Endosome maturation depends on membrane contact sites (MCSs) formed between endoplasmic reticulum (ER) and endolysosomes (LyLEs). The mechanism underlying lipid supply for this process and its pathophysiological relevance remains unclear, however. Here, we identify PDZD8—the mammalian ortholog of a yeast ERMES subunit—as a protein that interacts with protrudin, which is located at ER-LyLE MCSs. Protrudin and PDZD8 promote the formation of ER-LyLE MCSs, and PDZD8 shows the ability to extract various lipids from the ER. Overexpression of both protrudin and PDZD8 in HeLa cells, as well as their depletion in mouse primary neurons, impairs endosomal homeostasis by inducing the formation of abnormal large vacuoles reminiscent of those apparent in spastin- or REEP1-deficient neurons. The protrudin-PDZD8 system is also essential for the establishment of neuronal polarity. Our results suggest that protrudin and PDZD8 cooperatively promote endosome maturation by mediating ER-LyLE tethering and lipid extraction at MCSs, thereby maintaining neuronal polarity and integrity.

These data, although based on a limited number of patients and samples, suggest that administration of B. breve strain Yakult could be an effective approach for achieving clinical benefits in immunocompromised hosts by improving their intestinal environments.

Summary In order to clarify the probiotic features of immunomodulation, cytokine production by murine spleen and Peyer’s patch (PP) cells was examined in response to probiotic and pathogenic bacteria. In spleen cells, probiotic Lactobacillus casei induced interleukin (IL)‐12 production by CD11b+ cells more strongly than pathogenic Gram‐positive and Gram‐negative bacteria and effectively promoted the development of T helper (Th) type 1 cells followed by high levels of secretion of interferon (IFN)‐γ. Although the levels of IL‐12 secreted by PP cells in response to L. casei were lower in comparison with spleen cells, Th1 cells developed as a result of this low‐level induction of IL‐12. However, IFN‐γ secretion by the L. casei‐induced Th1 cells stimulated with a specific antigen was down‐regulated in PP cells. Development of IL‐17‐producing Th17 cells was efficiently induced in PP cells by antigen stimulation. Lactobacillus casei slightly, but significantly, inhibited the antigen‐induced secretion of IL‐17 without a decrease in the proportion of Th17 cells. No bacteria tested induced the development of IL‐10‐producing, transforming growth factor‐β‐producing or Foxp3‐expressing regulatory T cells, thus suggesting that certain probiotics might regulate proinflammatory responses through as yet unidentified mechanisms in PP cells. These data show probiotic L. casei to have considerable potential to induce IL‐12 production and promote Th1 cell development, but the secretion of proinflammatory cytokines such as IL‐12 and IL‐17 may be well controlled in PP cells.

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Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca 2ϩ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP ϩ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca 2ϩ mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 Ϯ 0.04 vs. 0.05 Ϯ 0.01 m/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types. neuroimmunity; substance P; neurokinin-1 receptor; CD63; granule tracking ANALYSES OF NERVE-MAST CELL INTERACTIONS have been of significant importance in unequivocally establishing the concept of bidirectional neuroimmune communication (13,25). In addition to the anatomic association of mast cells and nerve fibers in many tissues (3,20), numerous studies have shown that mast cell activation can be evoked by nerve stimulation or the application of neurotransmitters and that mast cell-derived mediators can influence neuronal activity (7,18,24). For instance, the neuropeptide substance P has been shown to cause mast cell degranulation when used at high doses, whereas exposure to picomolar concentrations of substance P primes the mast cell, lowering the degranulation stimulation threshold to a second stimulus (10). We have used an in vitro model of mast cell-nerve interaction, composed of cocultures of the mucosal mast cell-like rat basophilic leukemic (RBL) cells and neurite-sprouting murine superior cervical ganglia (5). We showed that nerve-mast cell communication did not require transduction by an intermediate cell and that the RBL cell activation response following neurite stimulation was mediated largely via substance P release and through neurokinin (NK)-1 receptors (21). Here, we sought to further examine neurite-RBL cell interactions, to determine whether neuroninduced mast cell activation is a generalized whole cell response or if there is an additional level of subtlety to the neurite-RBL cell interaction that occurs at the specific...