A case of a vaginal Brenner tumor without a gland mimicking a borderline tumor: unusual morphology and diagnostic pitfalls.

Brenner tumor is a rare neoplasm of the vagina. This tumor is diagnosed according to the criteria of ovarian tumors. We report here a 64-year-old postmenopausal woman with a 2.0-cm sessile vaginal polyp for 9 years. Microscopic examination showed unusual features of no gland appearing in the tumor, but the other two characteristic components of transitional islands and dense fibrous stroma were observed. The tumor was diagnosed as a vaginal Brenner tumor on the basis of the definition proposed by the World Health Organization classification of female reproductive organ tumors. In our case, part of the epithelial nests of the Brenner tumor showed basaloid cell differentiation with peripheral palisading, and irregular papillary hyperplasia was observed around the epithelial nests similar to a borderline tumor. However, no mitotic activity or nuclear atypia was present in either the epithelial or stromal components. The presence of epithelial nests requires attention in the medical history of the patient. Our patient did not have a history of primary urothelial carcinoma. Our patient's benign vaginal Brenner tumor with different morphological characteristics supports the current notion that Walthard nests might act as possible precursor lesions.

Functions and mechanisms of chemokine receptor 7 in tumors of the digestive system.

Chemokine (C-X-C motif) receptor 7 (CXCR7), recently termed ACKR3, belongs to the G protein-coupled cell surface receptor family, binds to stromal cell-derived factor-1 [SDF-1, or chemokine (C-X-C motif) ligand 12] or chemokine (C-X-C motif) ligand 11, and is the most common chemokine receptor expressed in a variety of cancer cells. SDF-1 binds to its receptor chemokine (C-X-C motif) receptor 4 (CXCR4) and regulates cell proliferation, survival, angiogenesis and migration. In recent years, another new receptor for SDF-1, CXCR7, has been discovered, and CXCR7 has also been found to be expressed in a variety of tumor cells and tumor-related vascular endothelial cells. Many studies have shown that CXCR7 can promote the growth and metastasis of a variety of malignant tumor cells. Unlike CXCR4, CXCR7 exhibits a slight modification in the DRYLAIV motif and does not induce intracellular Ca2+ release following ligand binding, which is essential for recruiting and activating G proteins. CXCR7 is generally thought to work in three ways: (1) Recruiting ß-arrestin 2; (2) Heterodimerizing with CXCR4; and (3) Acting as a "scavenger" of SDF-1, thus lowering the level of SDF-1 to weaken the activity of CXCR4. In the present review, the expression and role of CXCR7, as well as its prognosis in cancers of the digestive system, were investigated.

Primary hepatic neuroendocrine carcinoma coexisting with distal cholangiocarcinoma: A case report and review of the literature.

INTRODUCTION: Although primary hepatic neuroendocrine carcinomas, whose prognostic mechanisms remain unclear, are rare, coexistence of neuroendocrine carcinomas and other tumors is rarer. In this report, we describe a unique case of coexistence between primary hepatic neuroendocrine carcinoma and a distal cholangiocarcinoma in the pancreas. PATIENT CONCERNS: A 64-year-old woman with a history of diabetes, but none of hepatitis, was admitted to hospital because of intermittent epigastric distension and pain discomfort for more than 1 month aggravated 1 day. A contrast-enhanced computed tomography (CT) scan of the upper abdomen and abdominal magnetic resonance imaging (MRI) revealed a thickening of the bile duct wall in the middle and lower segment of common bile duct and the corresponding lumen is narrow and low-density tumors with ring enhancement (1.83 cm × 1.9 cm) in lobi hepatis dexte. DIAGNOSIS: Primary neuroendocrine carcinoma of the liver was diagnosed to be coexisting with a distal cholangiocarcinoma, which had invaded the pancreas. Immunohistochemical examination revealed that the neoplastic cells strongly expressed chromogranin A, synaptophysin, and CD56 proteins. The tumor cells did not express HepPar-1, glypican-3, S-100, CK7, and CK19 in the liver tumor. A distal bile duct in pancreatic tissues shows the characteristics of typical bile duct carcinoma, as an invasion of carcinoma is also seen in the pancreatic tissues. Gastrointestinal endoscopy, chest and abdominal CT, abdominal MRI, and positron emission tomography (PET)-CT were used to exclude metastatic neuroendocrine tumors of the liver. INTERVENTIONS: Resection of the pancreas-duodenum, the right anterior lobe of the liver, and regional lymph nodes was performed in patients. OUTCOMES: The patient had survived for 5 months after the operation. CONCLUSION: A unique case of a coexistence of primary hepatic neuroendocrine carcinoma and a distal cholangiocarcinoma, which had invaded the pancreas. No treatment guidelines are established for the treatment of the unique case.

Dual Role of Hepatic Macrophages in the Establishment of the Echinococcus multilocularis Metacestode in Mice.

Echinococcus multilocularis larvae, predominantly located in the liver, cause a tumor-like parasitic disease, alveolar echinococcosis (AE), that is characterized by increased infiltration of various immune cells, including macrophages, around the lesion that produces an "immunosuppressive" microenvironment, favoring its persistent infection. However, the role of hepatic macrophages in the host defense against E. multilocularis infection remains poorly defined. Using human liver tissues from patients with AE and a hepatic experimental mouse model of E. multilocularis, we investigated the phenotype and function of hepatic macrophages during the parasite infection. In the present study, we found that a large number of CD68+ macrophages accumulated around the metacestode lesion in the liver of human AE samples and that both S100A9+ proinflammatory (M1 phenotype) and CD163+ anti-inflammatory (M2 phenotype) macrophages were significantly higher in close liver tissue (CLT) than in distant liver tissue (DLT), whereas M2 macrophages represent the dominant macrophage population. Furthermore, E. multilocularis-infected mice exhibited a massive increase in macrophage (F4/80+) infiltration in the liver as early as day 5, and the infiltrated macrophages were mainly monocyte-derived macrophages (CD11bhi F4/80int MoMFs) that preferentially differentiated into the M1 phenotype (iNOS+) at the early stage of E. multilocularis infection and then polarized to anti-inflammatory macrophages of the M2 phenotype (CD206+) at the chronic stage of infection. We further showed that elimination of macrophages by treatment of mice with clodronate-liposomes before E. multilocularis infection impaired worm expulsion and was accompanied by a reduction in liver fibrosis, yielding a high parasite burden. These results suggest that hepatic macrophages may play a dual role in the establishment and development of E. multilocularis metacestodes in which early larvae clearance is promoted by M1 macrophages while persistent metacestode infection is favored by M2 macrophages.

Thioredoxin peroxidase secreted by Echinococcus granulosus (sensu stricto) promotes the alternative activation of macrophages via PI3K/AKT/mTOR pathway.

BACKGROUND: Larvae of Echinococcus granulosus (sensu lato) dwell in host organs for a long time but elicit only a mild inflammatory response, which indicates that the resolution of host inflammation is necessary for parasite survival. The recruitment of alternatively activated macrophages (AAMs) has been observed in a variety of helminth infections, and emerging evidence indicates that AAMs are critical for the resolution of inflammation. However, whether AAMs can be induced by E. granulosus (s.l.) infection or thioredoxin peroxidase (TPx), one of the important molecules secreted by the parasite, remains unclear. METHODS: The activation status of peritoneal macrophages (PMs) derived from mice infected with E. granulosus (sensu stricto) was analyzed by evaluating the expression of phenotypic markers. PMs were then treated in vivo and in vitro with recombinant EgTPx (rEgTPx) and its variant (rvEgTPx) in combination with parasite excretory-secretory (ES) products, and the resulting activation of the PMs was evaluated by flow cytometry and real-time PCR. The phosphorylation levels of various molecules in the PI3K/AKT/mTOR pathway after parasite infection and antigen stimulation were also detected. RESULTS: The expression of AAM-related genes in PMs was preferentially induced after E. granulosus (s.s.) infection, and phenotypic differences in cell morphology were detected between PMs isolated from E. granulosus (s.s.)-infected mice and control mice. The administration of parasite ES products or rEgTPx induced the recruitment of AAMs to the peritoneum and a notable skewing of the ratio of PM subsets, and these effects are consistent with those obtained after E. granulosus (s.s.) infection. ES products or rEgTPx also induced PMs toward an AAM phenotype in vitro. Interestingly, this immunomodulatory property of rEgTPx was dependent on its antioxidant activity. In addition, the PI3K/AKT/mTOR pathway was activated after parasite infection and antigen stimulation, and the activation of this pathway was suppressed by pre-treatment with an AKT/mTOR inhibitor. CONCLUSIONS: This study demonstrates that E. granulosus (s.s.) infection and ES products, including EgTPx, can induce PM recruitment and alternative activation, at least in part, via the PI3K/AKT/mTOR pathway. These results suggest that EgTPx-induced AAMs might play a key role in the resolution of inflammation and thereby favour the establishment of hydatid cysts in the host.

Evaluation of the diagnostic value of the immunoblotting and ELISA tests using recombinant Em18 antigen in human alveolar echinococcosis from Xingjiang China.

Alveolar echinococcosis (AE) is a prevalent epidemic in the northern hemisphere, especially in central Europe and western China. Serum diagnosis is important for patients with AE, especially during the first screening. The present study purified the recombinant Em18-GST (rEm18-GST), and detected its diagnostic performance in human alveolar echinococcosis patients of Xinjiang, China with immunoblotting (IB) and enzyme-linked immunosorbent assay (ELISA). Serum samples were collected from 50 patients with AE, 222 patients with cystic echinococcosis (CE), 158 patients with other unrelated infections and 106 healthy individuals. The IB results showed that serum samples of 47 patients with AE and 12 patients with CE were rEm18-positive. However, only one sample from patients with cancer showed a cross-reaction with rEm18 in IB. The overall sensitivity was 94%, and the total specificity was 96.58%. For the rEm18 results using ELISA, the sera of 46 patients with AE were positive, and the overall sensitivity was 92%. In conclusion, compared with imaging tools, including computed tomography, magnetic resonance imaging and positron emission tomography, rEm18 has considerable advantages for AE serodiagnosis.

CXCR7/CXCL12 axis is involved in lymph node and liver metastasis of gastric carcinoma.

AIM: To investigate the role of CXC chemokine receptor (CXCR)-7 and CXCL12 in lymph node and liver metastasis of gastric carcinoma. METHODS: In 160 cases of gastric cancer, the expression of CXCR7 and CXCL12 in tumor and matched tumor-adjacent non-cancer tissues, in the lymph nodes around the stomach and in the liver was detected using immunohistochemistry to analyze the relationship between CXCR7/CXCL12 expression and clinicopathological features and to determine whether CXCR7 and CXCL12 constitute a biological axis to promote lymph node and liver metastasis of gastric cancer. Furthermore, the CXCR7 gene was silenced and overexpressed in human gastric cancer SGC-7901 cells, and cell proliferation, migration and invasiveness were measured by the MTT, wound healing and Transwell assays, respectively. RESULTS: CXCR7 expression was up-regulated in gastric cancer tissues (P = 0.011). CXCR7/CXCL12 expression was significantly related to high tumor stage and lymph node (r = 0.338, P = 0.000) and liver metastasis (r = 0.629, P = 0.000). The expression of CXCL12 in lymph node and liver metastasis was higher than that in primary gastric cancer tissues (χ2 = 6.669, P = 0.010; χ2 = 25379, P = 0.000), and the expression of CXCL12 in lymph node and liver metastasis of gastric cancer was consistent with the positive expression of CXCR7 in primary gastric cancer (r = 0.338, P = 0.000; r = 0.629, P = 0.000). Overexpression of the CXCR7 gene promoted cell proliferation, migration and invasion. Silencing of the CXCR7 gene suppressed SGC-7901 cell proliferation, migration and invasion. Human gastric cancer cell lines expressed CXCR7 and showed vigorous proliferation and migratory responses to CXCL12. CONCLUSION: The CXCR7/CXCL12 axis is involved in lymph node and liver metastasis of gastric cancer. CXCR7 is considered a potential therapeutic target for the treatment of gastric cancer.

Neuroendocrine Neoplasms of the Major Duodenal Papilla With Focus on Histopathological Features and Prognosis.

Neuroendocrine neoplasms (NENs) are generally indolent and progress slowly. However, NENs of major duodenal papilla are uncommon. We retrospectively assessed relevant clinicopathological findings in 9 consecutive patients treated for major duodenal papilla NENs by pancreaticoduodenectomy in our hospital from 2009 to 2013. Eight of the 9 patients (89%) presented with painless obstructive jaundice and one with intermittent fever, attributable to pancreatitis, without jaundice. The diagnostic accuracy was 75% (6/8) for biopsies obtained under duodenoscope guidance. Enhanced multidetector computed tomography detected 89% (8/9) of tumors. Patients with uncertain preoperative diagnoses all underwent intraoperative frozen section pathological diagnosis. Tumor cells extended to at least the muscularis propria in all patients. There were 5 neuroendocrine tumors, 2 neuroendocrine carcinomas, and 2 mixed adenoneuroendocrine carcinomas. Two, 4, and 3 cases were grades 1, 2, and 3, respectively. Grade 3 tumor patients had poor prognoses with tumor recurrence or metastasis within 2 months and all died within 1 year. The overall survival rate (1 year) of grade 3 was lower than in grades 1 and 2 (P < .05). Patients with grade 1 tumors had a similar prognosis to grade 2 (P > .05). To date, only 4 cases of this tumor have been reported in the Chinese literature. The prognosis can be predicted accurately by histopathological features accordingly to the World Health Organization 2010 classification. Multiple imaging techniques and pathological examination should be carried out appropriately to diagnose the disease early.

Perinodular ductular reaction/epithelial cell adhesion molecule loss in small hepatic nodules.

AIM: To investigate if loss of epithelial cell adhesion molecule (EpCAM) is associated with microinvasion in hepatocellular carcinomas (HCCs) in the presence of chronic hepatitis B. METHODS: The expression of EpCAM, cytokeratin 7 (CK7) and CK19 in 112 hepatic nodules was studied, including 20 HCCs with nodules ≤ 3 cm, 26 HCCs with nodules > 3 cm, 20 high-grade dysplastic nodules, 26 cirrhotic, large regenerative nodules and 20 cases of cirrhosis. RESULTS: Membranes of ductular reaction (DR) hepatobiliary cells, interlobular bile duct and some hepatic cells were positive for EpCAM expression. Active expression of DR/EpCAM was observed in the majority of noninvasive nodules (50/66, 75.76%); however, expression was absent in the major area of invasion in HCCs (42/46, 91.30%). DR/EpCAM loss in HCCs ≤ 3 cm was higher than in high-grade dysplastic nodules (HGDNs) (P < 0.05), cirrhotic, large regenerative nodules and cirrhosis (P < 0.01). Furthermore, patients (20 HCCs ≤ 3 cm, 26 HCCs > 3 cm, 20 HGDNs) with DR/EpCAM expression had a higher overall survival rate (P < 0.01) and lower early recurrence rate (P < 0.01). DR/EpCAM expression showed a close relationship with DR/CK7 and DR/CK19 expression (P < 0.01). The area under the receiver operating characteristic (ROC) curve of DR/EpCAM was similar to that of DR/CK7 and DR/CK19 (P > 0.05). The diagnostic specificity and diagnostic accuracy were both increased when DR/EpCAM, DR/CK7 and DR/CK19 were combined (P < 0.01). CONCLUSION: DR/EpCAM loss may be a useful marker for determining microinvasion in HCCs ≤ 3 cm, but also for predicting prognosis.

[Echinococcus multilocularis infection leads to increased expression of proliferation and anti-apoptosis factors in liver cells in BALB/c mice].

OBJECTIVE: To investigate the effects of Echinococcus multilocularis on host liver cell proliferation in vivo using a BALB/c mouse alveolar hydatid infection model. METHODS: Sixty-five 8-10-week-old female BALB/c mice were randomly divided into an experimental group (n = 40) and a control group (n = 25) and administered an abdominal injection into the left liver lobe of E. multilocularis protoscolices in saline solution or saline solution alone, respectively. At post-injection day 2, 8, 30, 60, and 90, liver samples were collected for analysis of lesions and lesion-adjacent tissue by hematoxylin-eosin staining and differential expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin A, and cyclin B1 by immunohistochemical staining. The significance of intergroup differences was assessed by Student's t-test. RESULTS: The control group showed normal liver histology at all time points. The experimental group developed E. multilocularis lesions that showed increased severity of pathological features, such as inflammatory cell invasion, steatosis and fibrous connective tissue hyperplasia, over time. At post-injection days 2 and 8, enlarged, binuclear and apocyte hepatocytes were observed close to the lesions. At post-injection days 30, 60, and 90, the number of hepatocytes expressing PCNA progressively increased in the experimental group, and the numbers were significantly higher than in the control group (7.01 +/- 1.89 vs. 1.03 +/- 0.52, 8.41 +/- 2.80 vs. 0.93 +/- 0.31, and 13.4 +/- 4.43 vs. 1.07 +/- 0.94; all P < 0.05). The same progressively increasing trend was seen in the number of hepatocytes expressing CyclinD1, but was only significantly different from controls at post-injection days 30 and 60 (6.73 +/- 2.52 vs. 0.48 +/- 0.43 and 8.22 +/- 3.09 vs. 0.55 +/- 0.34; both P < 0.05). In contrast, the number of hepatocytes expressing cyclin A was significantly increased at post-injection day 30 and then showed a decreasing trend at days 60 and 90, although the numbers of expressing cells remained significantly higher than control levels at all time points (7.75 +/- 3.05 vs. 0.69 +/- 0.36, 3.42 +/- 1.80 vs. 1.14 +/- 0.42, and 3.03 +/- 1.50 vs. 0.69 +/- 0.31; all P < 0.05). The number of hepatocytes expressing CyclinB1 in the experimental group was less robust than the other cyclins (with a general temporal trend of increase followed by decrease), but the differential expression was not significantly different from the control levels at any time point. CONCLUSION: E. multilocularis infection may promote the expression of host factors related to proliferation and anti-apoptosis in liver. This pathogen-mediated modulation of host cell-survival mechanisms may provide a rationale explanation for the clinical observations of hepatomegaly and the unexpected survival of alveolar echinococcosis patients following major hepatic resection.

[Diagnostic value of absent ductular reaction at hepatocellular-stromal boundaries in early stage hepatocellular carcinoma].

OBJECTIVE: To investigate the role of absent ductular reaction (DR) at hepatocellular-stromal boundaries in early stage hepatocellular carcinoma (HCC) with cirrhosis in patients with chronic hepatitis B. METHODS: Cytokeratin (CK)7 and CK19 expression was detected by the SP immunohistochemistry method in 112 hepatic nodules taken from 20 cases of early HCC, 26 cases of HCC with nodules more than 3 cm, 20 cases of high-grade dysplastic nodule (HGDN), 26 cases of low-grade dysplastic nodule (LGDN), and 20 cases of cirrhosis (CIR). DR/CK7 and DR/CK19 were assessed separately on a semi-quantitative scale and statistically analyzed. RESULTS: The mean age of the patients in the study was 53.71 years-old, and the study population consisted of 73 males and 39 females. The follow-up time ranged from 3 to 90 months. Positive CK7 and CK19 staining was detected in the cytoplasm of DR-positive hepatobiliary cells, interlobular bile duct, and a portion of hepatic cells. All of the DR/CK7- and DR/CK19-positive cells were localized around the non-invasive nodules. Specimens with focal or diffuse DR/CK7- and DR/CK19-loss had more robust stromal invasion. Specimens from early HCC cases showed greater DR/CK19 loss than specimens from HGDN cases, LGDN cases and CIR cases (all P less than 0.01). DR/CK7 loss of early HCC was less than HCC with nodules more than 3 cm (P less than 0.05), and more than LGDN cases and CIR cases (both P less than 0.01).The area under the receiver operating characteristic curve of DR/CK7 was very similar to that of DR/CK19 (P more than 0.05). Pearson's correlation analysis indicated that DR/CK7 and DR/CK19 were positively correlated with tumor-free time (P less than 0.01) and negatively correlated with early recurrence time as well as death rate (both P less than 0.01). Furthermore, cases showing DR/CK7 or DR/CK19 loss had lower overall survival rate and tumor-free survival rate (P less than 0.01) and higher early recurrence rate (P less than 0.01). CONCLUSION: DR/CK7 and DR/CK19 immunostaining may help to distinguish non-invasive HGDNs from both minimally-invasive and overtly-invasive HCCs by identifying small foci of invasion and predicting increased risk of invasiveness.

The status of phosphorylated p38 in esophageal squamous cell carcinoma.

The p38 mitogen-activated protein kinase (MAPK) is a member of the MAPK family, which is initially found to be activated by stress stimuli, proinflammatory cytokines, and growth factors. However, its role in the pathogenesis of esophageal squamous cell carcinoma (ESCC) is largely unkown, so we investigate the role of phosphorylated p38 (p-p38) MAPK in ESCC. First of all, in vitro cell line ECa109, SB203580 as selective inhibitor of p38, can suppress the growth of esophageal cancer cell in a dose- and time-dependent way, suggesting that ECa109 cell growth and proliferation was closely associated with p-p38. Using western-blot analysis of fresh 16 paired surgically resected ESCC and matched non-tumor adjacent tissues (NAT), we showed that p-p38 was significantly expressed higher in NAT compared to ESCC. Moreover, expressions of p-p38 were further confirmed by 162 paired of formalin-fixed paraffin-embedded (FFPE) ESCC and NAT by immunohistochemistry, the same trend result was obtained through statistical analysis that there was increased expression of p-p38 in NAT as compared with ESCC (P < 0.01), and expression of p-p38 was not significantly associated with lymph nodes metastasis (P > 0.05) and ESCC differentiation degree (P > 0.05). Taken together, all the results we obtained demonstrated that p-p38 plays a key role in the malignant transformation of ESCC.

[Effect of Echinococcus granulosus cyst fluid on the proliferation of HepG2 cells].

OBJECTIVE: To investigate whether Echinococcus granulosus cyst fluid-infected host liver cells had differential expression of mitogen-activated protein kinases (MAPKs) or differential cell cycle activity. METHODS: Human liver cells cultured with different concentrations of hydatid cyst fluid (HCF) were tested by the MTT method to determine effects on proliferation. The cell cycle was assessed by flow cytometry. Western blotting was used to detect changes in protein expressions of p-ERK, PCNA, cyclin-A, cyclin-B1, cyclin-D1, and cyclin-E. RESULTS: Forty-eight, 72 and 96 h of HCF at 15%, 30% and 60% concentrations in the cell media significantly promoted cell proliferation (F=67.845, P less than 0.01) and compared to controls (P less than 0.05). Cells exposed to 15% HCF for 48 h showed significantly induced expression of p-ERK (F=1.916, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 15% HCF for 24 h showed significantly induced expression of cyclin-Dl (F=3.901, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 15% HCF for 48 h or 30% HCF for 72 h showed significantly induced expression of PCNA (F=91.140, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 15% HCF for 48 h or 30% HCF for 72 h shed significantly induced expression of cyclin-A (F=18.587, P=0.002), higher than controls (P less than 0.01). Cells exposed to 15% HCF for either 48 h or 72 h showed significantly induced expression of cyclin-B1 (F=2.064, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 30% HCF for 96 h showed significantly induced expression of cyclin-E (F=1.068, P less than 0.01), higher than controls (P less than 0.01). CONCLUSION: Hydatid cyst fluid exerts no inhibitory effect on primary cultured host liver cells, but may promote cellular proliferation.

Molecular characterization of a signal-regulated kinase homolog from Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs of the host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage. Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK. METHODS: DNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence. RESULTS: We cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices. CONCLUSIONS: We have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and protoscolex. These findings will be useful in further study of the biological functions of the gene in the growth and development of Echinococcus and will contribute to research on novel anti-echinococcosis drug targets.

[Echinococcus granulosus recombinant antigen B induced IDO expression in mouse bone marrow-derived dendritic cells in vitro].

OBJECTIVE: To observe the expression of indoleamine 2,3-dioxygenase (IDO) in mouse bone marrow-derived dendritic cells (DCs) after adding Echinococcus granulosus recombinant antigen B (rAgB) in vitro. METHODS: CD11c+ DCs generated from bone marrow precursor cells of C57BL/6 mice and cultured in the presence of recombinant mouse GM-CSF (rmGM-CSF). The morphology of DCs was observed by inverted microscope and scanning electronic microscope. The level of I-A/I-E, CD40, CD80, and CD86 on DCs were determined by flow cytometry. T cell proliferation induced by DCs were evaluated by using mixed lymphocyte reaction (MLR) assay. At day 6 post culture, the immature DCs were collected, and part of the immature DCs stimulated with lipopolysaccharide (LPS) for 24 h were examined by flow cytometry. Immature DCs were divided into 3 groups: negative control group, positive control group (rmIFN-gamma, 1000 U/ml) and rAgB group. Immature DCs of positive control group and rAgB group were induced with 1000 U/ml rmIFN-gamma and 15 microg/ml rAgB, respectively. IDO expression in DCs was examined 24 h after induction using immunohistochemical method and Western blotting. RESULTS: More than 80% CD11c+ DCs were harvested. The typical DCs were observed under inverted microscope and scanning electronic microscope. The level of CD40, CD80, and IA/IE (MHC II) in mature DCs group was significantly higher than that of immature DCs group (P < 0.05). In MLR, mitomycin-treated DCs can stimulate T lymphocytes proliferation activity. There were significantly differences in IDO expression in the negative control group [(4.544 +/- 1.752)%], positive control group [(20.464 +/- 4.452)%] and rAgB group [(11.148 +/- 1.966)%] (P < 0.05). Western blotting result indicated that the ratio of IDO/GAPDH in rAgB group (0.573 +/- 0.129) was significantly higher than that of negative group (0.229 +/- 0.085) (P < 0.05), and there were no significant difference in the ratio of IDO/GAPDH between IFN-gamma group (0.794 +/- 0.114) and rAgB group (P > 0.05). CONCLUSION: rAgB can induce IDO expression in bone marrow-derived dendritic cells in vitro.

[The relationship between abnormity of synaptonemal complex and male fertility impairment in human].

It was said that 11% of all men showed their infertility. The genetic origins of male infertility may be classified into three main groups: chromosome abnormalities, microdeletions and gene mutations. Growing literature has shown that spermatogenesis failure or reproductive failure (pregnancy wastages) occurred in male carriers of chromosomal distortion/aberration. But the mechanisms remain largely unsolved. Synaptonemal complex (SC) of human spermatocytes from such carriers offers new information. This review aims to summarize recent development of SC analysis for male infertile diagnosis and sum up our results obtained recently. The relationship between male infertility/sterility and SC abnormity was discussed and reviewed as following five aspects. (1) The association of XY-bivalent and the rearranged autosomes interfere with or affect, by their contact, X chromosome normal functions. (2) Male infertility is related to the incomplete pairing in break regions of rearranged autosomes. (3) SC fragmentation, lateral elements (LEs) swelling and pairing disorder result in spermatogenic failure. (4) This heterosynapsis at early stage of meiosis in rearranged autosomes, results in unbalanced germs and pregnancy wastages. (5) Gene mutations of SC proteins result in male infertility.

[Analysis of synaptonemal complex from a carrier with 46,XY,t(11;18) balanced translocation].

Over ten years ago, two male patients of pregnancy wastage, who were the carriers of 4;6 and 4;13 reciprocal translocations, respectively, were analyzed with synaptonemal complexes (SCs) by de Perdigo et al. They suggested that the pregnancy wastage should be caused by heterosynapsis, which reciprocal translocations resulted in. The heterosynapsis might avoid spermatogenesis failure, but easily induced non-disjunction of some chromosomes and led to occurrence of unbalanced gametes. A new male patient of the pregnancy wastage was detected in China three years ago. Karyotyping of the patient showed 11;18 reciprocal translocation. Analysis of synaptonemal complexes (SCs) of the patient was performed using the EM technology of SC surface spreading, SDS treatment, AgNO3 staining. The SCs of 30 spermatocytes at pachytenes were analyzed. The SCs in electron micrographs were measured by the method published by de Perdigo et al. (1991). The results showed that each of the observed spermatocytes displayed 20 autosomal bivalents, a quadrivalent 11;18 and a sexual bivalent. Of the 30 spermatocytes, 21 each exhibited a quadrivalent of adequate quality for measurement and interpretation. The 21 quadrivalents were classified into three types: type I, quadrivalents with fully paired arms with the expected cross-configuration, two quadrivalents were of this type. Type II, quadrivalents with an evident heterosynapsis, forming five SC segments (four fully pairing arms and one middle pairing region), six quadrivalents were of this type. Type III, quadrivalents with four paired arms, but with asynapsed regions around the breakpoints, thirteen quadrivalents were of this type. Based on configuration, quadrivalents were classified into cis-configuration with above three types and trans-configuration with above two types except type I. Each of the analyzed quadrivalents showed four synaptic arms, which respectively paired between chromosome 11 and t18 (the translocated 18;11), 18 and t18, 18 and t11 (the translocated 11;18), and 11 and t11, and the arms were designated as A, B, C, and D correspondingly. The measurements of A and D as well as B and C observed in different cells were taken by calculating the percentages of their lengths in comparison to the length of chromosome 11 and 18, respectively. So were the middle synaptic regions. Measurements of SCs revealed that the location of the putative breakpoints estimated from the fully paired quadrivalents were different from those determined by G-band analysis for mitotic chromosomes. The length of the paired and unpaired segments varied from one quadrivalent to another. For instance, in the case of fully paired A and D arms, the breakpoint was located near to 45.59% and 38.53% of the length of the normal chromosome 11, the breakpoint from the mitotic chromosome was estimated to be near to 45%. The location of the putative breakpoints estimated from the No. 1 quadrivalent was consistent with the breakpoint from the mitotic chromosome. This suggested that only this quadrivalent showed complete homologous pairing. Obviously, the lengths of middle pairing region in type II quadrivalent were different from one quadrivalent to another. The middle pairing region/the length of the normal chromosome 11 is 4.11% - 18%, that/the length of the normal chromosome 18 is 8% - 41.1%. The middle pairing regions should be the heterologous pairing regions in quadrivalents. The quadrivalents with unsynaptic regions also showed partially heterologous synapsis. In 14 of 21 quadrivalents, the paired regions overlapped the mitotic breakpoint position in chromosome 11. In 20 of 21 quadrivalents, the paired regions overlapped the chromosome 18 of mitotic breakpoint position in one or two arms. As synapsis can proceed homologously only up to the breakpoints, a heterosynapsis apparently occurred in 20 quadrivalents. The length of the paired and unpaired segments varied from one quadrivalent to another, and the measurements of the arms of the quadrivalents could not be used to determine breakpoints. The unpaired segment showed a thick and sometimes a split aspect similar to that of the sexual bivalent. Of the 20 quadrivalents with heterosynapsis, four were at early pachytene stages, fourteen at the middle and two at the late, respectively. The heterosynapsis, which occurred on the early pachytene, was without previous homosynapsis. This was different from the classical "synaptic adjustment" during the late pachrtene of spermatocytes. Many researches on reciprocal translocation associated with pregnancy wastage have been reported. It is most possible that the wastage detected in this study was related to the t(11;18). Normal synapsis finishes at early pachytene, while in the observed proband, most of the quadrivalents still left unsynaptic parts during middle and late pachytene stages. Obviously, the synapsis was delayed or incomplete. The unsynaptic regions distributed mainly in the central regions. The central regions perhaps completed the synapsis more late, therefore they had more chances to leave the unsynaptic parts. In addition, the results of SC analysis showed that heterosynapsis occurred wildly and was exhibited from early to late pachytene stages. It has been reported that crossing over could be inhibited by heterosynapsis in mice, man and boar, and normal segregation needs existence of crossing over. Therefore, heterosynapsis may interfere in normal segregation. Both unsynapsis and heterosynapsis could induce incidence of unbalanced gametes. In general, the unbalanced gametes are lethal and could not take part in fertilization, once they fertilize with normal eggs, the risk of pregnancy wastage or other genetic disorders will exist certainly. Our results showed that the heterosynapsis occurred widely, so a poor risk of pregnancy wastages caused by reciprocal translocation probably is not unusual.

Primary targeting of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha against hepatocellular carcinoma.

AIM: To obtain human recombinant Fv-immunotoxin hscFv(25)-mTNFalpha (mutant human TNFalpha fused to human scFv(25)) against hepatocellular carcinoma (HCC). METHODS: Two relevant sites of enzymatic digestion were added to mTNFalpha by PCR. MTNFalpha was linked to the 3' end of hscFv(25) in pGEX4T-1 vector. This anti-HCC recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was expressed in Escherichia coli and purified from inclusions. After purified by glutathione-S-transferase affinity chromatography and thrombin digestion, it was identified by electrophoresis and Western blot. And then, the purified recombinant Fv-immunotoxin was injected into nude mice with HCC xenografts through their tail veins. MTNFalpha protein and PBS were used as control at the same time. After treated for two weeks, nude mice were executed. The bulk and weight of tumors were observed. The tumor tissues were stained by immunohistochemical method with TNFalpha antibody. RESULTS: The expression ratio of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was 12% of bacterial protein. The result of tumor restraining trials of hscFv(25)-mTNFalpha showed 2/5 complete remission and 3/5 partial remission. mTNFalpha restraining trials showed 5/5 partial remission. The therapeutic result of hscFv(25)-mTNFalpha was better than that of mTNFalpha (F=8.70, P<0.05). The hscFv(25)-mTNFalpha remedial tumor tissues were positive for TNFalpha by immunohistochemical staining. The positive granules mainly existed in the cytoplasm of tumor cell. CONCLUSION: Recombinant Fv-immunotoxin hscFv(25)-mTNFalpha has better therapeutic effect than mTNFalpha. It can inhibit the cellular growth of HCC and has some potential of clinical application.

Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus.

AIM: Quasispecies of hepatitis C virus (HCV) are the foundation for rapid sequence evolution of HCV to evade immune surveillance of hosts. The consensus sequence evolution of a segment of HCV NS3 region, which encompasses putative cytotoxic T cell epitopes, was evaluated. METHODS: Three male patients, infected with HCV through multiple transfusions, were identified from clinical symptoms and monitored by aminotransferase for 60 months. Blood samples taken at months 0, 32, and 60 were used for viral RNA extraction. A segment of HCV NS3 region was amplified from the RNA extraction by RT-PCR and subjected to subcloning and sequencing. HLA types of these three patients were determined using complement-dependent microlymphocytotoxic assay. CTL epitopes were predicted using MHC binding motifs. RESULTS: No patient had clinical symptoms or elevation of aspartate/alanine aminotransferase. Two patients showed positive HCV PCR results at all 3 time points. The other one showed a positive HCV PCR result only at month 0. A reported HLA-A2-restricted CTL epitope had no alteration in the HLA-A2-negative carrier over 60 months. In the HLA-A2-positive individuals, all the sequences from 0 month 0 showed an amber mutation on the initial codon of the epitope. Most changes of consensus sequences in the same patient occurred on predicted cytotoxic T cell epitopes. CONCLUSION: Amber mutation and changes of consensus sequence in HCV NS3 region may be related to viral immune escape.