Knockdown of ELMO3 Suppresses Growth, Invasion and Metastasis of Colorectal Cancer.

The engulfment and cell motility (ELMOs) family of proteins plays a crucial role in tumor cell migration and invasion. However, the function of ELMO3 is poorly defined. To elucidate its role in the development and progression of colorectal cancer (CRC), we examined the expression of ELMO3 in 45 cases of paired CRC tumor tissues and adjacent normal tissues. Furthermore, we assessed the effect of the knockdown of ELMO3 on cell proliferation, cell cycle, migration, invasion and F-actin polymerization in HCT116 cells. The result shows that the expression of ELMO3 in CRC tissues was significantly increased in comparison to the adjacent normal colorectal tissues. Moreover, this overexpression was associated with tumor size (p = 0.007), tumor differentiation (p = 0.001), depth of invasion (p = 0.009), lymph node metastasis (p = 0.003), distant metastasis (p = 0.013) and tumor, node, metastasis (TNM)-based classification (p = 0.000). In in vitro experiments, the silencing of ELMO3 inhibited cell proliferation, invasion, metastasis, and F-actin polymerization, and induced Gap 1 (G1) phase cell cycle arrest. Our study demonstrates that ELMO3 is involved in the processes of growth, invasion and metastasis of CRC, and could be used a potential molecular diagnostic tool or therapy target of CRC.

Control of Methicillin-Resistant Staphylococcus aureus Pneumonia Utilizing TLR2 Agonist Pam3CSK4.

The spread of methicillin-resistant Staphylococcus aureus (MRSA) is a critical health issue that has drawn greater attention to the potential use of immunotherapy. Toll-like receptor 2 (TLR2), a pattern recognition receptor, is an essential component in host innate defense system against S. aureus infection. However, little is known about the innate immune response, specifically TLR2 activation, against MRSA infection. Here, we evaluate the protective effect and the mechanism of MRSA murine pneumonia after pretreatment with Pam3CSK4, a TLR2 agonist. We found that the MRSA-pneumonia mouse model, pretreated with Pam3CSK4, had reduced bacteria and mortality in comparison to control mice. As well, lower protein and mRNA levels of TNF-α, IL-1ß and IL-6 were observed in lungs and bronchus of the Pam3CSK4 pretreatment group. Conversely, expression of anti-inflammatory cytokine IL-10, but not TGF-ß, increased in Pam3CSK4-pretreated mice. Our additional studies showed that CXCL-2 and CXCL1, which are necessary for neutrophil recruitment, were less evident in the Pam3CSK4-pretreated group compared to control group, whereas the expression of Fcγ receptors (FcγⅠ/Ⅲ) and complement receptors (CR1/3) increased in murine lungs. Furthermore, we found that increased survival and improved bacterial clearance were not a result of higher levels of neutrophil infiltration, but rather a result of enhanced phagocytosis and bactericidal activity of neutrophils in vitro and in vivo as well as increased robust oxidative activity and release of lactoferrin. Our cumulative findings suggest that Pam3CSK4 could be a novel immunotherapeutic candidate against MRSA pneumonia.

Overexpression of HMGB1 A-box reduced lipopolysaccharide-induced intestinal inflammation via HMGB1/TLR4 signaling in vitro.

AIM: To investigate the inhibitory effects and mechanism of high mobility group box (HMGB)1 A-box in lipopolysaccharide (LPS)-induced intestinal inflammation. METHODS: Overexpression of HMGB1 A-box in human intestinal epithelial cell lines (SW480 cells) was achieved using the plasmid pEGFP-N1. HMGB1 A-box-overexpressing SW480 cells were stimulated with LPS and co-culturing with human monocyte-like cell lines (THP-1 cells) using a Transwell system, compared with another HMGB1 inhibitor ethyl pyruvate (EP). The mRNA and protein levels of HMGB1/toll-like receptor (TLR) 4 signaling pathways [including HMGB1, TLR4, myeloid differentiation factor88 (MYD88), Phosphorylated Nuclear Factor κB (pNF-κB) p65] in the stimulated cells were determined by real-time polymerase chain reaction and Western blotting. The levels of the proinflammatory mediators [including HMGB1, interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α] in the supernatants of the stimulated cells were determined by ELISA. RESULTS: EP downregulated the mRNA and protein levels of HMGB1, inhibited the TLR4 signaling pathways (TLR4, MYD88 and pNF-κB p65) and reduced the secretion of proinflammatory mediators (HMGB1, IL-1ß, IL-6 and TNF-α) in the SW480 and THP-1 cells activated by LPS but not in the unstimulated cells. Activated by LPS, the overexpression of HMGB1 A-box in the SW480 cells also inhibited the HMGB1/TLR4 signaling pathways and reduced the secretion of these proinflammatory mediators in the THP-1 cells but not in the transfected and unstimulated cells. CONCLUSION: HMGB1 A-box, not only EP, can reduce LPS-induced intestinal inflammation through inhibition of the HMGB1/TLR4 signaling pathways.

Construction and characterization of recombinant attenuated Salmonella typhimurium expressing the babA2/ureI fusion gene of Helicobacter pylori.

AIM: This study aimed to construct a live attenuated Salmonella typhimurium strain harbouring the Helicobacter pylori babA2 and ureI fusion gene, and to evaluate its immunogenicity. METHODS: The babA2 and ureI fusion gene were cloned on an asd+ vector pYA3342 and expressed in attenuated S. typhimurium strain x8501 (Δasd). The level of babA2 and ureI fusion protein expression in S. typhimurium x8501 was examined by RT-PCR and Western blot tests. Stability of the recombinant x8501 (pYA3342/babA2/ureI) was determined after incubation for five days in vitro. RESULTS: The fusion gene, composed of 2860 base pairs, was inserted into the recombinant vector, as indicated by PCR amplification, endonuclease digestion and sequencing. Compared with the GenBank database, homologies of amino-acid sequences of the cloned babA2 and ureI were 100% and 97%, respectively. Recombinant fusion protein was recognized by commercial antibodies for whole-cell lysate of H. pylori. Furthermore, plasmids were able to stably reside in host bacteria. CONCLUSION: A prokaryotic expression system, recombinant live attenuated S. typhimurium expressing the H. pylori babA2 and ureI fusion gene, was successfully constructed, and the expressed fusion protein showed satisfactory immunoreactivity, thus offering a new candidate for prophylactic and therapeutic vaccines against H. pylori.

[Cellular immunity induced by H.pylori vaccine with chitosan as adjuvant].

AIM: To study the cellular immunity induced by H.pylori vaccine with chitosan as adjuvant and the mechanism of immunological protection. METHODS: BALB/c mice were randomly divided into nine groups and immunized by (1)PBS alone, (2)chitosan solution alone, (3)chitosan particles alone, (4)H.pylori antigen alone, (5)H.pylori antigen plus chitosan solution, (6)H.pylori antigen plus chitosan particles, (7)H.pylori antigen plus cholera toxin (CT), (8)H.pylori antigen plus chitosan solution and CT, (9)H.pylori antigen plus chitosan particles and CT orally respectively once a week for four weeks. At 4 weeks after the last immunization, mice were challenged by alive H.pylori (1x10(9) CFU/mL) twice at two days intervals. Before and after the challenge, mice were killed in batches and the H.pylori-infection in gastric mucosa was detected by H.pylori culture and Giemsa stain. ELISA and HE stain were used to detect IL-2, IL-4, IL-10 levels and pathologic change in gastric mucosa. RESULTS: (1)In the groups with chitosan as an adjuvant, 60% mice could achieve immunological protection, which was consistent with using CT as an adjuvant (58.33%), and was more than that when using H.pylori antigen alone or without H.pylori antigen. (2)After challenge, the IL-2 levels in gastric mucosa in the groups with adjuvants were significantly higher than those in the control group (P<0.001-0.05). Moreover, IL-2 levels in the groups with adjuvants after challenge were significantly higher than those before challenge (P<0.05). Before challenge, the IL-10 and IL-4 levels in gastric mucosa were significantly higher in the groups with chitosan as adjuvant than those in non-adjuvant groups (P<0.05). After challenge, IL-10 levels were significantly higher in the groups with chitosan particles as adjuvant than those in other groups (P<0.05); IL-4 levels were significantly higher in the groups with chitosan particles as an adjuvant than those in the group with CT as an adjuvant, and those in the group with chitosan solution as an adjuvant were significantly higher than those in control group, non-adjuvant group and the groups with CT (P<0.05). IL-10 and IL-4 in the groups with adjuvants after challenge were significantly lower than those before challenge (P<0.05). (3)The degree of inflammation in gastric mucosa was significantly lower in the groups with chitosan and chitosan particles as adjuvant than those with CT as adjuvant(P<0.05). CONCLUSION: (1)H.pylori vaccine with chitosan as an adjuvant has the immune protective effect against H.pylori infection. (2)H.pylori vaccine with chitosan as an adjuvant could reverse the inhibition of Th2 induced by H.pylori infection and recover the Th1/Th2 imbalance, which might contribute to the immune protection against H.pylori. (3)The rate of gastritis induced by H.pylori vaccine with chitosan as adjuvant was significantly lower than those with CT as adjuvant.

[Study on the expression of TLR4 on splenocytes and its relation with CD4(+) CD25(+) regulatory T cells in peripheral blood in severely burnt rats].

AIM: To investigate the expression of Toll like receptor 4(TLR4) on splenocytes and TNF-alpha mRNA, the dynamic changes of CD4(+) CD25(+) regulatory T cell(Treg) and endotoxin(ET) in peripheral blood of burnt rats during the early phase, and to explore the mechanism against inflammatory reaction in intestine-derived infection after burn. METHODS: 64 SD male rats were randomly separated into control group and burn model groups. Rats of burn model group were burnt with vapor under 3 mPa pressure and 108 degrees Celsius temperature for 8 seconds to achieve deep partial-thickness burn, and a 30% total body surface area (TBSA) burn model was made. Rats were sacrificed before and 2, 5, 8, 12, 24, 48 and 72 h after burn, and the TLR4 and TNF-alpha mRNA expression in splenocytes were measured at various intervals by RT-PCR. The expression of TLR4 protein was measured by Western blot, and the percentage of Treg cells in CD4(+) T cells was detected by flow cytometry (FCM), and the LPS concentration of plasma was detected by limulus lysate test. RESULTS: The expression of TLR4 mRNA, TNF-alpha mRNA, TLR4 protein and the levels of Treg, ET were significantly increased at some times points after burn. The expression of TLR4 mRNA and protein reached the peak at 8 h, whereas the TNF-alpha mRNA at 12 h, and Treg and ET at 8 h. The peak values of them were 3.66+/-0.51, 2.27+/-0.19, 1.65+/-0.23, 63.19+/-12.65% and 11.68+/-1.71 Eu/mL respectively, which were of significant difference when compared with the control group (P<0.01). The expression of TLR4 mRNA showed a positive correlation with that of Treg, ET and TNF-alpha mRNA (r=0.898, 0.811, 0.462, P<0.01). CONCLUSION: Treg might play a major role in the process of immune regulation in rats after burn, the mechanism of which may be correlated with the increase of LPS-TLR4 signal transduced by intestine-derived infection.

[Anti-Helicobacter pylori effect and regulation of T helper response of chitosan].

OBJECTIVE: To study the anti-Helicobacter pylori (Hp) effect and the regulation of T helper (Th) response of chitosan. METHODS: Hp infected grade one female BALB/c mice model was established by inoculating Hp Sydney strain 1 and the mice were randomly divided into eight groups and administrated with (1) Arabia glue solution (control group), (2) omeprazole, (3) amoxicillin, (4) amoxicillin plus omeprazole, (5) chitosan, (6) chitosan plus omeprazole, (7) chitosan plus amoxicillin, (8) chitosan plus amoxicillin plus omeprazole respectively twice daily for 14 consecutive days. Four weeks after the last administration, these mice were all killed and samples of gastric mucosa were embedded in paraffin, sectioned and assayed with Giemsa stain. The remaining gastric mucosa was used to quantitatively culture Hp. An quantitative ELISA was used to detect IL-2, IFNgamma, IL-12, IL-4, IL-10 content in gastric mucosa. RESULTS: The eradication rate of Hp was 0, 0, 41.7%, 58.3%, 58.3%, 66.7%, 83.3% and 91.7% respectively among these eight groups (P < 0.01). The Hp colony density in the (1) group and (2) group was significantly higher than that in the other six groups (P < 0.05). The Hp colony density in (3) group was significantly higher than that in (6) group, (7) group and (8) group (P < 0.05) and that in (4) group was significantly higher than that in (8) group (P < 0.05). There was no difference in the content of IL-2 in the gastric mucosa among these eight groups (P > 0.05). The content of IFNgamma, IL-12, IL-4 and IL-10 in the gastric mucosa in groups with chitosan was significantly higher than that in groups without chitosan (P < 0.05). CONCLUSIONS: Chitosan has anti-Hp effect and synergism with amoxicillin in vivo. Chitosan can up-regulate Th1 and Th2 response.

Th immune response induced by H pylori vaccine with chitosan as adjuvant and its relation to immune protection.

AIM: To study the immunological protective effect of H pylori vaccine with chitosan as an adjuvant and its mechanism. METHODS: Female BALB/c mice were randomly divided into seven groups and orally immunized respectively with PBS, chitosan solution, chitosan particles, H pylori antigen, H pylori antigen plus cholera toxin (CT), H pylori antigen plus chitosan solution, H pylori antigen plus chitosan particles once a week for four weeks. Four weeks after the last immunization, the mice were challenged twice by alive H pylori (1 x 10(9) CFU/mL) and sacrificed. Part of the gastric mucosa was embedded in paraffin, cut into sections and assayed with Giemsa staining. Part of the gastric mucosa was used to quantitatively culture H pylori. ELISA was used to detect cytokine level in gastric mucosa and anti- H pylori IgG1, IgG2a levels in serum. RESULTS: In the groups with chitosan as an adjuvant, immunological protection was achieved in 60% mice, which was significantly higher than in groups with H pylori antigen alone and without H pylori antigen (P < 0.05 or 0.001). Before challenge, the level of IFN and IL-12 in gastric mucosa was significantly higher in the groups with chitosan as an adjuvant than in the control group and the group without adjuvant (P < 0.05 or 0.005). After challenge, the level of IFN and IL-12 was significantly higher in the groups with adjuvant than in the groups without adjuvant and antigen (P < 0.05 or 0.001). Before challenge, the level of IL-2 in gastric mucosa was not different among different groups. After challenge the level of IL-2 was significantly higher in the groups with adjuvant than in the control group (P < 0.05 or 0.001). Before challenge, the level of IL-10 in gastric mucosa was significantly higher in the groups with chitosan as an adjuvant than in other groups without adjuvant (P < 0.05 or 0.01). After challenge, the level of IL-10 was not different among different groups. Before challenge, the level of IL-4 in gastric mucosa was significantly higher in the groups with chitosan as an adjuvant than in other groups without adjuvant (P < 0.05). After challenge, the level of IL-4 was significantly higher in the groups with chitosan particles as an adjuvant than in the group with CT as an adjuvant (P < 0.05), and in the group with chitosan solution as an adjuvant, the level of IL-4 was significantly higher than that in control group, non-adjuvant group and the groups with CT (P < 0.05 or 0.001). The ratio of anti- H pylori IgG2a/IgG1 in serum was significantly lower in the groups with chitosan as an adjuvant than in the groups with CT as an adjuvant or without adjuvant (P < 0.01). CONCLUSION: H pylori vaccine with chitosan as an adjuvant can protect against H pylori infection and induce both Th1 and Th2 type immune response.

[The influence of apoptosis of lymphocytes of Peyer's patches on the pathogenesis of gut barrier damage in severely scalded mice].

OBJECTIVE: To investigate the changes in cellular apoptosis of Peyer's patches in severely scalded mice, and to explore its role in the pathogenesis of gut barrier damage. METHODS: Forty BALB/c mice were randomly divided into normal control, 12 post-scald hour (12PSH), 24PSH and 72PSH groups, with 10 in each group. The mice in all PSH groups were inflicted with 20% TBSA full-thickness scald on the back. The mice in all the groups were sacrificed at different time points, and Peyer's patches were harvested from all the mice for HE staining, DNA gel electrophoresis, and flow cytometry ( FCM) examination with FITC conjugated Annexin-v and propidium iodide( PI) staining of cells. RESULTS: HE staining revealed that there were relatively abundant apoptotic cells scattering in Peyer's patches of scalded mice . DNA electrophoresis of Peyer's patches revealed typical " ladder" pattern at all indicated time points in scalded mice. Apoptotic percentage of detached Peyer's patches cells in control and scalded group were (4. 9+/-2. 1)% , (26.7+/-3. 1)% , (21.6 +/-4.0)% ,(12. 8 +/-2.0)% , respectively, and the percentage reached the peak at 12 PSH. CONCLUSION: Apoptosis is a principle modality of cell death of small intestinal Peyer's patches lymphocytes in severely scalded mice, and it might contribute to immunity barrier failure of intestinal wall after severe thermal injury.

[The role of local immune response in gastric mucosa in the protection induced by Helicobacter pylori vaccine with chitosan as adjuvant].

OBJECTIVE: To study the immunological protection of Helicobacter pylori (H. pylori) vaccine with chitosan as adjuvant and it's mechanism. METHODS: One-grade female BALB/c mice were randomly divided into nine groups and immunized by (1) PBS alone, (2) chitosan solution alone, (3) chitosan particles alone, (4) H. pylori antigen alone, (5) H. pylori antigen plus chitosan solution, (6) H. pylori antigen plus chitosan particles, (7) H. pylori antigen plus CT, (8) H. pylori antigen plus chitosan solution and cholera toxin (CT), (9) H. pylori antigen plus chitosan particles and CT orally respectively once a week for four weeks. At 4 weeks after the last immunization, these mice were challenged by alive H. pylori (1 x 10(9)/ml) twice at two days intervals. At 4 weeks after the last challenge, these mice were all killed and Gastric mucosa were embedded in paraffin, sectioned and assayed with Giemsa stain. The other gastric mucosa were used to quantitatively culture H. pylori. An ELISA was used to detect anti-H. pylori IgA in saliva and gastric mucosa and a quantitative ELISA was used to detect IL-2, IL-4, IL-10 content in gastric mucosa, and SP immunohistochemical method was used to detect secretory immunoglobulin A (sIgA) in gastric mucosa. RESULTS: (1) In the groups with chitosan as adjuvant, 60% mice could achieve immunological protection, which was according to that with CT as adjuvant (58.33%), and was significantly higher than H. pylori antigen alone and other groups without H. pylori antigen (P < 0.01 or P < 0.05). While the rates of protection in the groups with chitosan plus CT as adjuvant were 84.62%, 85.71% and the H. pylori colonization score in it was significantly lower than that in the groups with CT as adjuvant and without adjuvants (P < 0.01 or P < 0.05). (2) the labeling index for sIgA-positive lumen of glands and special anti-H. pylori IgA levels in gastric mucosa in the groups with chitosan as an adjuvant had no difference with those in the group with CT as an adjuvant (P > 0.05) and were significantly higher than those in non-adjuvant groups, while those in the groups with chitosan plus CT were significantly higher than those in the group with CT as an adjuvant (P < 0.01 or P < 0.05). (3) Before challenge, the content of IL-2 in gastric mucosa were no different among different groups (P > 0.05). After challenge the content of IL-2 were significantly higher in the groups with adjuvant than those in the control group (P < 0.01 or P < 0.05), Moreover, those in the groups with antigen after challenge were significantly higher than those before challenge (P < 0.05). (4) Before challenge, the content of IL-10 in gastric mucosa were significantly higher in the groups with chitosan as adjuvant than those in the control group and the group without adjuvant (P < 0.01 or P < 0.05). After challenge, the content of IL-10 were no different among different groups (P > 0.05). Moreover, those in the groups with adjuvant after challenge were significantly lower than those before challenge (P < 0.01). (5) Before challenge, the content of IL-4 in gastric mucosa were significantly higher in the groups with chitosan as adjuvant than those in the control group and the group without adjuvant (P < 0.05), After challenge, the content of IL-4 were significantly higher in the groups with chitosan particles as an adjuvant than those in the group with CT as an adjuvant (P < 0.05), and those in the group with chitosan solution as an adjuvant were significantly higher than those in control group, non-adjuvant group and the groups with CT (P < 0.01 or P < 0.05), Moreover, those in the groups with adjuvant after challenge were significantly lower than those before challenge (P < 0.01 or P < 0.05). CONCLUSIONS: (1) H. pylori vaccine with chitosan as adjuvant could protect against H. pylori infection, this suggested that chitosan could be a mucosa adjuvant of H. pylori vaccine, and it could effectively elicit special humoral immune response of systemic and local mucosa, which might be one of its protective mechanism. (2) H. pylori vaccine with chitosan as adjuvant may induce both Th1 and Th2 type immune response, and after challenge it could reverse the inhibition of Th2 induced by H. pylori infection and recover the Th1/Th2 imbalance. which might contribute to the immune protection against H. pylori.