Effects of different doses of dexmedetomidine on levels of serum claudin-1 and diamine oxidase in patients undergoing laparoscopic radical resection of gynecological malignant tumors / 肿瘤研究与临床

Objective:To explore the effect of different doses of dexmedetomidine (Dex) on levels of tight-junction protein claudin-1 and diamine oxidase (DAO) in patients undergoing laparoscopic radical resection of gynecological malignant tumors.Methods:A total of 60 patients with gynecological malignant tumors who were scheduled to undergo laparoscopic radical resection under general anesthesia from January 2019 to January 2020 in the Second Hospital of Shanxi Medical University were selected, including 43 cases of cervical cancer (stageⅠ-Ⅱ A), 9 cases of ovarian cancer (stageⅠ A-Ⅲ C), and 8 cases of endometrial carcinoma (stageⅠ). Accroding to the random number table method, the patients were divided into control group (group C), low-dose Dex group (group D 1) and high-dose Dex group (group D 2), with 20 cases in each group. Patients in group D 1 were given Dex 0.5 μg·kg -1·h -1 by constant rate intravenous infusion pump after induction until 30 min before the end of operation. Patients in group D 2 were given Dex 1.0 μg·kg -1·h -1 by constant rate intravenous infusion pump after induction until 30 min before the end of operation. Group C adopted the same calculation method and received the same amount of 0.9% sodium chloride solution by infusion pump. At 10 min before induction (T 1), 1 hour after pneumoperitoneum (T 2) and 12 hours after pneumoperitoneum (T 3), 5 ml of brachial vein blood was collected from the patients, and the levels of claudin-1 protein, DAO and blood glucose were measured. Results:At T 1, T 2 and T 3, the expression levels of claudin-1 in group C were (77.05±17.61) pg/ml, (66.76±12.97) pg/ml and (55.93±12.71) pg/ml, and the difference was statistically significant ( F = 10.449, P<0.05); the expression levels of DAO in group C were (4.83±0.93) ng/ml, (5.62±1.01) ng/ml and (5.98±1.21) ng/ml, and the difference was statistically significant ( F = 6.139, P < 0.05); the levels of blood glucose in group C were (4.82±0.66) mmol/L, (7.55±0.94) mmol/L and (6.51±0.54) mmol/L, and the difference was statistically significant ( F = 70.197, P < 0.05). At T 2, the expression level of claudin-1 in group D 1 was (69.12±13.02) pg/ml, which was not significantly different from group C ( t = -0.575, P > 0.05); the expression level of claudin-1 in group D 2 was (76.36±14.89) pg/ml, which was higher than that in group C, and the difference was statistically significant ( t = -2.175, P < 0.05). At T 3, the expression levels of claudin-1 in group D 1 and group D 2 were (66.14±14.36) pg/ml and (73.37±16.93) pg/ml, which were higher than that in group C, and the differences were statistically significant ( t values were -2.380 and -3.682, both P < 0.05). The expression levels of DAO in group D 1 and group D 2 were (5.02±0.84) ng/ml and (4.91±0.93) ng/ml at T 2, and (5.29±0.86) ng/ml and (5.20±0.98) ng/ml at T 3, which were lower than those in group C, and the differences were statistically significant ( t values were 2.051, 2.295, 2.079 and 2.285, all P < 0.05). The levels of blood glucose in group D 1 and group D 2 were (7.10±0.66) mmol/L and (6.77±0.97) mmol/L at T 2, and (5.95±0.94) mmol/L and (5.93±0.74) mmol/L at T 3, which were lower than those in group C, and the differences were statistically significant ( t values were 2.565, 5.374, 2.293 and 2.765, all P < 0.05). Conclusion:Continuous infusion of Dex can inhibit the stress response caused by long-term CO 2 pneumoperitoneum in laparoscopic radical resection of gynecological malignant tumors, and adjust the changes of expression levels of claudin-1 protein and DAO, reduce the damage of intestinal mucosal cells, facilitate the recovery of intestinal function, and the effect of high-dose Dex is better than low-dose Dex.

Expression and significance of tight junction protein 3 in colorectal cancer / 中国基层医药

Objective@#To analyze the expression of tight junction protein 3(claudin-3) in colorectal cancer and its relationship with the development, metastasis and prognosis of colorectal cancer.@*Methods@#From February 2013 to February 2015, 78 patients with colorectal cancer operated in the People's Hospital of Sanmen County were selected in this study.The tissues of colorectal cancer and adjacent tissues were collected and claudin-3 expression was detected.The relationship between claudin-3 and clinicopathological features of colorectal cancer was analyzed.@*Results@#The positive rate of claudin-3 in cancer tissues(83.33%) was higher than that in paracancerous tissues(48.72%), and the difference was statistically significant(χ2=20.832, P<0.01). The positive rate of claudin-3 in the poorly differentiated group(100.00%) was significantly higher than that in the well-differentiated group(85.71%) and the well-differentiated group(52.63%)(χ2=19.209, P<0.01). The claudin-3 positive rate with lymphatic metastasis(97.30%) was higher than that without lymphatic metastasis(70.73%), and the difference was statistically significant(χ2=9.882, P<0.01). The claudin-3 positive rate in patients with less than 3 years of survival (91.11%) was higher than that in patients with more than 3 years of survival(72.73%)(χ2=4.633, P<0.05).@*Conclusion@#The expression of claudin-3 is related to the occurrence, development and lymphatic metastasis of colorectal cancer, and can predict the prognosis of the patients.

Effects of sodium butyrate on intestinal barrier of severe scald mice and the related mechanism / 中华烧伤杂志

Objective@#To investigate the effects of sodium butyrate on intestinal barrier of the severe scald mice and the related mechanism.@*Methods@#Eighteen C57BL/6 female mice, aged eight to twelve weeks, were divided into sham scald group, pure scald group, and scald+ sodium butyrate group according to random number table, with 6 mice in each group. Back of each mouse in pure scald group and scald+ sodium butyrate group were immersed into 90 ℃ water for 9 s, causing full-thickness scald of 30% total body surface area, while back of each mouse in sham scald group were immersed into 37 ℃ water for 9 s, causing sham injury. All of the mice in 3 groups were intraperitoneally injected with 1 mL sterile lactated Ringer′s solution immediately after injury. Besides, mice in scald+ sodium butyrate group were intraperitoneally injected with 300 mg/kg sodium butyrate at 30 min before injury and immediately after injury, while mice in sham scald group and pure scald group were intraperitoneally injected with the same volume of sterile phosphate buffer solution. At post injury hour (PIH) 24, portal vein of mice in 3 groups was harvested, intestinal permeability was measured by fluorescin isothiocyanate-dextran fluorescence probe tracing method, then lileal tissue of mice in 3 groups was harvested, protein expressions of zonula occludens l (ZO-1), occludin, claudin-1, claudin-2, nucleotide-binding oligomerization domain-containing protein-like receptor family pyrin domain containing 3 (NLRP3), interleukin-1β (IL-1β), and IL-18 were detected by Western blotting, and distribution of ZO-1 in intestinal mucosa was observed by indirect immunofluorescence. Data were processed with one-way analysis of variance, least-significant difference test, and Bonferroni correction.@*Results@#(1) At PIH 24, the intestinal permeability of mice in sham scald group, pure scald group, and scald+ sodium butyrate group was 0.88±0.19, 2.62±0.48, 1.23±0.16, respectively. Compared with that in sham scald group, the intestinal permeability of mice in pure scald group was significantly elevated (P<0.01), while the intestinal permeability of mice in scald+ sodium butyrate group showed no obvious change (P>0.05). Compared with that in pure scald group, the intestinal permeability of mice in scald+ sodium butyrate group was significantly decreased (P<0.01). (2) At PIH 24, compared with those in sham scald group, the protein expressions of ZO-1, occludin, and claudin-1 of mice in pure scald group and scald+ sodium butyrate group were significantly decreased (P<0.05), while the protein expression of claudin-2 was significantly increased (P<0.05). At PIH 24, compared with those of pure scald group, the protein expressions of ZO-1 and occludin of mice in scald+ sodium butyrate group were significantly elevated (P<0.05), while the protein expression of claudin-2 was significantly decreased (P<0.05), the protein expression of claudin-1 showed no significant difference (P>0.05). (3) At PIH 24, compared with those in sham scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in pure scald group and scald+ sodium butyrate group were significantly increased (P<0.05). Compared with those of pure scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in scald+ sodium butyrate group were significantly decreased (P<0.05). (4) At PIH 24, ZO-1 in intestinal mucosa of mice in sham scald group was distributed smoothly, continuously and homogeneously along the membrane. ZO-1 in intestinal mucosa of mice in pure scald group was distributed unsmoothly with breaks. The distribution of ZO-1 in intestinal mucosa of mice in scald+ sodium butyrate group was ameliorated compared with that in pure scald group.@*Conclusions@#Sodium butyrate can inhibit the activation of NLRP3 inflammasome and decrease the production of IL-1β and IL-18 in intestinal mucosa of severe scald mice, which protects the intestinal barrier function by alleviating the alteration of tight junction protein expression and localization.

CLAUDINS 1, 3, 4, 7 AND 10-YEAR SURVIVAL IN TRIPLE-NEGATIVE BREAST TUMORS / Claudinas 1, 3, 4, 7 e sobrevida de 10 anos em tumores de mama triplo-negativos

Purpose: Breast cancer is a major cause of morbidity and mortality and is known to be a heterogeneous disease. The clinical and molecular characterization of its subtypes is critical to guide its prognosis and treatment. The study of the expression of Claudins (CLDN) might help in the characterization of these tumors. This study investigated the association of expression of CLDN-1, CLDN-3, CLDN-4 and CLDN-7 with 10-year survival in a series of triple-negative breast cancers. Methods: Eighty triple negative tumors were analyzed by automated immunohistochemistry for CLDN-1, CLDN-3, CLDN-4 and CLDN-7. The immunohistochemical expression was assessed by the H-Score (intensity multiplied by the percentage of staining on membrane). The associations between the expression of CLDN and 10-year survival were evaluated by Kaplan-Meier curves and Cox regressions. Results: Positive expression (H-score ≥50) of CLDN-1, CLDN-3, CLDN-4 and CLDN-7 were observed in 41.3, 77.5, 67.5 and 18.8% of the cohort, respectively. Patients with positive CLDN-1 expression had a significant lower survival than their counterparts [HR=2.37 (95%CI 1.19­4.72)]. Further, CLDN-3 was inversely associated with overall survival. Patients with positive expression of CLDN-1 and negative expression of CLDN-3 had a HR 10.4 (95%CI 3.40­31.8) higher than patients with negative expression of CLDN-1 and positive expression of CLDN-3. Neither CLDN-4 nor CLDN-7 expression was associated with 10-year survival. Conclusions: Differential expression of CLDN can help in clinicopathological characterization of triple-negative tumors. Moreover, CLDN-1 and CLDN-3 appear to be important prognostic factors for these tumors.

Objetivo: O câncer de mama é uma das principais causas de morbidade e mortalidade, conhecido por ser uma doença heterogênea. A caracterização clínica e molecular de seus subtipos é fundamental para orientar seu prognóstico e tratamento. O estudo da expressão de claudinas (CLDN) pode auxiliar na caracterização desses tumores. Este estudo investigou a associação da expressão de CLDN-1, CLDN-3, CLDN-4 e CLDN-7 com 10 anos de sobrevida em uma série de cânceres de mama triplo-negativos. Métodos: Oitenta tumores triplo-negativos foram analisados por imuno-histoquímica automatizada para CLDN-1, CLDN-3, CLDN-4 e CLDN-7. A expressão imuno-histoquímica foi avaliada pelo escore H (intensidade multiplicada pela porcentagem de coloração na membrana). As associações entre a expressão de CLDN e a sobrevida em 10 anos foram avaliadas pelas curvas de Kaplan-Meier e regressões de Cox. Resultados: Foi observada expressão positiva (escore H ≥ 50) de CLDN-1, CLDN-3, CLDN-4 e CLDN-7 em 41,3, 77,5, 67,5 e 18,8% da coorte, respectivamente. Pacientes com expressão positiva de CLDN-1 tiveram uma sobrevida significativamente menor do que suas contrapartes [HR = 2,37 (IC 95% 1,19-4,72)]. Além disso, o CLDN-3 foi inversamente associado à sobrevida global. Pacientes com expressão positiva de CLDN-1 e expressão negativa de CLDN-3 tiveram uma FC 10,4 (IC 95% 3,40­31,8) vezes maior do que pacientes com expressão negativa de CLDN-1 e expressão positiva de CLDN-3. Nem a expressão de CLDN-4 nem de CLDN-7 foi associada a uma sobrevida de 10 anos. Conclusões: A expressão diferencial de CLDN pode ajudar na caracterização clinico-patológica de tumores triplo-negativos. Além disso, CLDN-1 e CLDN-3 parecem ser importantes fatores prognósticos para esses tumores.

Effect of urban atmospheric fine particulate matter PM2.5 on the expression of skin barrier-associated proteins in human keratinocytes / 中华皮肤科杂志

Objective@#To evaluate the effect of atmospheric fine particulate matter (PM2.5) on the expression of skin barrier-associated proteins and proinflammatory cytokines in human keratinocytes.@*Methods@#Atmospheric PM2.5 samples were given by Professor Yufeng Zhou in Children′s Hospital of Fudan University. Human primary keratinocytes were isolated from circumcised foreskins of 5 males, and subjected to culture. These human primary keratinocytes were divided into several groups to be stimulated with PM2.5 at different concentrations of 0 (control group) , 10, 50, 100, 200 mg/L for 24 hours, and cell counting kit (CCK) -8 assay was performed to determine the survival rates of keratinocytes. Fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of filaggrin, keratin-14 and claudin-1 in these keratinocytes respectively, and enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of interleukin (IL) -1α, thymic stromal lymphopoietin (TSLP) and IL-33 in the culture supernatant of these keratinocytes. Statistical analysis was carried out with SPSS13.0 software by using one-way analysis of variance, least significant difference (LSD) -t test and Pearson correlation analysis.@*Results@#After 24-hour treatment with different concentrations of PM2.5, there was no significant difference in the survival rate of keratinocytes between the 10-mg/L PM2.5 group and control group (P > 0.05) , while the survival rates of keratinocytes were significantly lower in the 50-, 100-, 200-mg/L PM2.5 groups than in the control group (all P < 0.05) . After the treatment with 50 mg/L PM2.5, the cell survival rate gradually decreased and then remained stable along with the increase of PM2.5 treatment duration, and the cell survival rate at 24 hours was 72.37% ± 3.12%. After 24-hour treatment with PM2.5, the mRNA expression of filaggrin and keratin-14 was significantly higher in both the 10- and 50-mg/L PM2.5 groups (filaggrin: 1.27 ± 0.15, 1.32 ± 0.09 respectively; keratin-14: 1.15 ± 0.13, 1.08 ± 0.16 respectively) than in the control group (all P < 0.05) , but lower in both the 100- and 200-mg/L PM2.5 groups (filaggrin: 0.84 ± 0.11, 0.42 ± 0.12 respectively; keratin-14: 0.67 ± 0.09, 0.74 ± 0.11 respectively) than in the control group (all P < 0.05) . The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly decreased mRNA expression of claudin-1 compared with the control group (all P < 0.05) . Western blot analysis showed that filaggrin expression was significantly higher in the 50- and 100-mg/L PM2.5 groups than in the control group (both P < 0.05) , while no significant difference was observed between the 10-, 200-mg/L PM2.5 groups and the control group (both P > 0.05) . The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly increased keratin-14 expression compared with the control group (all P < 0.05) . The claudin-1 expression did not differ between the 10-mg/L PM2.5 group and control group (P = 0.87) , but significantly higher in the 50-, 100- and 200-mg/L PM2.5 groups than in the control group (all P < 0.05) . The stimulation with PM2.5 at 10, 50, 100 and 200 mg/L could induce an increase in the supernatant levels of TSLP, IL-1α and IL-33 (all P < 0.01) . Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP, IL-1α and IL-33 were positively correlated with the concentration of PM2.5 (r = 0.57, 0.67, 0.91 respectively, all P < 0.05) .@*Conclusion@#The exposure to PM2.5 can induce decreased survival rate of keratinocytes, aberrant expression of filaggrin, keratin-14 and claudin-1, and elevated secretion of the proinflammatory cytokines TSLP, IL-1α and IL-33, which may lead to impaired skin barrier function.

Effect of urban atmospheric fine particulate matter PM2.5 on the expression of skin barrier-associated proteins in human keratinocytes / 中华皮肤科杂志

Objective To evaluate the effect of atmospheric fine particulate matter(PM2.5)on the expression of skin barrier-associated proteins and proinflammatory cytokines in human keratinocytes. Methods Atmospheric PM2.5 samples were given by Professor Yufeng Zhou in Children' s Hospital of Fudan University. Human primary keratinocytes were isolated from circumcised foreskins of 5 males, and subjected to culture. These human primary keratinocytes were divided into several groups to be stimulated with PM2.5 at different concentrations of 0(control group), 10, 50, 100, 200 mg/L for 24 hours, and cell counting kit(CCK)-8 assay was performed to determine the survival rates of keratinocytes. Fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of filaggrin, keratin-14 and claudin-1 in these keratinocytes respectively, and enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of interleukin (IL)-1α, thymic stromal lymphopoietin(TSLP)and IL-33 in the culture supernatant of these keratinocytes. Statistical analysis was carried out with SPSS13.0 software by using one-way analysis of variance, least significant difference (LSD)- t test and Pearson correlation analysis. Results After 24 - hour treatment with different concentrations of PM2.5, there was no significant difference in the survival rate of keratinocytes between the 10-mg/L PM2.5 group and control group (P > 0.05), while the survival rates of keratinocytes were significantly lower in the 50-, 100-, 200-mg/L PM2.5 groups than in the control group(all P<0.05). After the treatment with 50 mg/L PM2.5, the cell survival rate gradually decreased and then remained stable along with the increase of PM2.5 treatment duration, and the cell survival rate at 24 hours was 72.37％ ± 3.12％. After 24-hour treatment with PM2.5, the mRNA expression of filaggrin and keratin-14 was significantly higher in both the 10- and 50-mg/L PM2.5 groups (filaggrin: 1.27 ± 0.15, 1.32 ± 0.09 respectively;keratin-14:1.15 ± 0.13, 1.08 ± 0.16 respectively)than in the control group(all P<0.05), but lower in both the 100- and 200-mg/L PM2.5 groups (filaggrin: 0.84 ± 0.11, 0.42 ± 0.12 respectively;keratin-14:0.67 ± 0.09, 0.74 ± 0.11 respectively)than in the control group(all P<0.05). The 10-, 50-, 100-and 200-mg/L PM2.5 groups showed significantly decreased mRNA expression of claudin-1 compared with the control group(all P < 0.05). Western blot analysis showed that filaggrin expression was significantly higher in the 50- and 100-mg/L PM2.5 groups than in the control group (both P < 0.05), while no significant difference was observed between the 10-, 200-mg/L PM2.5 groups and the control group(both P > 0.05). The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly increased keratin-14 expression compared with the control group(all P<0.05). The claudin-1 expression did not differ between the 10-mg/L PM2.5 group and control group (P = 0.87), but significantly higher in the 50-, 100- and 200-mg/L PM2.5 groups than in the control group(all P<0.05). The stimulation with PM2.5 at 10, 50, 100 and 200 mg/L could induce an increase in the supernatant levels of TSLP, IL-1αand IL-33(all P<0.01). Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP, IL-1αand IL-33 were positively correlated with the concentration of PM2.5(r=0.57, 0.67, 0.91 respectively, all P < 0.05). Conclusion The exposure to PM2.5 can induce decreased survival rate of keratinocytes, aberrant expression of filaggrin, keratin-14 and claudin-1, and elevated secretion of the proinflammatory cytokines TSLP, IL-1αand IL-33, which may lead to impaired skin barrier function.

Effects of short chain fatty acid on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide and the related mechanism / 中华烧伤杂志

Objective@#To investigate the effects of short chain fatty acid (SCFA) on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide (LPS) and the related mechanism.@*Methods@#The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. Cells were divided into control group, LPS group, and SCFA+ LPS group according to the random number table. Cells in control group were only routinely cultured with DMEM medium. Cells in LPS group were cultured with DMEM medium and LPS with final mass concentration of 10 μg/mL. Cells in SCFA+ LPS group were cultured with DMEM medium, LPS and SCFA (consisting of 0.5 mmol/L acetate, 0.01 mmol/L propionate, and 0.01 mmol/L butyrate) with final mass concentration of 10 μg/mL. At post culture hour (PCH) 0, 1, 2, 6, 12, and 24, transepithelial electrical resistance (TER) of cells was determined with an ohmmeter, with sample number of 72. Another portion of cells were divided and treated as above, and then Western blotting was employed to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, and claudin-1 at PCH 24, with sample number of 6. Another portion of cells were divided and treated as above and then immunofluorescence was used to observe cellular morphology and distribution of ZO-1. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction.@*Results@#(1) Compared with that in control group, TER of cells in LPS group was significantly reduced from PCH 1 to 24 (P<0.01), while TER of cells in SCFA+ LPS group showed no obvious change (P>0.05). TER of cells in SCFA+ LPS group was significantly higher than that in LPS group from PCH 1 to 24 (P<0.01). (2) Compared with the protein expressions of ZO-1, occludin, and claudin-1 of cells in control group (1.25±0.10, 1.17±0.04, and 1.24±0.20), those of cells in LPS group (0.74±0.23, 0.76±0.11, and 0.77±0.11) at PCH 24 were significantly decreased (P<0.05), while those of cells in SCFA+ LPS group (1.23±0.46, 1.05±0.09, and 1.01±0.13) showed no significant differences (P>0.05). Protein expressions of occludin and claudin-1 of cells in SCFA+ LPS group were significantly higher than those in LPS group at PCH 24 (P<0.05). Protein expression of ZO-1 of cells in SCFA+ LPS group was higher than that in LPS group at PCH 24 with no significant difference (P>0.05). (3) At PCH 24, cells in control group were compact in arrangement with pebble-like appearance, and ZO-1 was distributed smoothly and continuously along the cell membrane. In LPS group, cells were sparse in arrangement with change in appearance, and ZO-1 was distributed uncontinuously along the cell membrane with curls and breaks. In SCFA+ LPS group, the appearance of cells and distribution of ZO-1 were remarkably ameliorated compared with those in LPS group.@*Conclusions@#SCFA can alleviate the barrier disruption of human intestinal epithelial cell induced by LPS through interdicting the abnormal distribution of ZO-1 and decrease of TER and tight junction proteins′ expressions.

Intestinal mucosal barrier damage and the development of cachexia in colon cancer patients / 中华普通外科杂志

Objective To investigate the association of bacterial translocation (BT) with cachexia in colonic cancer patients.Methods From September 2015 to May 2017 the clinical data of 292 colon cancer patients were studied at Qingdao Municipal Hospital.The bacteria in peripheral blood and mesenteric lymph nodes were detected by bacterial culture,and the bacterial DNA in peripheral blood was detected by PCR technique to determine the occurrence of bacterial translocation.Intestinal epithelial T-cell subsets and NK cells were evaluated using flow cytometry.Western blot and immunofluorescence were used to check tight junction proteins Occludin,Claudins-2,Zonula occluden-2 in intestinal epithelium.Fluorescence in situ hybridization and immunohistochemistry were used to detect the translocated bacteria and endotoxin.Results Compared with noncachectic patients,cachectic patients had a significandy higher BT ratio (27.8％ vs.7.2％,x2 =20.871,P ＜ 0.001).BT in the intestinal mucus layer was associated with lower levels of T-cell subsets and NK cells in the intestinal epithelium in BT(+) patients (CD3 + T:36.69％ ±5.87％ vs.41.63％ ±5.03％,CD4+T:44.08％ ±5.12％ vs.49.58％ ±7.01％,CD8+T:65.68％ ±5.51％ vs.61.43％ ± 5.58％,CD4+ T/CD8+ T:0.71％ ± 0.21％ vs.0.91％ ±0.23％,NK:27.86％ ± 3.93％ vs.34.69％ ± 4.52％,all P ＜ 0.01).Endotoxin was detected within the small intestinal wall in BT(+) patients and claudin-2 expression increased (0.63 ± 0.13 vs.0.21-± 0.06,t =-2.936,P ＜ 0.01),whereas Occludin and Zonula occluden-2 expressions decreased (0.37 ± 0.13 vs.0.84±0.17,0.16±0.07 vs.0.58±0.19,t=2.151,2.111,bothP＜0.05).Conclusions BTmay contribute to the development of colon cancer cacheria,and tight junction could be the gateway of BT.

Changes of Expressions of Claudin-1,-2,-4 in Experimental Colitis Rats / 胃肠病学

Background:Ulcerative colitis (UC)is a chronic inflammatory disorder. Studies have shown that intestinal injury of UC is related to changes of tight junction proteins. Aims:To investigate the expressions and localizations of tight junction protein claudin-1,-2,-4. Methods:Forty female Wistar rats were randomly divided into normal control group and model group. Rats in model group received 7. 5 mg/ mL oxazolone enema to induce experimental colitis. Rats received 0. 9%NaCl solution enema were served as normal controls. Macroscopic score and histological score were assessed. ELISA was used to determine serum and colon tissue inflammatory factor TNF-α,IL-4,IL-5,IL-10 levels. Protein expressions of tight junction protein claudin-1,-2,-4 were measured by immunohistochemistry and Western blotting. mRNA expressions of claudin-1,-2,-4 were determined by real-time PCR. Results:Compared with normal control group,macroscopic score and histological score were significantly increased (P < 0. 05),serum and colon tissue IL-4 and IL-5 levels were significantly increased (P < 0. 05)in model group,however,no significant differences in TNF-α and IL-10 levels were found between the two groups (P > 0. 05). mRNA and protein expressions of claudin-1,-4 were significantly decreased in model group than in normal control group (P < 0. 05),while mRNA and protein expressions of claudin-2 were significantly increased (P < 0. 05). Conclusions:Changes of distributions and expressions of tight junction protein claudin-1,-2,-4 are found in experimental colitis rats,which may lead to impaired epithelial barrier,and might be served as potential target for treatment of UC.

Changes of Expressions of Claudin-1,-2,-4 in Experimental Colitis Rats / 胃肠病学

Background:Ulcerative colitis (UC)is a chronic inflammatory disorder. Studies have shown that intestinal injury of UC is related to changes of tight junction proteins. Aims:To investigate the expressions and localizations of tight junction protein claudin-1,-2,-4. Methods:Forty female Wistar rats were randomly divided into normal control group and model group. Rats in model group received 7. 5 mg/ mL oxazolone enema to induce experimental colitis. Rats received 0. 9%NaCl solution enema were served as normal controls. Macroscopic score and histological score were assessed. ELISA was used to determine serum and colon tissue inflammatory factor TNF-α,IL-4,IL-5,IL-10 levels. Protein expressions of tight junction protein claudin-1,-2,-4 were measured by immunohistochemistry and Western blotting. mRNA expressions of claudin-1,-2,-4 were determined by real-time PCR. Results:Compared with normal control group,macroscopic score and histological score were significantly increased (P < 0. 05),serum and colon tissue IL-4 and IL-5 levels were significantly increased (P < 0. 05)in model group,however,no significant differences in TNF-α and IL-10 levels were found between the two groups (P > 0. 05). mRNA and protein expressions of claudin-1,-4 were significantly decreased in model group than in normal control group (P < 0. 05),while mRNA and protein expressions of claudin-2 were significantly increased (P < 0. 05). Conclusions:Changes of distributions and expressions of tight junction protein claudin-1,-2,-4 are found in experimental colitis rats,which may lead to impaired epithelial barrier,and might be served as potential target for treatment of UC.

Clinicopathologic and prognosis features of Claudin-low breast cancers / 中华病理学杂志

Objective@#To investigate the clinicopathologic and prognostic features of Claudin-low breast cancers (CLBC).@*Methods@#Tissue microarray sections were scored semiquantitatively for the immunohistochemical expression of claudin-1, -3, -4, -7 and -8 in 233 cases of invasive breast cancers collected from Qingdao Central Hospital from January 2010 to December 2011.@*Results@#The expression rate of Claudin-3 (72/212, 33.9%) and -4 (56/212, 45.2%) was most similar, and Claudin-4 showed the highest expression. Twenty one cases (21/212, 9.0%) were diagnosed as CLBC, with triple-negative breast cancer (TNBC) accounted for the highest proportion (11/21, 52.4%). Among the CLBC cases, the invasive carcinoma no special type (66.7%, 14/21) and metaplastic carcinoma (14.3%, 3/21) were mostly seen, while metaplastic squamous carcinoma did not show Claudin-low pattern. Compared to the non CLBC in this cohort, CLBC had higher proportion of histologic grade 3 and tumors larger than 2 cm, and the proportions were slightly lower than TNBC. Patients with CLBC had lower 5 year disease-free(P>0.05) and overall survival rates(P=0.018).@*Conclusion@#CLBC shows distinct clinicopathologic and prognostic features comparing to other subtypes, and is associated with poor prognosis.

Skin barrier in atopic dermatitis: beyond filaggrin

Abstract: Atopic dermatitis is a chronic inflammatory skin disease with a complex pathogenesis, where changes in skin barrier and imbalance of the immune system are relevant factors. The skin forms a mechanic and immune barrier, regulating water loss from the internal to the external environment, and protecting the individual from external aggressions, such as microorganisms, ultraviolet radiation and physical trauma. Main components of the skin barrier are located in the outer layers of the epidermis (such as filaggrin), the proteins that form the tight junction (TJ) and components of the innate immune system. Recent data involving skin barrier reveal new information regarding its structure and its role in the mechanic-immunological defense; atopic dermatitis (AD) is an example of a disease related to dysfunctions associated with this complex.

Identificação de subtipos moleculares baseada no perfil imunoistoquímico de carcinomas mamários triplo-negativos em mulheres com idade até 45 anos e sua distribuição nas diferentes regiões geográficas do Brasil / Identification of molecular subtypes based on immunohistochemical profile of triple-negative breast cancer (TNBCs) in women aged up to 45 years and its distribution in different geographical regions of Brazil

Carcinomas mamários triplo-negativos correspondem ao grupo heterogêneo de neoplasias mamárias caracterizadas pela ausência de expressão dos receptores de estrogênio e progesterona e sem amplificação ou superexpressão do HER2. São mais prevalentes em mulheres jovens e em afrodescendentes. Eles se associam frequentemente ao fenótipo basal-símile determinado geneticamente, entretanto, incluem também outros tipos moleculares intrínsecos. Metodologias de análise genética de novas gerações têm permitido sua estratificação em subgrupos distintos, o que justifica a heterogeneidade clínica deste grupo de neoplasias. A identificação desses subgrupos através de marcadores imunoistoquímicos de aplicação prática ainda é pouco explorada, embora seja uma ferramenta promissora na sua estratificação e determinação de alvos terapêuticos. OBJETIVOS: Nosso objetivo é explorar os perfis imunoistoquímicos dos carcinomas mamários triplo-negativos em mulheres com idade até 45 anos e investigar possíveis diferenças entre as cinco regiões geográficas brasileiras. MÉTODOS: Selecionamos 118 amostras de tumores de pacientes com idade até 45 anos, com carcinoma invasivo, blocos de parafina disponíveis e perfil imunoistoquímico triplo-negativo, procedentes das cinco regiões geográficas. Estes casos foram revisados quanto à determinação de tipo e grau histológico e as seguintes características anatomopatológicas: contorno do tumor, presença e fração do componente "in situ", embolização vascular peritumoral, tipo e grau da reação estromal, presença de necrose tumoral e formação de túbulos pela neoplasia. Foram selecionadas áreas representativas do tumor para construção de blocos de microarranjos de tecido para estudo imunoistoquímico. Foram pesquisados os seguintes marcadores: citoqueratinas basais 5/6 e 14, citoqueratinas luminais 8 e 18, receptor do fator de crescimento epidérmico (EGFR ou HER1), receptor de androgênio, e-caderina, catenina-beta,...

Triple-negative breast carcinomas (TNBC) correspond to a heterogeneous group of neoplasia characterized by the lack of expression of estrogen and progesterone receptors, and by the absence of amplification or overexpression of HER2. They are more prevalent among African descendants and younger women. They are often associated with the basal-like genetic phenotype; however, other intrinsic molecular types are included. Genomic analyses of next-generation methods have allowed stratification of TN breast carcinomas into distinct subgroups, explaining the clinical heterogeneity of this group of neoplasias. The identification of these subgroups by immunohistochemical markers is not well explored, although it represents a potential useful tool for the stratification and determination of therapeutic targets. OBJECTIVES: Our aim is to explore the TNBC immunohistochemical profile in patients of 45 year-old or younger, and to investigate possible differences among the five Brazilian geographic regions. METHODS: We've selected 118 samples of tumors from patients up to 45 years-old from five Brazilian geographic regions, with invasive carcinoma, available paraffin blocks, and triple-negative immunohistochemical profile. All the cases were reviewed as for determination of histologic type and grading, and the following pathological features: tumor contour, presence and percentage of in situ component, peritumoral vascular embolization, type and grade of stromal reaction, presence of tumoral necrosis, and tubule formation by neoplasia. Representative tumor areas were selected for tissue microarray construction for immunohistochemical study. The following markers were studied: Basal cytokeratins 5/6 and 14; luminal cytokeratins 8 and 18; Epidermal growth factor receptor (EGFR or HER1); androgen receptor; e-cadherin; catenin-beta; claudins (3,4 and 7); vimentin; smooth-muscle actin; p63, ALDH1, and Ki-67. According to the expression of the markers, the tumors...

Effect of caveolin-1 on renal injury and the expression of tight junction protein in MRL/lpr mice kidney / 中华风湿病学杂志

Objective To study the effect of caveolin-1 on renal injury and the expression of tight junction proteins in MRL/lpr mice kidney.Methods The mice were divided into 4 groups:5 mice in the normal control group (BALB/c mice);the MRL/lpr lupus mice (n=18) were randomly divided into the MRL/lpr group in which 6 mice were included;the negative control group in which 6 mice were included;the caveolin1 transfection group in which 6 mice were included.The changes of urine protein,the levels of urea (BUN) and creatinine (Cr) were detected.The expressions of claudin-5,occludin,ZO-1 and caveolin-1 protein were determined by western bloting.Analysis of variance was used to determine statistical significant differences between the two groups.A significance level of 0.05 was considered as signigicant.Results Compared with the control group,24 h urine protein [(2 894±437) mg,(412±72) mg],BUN [(8.7±1.5) mmol/L,(6.9±0.4) mmol/L],Cr [(106±22) μmol/L,(85±4) μmol/L] were significantly increased,level of caveolin-1 protein increased (265±17,61±6),the level of occludin (114±12,190±12),claudin-5 (60±5,80±6) and ZO-1 (98±11,206±15) protein decreased in the MRL/lpr group (P＜0.05).After caveolin-1 transfection,the levels of urinary protein [(1 253±249) mg,(2 894±437) mg],BUN [(6.5±1.3) mmol/L,(8.7±1.5) mmol/L],Cr [(78±17) μmol/L,(106±22)μmol/L] were significantly decreased,and the levels of occludin (218±16,114±12),claudin-5 (87±6,60±5)ZO-1 (313±17,98±11) were increased (P＜0.05).Conclusion The expression of caveolin-1 protein in the renal tissues of lupus nephritis increases.Caveolin-1 can reduce the expression of tight junction proteins and contribute to progres-sion of lupus nephritis.

Claudin-1, -2, -4, and -5: comparison of expression levels and distribution in equine tissues

Claudins, which are known as transmembrane proteins play an essential role in tight junctions (TJs) to form physical barriers and regulate paracellular transportation. To understand equine diseases, it is helpful to measure the tissue-specific expression of TJs in horses. Major equine diseases such as colic and West Nile cause damage to TJs. In this study, the expression level and distribution of claudin-1, -2, -4, and -5 in eight tissues were assessed by Western blotting and immunohistochemistry methods. Claudin-1 was primarily identified in the lung, duodenum, and uterus, claudin-2 was evenly observed in equine tissues, claudin-4 was abundantly detected in the liver, kidney and uterus, and claudin-5 was strongly expressed in the lung, duodenum, ovary, and uterus, as determined by Western blotting method. The localization of equine claudins was observed by immunohistochemistry methods. These findings provide knowledge regarding the expression patterns and localization of equine claudins, as well as valuable information to understand tight junction-related diseases according to tissue specificity and function of claudins in horses.

Protective effect of ulinastatin on intestinal barrier function of septic rats / 中华临床感染病杂志

Objective To observe the protective effect of ulinastatin (UTI) on the intestinal barrier function of septic rats.Methods Septic rat model was established using Sprague-Dawley rats by cecal ligation and puncture (CLP) method.Thirty Sprague-Dawley rats were randomly divided into 3 groups (n =10 for each grop) : sham group, septic group and UTI group.All rats received intraperitoneal injections of 0.9％ saline (10 mL/kg) after and 8 h after surgery.In UTI group, UTI (10 × 104 U/kg in 10 mL/kg saline) was injected after and 8 h after surgery.Collect blood samples after 0, 8, 12 h after surgery to examine levels of procalcitonin (PCT), intestinal fatty acid binding protein (iFABP) and diamine oxidase (DAO) by enzyme-linked immunosorbent assay (ELISA) method.Rats were killed 12 h after surgery to collect intestine tissue samples.Pathological changes of intestine were observed under microscopy, and the expression of tight junction protein-1 (ZO-1) and occludin were analyzed by Western blot.Results In sham group, the mucosa structure was complete and the shape was normal, and villi stood neatly.In septic group, intestinal was expanded, intertinal mucosal was atrophic, villi were scanty.An inflammatory infiltrate with numerous nuetrophils was found in the mucosal.In UTI group, the level of severity was relatively slight.The relative optical density of Western blot images were decreased on ZO-1 and occludin in CLP and UTI groups, and decreased more in CLP group (F =43.15 and 52.23, P ＜ 0.05).At 0h after surgery, the plasma values of PCT, iFABP and DAO were similar in three groups (F =11.17, 22.45 and 13.58, P ＞ 0.05).At 8h and 12h after surgery, values of PCT, iFABP and DAO in septic and UTI groups were much higher than those in the sham group, and those in UTI group were also significantly higher than those in septic group (F8h=85.26, 44.59 and 101.47, F12h =59.44, 49.26 and 69.57, all P＜0.05).PCT, iFABP and DAO levels were first increased and then fell down in sham group, those in septic group were keeping increasing, and those in UTI group were first increased and then kept stable.Conclusion UTI shows protective effect to intestinal barrier function in rats with sepsis.

Expression of claudin, paxillin and FRA-1 in non-nodular breast lesions in association with microcalcifications / Expressão de claudina, paxilina e FRA-1 em lesões mamárias não nodulares associadas a microcalcificações

CONTEXT AND OBJECTIVE The possible role of adhesion molecules in early breast carcinogenesis has been shown in the literature. We aimed to analyze early adhesion imbalances in non-nodular breast lesions and their association with precursor lesions, in order to ascertain whether these alterations exist and contribute towards early carcinogenesis. DESIGN AND SETTING Retrospective cross-sectional study based on medical records at a private radiological clinic in São Paulo, Brazil. METHODS We retrospectively reviewed the medical records of all consecutive women attended between August 2006 and July 2007 who presented mammographic evidence of breast microcalcifications classified as Breast Imaging Reporting and Data System Atlas (BI-RADS) type 4. These women underwent stereotaxic biopsy. Clinical, radiological and pathological data were collected, and immunohistochemical assays searched for claudin, paxillin, FRA-1 and HER-2. RESULTS Over this period, 127 patients were evaluated. Previous BI-RADS diagnoses showed that 69 cases were in category 4A, 47 in 4B and 11 in 4C. Morphological assessment showed benign entities in 86.5%. Most of the benign lesions showed preserved claudin expression, associated with paxillin (P < 0.001). Paxillin and HER-2 expressions were correlated. FRA-1 expression was also strongly associated with HER-2 expression (P < 0.001). CONCLUSIONS Although already present in smaller amounts, imbalance of adhesion molecules is not necessarily prevalent in non-nodular breast lesions. Since FRA-1 expression reached statistically significant correlations with radiological and morphological diagnoses and HER-2 status, it may have a predictive role in this setting. .

CONTEXTO E OBJETIVO A literatura tem mostrado a importância de moléculas de adesão na carcinogênese precoce de mama. Objetivamos analisar desequilíbrios precoces de adesão em lesões não nodulares da mama e associação com lesões precursoras, a fim de verificar se essas alterações existem e contribuem com a carcinogênese. TIPO DE ESTUDO E LOCAL Estudo retrospectivo baseado em prontuários médicos, numa clínica radiológica privada em São Paulo, Brasil. MÉTODOS Revisamos retrospectivamente prontuários de todas as mulheres consecutivamente atendidas com evidência mamográfica de microcalcificações mamárias, classificadas como tipo 4 do Breast Imaging Reporting and Data System Atlas (BI-RADS) entre agosto de 2006 e julho de 2007. Elas foram submetidas a biópsia estereotáxica. Dados clínicos, radiológicos e histopatológicos foram coletados e ensaios de imunoistoquímica procuraram por claudina, paxilina, HER-2 e FRA-1. RESULTADOS No período, 127 pacientes foram avaliadas. Diagnósticos de BI-RADS anteriores tinham 69 casos na categoria 4A, 47 em 4B, e 11 em 4C. A avaliação morfológica mostrou entidades benignas em 86,5%. A maioria das lesões benignas mostrou expressão preservada de claudina, associada a paxilina (P < 0,001). Expressões de paxilina e HER-2 foram correlacionadas. Expressão de FRA-1 associou-se à de HER-2 (P < 0,001). CONCLUSÕES Embora já presente em menor quantidade, o desequilíbrio de moléculas de adesão não é necessariamente prevalente em lesões mamárias nodulares e talvez a expressão de FRA-1 possa ter um papel preditivo neste cenário, uma vez que atingiu correlações ...

Regulation of claudin-4 gene expression in endometrial adenocarcinoma Ishikawa cell line by progesterone / 中华妇产科杂志

ObjectiveTo explore the regulation of claudin-4 expression in endometrial adenocarcinoma cell lines by progesterone.Methods Ishikawa cells were treated with various concentrations of megestrol acetate (MA:2,5,10,15,20 mg/L).After cultured for 24,48 and 72 hours,cells growth were measured by methyl thiazolyl tetrazolium (MTT).The group of Ishikawa cells incubated with MA at the 50％ inhibitory concentration ( IC50 ) was selected for cell apoptosis assay by using transmission electron microscopy and flow cytometry method.Real-time PCR and western blot were used for detecting the mRNA and protein expression levels of claudin-4.The localization of claudin-4 was examined by immunofluorescent staining.Results The inhibitory effects of megestrol acetate on the growth of Ishikawa cells were dosedependent and time-dependent.IC50 of MA on Ishikawa cells was 15 mg/L after incubated for 72 hours.After MA treatment,Ishikawa cells showed shrinkage,nuclear chromatin condensation,fractures of nuclear membrane and endoplasmic reticulum expansion,even round apoptotic bodies were found.The apoptosis rate of cells before MA treatment was (0.076 ±0.024)％,and the rate was (3.934 ±0.816)％ by MA treated for 72 hours,in which there were signicant difference( P ＜ 0.05 ).The relative quantification of claudin-4 mRNA and protein of the cells before MA treatment were 0.64 ± 0.20 and 0.94 ± 0.18,while they were 0.47 -0.15 and 0.62 ±0.15 after MA treated.The expression of claudin-4 was significantly decreased after MA treatment ( P ＜ 0.05 ).The localization of claudin-4 transferred from cytomembrane to cytoplasm and nucleus after MA treatment.Conclusions MA could inhibite the growth of Ishikawa cells,in which the mechanism may be decrease the expression of claudin-4 and the apoptosis of cells.The distribution change of claudin-4 may be related to the anti-cancer effect of progesterone.

Apical acidity decreases inhibitory effect of omeprazole on Mg2+ absorption and claudin-7 and -12 expression in Caco-2 monolayers

Clinical studies reported hypomagnesaemia in long-term omeprazole usage that was probably due to intestinal Mg2+ wasting. Our previous report demonstrated the inhibitory effect of omeprazole on passive Mg2+ transport across Caco-2 monolayers. The present study aimed to identify the underlying mechanism of omeprazole suppression of passive Mg2+ absorption. By using Caco-2 monolayers, we demonstrated a potent inhibitory effect of omeprazole on passive Mg2+, but not Ca2+, transport across Caco-2 monolayers. Omeprazole shifted the %maximum passive Mg2+ transport-Mg2+ concentration curves to the right, and increased the half maximal effective concentration of those dose-response curves, indicating a lower Mg2+ affinity of the paracellular channel. By continually monitoring the apical pH, we showed that omeprazole suppressed apical acid accumulation. Neomycin and spermine had no effect on passive Mg2+ transport of either control or omeprazole treated monolayers, indicating that omeprazole suppressed passive Mg2+ transport in a calcium sensing receptor (CaSR)-independent manner. The results of western blot analysis showed that omeprazole significantly suppressed claudin (Cldn)-7 and -12, but not Cldn-2, expression in Caco-2 cells. By using apical solution of pH 5.5, 6.0, 6.5, and 7.0, we found that apical acidity markedly increased passive Mg2+ transport, Mg2+ affinity of the paracellular channel, and Cldn-7 and -12 expression in Caco-2 monolayers. Apical acidity abolished the inhibitory effect of omeprazole on passive Mg2+ transport and Cldn-7 and -12 expression. Our results provided the evidence for the regulation of intestinal passive Mg2+ absorption by luminal acidity-induced increase in Cldn-7 and -12 expression.

Role of Estrogen Receptor-alpha in the Regulation of Claudin-6 Expression in Breast Cancer Cells / 한국유방암학회지

PURPOSE: In our previous studies we showed that upregulating claudin-6 (CLDN6) expression may contribute to preventing breast cancer, and that 17beta-estradiol induces a concentration- and time-related effect on CLDN6 mRNA and protein expression in MCF-7 cells. However, the mechanisms of 17beta-estradiol regulation of CLDN6 are still unclear. We determined the role of estrogen receptors in the regulation of CLDN6 expression in human breast cancer tissues and a cell line. METHODS: CLDN6, estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) expression in breast cancer tissues were examined using immunohistochemistry. The human breast cancer cell line, MCF-7, which expresses ERalpha but not ERbeta was used. CLDN6 and ERalpha expression were measured by reverse transcriptase-PCR, Western blotting and immunofluorescent staining. Treatments with propyl pyrazole triol (PPT) and ICI 182, 780 (ICI) were performed. RESULTS: The results revealed that CLDN6 expression was related to ERalpha in breast cancer tissues (p=0.033). PPT, an ERalpha-selective ligand, upregulated CLDN6 expression at 10-5 mol/L after 24 hours. The effect of PPT on regulating CLDN6 expression in MCF-7 cells was blocked by ICI. CONCLUSION: These findings suggest that Eralpha reulates CLDN6 expression in breast cancer tissues and that 17beta-estradiol induces CLDN6 expression through an ERalpha pathway in MCF-7 cells.