European recommendations and quality assurance for cytogenomic analysis of haematological neoplasms.

Cytogenomic investigations of haematological neoplasms, including chromosome banding analysis, fluorescence in situ hybridisation (FISH) and microarray analyses have become increasingly important in the clinical management of patients with haematological neoplasms. The widespread implementation of these techniques in genetic diagnostics has highlighted the need for guidance on the essential criteria to follow when providing cytogenomic testing, regardless of choice of methodology. These recommendations provide an updated, practical and easily available document that will assist laboratories in the choice of testing and methodology enabling them to operate within acceptable standards and maintain a quality service.

Sexual dimorphism in relation to adipose tissue and intrahepatocellular lipid deposition in early infancy.

Sexual dimorphism in adiposity is well described in adults, but the age at which differences first manifest is uncertain. Using a prospective cohort, we describe longitudinal changes in directly measured adiposity and intrahepatocellular lipid (IHCL) in relation to sex in healthy term infants. At median ages of 13 and 63 days, infants underwent quantification of adipose tissue depots by whole-body magnetic resonance imaging and measurement of IHCL by in vivo proton magnetic resonance spectroscopy. Longitudinal data were obtained from 70 infants (40 boys and 30 girls). In the neonatal period girls are more adipose in relation to body size than boys. At follow-up (median age 63 days), girls remained significantly more adipose. The greater relative adiposity that characterises girls is explained by more subcutaneous adipose tissue and this becomes increasingly apparent by follow-up. No significant sex differences were seen in IHCL. Sex-specific differences in infant adipose tissue distribution are in keeping with those described in later life, and suggest that sexual dimorphism in adiposity is established in early infancy.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Post-transplant T cell chimerism predicts graft versus host disease but not disease relapse in patients undergoing an alemtuzumab based reduced intensity conditioned allogeneic transplant.

In this multicentre retrospective study we have studied the impact of T cell chimerism on the outcome of 133 patients undergoing an alemtuzumab based reduced intensity conditioning allograft (RIC). The median age of the patients was 50 years (range 42-55 years). 77 patients were transplanted using an HLA identical sibling donor while 56 patients received a fully matched volunteer unrelated donor graft. 64 patients had a lymphoid malignancy and 69 were transplanted for a myeloid malignancy. 38 patients (29%) relapsed with no significant difference in risk of relapse between patients developing full donor and mixed donor chimerism in the T-cell compartment on D+90 and D+180 post transplant. Day 90 full donor T cell chimerism correlated with an increased incidence of acute GVHD according to NIH criteria (p=0.0004) and the subsequent development of chronic GVHD. Consistent with previous observations, our results confirmed a correlation between the establishment of T cell full donor chimerism and acute GVHD in T deplete RIC allografts. However our study failed to identify any correlation between T cell chimerism and relapse risk and challenge the use of pre-emptive donor lymphocyte infusions (DLI) in patients with mixed T cell chimerism transplanted using an alemtuzumab based RIC regimen.

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi).

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.

Rhesus monkeys mislocalize saccade targets flashed for 100ms around the time of a saccade.

Humans and monkeys mislocalize targets flashed around the time of a saccade. Here, we present data from three monkeys on a double-step task with a 100ms target duration. All three subjects mislocalized targets that were flashed around the time of the first saccade, in spite of long intersaccadic intervals. The error was consistently in the direction opposite that of the saccade, and occurred in some cases when the target presentation was entirely presaccadic. This is inconsistent with a theory invoking a damped representation of eye position, but it is consistent with the hypothesis that it is due to an error in peri-saccadic remapping.

Contaminant-associated disruption of vitamin A and its receptor (retinoic acid receptor alpha) in free-ranging harbour seals (Phoca vitulina).

Polychlorinated biphenyls (PCBs) have been associated with a number of toxic effects in marine mammals such as endocrine disruption and immunotoxicity that, in turn, are widely thought to have contributed to population level impacts including reproductive failure and outbreaks of disease. In this study, the dietary hormone vitamin A and expression levels of one of its receptors, retinoic acid receptor alpha (RARalpha), were used as biomarkers of PCB-associated health effects in harbour seals. Harbour seal pups (n=24) were live-captured in coastal British Columbia, Canada, and Washington State, USA, and sampled for whole blood (to obtain peripheral blood mononuclear cells, PBMCs) and blood plasma, as well as biopsies of blubber and skin. Concentrations of circulatory vitamin A (retinol) in plasma and stored vitamin A in blubber were negatively associated with blubber PCB concentrations (R=-0.518, p=0.013 and R=-0.645, p=0.009, respectively). However, vitamin A concentrations in skin, an important target tissue, remained constant, which likely reflects a compensatory transfer from blubber to maintain physiological functions. In addition, we characterized the harbour seal RARalpha, and investigated its expression levels as a potential biomarker in seals. RARalpha expression in blubber, but not on PBMCs, was elevated in more contaminated animals (R=0.580, p=0.009). This may represent a direct contaminant-related effect, or, a compensation for the contaminant-related disruption of (circulatory and/or blubber) hormone levels. Since vitamin A is critical to developmental, reproductive and immunological health, our observations of a contaminant-related disruption of its physiology in free-ranging seals may portend population level consequences. Vitamin A concentrations and RARalpha expression levels can therefore represent relevant and sensitive biomarkers of PCB-associated toxic effects in toxicological studies of marine mammals.

Relationship of deep white matter hyperintensities and cerebral blood flow in severe carotid artery stenosis.

Leukoaraiosis (LA) has been associated with abnormalities of both large and small blood vessels. This study attempts to clarify the pathogenesis of LA by testing the hypothesis that increased frequency of LA with occlusive extra-cranial arterial disease results directly from global reduction in cerebral blood flow (CBF). Thirty-five normal subjects and 55 patients with carotid stenosis (>70%) were studied using MR. CBF was measured using phase contrast MR angiography and LA was scored using previously validated scoring system. Patients were divided into those with evidence of previous infarction on MRI and those without. LA was more severe in patients than in normal subjects (P<0.01) and correlated with age in normal subjects but not in patients. CBF in patients with (809+/-214 ml/min) and without infarction (mean 792+/-181 ml/min) was significantly lower than in normal subjects (mean 1073+/-194 ml/min). There was no correlation between the severity of LA and measured CBF in any group. The severity of LA is greater in patients with severe carotid stenosis but is not correlated to reductions in CBF. This suggests that microvascular abnormality is the dominant pathogenetic factor in LA even in the presence of severe stenotic/occlusive large vessel disease.

Treatment response in late-onset depression: relationship to neuropsychological, neuroradiological and vascular risk factors.

BACKGROUND: Late-onset depressive disorder is associated with white matter lesions and neuropsychological deficits that in some studies are linked to a poorer outcome for depression. Some white matter lesions may be vascular in origin. This study investigated the relationship between response or non-response to antidepressant monotherapy and neuropsychological function, structural brain measures and vascular factors. METHOD: This was a case control study. Fifty patients with late-onset major depressive disorder (29 who were responders to antidepressant monotherapy and 21 who were not) were compared with 35 non-depressed control subjects. Measures included assessment of vascular risk factors, neuropsychological testing and a magnetic resonance imaging (MRI) scan. RESULTS: After adjustment for depressed mood and medication at evaluation, both patient groups had significantly more impairment compared to control subjects on verbal learning tasks involving immediate or delayed recall. Patients who did not respond to antidepressant monotherapy had significantly poorer performance than controls on tests involving visuospatial ability, language, word recognition and tests of executive function, whereas there were no differences between control subjects and responders. On two tests of executive function (verbal fluency and the Stroop test) non-responders scored significantly worse than responders. There were no significant group differences on MRI measures of atrophy or of white matter lesions apart from a higher periventricular hyperintensity score in non-responders compared to controls. There were no group differences on measures of vascular disease. CONCLUSION: The results lend support to the emerging evidence that resistance to treatment in late-onset depression may be associated with impaired executive function. Subtle cerebrovascular mechanisms may be involved.

Right-hemisphere encephalopathy in elderly subjects with schizophrenia: evidence from neuropsychological and brain imaging studies.

RATIONALE: Cognitive impairment is a recognised feature of schizophrenia. Elderly patients with early-acquired schizophrenia are seriously affected, with a proportion of them showing clinically significant dementia, not accounted for by any recognized degenerative processes common in this age group, such as Alzheimer's disease. Progression of cognitive deficits is described in elderly institutionalised patients, but disputed amongst community dwelling subjects. The pattern of cognitive deficits in this age group is not yet clearly defined, although there is some evidence that it differs from that in Alzheimer's disease. There is little evidence of any underlying specific brain abnormality. OBJECTIVES: To characterize the neuropsychological deficits in elderly schizophrenia patients and distinguish them from those in Alzheimer's disease. To establish the presence of underlying structural brain abnormality using MRI. METHODS: Twenty-eight elderly schizophrenia patients with onset before the age of 45 years carried out neuropsychology tests. Twelve scored in the dementia range and were compared with 16 equally impaired patients with early Alzheimer's disease. Thirteen of the schizophrenia patients consented to brain MRI. The imaging data were analysed using a newly developed automated method of measuring CSF volume distributions and compared with data from 30 age-matched normal controls. RESULTS: The schizophrenia group was more impaired on visuo-spatial tasks than the Alzheimer's group but less impaired on corresponding verbal tasks, despite similar overall cognitive impairment. The MR scans revealed right-sided enlargement of ventral CSF spaces in the schizophrenia patients especially in the posterior third, and this correlated with their impaired performance on visuo-spatial tasks. CONCLUSIONS: The results suggest that right hemisphere impairment underlies the specific profile of cognitive impairment in elderly patients with schizophrenia.

Dead? Or just cold: profoundly hypothermic patient with no signs of life.

A 37 year old man was found in his garden cold with no signs of life. Pupils were fixed and dilated. Electrocardiography showed asystole initially. The paramedic crew started cardiopulmonary resuscitation and transferred him to the accident and emergency department. His temperature was 17.0 degrees C. Cardiopulmonary resuscitation was continued for three hours before rewarming using partial cardiopulmonary bypass. He eventually regained spontaneous cardiac output and made a full neurological recovery. Hypothermic patients with no evidence of life cannot be assumed to be dead as there is a chance of full recovery when fully warmed.

Seroprevalence of Toxoplasma gondii in harbor seals (Phoca vitulina) in southern Puget Sound, Washington.

As part of the Puget Sound Ambient Monitoring Program of the Washington Department of Fish and Wildlife, serum samples from 380 harbor seals (Phoca vitulina) were tested for antibodies to Toxoplasma gondii in the modified agglutination test (MAT) incorporating formalin-fixed tachyzoites and mercaptoethanol. Antibodies to T. gondii were found in 29 of 380 (7.6%) seals with titers of 1:25 in 13, 1:50 in 14, and > or = 1:500 in 2 seals. Results indicate natural exposure of these wild marine mammals to T. gondii oocysts.

Utilization of medical services and quality of life among low-income patients with generalized anxiety disorder attending primary care clinics.

OBJECTIVE: Anxiety disorders appear to influence morbidity and medical utilization. However, little is known about the relationship between Generalized Anxiety Disorder, quality of life, and medical utilization, especially among low-income patients. The goals of this investigation were to 1) determine if low-income patients with GAD utilize medical services more than patients with other Axis I diagnoses, or no psychopathology, and 2) compare the health-related quality of life of these three groups. METHOD: Participants were randomly recruited from public primary care clinics and administered intake assessments of demographics, stress, and health-related self-report questionnaires. At the end of the first year a structured psychiatric interview was administered (N = 431). Over the second year, patients (n = 360) were administered a health-related quality of life measure every three months for four assessments. Medical charts were abstracted to collect information about chronic illnesses and visits to outpatient clinics and the emergency department during the two years. RESULTS: Patients were predominantly middle-aged, low-income, uninsured African-American females. In this low-income sample, patients with GAD utilized the emergency department more and reported poorer quality of life than patients with other Axis I disorders and patients without any psychopathology. CONCLUSION: Low-income patients with GAD utilize the emergency department more and report poorer quality of life than patients with other Axis I disorders and patients without any psychopathology. Programs to identify and treat patients with GAD may yield improvements in quality of life, as well as reduce emergency department utilization.

Neoplastic transformation by Notch requires nuclear localization.

Notch proteins are plasma membrane-spanning receptors that mediate important cell fate decisions such as differentiation, proliferation, and apoptosis. The mechanism of Notch signaling remains poorly understood. However, it is clear that the Notch signaling pathway mediates its effects through intercellular contact between neighboring cells. The prevailing model for Notch signaling suggests that ligand, presented on a neighboring cell, triggers proteolytic processing of Notch. Following proteolysis, it is thought that the intracellular portion of Notch (N(ic)) translocates to the nucleus, where it is involved in regulating gene expression. There is considerable debate concerning where in the cell Notch functions and what proteins serve as effectors of the Notch signal. Several Notch genes have clearly been shown to be proto-oncogenes in mammalian cells. Activation of Notch proto-oncogenes has been associated with tumorigenesis in several human and other mammalian cancers. Transforming alleles of Notch direct the expression of truncated proteins that primarily consist of N(ic) and are not tethered to the plasma membrane. However, the mechanism by which Notch oncoproteins (generically termed here as N(ic)) induce neoplastic transformation is not known. Previously we demonstrated that N1(ic) and N2(ic) could transform E1A immortalized baby rat kidney cells (RKE) in vitro. We now report direct evidence that N1(ic) must accumulate in the nucleus to induce transformation of RKE cells. In addition, we define the minimal domain of N1(ic) required to induce transformation and present evidence that transformation of RKE cells by N1(ic) is likely to be through a CBF1-independent pathway.

Central GABA activation and behaviors evoked by tail-pinch stress in the rat.

Experiments were conducted to evaluate the possibility that central GABA(A) receptors are involved in the stress response of rats. Separate groups of animals were implanted bilaterally with cannulae in the lateral cerebral ventricle, substantia nigra, and anterior to the rostral margin of the substantia nigra. Microinjections of the GABA(A) agonist muscimol into each of these areas augmented the stress response evoked by moderate tail pinch. Although consistent changes in the amount of food eaten in response to stress were not observed, stress-evoked gnawing was significantly increased by muscimol at all three sites. Additionally, intraventricular muscimol resulted in an enhancement of stress-evoked oral stereotypy, revolution (escape behavior), and vocalization. The data suggest that a GABAergic component exists in the central mediation of stress. The results are discussed in regard to possible interactions between GABA and central dopamine systems.

cDNA isolation, genomic structure, regulation, and chromosomal localization of human lung Kruppel-like factor.

Lung Kruppel-like factor (LKLF) is a zinc finger transcription factor critical for embryonic development. We have previously identified and isolated the mouse LKLF gene and examined its role using gene targeting. In this report, we describe the isolation and molecular characterization of the human homolog of murine LKLF. The human and mouse LKLF homologs exhibit an 85% nucleotide identity and share 90% amino acid similarity. Furthermore, the 5' sequence in the proximal promoter region and 3' untranslated region are also conserved between the two species. Of particular interest is the finding that while sequences in the proximal promoter have diverged between mouse and human, a region of 75 nucleotides is essentially identical. Site-directed mutagenesis in this region impairs the ability of the LKLF promoter to drive reporter gene expression, indicating that it represents a novel transcriptional element important in the regulation of LKLF gene expression. The activation domain is highly proline-rich and, similar to mouse LKLF, contains 22% proline residues. The human LKLF transcriptional unit is located in a genomic region of approximately 3 kb on chromosome 19p13.1. This region of chromosome 19 is known to contain genes involved in various human diseases. Like mouse LKLF, human LKLF consists of three exons that are interrupted by two small introns. The locations of intron/exon boundaries and splice sites are conserved between two homologs. Northern analysis shows that LKLF is expressed in lung in addition to heart, skeletal muscle, placenta, and pancreas. The isolation and chromosomal mapping of human LKLF will make it possible to initiate studies devoted to assess the involvement of this gene in human disease(s).

Clinical evaluation of the new Pall leucocyte-depleting blood cardioplegia filter (BC1).

It is now widely acknowledged that autologous leucocytes are inappropriately activated during cardiopulmonary bypass (CPB). Removal of these activated leucocytes has been proposed as a clinical intervention. Several papers have recently reported benefits of systemic leucocyte depletion during CPB. There is also evidence that leucocyte-depleted blood cardioplegia is advantageous in the globally ischaemic human heart transplant setting. Recently, a new leucocyte-depleting filter for blood cardioplegia has been developed (Pall, BC1). In this paper, we report on the safety and efficiency of this device in the clinical situation. Fourteen patients undergoing routine cardiac surgery were recruited into this study. The BC1 blood cardioplegia filter was found to be an efficient leucocyte-depleting device, removing in excess of 70% (p = 0.001) of white blood cells, on average, from up to 5.3 litres of blood cardioplegia. The filter removed a small proportion of platelets (typically 11.3%), however, this was not statistically significant and no bleeding problems were encountered. Red cell removal was negligible and was not statistically significant, and no evidence of haemolysis was noted. The filter offered a very low resistance to flow with a mean pressure drop (deltaP) of 10.8 mmHg at a mean flow rate of 315 ml/min. We conclude that the Pall BC1 filter is a safe and efficient device for use with blood cardioplegia.