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.

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.

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.

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.

The assessment of fatigue: Psychometric qualities and norms for the Checklist individual strength.

OBJECTIVE: The Checklist Individual Strength (CIS) measures four dimensions of fatigue: Fatigue severity, concentration problems, reduced motivation and activity. On the fatigue severity subscale, a cut-off score of 35 is used. This study 1) investigated the psychometric qualities of the CIS; 2) validated the cut-off score for severe fatigue and 3) provided norms. METHODS: Representatives of the Dutch general population (n=2288) completed the CIS. The factor structure was investigated using an exploratory factor analysis. Internal consistency and test-retest reliability were determined. Concurrent validity was assessed in two additional samples by correlating the CIS with other fatigue scales (Chalder Fatigue Questionnaire, MOS Short form-36 Vitality subscale, EORTC QLQ-C30 fatigue subscale). To validate the fatigue severity cut-off score, a Receiver Operating Characteristics analysis was performed with patients referred to a chronic fatigue treatment centre (n=5243) and a healthy group (n=1906). Norm scores for CIS subscales were calculated for the general population, patients with chronic fatigue syndrome (CFS; n=1407) and eight groups with other medical conditions (n=1411). RESULTS: The original four-factor structure of the CIS was replicated. Internal consistency (α=0.84-0.95) and test-retest reliability (r=0.74-0.86) of the subscales were high. Correlations with other fatigue scales were moderate to high. The 35 points cut-off score for severe fatigue is appropriate, but, given the 17% false positive rate, should be adjusted to 40 for research in CFS. CONCLUSION: The CIS is a valid and reliable tool for the assessment of fatigue, with a validated cut-off score for severe fatigue that can be used in clinical practice.

Patient Education in a 14-month Randomised Trial Fails to Improve Adherence in Ulcerative Colitis: Influence of Demographic and Clinical Parameters on Non-adherence.

BACKGROUND AND AIM: Recent observational studies document that non-adherence to mesalamine therapy during remission is frequent. We aimed to investigate patient impact of patient education using objective assessments of adherence. METHODS: A 14-month randomised, prospective clinical trial of adherence to mesalamine was conducted in 248 patients with ulcerative colitis [UC], Colitis Activity Index [CAI] ≤ 9, receiving standard care [n = 122] versus a standardised patient education programme [n = 126]. Primary endpoint was adherence at all visits (5-aminosalicylic acid [5-ASA] urine levels). Secondary endpoints included quality of life (inflammatory bowel disease questionnaise [IBDQ]), disease activity, partial adherence, and self-assessment of adherence. RESULTS: Patient allocation was well balanced. Baseline non-adherence was high in quiescent/mildly active UC [52.4%] without difference between the groups (52.4% of patients in the education group versus 52.5% in the standard care group [p = 0.99]). No difference between the intervention group and standard care was seen in IBDQ, partial adherence, self-assessment of adherence, or therapy satisfaction at all visits. We suggest a model in which individual risks for non-adherence are driven by patients with young age, short disease duration, and low education levels. CONCLUSIONS: Non-adherence is frequent in a population with quiescent/mildly active UC. Although more than 25% of the population was not in remission at the various time points, no relationship between disease activity and adherence was seen over the 14-month observation period. Physicians should maximise their efforts to motivate high-risk patients for adherence. Future trials should use objective exposure assessments to examine the impact of continuous education and consultations on the background of individual risks to develop non-adherence.

Cognitive behaviour therapy for chronic fatigue syndrome: Differences in treatment outcome between a tertiary treatment centre in the United Kingdom and the Netherlands.

OBJECTIVE: Cognitive behaviour therapy (CBT) reduces fatigue and disability in chronic fatigue syndrome (CFS). However, outcomes vary between studies, possibly because of differences in patient characteristics, treatment protocols, diagnostic criteria and outcome measures. The objective was to compare outcomes after CBT in tertiary treatment centres in the Netherlands (NL) and the United Kingdom (UK), using different treatment protocols but identical outcome measures, while controlling for differences in patient characteristics and diagnostic criteria. METHODS: Consecutively referred CFS patients who received CBT were included (NL: n=293, UK: n=163). Uncontrolled effect sizes for improvement in fatigue (Chalder Fatigue Questionnaire), physical functioning (SF-36 physical functioning subscale) and social functioning (Work and Social Adjustment Scale) were compared. Multiple regression analysis was used to examine whether patient differences explained outcome differences between centres. RESULTS: Effect sizes differed between centres for fatigue (Cohen's D NL=1.74, 95% CI=1.52-1.95; UK=0.99, CI=0.73-1.25), physical functioning (NL=0.99, CI=0.81-1.18; UK=0.33, CI=0.08-0.58) and social functioning (NL=1.47, CI=1.26-1.69; UK=0.61, CI=0.35-0.86). Patients in the UK had worse physical functioning at baseline and there were minor demographic differences. These could not explain differences in centre outcome. CONCLUSION: Effectiveness of CBT differed between treatment centres. Differences in treatment protocols may explain this and should be investigated to help further improve outcomes.

Pharmacological treatment with L-DOPA may reduce striatal dopamine transporter binding in in vivo imaging studies.

Numerous neurologic and psychiatric conditions are treated with pharmacological compounds, which lead to an increase of synaptic dopamine (DA) levels. One example is the DA precursor L-3,4-dihydroxyphenylalanine (L-DOPA), which is converted to DA in the presynaptic terminal. If the increase of DA concentrations in the synaptic cleft leads to competition with exogenous radioligands for presynaptic binding sites, this may have implications for DA transporter (DAT) imaging studies in patients under DAergic medication. This paper gives an overview on those findings, which, so far, have been obtained on DAT binding in human Parkinson's disease after treatment with L-DOPA. Findings, moreover, are related to results obtained on rats, mice or non-human primates. Results indicate that DAT imaging may be reduced in the striata of healthy animals, in the unlesioned striata of animal models of unilateral Parkinson's disease and in less severly impaired striata of Parkinsonian patients, if animal or human subjects are under acute or subchronic treatment with L-DOPA. If also striatal DAT binding is susceptible to alterations of synaptic DA levels, this may allow to quantify DA reuptake in analogy to DA release by assessing the competition between endogenous DA and the administered exogenous DAT radioligand.

Focus on GABA(A) receptor function. A comparative analysis of in vivo imaging studies in neuropsychiatric disorders.

UNLABELLED: Impairment of GABA(A) receptor function is increasingly recognized to play a major role in the pathophysiology of neuropsychiatric diseases including anxiety disorder (AD), major depressive disorder (MDD) and schizophrenia (SZ). PATIENTS, METHOD: We conducted a PUBMED search, which provided a total of 23 in vivo investigations with PET and SPECT, in which GABA(A) receptor binding in patients with the primary diagnosis of AD (n = 14, 160 patients, 172 controls), MDD (n = 2, 24 patients, 28 controls) or SZ (n = 6, 77 patients, 90 controls) was compared to healthy individuals. RESULTS: A retrospective analysis revealed that AD, MDD and SZ differed as to both site(s) and extent(s) of GABAergic impairment. Additionally, it may be stated that, while the decline of GABA(A) receptor binding AD involved the whole mesolimbocortical system, in SZ it was confined to the frontal and temporal cortex. CONCLUSION: As GABA is known to inhibit dopamine and serotonin, GABAergic dysfunction may be associated with the disturbances of dopaminergic and serotonergic neurotransmission in neuropsychiatric disorders.

[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.

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.

Targeting insulin-like growth factor 1 leads to amelioration of inflammatory demyelinating disease.

In patients with multiple sclerosis (MS) and in mice with experimental autoimmune encephalomyelitis (EAE), proliferating autoreactive T cells play an important role in the pathogenesis of the disease. Due to the importance of these myelin-specific T cells, these cells have been therapeutic targets in a variety of treatments. Previously we found that Lenaldekar (LDK), a novel small molecule, could inhibit exacerbations in a preclinical model of MS when given at the start of an EAE exacerbation. In those studies, we found that LDK could inhibit human T cell recall responses and murine myelin responses in vitro. In these new studies, we found that LDK could inhibit myelin specific T cell responses through the insulin-like growth factor-1 receptor (IGF-1R) pathway. Alteration of this pathway led to marked reduction in T cell proliferation and expansion. Blocking this pathway could account for the observed decreases in clinical signs and inflammatory demyelinating disease, which was accompanied by axonal preservation. Our data indicate that IGF-1R could be a potential target for new therapies for the treatment of autoimmune diseases where autoreactive T cell expansion is a requisite for disease.

Endoscopic ultrasound of the colon for the differentiation of Crohn's disease and ulcerative colitis in comparison with healthy controls.

BACKGROUND: Diagnosis of inflammatory bowel disease (IBD) is based on clinical presentation, colonoscopy and histology. Differentiation of Crohn's disease (CD) and ulcerative colitis (UC) can be difficult in some patients. Endoscopic ultrasound (EUS) provides high resolution images of the gastrointestinal wall (GI) and may be an alternative to differentiate CD/UC. AIM: EUS of the GI layers in patients with IBD and healthy controls (HC) for the differential diagnosis of UC/CD in a prospective, blinded study. METHODS: Consecutive patients with CD, UC or HC underwent EUS in the mid sigmoid colon with a forward-viewing radial echoendoscope. Mucosal, submucosal, total wall thickness (TWT) and locoregional lymphnodes (LN) were assessed by EUS in a blinded fashion. TWT was correlated with macroscopic IBD scores and histological inflammation scores. RESULTS: Total wall thickness of 61 HC was 1.71 ± 0.02 mm, and 3.51 ± 0.15 mm in n = 52 with active IBD. In patients with active UC significant thickening of the mucosa was observed but nearly normal submucosa and m.propria. In active CD significant thickening of the submucosal layer was seen with nearly normal mucosa and m.propria [MucosaUC  = 2.08 ± 0.11 mm, MucosaCD  = 1.32 ± 0.17 mm (P = 0.0001); SubmucosaUC  = 1.01 ± 0.08 mm, SubmucosaCD  = 2.01 ± 0.22 mm (P = 0.0001)]. In 73.7% of patients with active CD, but in none with UC, paracolonic lymph nodes were detected. When mucosal-submucosal and TWT and LNs were combined, the sensitivity was 92.3% for the differentiation of active UC/CD. There was a strong correlation of TWT with histological inflammation scores (UC: r = 0.43; CD: r = 0.69). CONCLUSIONS: Increased total wall thickness has a high positive predictive value for active IBD. EUS can differentiate active UC from CD and quantify the level of colonic inflammation.

DiGeorge syndrome gene tbx1 functions through wnt11r to regulate heart looping and differentiation.

DiGeorge syndrome (DGS) is the most common microdeletion syndrome, and is characterized by congenital cardiac, craniofacial and immune system abnormalities. The cardiac defects in DGS patients include conotruncal and ventricular septal defects. Although the etiology of DGS is critically regulated by TBX1 gene, the molecular pathways underpinning TBX1's role in heart development are not fully understood. In this study, we characterized heart defects and downstream signaling in the zebrafish tbx1(-/-) mutant, which has craniofacial and immune defects similar to DGS patients. We show that tbx1(-/-) mutants have defective heart looping, morphology and function. Defective heart looping is accompanied by failure of cardiomyocytes to differentiate normally and failure to change shape from isotropic to anisotropic morphology in the outer curvatures of the heart. This is the first demonstration of tbx1's role in regulating heart looping, cardiomyocyte shape and differentiation, and may explain how Tbx1 regulates conotruncal development in humans. Next we elucidated tbx1's molecular signaling pathway guided by the cardiac phenotype of tbx1(-/-) mutants. We show for the first time that wnt11r (wnt11 related), a member of the non-canonical Wnt pathway, and its downstream effector gene alcama (activated leukocyte cell adhesion molecule a) regulate heart looping and differentiation similarly to tbx1. Expression of both wnt11r and alcama are downregulated in tbx1(-/-) mutants. In addition, both wnt11r (-/-) mutants and alcama morphants have heart looping and differentiation defects similar to tbx1(-/-) mutants. Strikingly, heart looping and differentiation in tbx1(-/-) mutants can be partially rescued by ectopic expression of wnt11r or alcama, supporting a model whereby heart looping and differentiation are regulated by tbx1 in a linear pathway through wnt11r and alcama. This is the first study linking tbx1 and non-canonical Wnt signaling and extends our understanding of DGS and heart development.

Discovery of biologically active oncologic and immunologic small molecule therapies using zebrafish: overview and example of modulation of T cell activation.

Zebrafish models continue to gain popularity as in vivo models for drug discovery. Described in this overview are advantages and challenges of zebrafish drug screening, as well as a novel in vivo screen for immunomodulatory compounds using transgenic, T cell reporting zebrafish larvae designed for discovery of compounds targeting T cell leukemia. This assay system allows rapid screening of large numbers of compounds while avoiding the pitfalls of assays based on cell cultures, which lack biologic context and are afflicted by genomic instability. The rationale for this approach is based on similarities of immature normal T cells and developmentally arrested, malignant lymphoblasts in mammalian species. The screening algorithm has been used to identify a nontoxic compound with activity in both acute leukemia models and models of multiple sclerosis, demonstrating the utility of this screening procedure.

Genomic amplification of an endogenous retrovirus in zebrafish T-cell malignancies.

Genomic instability plays a crucial role in oncogenesis. Somatically acquired mutations can disable some genes and inappropriately activate others. In addition, chromosomal rearrangements can amplify, delete, or even fuse genes, altering their functions and contributing to malignant phenotypes. Using array comparative genomic hybridization (aCGH), a technique to detect numeric variations between different DNA samples, we examined genomes from zebrafish (Danio rerio) T-cell leukemias of three cancer-prone lines. In all malignancies tested, we identified recurring amplifications of a zebrafish endogenous retrovirus. This retrovirus, ZFERV, was first identified due to high expression of proviral transcripts in thymic tissue from larval and adult fish. We confirmed ZFERV amplifications by quantitative PCR analyses of DNA from wild-type fish tissue and normal and malignant D. rerio T cells. We also quantified ZFERV RNA expression and found that normal and neoplastic T cells both produce retrovirally encoded transcripts, but most cancers show dramatically increased transcription. In aggregate, these data imply that ZFERV amplification and transcription may be related to T-cell leukemogenesis. Based on these data and ZFERV's phylogenetic relation to viruses of the murine-leukemia-related virus class of gammaretroviridae, we posit that ZFERV may be oncogenic via an insertional mutagenesis mechanism.

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.

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.

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.