscale fatigue/inertia, MAF and the recently developed BRAF-MDQ were evaluated by rheumatologists, nurses and RA patients in a Delphi procedure. Items were selected for development of the item bank/CAT if rated as adequate by at least 80% of the participants (when 50% or less they were excluded). On the basis of participants' comments, remaining items were re-worded and re-evaluated in the following round. The procedure stopped when all items were selected or rejected. RESULTS: Ten rheumatologists, 20 nurses and 15 RA patients participated. After the first round, 40% of the traditional items and 60% of the BRAF-MDQ items were directly selected and 3 items of the traditional questionnaires and 1 item of the BRAF-MDQ were directly excluded. Remaining items were re-worded, eight of which were presented for re-evaluation in the second round. Finally, 90% of the items from the traditional questionnaires and 95% of the items from the new BRAF-MDQ were included in our item pool. CONCLUSIONS: Fifty-five of the 60 items (92%) from fatigue questionnaires proved to have good content validity and were feasible for use in the Netherlands, some after adaptation.

Mitoferrin is essential for erythroid iron assimilation.

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.

The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish.

The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.

Examination of the current top candidate genes for AD in a genome-wide association study.

With the advent of technologies that allow simultaneous genotyping of thousands of single-nucleotide polymorphisms (SNPs) across the genome, the genetic contributions to complex diseases can be explored at an unprecedented detail. This study is among the first to apply the genome-wide association study (GWAS) approach to Alzheimer disease (AD). We present our GWAS results from the German population for genes included in the 'Top Results' list on the AlzGene database website. In addition to the apolipoprotein E locus, we identified nominally significant association signals in six of the ten genes investigated, albeit predominantly for SNPs other than those already published as being disease associated. Further, all of the four AD genes previously identified through GWAS also showed nominally significant association signals in our data. The results of our comparative study reinforce the necessity for replication and validation, not only of GWAS but also of candidate gene case-control studies, in different populations. Furthermore, cross-platform comparison of genotyping results can also identify new association signals. Finally, our data confirm that GWAS, regardless of the platform, are valuable for the identification of genetic variants associated with AD.

In vivo imaging neurotransmitter function. The rat 6-hydroxydopamine model and its relevance for human Parkinson's disease.

This article gives an overview of those small animal imaging studies which have been conducted on neurotransmitter function in the rat 6-hydoxydopamine (6-OHDA) model of Parkinson's disease, and discusses findings with respect to the outcome of clinical studies on Parkinsonian patients.

Subclinical Pulmonary Involvement in Active IBD Responds to Biologic Therapy.

OBJECTIVE: Increased mortality from respiratory diseases was observed in epidemiological studies of patients with ulcerative colitis [UC] as a potentially underestimated extraintestinal manifestation. We therefore investigated the presence of pulmonary manifestations of inflammatory bowel disease [IBD] and the potential effect of tumour necrosis factor alpha [TNF-α] inhibitors on pulmonary function tests [PFT] in a prospective, longitudinal study. METHODS: In all, 92 consecutive patients with IBD (49 Crohn´s disease [CD], 43 UC) and 20 healthy controls were recruited. Fifty patients with IBD were in remission, and 42 had active disease with 22 of these being examined before and 6 weeks after initiating anti-TNF therapy. Pulmonary function tests [PFT] were evaluated using the Medical Research Council [MRC] dyspnoea index and a standardized body plethysmography. IBD activity was assessed using Harvey-Bradshaw index for CD and partial Mayo score for UC. Data are presented as mean ± standard error of the mean [SEM]. RESULTS: Patients with active IBD showed significant reduction of PFT. Forced expiration [Tiffeneau index] values [FEV1%] were significantly reduced in IBD patients with active disease [78.8 ± 1.1] compared with remission [86.1 ± 0.9; p = 0.0002] and with controls [87.3 ± 1.3; p = 0.001]. Treatment with anti-TNF induced a significant relief in obstruction [p = 0.003 for FEV1% in comparison with baseline levels]. The level of pulmonary obstruction significantly correlated with clinical inflammation scores [HBI or Mayo]. CONCLUSIONS: PATIENTS: with active IBD present with significant obstructive abnormalities in their PFTs. Obstruction is related to inflammatory activity, with anti-TNF improving PFTs. Pulmonary obstruction and possibly chronic bronchopulmonary inflammation is an overlooked problem in active IBD that is probably obscured by intestinal symptoms.

The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function.

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. In the absence of CCT-controlled protein folding, Th2 polarization diverges from normal differentiation with paradoxical continued IFN-γ expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.

Characterization of the zebrafish T cell receptor beta locus.

Zebrafish (Danio rerio) has become an increasingly important model for immunological study. Its immune system is remarkably similar to that of mammals and includes both the adaptive and innate branches. Zebrafish T cells express functional T cell receptors (TCR), and all four TCR loci are present within the genome. Using 5'-rapid amplification of cDNA ends, we cloned and sequenced zebrafish TCRbeta transcripts. TCRbeta VDJ coding joints demonstrate conservation of mechanisms used by other vertebrate species to increase junctional diversity. Using the sequences obtained, along with previously published data, we comprehensively annotated the zebrafish TCRbeta locus. Overall, organization of the locus resembles that seen in mammals. There are 51 V segments, a single D segment, 27 Jbeta1 segments, a single Jbeta2 segment, and two constant regions. This description of the zebrafish TCRbeta locus has the potential to enhance immunological research in zebrafish and further our understanding of mammalian TCR repertoire generation.

Polymer Topology Determines the Formation of Protein Corona on Core-Shell Nanoparticles.

Linear and cyclic poly(2-ethyl-2-oxazoline) (PEOXA) adsorbates provide excellent colloidal stability to superparamagnetic iron oxide nanoparticles (FexOy NPs) within protein-rich media. However, dense shells of linear PEOXA brushes cannot prevent weak but significant attractive interactions with human serum albumin. In contrast, their cyclic PEOXA counterparts quantitatively hinder protein adsorption, as demonstrated by a combination of dynamic light scattering and isothermal titration calorimetry. The cyclic PEOXA brushes generate NP shells that are denser and more compact than their linear counterparts, entirely preventing the formation of a protein corona as well as aggregation, even when the lower critical solution temperature of PEOXA in a physiological buffer is reached.

Systematic expression profiling of innate immune genes defines a complex pattern of immunosenescence in peripheral and intestinal leukocytes.

Immunosenescence is characterized by a quantitative decline of adequate immune responses, which renders the elderly individual particularly susceptible to bacterial, viral and fungal pathogens. Whereas changes of the aging adaptive immune system (for example, reduced immunoglobulin secretion) have been extensively characterized, alterations of the innate immune system are still poorly understood. The aim of the present study was to systematically examine mRNA expression levels of innate immune genes and proinflammatory cytokines in peripheral and intestinal leukocytes of subjects of different ages. In both, whole blood samples and in colonic biopsies most of the Toll-like receptors (TLRs) and nucleotide-binding and oligomerization domain-like receptors (NLRs) transcript levels were significantly downregulated in elderly subjects (90-99 years). Older individuals, when compared to the younger, exhibited an increased expression and/or secretion of proinflammatory cytokines by peripheral and intestinal leukocytes as well as an increased level of nuclear factor-kappaB activation in colonic biopsies. The observed downregulation of TLRs and NLRs during the aging process may contribute to the lack of effective recognition of invading pathogens or the commensal flora. This effect results in aberrant secondary immune cell activation and could significantly contribute to morbidity and mortality at advanced age.

From model cell line to in vivo gene expression: disease-related intestinal gene expression in IBD.

Crohn's disease (CD) and ulcerative colitis (UC) are subforms of inflammatory bowel diseases (IBD). Genetic and environmental factors influencing the onset and course of the diseases have been recently identified. This study uses a two-step approach to detect genes involved in the pathogenesis of IBD by microarray analysis and real-time PCR (RT-PCR). In a first step, microarray expression screening was used to obtain tumour necrosis factor-alpha (TNF-alpha) induction profiles of two human cell lines to represent the tissue cell types involved in IBD. In a second step, a subset of differentially expressed genes was examined by real-time PCR in intestinal biopsy samples of normal controls (NC) compared with UC and CD patients, as well as to a cohort of patients suffering from intestinal diseases other than IBD. Data were obtained from 88 CD, 88 UC, 53 non-IBD patients (inflammatory control), DC and 45 NC individuals. The experimental design enabled the identification of disease-specific expressed genes. DnaJ (Hsp40) homologue, subfamily B, member 5 (DNAJB5) was downregulated in intestinal biopsy samples of the UC cohort compared with NC. A difference in JUNB expression levels was observed by comparing biopsy samples from inflamed and non-inflamed areas of UC patients. Transcript expression differences between IBD and control cohorts were found by examining histamine N-methyltransferase (HNMT), interleukin-1A (IL-1A) and proplatelet basic protein (PPBP) expression. The experimental procedure represents an approach to identify disease-relevant genes, which is applicable to any disease where appropriate model systems are available.

Immunology and zebrafish: spawning new models of human disease.

The zebrafish has emerged as a powerful new vertebrate model of human disease. Initially prominent in developmental biology, the zebrafish has now been adopted into varied fields of study including immunology. In this review, we describe the characteristics of the zebrafish, which make it a versatile model, including a description of its immune system with its remarkable similarities to its mammalian counterparts. We review the zebrafish disease models of innate and adaptive immunity. Models of immune system malignancies are discussed that are either based on oncogene over-expression or on our own forward-genetic screen that was designed to identify new models of immune dysregulation.

Detection of diverse bacterial signatures in atherosclerotic lesions of patients with coronary heart disease.

BACKGROUND: Bacterial infection has been discussed as a potential etiologic factor in the pathophysiology of coronary heart disease (CHD). This study analyzes molecular phylogenies to systematically explore the presence, frequency, and diversity of bacteria in atherosclerotic lesions in patients with CHD. METHODS AND RESULTS: We investigated 16S rDNA signatures in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients with CHD, control material from postmortem patients (n=15), and heart-beating organ donors (n=11) using clone libraries, denaturating gradient gel analysis, and fluorescence in situ hybridization. Bacterial DNA was found in all CHD patients by conserved PCR but not in control material or in any of the normal/unaffected coronary arteries. Presence of bacteria in atherosclerotic lesions was confirmed by fluorescence in situ hybridization. A high overall bacterial diversity of >50 different species, among them Staphylococcus species, Proteus vulgaris, Klebsiella pneumoniae, and Streptococcus species, was demonstrated in >1500 clones from a combined library and confirmed by denaturating gradient gel analysis. Mean bacterial diversity in atheromas was high, with a score of 12.33+/-3.81 (range, 5 to 22). A specific PCR detected Chlamydia species in 51.5% of CHD patients. CONCLUSIONS: Detection of a broad variety of molecular signatures in all CHD specimens suggests that diverse bacterial colonization may be more important than a single pathogen. Our observation does not allow us to conclude that bacteria are the causative agent in the etiopathogenesis of CHD. However, bacterial agents could have secondarily colonized atheromatous lesions and could act as an additional factor accelerating disease progression.

Method for isolation of PCR-ready genomic DNA from zebrafish tissues.

Here we describe a method for the isolation of PCR-ready genomic DNA from various zebrafish tissues that is based on a previously published murine protocol. The DNA solutions are of sufficient quality to allow PCR detection of transgenes from all commonly used zebrafish tissues. In sperm, transgene amplification was successful even when diluted 1000-fold, allowing easy identification of transgenic founders. Given its speed and low cost, we anticipate that the adoption of this method will streamline DNA isolation for zebrafish research.