Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells.

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro. This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage.

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility adjustments. Conventionally, their contractility is gauged by observing morphological shifts and nearby capillary diameter changes under specific circumstances. Yet, post-tissue fixation, evaluating vitality and ensuing pericyte contractility of imaged brain pericytes becomes compromised. Similarly, genetically labeling brain pericytes falls short in distinguishing between viable and non-viable pericytes, particularly in neurologic conditions like subarachnoid hemorrhage (SAH), where our preliminary investigation validates brain pericyte demise. A reliable protocol has been devised to surmount these constraints, enabling simultaneous fluorescent tagging of both functional and non-functional brain pericytes in brain sections. This labeling method allows high-resolution confocal microscope visualization, concurrently marking the brain slice microvasculature. This innovative protocol offers a means to appraise brain pericyte contractility, its impact on capillary diameter, and pericyte structure. Investigating brain pericyte contractility within the SAH context yields insightful comprehension of its effects on cerebral microcirculation.

Early Follow-Up of Arthroscopic Latarjet Procedure with Screw or Suture-Button Fixation for Recurrent Anterior Shoulder Instability.

OBJECTIVE: To evaluate the early clinical and radiographic results of arthroscopic Latarjet procedure using screw or suture-button fixation in patients with recurrent anterior shoulder dislocation. METHODS: Twelve patients who underwent arthroscopic Latarjet procedure between January 2015 and December 2018 at our institution were retrospectively studied. Data of the patients' history, including age, gender, side of affected arm, body mass index (BMI), and the number of dislocations since fist dislocation were collected. Preoperative and postoperative clinical follow-up data were evaluated using Walch-Duplay score, American Shoulder and Elbow Society (ASES) score, and modified Rowe score. Active external rotation and active internal rotation at 90° of abduction as well as active elevation were evaluated preoperatively and postoperatively. The position and healing condition of the transferred coracoid bony graft were also assessed using computed tomography (CT) and Mimics 19.0 software. RESULTS: Mean follow-up was 24.9 months (range, 13 to 53 months) of all patients. At final follow-up, the average ASES score (preoperative vs postoperative values) had improved from 68.9 ± 7.9 to 91.1 ± 6.1 in screw fixation group and 68.9 ± 8.9 to 87.5 ± 6.7 in suture-button fixation group; the average Rowe score (preoperative vs postoperative values) had improved from 25.0 ± 8.4 to 92.5 ± 4.2 in screw fixation group and 21.7 ± 13.7 to 93.3 ± 4.1 in suture-button fixation group; the average of Walch-Duplay score (preoperative vs postoperative values) had improved from 12.5 ± 15.1 to 91.7 ± 4.1 in screw fixation group and 18.3 ± 20.7 to 88.3 ± 7.5 in button fixation group. The forward flexion was 175.0° ± 8.4° preoperatively and 178.3° ± 4.1° postoperatively in screw fixation group while 174.8° ± 10.2° preoperatively and 175.0° ± 5.5° postoperatively in suture-button fixation group. The active external rotation was 77.5° ± 5.2° preoperatively and 71.7° ± 4.1° postoperatively in screw fixation group while 72.5° ± 6.9° preoperatively and 68.3° ± 7.5° postoperatively in suture-button fixation group. The average of active internal rotation was 66.7° ± 6.1° preoperatively and 67.5° ± 6.1° postoperatively in screw fixation group while 68.3° ± 11.3° preoperatively and 66.7° ± 7.5° postoperatively in suture-button fixation group. In postoperative CT scan, 91.7% grafts midline center were located at or under the equator in the en face view; 75% of the bone blocks were flush to the glenoid face in the axial view, with only two grafts exhibiting slight medial placement in screw fixation group (33.3%) and one graft exhibiting slight lateral placement in suture-button fixation group (16.7%). All grafts achieved bone union. Graft absorption mostly occurred outside of the "best-fit" circle. The average bony absorption rates of the coracoid grafts were 25.2% and 10.18% in screw fixation group and suture-button fixation group, respectively, at 6 months postoperative follow-up. CONCLUSION: Both suture-button fixation and screw fixation techniques in arthroscopic Latarjet procedure revealed excellent clinical outcomes with low complication rates in the early follow-up. The suture-button fixation exhibited a flexible fixation pattern that allowed for self-correction to some extent, even slight lateralization could finally remodel over time.

Analysis of factors affecting the prognosis of COVID-19 patients and viral shedding duration.

The clinical characteristics of patients with COVID-19 were analysed to determine the factors influencing the prognosis and virus shedding time to facilitate early detection of disease progression. Logistic regression analysis was used to explore the relationships among prognosis, clinical characteristics and laboratory indexes. The predictive value of this model was assessed with receiver operating characteristic curve analysis, calibration and internal validation. The viral shedding duration was calculated using the Kaplan-Meier method, and the prognostic factors were analysed by univariate log-rank analysis and the Cox proportional hazards model. A retrospective study was carried out with patients with COVID-19 in Tianjin, China. A total of 185 patients were included, 27 (14.59%) of whom were severely ill at the time of discharge and three (1.6%) of whom died. Our findings demonstrate that patients with an advanced age, diabetes, a low PaO2/FiO2 value and delayed treatment should be carefully monitored for disease progression to reduce the incidence of severe disease. Hypoproteinaemia and the fever duration warrant special attention. Timely interventions in symptomatic patients and a time from symptom onset to treatment <4 days can shorten the duration of viral shedding.

The role of carotid stenosis ultrasound scale in the prediction of ischemic stroke.

INTRODUCTION: To improve the accuracy of ultrasound techniques for the assessment of carotid stenosis, we designed a novel carotid artery stenosis ultrasound scale (CASUS), and evaluated its accuracy, reliability, and its value in predicting the occurrence of cardiovascular and cerebrovascular diseases in a prospective study. METHODS: A total of 750 patients with first-time ischemic stroke and hospitalized within 24 h were enrolled in the study. Using color Doppler ultrasound (CDUS), the degree of stenosis and blood flow (BF) in bilateral internal carotid arteries (ICA) and the V1-V3 segment of vertebral arteries (VA) was assessed. Cubic simulation curves for BF and global blood flow (GBF) over the stenosis score (SS), total stenosis score (TSS), and radiological imaging- total stenosis score (RI-TSS) were fitted and compared. The receiver operating characteristic (ROC) curves using TSS, RI-TSS, or GBF to predict various ischemic stroke endpoints were also analyzed and compared. RESULTS: There was a linear relationship between SS and BF both ICA and VA (R2 were 0.734 and 0.783, respectively, both P < 0.05). Both TSS and RI-TSS with GBF showed an inverse "S" curve relationship (R2 was 0.839 and 0.843, all P < 0.05). The AUC values of TSS-based and RI-TSS-based predictions of each endpoint were all greater than 0.7 (all P < 0.05), but the differences of the AUC values between TSS, RI-TSS, and GBF were not statistically significant (all P > 0.05). CONCLUSIONS: The novel CASUS can better reflect the level of cerebral reperfusion in patients with ischemic stroke and can better predict the occurrence of cardiovascular and cerebrovascular diseases.

Mutation spectrum of the bestrophin-1 gene in a large Chinese cohort with bestrophinopathy.

BACKGROUND: Bestrophin-1 (BEST1) gene is associated with a wide range of ocular phenotypes, collectively termed as bestrophinopathy. The aim of the current study was to identify the mutation spectrum of BEST1 in a large cohort of Chinese patients with bestrophinopathy. METHODS: Patients clinically suspected of bestrophinopathy were screened using multigene panel testing. All BEST1 variants were confirmed by Sanger sequencing, and validated in the families. FINDINGS: A total of 92 patients (Best vitelliform macular dystrophy (BVMD)=77; autosomal recessive bestrophinopathy (ARB)=15) from 58 unrelated families of Chinese origin and their available family members (n=65) were recruited. Overall, 39 distinct disease-causing BEST1 variants were identified, including 13 novel variants, and two reported variants but novel for ARB. Of them, 14 were associated with ARB, 23 with BVMD and two (c.604C>T and c.898G>A) with both BVMD and ARB. Most mutations associated with BVMD were missense (97.78%), while ARB was associated with more complex mutations, including missense (88.46%), splicing effect (3.85%), and frameshifts (15.38%). BEST1 hotspots were c.898G>A and c.584C>T among BVMD and ARB patients, respectively. Hot regions were located in exons 8, 2 and 6 in BVMD patients, and in exons 5 and 7 in ARB patients. The overall penetrance of BEST1 in our cohort was 71.30%, no de novo mutations were identified. CONCLUSION: This is the largest study to date that provides major population-based data of the BEST1 mutation spectrum in China. Our results can serve as a well-founded reference for genetic counselling for patients with bestrophinopathy of Chinese origin.

Expanding the clinical and genetic spectrum of Heimler syndrome.

BACKGROUND: Heimler syndrome (HS) is a rare hereditary systemic disorder, partial clinically overlapping with Usher syndrome. So far, our knowledge of HS is very limited, many cases are misdiagnosed or may not even be diagnosed at all. This study aimed to analyze the clinical and genetic characteristics of HS, and to evaluate potential phenotype-genotype correlations. RESULTS: Two HS cases caused by PEX1 mutations were identified, and a novel likely pathogenic mutation, PEX1 c.895_896insTATA, was found. The main ophthalmic finding of the two patients was consistent with retinitis pigmentosa accompanied by cystoid macular edema, but short axial length and hyperopia were also observed as two previously unreported ocular phenotypes. Analysis of the literature showed that of the 29 HS patients previously reported, 12 had PEX6 mutations, 10 had PEX1 mutations, two had PEX26 mutations, and the remaining patients were not genetically tested. Three novel genotype-phenotype correlations were revealed from analysis of these patients. First, most genotypes of every HS patient include at least one missense variant; second, at least one mutation in PEX1 or PEX6 gene affects the AAA-ATPase region in every HS patient with retinal dystrophy, suggesting AAA-ATPase region is a hypermutable region in patients with a retinal dystrophy; third, there are no significant differences between PEX1-, PEX6-, and PEX26-associated phenotypes. CONCLUSION: Next-generation sequencing is important for the diagnosis of HS. This study expands the clinical and genetic spectrum of HS, and provides additional insights into genotype-phenotype correlations, which is vital for accurate clinical practice, genetic counseling, and pathogenesis studies.

ABCA4 Gene Screening in a Chinese Cohort With Stargardt Disease: Identification of 37 Novel Variants.

Purpose: To clarify the mutation spectrum and frequency of ABCA4 in a Chinese cohort with Stargardt disease (STGD1). Methods: A total of 153 subjects, comprising 25 families (25 probands and their family members) and 71 sporadic cases, were recruited for the analysis of ABCA4 variants. All probands with STGD1 underwent a comprehensive ophthalmologic examination. Overall, 792 genes involved in common inherited eye diseases were screened for variants by panel-based next-generation sequencing (NGS). Variants were filtered and analyzed to evaluate possible pathogenicity. Results: The total variant detection rate of at least one ABCA4 mutant allele was 84.3% (129/153): two or three disease-associated variants in 86 subjects (56.2%), one mutant allele in 43 subjects (28.1%), and no variants in 24 subjects (15.7%). Ninety-six variants were identified in the total cohort, which included 62 missense (64%), 15 splicing (16%), 11 frameshift (12%), 6 nonsense (6%), and 2 small insertion or deletion (2%) variants. Thirty-seven novel variants were found, including a de novo variant, c.4561delA. The most prevalent variant was c.101_106delCTTTAT (10.5%), followed by c.2894A > G (6.5%) and c.6563T > C (4.6%), in STGD1 patients from eastern China. Conclusion: Thirty-seven novel variants were detected using panel-based NGS, including one de novo variant, further extending the mutation spectrum of ABCA4. The common variants in a population from eastern China with STGD1 were also identified.

Genetic and Clinical Findings in a Large Cohort of Chinese Patients with Suspected Retinitis Pigmentosa.

PURPOSE: To characterize the genetic landscape of patients with suspected retinitis pigmentosa (RP) in the Chinese population. DESIGN: Cohort study. PARTICIPANTS: A total of 1243 patients of Chinese origin with clinically suspected RP and their available family members (n = 2701) were recruited. METHODS: All patients and available family members were screened using multigene panel testing (including 586 eye disease-associated genes), followed by clinical variant interpretation. MAIN OUTCOME MEASURES: Diagnostic yield, the 17 most commonly implicated genes, age at onset, de novo mutations, and clinical usefulness of genetic testing. RESULTS: Overall, 72.08% of patients received a molecular diagnosis, and the 17 top genes covered 75.63% of diagnostic cases. Diagnostic yield was higher among patients in the early-onset subgroup (≤5 years old, 79.58%) than in the childhood or adolescence-onset subgroup (6-16 years old, 73.74%) and late-onset subgroup (≥17 years old, 65.99%). Moreover, different genes associated with different onset ages and subgroups with different onset ages showed a diverse mutation spectrum. Only 11 de novo mutations (3.18%) were identified. Furthermore, 16.84% of the patients who received a molecular diagnosis had refinement of the initial clinical diagnoses, and the remaining 83.16% received definite genetic subtypes of RP. CONCLUSIONS: This large cohort study provides population-based data of the genome landscape of patients with suspected RP in China. The diagnostic yield was significantly higher than that in previous studies, and the mutation spectrum is completely different with other populations. Genetic testing improves the chance to establish a precise diagnosis, identifies features not previously determined, and allows a more accurate refinement of risk to family members. Our results not only expand the existing genotypic spectrum but also serve as an efficient reference for the design of panel-based genetic diagnostic testing and genetic counseling for patients with suspected RP in China.

[Analysis of clinical effects of iliolumbar fixation in treating sacrum fracture of Denis type II].

OBJECTIVE: To evaluate the clinical effects of iliolumbar fixation for the sacrum fractures of Denis type II. METHODS: The clinical data of 86 patients with sacrum fracture of Denis type II treated by iliolumbar fixation from January 2008 to January 2012 were retrospectively analyzed. There were 55 males and 31 females, aged from 17 to 55 years old with an average of 39.1 years. Among them, 73 cases complicated with pelvis fracture and 13 cases with acetabular fracture; 37 cases with sacral neurological symptoms and 49 cases without sacral neurological symptoms. Fracture healing time, nerve function, clinical function and complications were observed in the patients. RESULTS: In 86 cases, 6 cases were out of followed-up and 80 cases were followed up from 24 to 71 months with an average of 36 months. The mean fracture healing time was 13 weeks (ranged, 10 to 38 weeks). According to Gibbons scoring to evaluate the neurological function, preoperative nerve rehabilitation, lower limbs feeling, lower limbs activity,bladder and rectum function,total score respectively were 0.62 +/- 0.04, 1.54 +/- 0.35, 1.12 +/- 0.18, 0.23 +/- 0.01, 3.46 +/- 0.47 and postoperative respectively were 0.82 +/- 0.12, 0.36 +/- 0.04, 0.05 +/- 0.01, 0.03 +/- 0.01, 1.25 +/- 0.22, there were statistically significant differences between preoperative and postoperative (P < 0.05). According to Majeed scoring to evaluate the clinical function, postoperative pain, standing, sitting, sexual life, work ability, total score respectively were 22.54 +/- 4.02, 27.93 +/- 5.46, 8.47 +/- 3.61, 2.54 +/- 1.33, 16.46 +/- 4.34, 81.32 +/- 8.73, 60 cases got excellent results, 17 good, 3 fair. The main complications including fracture nonunion of 5 cases,deep incision infection of 1 case, and screw prominence resulting uncomfortable of 8 cases. CONCLUSION: Iliolumbar fixation has the advantages of stable fixation, satisfactory functional rehabilitation, less complications, and is a good method in treating sacrum fracture of Denis type II.

The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease.

In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1 (SIAH-1) in PC12 cells. 1-Methyl-4-phenylpyridinium (MPP(+)) treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased mRNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 mRNA expression. It also increased cell viability. Combined treatment with MPP(+) and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson's disease.

Silencing of SIAH1 in SH-SY5Y affects α-synuclein degradation pathway.

Seven in absentia homolog (SIAH) is a ubiquitin ligase that monoubiquitinates α-synuclein. Lewy bodies are characteristically rich in monoubiquitinated α-synuclein. We aimed to determine the effect of siRNA-SIAH1 on α-synuclein autophagy and UPS degradation in SH-SY5Y. SIAH1 expression was measured with real-time quantitative PCR and Western Blot. Cell proliferation was measured by CCK-8 assay; cell apoptosis assayed by flow cytometry. Relative protein expressions were measured by Western Blot. mRNA levels of relative protein were measured by real-time quantitative PCR. The expression of α-synuclein, LC3-II and SIAH1 were observed by confocal microscopy. We found: (1) Transfection efficiency of SIAH1-siRNA into SH-SY5 measured approximately 89% by flow cytometry. (2) siRNA silencing of SIAH1 promoted cellular proliferation and suppressed apoptosis. (3) Protein and mRNA expression of α-synuclein, LC3-II and p53 decreased after SIAH1 knockdown. E1 protein and mRNA levels increased after SIAH1 siRNA. These data show silencing SIAH1 increased cell proliferation and inhibited apoptosis in SH-SY5Y neuroblastoma cells. SIAH1 knockdown enhanced the clearance of non-aggregated α-synuclein by UPS. SIAH1 is a potential target for treatment of Parkinson's disease.

[Fundus fluorescein angiography in metastatic choroidal carcinomas and differentiating metastatic choroidal carcinomas from primary choroidal melanomas].

OBJECTIVE: To investigate the characteristics of fundus fluorescein angiography (FFA) in metastatic choroidal carcinomas and determine the value of FFA in differentiating metastatic choroidal carcinomas from primary choroidal melanomas. METHODS: It was a retrospective case series. The retrospective analysis of clinical data and FFA findings was performed in 23 eyes of 22 patients with metastatic choroidal carcinomas and 31 eyes of 31 patients with primary choroidal melanomas as the control. RESULTS: Ocular fundus findings of metastatic choroidal carcinomas were divided into three types: solitary flat (tumor thickness less than 3 mm), solitary elevated (tumor thickness more than 3 mm) or diffuse type. FFA of the three types showed hypofluorescence during the arterial phase and progressive hyperfluorescence during the subsequent phases. The border of the lesions revealed retinal capillary dilation during the arteriovenous phase and persistent pinpoint leakage throughout the angiogram. Retinal capillary dilation and pinpoint leakage were more frequently presented in the solitary flat type. Simultaneous visualization of retinal and tumor circulation (the so called double circulation) was more frequently presented in the solitary elevated type. Pinpoint leakage could be detected in 17 (73.91%) eyes of metastatic choroidal carcinomas and in 5 (16.13%) eyes of primary choroidal melanomas. The difference between the visibility of pinpoint leakage in metastatic choroidal carcinomas and primary choroidal melanomas was statistically significant (P = 0.0000). When pinpoint leakage of FFA was used to differentiate metastatic choroidal carcinomas from primary choroidal melanomas, the sensitivity, specificity, accuracy, positive and negative predictive values were 73.91%, 83.87%, 79.63%, 77.27%, 81.25% respectively. CONCLUSIONS: FFA is helpful for the diagnosis of metastatic choroidal carcinomas. Pinpoint leakage on the border of lesions has some value in differentiating metastatic choroidal carcinomas from primary choroidal melanomas.

[Clinical manifestations and gene analysis in one Chinese family with Best vitelliform macular dystrophy].

OBJECTIVE: To describe clinical phenotype in a Chinese family with Best vitelliform macular dystrophy (BVMD) and to identify the mutation of the VMD2 gene in this family. METHODS: It was a retrospective case analysis. Five patients (10 eyes) were diagnosed as BVMD by the fundus photography, EOG, fluorescein angiography (FFA) and optical coherence tomography (OCT). Their clinical data were analyzed retrospectively. Molecular genetic analysis was performed on DNA extracted from peripheral leucocytes of all patients and 2 unaffected family members. Exon 1 to 11 of the VMD2 gene were amplified by polymerase chain reaction for direct sequencing. RESULTS: The pedigree showed an autosomal dominant inheritance. Ten eyes from 5 patients were classified into Stage 0, II a, II b, III and IV with different clinical manifestations. Direct sequencing of all affected members revealed a T-->G transition at codon 301, producing Asp301Glu mutation of VMD2 gene. CONCLUSIONS: Asp301Glu mutation of the VMD2 gene is found in a Chinese family with BVMD. The phenotype of BVMD in this family belongs to geographic type. Molecular genetic approach may be useful for the proper diagnosis of BVMD.

[Clinical features of Best vitelliform macular dystrophy].

OBJECTIVE: To investigate the clinical features of Best vitelliform macular dystrophy (BVMD) in Chinese patients. METHODS: Ten consecutive patients (20 eyes) were diagnosed as BVMD by the fundus photography, EOG, fluorescein angiography (FFA) and optical coherence tomography (OCT). Their clinical data were analyzed retrospectively. RESULTS: Of the twenty eyes from ten patients, three eyes from three patients (age range 9-18 years, mean 12.33+/-4.93 years) in Stage II, two eyes from two patients (age range 9-18, mean 13.50+/-6.36 years) in Stage IIa, four eyes from two patients (age range 11-29 years, mean 20.00+/-10.39 years) in Stage III and eleven from six patients (age range 9-44 years, mean 27.09+/-14.02 years) in Stage IV were found at their first presentation to our hospital. OCT scan showed the broadening of the outer-retina-choroid-complex signal with the retinal elevation in Stage II. The moderately reflective material which represents the vitelliform material may accumulate forming a conical mound that would elevate the retinal sensory layer in Stage IIa. In 'pseudohypopon' or atrophy phase there may be a large volume of serous retinal detachment. If a fibrous macular or foveal atrophy was seen in the fundus photograph, the thinning of the outer-retina-choroid-complex signal with serous retinal detachment may be shown by OCT. CONCLUSIONS: The present observation is a first study on the clinical findings of Chinese BVMD patients. It supports the hypothesis that the yellowish material is located under the RPE. Long term evaluation with more patients should be done to acknowledge more characteristics of BVMD in Chinese patients.

[Smad4 and TGF-beta1 expression and clinical significance in bladder transitional cell carcinoma].

OBJECTIVE: To determine the expression of Smad4 and TGF-beta1 in bladder transitional cell carcinoma (BTCC), and to understand the mechanism of invasion, angiogenesis, and metastasis of BTCC. METHODS: The expressions of Smad4 and TGF-beta1 in samples of 42 human bladder carcinoma and 12 normal bladder mucosa tissues were determined with standard immunohistochemical analysis. We also analyzed the relationship among the expressions of Smad4 and TGF-beta1 and invasion, angiogenesis, and metastasis of BTCC, and the correlation between Smad4 and TGF-beta1. RESULTS: The positive rate of Smad4 in BTCC was significantly lower than those in normal bladder mucosa tissues (33.3% vs 83.3%, P < 0.01). The expressions of Smad4 in poorly differentiated, invasive, recurrent, or with lymph node metastasis of BTCCs were lower than those in well differentiated, superficial, nonrecurrent, or without lymph node metastasis ones (P <0.05). The positive rate of TGF-beta1 in BTCC was significantly lower than that in normal bladder mucosa tissues (64.3% vs 100%, P <0.01), which was positively correlated to that of Smad4 (P = 0.000). CONCLUSION: The expressions of Smad4 and TGF-beta1 in BTCC decrease with the increase in clinical stage, poor pathological grade, and the recurrence and metastasis of BTCC.