Varied immune responses of HBV-specific B cells in patients undergoing pegylated interferon-alpha treatment for chronic hepatitis B.

BACKGROUND & AIMS: The changes of HBV-specific B-cells in chronic hepatitis B (CHB) patients underwent pegylated interferon-alfa (PEG-IFNα) treatment and achieved functional cure remain unclear. We aimed to evaluate the alterations in HBV-specific B-cells during treatment and therefore explored the mechanism of functional recovery of HBsAg-specific B-cells. METHODS: We included 39 nucleos(t)ide analogues-treated CHB patients who received sequential combination therapy with PEG-IFNα and 8 treatment-naive CHB patients. HBV-specific B-cells were characterized ex vivo using fluorescent labeled HBsAg and HBcAg. The frequency, phenotype, and subsets of HBV-specific B-cells and follicular helper T cells (Tfh-cells) were detected using flow cytometry. The functionality of HBV-specific B-cells was quantified through ELISpot assays. RESULTS: During treatment, the fraction of activated memory B-cells (MBCs) among HBsAg-specific B-cells and the expression of IgG, CXCR3, and CD38 increased. Antibody-secretion capacity of HBsAg-specific B-cell was restored after treatment only in patients with a functional cure and it showed a positive correlation with serum hepatitis B surface antibody levels. The phenotype and function of HBsAg-specific B-cells differed between patients with and without functional cure. Patients with functional cure exhibited IgG+ classical MBCs and plasmablasts in HBsAg-specific B-cells. HBcAg-specific B-cells displayed both attenuated antibody secretion with reduced IgG expression and an IgM+ atypical type of MBCs after treatment, irrespective of with and without functional cure. The number of CD40L+ Tfh-cells increased after PEG-IFNα treatment and positively correlated with HBsAg-specific B-cell activation. CONCLUSIONS: After PEG-IFNα treatment, HBsAg- and HBcAg-specific B-cells exhibit various changes in antibody secretion. Their functional differences are reflected in the alterations in phenotypes and subtypes. The presence of CD40L+ Tfh-cells is associated with the active recovery of HBsAg-specific B-cells. IMPACT AND IMPLICATIONS: HBV-related complications and hepatocellular carcinoma remain the leading causes of mortality from chronic liver disease worldwide, and a cure is rarely achieved with antiviral therapies. Elucidating the immunological mechanisms underlying the functional cure of CHB patients offers a promising therapeutic strategy for viral clearance, such as therapeutic vaccine. We analyzed the alterations in HBV-specific B-cells in patients treated with PEG-IFNα and identified novel pathways for immunotherapeutic boosting of B cell immunity.

Albumin-bilirubin score is associated with response to pegylated interferon and nucleos(t)ide analogues in chronic hepatitis B patients.

BACKGROUND AND AIM: Recently, the role of albumin-bilirubin (ALBI) score in chronic hepatitis B (CHB) has not been well-understood. We aimed to investigate the association of ALBI score with natural history of chronic HBV infection and treatment response of CHB patients. METHODS: The ALBI score in a cohort of 849 individuals including 721 chronic HBV-infected patients naïve to anti-HBV treatment in different phases and 128 healthy controls were estimated. Additionally, the dynamic changes of ALBI score of 243 hepatitis B e antigen (HBeAg)-positive CHB patients treated with pegylated interferon-alpha (PEG-IFN-α) or nucleos(t)ide analogues (NAs) were tested for 72 weeks. RESULTS: ALBI score differed among phases, with the highest score in HBeAg-positive CHB patients, followed by HBeAg-negative CHB patients, HBeAg-positive chronic HBV infection, and HBeAg-negative chronic HBV infection. Besides, CHB patients harbouring high baseline ALBI score exhibited a relatively stronger therapeutic response to PEG-IFN-α or NAs. Moreover, the rate of HBeAg and HBsAg loss in patients with ALBI grade 2 was persistently higher than that in patients with ALBI grade 1 throughout the course of treatment. Furthermore, ALBI score was an independent predictor of sustained response achievement. The combined use of ALBI score, HBeAg and ALT could enhance the predictive value of treatment response. CONCLUSIONS: ALBI score differed significantly across the natural course of chronic HBV infection and was correlated with PEG-IFN-α and NAs treatment response in HBeAg-positive CHB patients, which suggested that ALBI score could be useful as an auxiliary clinical factor to determine the initiation of therapy and predict stronger antiviral treatment response.

Hyperbranched rolling circle amplification (HRCA)-based fluorescence biosensor for ultrasensitive and specific detection of single-nucleotide polymorphism genotyping associated with the therapy of chronic hepatitis B virus infection.

Detection of specific genes related to drug action can provide scientific guidance for personalized medicine. Taking the detection of a single-nucleotide polymorphism (SNP) genotyping related to the chronic hepatitis B virus (HBV) therapy as an example, a novel biosensor with high sensitivity and selectivity was developed based on the hyperbranched rolling circle amplification (HRCA) in this work. The single-base mutant DNA (mutDNA) sequence can perfectly hybridize with the specially designed discrimination padlock probe and initiate the HRCA reaction. Subsequently, a great abundant of double-strand DNA sequences were released and a strong fluorescence signal can be detected after adding SYBR Green I. In particular, the enhanced fluorescence intensity exhibits a linear relationship with the logarithm of mutDNA concentration ranging from 0.1 nM to 40 nM with a low detection limit of 0.05 nM. However, when there was even a single base mismatch in the target DNA, the HRCA was suppressed and fluorescence response process could not occur, resulting in a high selectivity of this biosensor. Moreover, this detection strategy also performs well in human serums, demonstrating its potential application in detecting SNPs in real biological samples.

Association of serum total cholesterol with pegylated interferon-α treatment in HBeAg-positive chronic hepatitis B patients.

BACKGROUND: Recent studies suggest that serum lipids are associated with pegylated interferon-alpha (PEG-IFN-α) treatment response in chronic hepatitis C patients. However, the role of serum lipids in influencing the outcome of HBV treatment is not well understood. This study aims to investigate the association of serum lipids with the response to interferon-alpha treatment for chronic hepatitis B (CHB) patients. METHODS: We dynamically measured 11 clinical serum lipid parameters of 119 hepatitis B e antigen (HBeAg)-positive CHB patients, including 53 patients who achieved sustained response (SR) and 66 patients who achieved non-response (NR) induced by PEG-IFN-α treatment for 48 weeks. RESULTS: The dynamic analysis showed that the baseline serum total cholesterol (TCHO) level was higher in the NR group than that in the SR group (P=0.004). Moreover, the correlation analysis demonstrated a significant positive correlation between TCHO and hepatitis B surface antigen (HBsAg) at baseline (P=0.009). In addition, CHB patients with high baseline TCHO levels exhibited higher HBV DNA, HBsAg, HBeAg and hepatitis B e antibody (HBeAb) levels during early treatment periods (weeks 0, 4, 12 and 24) than those with the low TCHO levels. Furthermore, the logistic regression analysis identified that baseline serum TCHO was a risk factor for NR achievement (OR=4.94; P=0.047). CONCLUSIONS: Our results indicated that serum TCHO was associated with PEG-IFN-α therapeutic response in HBeAg-positive CHB patients which suggested that serum TCHO could be useful as an auxiliary clinical factor to predict poor efficacy of PEG-IFN-α therapy.

The long non-coding RNA PTTG3P promotes cell growth and metastasis via up-regulating PTTG1 and activating PI3K/AKT signaling in hepatocellular carcinoma.

BACKGROUND: Dysfunctions of long non-coding RNA (lncRNAs) have been associated with the initiation and progression of hepatocellular carcinoma (HCC), but the clinicopathologic significance and potential role of lncRNA PTTG3P (pituitary tumor-transforming 3, pseudogene) in HCC remains largely unknown. METHODS: We compared the expression profiles of lncRNAs in 3 HCC tumor tissues and adjacent non-tumor tissues by microarrays. In situ hybridization (ISH) and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to assess the level of PTTG3P and prognostic values of PTTG3P were assayed in two HCC cohorts (n = 46 and 90). Artificial modulation of PTTG3P (down- and over-expression) was performed to explore the role of PTTG3P in tumor growth and metastasis in vitro and in vivo. Involvement of PTTG1 (pituitary tumor-transforming 1), PI3K/AKT signaling and its downstream signals were validated by qRT-PCR and western blot. RESULTS: We found that PTTG3P was frequently up-regulated in HCC and its level was positively correlated to tumor size, TNM stage and poor survival of patients with HCC. Enforced expression of PTTG3P significantly promoted cell proliferation, migration, and invasion in vitro, as well as tumorigenesis and metastasis in vivo. Conversely, PTTG3P knockdown had opposite effects. Mechanistically, over-expression of PTTG3P up-regulated PTTG1, activated PI3K/AKT signaling and its downstream signals including cell cycle progression, cell apoptosis and epithelial-mesenchymal transition (EMT)-associated genes. CONCLUSIONS: Our findings suggest that PTTG3P, a valuable marker of HCC prognosis, promotes tumor growth and metastasis via up-regulating PTTG1 and activating PI3K/AKT signaling in HCC and might represent a potential target for gene-based therapy.

Diagnostic value of preoperative inflammatory markers in patients with glioma: a multicenter cohort study.

OBJECTIVE Glioma is the most common form of brain tumor and has high lethality. The authors of this study aimed to elucidate the efficiency of preoperative inflammatory markers, including neutrophil/lymphocyte ratio (NLR), derived NLR (dNLR), platelet/lymphocyte ratio (PLR), lymphocyte/monocyte ratio (LMR), and prognostic nutritional index (PNI), and their paired combinations as tools for the preoperative diagnosis of glioma, with particular interest in its most aggressive form, glioblastoma (GBM). METHODS The medical records of patients newly diagnosed with glioma, acoustic neuroma, meningioma, or nonlesional epilepsy at 3 hospitals between January 2011 and February 2016 were collected and retrospectively analyzed. The values of NLR, dNLR, PLR, LMR, and PNI were compared among patients suffering from glioma, acoustic neuroma, meningioma, and nonlesional epilepsy and healthy controls by using nonparametric tests. Correlations between NLR, dNLR, PLR, LMR, PNI, and tumor grade were analyzed. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic significance of NLR, dNLR, PLR, LMR, PNI, and their paired combinations for glioma, particularly GBM. RESULTS A total of 750 patients with glioma (Grade I, 81 patients; Grade II, 208 patients; Grade III, 169 patients; Grade IV [GBM], 292 patients), 44 with acoustic neuroma, 271 with meningioma, 102 with nonlesional epilepsy, and 682 healthy controls were included in this study. Compared with healthy controls and patients with acoustic neuroma, meningioma, or nonlesional epilepsy, the patients with glioma had higher values of preoperative NLR and dNLR as well as lower values of LMR and PNI, whereas PLR was higher in glioma patients than in healthy controls and patients with nonlesional epilepsy. Subgroup analysis revealed a positive correlation between NLR, dNLR, PLR, and tumor grade but a negative correlation between LMR, PNI, and tumor grade in glioma. For glioma diagnosis, the area under the curve (AUC) obtained from the ROC curve was 0.722 (0.697-0.747) for NLR, 0.696 (0.670-0.722) for dNLR, 0.576 (0.549-0.604) for PLR, 0.760 (0.738-0.783) for LMR, and 0.672 (0.646-0.698) for PNI. The best diagnostic performance was obtained with the combination of NLR+LMR and dNLR+LMR, with AUCs of 0.777 and 0.778, respectively. Additionally, NLR (AUC 0.860, 95% CI 0.832-0.887), dNLR (0.840, 0.810-0.869), PLR (0.678, 0.641-0.715), LMR (0.837, 0.811-0.863), and PNI (0.740, 0.706-0.773) had significant predictive value for GBM compared with healthy controls and other disease groups. As compared with the Grade I-III glioma patients, the GBM patients had an AUC of 0.811 (95% CI 0.778-0.844) for NLR, 0.797 (0.763-0.832) for dNLR, 0.662 (0.622-0.702) for PLR, 0.743 (0.707-0.779) for LMR, and 0.661(0.622-0.701) for PNI. For the paired combinations, NLR+LMR demonstrated the highest accuracy. CONCLUSIONS The NLR+LMR combination was revealed as a noninvasive biomarker with relatively high sensitivity and specificity for glioma diagnosis, the differential diagnosis of glioma from acoustic neuroma and meningioma, GBM diagnosis, and the differential diagnosis of GBM from low-grade glioma.

Red blood cell distribution width levels correlate with liver fibrosis and inflammation: a noninvasive serum marker panel to predict the severity of fibrosis and inflammation in patients with hepatitis B.

We aimed to study whether red blood cell distribution width (RDW) could be one of the variables determining the extent of liver fibrosis and inflammation in patients with biopsy-proven hepatitis B. A total of 446 hepatitis B virus-infected patients who underwent liver biopsy were divided into 2 groups: absent or mild and moderate-severe according to the severity of liver fibrosis and inflammation. The independent variables that determine the severity of liver fibrosis and inflammation were explored. RDW values increased with progressive liver fibrosis and inflammation. After adjustments for other potent predictors, liver fibrosis (moderate-severe) was independently associated with RDW, platelet, and albumin (odds ratio = 1.121, 0.987, and 0.941, respectively), whereas increased odds ratios of significant inflammation were found for RDW, alanine aminotransferase, albumin, and PLT (odds ratio = 1.146, 1.003, 0.927, and 0.990, respectively). The sensitivity and specificity of model A were 70.0% and 62.9% for detection of significant liver fibrosis [area under the receiver-operating characteristic curve (AUC) = 0.713, P < 0.001]. The sensitivity and specificity of model B were 66.1% and 79.4% for predicting advanced liver inflammation (AUC = 0.765, P < 0.001). Compared with preexisting indicators, model A achieved the highest AUC, whereas model B showed a higher AUC than RDW to platelet ratio (0.670, P < 0.001) and FIB-4 (0.740, P = 0.32). RDW may provide a useful clinical value for predicting liver fibrosis and necroinflammation in hepatitis B-infected patients with other markers.

Construction of a capsule associated protein 10 gene eukaryotic expression vector for RNA interference and confirmation of biologic relevance.

BACKGROUND: The capsule associated protein 10 gene (cap10) is indispensible for the formation of the polysaccharide capsule, and is important in maintaining virulence of the Cryptococcus (C.) neoformans. In this study, we aimed to construct an short hairpin RNA (shRNA) expression vector targeting C. neoformans cap10 gene expression and confirm its biologic relevance. METHODS: A pair of oligonucleotides targeting the cap10 cDNA sequence was designed and synthesized. It was cloned into the plasmid psilencer4.1-CMV neo to construct an eukaryotic shRNA expression vector. The vector was transfected into C. neoformans cells using the LiAc method. The expression of cap10 was assessed by real-time fluorescence quantitative PCR. Groups of C. neoformans cells were incubated with murine macrophage-like J774A.1 cells, and the phagocytic indexes and ratios were determined by the microscopic observation method. RESULTS: The expression of cap10 in C. neoformans cells transfected with ps4.1 neo-cap10 ((175,535.00 ± 47,004.00) copies/µl) was lower than that of cells transfected with the empty vector ((512,698.89 ± 32,318.02) copies/µl) and mock transfected cells ((562,931.66 ± 65,928.41) copies/µl). The average phagocytic ratio and phagocytic index of J774A.1 cells following incubation with C. neoformans were higher for cells transfected with ps4.1 neo-cap10 (0.21 ± 0.02, (19.06 ± 1.66)%) than for the control experimental group (0.08 ± 0.02, (6.57 ± 1.23)%) and the blank experimental group ((0.07 ± 0.01), (5.89 ± 1.07)%) (P < 0.05). CONCLUSIONS: The cap10 shRNA vector was successfully prepared and transfected into C. neoformans cells. The effect of RNA interference on the expression of the C. neoformans cap10 gene is effective, and it can induce phagocytosis of C. neoformans.

Novel mutations of PRSS1 gene in patients with pancreatic cancer among Han population.

BACKGROUND: A high mortality rate of pancreatic cancer becomes a bottleneck for further treatment with long-term efficacy. It is urgent to find a new mean to predict the early onset of pancreatic cancer accurately. The authors hypothesized that genetic variants of cationic trypsinogen (PRSS1) gene could affect trypsin expression/function and result in abnormal activation of protease activated receptor-2 (PAR-2), then lead to pancreatic cancer. The aim of this study was to elaborate some novel mutations of PRSS1 gene in the patients with pancreatic cancer. METHODS: Totally 156 patients with pancreatic cancer and 220 unrelated individuals as controls were enrolled in this study. The mutations of PRSS1 gene were analyzed by direct sequencing. K-ras Mutation Detection Kit was used to find the general k-ras gene disorder in the pancreatic cancer tissue. Then the clinical data were collected and analyzed simultaneously. RESULTS: There were two patients who carried novel mutations which was IVS 3 + 157 G > C of PRSS1 gene in peripheral blood specimens and pancreatic cancer tissue. What's more, it was surprising to find a novel complicated mutation of exon 3 in PRSS1 gene (c.409 A > G and c.416 C > T) in another young patient. The complicated mutation made No. 135 and No. 137 amino acid transfer from Thr to Ala and Thr to Met respectively. No any mutation was found in the normal controls while no mutations of k-ras gene were detected in the three patients. CONCLUSION: Mutations of PRSS1 gene may be an important factor of pancreatic cancer.

Analysis of the ratio of mitchondrial DNA with A1555G mutant to wild type in deaf patients of Fujian province in China by a new method and its relationship with the severity of hearing loss.

BACKGROUND: It has been suggested that the ratio of mutant and wild type mitochondrial DNA may be related to its clinical phenotype. In this study, we developed a high sensitive real-time amplification refractory mutation system-quantitative polymerase chain reaction (RT-ARMS-qPCR) assay for quantitation of the mitochondrial DNA (mtDNA) with a mutated 1555 site, and explored the relationship between the ratio of mutated mtDNA and the severity of hearing loss of mitochondrial deafness (MD) patients of Fujian province in China. METHODS: An amplified mtDNA fragment containing the 1555 site was ligated into a vector to construct a plasmid DNA standard. An RT-ARMS-qPCR system was used to measure the mtDNA copy number containing wild-type and mutant sequences in a cohort of 126 MD patients of Fujian province in China. Combined with the clinical data, we explored the relationship between the ratio of mutated mtDNA and the severity of hearing loss of MD. RESULTS: The variation coefficient in the cohort was 1.21%, the interassay variation coefficient was 1.78%, and the linear range was 10(2) - 10(8) copies/µl for detecting a recombinant, wild-type plasmid. The primers amplified only the intended sequences. Mutation copy number correlated with the degree of deafness in sporadic cases with heteroplasmic mutations of mtDNA A1555G (R = 0.811, P = 0.003), but not in sporadic cases with homoplasmic mutations (R = 0.007, P = 0.989). The copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G in familial cases correlated with degree of deafness (R = 0.352, P = 0.023 and R = 0.90, P = 0.012, respectively). CONCLUSIONS: The RT-ARMS-qPCR system is suitable for determining the copy number of mtDNA fragments containing the A1555G mutation. The ratio of mutated mtDNA correlates with the severity of hearing loss of MD.

[Detection of deletional alpha-thalassemia from free fetal DNA in maternal plasma].

This study was aimed to develop a simple, rapid, accurate and wound-free single-tube multiplex polymerase chain reaction (PCR) assay, which can be used for molecular screening and prenatal diagnosis of 3 types of the commonest deletional alpha-thalassemias in Chinese population. The deletional alpha-thalassemias free fetal DNA of 50 anemic pregnant plasma were detected by means of single-tube multiplex PCR with 4 groups of primers designed by using the gap-PCR protocol. The results showed that 5 cases were found to be alpha-thalassemias in free fetal DNA of 50 anaemia pregnant plasma, out of them 3 cases were the Southeast Asia type of deletional alpha-thalassemias, 2 cases were alpha-(3.7) type of deletional alpha-thalassemias (rightward deletion). It is concluded that the single-tube multiplex PCR assay presented in this study is a simple and convenient, rapid and accurate method for detecting 3 common deletional alpha-thalassemias, and use of this method has an important significance for the deletional alpha-thalassemias to ensure the population health quality and reduce the social burden.

[Mitochondrial DNA A1555G mutation of seven families with nonsyndromic hearing loss].

OBJECTIVE: To study mitochondrial DNA (mtDNA) A1555G mutation in seven families with nonsyndromic hearing loss (NSHL). METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real time-amplification refractory mutation system-quantitative PCR (ARMS-qPCR) were applied to detect mtDNA A1555G mutation in seven NSHL families. Related clinical data were also collected and analyzed. RESULTS: The mtDNA A1555G mutation was detected in members from the maternal side, including heteroplasmy and homozygosis, others were negative for this mutation. The copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G correlated well with the degree of deafness (R = 0.341, P = 0.022 and R = 0.85, P = 0.015, respectively). CONCLUSION: The mutation rate of the mtDNA A1555G is high in the NSHL patients, the mutation type include heteroplasmy and homozygosis. There is significant correlation between the mtDNA A1555G copy number and the severity of hearing loss.

[Correlation between degree of mitochondrial DNA 1555 mutation and clinical phenotype of nonsyndromic hearing loss].

OBJECTIVE: To study the correlation between the number of mtDNA (mitochondrial DNA) copies containing mtDNA A1555G mutation site and phenotype and further elucidate the molecular genetic basis of phenotype diversity of nonsyndromic hearing loss. METHODS: Real time-amplification refractory mutation system-quantitative PCR was employed to detect the number of mtDNA copies in mild type and mutant type of mtDNA 1555. RESULTS: In the sporadic group, there was no significant correlation between mtDNA A1555G homogeneity mutation copies and phenotype (R = 0.001, P = 0.997) while significant correlation existed between mtDNA A1555G heteroplasmic mutations and phenotype (R = 0.771, P = 0.003). In the familial group there was significant correlation mtDNA 1555 homogeneity mutation copies and phenotype (R = 0.341, P = 0.022) and significant correlation existed between mtDNA 1555 heterogenicity mutation copies and phenotype (R = 0.85, P = 0.015). CONCLUSION: There is significant correlation between the mtDNA A1555G mutation copies and the severity of hearing loss.

Multisite mutations of the PRSS1 gene in a Chinese patient with chronic pancreatitis.

BACKGROUND: Chronic pancreatitis shows alterations in the trypsinogen gene (protease serine 1, PRSS1) in some individuals. The conversion of trypsinogen to trypsin in the pancreas is believed to be one of the causes of pancreatitis. This study was to identify the mutation of the PRSS1 gene in a Chinese patient with chronic pancreatitis and to analyze the clinical features of the disease. METHODS: In 6 patients with chronic pancreatitis and 120 normal controls, PRSS1 genes were amplified by the polymerase chain reaction and the products were analyzed by sequencing. RESULTS: Multisite mutations of PRSS1 were found in a patient with chronic pancreatitis. C to A mutation occurred in exon 3 of PRSS1, and T to A mutation in the same exon. These mutations were not found in normal controls or the patients with chronic pancreatitis. CONCLUSION: These are novel mutations in PRSS1.

Novel mutation and polymorphism of PRSS1 gene in the Chinese patients with hereditary pancreatitis and chronic pancreatitis.

BACKGROUND: Mutations in the cationic trypsinogen gene (PRSS1) have been detected in patients with hereditary pancreatitis (HP). This study investigated the prevalence of the R122H (c.365 G > A), A121T (c.361 G > A) and D162D (c.488 C > T) mutations or polymorphisms in the common, non-hereditary forms of chronic pancreatitis and in an HP family. METHODS: DNA was prepared from blood samples of 54 patients with chronic pancreatitis (35 alcoholic, 17 idiopathic and 2 hereditary) and 120 normal controls. The PRSS1 genes were amplified by polymerase chain reaction (PCR) and their products were analyzed by sequencing and related clinical data were also collected. RESULTS: A new polymorphism (c.488 C > T) of PRSS1 was found in 25 patients with chronic pancreatitis (including one affected member of the HP family) and six members of the normal controls. The C/T genotype was significantly increased in chronic pancreatitis (OR: 16.379, 95% CI: 5.7522 - 52.3663), the frequency of c.488 C > T change was in according with the Hardy-Weinberg equilibrium, but it doesn't affect the clinical phenotype. The commonly reported change of R122H (c.365 G > A) was not detected in any of the study subjects. c.361 G > A was found in 2 affected members and one unaffected carrier in an HP family. One of the affected members of an HP family had c.361 G > A mutation and polymorphism (c.488 C > T) in the PRSS1 gene at the same time. The patient's clinical values (C3, C4, CA19-9 and HbA1c) were higher than those of the other patients with chronic pancreatitis. The two patients with HP developed diabetes mellitus and their father died with pancreatic cancer. CONCLUSION: A new polymorphism (c.488 C > T) in the PRSS1 gene is associated with chronic pancreatitis, but it did not affect the clinical phenotype while the A121T (c.361 G > A) mutation in the gene shows a significant correlation in the patients with HP.

[A novel mutation of PRSS1 gene in a Chinese hereditary pancreatitis family].

We report the identification of a novel mutation in the protease serine 1 (PRSS1) gene in a hereditary pancreatitis (HP) family. Relevant clinical data were collected in this 24-member family. Moreover, the PRSS1 gene was amplified from the genomic DNA of the members with pancreatitis. The amplified products were analyzed by sequencing. A cytosine (C) to thymine (T) mutation in PRSS1 exon3 was detected in four affected members. This novel PRSS1 mutation may be an important factor associated with HP.

[Study on a new point mutation of nt7444 G-->A in the mitochondrial DNA in a type 2 diabetes mellitus family].

Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and direct sequencing were applied to detect a new point mutation of nt7444G-->A in the mitochondrial DNA in a type 2 diabetes mellitus family. The related clinical data were also collected and analyzed. mtDNA G7444A mutation in the cytochrome c oxidase I (COI) gene was found in 11 of 27 cases, all of whom were from the maternal side. Among them, 5 were confirmed to have type 2 diabetes mellitus, and one had impaired glucose tolerance. We conclude that the novel point mutation of mtDNA G7444A may be an independent factor associated with type 2 diabetes mellitus.

[Enhanced anti-tumor effect of dendritic cells modified by CIITA gene in hepatocellular carcinoma-bearing mice].

AIM: To study the effect of MHC class II transactivator(CIITA ) on the expression of MHC class I/II molecules, CD80 and CD86 molecules on dendritic cells(DCs), and explor the use of DCs modified by CIITA gene in the biotherapy of tumors. METHODS: We transfected the mouse CIITA gene by lipofectamine into DCs drived from mouse bone marrow. The expression of MHC class I/II, CD80, and CD86 molecules was detected by flow cytometry. 40 mice bearing hepatcellular carcinoma were divided into 4 groups: group 1 were treated with a para-tumor injection of PBS, group 2 DCs, group 3 modified DCs, and group 4 modified DCs pulsed with H22 tumor antigen. Then the anti-tumor effect of DCs modified by mCIITA was evaluated by measuring the tumor size. RESULTS: After transfection of mCIITA, the expression rate of MHC class I/II molecules of DCs increased from 74.2%/66.7% to 93.6%/91.4%, and that of CD80/CD86 increased from 52.3%/60.5% to 89.7%/91.5%. After immunization, the growth of H22 tumors in group 4 mice was significantly inhibited compared with that in other three groups (P<0.05 at 3, 4, 5, 6, and 7 weeks versus groups 1, 2; P<0.05 at 5, 6, 7 weeks versus group 3). CONCLUSION: The expression of MHC I/II, CD80 and CD86 on DCs was regulated markedly by transfection of mCIITA. mCIITA transfection also enhanced the anti-tumor effect of DCs. Our results provided a new method for biotherapy of tumors with DCs.

[Construction of mutants of MHC class II molecule transactivator gene capable of suppressing HLA-DR/DQ expression and exploration on their mechanism].

AIM: To construct MHC class II transactivator (CIITA) gene mutants which could suppress the expression of MHC class II molecule and explore its mechanism. METHODS: Restrictive enzymes digestion, PCR and synthesized oligonucleotide strands were used to construct three mutants including pcDNA3mCIITA2, pcDNA3mCIITA3 and pcDNA3mCIITA4. These mutants and pcDNA3 vector were transfected into Hela and Raji cell lines by lipofectamine. RT-PCR and flow cytometry were used to determine the changes of the inducible/constitutive expression of MHC class II-molecule. The mCIITA4 mutant was transferred to the tetracycline dose-dependent plasmid pUHD10-3. By adjusting the concentration of Dox in cultural circumstances, and regulating expressed level of external mutant C II TA, the relationship between the expression level of mC II TA4 mutant and that of MHC class II molecule was observed. RESULTS: pcDNA3mCIITA3 and pcDNA3mCIITA4 could significantly suppress the expression of HLA-DR/DQ gene in Hela and Raji cells, while pcDNA3 empty vector and pcDNA3mCIITA2 had no effect on the MHC class II expression. The inhibition rate of MHC class II molecule expression was directly proportional to the quantity of transfected CII TA mutant. CONCLUSION: The mutants pcDNA3 mCII TA3 and pcDNA3mCIITA4 can suppress the expression of MHC class II gene. Above date confirmed primarily that mutant C II TA suppressed the expression of MHC class II molecule by competitive binding with transactivator for the normal endogenous wild type C II TA molecule.