Determination of secretory granule maturation times in pancreatic islet β-cells by serial block-face electron microscopy.

It is shown how serial block-face electron microscopy (SBEM) of insulin-secreting β-cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the β-cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It was found that 7.

Luciferase-Based Detection of Antibodies for the Diagnosis of HPV-Associated Head and Neck Squamous Cell Carcinoma.

Point-of-care tests are needed for the screening of head and neck squamous cell carcinoma (HNSCC) and other malignancies. Luciferase immunoprecipitation systems (LIPS), employing light-emitting proteins, were used to examine serum antibodies against several cancer-associated targets in blood donor controls and subjects with colon cancer (CC) and HNSCC. The assessment of antibodies against the wild type p53 tumor antigen showed that approximately 25% of the CC and 20% of the HNSCC patients were seropositive.

Insulinoma-Associated-1: From Neuroendocrine Tumor Marker to Cancer Therapeutics.

Insulinoma-associated-1 (IA-1 or INSM1) encodes a zinc-finger transcription factor, which was isolated from a human insulinoma subtraction library, with specific expression patterns, predominantly in developing neuroendocrine tissues and tumors. INSM1 is key in early pancreatic endocrine, sympatho-adrenal lineage, and pan-neurogenic precursor development. gene ablation results in impairment of pancreatic β cells, catecholamine biosynthesis, and basal progenitor development during mammalian neocortex maturation.

Targeted deletion of Insm2 in mice result in reduced insulin secretion and glucose intolerance.

Background: Neurogenin3 (Ngn3) and neurogenic differentiation 1 (NeuroD1), two crucial transcriptional factors involved in human diabetes (OMIM: 601724) and islet development, have been previously found to directly target to the E-boxes of the insulinoma-associated 2 (Insm2) gene promoter, thereby activating the expression of Insm2 in insulin-secretion cells. However, little is known about the function of Insm2 in pancreatic islets and glucose metabolisms.

Methods: Homozygous Insm2 mice were generated by using the CRISPR-Cas9 method.

Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway.

The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated.

Ultrarapid Measurement of Diagnostic Antibodies by Magnetic Capture of Immune Complexes.

Rapid point-of-care, antibody-based testing is not currently available for the diagnosis of most autoimmune and infectious diseases. Here we report a simple, robust and ultrafast fluid-phase immunocapture method for clinical measurements of antibody levels. This method employs neodymium magnetic sticks that capture protein A/G-coated paramagnetic beads bound to antibody-luciferase-labeled antigen complexes.

Small cell lung cancer growth is inhibited by miR-342 through its effect of the target gene IA-2.

Background: Small cell lung cancers (SCLC) are tumors of neuroendocrine origin. Previous in vitro studies from our laboratory showed that SCLC expresses high levels of the transmembrane dense core vesicle protein IA-2 (islet cell antigen-2) as compared to normal lung cells. IA-2, through its effect on dense core vesicles (DCVs), is known to be involved in the secretion of hormones and neurotransmitters.

Stimulation of Toll-Like Receptors profoundly influences the titer of polyreactive antibodies in the circulation.

Polyreactive antibodies are a major component of the natural antibody repertoire and bind to a variety of structurally unrelated molecules. These antibodies are thought to provide a first line of defense against bacterial infections and play a major role in the clearance of apoptotic cells. What triggers the secretion of these antibodies has remained an enigma.

Partial reconstitution of humoral immunity and fewer infections in patients with chronic lymphocytic leukemia treated with ibrutinib.

Chronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia, which contributes to a high rate of infections and morbidity. Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), inhibits B-cell receptor signaling and is an effective, US Food and Drug Administration (FDA)-approved treatment of CLL. Inactivating germline mutations in BTK cause a severe B-cell defect and agammaglobulinemia.

The Ia-2β intronic miRNA, miR-153, is a negative regulator of insulin and dopamine secretion through its effect on the Cacna1c gene in mice.

Aims/hypothesis: miR-153 is an intronic miRNA embedded in the genes that encode IA-2 (also known as PTPRN) and IA-2β (also known as PTPRN2). Islet antigen (IA)-2 and IA-2β are major autoantigens in type 1 diabetes and are important transmembrane proteins in dense core and synaptic vesicles. miR-153 and its host genes are co-regulated in pancreas and brain.

Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells.

A combination of two-dimensional (2D) and three-dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set.

Polyreactive Antibodies: Function and Quantification.

Polyreactive antibodies, a major component of the natural antibody repertoire, bind with low affinity to a variety of structurally unrelated antigens. Many of these antibodies are germline or near germline in sequence. Little is known, however, about the function of these antibodies.

Pathophysiologic changes in IA-2/IA-2β null mice are secondary to alterations in the secretion of hormones and neurotransmitters.

IA-2 and IA-2β are transmembrane proteins of dense-core vesicles (DCV). The deletion of these proteins results in a reduction in the number of DCV and the secretion of hormones and neurotransmitters. As a result, this leads to a variety of pathophysiologic changes.

Impact of natural IgM concentration on gene therapy with adenovirus type 5 vectors.

Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner.

The minimal promoter region of the dense-core vesicle protein IA-2: transcriptional regulation by CREB.

Aims: IA-2 is a transmembrane protein found in the dense-core vesicles (DCV) of neuroendocrine cells and one of the major autoantigens in type 1 diabetes. DCV are involved in the secretion of hormones (e.g.

Quantitative analysis of mouse pancreatic islet architecture by serial block-face SEM.

We have applied serial block-face scanning electron microscopy (SBF-SEM) to measure parameters that describe the architecture of pancreatic islets of Langerhans, microscopic endocrine organs that secrete insulin and glucagon for control of blood glucose. By analyzing entire mouse islets, we show that it is possible to determine (1) the distributions of alpha and beta cells, (2) the organization of blood vessels and pericapillary spaces, and (3) the ultrastructure of the individual secretory cells. Our results show that the average volume of a beta cell is nearly twice that of an alpha cell, and the total mitochondrial volume is about four times larger.

Luciferase immunoprecipitation systems for measuring antibodies in autoimmune and infectious diseases.

Antibody profiles have the potential to revolutionize personalized medicine by providing important information related to autoimmunity against self-proteins and exposure to infectious agents. One immunoassay technology, luciferase immunoprecipitation systems (LIPS), harnesses light-emitting recombinant proteins to generate robust, high-quality antibody data often spanning a large dynamic range of detection. Here, we describe the general format of LIPS and discuss studies using the technology to measure autoantibodies in several human autoimmune diseases including type 1 diabetes, Sjögren's syndrome, systemic lupus erythematosus, and immunodeficiencies secondary to anticytokine autoantibodies.

The dense core vesicle protein IA-2, but not IA-2β, is required for active avoidance learning.

The islet-antigens IA-2 and IA-2β are major autoantigens in type-1 diabetes and transmembrane proteins in dense core vesicles (DCV). Recently we showed that deletion of both IA-2 and IA-2β alters the secretion of hormones and neurotransmitters and impairs behavior and learning. The present study was designed to evaluate the contribution to learning of each of these genes by using single knockout (SKO) and double knockout (DKO) mice in an active avoidance test.

Polyreactive antibodies plus complement enhance the phagocytosis of cells made apoptotic by UV-light or HIV.

Polyreactive antibodies are a major component of the natural antibody repertoire and are capable of binding a variety of structurally unrelated antigens. Many of the properties attributed to natural antibodies, in fact, are turning out to be due to polyreactive antibodies. In humans, each day, billions of cells undergo apoptosis.

Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 β: identification of miR-153 target genes with functions related to IA-2β in pancreas and brain.

Aims/hypothesis: We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2β. We also identified miR-153 target genes that correlated with IA-2β localisation and function.

Methods: A bioinformatics approach was used to identify miR-153's genomic organisation.

[Introduction to preventive and predictive medicine: past experience and future reality].

The active practical introduction of the achievements of genomics, proteomics, metabolomics, and bioinformatics brought about a fundamental change in views on the role and place of medicine in the structure of healthcare at the turn of the 1980s-1990s, by giving impetus to the development of the radically new health care area--preventive, predictive, and personalized medicine (PPPM).