Chunmei Cheng

Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis

CD4+CD25high regulatory T cells (Tregs) play a critical role in self‐tolerance, as seen in murine autoimmunity. Studies on Tregs in human autoimmunity have focused primarily on peripheral blood samples. A study targeting diseased tissue should identify direct relationships between Tregs and autoimmunity. Peripheral blood samples were collected from 91 patients with primary biliary cirrhosis (PBC), 28 immediate relatives, and 41 healthy controls, and Treg frequencies were determined as a percentage of CD4+CD25high T cells in CD4+TCR‐αβ+ T cells. A tissue‐targeted determination of frequency and distribution of FoxP3+ Tregs was also performed on 90 different liver tissue specimens exhibiting PBC (n = 52), chronic hepatitis C (CHC) (n = 30), and autoimmune hepatitis (AIH) (n = 8). Treg suppression studies were performed on 50 PBC patients and 27 controls. Patients with PBC demonstrated a relative reduction of Tregs compared with controls (P < .0002). Interestingly, a deficiency in CD4+CD25+ Tregs was also found in the daughters and sisters of PBC patients compared with controls (P < .0007). However, functional studies did not reveal a global PBC Treg defect. The level of FoxP3‐expressing Tregs was markedly lower in affected PBC portal tracts compared with CHC and AIH (P < .001). In addition, the CD8+T cell/FoxP3+ Treg ratio was significantly higher in livers of late‐stage PBC compared with those of CHC (P < .001) and early‐stage AIH (P < .001). In conclusion, these data provide support for a genetic modulation of Treg frequency and illustrate the role Tregs play in the loss of tolerance in PBC. (HEPATOLOGY 2006;43:729–737.)

Survivin expression in tumor cell nuclei is predictive of a favorable prognosis in gastric cancer patients

One hundred and thirty three gastric cancer cases were investigated immunohistochemically to clarify the biological role of survivin in gastric cancer cells using a commercially available anti-survivin antibody (SURV11A). Five gastric cancer cell lines were employed to assess localization of survivin by reverse-transcription-polymerase chain reaction (RT-PCR) Southern blotting, Western blotting and immunofluorescence, signals being found in both nucleus and cytoplasm. Survivin nuclear staining of gastric cancer cells was evident in 109 of 133 cases (82.0%) and associated with a favorable prognosis, being an independent prognosticator on multivariate analysis. Survivin nuclear positivity also correlated with younger age and lower incidence of vessel cancer invasion. In contrast, survivin cytoplasmic positivity was noted in 117 cases (88.0%) and did not correlate with any factor of progression or prognosis. The results indicate that survivin is present in the majority of gastric cancer cells but a nuclear localization may play an important physiological role in hindering tumor progression.

NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti–PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9–10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.

Expression profiling of endogenous secretory receptor for advanced glycation end products in human organs

The receptor for advanced glycation end products (RAGE) is a cell surface multiligand receptor of the immunoglobulin superfamily, which participates in physiological and pathological processes such as neuronal development, diabetes, inflammation, neurodegenerative disorders, and cancer. A novel splice variant of RAGE-endogenous secretory decoy form (esRAGE) was recently identified and is thought to be a prospective candidate to modify these RAGE-associated conditions. Here, we investigated the expression and distribution of esRAGE and RAGE proteins with domain-specific antibodies. We studied a wide variety of adult normal human preparations obtained from surgical and autopsy specimens using a tissue microarray technique. The results revealed that esRAGE was widely distributed and we classified its expression into four patterns. In pattern A, the cytoplasm is stained diffusely in neurons, vascular endothelium, pneumocytes, mesothelium, pancreatic β cells, and macrophages/monocytes. In pattern B, dot-like granules are stained in the supranuclear regions facing the luminal surface of the bile ducts, salivary glands, digestive tracts, renal tubules, prostate, skin, thyroid, and bronchioles. Pattern C is represented by diffuse staining in the stromal area of the arterial walls. Pattern D shows diffuse and strong staining of secreted materials such as thyroidal colloid, crystals in renal tubular lumen, and glandular lumen in prostate. This study provides, for the first time, a histopathological basis for understanding the physiological roles of esRAGE in humans, and will contribute to elucidating the participation of esRAGE in pathological processes and to exploring novel diagnostic and therapeutic concepts.

Cholesterol-fed rabbit as a unique model of nonalcoholic, nonobese, non-insulin-resistant fatty liver disease with characteristic fibrosis

BackgroundThe number of patients suffering from metabolic syndrome is increasing rapidly. Metabolic syndrome causes severe pathological changes in various organs, including the liver, and its main phenotype is nonalcoholic fatty liver disease (NAFLD). NAFLD has a broad spectrum ranging from simple fatty change to severe steatohepatitis with marked fibrosis. Recently, several experimental animal models for NAFLD have been proposed. However, most were established by rather artificial conditions such as genetic alteration. In the present study, we tried to establish a unique animal model mimicking some of the physiopathological features of NAFLD using high-cholesterol-fed rabbits.MethodsMale rabbits fed with standard rabbit food containing 1% cholesterol for 8 weeks and 12 weeks were compared to controls (six rabbits/group). The weight of food was strictly restricted to 100 g/rabbit per day.ResultsBody weights and fasting plasma insulin levels showed no significant differences among the groups. In contrast, characteristic fine fibrosis was extended from perivenular to pericellular areas, and microvesicular fatty change with ballooning degeneration was observed in perivenular areas in livers of the cholesterol-fed rabbits. Increase of serum cholesterol level, activation of hepatic stellate cells, and exposure to oxidative stress were also recognized.ConclusionsCholesterol-fed rabbits share several physiopathological features of NAFLD. Because this model did not show insulin resistance or obesity, it may be useful for elucidating the mechanism of NAFLD related mainly to hyperlipidemia.

Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt’s lymphoma cells

Purpose: Epstein-Barr virus (EBV) is associated with the development of several lymphoid and epithelial malignancies, including Burkitt’s lymphoma. The EBV latent protein, EBV Nuclear Antigen 1 (EBNA1), is detectable in almost all types of EBV-associated tumors and is essential for replication and maintenance of the latent episome of EBV. We here examined whether the RNA interference (RNAi) technique could be employed to suppress expression of EBNA1 in EBV-positive Burkitt’s lymphoma cells. Methods: A Raji cell line expressing small hairpin RNAs (shRNAs) against EBNA1 was established and EBNA1 mRNA level was determined by real-time RT-PCR analysis. We investigated the effects of EBNA1 silence on lymphoma cell growth and cell cycle progression. Results: Transfection of an EBNA1 RNAi plasmid resulted in substantial loss of EBNA1 mRNA and significantly inhibited proliferation of Raji cells relative to the control plasmid case. Suppression of EBNA1 was also associated with downregulation of EBV oncogene EBNA2, a decreased PCNA labeling index and increased G0/G1 fraction in cell cycle analysis. Conclusions: These findings point to potential therapeutic applications for vector-mediated siRNA delivery to control EBV-associated malignant disorders.

Identification of mouse orthologue of endogenous secretory receptor for advanced glycation end-products: structure, function and expression

The cell-surface RAGE [receptor for AGE (advanced glycation end-products)] is associated with the development of diabetic vascular complications, neurodegenerative disorders and inflammation. Recently, we isolated a human RAGE splice variant, which can work as a decoy receptor for RAGE ligands, and named it esRAGE (endogenous secretory RAGE). In the present study, we have isolated the murine equivalent of esRAGE from brain polysomal poly(A)+ (polyadenylated) RNA by RT (reverse transcription)-PCR cloning. The mRNA was generated by alternative splicing, and it encoded a 334-amino-acid protein with a signal sequence, but lacking the transmembrane domain. A transfection experiment revealed that the mRNA was actually translated as deduced to yield the secretory protein working as a decoy in AGE-induced NF-kappaB (nuclear factor kappaB) activation. RT-PCR and immunoblotting detected esRAGE mRNA and protein in the brain, lung, kidney and small intestine of wild-type mice, but not of RAGE-null mice. The esRAGE expression was increased in the kidney of diabetic wild-type mice. The present study has thus provided an animal orthologue of esRAGE for clarification of its roles in health and disease.

Detection of JC virus DNA sequences in colorectal cancers in Japan

JC virus (JCV), a ubiquitous polyoma virus that commonly infects humans, was first identified as the etiologic agent for the fetal demyelinating disease, progressive multifocal leukoencephalopathy. Recently, a number of reports have documented detection of JCV in samples derived from several types of neural as well as non-neural human tumors. It has been suggested that oncogenicity of JCV depends on a T antigen having a strict structural homology to the T antigen of simian virus 40. To clarify whether JCV might have a potential role with regard to colorectal cancers, we investigated the presence of its genome in a series of cases along with colorectal adenomas and normal colonic mucosa, targeting T antigen, VP and agnoprotein by nested polymerase chain reaction and Southern blotting and T antigen by immunohistochemistry. While VP and agnoprotein were not found in any of the samples examined, T antigen was detected in 6 of 23 colorectal cancers (26.1%) and 1 of 21 adenomas (4.8%), but none of 20 samples of normal colonic mucosa. No clear and diffuse staining with anti-T-antigen antibodies (1:100) could be detected, and there was no correlation with CD20-positive cells, which might have indicated JCV latent infection of B lymphocytes. Presence of T antigen did not influence clinicopathological variables, including survival. In one colonic cancer case positive for T antigen together with lymph node metastasis, DNA extracted from cancer cells in the lymph node revealed no detection of T antigen. Our results are in the intermediate position between the high T antigen rate (81%) in one report and the lack of it (0%) in another focused on colon cancers. It was concluded that T antigen might be integrated in cancer cells in approximately one fourth of Japanese colon cancer cases without clear and diffuse expression of the protein, suggesting a possible role in oncogenesis which might involve a hit-and-run mechanism.

Endogenous secretory receptor for advanced glycation endproducts as a novel prognostic marker in chondrosarcoma.

Chondrosarcoma, the second most frequent primary malignant bone tumor, is classified into 3 grades according to histologic criteria of malignancy. However, a low‐grade lesion can be difficult to distinguish from a benign enchondroma, whereas some histologically low‐grade lesions may carry a poor prognosis. The receptor for advanced glycation endproducts (RAGE) and its ligand, high‐mobility group box‐1 (HMGB1), was quantified in enchondromas and chondrosarcomas to determine whether these markers were associated with histological malignancy and prognosis.

Expression of KAI1 and tenascin, and microvessel density are closely correlated with liver metastasis of gastrointestinal adenocarcinoma

Aim: To seek good markers to predict invasion and metastasis of gastrointestinal adenocarcinoma (GIA). Methods: Expression of KAI1 and tenascin were examined on tissue microarrays containing gastric adenocarcinoma (n = 98), colorectal adenocarcinoma (n = 125), gastric adjacent non-cancerous mucosa (n = 95) and colorectal adjacent non-cancerous mucosa (n = 112) by immunostaining. Microvessel density (MVD) in GIA was labelled using anti-CD34 antibody by immunostaining. Expression of KAI1 and tenascin, and MVD were compared with clinicopathological features of tumours, including PTEN (phosphatase and tensin homology deleted from human chromosome 10) and EMMPRIN (extracellular matrix metalloproteinase inducer) expression. Results: KAI1 expression was higher in GIAs than in their adjacent non-cancerous mucosa (p<0.05). KAI1 and tenascin expression showed a significantly negative association with liver metastasis of GIA (p<0.05), but not with depth of invasion, venous invasion or lymph node metastasis (p>0.05). A significantly negative relationship was observed between EMMPRIN and tenascin expression in GIA (p<0.05). MVD was positively correlated with depth of invasion, venous invasion, lymph node metastasis and liver metastasis of tumours (p<0.05), whereas it was negatively correlated with PTEN expression (p<0.05). Conclusions: Up-regulated KAI1 expression may play an important part in malignant transformation of gastrointestinal epithelial cells. Reduced expression of KAI1 and tenascin might underlie the molecular basis of liver metastasis of GIA. Angiogenesis is a key event in the invasion and metastasis of GIA. These markers might be used to indicate liver metastasis of GIA in clinicopathological practice.