The length of survival among patients with follicular lymphoma correlates with the molecular features of nonmalignant immune cells present in the tumor at diagnosis.

SUMMARY Neuronal arborization is regulated by cell autonomous and non-autonomous mechanisms including endosomal signaling via BDNF/TrkB. The endosomal Na+/H+ exchanger 6 (NHE6) is mutated in a new autism-related disorder. NHE6 functions to permit proton leak from endosomes yet the mechanisms causing disease are unknown. We demonstrate that loss of NHE6 results in over-acidification of the endosomal compartment and attenuated TrkB signaling. Mouse brains with disrupted NHE6 display reduced axonal and dendritic branching, reduced synapse number and circuit strength. Site-directed mutagenesis shows that the proton leak function of NHE6 is required for neuronal arborization. We find that TrkB receptor co-localizes to NHE6-associated endosomes. TrkB protein and phosphorylation are reduced in NHE6 mutant neurons in response to BDNF signaling. Finally, exogenous BDNF rescues defects in neuronal arborization. We propose that NHE6 mutation leads to circuit defects that are in part due to impoverished neuronal arborization that may be treatable by enhanced TrkB signaling.

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Type 1 diabetes results from the autoimmune destruction of insulin-producing pancreatic ␤-cells by cytotoxic Tlymphocytes (CTLs). In humans, few ␤-cell epitopes have been reported, thereby limiting the study of ␤-cell-specific CTLs in type 1 diabetes. To identify additional epitopes, HLA class I peptide affinity algorithms were used to identify a panel of peptides derived from the ␤-cell proteins islet amyloid polypeptide (IAPP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulin, insulinoma-associated antigen 2 (IA-2), and phogrin that were predicted to bind HLA-A*0201. Peripheral blood mononuclear cells from 24 HLA-A*0201 recent-onset type 1 diabetic patients and 11 nondiabetic control subjects were evaluated for ␥-interferon secretion in response to peptide stimulation in enzyme-linked immunospot assays. We identified peptides IAPP9-17, IGRP215-223, IGRP152-160, islet IA-2(172-180), and IA-2(482-490) as novel HLA-A*0201-restricted T-cell epitopes in type 1 diabetic patients. Interestingly, we observed a strong inverse correlation between the binding affinity of ␤-cell peptides to HLA-A*0201 and CTL responses against those peptides in recent-onset type 1 diabetic patients. In addition, we found that self-reactive CTLs with specificity for an insulin peptide are frequently present in healthy individuals. These data suggest that many ␤-cell epitopes are recognized by CTLs in recentonset type 1 diabetic patients. These epitopes may be important in the pathogenesis of type 1 diabetes. Diabetes 55: 3068 -3074, 2006

Cytotoxic T-lymphocytes (CTLs) are considered to be essential for ␤-cell destruction in type 1 diabetes. However, few islet-associated peptides have been demonstrated to activate autoreactive CTLs from type 1 diabetic subjects. In an effort to identify novel epitopes, we used matrixassisted algorithms to predict peptides of glial fibrillary acidic protein (GFAP), prepro-islet amyloid polypeptide (ppIAPP), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) that likely bind to HLA-A*0201 with a strong affinity and contain a COOHterminal proteasomal cleavage site. Seven peptides stabilized HLA-A*0201 expression in binding assays and were used to stimulate peripheral blood mononuclear cells and were evaluated for granzyme B secretion. We found that 5 of 13 type 1 diabetic subjects and 4 of 6 antibody-positive relatives exhibited greater numbers of granzyme B-secreting cells in response to at least one putative epitope compared with healthy control subjects. The most prevalent responses in antibody-positive and type 1 diabetic subjects were to ppIAPP(9-17). Other peptides recognized by type 1 diabetic or antibody-positive subjects included GFAP(143-151), IGRP(152-160), and GFAP(214-222). These data implicate peptides of ppIAPP, GFAP, and IGRP as CTL epitopes for a heterogenous CD8 ؉ T-cell response in type 1 subjects and antibody-positive relatives. Diabetes

Luminal pH is an important functional feature of intracellular organelles. Acidification of the lumen of organelles such as endosomes, lysosomes, and the Golgi apparatus plays a critical role in fundamental cellular processes. As such, measurement of the luminal pH of these organelles has relevance to both basic research and translational research. At the same time, accurate measurement of intraorganellar pH in living cells can be challenging and may be a limiting hurdle for research in some areas. Here, we describe three powerful methods to measure rigorously the luminal pH of different intracellular organelles, focusing on endosomes, lysosomes, and the Golgi apparatus. The described methods are based on live imaging of pH-sensitive fluorescent probes and include: (1) A protocol based on quantitative, ratiometric measurement of endocytosis of pH-sensitive and pH-insensitive fluorescent conjugates of transferrin; (2) A protocol for the use of proteins tagged with a ratiometric variant of the pH-sensitive intrinsically fluorescent protein pHluorin; and (3) A protocol using the fluorescent dye LysoSensor™. We describe necessary reagents, key procedures, and methods and equipment for data acquisition and analysis. Examples of implementation of the protocols are provided for cultured cells derived from a cancer cell line and for primary cultures of mouse hippocampal neurons. In addition, we present strengths and weaknesses of the different described intraorganellar pH measurement methods. These protocols are likely to be of benefit to many researchers, from basic scientists to those conducting translational research with a focus on diseases in patient-derived cells.