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Plant phloem sap is an extreme diet for animals, partly because of its high and variable sugar content. The physiological and feeding traits of the pea aphid Acyrthosiphon pisum that define the upper and lower limits to the range of dietary sucrose concentrations utilised by this insect were determined principally using chemically defined diets containing 0.125-1.5 mol l-1 sucrose. On the diets with 0.125 mol l-1 and 1.5 mol l-1 sucrose, the aphids died as larvae within 8 and 14 days of birth, respectively. On the other diets, 60-96% of aphids developed to adulthood, and the 0.5 mol l-1 and 0.75 mol I-1 diets supported the highest fecundity. The diet with 0.125 mol l-1 sucrose was ingested at 36% of the rate of the 0.25 mol I-1 sucrose diet, but >90% of ingested sucrose-carbon was assimilated on both diets. This suggests that the lower limit is dictated by the aphid feeding response, specifically, a requirement for a minimal concentration of sucrose for sustained feeding. The haemolymph osmotic pressure of aphids on diets with 0.125-1.5 mol I-1 sucrose was up to 68% higher than on 0.125-1.0 mol l-1 sucrose diets, but diet consumption and sucrose-carbon assimilation was not reduced on the very high sucrose diets relative to 1.0 mol l-1 sucrose. This suggests that failure of the osmoregulatory capacity of the insects on high sucrose diets may define the upper limit to the range of dietary sucrose utilised by the aphids. The mean haemolymph osmotic pressure of aphids on plants with phloem sap containing 0.37-0.97 mol l-1 sucrose was 1.61±0.063 MPa (mean ± s.e.m.), not significantly different from that (1.47±0.059 MPa) on diets with 0.25-1.0 mol l-1 sucrose. It is concluded that the osmoregulatory response of aphids to diets and plants are comparable, and, more generally, that the feeding and osmoregulatory capabilities of the aphids are compatible with the phloem sugar levels commonly encountered by aphids feeding on plants. [ABSTRACT FROM AUTHOR]

Abstract. The fate of radioactively labelled amino acids injected into the haemolymph of the aphid Aphis fabae was investigated. Radioactivity from each of L-[U-14C]-glutamic acid, L-[U-14C]-serine and L-[U-14C]-threonine in the aphid tissues declined exponentially, at rates of 32, 9.3 and 1.0 pmol/aphid/min, respectively. For 14C-glutamic acid, radioactivity lost from the aphids was recovered quantitatively as carbon dioxide, and radioactivity in aphid saliva and honeydew was undetectable. When expressed on a per unit aphid biomass basis, the rate of respiratory loss of glutamic acid from aphids reared on chemically-defined diets was more than double that of aphids reared on the host plant, Vicia faba. It is concluded that respiration is a quantitatively important component to the aphid metabolism of glutamic acid and other amino acids. [ABSTRACT FROM AUTHOR]

Introduction: In this study, we investigated biomarkers in a midlife, racially diverse, at-risk cohort to facilitate early identification and intervention. We examined neuroimaging measures, including resting state functional magnetic resonance imaging (fMRI), white matter hyperintensity vo (WMH), and hippocampal volumes, alongside cerebrospinal fluid (CSF) markers., Methods: Our data set included 76 cognitively unimpaired, middle-aged, Black Americans (N = 29, F/M = 17/12) and Non-Hispanic White (N = 47, F/M = 27/20) individuals. We compared cerebrospinal fluid phosphorylated tau141 and amyloid beta (Aβ)42 to fMRI default mode network (DMN) subnetwork connectivity, WMH volumes, and hippocampal volumes., Results: Results revealed a significant race × Aβ42 interaction in Black Americans: lower Aβ42 was associated with reduced DMN connectivity and increased WMH volumes regions but not in non-Hispanic White individuals., Discussion: Our findings suggest that precuneus DMN connectivity and temporal WMHs may be linked to Alzheimer's disease risk pathology during middle age, particularly in Black Americans., Highlights: Cerebrospinal fluid (CSF) amyloid beta (Aβ)42 relates to precuneus functional connectivity in Black, but not White, Americans. Higher white matter hyperintensity volume relates to lower CSF Aβ42 in Black Americans. Precuneus may be a hub for early Alzheimer's disease pathology changes detected by functional connectivity., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

The antibody repertoire (Rep-seq) sequencing revolutionized the diversity of antigen B cell receptor studies, allowing deep and quantitative analysis to decipher the role of adaptive immunity in health and disease. Particularly, horse (Equus caballus) polyclonal antibodies have been produced and used since the century XIX to treat and prophylaxis diphtheria, tuberculosis, tetanus, pneumonia, and, more recently, COVID-19. However, our knowledge about the horse B cell receptors repertories is minimal. We present a deep horse antibody heavy chain repertoire (IGH) characterization of non-infected horses using NGS (Next generation sequencing). This study obtained a mean of 248,169 unique IgM clones and 66,141 unique IgG clones from four domestic adult horses. Rarefaction analysis showed sequence coverage was between 52 % and 82 % in IgM and IgG isotypes. We observed that besides horses antibody can use all functional IGHV genes, around 80 % of their antibodies use only three IGHV gene segments, and around 55 % use only one IGHJ gene segment. This limited VJ diversity seems to be compensated by the junctional diversity of these antibodies. We observed that the junctional diversity in horse antibodies is widespread, present in more than 90 % of horse antibodies. Besides this, the length of this region seems to be higher in horse antibodies than in other species. N1 and N2 nucleotides addition range from 0 to 111 nucleotides. In addition, around 45 % of the antibody clones have more than ten nucleotides in both the N1 and N2 junction regions. This diversity mechanism may be one of the most important in providing variability to the equine antibody repertoire. This study provides new insights regarding horse antibody composition, diversity generation, and particularities compared to other species, such as the frequency and length of N nucleotide addition. This study also points out the urgent need to better characterize TdT in horses and other species to better understand antibody repertoire characteristics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Purpose of the Review: Older adults with major depressive disorder are particularly vulnerable to MDD-associated adverse cognitive effects including slowed processing speed, decreased attention, and executive dysfunction. The purpose of this review is to describe the approach to a clinical neuropsychological evaluation in older adults with MDD. Specifically, this review compares and contrasts neurocognitive screening and clinical neuropsychological evaluation procedures and details the multiple components of the clinical neuropsychological evaluation., Recent Findings: Research has shown that neurocognitive screening serves a useful purpose to provide an acute and rapid assessment of global cognitive function; however, it has limited sensitivity and specificity. The clinical neuropsychological evaluation process is multifaceted and encompasses a review of available medical records, neurobehavioral status and diagnostic interview, comprehensive cognitive and clinical assessment, examination of inclusion and diversity factors as well as symptom and performance validity, and therapeutic feedback. As such, the evaluation provides invaluable information on multiple cognitive functions, establishes brain and behavior relationships, clarifies neuropsychiatric diagnoses, and can inform the etiology of cognitive impairment. Clinical neuropsychological evaluation plays a unique and critical role in integrated healthcare for older adults with MDD. Indeed, the evaluation can serve as a nexus to synthesize information across healthcare providers in order to maximize measurement-based care that can optimize personalized medicine and overall health outcomes., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Treatment on risk adapted intensive pediatric protocols has improved outcome for teenagers and young adults (TYA) with T-cell acute lymphoblastic leukemia (T-ALL). Understanding the biology of disease in this age group and the genetic basis of relapse is a key goal as patients with relapsed/refractory disease have poor outcomes with conventional chemotherapy and novel molecular targets are required. This study examines the question of whether TYA T-ALL has a specific biological-molecular profile distinct from pediatric or adult T-ALL., Methods: Genomic characterization was undertaken of a retrospective discovery cohort of 80 patients aged 15-26 years with primary or relapsed T-ALL, using a combination of Genome-Wide Human SNP Array 6.0, targeted gene mutation and promoter methylation analyses. Findings were confirmed by MLPA, real-time quantitative PCR, and FISH. Whole Exome Sequencing was performed in 4 patients with matched presentation and relapse to model clonal evolution. A prevalence analysis was performed on a final data set of 1,792 individual cases to identify genetic lesions with age specific frequency patterns, including 972 pediatric (1-14 years), 439 TYA (15-24 years) and 381 adult (≥25 years) cases. These cases were extracted from 19 publications with comparable genomic data identified through a PubMed search., Results: Genomic characterization of this large cohort of TYA T-ALL patients identified recurrent isochromosome 7q i(7q) in our discovery cohort (n = 3). Prevalence analysis did not identify any age specific genetic abnormalities. Genomic analysis of 6 pairs of matched presentation - relapsed T-ALL established that all relapses were clonally related to the initial leukemia. Whole exome sequencing analysis revealed recurrent, targetable, mutations disrupting NOTCH, PI3K/AKT/mTOR, FLT3, NRAS as well as drug metabolism pathways., Conclusions: All genetic aberrations in TYA T-ALL occurred with an incidence similar or intermediate to that reported in the pediatric and adult literature, demonstrating that overall TYA T-ALL exhibits a transitional genomic profile. Analysis of matched presentation - relapse supported the hypothesis that relapse is driven by the Darwinian evolution of sub-clones associated with drug resistance (NT5C2 and TP53 mutations) and re-iterative mutation of known key T-ALL drivers, including NOTCH1., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Time-of-Flight (ToF) sensors and stereo vision systems are two of the most diffused depth acquisition devices for commercial and industrial applications. They share complementary strengths and weaknesses. For this reason, the combination of data acquired from these devices can improve the final depth estimation accuracy. This paper introduces a dataset acquired with a multi-camera system composed by a Microsoft Kinect v2 ToF sensor, an Intel RealSense R200 active stereo sensor and a Stereolabs ZED passive stereo camera system. The acquired scenes include indoor settings with different external lighting conditions. The depth ground truth has been acquired for each scene of the dataset using a line laser. The data can be used for developing fusion and denoising algorithms for depth estimation and test with different lighting conditions. A subset of the data has already been used for the experimental evaluation of the work "Stereo and ToF Data Fusion by Learning from Synthetic Data"., (© 2019 The Author(s).)

Somatic genetic abnormalities are initiators and drivers of disease and have proven clinical utility at initial diagnosis. However, the genetic landscape and its clinical utility at relapse are less well understood and have not been studied comprehensively. We analyzed cytogenetic data from 427 children with relapsed B-cell precursor ALL treated on the international trial, ALLR3. Also we screened 238 patients with a marrow relapse for selected copy number alterations (CNAs) and mutations. Cytogenetic risk groups were predictive of outcome postrelapse and survival rates at 5 years for patients with good, intermediate-, and high-risk cytogenetics were 68%, 47%, and 26%, respectively (P < .001). TP53 alterations and NR3C1/BTG1 deletions were associated with a higher risk of progression: hazard ratio 2.36 (95% confidence interval, 1.51-3.70, P < .001) and 2.15 (1.32-3.48, P = .002). NRAS mutations were associated with an increased risk of progression among standard-risk patients with high hyperdiploidy: 3.17 (1.15-8.71, P = .026). Patients classified clinically as standard and high risk had distinct genetic profiles. The outcome of clinical standard-risk patients with high-risk cytogenetics was equivalent to clinical high-risk patients. Screening patients at relapse for key genetic abnormalities will enable the integration of genetic and clinical risk factors to improve patient stratification and outcome. This study is registered at www.clinicaltrials.org as #ISCRTN45724312., (© 2016 by The American Society of Hematology.)

Glucocorticoid (GC) resistance is a continuing clinical problem in childhood acute lymphoblastic leukaemia (ALL) but the underlying mechanisms remain unclear. A proteomic approach was used to compare profiles of the B-lineage ALL GC-sensitive cell line, PreB 697, and its GC-resistant sub-line, R3F9, pre- and post-dexamethasone exposure. PAX5, a transcription factor critical to B-cell development was differentially regulated in the PreB 697 compared to the R3F9 cell line in response to GC. PAX5 basal protein expression was less in R3F9 compared to its GC-sensitive parent and confirmed to be lower in other GC-resistant sub-lines of Pre B 697 and was associated with a decreased expression of the PAX5 transcriptional target, CD19. Gene set enrichment analysis showed that increasing GC-resistance was associated with differentiation from preB-II to an immature B-lymphocyte stage. GC-resistant sub-lines were shown to have higher levels of phosphorylated JNK compared to the parent line and JNK inhibition caused re-sensitization to GC. Exploiting this maturation may be key to overcoming GC resistance and targeting signalling pathways linked to the maturation state, such as JNK, may be a novel approach., (© 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.)

For most children who relapse with acute lymphoblastic leukemia (ALL), the prognosis is poor, and there is a need for novel therapies to improve outcome. We screened samples from children with B-lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (www.clinicaltrials.gov, #NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3, and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high-risk features including early relapse, central nervous system (CNS) involvement, and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wild type (WT); however, by using more sensitive allelic-specific assays, low-level mutated subpopulations were found in many cases, suggesting that they survived up-front therapy and subsequently emerged at relapse. Preclinical evaluation of the mitogen-activated protein kinase kinase 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway-mutated ALL compared with WT cells both in vitro and in an orthotopic xenograft model engrafted with primary ALL; in the latter, reduced RAS-mutated CNS leukemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway-mutated relapsed ALL., (© 2014 by The American Society of Hematology.)

Casitas B-lineage lymphoma (CBL) proteins are RING finger ubiquitin E3 ligases that attenuate the signaling of receptor tyrosine kinases and are mutated in a number of myeloid disorders. In this study, mutational screening of the linker-RING domains of CBL and CBLB was performed by denaturing high performance liquid chromatography in a cohort of diagnostic (n = 180) or relapse (n = 46) samples from children with acute lymphoblastic leukemia. Somatic mutations were identified in three children, giving an overall incidence of 1.7% and involved small deletions affecting the intron/exon boundaries of exon 8, leading to skipping of exon 8 and abolishing E3 ligase function. Mutated primary samples were associated with constitutive activation of the RAS pathway and sensitivity to MEK inhibitors was shown. Thus, mutation of CBL is an alternative route to activate the RAS pathway and may identify children who are candidates for MEK inhibitor clinical trials., (Copyright © 2011 Wiley-Liss, Inc.)

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that has a dismal outcome when treated with standard therapy. For improved diagnosis and risk stratification, the initiating genetic events need to be elucidated. To investigate the genetic basis of BCP-ALL, genomes of 94 iAMP21 patients were interrogated by arrays, FISH, and multiplex ligation-dependent probe amplification. Most copy number alterations targeted chromosome 21, reinforcing the complexity of this chromosome. The common region of amplification on chromosome 21 was refined to a 5.1-mb region that included RUNX1, miR-802, and genes mapping to the Down syndrome critical region. Recurrent abnormalities affecting genes in key pathways were identified: IKZF1 (22%), CDKN2A/B (17%), PAX5 (8%), ETV6 (19%), and RB1 (37%). Investigation of clonal architecture provided evidence that these abnormalities, and P2RY8-CRLF2, were secondary to chromosome 21 rearrangements. Patient outcome was uniformly poor with standard therapy irrespective of the presence or absence of these changes. This study has provided evidence that chromosome 21 instability is the only anomaly among those so far investigated that is common to all iAMP21 patients, and therefore the initiating event is likely to be found among the complex structural rearrangements of this abnormal chromosome.

The mismatch repair (MMR) pathway is a post-replicative DNA repair process and MMR deficiency is a common feature of ALL cell lines. In this study we have investigated MMR deficiency in a large cohort of primary relapsed ALL (n=40) and investigated coding microsatellites (MS) of the lymphoid transcription factors, PAX5 and IKZF1 as downstream target genes. Only one patient showed MMR deficiency, as evidenced by microsatellite instability, which was acquired at relapse and was associated with reduced expression of both MLH1 and MSH2. Coding MS in candidate target genes including PAX5, IKZF1, BAX and TGFBRII were all wild type in this patient but the MMR-deficient cell line REH, was confirmed to have a coding MS in both PAX5 and TGFBRII. Whilst MMR deficiency is not highly prevalent in primary ALL, optimisation of the drug regimen to omit/replace thioguanines should be considered for children with MMR deficiency and/or reduced expression of key pathway components., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Flow cytometric minimal residual disease (MRD) monitoring could become more powerful if directed towards the disease-maintaining leukemic stem cell (LSC) compartment. Using a cohort of 48 children with B-lineage acute lymphoblastic leukemia (ALL), we sought the newly proposed candidate-LSC population, CD34(+)CD38(low)CD19(+), at presentation and in end of induction bone marrow samples. We identified the candidate LSC population in 60% of diagnostic samples and its presence correlated with expression of CD38, relative to that of normal B-cell progenitors. In addition, the candidate LSC was not detectable in all MRD positive samples. The absence of the population in 40% of diagnostic and 40% of MRD positive samples does not support the use of this phenotype as a generic biomarker to track LSCs and suggests that this phenotype may be an artifact of CD38 underexpression rather than a biologically distinct LSC population. ClinicalTrials.gov Identifier: NCT00222612.

We report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11;q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.

Minimal residual disease detection, used for clinical management of children with acute lymphoblastic leukemia, can be performed by molecular analysis of antigen-receptor gene rearrangements or by flow cytometric analysis of aberrant immunophenotypes. For flow minimal residual disease to be incorporated into larger national and international trials, a quality assured, standardized method is needed which can be performed in a multi-center setting. We report a four color, flow cytometric protocol established and validated by the UK acute lymphoblastic leukemia Flow minimal residual disease group. Quality assurance testing gave high inter-laboratory agreement with no values differing from a median consensus value by more than one point on a logarithmic scale. Prospective screening of B-ALL patients (n=206) showed the method was applicable to 88.3% of patients. The minimal residual disease in bone marrow aspirates was quantified and compared to molecular data. The combined risk category concordance (minimal residual disease levels above or below 0.01%) was 86% (n=134). Thus, this standardized protocol is highly reproducible between laboratories, sensitive, applicable, and shows good concordance with molecular-based analysis.

Inactivation of the tumor suppressor gene, CDKN2A, can occur by deletion, methylation, or mutation. We assessed the principal mode of inactivation in childhood acute lymphoblastic leukemia (ALL) and frequency in biologically relevant subgroups. Mutation or methylation was rare, whereas genomic deletion occurred in 21% of B-cell precursor ALL and 50% of T-ALL patients. Single nucleotide polymorphism arrays revealed copy number neutral (CNN) loss of heterozygosity (LOH) in 8% of patients. Array-based comparative genomic hybridization demonstrated that the mean size of deletions was 14.8 Mb and biallelic deletions composed a large and small deletion (mean sizes, 23.3 Mb and 1.4 Mb). Among 86 patients, only 2 small deletions were below the resolution of detection by fluorescence in situ hybridization. Patients with high hyperdiploidy, ETV6-RUNX1, or 11q23/MLL rearrangements had low rates of deletion (11%, 15%, 13%), whereas patients with t(9;22), t(1;19), TLX3, or TLX1 rearrangements had higher frequencies (61%, 42%, 78%, and 89%). In conclusion, CDKN2A deletion is a significant secondary abnormality in childhood ALL strongly correlated with phenotype and genotype. The variation in the incidence of CDKN2A deletions by cytogenetic subgroup may explain its inconsistent association with outcome. CNN LOH without apparent CDKN2A inactivation suggests the presence of other relevant genes in this region.

The search for target genes involved in unbalanced acquired chromosomal abnormalities has been largely unsuccessful, because the breakpoints of these rearrangements are too variable. Here, we use the example of dicentric chromosomes in B cell precursor acute lymphoblastic leukemia to show that, despite this heterogeneity, single genes are targeted through a variety of mechanisms. FISH showed that, although they were heterogeneous, breakpoints on 9p resulted in the partial or complete deletion of PAX5. Molecular copy number counting further delineated the breakpoints and facilitated cloning with long-distance inverse PCR. This approach identified 5 fusion gene partners with PAX5: LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21), and C20orf112 (20q11.1). In each predicted fusion protein, the DNA-binding paired domain of PAX5 was present. Using quantitative PCR, we demonstrated that both the deletion and gene fusion events resulted in the same underexpression of PAX5, which extended to the differential expression of the PAX5 target genes, EBF1, ALDH1A1, ATP9A, and FLT3. Further molecular analysis showed deletion and mutation of the homologous PAX5 allele, providing further support for the key role of PAX5. Here, we show that specific gene loci may be the target of heterogeneous translocation breakpoints in human cancer, acting through a variety of mechanisms. This approach indicates an application for the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent and few genes have been identified. It can be extrapolated that this strategy will reveal that the same mechanisms operate in cancer pathogenesis in general.

Deregulation of the RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling cascade is often caused by somatic mutations in genes encoding proteins which influence the activity of this pathway and include NRAS, KRAS2, FLT3, PTPN11, and BRAF. We report the first comprehensive mutational screen of key exons of these genes in a large cohort of unselected acute lymphoblastic leukemia (ALL) cases at diagnosis (n = 86) and in a more selected cohort at disease recurrence (n = 47) using the sensitive method of denaturing high-performance liquid chromatography. We show that somatic mutations that deregulate the pathway constitute one of the most common genetic aberrations in childhood ALL (cALL), being found in 35% of diagnostic and 25% of relapse samples. In matched presentation/relapse pairs, mutations predominating at relapse could be shown to be present at very low levels at diagnosis using allele-specific PCR, thus implicating the mutated clone in disease progression. Importantly, in primary samples, we show that mutations are associated with activated ERK and differential cytotoxicity to MEK-ERK inhibitors was shown for some patients. Inhibitors of the pathway, which are currently undergoing clinical trial, may be a novel therapeutic option for cALL, particularly at relapse.

Glucocorticoids (GCs) specifically induce apoptosis in malignant lymphoblasts and are thus pivotal in the treatment of acute lymphoblastic leukemia (ALL). However, GC-resistance is a therapeutic problem with an unclear molecular mechanism. We generated approximately 70 GC-resistant sublines from a GC-sensitive B- and a T-ALL cell line and investigated their mechanisms of resistance. In response to GCs, all GC-resistant subclones analyzed by real-time polymerase chain reaction (PCR) showed a deficient up-regulation of the GC-receptor (GR) and its downstream target, GC-induced leucine zipper. This deficiency in GR up-regulation was confirmed by Western blotting and on retroviral overexpression of GR in resistant subclones GC-sensitivity was restored. All GC-resistant subclones were screened for GR mutations using denaturing high-pressure liquid chromatography (DHPLC), DNA-fingerprinting, and fluorescence in situ hybridization (FISH). Among the identified mutations were some previously not associated with GC resistance: A484D, P515H, L756N, Y663H, L680P, and R714W. This approach revealed three genotypes, complete loss of functional GR in the mismatch repair deficient T-ALL model, apparently normal GR genes in B-ALLs, and heterozygosity in both. In the first genotype, deficiency in GR up-regulation was fully explained by mutational events, in the second by a putative regulatory defect, and in the third by a combination thereof. In all instances, GC-resistance occurred at the level of the GR in both models.