The Leu 7+ cell population of the human pancreas: preferential distribution and morphology

Anti-Leu 7 is a monoclonal antibody which recognizes an antigenic determinant present on the surface of a subset of human large granular lymphocytes and on central and peripheral neural elements. Furthermore it cross-reacts with an intracellular protein of secretory granule matrix in neuroendocrine cells. The presence of Leu 7+ cells has been studied in lymphoid and non-hemopoietic organs. We have analyzed the Leu 7 positivity in six pancreata from cadaver donors by means of immunocytochemical methods. Leu 7+ cells were found to be also present in the exocrine portion of the organ in which they represent a nonhomogeneous cellular population. In fact, two different types of Leu 7+ elements populate the exocrine pancreas: a, Leu 7+ cells showing an intracellular granule positivity; b, Leu 7+ cells showing surface positivity. The endocrine pancreas, in contrast, contains the majority (85%) of Leu 7+ elements, belonging to the intracellular positive type only.     

Epitope mapping by solution NMR spectroscopy

Antibodies play an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their epitopes, that is, the region that they recognize on their target molecule, is useful for purposes ranging from molecular biology research to vaccine design and intellectual property protection. Solution NMR spectroscopy is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to epitope discovery. Here, we illustrate how NMR epitope mapping can be used to rapidly and accurately determine protein antigen epitopes. The basic concept is that differences in the NMR signal of an antigen free or bound by an antibody will identify epitope residues. NMR epitope mapping provides more detailed information than mutagenesis or peptide mapping and can be much more rapid than X‐ray crystallography. Advantages and drawbacks of this technique are discussed together with practical considerations. Copyright © 2015 John Wiley & Sons, Ltd.     

An interaction between PRRT2 and Na+/K+ ATPase contributes to the control of neuronal excitability

Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na⁺/K⁺ ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.Subject terms: Proteomics, Cellular neuroscience, Molecular neuroscience, Paediatric neurological disorders     

Similarity of the nocturnal profile of serum melatonin at early puberty and early adulthood.

In the present study we determined the nocturnal profile of serum melatonin (MT) concentrations in 10 short normal children at Tanner stage I-II of pubertal development (12.5-14.9 yrs) and in 6 young adults (24-29 yrs). Blood was collected every 30 min from 00(00) to 06(00). We did not find any significant difference in the nocturnal profile of serum MT, as gauged by the comparison of MT concentrations at any time-point tested as well as of the transverse means (84.2 +/- 36.0 pg/ml [M +/- SD] in the children vs 78.7 +/- 10.8 pg/ml in the adults). Mean serum melatonin concentration was not correlated to sex hormone concentration or body surface area. Our findings do not support the view that MT concentrations fall at the beginning of pubertal development and that changes in body size may be the reason for age-dependent changes of serum MT concentrations.     

Characterization with dithiothreitol (DTT) of IgM auto-lymphocytotoxic antibodies in dialyzed patients awaiting kidney transplants

A group of 42 sensitized dialysis patients showing high reactivity (81%-100%) against a random panel of peripheral blood lymphocytes (PBL), were analyzed for the presence of autoreactive lymphocytotoxic antibodies. The test was performed at different temperatures (4 degrees C, 22 degrees C, 37 degrees C) with dithiothreitol (DTT) against autologous PBL, EBV-induced autologous B lymphoblasts (A-BCL), K562 cells and T lymphocytes. Thirteen of 42 patients had IgM auto-lymphocytotoxic antibodies. The broadly reactive IgM autoantibodies could be inactivated by treatment with DTT 5 mM and allowed the identification of the presence of autoantibodies alone or in combination with anti-IgG alloantibodies.     

Histochemical study on human germinal centre, mantle-zone and extra-follicular area lymphoid cell subpopulations. Immunological and cytochemical correlations with lymphomatous cells, peripheral normal and leukemic lymphocytes

Tissue sections of lymph nodes, appendices and tonsils, together with smears of immunologically separated peripheral lymphoid cells from a B-CLL and lymphomatous cells from an immunocytoma were submitted to combined enzyme cytochemical investigations with acid alpha-naphthyl acetate esterase (ANAE), beta-glucuronidase (B-G), acid phosphatase (AcPh), adenosine triphosphatase (ATPase), a,d 5'nucleotidase (5'N). T-cells were Acph+, ATPase- and 5'N-. The vast majority of T- and B-cells displayed ANAE and B-G activities with two distinct staining patterns (T-like and B-like pattern). A high proportion of lymphoid cells in the germinal centre (G.C.) and the vast majority of lymphoid cells in the mantle-zone (M.Z.) were shown to belong to B-cell system because of the expression of ATPase and 5'N in their membranes. Some lymphoid cells positive for ANAE and B-G with a B-like pattern and for AcPh were recognizable in the G.C. In the M.Z. only a few lymphoid cells being ANAE+, with a T-like pattern, and AcPh+ were shown to belong to the T-cell system. In contrast, in this zone a high proportion of small lymphoid cells (64% +/- 10%) showed ANAE activity, mostly with B-like pattern. Therefore, these findings indicate that in the M.Z. a high proportion of B-cells ATPase+ and 5'N+ also display ANAE activity. By comparison of the results obtained from lymphoid tissue sections, B-CLL and immunocytoma cell suspensions and normal circulating lymphocytes we can conclude that B-ANAE-positive cells of the M.Z. do not usually appear in the peripheral blood. They circulate in large numbers only in some pathological conditions (like our reported B-CLL). Therefore, B-ANAE-positive lymphoid cells of the mantle, with a B-like staining pattern, include a wide range of subsets which exclude large lymphoid cells and plasma cells.