Routine proteinuria monitoring for bevacizumab in patients with gynecologic malignancies.

BACKGROUND: Proteinuria leading to nephrotic syndrome is a rare adverse event arising from treatment with bevacizumab. There is limited evidence to guide the frequency and appropriate test for monitoring for proteinuria. The purpose of this study was to determine the prevalence and severity of proteinuria during bevacizumab administration to patients with gynecologic malignancies, and to evaluate risk factors associated with this toxicity; a secondary objective was to evaluate the cost of routine proteinuria monitoring to assess for opportunities of cost containment that could change clinical practice. METHODS: A retrospective chart review was performed at an academic gynecologic oncology clinic. Women over 18 years of age with a diagnosed gynecologic malignancy were evaluated for the development of proteinuria while receiving bevacizumab treatment as measured by a urine protein-to-creatinine ratio. Patient and disease-specific risk factors were evaluated using logistic regression to determine correlations of risk factors to development of proteinuria. Cost assessment was performed using institution-specific data for urine laboratory tests. RESULTS: Eighty-nine patients were identified, and the overall prevalence of proteinuria of any grade was 35%. The mean number of bevacizumab cycles was 13 (2-64 cycles). The majority of patients experienced grade 1 proteinuria (70%, 62 patients). Grade 3 proteinuria was observed in two patients (2%). There was a trend toward increased bevacizumab cycles associated with increased grade proteinuria (p = 0.053), however there were no factors significantly associated with the development of proteinuria as measured by urine protein-to-creatinine ratio. CONCLUSION: Monitoring of urine protein-to-creatinine ratios with each cycle may be unnecessary due to the low prevalence of grade 3 proteinuria observed. Additionally, urine protein-to-creatinine ratios may not provide adequate assessment of proteinuria toxicity associated with bevacizumab therapy. Potential cost savings opportunities for the institution can be realized with a cost-reductive monitoring algorithm that will utilize less costly laboratory techniques for patients at high risk of developing proteinuria.

Alternate endpoints and clinical outcome assessments in pediatric ulcerative colitis registration trials.

OBJECTIVES: Presently, there is no consensus on endpoint measures to assess clinical outcomes for pediatric ulcerative colitis (UC). This study reviewed the endpoints used in the registration trials of approved drugs for pediatric UC. METHODS: The primary efficacy endpoints of all registration trials completed from 1950 to 2008 that led to Food and Drug Administration approval for indications in pediatric and adult UC were reviewed. RESULTS: Colazal and Remicade have been approved for pediatric UC indication, and clinical response was used as a primary endpoint in these registration trials. The clinical response in the adult Colazal trials was defined as a reduction of rectal bleeding and improvement in at least one of the other assessed symptoms (stool frequency, patient functional assessment, abdominal pain, sigmoidoscopic grade, and physician's global assessment) assessed by the Sutherland UC Activity Index. The pediatric Colazal trial defined clinical response using the Modified Sutherland UC Activity Index, which excluded abdominal pain and functional assessment. Both adult and pediatric Remicade trials used clinical response defined by the Mayo score as the primary endpoint. The Pediatric Ulcerative Colitis Activity Index was used to measure various secondary endpoints in the pediatric Remicade trial. CONCLUSIONS: Pediatric-specific endpoints were used, but outcome measures and definition of clinical response were not consistent in pediatric UC trials. Consensus on the definition of successful treatment outcome (clinical response and/or remission) and collaboration in the development of well-defined and reliable measures of signs and symptoms for use in conjunction with endoscopic parameters of mucosal healing will facilitate pediatric drug development.

The initiation of liver development is dependent on Foxa transcription factors.

The specification of the vertebrate liver is thought to occur in a two-step process, beginning with the establishment of competence within the foregut endoderm for responding to organ-specific signals, followed by the induction of liver-specific genes. On the basis of expression and in vitro studies, it has been proposed that the Foxa transcription factors establish competence by opening compacted chromatin structures within liver-specific target genes. Here we show that Foxa1 and Foxa2 (forkhead box proteins A1 and A2) are required in concert for hepatic specification in mouse. In embryos deficient for both genes in the foregut endoderm, no liver bud is evident and expression of the hepatoblast marker alpha-fetoprotein (Afp) is lost. Furthermore, Foxa1/Foxa2-deficient endoderm cultured in the presence of exogenous fibroblast growth factor 2 (FGF2) fails to initiate expression of the liver markers albumin and transthyretin. Thus, Foxa1 and Foxa2 are required for the establishment of competence within the foregut endoderm and the onset of hepatogenesis.

Ethical considerations in conducting pediatric research.

The critical need for pediatric research on drugs and biological products underscores the responsibility to ensure that children are enrolled in clinical research that is both scientifically necessary and ethically sound. In this chapter, we review key ethical considerations concerning the participation of children in clinical research. We propose a basic ethical framework to guide pediatric research, and suggest how this framework might be operationalized in linking science and ethics. Topics examined include: the status of children as a vulnerable population; the appropriate balance of risk and potential benefit in research; ethical considerations underlying study design, including clinical equipoise, placebo controls, and non-inferiority designs; the use of data monitoring committees; compensation; and parental permission and child assent to participate in research. We incorporate selected national (USA) and international guidelines, as well as regulatory approaches to pediatric studies that have been adopted in the USA, Canada, and Europe.

Unintentional Epidural Anesthesia Mimicking Cauda Equina Syndrome from Suprafascial Injection of Liposomal Bupivacaine After Lumbar Foraminotomy: A Case Report.

CASE: This case report describes a 46-year-old woman undergoing right-sided L5 to S1 decompression who received liposomal bupivacaine (LB) for postoperative analgesia and developed unintentional epidural anesthesia with symptoms mimicking cauda equina syndrome. The patient's symptoms resolved 72 hours postoperatively, approximately the length that LB typically lasts. At the 16-month follow-up, the patient demonstrated complete neurological function with no lower extremity strength or sensation deficits. CONCLUSIONS: Tracking of LB into the epidural space after lumbar surgery may cause transient epidural anesthesia with symptoms that mimic cauda equina syndrome.

The MODY1 gene HNF-4alpha regulates selected genes involved in insulin secretion.

Mutations in the gene encoding hepatocyte nuclear factor-4alpha (HNF-4alpha) result in maturity-onset diabetes of the young (MODY). To determine the contribution of HNF-4alpha to the maintenance of glucose homeostasis by the beta cell in vivo, we derived a conditional knockout of HNF-4alpha using the Cre-loxP system. Surprisingly, deletion of HNF-4alpha in beta cells resulted in hyperinsulinemia in fasted and fed mice but paradoxically also in impaired glucose tolerance. Islet perifusion and calcium-imaging studies showed abnormal responses of the mutant beta cells to stimulation by glucose and sulfonylureas. These phenotypes can be explained in part by a 60% reduction in expression of the potassium channel subunit Kir6.2. We demonstrate using cotransfection assays that the Kir6.2 gene is a transcriptional target of HNF-4alpha. Our data provide genetic evidence that HNF-4alpha is required in the pancreatic beta cell for regulation of the pathway of insulin secretion dependent on the ATP-dependent potassium channel.

Analysis of genetic variation in Ashkenazi Jews by high density SNP genotyping.

BACKGROUND: Genetic isolates such as the Ashkenazi Jews (AJ) potentially offer advantages in mapping novel loci in whole genome disease association studies. To analyze patterns of genetic variation in AJ, genotypes of 101 healthy individuals were determined using the Affymetrix EAv3 500 K SNP array and compared to 60 CEPH-derived HapMap (CEU) individuals. 435,632 SNPs overlapped and met annotation criteria in the two groups. RESULTS: A small but significant global difference in allele frequencies between AJ and CEU was demonstrated by a mean FST of 0.009 (P < 0.001); large regions that differed were found on chromosomes 2 and 6. Haplotype blocks inferred from pairwise linkage disequilibrium (LD) statistics (Haploview) as well as by expectation-maximization haplotype phase inference (HAP) showed a greater number of haplotype blocks in AJ compared to CEU by Haploview (50,397 vs. 44,169) or by HAP (59,269 vs. 54,457). Average haplotype blocks were smaller in AJ compared to CEU (e.g., 36.8 kb vs. 40.5 kb HAP). Analysis of global patterns of local LD decay for closely-spaced SNPs in CEU demonstrated more LD, while for SNPs further apart, LD was slightly greater in the AJ. A likelihood ratio approach showed that runs of homozygous SNPs were approximately 20% longer in AJ. A principal components analysis was sufficient to completely resolve the CEU from the AJ. CONCLUSION: LD in the AJ versus was lower than expected by some measures and higher by others. Any putative advantage in whole genome association mapping using the AJ population will be highly dependent on regional LD structure.

Regeneration of pancreatic islets after partial pancreatectomy in mice does not involve the reactivation of neurogenin-3.

Understanding the factors and mechanisms involved in beta-cell regeneration will guide therapeutic efforts to augment beta-cell mass in patients with diabetes. Neurogenin-3 (Ngn3) is a bHLH transcription factor that responds to Notch signaling and whose expression marks endocrine progenitors. During fetal development, all endocrine cells are derived from Ngn3(+) precursors. Although expression of Ngn3 in the adult pancreas has not been reported, it has been suggested that islet regeneration in adult organisms recapitulates embryonic developmental pathways. Here, we investigated whether beta-cell regeneration in adult mice recapitulates the embryonic pathway involving Ngn3 activation. Despite full recovery of beta-cell mass after 50% partial pancreatectomy (Ppx) in BALB/c mice, no pancreatic Ngn3 immunoreactivity was detected, even when the beta-cell trophic glucagon-like peptide-1 receptor agonist exendin-4 was administered after the procedure. Even when we used the stable expression of enhanced green fluorescent protein (EGFP) in Ngn3(EGFP/+) mice to trace Ngn3 expression after Ppx, no pancreatic Ngn3 expression was detected. Although ectopic expression of Ngn3 can promote an endocrine transcriptional program in adult cells and may thus have therapeutic potential in the development of surrogate beta-cells, our studies indicate that a reactivation of endogenous Ngn3 expression is not required for adult beta-cell regeneration in vivo.

Foxa2 controls Pdx1 gene expression in pancreatic beta-cells in vivo.

Differentiation of early foregut endoderm into pancreatic endocrine and exocrine cells depends on a cascade of gene activation events controlled by various transcription factors. Prior in vitro analysis has suggested that the forkhead/winged helix transcription factor Foxa2 (formerly HNF-3beta) is a major upstream regulator of Pdx1, a homeobox gene essential for pancreatic development. Pdx1 is also essential for the maintenance of glucose homeostasis, as its human orthologue, IPF-1, is mutated in a subset of patients with early-onset type 2 diabetes (MODY4). To analyze the Foxa2/Pdx1 regulatory cascade during pancreatic beta-cell differentiation, we used conditional gene ablation of Foxa2 in mice. We demonstrated that the deletion of Foxa2 in beta-cell-specific knockout mice results in downregulation of Pdx1 mRNA and subsequent reduction of PDX-1 protein levels in islets. These data represent the first in vivo demonstration that Foxa2 acts upstream of Pdx1 in the differentiated beta-cell.

Functional genomics of the endocrine pancreas: the pancreas clone set and PancChip, new resources for diabetes research.

Over the past 5 years, microarrays have greatly facilitated large-scale analysis of gene expression levels. Although these arrays were not specifically geared to represent tissues and pathways known to be affected by diabetes, they have been used in both type 1 and type 2 diabetes research. To prepare a tool that is particularly useful in the study of type 1 diabetes, we have assembled a nonredundant set of 3,400 clones representing genes expressed in the mouse pancreas or pathways known to be affected by diabetes. We have demonstrated the usefulness of this clone set by preparing a cDNA glass microarray, the PancChip, and using it to analyze pancreatic gene expression from embryonic day 14.5 through adulthood in mice. The clone set and corresponding array are useful resources for diabetes research.

Transcriptional program of the endocrine pancreas in mice and humans.

The Endocrine Pancreas Consortium was formed in late 1999 to derive and sequence cDNA libraries enriched for rare transcripts expressed in the mammalian endocrine pancreas. Over the past 3 years, the Consortium has generated 20 cDNA libraries from mouse and human pancreatic tissues and deposited >150,000 sequences into the public expressed sequence tag databases. A special effort was made to enrich for cDNAs from the endocrine pancreas by constructing libraries from isolated islets. In addition, we constructed a library in which fetal pancreas from Neurogenin 3 null mice, which consists of only exocrine and duct cells, was subtracted from fetal wild-type pancreas to enrich for the transcripts from the endocrine compartment. Sequence analysis showed that these clones cluster into 9,464 assembly groups (approximating unique transcripts) for the mouse and 13,910 for the human sequences. Of these, >4,300 were unique to Consortium libraries. We have assembled a core clone set containing one cDNA for each assembly group for the mouse and have constructed the corresponding microarray, termed "PancChip 4.0," which contains >9,000 nonredundant elements. We show that this PancChip is highly enriched for genes expressed in the endocrine pancreas. The mouse and human clone sets and corresponding arrays will be important resources for diabetes research.

Adverse Events in Very Young Children Prescribed Psychotropic Medications: Preliminary Findings from an Acute Clinical Sample.

OBJECTIVE: The present study used the Pediatric Adverse Events Rating Scale (PAERS) to provide a systematic assessment of adverse events (AEs) related to psychotropic medication use in a clinical sample of young children attending a specialized, early childhood partial hospital program. Study goals were as follows: 1) To describe the frequency and types of specific psychotropic medication-related AEs experienced by very young children (ages 3-7 years) in an acute clinical sample, and 2) to identify the psychotropic medication(s) and/or class(es) associated with the highest frequency of AEs. METHODS: Participants were 158 children (118 males; ages 36-95 months, mean=66 months, SD=14.6 months) who presented to a hospital-based day treatment program for young children with severe emotional and behavioral problems, and were prescribed a psychotropic medication at any point during the hospitalization. Data on AEs related to psychotropic medication were collected using the PAERS from 2011 to 2014. RESULTS: The percentages of children who experienced one or more AEs attributed to a psychiatric medication ranged from 0 (sertraline, melatonin) to 41.2% (fluoxetine), with wide variability in the types AEs reported. The overall frequencies of events caused by a stimulant were similar across the two medications examined (21.4% and 27.7% for mixed amphetamine salts and methylphenidate, respectively), with mood-related difficulties and decreased appetite being the most common AEs reported. The frequencies of AEs caused by an α agonist were also similar across the two medications examined (9.8% and 17.2% for guanfacine and clonidine, respectively), with fatigue as the most commonly reported AE. With respect to the selective serotonin reuptake inhibitor (SSRI) class, there was a trend for fluoxetine to be associated with more AEs (41.2%) than sertraline (for which no AEs were reported). The most common AEs reported for fluoxetine were impulsivity and poor concentration. CONCLUSIONS: The data presented here support existing literature reporting differences in AEs between age groups. More rigorous studies are warranted to further examine the types and frequencies of AEs related to psychotropic medications in very young children.

Inflammatory Factors Mediate Vulnerability to a Social Stress-Induced Depressive-like Phenotype in Passive Coping Rats.

BACKGROUND: Coping strategy impacts susceptibility to psychosocial stress. The locus coeruleus (LC) and dorsal raphe (DR) are monoamine nuclei implicated in stress-related disorders. Our goal was to identify genes in these nuclei that distinguish active and passive coping strategies in response to social stress. METHODS: Rats were exposed to repeated resident-intruder stress and coping strategy determined. Gene and protein expression in the LC and DR were determined by polymerase chain reaction array and enzyme-linked immunosorbent assay and compared between active and passive stress-coping and unstressed rats. The effect of daily interleukin (IL)-1 receptor antagonist before stress on anhedonia was also determined. RESULTS: Rats exhibited passive or active coping strategies based on a short latency (SL) or longer latency (LL) to assume a defeat posture, respectively. Stress differentially regulated 19 and 26 genes in the LC and DR of SL and LL rats, respectively, many of which encoded for inflammatory factors. Notably, Il-1ß was increased in SL and decreased in LL rats in both the LC and DR. Protein changes were generally consistent with a proinflammatory response to stress in SL rats selectively. Stress produced anhedonia selectively in SL rats and this was prevented by IL-1 receptor antagonist, consistent with a role for IL-1ß in stress vulnerability. CONCLUSIONS: This study highlighted distinctions in gene expression related to coping strategy in response to social stress. Passive coping was associated with a bias toward proinflammatory processes, particularly IL-1ß, whereas active coping and resistance to stress-related pathology was associated with suppression of inflammatory processes.

Clinical endocrinology and metabolism. Development of gut endocrine cells.

During development, the definitive endoderm differentiates into several gastrointestinal epithelial lineages, including enteroendocrine cells. The enteroendocrine lineage consists of at least 15 different cell types that are categorized based on their morphology, location and peptide hormone expression. The mechanisms regulating enteroendocrine cell differentiation are likely to be critical not only in embryonic development, but also during the constant renewal of gut epithelia in the adult. The identification of transcription factors and regulatory DNA elements required for cell type-specific gene expression in various endocrine cell types has broadened our understanding of the regulatory networks controlling the spatial and temporal activation of enteroendocrine differentiation programs. This chapter will review recent studies of transcription factors during enteroendocrine cell differentiation, with a focus on the central role for the Notch signaling pathway in enteroendocrine cell fate decisions.

Characteristics of community mental health clinics associated with treatment engagement.

OBJECTIVES: Past literature documents many individual predictors of treatment engagement among mental health clients in community settings, but few studies have examined clinic characteristics that may be associated with treatment engagement. With data from a patient activation and self-management trial, this study examined the variation in demographic and clinic characteristics across community mental health clinics and whether this variation predicted differences in treatment engagement in mental health services. METHODS: Chart reviews were conducted for 638 clients of 12 community mental health clinics. Client attendance records were collected for a one-year period to examine engagement (defined as the ratio of kept versus scheduled appointments). Adjusting for client variability, the investigators examined which clinic-level characteristics were associated with treatment engagement. RESULTS: Clinics varied significantly in their clients' demographic characteristics and engagement in mental health care. Providing case management and offering transportation vouchers or free parking at the clinic were associated with lower engagement. However, offering outreach was associated with greater engagement. CONCLUSIONS: The results of this study suggest that certain clinic characteristics are associated with engagement in mental health services. These results demonstrate the difficulties faced by community mental health clinics in reducing no-show rates even in the face of strong efforts to improve engagement.

Manganese-enhanced magnetic resonance imaging (MEMRI) reveals brain circuitry involved in responding to an acute novel stress in rats with a history of repeated social stress.

Responses to acute stressors are determined in part by stress history. For example, a history of chronic stress results in facilitated responses to a novel stressor and this facilitation is considered to be adaptive. We previously demonstrated that repeated exposure of rats to the resident-intruder model of social stress results in the emergence of two subpopulations that are characterized by different coping responses to stress. The submissive subpopulation failed to show facilitation to a novel stressor and developed a passive strategy in the Porsolt forced swim test. Because a passive stress coping response has been implicated in the propensity to develop certain psychiatric disorders, understanding the unique circuitry engaged by exposure to a novel stressor in these subpopulations would advance our understanding of the etiology of stress-related pathology. An ex vivo functional imaging technique, manganese-enhanced magnetic resonance imaging (MEMRI), was used to identify and distinguish brain regions that are differentially activated by an acute swim stress (15 min) in rats with a history of social stress compared to controls. Specifically, Mn(2+) was administered intracerebroventricularly prior to swim stress and brains were later imaged ex vivo to reveal activated structures. When compared to controls, all rats with a history of social stress showed greater activation in specific striatal, hippocampal, hypothalamic, and midbrain regions. The submissive subpopulation of rats was further distinguished by significantly greater activation in amygdala, bed nucleus of the stria terminalis, and septum, suggesting that these regions may form a circuit mediating responses to novel stress in individuals that adopt passive coping strategies. The finding that different circuits are engaged by a novel stressor in the two subpopulations of rats exposed to social stress implicates a role for these circuits in determining individual strategies for responding to stressors. Finally, these data underscore the utility of ex vivo MEMRI to identify and distinguish circuits engaged in behavioral responses.

Cellular adaptations of dorsal raphe serotonin neurons associated with the development of active coping in response to social stress.

BACKGROUND: Social stress is a risk factor for affective disorders for certain vulnerable individuals. Stress and depression are linked in part through regulation of the dorsal raphe (DR)-serotonin (5-HT) system by the stress-related neuropeptide, corticotropin-releasing factor (CRF). We used a rat social stress model that shows individual differences in coping strategies to determine whether differences in CRF-5-HT interactions underlie individual differences in the vulnerability to social stress. METHODS: Rats were exposed to the resident-intruder model of social stress for 5 days. In vivo single-unit recordings assessed DR-5-HT neuronal responses to CRF and immunoelectron microscopy assessed CRF1 and CRF2 cellular localization 24 hours after the last stress. RESULTS: Rats responded to social stress passively, assuming defeat with short latencies (48%), or actively, with proactive behaviors and longer defeat latencies (LL, 52%). Whereas CRF (30 ng, intra-DR) inhibited 5-HT neuronal activity of control and SL rats, it activated 5-HT neurons of LL rats, an effect that was CRF2-mediated. Consistent with this, social stress promoted CRF1 internalization together with CRF2 recruitment to the plasma membrane of DR neurons selectively in LL rats. CONCLUSIONS: These data suggest that a proactive coping strategy toward social stress is associated with a redistribution of CRF1 and CRF2 in DR-5-HT neurons that primes the system to be activated by subsequent stress. The lack of this adaptation in passive coping rats may contribute to their depressive-like phenotype. These studies provide a cellular mechanism for individual differences in stress responses and consequences.

Imaging pancreatic beta-cells in the intact pancreas.

We have developed a method to visualize fluorescent protein-labeled beta-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the beta-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, beta-cell clusters, and single beta-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary beta-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red beta-cells. The imaging technique that we have developed, coupled with the transgenic mice in which beta-cells and beta-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states.

Foxa2 is required for the differentiation of pancreatic alpha-cells.

The differentiation of insulin-producing beta-cells has been investigated in great detail; however, little is known about the factors that delineate the second-most abundant endocrine lineage, the glucagon-producing alpha-cell. Here we utilize a novel YAC-based Foxa3Cre transgene to delete the winged helix transcription factor Foxa2 (formerly HNF-3beta) in the pancreatic primordium during midgestation. The resulting Foxa2(loxP/loxP); Foxa3Cre mice are severely hypoglycemic and die within the first week of life. Mutant mice are hypoglucagonemic secondary to a 90% reduction of glucagon expression. While the number of mature glucagon-positive alpha-cells is dramatically reduced, specification of alpha-cell progenitors is not affected by Foxa2 deficiency. By marker gene analysis, we show that the expression of the alpha-cell transcription factors Arx, Pax6, and Brn4 does not require Foxa2 in the transcriptional hierarchy governing alpha-cell differentiation.