[PD-1/PD-L1 expressions in liver tissues of patients with chronic HBV infection].

OBJECTIVE: To detect and compare the PD-1/PD-L1 (programmed death 1/programmed death 1 ligand) expressions in the liver tissues of chronic HBV infection patients in immune tolerant phase and those in immune clearance phase. METHODS: Liver biopsy samples were divided into two groups: 25 samples from patients in immune clearance phase and 19 samples from patients in immune tolerant phase. PD-1/PD-L1 expressions on T lymphocytes in these liver biopsy specimens were detected by immunohistochemistry method. Percentage of PD-1/PD-L1 positive cells among CD3 positive cells was calculated by semi-quantitative evaluation. Differences between the two groups were statistically analyzed. RESULTS: PD-1/PD-L1 expressions were significantly higher in the patients in immune tolerant phase as compared to that in immune active phase (P < 0.05). No statistical difference found between the two groups for PD-L1 expression in Kupffer cells (P > 0.05). CONCLUSION: PD-1/PD-L1 expression level can reflect the immune functions of chronic hepatitis B patients.

Gpc-3 is a notable diagnostic, prognostic and a latent targeted therapy marker in hepatocellular carcinoma.

BACKGROUND/AIMS: To describe the features of the Gpc-3, explore the significance and value about the role of Gpc-3 in pathological diagnosis and prognostic evaluation of hepatocellular carcinoma. METHODOLOGY: Take an overview of Gpc-3 expression in hepatocellular carcinoma and its expression of the relationship between the clinicopathological features of hepatocellular carcinoma, analysis the expression of Gpc-3 in liver cell adenoma, heterosexual hyperplasia and hepatitis C. RESULTS: The expression of GPC-3 has a certain amount of specificity and sensitivity in hepatocellular carcinoma. CONCLUSIONS: Gpc-3 is not only a diagnostic and prognostic marker in hepatocellular carcinoma, but also is expected to be an ideal target for the therapy of hepatocellular carcinoma.

Glutamine synthetase as an early marker for hepatocellular carcinoma based on proteomic analysis of resected small hepatocellular carcinomas.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis. Because small HCCs possess most of the characteristics of early HCC, we investigated small HCCs to screen potential biomarkers for early diagnosis. METHODS: Proteins were extracted from 10 sets of paired tissue samples from HBV-infected small-HCC patients. The extracted proteins were well resolved by two-dimensional electrophoresis. These HCC-associated proteins were then identified by MALDI-TOF/TOF MS following image analysis. Western blotting and immunohistochemistry were used to assess glutamine synthetase (GS) and phenazine biosynthesis-like domain-containing protein (PBLD) expression in liver tissue. Enzyme-linked immunosorbent assays in 152 serum samples (from 49 healthy donors, 24 patients with liver cirrhosis, and 79 with HCC) were used to further assess the significance of GS clinically. RESULTS: Fifteen up-regulated and three down-regulated proteins were identified. Western blotting confirmed GS overexpression and decreased PBLD expression in liver tissue. Immunohistochemistry showed that GS was expressed in 70.0% (84/120) of HCCs and 35.8% (43/120) of nontumor tissues; PBLD was expressed in 74.2% (89/120) of nontumor tissues and 40.8% (49/120) of HCCs. The Chi-square test showed significant expression differences between HCCs and adjacent tissues. Consistent with this, serum GS levels in HCC patients were significantly higher than those in liver cirrhosis patients and healthy donors, while the latter two groups were also significantly different. In addition, a diagnostic cutoff value of 2.6 mg/ml was used for GS; it was elevated in 19 (76.0%) of 25 HCC patients with AFP

[The prediction and validation of liver fibrosis by a noninvasive model and validation in patients with chronic hepatitis B].

OBJECTIVE: To develop a simple model for the noninvasive diagnosis of liver fibrosis in patients with chronic hepatitis B and to testify its diagnostic value. METHODS: One hundred and ninety patients with chronic hepatitis B who had undergone liver biopsy were divided into 2 groups: one for developing the model (n = 110) and one for validation (n= 80). Histological staging of liver fibrosis, assessed blindly and independently by 2 pathologists, was determined according to Scheuer fibrosis score. Twenty markers involved in the study were analyzed initially in the estimation group to derive a predictive model to discriminate the stages of fibrosis. The model created was then assessed with receiver operating characteristic curve (ROC) analysis. It was also applied to the validation group to test its accuracy. RESULTS: Haptoglobin (HPT), gamma-glutamyl transpeptidase (GGT) and platelet were identified by logistic regression analysis as independent factors of fibrosis. A model developed from the above three markers was established to predict the stage of fibrosis(S). In ROC analysis, the area under curve (AUC) for identifying S > or =1, S > or = 2, S > or = 3 and S =4 was 0.832, 0.835, 0.820 and 0.843 respectively. The model had a similar AUC in the validation group without statistically significant difference. Using a cut-off of <0. 18, significant fibrosis (S > or = 2) could be excluded in 27 patients of the total patient population (negative predictive value 90%). Similarly, applying a cut-off > or = 0.70, significant fibrosis could be identified correctly in 67 patients of the total patient population (positive predictive value 82.7%). The model had a high level of diagnostic value in patients with HBeAg-positive chronic hepatitis B as well as in patients with HBeAg-negative chronic hepatitis B (AUC for identifying S > or = 2, 0.857 vs 0.802). Restricting biopsy to patients with intermediate scores ( > or = 0.70 and <0.18) may prevent liver biopsies in 58.4% of the patients while maintaining 84.7% accuracy. CONCLUSIONS: A model including HPT, GGT and platelet is a simple and reliable index for predicting significant fibrosis in patients with HBeAg-positive chronic hepatitis B as well as in patients with HBeAg-negative chronic hepatitis B.

[Detection of CD4+CD25+ regulatory T cells in liver tissues of fibrosing cholestatic hepatitis after liver and kidney transplantation].

OBJECTIVES: To study the expression and distribution of CD4+CD25+ regulatory T cells (Treg) in liver tissues of patients with fibrosing cholestatic hepatitis (FCH) after liver and kidney transplantation and to investigate their roles in the pathogenesis of FCH. METHODS: Liver biopsy specimens from five patients with FCH were studied histopathologically. A specific marker for CD4+CD25+ regulatory T cells in those specimens was detected with anti-FOXP3 monoclonal antibody by immunohistochemistry. Apoptoses of hepatocytes were detected with in situ apoptosis detection TUNEL kit. RESULTS: Fibrosis in portal and around portal areas, cholestasis in some of the hepatocytes and canaliculi, widespread ballooning and ground-glass appearance of liver cells, and positivity of HBsAg and HBcAg and Pre-S1 protein were seen in the livers of all cases. The positive signal of FOXP3 was located in the cytoplasm of lymphocytes and the positive cells were mainly aggregated in the portal areas as well as occasionally appearing in the hepatic sinusoids. There were many more apoptotic hepatocytes near the portal areas. CONCLUSION: Fibrosing cholestatic hepatitis has specific pathological characteristics which might be caused by high expressions of FOXP3 in liver tissues.

Influence of HBcAg in liver cell plasma on expression of transforming growth factor-beta 1 in liver tissue of low-grade chronic hepatitis B patients.

AIM: To study the influence of HBcAg on the expression of transforming growth factor-beta 1 (TGF-beta1) in liver tissue of low-grade chronic hepatitis B (CHB) patients. METHODS: The expression of TGF-beta1 and HBcAg in liver samples from 93 low-grade CHB patients was detected by immunohistochemistry and valuated by semi-quantitative scoring. RESULTS: In the 93 low-grade CHB patients, HBcAg was expressed in cell plasma but not in the liver tissue. There was no significant difference between the two groups. CONCLUSION: The expression of TGF-beta1 is not related with HBcAg expressed as plasma type in the tissues of low-grade CHB patients.

[Pathology of AIDS-related lymphadenopathy and its relationship to expression of CD4 + CD25 + regulatory T cells in lymphoid node tissue].

OBJECTIVE: To observe the pathology of AIDS-related lymphadenopathy and its relationship to the expression and distribution of CD4 + CD25 + regulatory T cells in lymphoid node tissue. METHODS: Totally 22 biopsy and 13 autopsy lymphoid node tissues from HIV-positive patients were examined under microscopy and pathological staging was performed. Specific marker for CD4 + CD25 + regulatory T cells in lymphoid node tissue was detected with anti-Foxp3 monoclonal antibody by immunohistochemistry. RESULTS: Among all the 35 specimens, 5, 4, 14, and 12 specimens were histopathologically staged from 1 to 4, respectively. FoxP3 were detected in all lymphoid node tissues. The distribution of FoxP3-positive lymphocytes were mainly in intermediate zone of follicle and cortical area in stages 1 and 2. The counts of FoxP3-positive lymphocytes remarkably decreased in stages 3 and 4, following depletion of lymphocytes. CONCLUSIONS: CD4 + CD25 + regulatory T cells exist in lymphoid node tissue of patients with HIV infection. Their amounts decrease or deplete along with the progression of AIDS-related lymphadenopathy.

[A pathological study on liver tissues of patients with HIV infection].

OBJECTIVE: To study the pathological changes of the liver tissues of patients with HIV infection. METHODS: 14 biopsy and 12 autopsy liver tissues were examined histologically. HIV-1 related antigen of outer membrane protein gp120 and capsid protein p24 were examined with their corresponding monoclonal antibodies by immunohistochemistry. RESULTS: In the biopsy group, cytomegalic virus (CMV) infection was found in one (1/14) case, outer membrane protein gp120 and/or capsid protein p24 antigen were detected in Kupffer cells and in some of the lymphocytes in 11 cases. All the hepatocytes were negative for outer membrane protein gp120 and capsid protein p24 antigens. In the autopsy group, there were 5 (5/12) cases of liver tissues with CMV infection and 5 cases each with mycobacterium and Toxoplasma gondii infection. Capsid protein p24 was detected in liver tissues in 3 cases. CONCLUSION: There is HIV infection in liver tissue of patients with HIV. The rate of opportunistic infections in liver biopsy samples was lower than that in the autopsy liver tissues of patients with HIV.

A clinicopathological study of three cases of severe acute respiratory syndrome (SARS).

AIMS: The severe acute respiratory syndrome (SARS) caused a large outbreak of atypical pneumonia in Beijing, China from early March 2003. We report the pathological features from three patients who died of SARS. METHODS: Autopsies were performed on three patients who died 9-15 days after the onset of the illness, and the clinical and laboratory features reviewed. Tissue sections were stained with haematoxylin and eosin (H&E), and in situ reverse transcriptase polymerase chain reaction (RT-PCR) on lung sections was performed using SARS coronavirus-specific primers. RESULTS: The typical gross pathological change in the lungs was diffuse haemorrhage on the lung surface. Histopathological examination revealed serous, fibrinous and haemorrhagic inflammation in most pulmonary alveoli, with capillary engorgement and some capillary microthrombosis. The pulmonary alveoli were thickened with interstitial mononuclear inflammatory infiltrates, diffuse alveolar damage, desquamation of pneumocytes and hyaline-membrane formation; fibrinoid material and erythrocytes were present in alveolar spaces. There were thromboemboli in some bronchial arterioles. Haemorrhagic necrosis and reduced numbers of lymphocytes were observed in lymph nodes and spleen. In situ RT-PCR detected SARS coronavirus RNA in type II alveolar cells, interstitial cells and bronchiolar epithelial cells from all three patients. CONCLUSIONS: Severe immunological damage in lung tissue is responsible for the clinical features of SARS.

Changes in serum and histology of patients with chronic hepatitis B after interferon alpha-2b treatment.

AIM: Chronic hepatitis B is a serious health problem. Interferon has long been used to treat Chronic hepatitis B. To evaluate the effects of interferon on chronic hepatitis B better, we designed the study to investigate the changes in sera and liver histology of patients with chronic hepatitis B after interferon alpha-2b treatment. METHODS: Twenty-four patients with chronic hepatitis B were enrolled in this study. They all received interferon alpha-2b treatment as following: 3 million units, i.m. t.i.w., for 18 weeks. Sera of all patients were obtained respectively for evaluation of ALT, HBsAg, HBcAg, HBeAg, HBV DNA and TIMP-1 before and after interferon treatment, also a liver biopsy pre- and post-treatment was performed for comparison of HAI, HBsAg, HBcAg, HBeAg, TIMP-1 and activated HSC in the liver tissue. RESULTS: Patients who had normalization of serum ALT and seroconversion of HBeAg and/or HBV DNA (blot hybridization) after treatment were defined as responders. The response rate in this study group was 37.5 % (7/24). Compared to pretreatment, the serum HBV DNA and TIMP-1 decreased significantly (P<0.05), so did the HAI, HBcAg, HBeAg, TIMP-1 and activated HSC (P<0.05). CONCLUSION: The significant decrease in HBV DNA in sera, the seroconversion of HBeAg, and the decrease of viral expression in liver indicated that interferon alpha-2b treatment can inhibit viral replication. The normalization of ALT in sera and the improvement of HAI in liver showed that interferon alpha-2b can improve the liver histology of patients with chronic hepatitis B. At the same time, interferon alpha-2b treatment can reduce the TIMP-1 in serum and liver and decrease the number of activated HSC, which may alleviate or inhibit hepatic fibrosis. Although the response rate was unsatisfactory, interferon play a beneficial role on patients with chronic hepatitis B in other respects. We still need further studies to improve the therapy effects.

Significant correlation between expression level of HSP gp96 and progression of hepatitis B virus induced diseases.

AIM: Gp96, also known as Grp94, is a member of heat shock protein (HSP) family and binds repertoires of peptides thereof eliciting peptide-specific T cell immune responses. It predominantly locates inside the endoplasmic reticulum (ER) with some cell surface expression in certain cancerous cells. Previous studies have shown that gp96 expression level was up-regulated in tumor cells, including hepatocellular carcinoma (HCC). However, relationship between the extent of gp96 expression and disease progression especially HBV-induced chronic infection, cirrhosis and hepatocellular carcinoma, has not been addressed before. As primary HCC can be induced and progressed from chronic hepatitis B virus (HBV) infection and HBV-induced cirrhosis, we designed an immunohistochemical experiment to test the correlation between gp96 expression level and HBV-induced disease progression, from chronic HBV infection, cirrhosis to HCC. METHODS: We chose liver samples from different patients of hepatitis B virus induced diseases, including chronic hepatitis B (77 patients), cirrhosis (27 patients) and primary HCC (30 patients), to test the expression level of gp96 in different affected groups. Formalin-fixed, and paraffin-embedded liver tissues taken from these patients were immuno-stained by using an anti-gp96 monoclonal antibody for the expression level of gp96 protein in the sections. In addition, Western blotting of whole cell lysates derived from established human embryonic liver cell lines and several human HCC cell lines (Huh7, HepG2, SSMC-7721) was compared with the expression of gp96. RESULTS: We found that the extent of elevated gp96 expression was significantly correlated with the disease progression, and was the highest in HCC patients, lowest in chronic HBV infection and was that of the cirrhosis in the middle. CONCLUSION: Increased expression of gp96 might be used as a diagnostic or prognostic bio-marker for the HBV infection and HBV-induced diseases.

Clinicopathological study on TTV infection in hepatitis of unknown etiology.

AIM: To investigate the state of infection, replication site, pathogenicity and clinical significance of transfusion transmitted virus (TTV) in patients with hepatitis, especially in patients of unknown etiology. METHODS: Liver tissues taken from 136 cases of non-A non-G hepatitis were tested for TT virus antigen and nucleic acid by in situ hybridization (ISH) and nested-polymerase chain reaction (PCR). Among them, TT virus genome and its complemental strand were also detected in 24 cases of autopsy liver and extrahepatic tissues with ISH. Meanwhile, TTV DNA was detected in the sera of 187 hepatitis patients by nested-PCR. The pathological and clinical data of the cases infected with TTV only were analyzed. RESULTS: In liver, the total positive rate of TTV DNA was 32.4% and the positive signals were located in the nuclei of hepatocytes. In serus, TTV DNA was detected in 21.4% cases of hepatitis A-G, 34.4% of non-A non-G hepatitis and 15% of healthy donors. The correspondence rate of TTV DNA detection between liver tissue with ISH and sera with PCR was 63.2% and 89.3% in the same liver tissues by ISH and by PCR, respectively. Using double-strand probes and single-strand probes designed to detect TTV genome, the correspondence rate of TTV DNA detected in liver and extrahepatic tissues was 85.7%. Using single-strand probes, TTV genome could be detected in liver and extrahepatic tissues by PCR, but its complemental strands (replication strands) could be observed only in livers. The liver function of most cases infected with TTV alone was abnormal and the liver tissues had different pathological damage such as ballooning, acidophilia degeneration, formation of apoptosis bodies and focus of necrosis, but the inflammation in the lobule and portal area was mild. CONCLUSION: The positive rate of TTV DNA among cases of hepatitis was higher than that of donors, especially in patients with non-A non-G hepatitis, but most of them were coinfected with other hepatitis viruses. TTV can infect not only hepatocytes, but also extrahepatic tissues. However, the chief replication place may be liver. The infection of TTV may have some pathogenicity. Although the pathogenicity is comparatively weak, it can still damage the liver tissues. The lesions in acute hepatitis (AH) and chronic hepatitis (CH) are mild, but in severe hepatitis (SH), it can be very serious and cause liver function failure, therefore, we should pay more attention to TTV when studying the possible pathogens of so-called "liver hepatitis of unknown etiology".

[Clinical manifestation, treatment, and outcome of severe acute respiratory syndrome: analysis of 108 cases in Beijing].

OBJECTIVE: To investigate the clinical manifestations, treatment, and outcome of severe acute respiratory syndrome (SARS). METHODS: The clinical data of 108 SARS in-patients were analyzed. RESULTS: Among the 108 cases, 35 males (32.4%) and 73 females (67.6%), aged 37 +/- 9 (range 13 approximately 83 years), most were white-collar workers, medical workers accounting for 28.7%. 87.0% had a definite contact history. 20.4% were complicated by other internal diseases. The main clinical manifestations included fever, cough, pectoralgia, chest distress, headache, etc. Involvement of multiple organs was often. The incubation period was 2 approximately 14 days. The course included early stage, progressive stage, climax, and convalescence. Mild type accounted for 4.6%, common type 39.8%, severe type 20.4%, and extremely severe type 35.2%. At the early stage, white blood cell count, lymphocyte count and ratio of lymphocyte, proalbumin, transferring, CD(3)(+), CD(4)(+), CD(8)(+) cell count were remarkably decreased, and C-reactive protein, alpha-acid glycoprotein, and alpha(2)-globulin were remarkably increased. X-ray chest film showed solitary or multiple local exudative changes, mostly in pulmonary lower field. During the progressive stage the hyaline or consolidation images were enlarged. High solution CT showed solitary or multiple cotton wadding like images and ground glass-like or consolidation images. Hypoxemia was common. At the progressive stage application of adrenocortical hormones and non-invasive mechanical ventilation helped stop the progress of disease. Ninety-four cases were discharged, 14 cases died. Traditional Chinese medicine (TCM) regards SARS as one of epidemic febrile diseases. Treatment by combination of TCM and Western medicine was effective. CONCLUSION: SARS mainly affects youth and people in their prime of life. It has its own characteristic clinical manifestations. The classification system of 5 types and 4 stages helps judge the condition. Treatment by combination of TCM and Western medicine is encouraged.

[A clinicopathological study on 3 cases of severe acute respiratory syndrome].

OBJECTIVE: To study the pathological characteristics of severe acute respiratory syndrome (SARS) and its relationship to clinical manifestation. METHODS: Tissue specimens from 3 autopsy cases of diagnosed SARS were studied under microscopy and the clinical data were reviewed. RESULTS: The typical pathological changes of lungs were diffuse hemorrhage on surface. A mixture features of serous, fibrinous and hemorrhagic inflammation were seen in most pulmonary alveoli with engorgement of capillary and there were microthrombosis in some capillary. Pulmonary alveoli became thick with interstitial mononuclear inflammatory infiltration, diffused alveoli damage, desquamation of pneumocytes and hyaline-membrane formation. Fibrinoid materials and erythrocytes could be found in alveolar spaces. There were thrombo-embolisms in some bronchial artery. Meanwhile, haemorrhagic necrosis was showed in lymph nodes and spleen with attenuation of lymphocytes. Other atypical pathological changes, such as hydropic degeneration, fatty degeneration, interstitial cell proliferation and some lesions observed in liver, heart, kidney, pancreas may have existed before the hospitalization. CONCLUSION: Severe damages of pulmonary and immunological system damage are responsible for clinical features of SARS and may lead to death of patients.

[Liver histological changes after lamivudine treating in chronic hepatitis B patients with HBeAg positive].

OBJECTIVES: To investigate the histological changes in liver biopsy tissues taken from chronic hepatitis B patients with HBsAg and HBeAg positive and ALT abnormal after lamivudine therapy for one year. METHODS: Lamivudine was given orally at the dose of 100 mg once a day for one year. 101 patients were enrolled into this open-label study. Paired liver biopsies from patients with hepatitis B before and after therapy with lamivudine were studied. Blinded biopsies were evaluated by a histopathologist and scored according to Knodell's histology activity index(HAI). RESULTS: 53.5% (54/101), 51.5% (52/101) and 31.7% (32/101) patients had a reduction of their total hepatic HAI score, necroinflammation and fibrosis scores by >or=2 points or 1 points at the end of one year of lamivudine therapy, compared with their pretreatment values, respectively. There were significant reduction of HAI score, necroinflammation and fibrosis scores from 8.0+/-4.7 to 5.2+/-3.3 (t=7.358, P<0.01), from 5.9+/-3.8 to 3.6+/-2.5 (t=7.298, P<0.01), and from 2.1+/-1.2 to 1.6+/-1.2 (t=3.827, P<0.01), respectively. The histological improvement was independent on the HBeAg seroconvertion during the therapy. CONCLUSION: Significant improvement in liver histology, both necroinflammation and fibrosis, can be obtained in the majority of patients treated with lamivudine for one year.

[Detection of hepatic progenitor cells in patients with severe hepatitis and their distribution].

OBJECTIVES: To identify hepatic progenitor cells (HPCs) in patients with severe hepatitis (SH) by detecting their markers and investigate the features of their distribution and location. METHODS: Liver tissues taken from 59 SH patients were tested for the receptor of stem cell factor (c-kit), pi-class glutathione S-transferase (GST-pi), cluster of differentiation 34 (CD34), cytokeratin 19 (CK19), cytokeratin 18 (CK18) and alpha fetoprotein (AFP) by immunohistochemistry (IHC). Meanwhile, 58 patients with acute or chronic hepatitis were also detected to act as controls. RESULTS: Hepatic progenitor cells could be seen in SH patients. Most of them existed as ductular cells that had been called "typical ductular proliferation (ADP)" or "typical ductular reaction" in previous research. These ductular cells were mainly located at the portal areas, fibro septa, periportal parenchyma and the border of the pseudolobuli and inflammatory foci. Further, c-kit, GST-pi, CK19 and CK18, but not CD34 and AFP could be detected in these cells. Another kind of HPC was the small hepatocyte-like cell (SHLC), which could express c-kit, GST-pi, and CK18, but not CK19, CD34 and AFP. The semi-quantitative analysis showed that the scope of ADP in SH patients was significantly larger than that in acute and chronic hepatitis patients (chi2= 63.62, P<0.05), and the scope of ADP in subacute severe hepatitis and chronic severe hepatitis patients was also significantly larger than that in acute severe hepatitis patients. CONCLUSION: In the course of regeneration of viral hepatitis, different types of pathology have different features. In acute and chronic hepatitis (G1-2), the regeneration is mainly owing to the proliferation of mature hepatocytes, and in chronic hepatitis (G3-4), there is the participation of HPCs, although they are limited. In severe hepatitis, however, since the replicative capacity of normal hepatocytes is impaired or prohibited, liver regenerates and restores mainly by the means of hepatic stem cells activation and proliferation. But the hepatic stem cells don't differentiate into their mature functional compartments directly at all. There are several intermediary or transition populations. In human severe hepatitis, they are mainly ductular cells, and parts of them are small hepatocyte-like cells.