Graduate anatomy education: How are we training the future generations of anatomy educators?

The teaching of anatomy is relevant to many fields and anatomy teachers are in demand. Individuals with a graduate anatomy education are some of the most sought-after candidates to fill open teaching positions, but it is unclear as to what constitutes a graduate anatomy education. The purpose of this study was to investigate the components of a graduate anatomy education in the United States. A survey regarding the components of doctoral, master's, and graduate certificate programs was distributed to program directors and department chairs at 71 US institutions. Respondents indicated that there were 17 doctoral, 28 master's, and 9 graduate certificate programs. Students completed coursework in all the traditional anatomical subdisciplines in approximately half of doctoral (53%) and master's (57%) programs, though the number was lower in graduate certificate programs (22%). In comparison, within 12 programs (5 doctoral, 4 master's, and 3 graduate certificate) students were required to complete coursework in less than 2 anatomical subdisciplines. Required coursework outside the subdisciplines usually involved educational theories and practices (61% of programs), research methods (52% of programs), and/or physiology (37% of programs). Respondents indicated that most programs (81%) were designed to prepare their students to teach. It appears that graduate anatomy training likely involves gross anatomy coursework, coursework in another anatomical subdiscipline, and coursework in educational theories and practices. Given the likely decline in the number of doctoral-level anatomy programs from 21 to 19, serious consideration should be given to hiring teaching candidates with master's or graduate certificate training in anatomy.

Targeting uPA-uPAR interaction to improve intestinal epithelial barrier integrity in inflammatory bowel disease.

BACKGROUND: Loss of intestinal epithelial barrier integrity is a critical component of Inflammatory Bowel Disease (IBD) pathogenesis. Co-expression regulation of ligand-receptor pairs in IBD mucosa has not been systematically studied. Targeting ligand-receptor pairs which are induced in IBD mucosa and function in intestinal epithelial barrier integrity may provide novel therapeutics for IBD. METHODS: We performed transcriptomic meta-analysis on public IBD datasets combined with cell surface protein-protein-interaction (PPI) databases. We explored primary human/mouse intestinal organoids and Caco-2 cells for expression and function studies of uPA-uPAR (prime hits from the meta-analysis). Epithelial barrier integrity was measured by Trans-Epithelial Electrical Resistance (TEER), FITC-Dextran permeability and tight junction assessment. Genetic (CRISPR, siRNA and KO mice) and pharmacological (small molecules, neutralizing antibody and peptide inhibitors) approaches were applied. Mice deficient of uPAR were studied using the Dextran Sulfate Sodium (DSS)-induced colitis model. FINDINGS: The IBD ligand-receptor meta-analysis led to the discovery of a coordinated upregulation of uPA and uPAR in IBD mucosa. Both genes were significantly upregulated during epithelial barrier breakdown in primary intestinal organoids and decreased during barrier formation. Genetic inhibition of uPAR or uPA, or pharmacologically blocking uPA-uPAR interaction protects against cytokine-induced barrier breakdown. Deficiency of uPAR in epithelial cells leads to enhanced EGF/EGFR signalling, a known regulator of epithelial homeostasis and repair. Mice deficient of uPAR display improved intestinal barrier function in vitro and during DSS-induced colitis in vivo. INTERPRETATION: Our findings suggest that blocking uPA-uPAR interaction via pharmacological agents protects the epithelial barrier from inflammation-induced damage, indicating a potential therapeutic target for IBD. FUNDING: The study was funded by Boehringer Ingelheim.

The intervention composed of aerobic training and non-exercise physical activity (I-CAN) study: Rationale, design and methods.

Recent data has suggested that prolonged sedentary behavior is independent risk factor for cardiovascular and all-cause mortality independent of adequate amounts of moderate to vigorous physical activity. However, few studies have prospectively evaluated if exercise training and increasing non-exercise physical activity leads to greater reduction in cardiometabolic risk compared to aerobic training alone. The purpose of the Intervention Composed of Aerobic Training and Non-Exercise Physical Activity (I-CAN) study is to determine whether a physical activity program composed of both aerobic training (consistent with public health recommendations) and increasing non-exercise physical activity (3000 steps above baseline levels) leads to enhanced improvements in waist circumference, oral glucose tolerance, systemic inflammation, body composition, and fitness compared to aerobic training alone in obese adults (N=45). Commercially available accelerometers (Fitbits) will be used to monitor physical activity levels and behavioral coaching will be used to develop strategies of how to increase non-exercise physical activity levels. In this manuscript, we describe the design, rationale, and methodology associated with the I-CAN study.

Modulation of diabetes in NOD mice by GAD65-specific monoclonal antibodies is epitope specific and accompanied by anti-idiotypic antibodies.

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells. Here we show that administration of a human monoclonal antibody (b96.11) specific to the 65-kDa isoform of glutamate decarboxylase (GAD65) to prediabetic non-obese diabetic (NOD) mice significantly delays the onset of autoimmune diabetes. We found this effect to be epitope-specific, as only b96.11 showed this therapeutic property, while a GAD65-specific human monoclonal control antibody (b78) derived from the same patient, but specific to a different determinant of GAD65, had no significant effect on the progression of disease. Administration of b96.11 or b78 to NOD mice was accompanied by the generation of anti-idiotypic antibodies. Importantly, the induced anti-idiotypic antibodies were specific for the immunizing antibody and blocked the binding of GAD65 by the respective antibody. These findings suggest a potential role for the internal image of the GAD65 determinant recognized by b96.11 in the anti-idiotypic antibody, supporting an immunomodulatory role for GAD65-specific autoantibodies, as originally postulated by Jerne.

Association of amino-terminal-specific antiglutamate decarboxylase (GAD65) autoantibodies with beta-cell functional reserve and a milder clinical phenotype in patients with GAD65 antibodies and ketosis-prone diabetes mellitus.

CONTEXT: We previously characterized patients presenting with diabetic ketoacidosis prospectively into four subgroups of ketosis-prone diabetes mellitus (KPDM), based on the presence or absence of beta-cell autoimmunity (A+ or A-) and beta-cell functional reserve (B+ or B-). The A+B- KPDM subgroup comprises patients with classic, autoimmune type 1 diabetes, whereas the A+B+ KPDM subgroup has only partial beta-cell loss and a distinct clinical phenotype. OBJECTIVE: We hypothesized that epitope specificity of autoantibodies directed against the 65-kDa isoform of glutamate decarboxylase (GAD65) reflects differences in beta-cell destruction. DESIGN: Sera of sequential GAD65Ab-positive KPDM patients admitted for diabetic ketoacidosis (n = 36) were analyzed for their epitope recognition using five GAD65-specific recombinant Fab and their ability to inhibit GAD65 enzymatic activity. All patients were followed longitudinally to assess beta-cell functional reserve and insulin dependence. RESULTS: Binding to an amino-terminal epitope defined by monoclonal antibody DPD correlated positively with fasting serum C-peptide levels at baseline (P = 0.0008) and after 1 yr (P = 0.007). Binding to the DPD-defined epitope also correlated positively with area under the curve for C-peptide after glucagon stimulation (P = 0.007) and with homeostasis model assessment percent B at 1 yr (P = 0.03). Binding to the DPD-defined epitope was significantly stronger in A+B+ than in A+B- patients (P = 0.001). Sera of 16 patients (44%) significantly inhibited GAD65 enzymatic activity, but this did not correlate with beta-cell function. CONCLUSION: DPD-defined epitope specificity is correlated directly with preserved beta-cell functional reserve in GAD65Ab-positive patients and is associated with the milder clinical phenotype of A+B+ KPDM.

A highly sensitive immunoassay resistant to autoantibody interference for detection of the diabetes-associated autoantigen glutamic acid decarboxylase 65 in blood and other biological samples.

BACKGROUND: Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet beta cells. GAD65 may also be released by transplanted islets undergoing immunological rejection. To test hypotheses regarding the utility of GAD65 as a biomarker for transplant rejection or diabetes-associated islet damage and also regarding the timing and instigators of GAD65 release in humans or animal models, a sensitive assay capable of measuring GAD65 in serum or plasma will be necessary. Ideally, this assay would also be resistant to interference by anti-GAD65 autoantibodies. METHODS: A novel, magnetic bead-based assay was developed based on GAD65 capture by a monoclonal antibody directed to the only region of the protein known not to be significantly targeted by autoantibodies. A subsequent denaturation step allows sensitive immunodetection to proceed using anti-GAD65 polyclonal antibodies that would otherwise potentially be blocked by bound autoantibodies. RESULTS: The GAD65 assay worked equally well with serum and plasma as with a solution of bovine serum albumin (BSA). The limit of blank was 31 pg/mL and did not differ significantly in the BSA solution (27 pg/mL). Mean recovery of GAD65 from the plasma of control subjects and GAD65 autoantibody-positive and -negative subjects with type 1 diabetes was 101 +/- 4.6%, 88 +/- 7.8%, and 99 +/- 7.0% (+/- SEM), respectively. The assay was used to quantify both recombinant GAD65 and the GAD65 content of human and rodent islets and other tissue extracts that were added to human plasma samples. CONCLUSIONS: A sensitive, autoantibody-resistant GAD65 assay has been developed that is compatible with detection in serum and plasma and therefore will likely also work with a variety of other biologic fluids. This assay may enable the use of circulating GAD65 as a biomarker of islet damage or transplant rejection and will facilitate in vivo studies of the pathogenesis of anti-GAD65 autoreactivity.

Analysis of GAD65 autoantibodies in Stiff-Person syndrome patients.

Autoantibodies to the 65-kDa isoform of glutamate decarboxylase GAD65 (GAD65Ab) are strong candidates for a pathological role in Stiff-Person syndrome (SPS). We have analyzed the binding specificity of the GAD65Ab in serum and cerebrospinal fluid (CSF) of 12 patients with SPS by competitive displacement studies with GAD65-specific rFab-derived from a number of human and mouse mAbs specific for different determinants on the Ag. We demonstrate considerable differences in the epitope specificity when comparing paired serum and CSF samples, suggesting local stimulation of B cells in the CSF compartment of these patients. Moreover, these autoantibodies strongly inhibit the enzymatic activity of GAD65, thus blocking the formation of the neurotransmitter gamma-aminobutyric acid. The capacity of the sera to inhibit the enzymatic activity of GAD65 correlated with their binding to a conformational C-terminal Ab epitope. Investigation of the inhibitory mechanism revealed that the inhibition could not be overcome by high concentrations of glutamate or the cofactor pyridoxal phosphate, suggesting a noncompetitive inhibitory mechanism. Finally, we identified a linear epitope on amino acids residues 4-22 of GAD65 that was recognized solely by autoantibodies from patients with SPS but not by serum from type 1 diabetes patients. A mAb (N-GAD65 mAb) recognizing this N-terminal epitope was successfully humanized to enhance its potential therapeutic value by reducing its overall immunogenicity.