IL-12 improves the anti-HCC efficacy of dendritic cells loaded with exosomes from overexpressing Rab27a tumor cells.

Determining the appropriate source of antigens for optimal antigen presentation to T cells is a major challenge in designing dendritic cell (DC) -based therapeutic strategies against hepatocellular carcinoma (HCC). Tumor-derived exosomes (Tex) express a wide range of tumor antigens, making them a promising source of antigens for DC vaccines. As reported, the exosomes secreted by tumor cells can inhibit the antitumor function of immune cells. In this study, we transfected hepatocellular carcinoma cells with Rab27a to enhance the yield of exosomes, which were characterized using transmission electron microscopy and Western blot analysis. We found that Tex secreted by overexpressing Rab27a Hepatocellular carcinoma cell lines pulsed DC is beneficial for the differentiation and maturation of DCs but inhibits the secretion of the IL-12 cytokine. Consequently, we developed a complementary immunotherapy approach by using Tex as an antigen loaded onto DCs, in combination with the cytokine IL-12 to induce antigen-specific cytotoxic T lymphocytes （CTLs）. The results indicated that the combination of DC-Tex and IL-12 was more effective in stimulating T lymphocyte proliferation, releasing IFN-γ， and enhancing cytotoxicity compared to using exosomes or IL-12 alone. Additionally, the inclusion of IL-12 also compensated for the reduced IL-2 secretion by DCs caused by Tex. Moreover, in a BALB/c nude mice model of hepatocellular carcinoma, CTLs induced by DC-Tex combined with IL-12 maximized the tumor-specific T-cell immune effect and suppressed tumor growth. Thus, Tex provides a novel and promising source of antigens, with cytokines compensating for the shortcomings of Tex as a tumor antigen. This work helps to clarify the role of exosomes in tumor immunotherapy and may offer a safe and effective prospective strategy for the clinical application of exosome-based cellular immunotherapy.

Age-Related Changes in Neurons and Satellite Glial Cells in Mouse Dorsal Root Ganglia.

The effects of aging on the nervous system are well documented. However, most previous studies on this topic were performed on the central nervous system. The present study was carried out on the dorsal root ganglia (DRGs) of mice, and focused on age-related changes in DRG neurons and satellite glial cells (SGCs). Intracellular electrodes were used for dye injection to examine the gap junction-mediated coupling between neurons and SGCs, and for intracellular electrical recordings from the neurons. Tactile sensitivity was assessed with von Frey hairs. We found that 3-23% of DRG neurons were dye-coupled to SGCs surrounding neighboring neurons in 8-24-month (Mo)-old mice, whereas in young adult (3 Mo) mice, the figure was 0%. The threshold current for firing an action potential in sensory neurons was significantly lower in DRGs from 12 Mo mice compared with those from 3 Mo mice. The percentage of neurons with spontaneous subthreshold membrane potential oscillation was greater by two-fold in 12 Mo mice. The withdrawal threshold was lower by 22% in 12 Mo mice compared with 3 Mo ones. These results show that in the aged mice, a proportion of DRG neurons is coupled to SGCs, and that the membrane excitability of the DRG neurons increases with age. We propose that augmented neuron-SGC communications via gap junctions are caused by low-grade inflammation associated with aging, and this may contribute to pain behavior.

Systemic chemotherapy and sequential locoregional radiotherapy in initially metastatic nasopharyngeal carcinoma: Retrospective analysis with 821 cases.

BACKGROUND: We designed this retrospective study to explore the best treatment modality for patients with initially metastatic nasopharyngeal carcinoma (NPC). METHODS: From 2008 to 2017, 821 patients were enrolled. Treatment modalities and prognostic factors were analyzed. RESULTS: Compared with chemotherapy alone and radiotherapy-based treatment, systemic chemotherapy-sequential locoregional radiotherapy to the nasopharyngeal primary tumor site were associated with a significantly increased 3-year overall survival (OS) rate (40.3%, 11.7%, and 22.9%, P < .001). The overall response rate of the paclitaxel combined with platinum and fluorouracil (TPF) regimen as first-line chemotherapy was higher than that of the paclitaxel plus platinum (TP) regimen (78.2% vs 70.0%, P = .038). A better OS was achieved in the TPF group compared to doublet drug regimens (3-year OS, 35.7% vs 25.3%, P < .001). CONCLUSIONS: Systemic chemotherapy-sequential locoregional radiotherapy may prolong OS and progression-free survival for selected patients with initially metastatic NPC.

Dye coupling among satellite glial cells in mammalian dorsal root ganglia.

Dorsal root ganglia (DRG) are key elements in sensory signaling under physiological and pathological conditions. Little is known about electrical coupling among cells in these ganglia. In this study, we injected the fluorescent dye Lucifer yellow (LY) into single cells to examine dye coupling in DRG. We found no dye coupling between neurons or between neurons and their attendant satellite glial cells (SGCs). In mouse DRG, we observed that in 26.2% of the cases SGCs that surround a given neuron were dye coupled. In only 3.2% of the cases SGCs that make envelopes around different neurons were coupled. The data from mouse ganglia were very similar to those from rat and guinea pig DRG. The results obtained by injection of the tracer biocytin were very similar to those observed with LY. The coupling incidence within the envelopes increased 3.1-fold by high extracellular pH (8.0), but coupling between envelopes was not affected. Acidic pH (6.8) reduced the coupling. High extracellular K+ (9.4 mM) increased the coupling 2.4-fold and 4.7-fold within and between envelopes, respectively. Low extracellular Ca2+ (0.5, 1.0 mM) partly reversed the effect of high K+ on coupling. The results showed that SGCs in mammalian sensory ganglia are connected by gap junctions. This coupling is very sensitive to changes in pH, and can therefore be modulated under various physiological and pathological conditions. The dependence of the coupling on extracellular K+ and Ca2+ suggests that the permeability of gap junctions can be altered by physiological and pharmacological stimuli.

Regeneration of myenteric plexus in the mouse colon after experimental denervation with benzalkonium chloride.

Recent reports suggest a far greater plasticity in nerve tissue than previously believed. As the digestive tract is exposed to a variety of insults, this question is relevant to enteric nerves, but little is known about their ability to recover from damage. To address this problem, we ablated the myenteric plexus of the mouse colon with the detergent benzalkonium chloride (BAC) and followed the ensuing morphologic changes for up to 60 days by using light- and electron microscopy. We found that, 2 days after BAC application, the treated area was essentially devoid of intact nerve elements. From day 7, new nerve fibers were observed within the denervated region. This growth progressed until, at days 30-60, newly grown nerve fibers were present in most of this region, and the pattern of muscle innervation was similar to the normal one. At least part of these fibers originated at neurons within intact ganglia surrounding the denervated region. The cross-sectional area of neurons near the denervated region at day 14 was 52% greater than controls. Glial cells were closely associated with the regenerating nerve fibers. From day 14 onward, we observed undifferentiated cells and differentiating neurons in ganglia surrounding the denervated region, and by day 30, new neurons were present in the myenteric region, along with regenerating nerve fibers. We conclude that the myenteric plexus is endowed with a considerable ability of regeneration and plasticity. The results provide evidence for the presence of stem cells and for an adult neurogenesis in this plexus.

The effects of axotomy on neurons and satellite glial cells in mouse trigeminal ganglion.

Damage to peripheral nerves induces ectopic firing in sensory neurons, which can contribute to neuropathic pain. As most of the information on this topic is on dorsal root ganglia we decided to examine the influence of infra-orbital nerve section on cells of murine trigeminal ganglia. We characterized the electrophysiological properties of neurons with intracellular electrodes. Changes in the coupling of satellite glial cells (SGCs) were monitored by intracelluar injection of the fluorescent dye Lucifer yellow. Electrophysiology of SGCs was studied with the patch-clamp technique. Six to eight days after axotomy, the percentage of neurons that fire spontaneously increased from 1.6 to 12.8%, the membrane depolarized from -51.1 to -45.5 mV, the percentage of cells with spontaneous potential oscillations increased from 19 to 37%, the membrane input resistance decreased from 44.4 to 39.5 MOmega, and the threshold for firing an action potential decreased from 0.61 to 0.42 nA. These changes are consistent with increased neuronal excitability. SGCs were mutually coupled around a given neuron in 21% of the cases, and to SGCs around neighboring neurons in only 4.8% of the cases. After axotomy these values increased to 37.1 and 25.8%, respectively. After axotomy the membrane resistance of SGCs decreased from 101 MOmega in controls to 40 MOmega, possibly due to increased coupling among these cells. We conclude that axotomy affects both neurons and SGCs in the trigeminal ganglion. The increased neuronal excitability and ectopic firing may play a major role in neuropathic pain.

Microarray analysis of gene expression profiles in the bovine mammary gland during lactation.

Mammary glands undergo functional and metabolic changes during virgin, lactation and dry periods. A total of 122 genes were identified as differentially expressed, including 79 up-regulated and 43 down-regulated genes during lactation compared with virgin and dry periods. Gene ontology analysis showed the functional classification of the up-regulated genes in lactation, including transport, biosynthetic process, signal transduction, catalytic activity, immune system process, cell death, and positive regulation of the developmental process. Microarray data clarified molecular events in bovine mammary gland lactation.

Gap junctions in dorsal root ganglia: possible contribution to visceral pain.

Peripheral injuries can lead to sensitization of neurons in dorsal root ganglia (DRGs), which can contribute to chronic pain. The neurons are sensitized by a combination of physiological and biochemical changes, whose full details are still obscure. Another cellular element in DRGs are satellite glial cells (SGCs), which surround the neurons, but little is known about their role in nociception. We investigated the contribution of SGCs to neuronal sensitization in isolated S1 DRGs from a mouse model of colonic inflammation induced by local application of dinitrosulfonate benzoate (DNBS). Retrograde labeling was used to identify DRG neurons projecting to the colon. Cell-to-cell coupling was determined by intracellular dye injection, and the electrical properties of the neurons were studied with intracellular electrodes. Pain behavior was assessed with von-Frey hairs. The dye injections showed that 10-12 days after DNBS application there was a 6.6-fold increase in gap junction-mediated coupling between SGCs surrounding adjacent neurons, and this occurred preferentially (another 2-fold increase) near neurons that project to the colon. Neuron-neuron coupling incidence increased from 0.7% to 12.1% by colonic inflammation. Inflammation led to an augmented neuronal excitability, and to a reduced pain threshold. Gap junction blockers abolished the inflammation-induced changes in SGCs and neurons, and significantly reversed the pain behavior. We propose that inflammation induces augmented cell coupling in DRGs that contributes to neuronal hyperexcitability, which in turn leads to visceral pain. Gap junction blockers may have potential as analgesic drugs.

Plasticity of interstitial cells of Cajal: a study of mouse colon.

Ablation of the myenteric plexus in mouse colon with the detergent benzalkonium chloride (BAC) is followed by considerable recovery of the nerves, indicating that this plexus is capable of regeneration and has plasticity. Interstitial cells of Cajal (ICC) are closely associated with enteric nerves, and the acquisition and maintenance of their adult phenotype are nerve-dependent. Little is known about the regenerative processes of ICC or about the possible dependence of these processes on neurons. To address these questions, we ablated the myenteric plexus in the mouse colon with BAC and followed changes in the adjacent ICC (ICC-MP) from day 2 to day 70 after treatment, by using c-kit-immunohistochemistry and electron microscopy. In the untreated area, c-kit-positive cells and ICC-MP with normal ultrastructural features were always present. The region partially affected by BAC contained some c-kit-positive cells, and either normal or vacuolated ICC-MP were observed by electron microscopy. Moreover, at days 60-70, ICC-MP with particularly extended rough endoplasmic reticulum were present in this area. In the treated area, either denervated or reinnervated, c-kit-positive cells were always absent. By day 14 after BAC treatment, nerve fibers had started to grow back into the treated region and, in the reinnervated area, cells with fibroblast-like features appeared and were seen to contact both nerve endings and smooth muscle cells and to acquire some typical ICC features. Thus, ICC are vulnerable to external insult but appear to have some ability to regenerate.

Morphological and electrophysiological changes in mouse dorsal root ganglia after partial colonic obstruction.

There is evidence that sensitization of neurons in dorsal root ganglia (DRG) may contribute to pain induced by intestinal injury. We hypothesized that obstruction-induced pain is related to changes in DRG neurons and satellite glial cells (SGCs). In this study, partial colonic obstruction was induced by ligation. The neurons projecting to the colon were traced by an injection of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate into the colon wall. The electrophysiological properties of DRG neurons were determined using intracellular electrodes. Dye coupling was examined with an intracellular injection of Lucifer yellow (LY). Morphological changes in the colon and DRG were examined. Pain was assessed with von Frey hairs. Partial colonic obstruction caused the following changes. First, coupling between SGCs enveloping different neurons increased 18-fold when LY was injected into SGCs near neurons projecting to the colon. Second, neurons were not coupled to other neurons or SGCs. Third, the firing threshold of neurons projecting to the colon decreased by more than 40% (P < 0.01), and the resting potential was more positive by 4-6 mV (P < 0.05). Finally, the number of neurons displaying spontaneous spikes increased eightfold, and the number of neurons with subthreshold voltage oscillations increased over threefold. These changes are consistent with augmented neuronal excitability. The pain threshold to abdominal stimulation decreased by 70.2%. Inflammatory responses were found in the colon wall. We conclude that obstruction increased neuronal excitability, which is likely to be a major factor in the pain behavior observed. The augmented dye coupling between glial cells may contribute to the neuronal hyperexcitability.