Breeding and preliminarily phenotyping of a congenic mouse model with alopecia areata.

In the current study, the alopecia areata gene was introduced into the C57BL/6 (B6) mouse through repeated backcrossing/intercrossing, and the allelic homozygosity of congenic AA(tj)mice (named B6.KM-AA) was verified using microsatellites. The gross appearance, growth characteristics, pathological changes in skin, and major organs of B6.KM-AA mice were observed. Counts and proportions of CD4⁺ and CD8⁺ T lymphocytes in peripheral blood were determined by flow cytometry. Results show that congenic B6.KM-AA mice were obtained after 10 generations of backcrossing/intercrossing. B6.KM-AA mice grew slower than B6 control mice and AA skin lesions were developed by four weeks of age. The number of hair follicles was reduced, but hair structures were normal. Loss of hair during disease progression was associated with CD4⁺ and CD8⁺ T lymphocytes infiltration peri-and intra-hair follicles. No pathological changes were found in other organs except for the skin. In the peripheral blood of B6.KM-AA mice, the percentage of CD4⁺ T cells was lower and percentage of CD8⁺ T cells higher than in control mice. These findings indicate that B6.KM-AA mice are characterized by a dysfunctional immune system, retarded development and T-cell infiltration mediated hair loss, making them a promising new animal model for human alopecia areata.

In vitro and in vivo characteristics of hepatic oval cells modified with human hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a multifunctional growth factor that controls cell scattering. It has been suggested that it regulates the proliferation of hepatic oval cells (HOCs). Using a HOC line that stably expresses the human HGF gene (hHGF), we investigated the in vitro proliferation and differentiation characteristics of hHGF-modified HOCs and explored their potential capacity for intrahepatic transplantation. A modified 2-acetylaminofluorene and partial hepatectomy (2-AAF/PH) model was established to activate the proliferation of oval cells in the rat liver. HOCs were transfected with the pBLAST2-hHGF plasmid and hHGF-carrying HOCs were selected based on blasticidin resistance. The level of hHGF secretion was determined via ELISA. Cell proliferation was determined using the MTT assay. Differentiation was induced by growth factor withdrawal. A two-cuff technique was used for orthotopic liver transplantation, and HOCs or hHGF-modified HOCs were transplanted into the recipients. The levels of biochemical indicators of liver function were measured after transplantation. An HOC line stably expressing hHGF was established. The transfected line showed greater hHGF secretion than normal HOCs. The hHGF gene promoted the proliferation capability of HOCs by reducing the peak time in vitro. The hHGF-modified HOCs differentiated into hepatocytes and bile duct epithelial cells upon growth factor withdrawal in vitro. In addition, hHGF-modified HOC transplantation significantly prolonged the median survival time (MST) and improved the liver function of recipients compared to HOC transplant recipients and nontransplanted controls. Our results indicate that hHGF-modified HOCs may have valuable properties for therapeutic liver regeneration after orthotopic liver transplantation.

Radical lymph node dissection and assessment: Impact on gallbladder cancer prognosis.

AIM: To investigate the lymph node metastasis patterns of gallbladder cancer (GBC) and evaluate the optimal categorization of nodal status as a critical prognostic factor. METHODS: From May 1995 to December 2010, a total of 78 consecutive patients with GBC underwent a radical resection at Liaocheng People's Hospital. A radical resection was defined as removing both the primary tumor and the regional lymph nodes of the gallbladder. Demographic, operative and pathologic data were recorded. The lymph nodes retrieved were examined histologically for metastases routinely from each node. The positive lymph node count (PLNC) as well as the total lymph node count (TLNC) was recorded for each patient. Then the metastatic to examined lymph nodes ratio (LNR) was calculated. Disease-specific survival (DSS) and predictors of outcome were analyzed. RESULTS: With a median follow-up time of 26.50 mo (range, 2-132 mo), median DSS was 29.00 ± 3.92 mo (5-year survival rate, 20.51%). Nodal disease was found in 37 patients (47.44%). DSS of node-negative patients was significantly better than that of node-positive patients (median DSS, 40 mo vs 17 mo, χ² = 14.814, P < 0.001), while there was no significant difference between N1 patients and N2 patients (median DSS, 18 mo vs 13 mo, χ² = 0.741, P = 0.389). Optimal TLNC was determined to be four. When node-negative patients were divided according to TLNC, there was no difference in DSS between TLNC < 4 subgroup and TLNC ≥ 4 subgroup (median DSS, 37 mo vs 54 mo, χ² = 0.715, P = 0.398). For node-positive patients, DSS of TLNC < 4 subgroup was worse than that of TLNC ≥ 4 subgroup (median DSS, 13 mo vs 21 mo, χ² = 11.035, P < 0.001). Moreover, for node-positive patients, a new cut-off value of six nodes was identified for the number of TLNC that clearly stratified them into 2 separate survival groups (< 6 or ≥ 6, respectively; median DSS, 15 mo vs 33 mo, χ² = 11.820, P < 0.001). DSS progressively worsened with increasing PLNC and LNR, but no definite cut-off value could be identified. Multivariate analysis revealed histological grade, tumor node metastasis staging, TNLC and LNR to be independent predictors of DSS. Neither location of positive lymph nodes nor PNLC were identified as an independent variable by multivariate analysis. CONCLUSION: Both TLNC and LNR are strong predictors of outcome after curative resection for GBC. The retrieval and examination of at least 6 nodes can influence staging quality and DSS, especially in node-positive patients.

Human hepatocyte growth factor (hHGF)-modified hepatic oval cells improve liver transplant survival.

Despite progress in the field of immunosuppression, acute rejection is still a common postoperative complication following liver transplantation. This study aims to investigate the capacity of the human hepatocyte growth factor (hHGF) in modifying hepatic oval cells (HOCs) administered simultaneously with orthotopic liver transplantation as a means of improving graft survival. HOCs were activated and isolated using a modified 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model in male Lewis rats. A HOC line stably expressing the HGF gene was established following stable transfection of the pBLAST2-hHGF plasmid. Our results demonstrated that hHGF-modified HOCs could efficiently differentiate into hepatocytes and bile duct epithelial cells in vitro. Administration of HOCs at the time of liver transplantation induced a wider distribution of SRY-positive donor cells in liver tissues. Administration of hHGF-HOC at the time of transplantation remarkably prolonged the median survival time and improved liver function for recipients compared to these parameters in the other treatment groups (P<0.05). Moreover, hHGF-HOC administration at the time of liver transplantation significantly suppressed elevation of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) levels while increasing the production of IL-10 and TGF-ß1 (P<0.05). HOC or hHGF-HOC administration promoted cell proliferation, reduced cell apoptosis, and decreased liver allograft rejection rates. Furthermore, hHGF-modified HOCs more efficiently reduced acute allograft rejection (P<0.05 versus HOC transplantation only). Our results indicate that the combination of hHGF-modified HOCs with liver transplantation decreased host anti-graft immune responses resulting in a reduction of allograft rejection rates and prolonging graft survival in recipient rats. This suggests that HOC-based cell transplantation therapies can be developed as a means of treating severe liver injuries.

[Phenotype analysis and mutant gene location of ventral yellow mouse (VY(Slac))].

The ventri-yellow pigmentation mouse (temporarily named VY(Slac)) arose spontaneously in the C57BL/6J inbred mouse strain, found and bred by Shanghai SLAC Laboratory Animal Co., Ltd. VY(Slac) presented a special phenotype marked by yellow coat on the ventral surface of neck and trunk that was without melanin deposition but maintained a normal structure. The number of melanocytes in epidermis and melanin in hair follicle of the abdominal skin of the mutant mouse were less than that of their background strain, while there was no significant difference between the dorsal skins of the two strains. This mutant phenotype was inherited as single-gene dominant inheritance, confirmed by genetic experiment, and there was no significant difference between VY(Slac) and B(6) for other biological parameters such as weight, anatomic and histological structures of major organs and blood physiology. When the linkage relationship between the genomic DNA samples of F(2) 48 mice (VY(Slac)D(2)F(1)×D(2)) and mutant phenotype were evaluated, the mutant gene was confirmed on chromosome 2 near D2Mit229. New microsatellite and SNP markers were selected to amplify genomic DNA samples of 196 F(2) mice and the mutant gene was narrowed down to 5.3 Mb region between rs13476833 and rs27310903 on chromosome 2. The preliminary results of our phenotype analysis and gene location provides a solid basis for further identification of this mutant gene.

Sinistral portal hypertension: clinical features and surgical treatment of chronic splenic vein occlusion.

OBJECTIVE: Sinistral, or left-sided, portal hypertension (SPH) is a rare cause of upper gastrointestinal hemorrhage. This retrospective analysis summarizes the clinical features of SPH and the results of surgical treatment. SUBJECTS AND METHODS: Between 2000 and 2009, patients from our hospital diagnosed with SPH were identified. Diagnosis of SPH was based on evidence of isolated splenic vein thrombosis, splenomegaly, gastroesophageal varices and exclusion of other causes of portal hypertension. RESULTS: Eight males and 5 females were included in the study, with a mean age of 43.5 ± 6.4 years (range: 31-68 years). Liver function was normal in all patients. Causes of SPH were chronic pancreatitis (n = 7), pancreatic cancer (n = 3), pancreatic cysts (n = 2) and neuroendocrine tumor (n = 1). The main clinical manifestations were gastrointestinal hemorrhage in 7 cases (53.8%), upper abdominal pain in 10 (76.9%) and hypersplenism in 12 (92.3%). All patients had splenomegaly and gastroesophageal varices. Twelve patients underwent splenectomy and 1 received surgical removal of a pancreatic cyst. No major gastrointestinal tract rebleed occurred after a mean follow-up of 46 months (±7 months). Two patients died of pancreatic cancer and 1 of acute myocardial infarction during follow-up. CONCLUSIONS: SPH should be suspected in patients with upper gastrointestinal varices as well as unexplained splenomegaly with normal liver function. Surgical intervention such as splenectomy offers a good long-term outcome in symptomatic patients.

Combined use of D-dimer and P-selectin for the diagnosis of splenic or portal vein thrombosis following splenectomy.

To investigate the predictive value of combined use of D-dimer and P-selectin for splenic or portal vein thrombosis (SPVT) after splenectomy. A prospective study was carried out in 82 patients who had undergone splenectomy for portal hypertension secondary to hepatic cirrhosis. Plasma levels D-dimer and P-selectin were measured before and after the surgery.27 (30.1%) patients developed SPVT following the portal hypertension surgery. The post-operative D-dimer and P-selectin levels in patients with SPVT were significantly higher than in those without PVT (n=55, P<0.01). The receiver-operated characteristics curves (ROC) of D-dimer and P-selectin showed a significant predictive value in SPVT (D-dimer, Az=0.880, P<0.01; P-slectin, Az=0.933, P<0.01). The sensitivity and specificity of D-dimer (>500 microg/mL) in diagnosing SPVT were 88.9% and 78.2% respectively. P-selectin >90ng/mL had an 85.2% sensitivity and 85.5% specificity for SPVT. The combination of the D-dimer and P-selectin criteria yielded an 82.0% sensitivity and 97.6% specificity for SPVT. In Conclusion, there was a significant elevation in plasma D-dimer and P-selectin in patients with post-operative SPVT. A combination of plasma D-dimer and P-selectin may be used as biomarkers to screen or diagnose SPVT following splenectomy.

[Effect of branch chain amino acid enriched formula on postoperative fatigue and nutritional status after digestive surgery].

OBJECTIVE: To evaluate the effect of branch chain amino acid (BCAA) enriched formula on nutritional status and postoperative fatigue for digestive surgery patients. METHODS: Forty patients who underwent digestive surgery were randomly received parenteral nutrition with either BCAA enriched (study group, n=20) or routine amino acid (control group, n=20) for seven consecutive days. Nitrogen balance,serum total protein, albumin, prealbumin, transferrin, retinol binding protein and postoperative fatigue score were monitored during the postoperative period. RESULTS: The cumulative postoperative fatigue scores were lower in BCAA group than that in the control group at the 4th, 5th and 7th day after operation (P< 0.05). Patients achieved positive nitrogen balance 2 days earlier in the study group than that in the control group, but there was no significant difference in cumulative nitrogen balance between the two groups. There was no significant difference in elevation of serum total protein, albumin, prealbumin, transferrin at the 7th day after operation between the two groups (P > 0.05), compared with those at the first day after operation. The serum level of retinol binding protein was higher in BCAA-enriched group than that in the control group (P=0.004). CONCLUSION: TPN with BCAA enriched formula can reduce postoperative fatigue score and improve the nutritional status for digestive surgery patients.

[Surgical treatments of preoperative unsuspected gallbladder carcinoma].

OBJECTIVE: To explore the surgical treatment of preoperative unsuspected gallbladder carcinoma (UGC) and its prognosis. METHODS: Eighteen patients of UGC admitted in our hospital from January 1996 to December 2003 were analyzed retrospectively. The clinicopathological characters and surgical treatment of UGC patients were compared with the preoperative diagnosed gallbladder carcinoma (DGC) patients admitted in the same period. The cumulative survival rate of the patients received radical resection in two groups was analyzed by Kaplan-Meier method. RESULTS: The serous layer was not invaded by tumor in 10 patients of UGC group (55.5%, 10/18), while the serous layer invaded and regional lymph node metastasis were detected in 39 patients of DGC group (90.7%, 39/43). Radical resection rate was 72.2% (13/18) in UGC group and 39.5% (17/43) in DGC group, and 5-year survival rates in those with radical resection were 54.6% and 23.5%, respectively (chi(L)(2) = 16.33, P < 0.01). Compared with the patients underwent palliative operation, the patients with radical resection has a longer median survival time (43.3 months vs 10.5 months, chi(L)(2) = 31.10, P < 0.01). CONCLUSIONS: The prognosis of UGC is better than that of DGC generally. Reoperation for UGC should be performed as soon as possible, and the prognosis can be improved by radical resection.

Metastasis of primary gallbladder carcinoma in lymph node and liver.

AIM: To evaluate the patterns with metastasis of gallbladder carcinoma in lymph nodes and liver. METHODS: A total of 45 patients who had radical surgery were selected. The patterns with metastasis of primary gallbladder carcinoma in lymph nodes and liver were examined histopathologically and classified as TNM staging of the American Joint Committee on Cancer. RESULTS: Of the 45 patients, 29 (64.4%) had a lymph node positive disease and 20 (44.4%) had a direct invasion of the liver. The frequency of involvement of lymph nodes was strongly influenced by the depth of the primary tumor (P = 0.0001). The postoperative survival rate of patients with negative lymph node metastasis was significantly higher than that of patients with positive lymph node metastasis (P = 0.004), but the postoperative survival rate of patients with N1 lymph node metastasis was not significantly different from that of patients with N2 lymph node metastasis (P = 0.3874). The postoperative survival rate of patients without hepatic invasion was significantly better than that of patients with hepatic invasion (P = 0.0177). CONCLUSION: Complete resection of the regional lymph nodes is important in advanced primary gallbladder carcinoma (PGC). The initial sites of liver spread are located mostly in segments IV and V. It is necessary to achieve negative surgical margins 2 cm from the tumor. In patients with hepatic hilum invasion, extended right hepatectomy with or without bile duct resection or portal vein resection is necessary for curative resection.

A case report of localized gastric amyloidosis.

AIM: To elucidate the clinical and laboratory features of localized gastric amyloidosis via a rare report along with a review of related literatures. METHODS: The clinical manifestations, laboratory results and surgical treatment of a female patient with localized gastric amyloidosis in our hospital were summarized. The relevant literatures were reviewed on the etiology, clinical features, diagnosis, treatment and prognosis of this disease. RESULTS: The patient was lack of specific clinical manifestations and positive laboratory results. Prior to the treatment, she was suspected to be of malignization from gastric ulcer by both gastroscopy and endoscopic ultrasonography, which was denied by the gastric biopsy. The patient was treated with subtotal gastrectomy and clearance of perigastric lymph nodes. The postoperative pathological diagnosis determined the lesion to be the deposition of amyloid materials in the gastric mucosa, submucosa and blood vessel walls with intestinal metaplasia and atrophy of the gastric glands, in which no malignant tumor was found. Congo red staining with prior potassium permanganate incubation confirmed the AA type of amyloid in this case. Multiple biopsies from esophagus, remnant stomach, duodenum, colon and bone marrow in the follow-up survey showed no amyloidal deposition in these tissues and organs. Up to the present, no signs of recurrence have been found in this patient. CONCLUSION: Localized gastric amyloidosis, being rare in incidence, should be considered in the differentiation of gastric tumors, in which biopsy is the only means to confirm the diagnosis. Currently, surgical resection of pathological tissue and circumambient lymph nodes may be a preferable therapeutic strategy for the localized amyloidosis to prevent possible complications. Although with a benign prognosis, gastric amyloidosis possesses a recurrent tendency as suggested by the literatures.