Search

Vagal afferents form the primary gut-to-brain neural axis, communicating signals that regulate gastrointestinal (GI) function and promote satiation, appetition and reward. Neurotrophin-4 (NT-4) is essential for the survival of vagal smooth muscle afferents of the small intestine, but not the stomach. Here we took advantage of near-complete labeling of GI vagal mucosal afferents in Nav1.8cre-Rosa26tdTomato transgenic mice to determine whether these afferents depend on NT-4 for survival. We quantified the density and distribution of vagal afferent terminals in the stomach and small intestine mucosa and their central terminals in the solitary tract nucleus (NTS) and area postrema in NT-4 knockout (KO) and control mice. NT-4KO mice exhibited a 75% reduction in vagal afferent terminals in proximal duodenal villi and a 55% decrease in the distal ileum, whereas, those in the stomach glands remained intact. Vagal crypt afferents were also reduced in some regions of the small intestine, but to a lesser degree. Surprisingly, NT-4KO mice exhibited an increase in labeled terminals in the medial NTS. These findings, combined with previous results, suggest NT-4 is essential for survival of a large proportion of all classes of vagal afferents that innervate the small intestine, but not those that supply the stomach. Thus, NT-4KO mice could be valuable for distinguishing gastric and intestinal vagal afferent regulation of GI function and feeding. The apparent plasticity of central vagal afferent terminals - an increase in their density - could have compensated for loss of peripheral terminals by maintaining near-normal levels of satiety signaling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34(+) bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics.

The presence of valine (V) at position 158 of FcgammaRllla (CD16) is known to improve clinical response to rituximab in indolent non-Hodgkin lymphoma (NHL). Little is known about the basic mechanisms for this observation. We examined natural killer (NK) cells from healthy donors representing the FcgammaRIIIa-158 polymorphic subgroups (V/V, V/F, and F/F) for gene transcript and cell surface CD16 expression, rituximab binding, and rituximab-dependent NK cell-mediated cytotoxicity. We observed higher levels of FcgammaRIIIa transcripts among individuals with the FcgammaRIIIa-158 V/V versus V/F or F/F genotype (P < .001); increased cell surface CD16 expression by quantitative flow cytometry on NK cells from individuals expressing at least one valine at FcgammaRIIIa-158 versus F/F (P = .029); as well as augmented rituximab binding and rituximab-mediated, antibody-dependent cellular cytotoxicity (ADCC). These results suggest that individuals expressing at least one valine at FcgammaRIIIa-158 might, in part, have better clinical outcomes due to increased CD16 expression, rituximab binding, and rituximab-mediated ADCC.

Sensory innervation of the gastrointestinal (GI) tract by the vagus nerve plays important roles in regulation of GI function and feeding behavior. This innervation is composed of a large number of sensory pathways, each arising from a different population of sensory receptors. Progress in understanding the functions of these pathways has been impeded by their close association with vagal efferent, sympathetic, and enteric systems, which makes it difficult to selectively label or manipulate them. We suggest that a genetic approach may overcome these barriers. To illustrate the potential value of this strategy, as well as to gain insights into its application, investigations of CNS pathways and peripheral tissues involved in energy balance that benefited from the use of gene manipulations are reviewed. Next, our studies examining the feasibility of using mutations of developmental genes for manipulating individual vagal afferent pathways are reviewed. These experiments characterized mechanoreceptor morphology, density and distribution, and feeding patterns in four viable mutant mouse strains. In each strain a single population of vagal mechanoreceptors innervating the muscle wall of the GI tract was altered, and was associated with selective effects on feeding patterns, thus supporting the feasibility of this strategy. However, two limitations of this approach must be addressed for it to achieve its full potential. First, mutation effects in tissues outside the GI tract can contribute to changes in GI function or feeding. Additionally, knockouts of developmental genes are often lethal, preventing analysis of mature innervation and ingestive behavior. To address these issues, we propose to develop conditional gene knockouts restricted to specific GI tract tissues. Two genes of interest are brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), which are essential for vagal afferent development. Creating conditional knockouts of these genes requires knowledge of their GI tract expression during development, which little is known about. Preliminary investigation revealed that during development BDNF and NT-3 are each expressed in several GI tract regions, and that their expression patterns overlap in some tissues, but are distinct in others. Importantly, GI tissues that express BDNF or NT-3 are innervated by vagal afferents, and expression of these neurotrophins occurs during the periods of axon invasion and receptor formation, consistent with roles for BDNF or NT-3 in these processes and in receptor survival. These results provide a basis for targeting BDNF or NT-3 knockouts to specific GI tract tissues, and potentially altering vagal afferent innervation only in that tissue (e.g., smooth muscle vs. mucosa). Conditional BDNF or NT-3 knockouts that are successful in selectively altering a vagal GI afferent pathway will be valuable for developing an understanding of that pathway's roles in GI function and food intake.

Leukemic cells from 30% of patients with acute myeloid leukemia (AML) have an activating mutation in the FLT3 (fms-like tyrosine kinase) gene, which represents a target for drug therapy. We treated 20 patients, each with mutant FLT3 relapsed/refractory AML or high-grade myelodysplastic syndrome and not believed to be candidates for chemotherapy, with an FLT3 tyrosine kinase inhibitor, PKC412 (N-benzoylstaurosporine), at a dose of 75 mg 3 times daily by mouth. The drug was generally well tolerated, although 2 patients developed fatal pulmonary events of unclear etiology. The peripheral blast count decreased by 50% in 14 patients (70%). Seven patients (35%) experienced a greater than 2-log reduction in peripheral blast count for at least 4 weeks (median response duration, 13 weeks; range, 9-47 weeks); PKC412 reduced bone marrow blast counts by 50% in 6 patients (2 of these to < 5%). FLT3 autophosphorylation was inhibited in most of the Corresponding patients, indicating in vivo target inhibition at the dose schedule used in this study. PKC412 is an oral tyrosine kinase inhibitor with clinical activity in patients with AML whose blasts have an activating mutation of FLT3, suggesting potential use in combination with active agents, such as chemotherapy.

The FLT3 receptor is activated by juxtamembrane insertion mutations and by activation loop point mutations in patients with acute myeloid leukemia (AML). In a systematic tyrosine kinase gene exon resequencing study, 21 of 24 FLT3 exons were sequenced in samples from 53 patients with AML, 9 patients with acute lymphoblastic leukemia (ALL), and 3 patients with myelodysplasia samples. Three patients had novel point mutations at residue N841 that resulted in a change to isoleucine in 2 samples and to tyrosine in 1 sample. Introduction of FLT3-N841I cDNA into Ba/F3 cells led to interleukin-3 (IL-3)-independent proliferation, receptor phosphorylation, and constitutive activation of signal transducer and activator of transcription 5 (STAT5) and extracellular regulatory kinase (ERK), suggesting that the N841I mutation confers constitutive activity to the receptor. An FLT3 inhibitor (PKC412) inhibited the growth of Ba/F3-FLT3N841I cells (IC(50) 10 nM), but not of wild-type Ba/F3 cells cultured with IL-3. PKC412 also reduced tyrosine phosphorylation of the mutant receptor and inhibited STAT5 phosphorylation. Examination of the FLT3 autoinhibited structure showed that N841 is the key residue in a hydrogen-bonding network that likely stabilizes the activation loop. These results suggest that mutations at N841 represent a significant new activating mutation in patients with AML and that patients with such mutations may respond to small-molecule FLT3 inhibitors such as PKC412.

Activating mutations of the FLT3 receptor tyrosine kinase are common in acute myelogenous leukemia (AML) but are rare in adult acute lymphoblastic leukemia (ALL). We have recently shown that FLT3 is highly expressed and often mutated in ALLs with rearrangement of the mixed lineage leukemia (MLL) gene on chromosome 11q23. Because hyperdiploid ALL samples also show high-level expression of FLT3, we searched for the presence of FLT3 mutations in leukemic blasts from 71 patients with ALL. The data show that approximately 25% (6 of 25) of hyperdiploid ALL samples possess FLT3 mutations, whereas only 1 of 29 TEL/AML1-rearranged samples harbored mutations (P =.04, Fisher exact test). Three mutations are novel in-frame deletions within a 7-amino acid region of the receptor juxtamembrane domain. Finally, 3 samples from patients whose disease would relapse harbored FLT3 mutations. These data suggest that patients with hyperdiploid or relapsed ALL might be considered candidates for therapy with newly described small-molecule FLT3 inhibitors.

Aberrant expression of transcription factor oncogenes such as HOX11, HOX11L2, TAL1/SCL, LYL1, LMO1, and LMO2 can be detected in lymphoblasts from up to 80% of patients with acute T-cell lymphoblastic leukemia (T-ALL). Transcriptional activation of these oncogenes in leukemic cells typically results from chromosomal rearrangements that place them next to highly active cis-acting transcriptional regulatory elements. However, biallelic activation of TAL1 in some T-ALL cases has been previously proposed. We have used allele-specific mRNA analysis to show that trans-acting mechanisms leading to biallelic overexpression of TAL1 are involved in 10 (42%) of 24 TAL1+ informative T-ALL cases, 2 (17%) of 12 HOX11+ informative cases, and 7 (64%) of 11 LMO2+ informative cases. We propose that aberrant expression of oncogenic transcription factors in a significant fraction of T-ALLs may result from loss of the upstream transcriptional mechanisms that normally down-regulate the expression of these oncogenes during T-cell development.

On the premise that empathy is important in patient care and nutrition counseling, the empathy levels of 217 clinical dietitians and 168 dietetic interns were measured using the Davis Interpersonal Reactivity Index. The Interpersonal Reactivity Index is designed to measure four aspects of empathy: empathic concern (EC), fantasy (FS), personal distress (PD), and perspective-taking (PT). Personal and professional characteristics were surveyed to determine possible influence on empathy. The instrument was mailed to 26 randomly selected internship programs. All of the dietetic interns and clinical dietitians who spend 80% of their time in patient care were requested to participate. The mean ages of the dietitians and interns were 35 and 25 years, respectively. Interns scored significantly higher on the EC (29.06, p less than .05) and FS (24.65, p less than .0005) scales than the dietitians (EC = 28.20, FS = 22.53). There was a highly significant negative correlation (r = -0.26, p less than .0001) between fantasy and age for dietitians and interns and between fantasy and experience for the dietitians (r = -0.23, p less than .0005). Dietitians and interns who had had graduate coursework in counselling, psychology, and/or educational psychology had significantly lower mean scores on the PD (17.14, p less than .05) and EC (27.82, p less than .05) scales and a significantly higher mean score on the PT (27.06, p less than .05) scale. Responses to an opinion question indicated that the mean for ideal percentage of time spent in direct patient contact was 70.1 for dietitians and 61.4 for interns, a highly significant difference (p less than .0001).(ABSTRACT TRUNCATED AT 250 WORDS)

A method for obtaining authentic human angiogenin utilizing an Escherichia coli recombinant expression system is described. A synthetic gene encoding angiogenin was placed into a vector for direct expression under the control of a modified E. coli trp promoter. The protein was produced by the bacteria in an insoluble form and purified to homogeneity by cation-exchange and reversed-phase HPLC following reduction/solubilization and reoxidation. The protein isolated was identified as Met-(-1) angiogenin by amino acid analysis and tryptic peptide mapping; the latter demonstrated that all three disulfide bonds had formed correctly. Both the enzymatic and angiogenic activities of the Met-(-1) protein were equivalent to those of native angiogenin. A Met-(-1) Leu-30 derivative of angiogenin was also isolated and found to be fully active. Conversion of Met-(-1) angiogenin to the authentic less than Glu-1 protein was achieved by treatment with Aeromonas aminopeptidase under conditions in which the new N-terminal glutamine readily cyclizes nonenzymatically. This aminopeptidase treatment may have more general applicability for removal of undesirable N-terminal methionine residues from foreign proteins expressed in bacteria.

A human placental cDNA library was screened using oligonucleotide probes based on the peptide sequence of the human class III alcohol dehydrogenase. An incomplete cDNA clone covering most of the coding sequence of class III alcohol dehydrogenase was isolated from a human placental cDNA library. This was subsequently used as a probe to obtain a full-length clone from a human testicular library. The cDNA sequence codes for a protein that is identical to the enzyme purified from human liver. Southern analysis of human genomic DNA suggests that it may contain more than a single copy per haploid genome.