Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes.

We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-ß and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell-cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.

Targeting survivin and p53 in pediatric acute lymphoblastic leukemia.

Despite advances in treatment and outcomes for patients with pediatric acute lymphoblastic leukemia (ALL), there continue to be subsets of patients who are refractory to standard chemotherapy and hematopoietic stem cell transplant. Therefore, novel gene targets for therapy are needed to further advance treatment for this disease. RNA interference technology has identified survivin as a potential therapeutic target. Survivin, a member of the inhibitor of apoptosis (IAP) proteins and chromosome passenger complex, is expressed in hematologic malignancies and overexpressed in relapsed pediatric ALL. Our studies show that survivin is uniformly expressed at high levels in multiple pediatric ALL cell lines. Furthermore, silencing of survivin expression in pediatric ALL cell lines as well as primary leukemic blasts reduces viability of these cells. This includes cell lines derived from patients with relapsed disease featuring cytogenetic anomalies such as t(12;21), Philadelphia chromosome t(9;22), t(1;19) as well as a cell line carrying t(17;19) from a patient with de novo ALL. Furthermore, inhibition of survivin increases p53-dependent apoptosis that can be rescued by inhibition of p53. Finally, a screen of randomly selected primary patient samples confirms that survivin-specific small interfering RNA and survivin-targeted drug, YM155, effectively reduce viability of leukemic blasts.

The Krüppel-like zinc finger protein GLIS3 transactivates neurogenin 3 for proper fetal pancreatic islet differentiation in mice.

AIMS/HYPOTHESIS: Mutations in GLIS3, which encodes a Krüppel-like zinc finger transcription factor, were found to underlie sporadic neonatal diabetes. Inactivation of Glis3 by gene targeting in mice was previously shown to lead to neonatal diabetes, but the underlying mechanism remains largely unknown. We aimed to elucidate the mechanism of action of GLIS family zinc finger 3 (GLIS3) in Glis3 ( -/- ) mice and to further decipher its action in in-vitro systems. METHODS: We created Glis3 ( -/- ) mice and monitored the morphological and biochemical phenotype of their pancreatic islets at different stages of embryonic development. We combined these observations with experiments on Glis3 expressed in cultured cells, as well as in in vitro systems in the presence of other reconstituted components. RESULTS: In vivo and in vitro analyses placed Glis3 upstream of Neurog3, the endocrine pancreas lineage-defining transcription factor. We found that GLIS3 binds to specific GLIS3-response elements in the Neurog3 promoter, activating Neurog3 gene transcription both directly, and synergistically with hepatic nuclear factor 6 and forkhead box A2. CONCLUSIONS/INTERPRETATION: These results indicate that GLIS3 controls fetal islet differentiation via direct transactivation of Neurog3, a perturbation that causes neonatal diabetes in mice.

Chromosomes with delayed replication timing lead to checkpoint activation, delayed recruitment of Aurora B and chromosome instability.

Certain chromosome rearrangements display a significant delay in chromosome replication timing (DRT) that is associated with a subsequent delay in mitotic chromosome condensation (DMC). DRT/DMC chromosomes are common in tumor cells in vitro and in vivo and occur frequently in cells exposed to ionizing radiation. A hallmark for these chromosomes is the delayed phosphorylation of serine 10 of histone H3 during mitosis. The chromosome passenger complex, consisting of multiple proteins including Aurora B kinase and INCENP is thought to be responsible for H3 phosphorylation, chromosome condensation and the subsequent segregation of chromosomes. In this report, we show that chromosomes with DRT/DMC contain phosphorylated Chk1, consistent with activation of the S-M phase checkpoint. Furthermore, we show that INCENP is recruited to the DRT/DMC chromosomes during all phases of mitosis. In contrast, Aurora B kinase is absent on DRT/DMC chromosomes when these chromosomes lack serine 10 phosphorylation of H3. We also show that mitotic arrest deficient 2 (Mad2), a member of the spindle assembly checkpoint, is present on DRT/DMC chromosomes at a time when the normally condensed chromosomes show no Mad2 staining, indicating that DRT/DMC activates the spindle assembly checkpoint. Finally, cells with DRT/DMC chromosomes have centrosome amplification, abnormal spindle assembly, endoreduplication and significant chromosome instability.

A DnaJ protein, apobec-1-binding protein-2, modulates apolipoprotein B mRNA editing.

Mammalian homologues of DnaJ proteins, also known as Hsp40 proteins, are co-chaperonins that complement Hsp70 chaperone function. Using the yeast two-hybrid system, we cloned an apolipoprotein (apo) B mRNA editing complementation protein, called apobec-1-binding protein-2 (ABBP-2), and found that it is a Class II DnaJ homologue. ABBP-2 binds to apobec-1, the mammalian apoB mRNA editase, via its J domain and neighboring G/F domain. It is a ubiquitously expressed protein, and, by transfection analysis of GFP-ABBP-2, we found that the protein is located in both the nucleus and cytosol of transfected cells, with predominance in the nucleus. Down-regulation of ABBP-2 expression in cultured cells inhibits endogenous apobec-1-mediated apoB mRNA editing. Like other Hsp40 proteins, ABBP-2 binds to Hsp70 and has ATPase-stimulating activity. Apobec-1-mediated apoB mRNA editing activity of in vitro tissue extracts requires the presence of Hsp70/ABBP-2. Although exogenously added ATP is not required for editing activity, removal of the endogenous ATP present in these extracts, which disrupts ABBP-2-Hsp70 interaction, completely inhibits editing. ABBP-2 differs from previously described auxiliary proteins (ABBP-1, ACF, and GRY-RBP) in that it does not contain any RNA recognition motifs. Not only is ABBP-2 required for efficient apoB mRNA editing, this newly discovered apobec-1-binding protein may help determine the subcellular distribution and trafficking of apobec-1 via its interaction with the chaperonin Hsp70.

Religion and mental health among women veterans with sexual assault experience.

OBJECTIVE: Religion has been shown to have a positive impact on well-being and to play an important role in coping with stressful life events. However, the buffering effect of religiosity on mental health, after a particularly stressful life event such as sexual assault, has not been studied. In this study we examined the buffering effect of religion on mental health and depression for women who report experiencing sexual assault while in the military. METHOD: The sample includes a nationally representative sample of 3,543 women veterans who use VA ambulatory care. Two dimensions of religiosity were used: organizational (frequency of religious service attendance) and subjective (the extent religious beliefs are a source of strength/comfort). Mental health was measured by the mental component summary (MCS) from the SF36 and depressive symptoms were measured by the Center for Epidemiologic Studies-Depression (CES-D) scale. RESULTS: Women veterans who reported experiencing sexual assault while in the military had lower mental health scores and higher levels of depression. Linear regression analysis indicated that these negative impacts diminished with increased frequency of religious service attendance, supporting the buffering effect of organizational religiosity on mental health and depression. Although the buffering effect of subjective religiosity was not evident, subjective religiosity was shown to be positively associated with better mental health in both groups of women with and without sexual assault experience in the military. CONCLUSIONS: Frequent religious service attendance buffers the negative impacts of sexual assault on mental health and depression of women veterans. The potential of integrating religiosity in designing interventions is discussed.

Risk adjustment for measuring health outcomes: an application in VA long-term care.

An empirically derived risk adjustment model is useful in distinguishing among facilities in their quality of care. We used Veterans Affairs (VA) administrative databases to develop and validate a risk adjustment model to predict decline in functional status, an important outcome measure in long-term care, among patients residing in VA long-term care facilities. This model was used to compare facilities on adjusted and unadjusted rates of decline. Predictors of decline included age, time between assessments, baseline functional status, terminal illness, pressure ulcers, pulmonary disease, cancer, arthritis, congestive heart failure, substance-related disorders, and various neurologic disorders. The model performed well in the development and validation databases (c statistics, 0.70 and 0.68, respectively). Risk-adjusted rates and rankings of facilities differed from unadjusted ratings. We conclude that judgments of facility performance depend on whether risk-adjusted or unadjusted decline rates are used. Valid risk adjustment models are therefore necessary when comparing facilities on outcomes.

Meta-analysis of binary data: which within study variance estimate to use?

We applied a mixed effects model to investigate between- and within-study variation in improvement rates of 180 schizophrenia outcome studies. The between-study variation was explained by the fixed study characteristics and an additional random study effect. Both rate difference and logit models were used. For a binary proportion outcome p(i) with sample size n(i) in the ith study, (circumflexp(i)(1-circumflexp(i))n)(-1) is the usual estimate of the within-study variance sigma(i)(2) in the logit model, where circumflexpi) is the sample mean of the binary outcome for subjects in study i. This estimate can be highly correlated with logit(circumflexp(i)). We used (macronp(i)(1-macronp)n(i))(-1) as an alternative estimate of sigma(i)(2), where macronp is the weighted mean of circumflexp(i)'s. We estimated regression coefficients (beta) of the fixed effects and the variance (tau(2)) of the random study effect using a quasi-likelihood estimating equations approach. Using the schizophrenia meta-analysis data, we demonstrated how the choice of the estimate of sigma(2)(i) affects the resulting estimates of beta and tau(2). We also conducted a simulation study to evaluate the performance of the two estimates of sigma(2)(i) in different conditions, where the conditions vary by number of studies and study size. Using the schizophrenia meta-analysis data, the estimates of beta and tau(2) were quite different when different estimates of sigma(2)(i) were used in the logit model. The simulation study showed that the estimates of beta and tau(2) were less biased, and the 95 per cent CI coverage was closer to 95 per cent when the estimate of sigma(2)(i) was (macronp(1-macronp)n(i))(-1) rather than (circumflexp(i)(1-circumflexp)n(i))(-1). Finally, we showed that a simple regression analysis is not appropriate unless tau(2) is much larger than sigma(2)(i), or a robust variance is used.

Two-hybrid cloning identifies an RNA-binding protein, GRY-RBP, as a component of apobec-1 editosome.

ApoB mRNA editing is mediated by an editosome complex with apobec-1 as its catalytic component. By yeast two-hybrid cloning using apobec-1 as bait we identified a 69.6-kDa RNA binding protein, GRY-RBP, that contains 3 RNA-recognition motifs (RRMs) as a novel apobec-1 associating protein. GRY-RBP may be an alternatively spliced species of NASP1, a protein of known function. GRY-RBP was shown to bind to apobec-1, the catalytic component of apoB mRNA editosome, in vivo and in vitro. Immunodepletion using a monospecific rabbit antibody abolished editing in apobec-1 expressing HepG2 S-100 extracts. GRY-RBD interacted with apobec-1 through its C-terminus. It contains three RRM (RNA recognition motifs) domains that are homologous to those found in human ACF (apobec-1 complementation factor). Phylogeny analysis of the RRM domain-containing proteins indicates that GRY-RBP clusters with hnRNP-R, ACF, and ABBP-1 (another apobec-1 binding protein). In addition to its involvement with apobec-1 editosome, the suggested cellular functions of GRY-RBD and its structural homologues include RNA transport and RNA secondary structure stabilization.

Calcium/calmodulin-dependent protein kinase II inhibitor protein: localization of isoforms in rat brain.

A second isoform of Ca2+/calmodulin-dependent-kinase II inhibitor protein (CaM-KIIN) has been identified using the yeast two-hybrid screen. The 1.8kb message encodes a 78 residue CaM-KIINalpha that is 65% identical in its putative open-reading frame and 95% identical in its inhibitory domain to the previously characterized CaM-KIINbeta. CaM-KIINalpha exhibits inhibitory properties towards recombinant mouse CaM-kinase IIalpha indistinguishable from CaM-KIINbeta. The 27 amino acid inhibitory peptide (CaM-KIINtide) derived from CaM-KIIN has the ability to inhibit brain CaM-kinase II activity from multiple organisms including rat, Drosophila and goldfish. Northern analysis of various rat tissues indicates that CaM-KIINalpha is specific to brain whereas CaM-KIINbeta message is also present in testis. In situ hybridization shows a general distribution of both isoforms in rat brain with stronger localization of CaM-KIINbeta in cerebellum and hindbrain and CaM-KIINalpha in frontal cortex, hippocampus and inferior colliculus. An antibody that recognizes both isoforms shows a distribution of CaM-KIIN in rat brain that correlates with immunoreactivity of CaM-kinase II. In cultured mature hippocampal neurons, CaM-KIIN is present in cell bodies and dendrites but, unlike CaM-kinase II, does not display punctate staining at synapses. These results suggest a localized function for CaM-KIIN in inhibiting specialized pools of CaM-kinase II.

Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice.

Obesity is a disorder of energy balance. Hormone-sensitive lipase (HSL) mediates the hydrolysis of triacylglycerol, the major form of stored energy in the body. Perilipin (encoded by the gene Plin), an adipocyte protein, has been postulated to modulate HSL activity. We show here that targeted disruption of Plin results in healthy mice that have constitutively activated fat-cell HSL. Plin -/- mice consume more food than control mice, but have normal body weight. They are much leaner and more muscular than controls, have 62% smaller white adipocytes, show elevated basal lipolysis that is resistant to beta-adrenergic agonist stimulation, and are cold-sensitive except when fed. They are also resistant to diet-induced obesity. Breeding the Plin -/- alleles into Leprdb/db mice reverses the obesity by ncreasing the metabolic rate of the mice. Our results demonstrate a role for perilipin in reining in basal HSL activity and regulating lipolysis and energy balance; thus, agents that inactivate perilipin may prove useful as anti-obesity medications.

Analyzing data with clumping at zero. An example demonstration.

This article demonstrates the use of two approaches to analyzing the relationship of multiple covariates to an outcome which has a high proportion of zero values. One approach is to categorize the continuous outcome (including the zero category) and then fit a proportional odds model. Another approach is to use logistic regression to model the probability of a zero response and ordinary least squares linear regression to model the non-zero continuous responses. The use of these two approaches was demonstrated using outcomes data on hours of care received from the Springfield Elder Project. A crude linear model including both zero and non-zero values was also used for comparison. We conclude that the choice of approaches for analysis depends on the data. If the proportional odds assumption is valid, then it appears to be the method of choice; otherwise, the combination of logistic regression and a linear model is preferable.

Apolipoprotein B: from editosome to proteasome.

Apolipoprotein (apo) B, the protein component of low-density lipoproteins (LDLs), has been under intense investigation for the last three decades. During the first decade after its initial description, most reports dealt with the physical-chemical characterization of apoB in its natural environment (i.e., intact LDL particles). A few studies dealing with attempts to elucidate the primary structure of apoB were published at this time (Deutsch et al., 1978; Bradley et al., 1980). However, most of these, in retrospect, represented heroic efforts that were doomed to failure because of the huge size and insoluble nature of apoB, once it is separated from its lipid environment. Indeed, during the 1970s, there was no universal agreement on the true molecular weight of the protein, which was not established until sometime into the second decade of apoB research (Yang et al., 1986b). The next 10 years were punctuated by breakthroughs on three different fronts in our understanding of apoB. The first exciting discovery was that apoB exists in two forms, apoB-100 and apoB-48 (Kane et al., 1980; Elovson et al., 1981). The next breakthrough was the elucidation of the primary structure of apoB-100 by a combination of cDNA cloning (Chen et al., 1986; Knott et al., 1986; Yang et al., 1986a) and direct peptide sequencing (Yang et al., 1986a, 1989). This decade of renaissance in apoB research was concluded by the elucidation of the structure of apoB-48. More important in terms of basic cellular molecular biology was the discovery of RNA editing, when apoB-48 was found to be the translation product of an edited apoB mRNA (Chen et al., 1987; Powell et al., 1987). RNA editing had just been described for a kinetoplastid protozoa the year before (Benne et al., 1986). ApoB mRNA editing was the first instance of RNA editing described in a higher eukaryote (Chan and Seeburg, 1995; Grosjean and Benne. 1998). The last decade, which brings us to the present, has been marked by studies that benefited from the breakthroughs of the 1980s. which enabled many different laboratories to examine various aspects of apoB structure, function, and expression. The function of apoB in vivo was analyzed in different animal models (e.g., transgenic animals that overexpress apoB) (Linton et al., 1993; Callow and Rubin, 1995; Veniant et al., 1997) and in knockout animals that have no functional apoB (Farese et al., 1995,1996; Huang et al., 1995,1996). Furthermore, the structure-function relationship of apoB has been investigated in mice that express site-specific apoB mutants (Callow and Rubin, 1995; Veniant et al., 1997: Borén et al., 1998). A breakthrough in a related area led to the identification and cloning of microsomal triglyceride transfer protein (MTP) (Wetterau and Zilversmitt, 1984: Wetterau et al., 1992; Sharp et al., 1993) and the demonstration that MTP is essential for apoB production (Gordon et al., 1994; Leiper et al., 1994). The absence of MTP was found to lead to the complete degradation of apoB, which harks back to an observation in 1987 that, even in the presence of MTP, a substantial proportion of newly synthesized apoB-100 undergoes intracellular degradation before secretion (Borchardt and Davis, 1987). Indeed, the intracellular degradation of apoB-100 is the major determinant of its production rate from the liver, since the transcription of apoB appears to be constitutive and not subject to much regulation (Pullinger et al., 1989). It was in 1996, almost a decade after the first description of apoB's destruction inside the cell, that the proteasome-ubiquitin pathway was found to be the major mechanism for the intracellular degradation of apoB-100 (Yeung et al., 1996). Another important development within the last decade was the cloning of APOBEC-1, the catalytic subunit of the apoB mRNA editing complex (editosome) (Teng et al., 1993). This chapter will review some of the major landmarks in apoB research in the last 10 to 15 years, concentrating mainl

Eye examinations for VA patients with diabetes: standardizing performance measures.

OBJECTIVE: To demonstrate the potential of the Health Plan Employer Data and Information Set (HEDIS) for the calculation of a performance measure for eye exams in the diabetic population using Veterans Health Administration (VA) administrative data. DESIGN: We calculated a 1-year HEDIS-defined patient denominator and three alternative denominators that considered coding factors in identifying a VA patient as diabetic. We calculated the HEDIS-defined numerator, along with alternative specifications that captured other types of eye exams. Finally, we supplemented national data with VA pharmacy and Medicare claims data to identify all VA diabetic patients at 14 selected VA facilities and to establish a more accurate picture of non-VA health care utilization. RESULTS: The national average annual HEDIS-defined eye exam rate in the VA was 26% in fiscal 1997 compared with 39% for managed care organizations. Medicare utilization raised this by 15 percentage points at 14 northeastern VA hospitals. Over 2 years, at least two-thirds of diabetic VA patients had some type of eye exam through VA or Medicare. CONCLUSION: A HEDIS measure of eye exams for VA patients with diabetes can be calculated using VA administrative data only. However, the question remains to what extent the denominator and numerator accurately and completely identify all diabetic patients using VA services and all appropriate eye exams. We recommend caution in interpreting the results of performance measurement across different health care sectors based on what we currently know are data system limitations.

Does diagnostic information contribute to predicting functional decline in long-term care?

BACKGROUND: Compared with the acute-care setting, use of risk-adjusted outcomes in long-term care is relatively new. With the recent development of administrative databases in long-term care, such uses are likely to increase. OBJECTIVES: The objective of this study was to determine the contribution of ICD-9-CM diagnosis codes from administrative data in predicting functional decline in long-term care. RESEARCH DESIGN: We used a retrospective sample of 15,693 long-term care residents in VA facilities in 1996. METHODS: We defined functional decline as an increase of > or =2 in the activities of daily living (ADL) summary score from baseline to semiannual assessment. A base regression model was compared to a full model enhanced with ICD-9-CM codes. We calculated validated measures of model performance in an independent cohort. RESULTS: The full model fit the data significantly better than the base model as indicated by the likelihood ratio test (chi2 = 179, df = 11, P <0.001). The full model predicted decline more accurately than the base model (R2 = 0.06 and 0.05, respectively) and discriminated better (c statistics were 0.70 and 0.68). Observed and predicted risks of decline were similar within deciles between the 2 models, suggesting good calibration. Validated R2 statistics were 0.05 and 0.04 for the full and base models; validated c statistics were 0.68 and 0.66. CONCLUSIONS: Adding specific diagnostic variables to administrative data modestly improves the prediction of functional decline in long-term care residents. Diagnostic information from administrative databases may present a cost-effective alternative to chart abstraction in providing the data necessary for accurate risk adjustment.

Successful resection of cecal hepatic metastasis extending into the right side of the heart under cardiopulmonary bypass.

Resection is the best hope for the cure of colorectal metastasis to the liver. However, surgery is indicated for only a few patients, especially those who have major vascular involvement. We report a 55-year-old woman with a liver metastasis from the cecum that showed a tumor thrombus in the right side of the heart. She had undergone laparoscopic right hemicolectomy for cecal cancer 6 months before, and presented with a palpable mass in the epigastrium. Abdominal ultrasonography, computed tomography, hepatic angiogram, and echocardiography showed a huge mass on the left lobe of the liver, with a tumor thrombus which extended to the right ventricle through the left hepatic vein and inferior vena cava. Tumor thrombectomy, through a right atriotomy, was success-fully performed under cardiopulmonary bypass, followed by left hepatic lobectomy. The patient's postoperative course was uneventful.

High polymorphism at the human melanocortin 1 receptor locus.

Variation in human skin/hair pigmentation is due to varied amounts of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eu- and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many mammals. We have sequenced the MC1R gene in 121 individuals sampled from world populations with an emphasis on Asian populations. We found variation at five nonsynonymous sites (resulting in the variants Arg67Gln, Asp84Glu, Val92Met, Arg151Cys, and Arg163Gln), but at only one synonymous site (A942G). Interestingly, the human consensus protein sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency (7%) in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. The MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon was sequenced to study the evolution of MC1R. The ancestral human MC1R sequence is identical to the human consensus protein sequence, while MC1R varies considerably among higher primates. A comparison of the rates of substitution in genes in the melanocortin receptor family indicates that MC1R has evolved the fastest. In addition, the nucleotide diversity at the MC1R locus is shown to be several times higher than the average nucleotide diversity in human populations, possibly due to diversifying selection.

Contributors to and mediators of psychological well-being for informal caregivers.

This article explores the relationships between caregiving stressors and caregiver well-being in a representative community sample of disabled elders and their informal caregivers. The direct and indirect effects of stressors and potential mediators on the outcome of caregiver psychological well-being, as measured by depression, were examined using path analysis. Potential mediators of the primary stressors on depression included mastery, emotional support; quality of relationship between the caregiver and the care recipient, formal service use and role overload. Findings indicate that the caregiving stressors (needs for care) led to caregiver depression indirectly through their effect on hours of care provided and the resulting caregiver perception of role overload. Quality of the caregiver/care recipient relationship mediated the relationship of the caregiving stressors and caregiver overload and depression. Finally, regardless of the level of primary stressors, caregivers with high levels of mastery or emotional support were at lower risk of depression. These findings can be used to inform the design of proactive caregiver interventions.