Dendrobium officinale Kinura et Migo glycoprotein promotes skin wound healing by regulating extracellular matrix secretion and fibroblast proliferation on the proliferation phase.

Dendrobium officinale Kinura et Migo (DOKM) has a variety of medicinal applications; however, its ability to promote wound healing has not been previously reported. The purpose of this study is to investigate the proliferative phase of the wound-healing effect of DOKM glycoprotein (DOKMG) in rats and to elucidate its mechanism of action in vitro. In the present study, the ointment mixture containing DOKMG was applied to the dorsal skin wounds of the full-thickness skin excision rat model, and the results showed that the wound healing speed was faster in the proliferative phase than vaseline. Histological analysis demonstrates that DOKMG promoted the re-epithelialization of wound skin. Immunofluorescence staining and quantitative polymerase chain reaction assays revealed that DOKMG promotes the secretion of Fibronectin and inhibits the secretion of Collagen IV during the granulation tissue formation period, indicating that DOKMG could accelerate the formation of granulation tissue by precisely regulating extracellular matrix (ECM) secretion. In addition, we demonstrated that DOKMG enhanced the migration and proliferation of fibroblast (3T6 cell) in two-dimensional trauma by regulating the secretion of ECM, via a mechanism that may implicate the AKT and JAK/STAT pathways under the control of epidermal growth factor receptor (EGFR) signalling. In summary, we have demonstrated that DOKMG promotes wound healing during the proliferative phase. Therefore, we suggest that DOKMG may have a potential therapeutic application for the treatment and management of cutaneous wounds.

Moringa oleifera Leaves Protein Enhances Intestinal Permeability by Activating TLR4 Upstream Signaling and Disrupting Tight Junctions.

Changes in intestinal mucosal barrier permeability lead to antigen sensitization and mast cell-mediated allergic reactions, which are considered to play important roles in the occurrence and development of food allergies. It has been suggested that protein causes increased intestinal permeability via mast cell degranulation, and we investigated the effect of camellia Moringa oleifera leaves protein on intestinal permeability and explored its role in the development of food allergies. The current study investigated the effect of M. oleifera leaves protein on intestinal permeability through assessments of transepithelial electrical resistance (TEER) and transmembrane transport of FITC-dextran by Caco-2 cells. The expression levels of Toll-like receptor 4 (TLR4), IL-8, Occludin, Claudin-1, and perimembrane protein family (ZO-1) were detected by real-time PCR and Western blotting. The effect of M. oleifera leaves protein on intestinal permeability was verified in mice in vivo. The serum fluorescence intensity was measured using the FITC-dextran tracer method, and the expression of tight junction proteins was detected using Western blotting. The results showed that M. oleifera leaves protein widened the gaps between Caco-2 cells, reduced transmembrane resistance, and increased permeability. This protein also reduced the mRNA and protein levels of Occludin, Claudin-1, and ZO-1. Animal experiments showed that intestinal permeability was increased, and that the expression of the tight junction proteins Occludin and Claudin-1 were downregulated in mice. This study shows that M. oleifera leaves protein has components that increase intestinal permeability, decrease tight junction protein expression, promote transmembrane transport in Caco-2 cells, and increase intestinal permeability in experimental animals. The finding that M. oleifera leaves active protein increases intestinal permeability suggests that this protein may be valuable for the prevention, diagnosis, and treatment of M. oleifera leaves allergy.

Development of RAG2 -/- IL2Rγ -/Y immune deficient FAH-knockout miniature pig.

Human hepatocyte transplantation for liver disease treatment have been hampered by the lack of quality human hepatocytes. Pigs with their large body size, longevity and physiological similarities with human are appropriate animal models for the in vivo expansion of human hepatocytes. Here we report on the generation of RAG2-/-IL2Rγ-/YFAH-/- (RGFKO) pigs via CRISPR/Cas9 system and somatic cell nuclear transfer. We showed that thymic and splenic development in RGFKO pigs was impaired. V(D)J recombination processes were also inactivated. Consequently, RGFKO pigs had significantly reduced numbers of porcine T, B and NK cells. Moreover, due to the loss of FAH, porcine hepatocytes continuously undergo apoptosis and consequently suffer hepatic damage. Thus, RGFKO pigs are both immune deficient and constantly suffer liver injury in the absence of NTBC supplementation. These results suggest that RGFKO pigs have the potential to be engrafted with human hepatocytes without immune rejection, thereby allowing for large scale expansion of human hepatocytes.

Pu-erh Tea Water Extract Mediates Cell Cycle Arrest and Apoptosis in MDA-MB-231 Human Breast Cancer Cells.

Pu-erh tea is believed to have health benefits, the growth inhibition activity of Pu-erh tea on breast cancer cell has not been investigated. In this study, we examined the activity of Pu-erh tea water extract on apoptosis and cell cycle arrest in the human breast adenocarcinoma cell line MDA-MB-231 and clarified its underlying mechanism of action. We found that Pu-erh tea extract inhibited cell proliferation and induced apoptosis in a dose-dependent manner. We also found that Pu-erh tea extract inhibited tumor cell growth within 24 h via accumulation of cells in S phase. Further experiments showed that at 24 h, Pu-erh tea extract up-regulated the expressions of P-p53 (Ser15), p21 and P-JNK and down-regulated the expressions of PCNA, CyclinD1 and CyclinE at the protein level in MDA-MB-231 cells. In particular, the JNK-specific inhibitor SP600125 restored the induction of P-JNK, P-p53 (Ser15), p21, CyclinD1 and CyclinE by Pu-erh tea extract. Our results indicate that Pu-erh tea water extract inhibits cell proliferation of MDA-MB-231 cells through the induction of apoptosis and the stimulation of cell cycle arrest, which is mediated via activation of the JNK-related pathway.

Constitutive lymphocyte transmigration across the basal lamina of high endothelial venules is regulated by the autotaxin/lysophosphatidic acid axis.

Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC's effect was abrogated by ATX inhibition, whereas LPA's effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte-HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state.

Temporal changes in cell marker expression and cellular infiltration in a controlled cortical impact model in adult male C57BL/6 mice.

BACKGROUND: Traumatic injury to the central nervous system (CNS) triggers a robust inflammatory response that leads to axonal damage and secondary degeneration of spared tissue. In contrast, some immune responses have neuroprotective effects. However, detailed information regarding the dynamics of immune responses after traumatic CNS injury is still unavailable. METHODS: In the present study, changes in the immune cells present in the injured brain, spleen, and cervical lymph nodes (CLNs), which are draining lymphatic organs from the CNS, were analyzed after controlled cortical impact (CCI) by flow cytometry and immunohistochemistry. RESULTS: The number of neutrophils and macrophages that infiltrated the injured brain immediately increased 1 d post-injury and declined rapidly thereafter. In the injured brain, resident microglia showed a bimodal increase during the first week and in the chronic phase (≥3 weeks) after injury. Increase in the Iba-1(+) microglia/macrophages was observed around the injured site. Morphologic analysis showed that Iba-1(+) cells were round at 1 week, whereas those at 3 weeks were more ramified. Furthermore, CD86(+)/CD11b(+) M1-like microglia increased at 4 weeks after CCI, whereas CD206(+)/CD11b(+) M2-like microglia increased at 1 week. These results suggest that different subsets of microglia increased in the acute and chronic phases after CCI. Dendritic cells and T cells increased transiently within 1 week in the injured brain. In the CLNs and the spleen, T cells showed dynamic changes after CCI. In particular, the alteration in the number of T cells in the CLNs showed a similar pattern, with a 1-week delay, to that of microglia in the injured brain. CONCLUSION: The data from this study provide useful information on the dynamics of immune cells in CNS injuries.

Constitutive plasmacytoid dendritic cell migration to the splenic white pulp is cooperatively regulated by CCR7- and CXCR4-mediated signaling.

Although the spleen plays an important role in host defense against infection, the mechanism underlying the migration of the innate immune cells, plasmacytoid dendritic cells (pDCs), into the spleen remains ill defined. In this article, we report that pDCs constitutively migrate into the splenic white pulp (WP) in a manner dependent on the chemokine receptors CCR7 and CXCR4. In CCR7-deficient mice and CCR7 ligand-deficient mice, compared with wild-type (WT) mice, substantially fewer pDCs were found in the periarteriolar lymphoid sheath of the splenic WP under steady-state conditions. In addition, the migration of adoptively transferred CCR7-deficient pDCs into the WP was significantly worse than that of WT pDCs, supporting the idea that pDC trafficking to the splenic WP requires CCR7 signaling. WT pDCs responded to a CCR7 ligand with modest chemotaxis and ICAM-1 binding in vitro, and priming with the CCR7 ligand enabled the pDCs to migrate efficiently toward low concentrations of CXCL12 in a CXCR4-dependent manner, raising the possibility that CCR7 signaling enhances CXCR4-mediated pDC migration. In agreement with this hypothesis, CCL21 and CXCL12 were colocalized on fibroblastic reticular cells in the T cell zone and in the marginal zone bridging channels, through which pDCs appeared to enter the WP. Furthermore, functional blockage of CCR7 and CXCR4 abrogated pDC trafficking into the WP. Collectively, these results strongly suggest that pDCs employ both CCR7 and CXCR4 as critical chemokine receptors to migrate into the WP under steady-state conditions.

Novel regulators of lymphocyte trafficking across high endothelial venules.

The physiological recruitment of circulating lymphocytes from the blood into secondary lymphoid tissues is an essential homeostatic mechanism for the immune system because it allows lymphocytes to encounter efficiently both their specific cognate antigen and the regulatory cells with which they need to interact, to initiate, maintain, and terminate immune responses appropriately. This constitutive lymphocyte trafficking is mediated by high endothelial venules (HEVs), which are present in secondary lymphoid tissues other than the spleen. There is growing evidence that lymphocyte trafficking across HEVs involves at least three steps, namely, (i) tethering/rolling, (ii) arrest/firm adhesion/intraluminal crawling, and (iii) transendothelial migration (TEM). Although the mechanisms underlying the first two steps have been determined relatively well, the mechanism regulating TEM is only partially understood. In particular, the molecular mechanism driving lymphocyte movement from the apical to the basolateral surface of the endothelial cells (ECs) of HEVs remains ill defined. This step is crucial for successful lymphocyte extravasation, and is thus an important target for therapeutic intervention in various immunological diseases. Here, we review the molecular mechanisms governing lymphocyte-HEV interactions, and highlight possible roles for two HEV proteins, i.e., nepmucin/CD300g and autotaxin, in lymphocyte TEM.

Constitutive expression of IDO by dendritic cells of mesenteric lymph nodes: functional involvement of the CTLA-4/B7 and CCL22/CCR4 interactions.

Dendritic cells (DCs) express the immunoregulatory enzyme IDO in response to certain inflammatory stimuli, but it is unclear whether DCs express this enzyme under steady-state conditions in vivo. In this study, we report that the DCs in mesenteric lymph nodes (MLNs) constitutively express functional IDO, which metabolizes tryptophan to kynurenine. In line with a previous report that regulatory T cells (Tregs) can induce IDO in DCs via the CTLA-4/B7 interaction, a substantial proportion of the MLN DCs were located in juxtaposition to Tregs, whereas this tendency was not observed for splenic DCs, which do not express IDO constitutively. When CTLA-4 was selectively deleted in Tregs, the frequency of IDO-expressing DCs in MLNs decreased significantly, confirming CTLA-4's role in IDO expression by MLN DCs. We also found that the MLN DCs produced CCL22, which can attract Tregs via CCR4, and that the phagocytosis of autologous apoptotic cells induced CCL22 expression in CCL22 mRNA-negative DCs. Mice genetically deficient in the receptor for CCL22, CCR4, showed markedly reduced IDO expression in MLN-DCs, supporting the involvement of the CCL22/CCR4 axis in IDO induction. Together with our previous observation that MLN DCs contain much intracytoplasmic cellular debris in vivo, these results indicate that reciprocal interactions between the DCs and Tregs via both B7/CTLA-4 and CCL22/CCR4 lead to IDO induction in MLN DCs, which may be initiated and/or augmented by the phagocytosis of autologous apoptotic cells by intestinal DCs. Such a mechanism may help induce the specific milieu in MLNs that is required for the induction of oral tolerance.

CXC chemokine ligand 12 promotes CCR7-dependent naive T cell trafficking to lymph nodes and Peyer's patches.

A number of chemokines, including CCL21, CCL19, CXCL12, and CXCL13, are coexpressed on the lumen or basal lamina of high endothelial venules (HEVs) in lymph nodes (LNs) and Peyer's patches (PPs), consistent with the idea that they might cooperate to regulate lymphocyte trafficking into these lymphoid tissues. In this study we report that CXCL12, acting through its receptor, CXCR4, cooperates with CCR7 ligands to promote T cell trafficking across HEVs. CXCL12 enhanced the CCR7-induced chemotaxis of wild-type but not CXCR4-deficient T cells in vitro at suboptimal concentrations of a CCR7 ligand, but without affecting the expression level or ligand-binding ability of CCR7. Real-time chemotaxis analysis showed that CXCL12 substantially shortened the lag time before cell migration began in vitro, but not the migration speed of T cells responding to suboptimal CCR7 ligand concentrations. In addition, CXCL12 augmented the CCR7 ligand-driven ERK phosphorylation and actin polymerization in T cells under the same conditions. In adoptive transfer experiments, CXCL12 promoted naive T cell trafficking to LNs and PPs in wild-type but not CCR7 ligand-deficient plt/plt recipient mice; this increased T cell trafficking was associated with enhanced binding of the T cells to HEVs and their subsequent migration into the LN parenchyma. Thus, CXCL12 synergizes with CCR7 ligands to promote T cell migration by sensitizing T cells through CXCR4, thus enabling them to respond to lower concentrations of CCR7 ligands. Such concerted action of chemokines provides an additional, previously unknown mechanism for efficient lymphocyte trafficking across HEVs into LNs and PPs.

Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions.

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.

CD4+CD25+ regulatory T cells in the small intestinal lamina propria show an effector/memory phenotype.

CD4(+)CD25(+) regulatory T cells (Tregs) have been implicated in the suppression of pathogenic responses to both self- and non-self-antigens in the intestine. However, their precise properties and functions in the gut, as well as the molecular basis of their recruitment to the gut, are poorly understood. Here, we found that most of the CD4(+)CD25(+) T cells in the small intestinal lamina propria (LP) express Foxp3 and exhibit an 'effector/memory' phenotype, CD44(hi)CD45RB(lo)CD62L(-), whereas only a minority of the Foxp3(+)CD4(+)CD25(+) T cells in the spleen and mesenteric lymph nodes showed this phenotype. The Tregs in the small intestinal LP (LP-Tregs) expressed higher levels of CCR4 and CCR9 and a substantially lower level of CCR7 than the Tregs in the spleen. In vitro, the LP-Tregs showed chemotaxis to CCL25/thymus-expressed chemokine. In addition, they showed efficient chemotaxis to the CCR4 ligands, CCL17/thymus and activation-regulated chemokine and CCL22/macrophage-derived chemokine, which are abundantly expressed by dendritic cells (DCs) in the small intestinal LP. In vivo, approximately 50% of the LP-Tregs were closely associated or in direct contact with LP-DCs. These findings demonstrate that LP-Tregs are phenotypically and functionally unique and raise the possibility that they are retained in the small intestinal LP through the action of CCL17 and CCL22, which are locally produced by LP-DCs.

Binding of lymphoid chemokines to collagen IV that accumulates in the basal lamina of high endothelial venules: its implications in lymphocyte trafficking.

Certain lymphoid chemokines are selectively and constitutively expressed in the high endothelial venules (HEV) of lymph nodes and Peyer's patches, where they play critical roles in the directional migration of extravasating lymphocytes into the lymphoid tissue parenchyma. How these chemokines are selectively localized and act in situ, however, remains unclear. In the present study, we examined the possibility that basal lamina-associated extracellular matrix proteins in the HEVs are responsible for retaining the lymphoid chemokines locally. Here we show that collagen IV (Col IV) bound certain lymphoid chemokines, including CCL21, CXCL13, and CXCL12, more potently than did fibronectin or laminin-1, but it bound CCL19 and CCL5 only weakly, if at all. Surface plasmon resonance analysis indicated that Col IV bound CCL21 with a low nanomolar K(D), which required the C-terminal region of CCL21. Col IV can apparently hold these chemokines in their active form upon binding, because the Col IV-bound chemokines induced lymphocyte migration efficiently in vitro. We found by immunohistochemistry that Col IV and CCL21, CXCL13, and CXCL12 were colocalized in the basal lamina of HEVs. When injected s.c. into plt/plt mice, CCL21 colocalized at least partially with Col IV on the basal lamina of HEVs in draining lymph nodes. Collectively, our results suggest that Col IV contributes to the creation of a lymphoid chemokine-rich environment in the basal lamina of HEVs by binding an array of locally produced lymphoid chemokines that promote directional lymphocyte trafficking from HEVs into the lymphoid tissue parenchyma.

Chemokines in tumor progression and metastasis.

Although chemokines have been thought of primarily as leukocyte attractants, a growing body of evidence indicates that they also contribute to a number of tumor-related processes, such as tumor cell growth, angiogenesis/angiostasis, local invasion, and metastasis. The current knowledge of the possible involvement of chemokines and their receptors in these cellular events are reviewed here. The operating mechanism of chemokines in relation to metastatic processes in vivo are also discussed.