Assessing Severity of Low Anterior Resection Syndrome After Intersphincteric Resection for Ultralow Rectal Cancer: A Pilot Study Using an Exploratory Instrument.

BACKGROUND: The Delphi consensus identified 8 symptoms and 8 consequences as the highest priorities for defining low anterior resection syndrome. OBJECTIVE: To describe an exploratory scoring instrument correlating the Delphi consensus on low anterior resection syndrome with functional and quality-of-life scores following intersphincteric resection for ultralow rectal cancer. DESIGN: This was a prospective pilot study. In accordance with the Wexner incontinence score, 5 frequency responses ranging from never (score 0) to always (score 4) were used to measure the severity of symptom- and consequence-specific variables. SETTINGS: Colorectal surgery referral center. PATIENTS: Among 161 eligible patients, 137 participants (85%) completed an electronic self-assessment survey regarding function and quality of life at scheduled follow-up, including 3 to 6, 12, and ≥24 months after ileostomy reversal. MAIN OUTCOME MEASURES: Outcome measures included patient-reported severity of the identified priorities, and their correlation with condition-specific quality of life. RESULTS: The most frequent symptom and consequence were "emptying difficulties" and "dissatisfaction with the bowels," respectively. Aside from "emptying difficulties," the proportions of negative symptom domains increased after reversal. In particular, neither the frequency responses nor the severity scores of "emptying difficulties" differed between groups. The percentages of "always" selection for consequence domains improved at 12-month follow-up, whereas a higher rate was observed at 24 months, except for "toilet dependence" and "dissatisfaction with the bowels." We found significant improvements in the summary score of the Fecal Incontinence Quality-of-Life Scale ( p = 0.04) and our exploratory instrument ( p = 0.009) but not in functional scores measured by traditional questionnaires. Furthermore, the condition-specific quality of life strongly correlated with the Delphi consensus severity score ( rs = -0.73). LIMITATIONS: Single-institution data and limited sample size. CONCLUSIONS: The important priorities identified by the Delphi consensus might enable a comprehensive overview and a better assessment of low anterior resection syndrome after intersphincteric resection. See Video Abstract . EVALE LA GRAVEDAD DEL SNDROME DE RESECCIN ANTERIOR BAJA DESPUS DE LA RESECCIN INTERESFINTRICA PARA EL CNCER DE RECTO ULTRABAJO UN ESTUDIO PILOTO QUE UTILIZA UN INSTRUMENTO EXPLORATORIO: ANTECEDENTES:El consenso Delphi identificó ocho síntomas y ocho consecuencias como las máximas prioridades para definir el síndrome de resección anterior baja.OBJETIVO:Describir un instrumento de puntuación exploratorio que correlaciona el consenso Delphi sobre el síndrome de resección anterior baja con puntuaciones funcionales y de calidad de vida después de la resección interesfinteriana para el cáncer de recto ultrabajo.DISEÑO:Este fue un estudio piloto prospectivo. De acuerdo con la puntuación de incontinencia de Wexner, se utilizaron cinco respuestas de frecuencia que van desde nunca (puntuación 0) hasta siempre (puntuación 4) para medir la gravedad de las variables específicas de los síntomas y las consecuencias.AJUSTES:Centro de referencia de cirugía colorrectal.PACIENTES:Entre 161 pacientes elegibles, 137 (85%) participantes completaron una encuesta electrónica de autoevaluación sobre la función y la calidad de vida en el seguimiento programado, incluidos 3 a 6, 12 y ≥ 24 meses después de la reversión de la ileostomía.MEDIDAS PRINCIPALES DE RESULTADO:Las medidas de resultado incluyeron la gravedad de estas prioridades informada por los pacientes, así como su correlación con la calidad de vida específica de la afección.RESULTADOS:El síntoma y la consecuencia más frecuentes fueron "dificultades para vaciar" e "insatisfacción con las deposiciones", respectivamente. Aparte de las "dificultades de vaciado", las proporciones de dominios de síntomas negativos aumentaron después de la reversión. En particular, tanto las respuestas de frecuencia como las puntuaciones de gravedad de las "dificultades para vaciar" no difirieron entre los grupos. Los porcentajes de "opción siempre" para los dominios de consecuencias mejoraron a los 12 meses de seguimiento, mientras que se observó una tasa más alta a los 24 meses después, excepto para "dependencia del baño" e "insatisfacción con los intestinos". Encontramos mejoras significativas en la puntuación resumida de la Escala de calidad de vida de incontinencia fecal ( p = 0,04) y nuestro instrumento exploratorio ( p = 0,009), pero no en las puntuaciones funcionales medidas con los cuestionarios tradicionales. Además, la calidad de vida específica de la condición se correlacionó fuertemente con la puntuación de gravedad del consenso Delphi (rs = -0,73).LIMITACIONES:Datos de una sola institución y tamaño de muestra limitado.CONCLUSIONES:Las importantes prioridades identificadas por el consenso Delphi podrían permitir una visión global y una mejor evaluación del síndrome de resección anterior baja después de la resección interesfintérica. (Traducción-Dr. Yesenia Rojas-Khalil ).

Conformal sphincteric resection for ultra-low rectal cancer located below the dentate line: A pilot report.

AIM: Sphincter-sparing surgery can be achieved in most cases of low rectal cancer with the development of intersphincteric resection. However, abdominoperineal resection is still inevitable for patients with tumours located below the dentate line. To address this, we have developed a procedure called conformal sphincteric resection (CSR) in which the corresponding part of the subcutaneous portion of the external anal sphincter and the perianal skin on the tumour side is removed to achieve a safe distal resection margin and lateral resection margin while the dentate line and the internal anal sphincter on the tumour-free side are preserved as much as possible, to achieve sphincter preservation without compromising oncological safety and functional acceptability, and to render tumour location no longer a contraindication for sphincter-sparing surgery. This is the first study to describe the concept, indication and surgical procedure of CSR and to report its preliminary surgical, oncological and functional results. METHODS: This is a retrospective, single-centre, single-arm pilot study conducted at Huashan Hospital, Fudan University. Demographic, clinicopathological, oncological and functional follow-up data were collected from 20 consecutive patients with rectal tumours located below the dentate line who underwent laparoscopic CSR by the same surgical team from June 2018 to March 2022. RESULTS: The mean distance of the tumour's lower edge from the anal verge was 13.1 ± 6.0 mm. The mean distal resection margin was 10.6 ± 4.3 mm. All circumferential resection margins were negative. There were no instances of perioperative mortality. The complication rate was 25% but all were Clavien-Dindo Grade I. Among the 20 cases, 17 were diagnosed with adenocarcinoma, one with squamous cell carcinoma and two with adenoma featuring high-grade intraepithelial neoplasia. Pathological TNM staging revealed two, seven, five, five and one case(s) in Stages 0, I, II, III and IV, respectively. The median follow-up period was 20 months (interquartile range 22 months), with no withdrawals. The overall and disease-free survival rates were both 95%. The mean Wexner incontinence score and low anterior resection syndrome score recorded 18 months following diverting ileostomy closure were 6.3 ± 3.8 and 27.3 ± 3.6, respectively. CONCLUSIONS: This study has proposed the CSR procedure for the first time, which is a technically feasible, oncologically safe and functionally acceptable procedure for carefully selected patients with rectal tumours located below the dentate line.

Prognostic value of TIMM50 expression in colorectal cancer.

Introduction: Translocase of the inner mitochondrial membrane 50 (TIMM50) is universally considered to play a key role in several malignancies. However, its role in predicting colorectal cancer (CRC) patient prognosis remains unclear. Material and methods: A total of 192 CRC patients (123 men and 69 women) who underwent radical resection participated in this study. The patients were followed up every 3 months after surgery for 5 years. TIMM50 expression in tumour tissues was measured by quantitative real-time PCR, Western blotting and immunohistochemistry. TIMM50 expression was studied to assess correlations with clinicopathological factors and survival time. Results: TIMM50 expression increased significantly in CRC tumour tissues. Moreover, high TIMM50 expression was related to pathologic stage (p = 0.043), N stage (p = 0.048) and distant metastasis (p = 0.015), but TIMM50 expression was not related to other clinical factors. A Kaplan-Meier survival analysis indicated that patients with low TIMM50 expression had a longer overall survival than those with high TIMM50 expression (p = 0.002). Furthermore, distant metastasis and high TIMM50 expression were confirmed as independent prognostic factors for the overall survival of CRC patients in a multivariate analysis (p = 0.003). Conclusions: TIMM50 may be a key factor for monitoring CRC and a new prognosis indicator for CRC patients.

miR-194 Inhibits the Proliferation of SW620 Colon Cancer Stem Cells Through Downregulation of SSH2 Expression.

PURPOSE: Colorectal cancer (CRC) stem cells are tumorigenic, capable of self-renewal, and resistant to therapy. Although the expression pattern and functions of micro RNA (miR)-194 in CRC cells have been widely investigated, little is known about its role in CRC stem cells. Therefore, the aim of this study was to investigate the potential role of miR-194 in CRC stem cells. MATERIALS AND METHODS: CRC stem cells were isolated from the SW620 colon cancer cell line using microbeads. The expression levels of miR-194 and slingshot 2 (SSH2) in CRC stem cells were detected by RT-PCR and Western blot. A luciferase reporter assay was performed to confirm that miR-194 directly targets SSH2. Proliferation of CRC stem cells was examined by colony formation and MTT assays. Apoptosis in CRC stem cells was detected by cell cycle and apoptosis assays. The role of miR-194 in tumor growth was determined in vivo. RESULTS: Cells positive for CD44 and CD133 accounted for approximately 88.7% of the isolated population after microbead isolation. We reveal for the first time that miR-194 expression is decreased in CRC stem cells. Specifically, miR-194 is involved in inhibiting the proliferation of CRC stem cells and promoting CRC stem cell apoptosis by directly targeting SSH2. Furthermore, overexpression of miR-194 resulted in blocking the G1/S transition, the induction of cellular apoptotic process, thereby suppressing the malignant behaviors of CRC stem cells. CONCLUSION: This study represents a novel characterization of miR-194 function in CRC stem cells, which may aid in the development of promising therapeutic strategies targeting CRC.

The Role Of Hepatic Stellate Cells In Promoting Liver Metastasis Of Colorectal Carcinoma.

PURPOSE: Colorectal cancer (CRC) is the most common malignancy in the gastrointestinal tract. The liver is the most common location of CRC metastases, which are the main causes of CRC-related death. However, the mechanisms underlying metastasis of CRC to the liver have not been characterized, resulting in therapeutic challenges. METHODS: The effects of hepatic stellate cells (HSCs) on T cells were evaluated using in vitro mixed lymphocyte reactions (MLRs) and cytokine production assays. HSC-induced CT26 cell migration and proliferation were evaluated in vitro and in vivo. RESULTS: HSCs induced T cell hypo-responsiveness, promoted T cell apoptosis, and induced regulatory T cell expansion in vitro. IL-2 and IL-4 were significantly lower in MLRs incubated with HSCs. Supernatants of MLRs with HSCs promoted CT26 cell proliferation and migration. Furthermore, the presence of HSCs increased the number of liver metastases and promoted proliferation of liver metastatic tumor cells in vivo. CONCLUSION: HSCs may contribute to an immunosuppressive liver microenvironment, resulting in a favorable environment for the colonization of CRC cells in the liver. These findings highlight a potential strategy for treatment of CRC liver metastases.

Intraoperative monitoring of pelvic autonomic nerves during laparoscopic low anterior resection of rectal cancer.

BACKGROUND: Some patients with low rectal cancer experience anorectal and urogenital dysfunctions after surgery, which can influence the long-term quality of life. In this study, we aimed to protect nerve function in such scenarios by performing intraoperative monitoring of pelvic autonomic nerves (IMPAN). PATIENTS AND METHODS: We retrospectively investigated a series of 87 patients undergoing laparoscopic low anterior resection of rectal cancer. Nerve-sparing was evaluated both visually and electrophysiologically. IMPAN was performed by stimulating the pelvic autonomic nerves under processed electromyography of the internal anal sphincter. Urination, defecation, sexual function, and the quality of life were evaluated using validated and standardized questionnaires preoperatively and at follow-up, 12 months after surgery. RESULTS: Among a total of 87 patients (53 male and 34 female patients), IMPAN with simultaneous electromyography of the internal anal sphincter was performed in 58 (66.7%) patients. Bilateral positive IMPAN results for both measurements, indicating successfully confirmed pelvic autonomic nerve preservation, were obtained in 45 (51.7%) patients. No significant difference was found in terms of urogenital and anorectal functions between preoperative and postoperative patients with bilateral positive IMPAN (P>0.05). Compared to preoperative patients with IMPAN (unilateral) or without IMPAN, these patients exhibited higher International Prostate Symptom Score, a lower International Index of Erectile Function-5, and a lower Female Sexual Function Index score at 12 months postoperatively (P<0.05). CONCLUSION: IMPAN is an appropriate method with which to laparoscopically protect nerve function.

Application of cryoablation to treat peritoneal carcinomatosis from gastric cancer in a rabbit model.

OBJECTIVES: Peritoneal carcinomatosis is one of the causes of death in patients with advanced gastric cancer. We assumed that cryoablation could be applied as adjuvant therapy to control peritoneal carcinomatosis from gastric cancer. METHODS: We investigated the feasibility of cryoablation technique in rabbit model using a novel cryoablation balloon probe. The cryozones were harvested 7 days after cryoablation for histological evaluation. The levels of cytokines in the peripheral blood of rabbits were also detected. RESULTS: The results demonstrated that cryoablation could be applied in a rabbit model of peritoneal carcinomatosis from gastric cancer. Seven days after cryoablation, necrotic tumor cells could be seen the cryozones. Higher level of IFN-γ was observed. The level of IL-10 was decreased after treatment. CONCLUSIONS: The findings provided the experimental basis for the future application of cryoablation in patients.

Advantages of Early Preventive Ileostomy Closure after Total Mesorectal Excision Surgery for Rectal Cancer: An Institutional Retrospective Study of 123 Consecutive Patients.

BACKGROUND/AIM: Preventive ileostomy is frequently constructed to minimizethe consequences of anastomotic leakage after resection of rectal cancer. There is no consensus regarding the best timing for temporary stoma closure after proctectomy for rectal cancer. This retrospective study sought to determine whether the timing of stoma closure influenced postoperative outcomes. METHODS: Subjects were 123 patients with rectal cancer undergoing laparoscopic or open total mesorectal excision surgery with preventive ileostomy from 2012 to 2015. They were divided into 2 groups according the timing of stoma closure: the standard group who had closure within 90 (60-120) days (n = 78) and the late group who had closure after 180 (150-210) days (n = 45). RESULTS: There was no significant difference in operative time, operative blood loss or postoperative complications between the 2 groups. Timing of postoperative fasting and length of hospital stay was similar in both groups. Adjuvant chemotherapy was not a risk factor for postoperative complications after stoma closure. CONCLUSIONS: There was no significant difference between different timings of temporary stoma closure in relation to postoperative complications. Delayed stoma closure showed no benefit in prevention of morbidity. Early closure is safe and can provide better quality of life for patients.

Clinical safety and outcomes of laparoscopic surgery versus open surgery for palliative resection of primary tumors in patients with stage IV colorectal cancer: a meta-analysis.

BACKGROUND: This study was performed to compare the clinical safety and outcomes of laparoscopic versus open surgery for primary tumors in patients with stage IV colorectal cancer. METHODS: Pertinent studies were selected from the MEDLINE, EMBASE, and Cochrane Library databases; references from published articles; and reviews. Both prospective and retrospective studies were included for the meta-analysis. Clinical outcomes included safety, complications, mortality, and survival. RESULTS: Six trials involving 1802 patients were included. The operative time was longer for laparoscopic than for open surgery (mean difference (MD) = 44.20, 95 % confidence interval (CI) 17.31-71.09, Z = 3.22, P = 0.001). Laparoscopic surgery was also associated with fewer postoperative complications (odds ratio 0.53, 95 % CI 0.37-0.78, Z = 3.29, P = 0.001) and less operative blood loss (MD = -65.40, 95 % CI -102.37 to -28.42, Z = 3.47, P = 0.0005). Median survival ranged from 11.4 to 30.1 months. The total hospital stay was 1.68 days shorter for laparoscopic than for open surgery (95 % CI -1.83 to -1.53, Z = 21.64, P < 0.00001). CONCLUSION: Laparoscopic surgery for palliative resection of stage IV colorectal cancer is associated with better perioperative outcomes than open surgery.

Prognostic factors and multidisciplinary treatment modalities for brain metastases from colorectal cancer: analysis of 93 patients.

BACKGROUND: The purpose of this study was to review patient characteristics and evaluate the potential factors affecting prognosis in cases of brain metastasis (BM) from colorectal cancer (CRC). METHODS: We retrospectively reviewed 93 cases of BM from CRC in our hospital. Patient demographics, neurologic symptoms, and location and number of BMs were recorded. Factors analyzed included: age; sex; Karnofsky performance score; number of BMs; presence of extracranial metastases; dimensions; location of tumors; treatment modalities. RESULTS: The overall 1- and 2-year survival rates were 27.7 and 9.9%. On multivariate analysis, the number of BMs, extracranial metastases and the initial treatment modalities were found to be independent prognostic factors for overall survival. Patients treated with surgical resection followed by WBRT or SRS had an improved prognosis relative to those treated with surgery alone (P=0.02 and P=0.02, respectively). No significance difference in survival rate was found between patients treated with SRS alone or SRS plus WBRT (P=0.11). CONCLUSIONS: Surgical resection of BMs from CRC in selected patients may help prolong survival. Additional radiotherapy following surgery is valuable in improving prognosis. Extracranial metastasis, multiple BM lesions and initial non operation can be considered as independent factors associated with poor prognosis.

Comparison of clinical safety and outcomes of early versus delayed laparoscopic cholecystectomy for acute cholecystitis: a meta-analysis.

OBJECTIVE: To compare the clinical safety and outcomes of early laparoscopic cholecystectomy versus delayed laparoscopic cholecystectomy for acute cholecystitis. METHODS: Pertinent studies were selected from the Medline, EMBASE, and Cochrane library databases, references from published articles, and reviews. Seven randomized controlled trials (early laparoscopic cholecystectomy versus delayed laparoscopic cholecystectomy) were selected. Conventional meta-analysis according to Cochrane Collaboration was used for the pooling of the results. RESULTS: Seven trials with 1106 patients were included. There was no significant difference between the two groups in terms of bile duct injury (Peto odds ratio 0.49 (95% confidence interval 0.05 to 4.72); P = 0.54) or conversion to open cholecystectomy (risk ratio 0.91 (95% confidence interval 0.69 to 1.20); P = 0.50). The total hospital stay was shorter by 4 days for early laparoscopic cholecystectomy (mean difference -4.12 (95% confidence interval -5.22 to -3.03) days; P < 0.00001). CONCLUSION: Early laparoscopic cholecystectomy during acute cholecystitis is safe and shortens the total hospital stay.

Clinical application of adoptive T cell therapy in solid tumors.

As an emerging therapeutic approach, adoptive T cell therapy shown promise in advanced solid malignancies. The results obtained in patients with metastatic melanoma and kidney cancer are encouraging because of the visible clinical benefits and limited adverse effects. Recently, the genetically-modified T cells expressing specific T cell receptors or chimeric antigen receptors are just now entering the clinical arena and show great potential for high avidity to tumor-associated antigens and long-lasting anti-tumor responses. However, continued investigations are necessary to improve the cell product quality so as to decrease adverse effects and clinical costs, and make adoptive T cell therapy a tool of choice for solid malignancies.

Expression of C4.4A is a potential independent prognostic factor for patients with gastric cancer.

C4.4A, a metastasis-associated gene, encodes a glycolipid-anchored membrane protein which is overexpressed in several human malignancies. However, there are few data available on C4.4A expression and its relationship with progression in gastric cancer. Our study was designed to explore the expression of C4.4A in gastric cancer and to correlate it with clinical outcome. C4.4A expression was studied by quantitative real-time RT-PCR and immunohistochemistry for assessment of correlations with clinicopathological factors. C4.4A mRNA expression was significantly up-regulated in gastric cancer as compared with noncancerous tissue (p<0.05)., being observed in 107 (88.4%) of the 121 gastric cancer cases by immunohistochemistry. We found that the expression of C4.4A mRNA was correlated with size of the tumor, depth of invasion, lymph node metastasis, distant metastasis and TNM stage. Moreover, patients with overexpression of C4.4A has a significantly worse survival (p<0.05). Further multivariable analysis indicated that the expression of C4.4A was an independent prognostic indicator for gastric cancer (p<0.05). In conclusion, overexpression of C4.4A correlates with metastatic potential of gastric cancer and C4.4A could be a novel independent prognostic marker for predicting outcome.

Recipient dendritic cells modified by RNA interference targeting CD80 and CD86 elicit T cell hyporesponsiveness via enhanced T cell apoptosis.

BACKGROUND: Despite extensive research, the mechanism of immature dendritic cells (DCs) induced immune hyporesponsiveness remains incompletely understood. METHODS: Recipient DCs from C3H mouse bone marrow cells were incubated with donor antigen from splenic lymphocytes of C57BL/6 mouse; these DCs were transfected with CD80/86 specific siRNA using lentiviral vectors. Flow cytometry was used to evaluate expression of CD80/86 on the antigen-pulsed recipient DCs. Immune regulatory activity was examined by mixed lymphocyte reaction, in which irradiated DCs were cultured with C3H spleen T cells. After the reaction, interleukin (IL)-2, IL-4, IL-10, and interferon (INF)-γ levels of mixed lymphocyte reaction culture supernatant were measured by enzyme-linked immunosorbent assay. The apoptotic T lymphocytes were identified by Annexin V and CD3 staining. RESULTS: There was a significant inhibition of CD80/86 expression in DCs transfected with CD80/86 lentiviral vectors compared with the control groups (P < 0.05), indicating the specificity of RNA interference. Enzyme-linked immunosorbent assay results showed a significant reduction of INF-γ, IL-2 and IL-10 in the CD80/86 lentivirus transfected group compared to the control groups (P < 0.05). There was no significant difference in IL-4 levels between the groups (P > 0.05). We also showed that CD80/86 low DCs loaded with alloantigen (1) stimulated low T cell proliferative responses via the indirect recognition pathway and (2) enhanced apoptotic activity (P < 0.05) in co-cultured T cells. CONCLUSIONS: Lentiviral vector transfection can effectively and specifically knock down target genes in DCs. The CD80/86 low DCs may show tolerogenic activity via induction of T-cell apoptosis, thereby modulating the activity of recipient-derived DCs. The use of this approach may potentially be clinically applicable.

Inhibition of arterial allograft intimal hyperplasia using recipient dendritic cells pretreated with B7 antisense peptide.

BACKGROUND: Low expression or absence of dendritic cell (DC) surface B7 molecules can induce immune tolerance or hyporesponse. Whether DCs could induce indirect allogeneic-specific cross-tolerance or hyporesponse to recipient T cells remains unclear. METHODS: Generated from C3H/He mice bone marrow cells pulsed with donor antigen from C57BL/6 mice, recipient DCs were incubated with B7 antisense peptide (B7AP). Immune regulatory activities were examined in vitro by a series of mixed lymphocyte reactions. Murine allogeneic carotid artery orthotopic transplantation was performed from C57BL/6 to C3H/He. Recipients were given B7AP-treated DCs 7 days before transplantation. Allograft pathological analysis was done 2 months after transplantation. RESULTS: B7AP-pretreated DCs markedly inhibited T-cell proliferation compared with untreated group. Pretreated T cells exhibited markedly reduced response to alloantigen versus third-party antigen. Pathological analysis of arterial allografts demonstrated significant reduction of intimal hyperplasia in B7-AP pretreated group versus control. CONCLUSION: Blockade of B7 molecules by B7AP could induce indirect allogeneic-specific hyporesponse and inhibit arterial allograft intimal hyperplasia, which may be involved in future strategies for human allograft chronic rejection.

Brain metastases from colorectal cancer: microenvironment and molecular mechanisms.

Colorectal cancer is one of the most common digestive tract malignancies in the world. Owing to the newer and more effective systemic therapies, the life of colorectal cancer patients can be remarkably prolonged, and the incidence of brain metastases is increasing. However, little is known about the underlying mechanisms of brain metastasis from colorectal cancer. Here we review the tumor microenvironment and metastasis associated molecules in brain metastases from colorectal cancer. A further understanding of these mechanisms will help us to propose better strategies for colorectal cancer patients with brain metastasis and improve their life quality.

[Differential analysis of protein profiles of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients].

OBJECTIVE: To identify the proteins which play key roles during the formation of cholesterol gallstone, differential analysis was carried out that the proteome of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients. METHODS: Vesicular and micellar phases were isolated by the density gradient ultracentrifugation method. Total proteins from the two phases were extracted, and the protein expressional profiles were established by two-dimensional electrophoresis respectively. The differentially expressed protein spots analyzed by ImageMaster two-dimensional electrophoresis analysis software were identified with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: The concentrations of proteins from vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were (1.5358 +/- 0.0682) mg/ml and (7.1222 +/- 0.2022) mg/ml (P < 0.01) respectively. The average matched protein spots were 120 +/- 24 and 198 +/- 37 in the two groups respectively. There were 72 +/- 16 matched spots in the two representative gels maps and the matched rate was 45.30%. Eight differentially expressed protein spots were identified from the two cholesterol-carrier phases. Among them, 6 were up-regulated with 2 down-regulated in vesicular phase compared with micellar phase. The abundance differentiation of RBP and HSA was confirmed by immunoblotting. CONCLUSIONS: The differential protein profiles of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were established and 8 differential protein spots were identified successfully. The data may be a basis for further screening the key regulators of formation of cholesterol gallstone.

Purification and characterization of 33.5 kDa vesicular protein in human bile.

AIM: The present study was undertaken to purify and partially characterize the 33.5-kilodalton (33.5 kDa) vesicular protein in human bile and to explore the possible molecular mechanisms of the initial crystal nucleation process. METHODS: The 33.5 kDa vesicular protein was isolated by ultracentrifugation and further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. The purified 33.5 kDa vesicular protein was subjected to N-terminal amino acid sequencing and amino acid analysis. Cholesterol crystallization activity was detected by cholesterol crystal growth assay. The sugar chain of the 33.5 kDa vesicular protein was analyzed by dot-immunobinding assay of lectin coupled to a peroxidase (HRP-DSA, HRP-ConA, HRP-WGA) and was deglycosylated using two different enzymatic approaches (N-deglycosylation and O-deglycosylation) to determine the molecular weight of the protein component, the type of linkage between polypeptide and carbohydrate components. RESULTS: The 33.5 kDa vesicular protein with complicated glycan was an extensively glycosylated (37.3%) monomer and these sugar chains strongly bound to DSA, but did not bind to ConA. Amino acid sequencing indicated that the protein was unique. The 33.5 kDa vesicular protein exhibited potent cholesterol crystallization promoting activity in vitro with derived crystal growth curve indices It, Ig, Ic presented as 0.57, 1.52, and 1.63 respectively. Both enzymatic proteolysis and N-deglycosylation of the protein removed all activity. CONCLUSION: These data suggest the 33.5 kDa vesicular protein may be responsible for the pathogenesis of cholesterol gallstone disease, and the sugar chains play an important role in pro-nucleating process.

[ELISA kit for detection of biliary vesicular protein and its preliminary clinical application].

OBJECTIVE: To establish a rapid and precise detective method of 33.5 kd vesicular protein and to screen an effective treatment of cholelithiasis. METHODS: Specific antibody of the biliary vesicular protein was obtained by immunizing rabbits and enzyme-linked immunosorbent assay (ELISA) kit was developed. The concentrations of 33.5 kd vesicular protein in serum and bile of gallstone patients and control were examined respectively. The effects of Cholagogue Dry Syrup and Eulektrol Capsule on decreasing 33.5 kd vesicular protein were also studied by ELISA kit. RESULTS: One-step ELISA equation was Y=0.035 X (r=0.99). The vesicular protein concentrations in serum and bile of cholesterol gallstone group [(179.8+/-97.9) mg/L and (213.4+/-70.1) mg/L respectively] were significantly (P<0.05) higher than in the pigment stone group and control. Data showed that, with 2-week administration, Cholagogue Dry Syrup significantly decreased both biliary and serum 33.5 kd vesicular protein of cholesterol gallstone patients, while Eulekrol Capsule and control groups didn't have the same results. CONCLUSION: The concentrations of 33.5 kd protein are different in cholesterol gallstone patients and healthy groups which might be related to cholesterol nucleation process. Cholagogue Dry Syrup is of cholagogic and litholytic effect by decreasing biliary lithogenesis.