high-throughput-flow-cytometry-cell-based-assay-to-detect-antibodies

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

The University of Sydney

Quantitative trait analysis may shed light on mechanisms regulating hematopoiesis in vivo. Strain-dependent variation existed among C57BL/6 (B6), DBA/2, and BXD recombinant inbred mice in the responsiveness of primitive progenitor cells to the early-acting cytokines kit ligand, flt3 ligand, and thrombopoietin. A significant quantitative trait locus was found on chromosome 2 that could not be confirmed in congenic mice, however, probably because of epistasis. Because it has been shown that alleles of unknown X-linked genes confer a selective advantage to hematopoietic stem cells in vivo in humans and in cats, we also analyzed reciprocal male D2B6F1 and B6D2F1 mice, revealing an X-linked locus regulating the responsiveness of progenitor and stem cells to early-acting factors. Among DBA/2, B6, and BXD recombinant inbred mice, correlating genetic variation was found in the absolute number and frequency of Lin(-)Sca1(++)kit(+) cells, which are highly enriched in hematopoietic progenitor and stem cells, and in the number of Lin(-)Sca1(++)kit(-) cells, a population whose biologic significance is unknown, suggesting that both populations are functionally related. Suggestive quantitative trait loci (QTLs) for the number of Lin(-)Sca1(++) cells on chromosomes 2, 4, and 7 were confirmed in successive rounds of mapping. The locus on chromosome 2 was confirmed in congenic mice. We thus demonstrated genetic variation in the response to cytokines critical for hematopoiesis in vivo and in the pool size of cells belonging to a phenotype used to isolate essentially pure primitive progenitor and stem cells, and we identified loci that may be relevant to the regulation of hematopoiesis in steady state.

Genetically determined variation in the number of phenotypically defined hematopoietic progenitor and stem cells and in their response to early-acting cytokines.

Elucidation of pathways involved in mouse strain-dependent variation in the hematopoietic stem cell (HSC) compartment may reveal novel mechanisms relevant in vivo. Here, we demonstrate genetically determined variation in the proliferation of lin-Sca1++kit+ (LSK) primitive hematopoietic progenitor cells in response to transforming growth factor-beta (TGF-beta) 2, the dose response of which was biphasic with a stimulatory effect at low concentrations. In contrast, the dose responses of TGF-beta1 or -beta3 were inhibitory and did not show mouse strain-dependent variation. A quantitative trait locus (QTL) for the effect of TGF-beta2 was identified on chromosome 4 overlapping with a QTL regulating the frequency of LSK cells. These overlapping QTL were corroborated by the observation that the frequency of LSK cells is lower in adult Tgfb2+/- mice than in wild-type littermates, indicating that TGF-beta2 is a genetically determined positive regulator LSK number in vivo. Furthermore, adult Tgfb2+/- mice have a defect in competitive repopulation potential that becomes more pronounced upon serial transplantation. In fetal TGF-beta2-deficient HSCs, a defect only appears after serial reconstitution. These data suggest that TGF-beta2 can act cell autonomously and is important for HSCs that have undergone replicative stress. Thus, TGF-beta2 is a novel, genetically determined positive regulator of adult HSCs.

Quantitative trait analysis reveals transforming growth factor-beta2 as a positive regulator of early hematopoietic progenitor and stem cell function.

The number of bone marrow hematopoietic stem and progenitor cells as defined by the lineage(-), Sca1(++), c-kit(+) (LSK) phenotype and their proliferative capacity in vitro are subject to quantitative genetic variation, and several quantitative trait loci (QTL) have been identified in young mice. Because some traits affecting hematopoiesis also change with age in a mouse strain-dependent fashion, we performed quantitative trait analysis in aged BXD recombinant inbred (RI) mice for the number and frequency of LSK cells, and for their proliferative capacity in vitro. Several novel QTL were identified. The number and frequency of LSK cells in old mice correlated inversely with lifespan. Furthermore, 4 of 7 lifespan QTL overlap with QTL contributing to the number, frequency, or proliferative capacity of LSK cells in young or old mice. Taken together, these data establish a close genetic, and perhaps functional, link between genetic variation in lifespan and characteristics of stem and progenitor cells.

Quantitative genetic variation in the hematopoietic stem cell and progenitor cell compartment and in lifespan are closely linked at multiple loci in BXD recombinant inbred mice.

TGF-beta is considered a negative regulator of hemopoietic stem and progenitor cells. We have previously shown that one TGF-beta isoform, TGF-beta2, is, in fact, a positive regulator of murine hemopoietic stem cell function in vivo. In vitro, TGF-beta2, but not TGF-beta1 and TGF-beta3, had a biphasic dose response on the proliferation of purified lin-Sca1(++)kit(+) (LSK) cells, with a stimulatory effect at low concentrations, which was subject to mouse strain-dependent variation. In this study we report that the stimulatory effect of TGF-beta2 on the proliferation of LSK cells increases with age and after replicative stress in C57BL/6, but not in DBA/2, mice. The age-related changes in the TGF-beta2 effect correlated with life span in BXD recombinant strains. The stimulatory effect of TGF-beta2 on the proliferation of LSK cells requires one or more nonprotein, low m.w. factors present in fetal calf and mouse sera. The activity of this factor(s) in mouse serum increases with age. Taken together, our data suggest a role for TGF-beta2 and as yet unknown serum factors in the aging of the hemopoietic stem cell compartment and possibly in organismal aging.

The positive regulatory effect of TGF-beta2 on primitive murine hemopoietic stem and progenitor cells is dependent on age, genetic background, and serum factors.

The mechanisms regulating thymic involution are unclear. In inbred mouse strains the rate of thymic involution and the function of the hematopoietic stem cell (HSC) compartment are subject to quantitative genetic variation. We have shown previously that transforming growth factor-beta2 (TGF-beta2) is a genetically determined positive regulator of HSCs. Here, we demonstrate that genetic variation in the rate of thymic involution correlates with genetic variation in the responsiveness of hematopoietic stem and progenitor cells to TGF-beta2. Corroborating these correlations, thymic cellularity and peripheral naive T-cell frequency were higher in old Tgfb2+/- mice than in wild-type littermates. The frequency of early T-cell precursors was increased in Tgfb2+/- mice, suggesting that TGF-beta2 affects the earliest stages of T-cell development in old mice. Reciprocal transplantation experiments indicated that TGF-beta2 expressed both in the (micro)environment and in the hematopoietic system can accelerate thymic involution; however, the age of the stem cells appeared irrelevant. Thus, although thymic involution is largely determined by the aged environment, TGF-beta2 plays a major modulatory role that is subject to genetic variation and is possibly mediated through its regulatory effects on early hematopoiesis.

Transforming growth factor-beta2 is involved in quantitative genetic variation in thymic involution.

We discovered a novel population of gammadelta T cells in the mouse that accumulates with age in hematopoietic organs, but not in epithelia. These cells are CD25low (an unusual phenotype for gammadelta T cells in the mouse); express higher levels of TCRgammadelta and CD44 than do CD25- gammadelta T cells; mainly express Vgamma2, Vgamma3, and Vgamma4 chains; and are largely quiescent. A very similar cell population appears in the late stages of fetal thymus organ cultures, suggesting that the accumulation of CD44 + CD25lowTCRgammadelta + cells is a response to stress induced by aging in vivo or by culture in vitro. The precursors of CD44 + CD25lowTCRgammadelta + cells are generated during fetal or very young adult life, as this population was undetectable in aged recipients of bone marrow from old or young donors. CD44 + CD25lowTCRgammadelta + cells may be a biomarker of aging, but could also play a role in the inflammatory changes that accompany aging.

Age-related accumulation of a novel CD44 + CD25lowgammadelta T-cell population in hematopoietic organs of the mouse.

Little is known about the psychosocial status of adolescents who undergo bariatric surgery. Our objective was to describe the psychological and behavioral characteristics of patients in this age group who underwent bariatric surgery at our institution.

Psychosocial status in adolescents undergoing bariatric surgery.

Over-the-wire exchange of tunneled dialysis catheters is the standard of care per K/DOQI guidelines for treating catheter-related bacteremia. However, Gram-positive bacteremia, specifically with staphylococcus species, may compromise over-the-wire exchange due to certain biological properties. This study addressed the effectiveness of over-the-wire exchange of staphylococcus-infected tunneled dialysis catheters compared with non-staphylococcus-infected tunneled dialysis catheters.

Staphylococcus-infected tunneled dialysis catheters: is over-the-wire exchange an appropriate management option?

The aim of this study was to determine the feasibility of automated detection of adrenal nodules, a common finding on CT, using a newly developed search engine that mines dictated radiology reports. To ensure Health Insurance Portability and Accountability Act compliance, we utilized a preexisting de-identified database of 32,974 CT reports from February 1, 2009 to February 28, 2010. Common adrenal descriptors from 29 staff radiologists were used to develop an automated rule-based algorithm targeting adrenal findings. Each sentence within the free text of reports was searched with an adapted NegEx negation algorithm. The algorithm was refined using a 2-week test period of reports and subsequently validated using a 6-week period. Manual review of the 3,693 CT reports in the validation period identified 222 positive reports while the algorithm detected 238 positive reports. The algorithm identified one true positive report missed on manual review for a total of 223 true positive reports. This resulted in a precision of 91% (217 of 238) and a recall of 97% (217 of 223). The sensitivity of the query was 97.3% (95% confidence interval (CI), 93.9-98.9%), and the specificity was 99.3% (95% CI, 99.1-99.6%). The positive predictive value of the algorithm was 91.0% (95% CI, 86.6-94.3%), and the negative predictive value was 99.8% (95% CI, 99.6-99.9%). The prevalence of true positive adrenal findings identified by the query (7.1%) was nearly identical to the true prevalence (7.2%). Automated detection of language describing common findings in imaging reports, such as adrenal nodules on CT, is feasible.

Development of automated detection of radiology reports citing adrenal findings.

Le but de cette &eacute;tude &eacute;tait de d&eacute;terminer la faisabilit&eacute; de la d&eacute;tection automatis&eacute;e des nodules surr&eacute;naliennes, une conclusion commune sur les CT, en utilisant un moteur de recherche nouvellement d&eacute;velopp&eacute;s que mines dict&eacute; des rapports de radiologie. Pour assurer la conformit&eacute; Health Insurance Portability and Accountability Act, nous avons utilis&eacute; une base de donn&eacute;es d&eacute;personnalis&eacute;e pr&eacute;existante des rapports CT 32 974 du 1er f&eacute;vrier 2009 au 28 f&eacute;vrier 2010. Communes descripteurs surr&eacute;naliennes de 29 radiologues de personnel ont servi &agrave; &eacute;laborer un algorithme fond&eacute; sur des r&egrave;gles automatis&eacute; ciblant les conclusions surr&eacute;naliennes. Chaque phrase dans le texte libre des rapports a &eacute;t&eacute; fouill&eacute; avec un algorithme de n&eacute;gation NegEx adapt&eacute;. L&#39;algorithme a &eacute;t&eacute; affin&eacute;e &agrave; l&#39;aide d&#39;une p&eacute;riode d&#39;essai de 2 semaines de rapports et par la suite valid&eacute; &agrave; l&#39;aide d&#39;une p&eacute;riode de 6 semaines. Examen manuel des rapports 3 693 CT dans la p&eacute;riode de validation identifi&eacute; 222 positifs alors que l&#39;algorithme d&eacute;tect&eacute;s positifs pour 238. L&#39;algorithme identifi&eacute; un rapport positif pur rat&eacute; sur examen manuel pour un total de 223 positifs pour vrais. Il en est r&eacute;sult&eacute; une pr&eacute;cision de 91 % (217 238) et un rappel de 97 % (217 de 223). La sensibilit&eacute; de la requ&ecirc;te &eacute;tait 97,3 % (intervalle de confiance &agrave; 95 % (IC), 93,9-98,9 %) et la sp&eacute;cificit&eacute; &eacute;tait de 99,3 % (IC &agrave; 95 %, 99,1-99,6 %). La valeur pr&eacute;dictive positive de l&#39;algorithme a &eacute;t&eacute; 91,0 % (IC &agrave; 95 %, 86,6-94,3 %), et la valeur pr&eacute;dictive n&eacute;gative &eacute;tait de 99,8 % (IC &agrave; 95 %, 99,6 &agrave; 99,9 %). La pr&eacute;valence de vrais r&eacute;sultats positifs de surr&eacute;naliennes identifi&eacute; par la requ&ecirc;te (7,1 %) &eacute;tait presque identique &agrave; la pr&eacute;valence r&eacute;elle (7,2 %). Automatis&eacute; de d&eacute;tection de langue d&eacute;crivant les r&eacute;sultats communs dans les rapports d&#39;imagerie, comme nodules surr&eacute;naliennes sur CT, est r&eacute;alisable.

D&eacute;veloppement de la d&eacute;tection automatis&eacute;e des rapports de radiologie citant les conclusions surr&eacute;naliennes.

Lo scopo di questo studio era di determinare la fattibilit&agrave; di rilevazione automatizzata dei noduli surrenali, una constatazione comune su CT, utilizzando un motore di ricerca di nuova concezione che miniere dettato radiologia rapporti. Per garantire la conformit&agrave; Health Insurance Portability and Accountability Act, abbiamo utilizzato un database preesistente identificato di CT 32.974 relazioni da 1&deg; febbraio 2009 al 28 febbraio 2010. Descrittori comuni surrenalici da 29 radiologi di personale sono stati utilizzati per sviluppare un algoritmo automatizzato basato su regole, targeting surrenale scoperte. Ogni frase all&#39;interno del testo libero di report &egrave; stato cercato con un algoritmo di negazione NegEx adattato. L&#39;algoritmo &egrave; stato perfezionato con un periodo di prova di 2 settimane di rapporti e successivamente convalidato utilizzando un periodo di 6 settimane. Revisione manuale dei rapporti 3.693 CT nel periodo di convalida identificato 222 rapporti positivi, mentre l&#39;algoritmo rilevato 238 rapporti positivi. L&#39;algoritmo identificato una vera relazione positiva perso sulla revisione manuale per un totale di 223 veri rapporti positivi. Ci&ograve; ha provocato una precisione del 91% (217 di 238) e un richiamo del 97% (217 di 223). La sensibilit&agrave; della query &egrave; stato 97,3% (95% intervallo di confidenza (CI), 93.9-98,9%) e la specificit&agrave; &egrave; stata 99,3% (95% CI, 99.1-99,6%). Il valore predittivo positivo dell&#39;algoritmo era 91.0% (95% CI, 86,6-94,3%), e il valore predittivo negativo era 99,8% (95% CI, 99,6-99,9%). La prevalenza di veri risultati positivi surrenali individuate dalla query (7,1%) era quasi identica alla vera diffusione (7,2%). Automatizzata di rilevamento della lingua che descrive i risultati comuni nei rapporti di imaging, come noduli surrenali su CT, &egrave; fattibile.

Jessica Langer

Institute for Neuroscience and Muscle Research,
The Kids Research Institute at the Children's Hospital at Westmead,
Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead

The University of Sydney

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum