LMP1 Gene Research Articles

Impaired Antigen Processing and Presentation Machinery is Associated with Immunotolerant State in Chronic Hepatitis B Virus Infection

The mechanism of hepatitis B virus (HBV)-specific T cell hyporesponsiveness in hepatitis Be antigen (HBeAg)-positive subjects is not well understood. Inefficient antigen processing and transport to major histocompatibility complex class I molecules, namely due to low molecular weight protein (LMP) 2 and 7 and transporter associated with antigen processing (TAP) 1 and 2 genes could be playing a role. Forty patients with chronic hepatitis B (CHB) infection, hepatitis B surface antigen, and HBeAg positive; 26 with raised (Gr. I) and 14 with persistently normal ALT levels (Gr. II) and 11 healthy controls (Gr. III) were studied. Total RNA was isolated from peripheral blood mononuclear cells and mRNA expression of TAP1, TAP2, LMP2, and LMP7 genes was analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction method. Gamma interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels were quantified by enzyme-linked immunosorbent assay (ELISA) using log-log and linear graphs, respectively. Group II CHB patients had significantly lower mRNA expression for TAP1 (p = 0.003) and LMP2 (p = 0.002) genes as compared to Gr. I patients. The mRNA expression of TAP2 and LMP7 genes was comparable between the groups. However, expression of TAP1 (p = 0.02), TAP2 (p = 0.035), and LMP2 (p = 0.041) was found to be significantly higher in Gr. III subjects compared to Gr. I and Gr. II patients. In Gr. I and II, the IFN-gamma {s54.2{9.4-165} pg/ml), (59.5{28.5-110} pg/ml)}, and TNF-alpha {12.0 (8.0-23.2)},{10.8(6.2-20.8)} pg/ml levels were comparable but were significantly (p = 0.00,0.004, respectively) higher than Gr. III subjects. Low expression of TAP1 and LMP2 suggests an important role of these genes in defective viral antigen processing in immune tolerant state of CHB patients. Higher IFN-gamma and TNF-alpha production in CHB are probably enough to potentiate liver injury but not enough to clear the chronic HBV infection. These novel observations could pave way for new therapeutic strategies for immune restoration in CHB infected patients.

Asymmetric Arginine dimethylation of Epstein–Barr virus nuclear antigen 2 promotes DNA targeting

The Epstein-Barr virus (EBV) growth-transforms B-lymphocytes. The virus-encoded nuclear antigen 2 (EBNA2) is essential for transformation and activates gene expression by association with DNA-bound transcription factors such as RBPJkappa (CSL/CBF1). We have previously shown that EBNA2 contains symmetrically dimethylated Arginine (sDMA) residues. Deletion of the RG-repeat results in a reduced ability of the virus to immortalise B-cells. We now show that the RG repeat also contains asymmetrically dimethylated Arginines (aDMA) but neither non-methylated (NMA) Arginines nor citrulline residues. We demonstrate that only aDMA-containing EBNA2 is found in a complex with DNA-bound RBPJkappa in vitro and preferentially associates with the EBNA2-responsive EBV C, LMP1 and LMP2A promoters in vivo. Inhibition of methylation in EBV-infected cells results in reduced expression of the EBNA2-regulated viral gene LMP1, providing additional evidence that methylation is a prerequisite for DNA-binding by EBNA2 via association with the transcription factor RBPJkappa.

Association of a specific ERAP1/ARTS1 haplotype with disease susceptibility in ankylosing spondylitis

Alterations in antigen processing have been proposed to play a significant role in the pathogenesis of ankylosing spondylitis (AS). A non-major histocompatibility complex gene encoding an endoplasmic reticulum aminopeptidase, ERAP1, has been implicated recently. This study assessed 13 coding single-nucleotide polymorphisms (SNPs) from 5 genes involved in antigen processing (ERAP1, TAP1, TAP2, LMP2, and LMP7) in 3 Canadian cohorts of patients with AS, to address the possibility of gene interactions in disease susceptibility. The study involved 992 AS cases and 1,437 controls from 3 centers (472 cases and 451 controls from Alberta, 138 cases and 392 controls from Newfoundland, and 382 cases and 594 controls from Toronto). Most of the patients with AS and healthy, unrelated controls were Caucasians of northern European descent. Single-marker and haplotype associations were determined using an allelic likelihood ratio test in UNPHASED, version 3.0.12, and the WHAP program, respectively. P values for significance of haplotype associations were calculated using a permutation test. A specific ERAP1 haplotype, rs27044/10050860/30187-CCT, was strongly associated with increased risk of AS in all 3 case-control cohorts (pooled odds ratio [OR] 1.81, 95% confidence interval [95% CI] 1.46-2.24; P=7x10(-8)), while a second specific ERAP1 haplotype, rs30187/26618/26653-CTG, reduced the disease risk (pooled OR 0.77, 95% CI 0.67-0.88; P=9x10(-5)). Significant associations were also noted for 3 ERAP1 SNP variants (rs10050860, rs30187, and rs26653), although no significant haplotype interaction between ERAP1 and TAP/LMP loci was evident. These data indicate that an AS disease locus may reside on a specific ERAP1 haplotype, and its effect is not multiplicative with contributions from TAP and LMP genes.

MALLORY DENK BODY FORMATION IS ASSOCIATED WITH AN INCREASE OF THE IMMUNOPROTEASOME AND A DECREASE OF THE 26S PROTEASOME

Mallory Denk body (MDB) formation is seen in the liver cells of alcoholic patients and in other non‐alcoholic diseases. Proteasome inhibition is hypothesized to be the one of causes of MDB formation. Mice fed the drug diethyl‐1, 4‐dihydro‐2, 4, 6‐trimethyl‐3, 5‐pyridinedicarboxylate (DDC) for 10 weeks is the experimental model used to study MDB formation. 20S proteasome chymotrypsin‐like activity was not changed in the liver of mice refed DDC. However, ATP‐stimulated 26S proteasome chymotrypsin‐like activity was decreased by DDC feeding. These results confirm the reduction of 26S‐dependent ubiquitin‐proteasome proteolysis, which is reflected in the accumulation of ubiquitinated cytokeratins. Three constitutively expressed subunits of the 20S proteasome are known to be replaced by the inducible subunits of the immunoproteasome LMP2, LMP7, and MECL‐1 when stimulated with IFNg. There was an up regulation of LMP2, LMP7 and MECL‐1 protein levels and gene expression in the liver of mice fed DDC. Gene expression and protein levels of FAT10, a member of the ubiquitin‐like modifier family, which is also induced by IFNg and TNFa, were induced by DDC feeding. The marked up regulation of FAT10 could account for the block of cytokeratin turnover by the 26S proteasome. These results support the concept that the proteasome population is switched from the 26S proteasome to the immunoproteasome. The lack of 26S proteasome formation causes an accumulation of altered cytokeratin proteins. Protein aggresomes such as MDB then form. Supported by NIH/NIAAA Grants 8116 and P50‐011999 Alcohol Center Grant, Liver/Pancreas.

A Chromosomally Encoded Virulence Factor Protects the Lyme Disease Pathogen against Host-Adaptive Immunity

Borrelia burgdorferi, the bacterial pathogen of Lyme borreliosis, differentially expresses select genes in vivo, likely contributing to microbial persistence and disease. Expression analysis of spirochete genes encoding potential membrane proteins showed that surface-located membrane protein 1 (lmp1) transcripts were expressed at high levels in the infected murine heart, especially during early stages of infection. Mice and humans with diagnosed Lyme borreliosis also developed antibodies against Lmp1. Deletion of lmp1 severely impaired the pathogen's ability to persist in diverse murine tissues including the heart, and to induce disease, which was restored upon chromosomal complementation of the mutant with the lmp1 gene. Lmp1 performs an immune-related rather than a metabolic function, as its deletion did not affect microbial persistence in immunodeficient mice, but significantly decreased spirochete resistance to the borreliacidal effects of anti-B. burgdorferi sera in a complement-independent manner. These data demonstrate the existence of a virulence factor that helps the pathogen evade host-acquired immune defense and establish persistent infection in mammals.

Single nucleotide polymorphisms in antigen processing machinery component ERAP1 significantly associate with clinical outcome in cervical carcinoma

Genetic variation of the antigen processing machinery (APM) components TAP2, LMP7, and ERAP1 is related to cervical carcinoma risk, although the relation with expression and clinical outcome remains unknown. We have investigated the occurrence of APM component single nucleotide polymorphisms (SNPs) in cervical carcinoma. Twelve nonsynonymous, coding SNPs in the TAP1, TAP2, LMP2, LMP7, and ERAP1 genes were genotyped in 75 cervical carcinoma patients with known APM component and HLA class I expression levels. Individual genotype distributions were assessed for association with APM component expression, various histopathological parameters and survival. Genotype distributions at the ERAP1-56 and ERAP1-127 loci were significantly associated with overall survival (OS); haplotype construction spanning these two SNPs revealed that the combination of a major allele at ERAP1-56 and a minor allele at ERAP1-127 was significantly associated with survival, homozygosity for this haplotype being associated with decreased OS (5-year survival 50% vs. 70 and 81% for complete absence or heterozygosity for this haplotype, respectively; P = 0.021). Heterozygosity for this haplotype was an independent predictor for better OS in multivariate analysis (HR = 0.219; P = 0.014). These data indicate that genetic variation in APM component genes, particularly ERAP1, is an important contributing factor in cervical carcinogenesis, progressive tumor growth and survival. The location of the ERAP1-127 SNP in the peptidase M1 domain of the ERAP1 aminopeptidase suggests the possibility of direct functional consequences of variation at this locus.

The evolution of Epstein-Barr virus inferred from the conservation and mutation of the virus glycoprotein gp350/220 gene

To study variations of Epstein-Barr virus (EBV), we analyzed the gp350/220 gene for several cell lines and Japanese wild isolates using direct sequencing. The N-terminal region was highly conserved in all EBVs except for Jijoye/P3HR-1 and a few isolates. The variation of the region coincided with EBV types A and B (also referred to as types 1 and 2) and were, respectively, designated as the types a and b. The type A/a was detected in most Japanese cell lines and wild isolates, and was classified as China1 type with latent membrane protein (LMP) 1 gene. The type B/b was detected in only a few wild isolates with the Med and China2 types. The C-terminus had more diversity than the N-terminus and lacked the divergence between types A/a and B/b. The phylogenetic analyses of the gp350/220 and LMP1 genes may suggest a mode of EBV evolution into types A/a and B/b and then to LMP1 subtypes.

High levels of Epstein–Barr virus DNA in latently infected gastric adenocarcinoma

Gastric adenocarcinoma is the second leading cause of cancer death worldwide. Epstein–Barr virus (EBV) is present in the malignant cells of approximately 10% of cases. It is unclear whether EBV is being missed in some gastric adenocarcinomas due to insensitive test methods or partial EBV genome loss. In this study, we screened 113 gastric adenocarcinomas from low- and high-incidence regions (United States and Central America) for the presence of EBV using a battery quantitative real-time PCR (Q-PCR) assays targeting disparate segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, BZLF1, EBER1) and histochemical stains targeting EBV-encoded RNA (EBER), the latent proteins LMP1 and LMP2, and the lytic proteins BMRF1 and BZLF1. EBV DNA was detected by Q-PCR in 48/75 United States cancers (64%) and in 38/38 Central American cancers (100%), which was a significant differrence. EBER was localized to malignant epithelial cells in 8/48 (17%) United States and 3/38 (8%) Central American cancers. Viral loads were considerably higher for EBER-positive vs EBER-negative cancers (mean 162 986 vs 62 EBV DNA copies per 100 000 cells). A viral load of 2000 copies per 100 000 cells is recommended as the threshold distinguishing EBER-positive from EBER-negative tumors. One infected cancer selectively failed to amplify the LMP2 gene because of a point mutation, whereas another cancer had an atypical pattern of Q-PCR positivity suggesting deletion of large segments of the EBV genome. Three different viral latency profiles were observed in the cancers based on constant expression of EBER and focal or variable expression of LMP1 or LMP2, without lytic protein expression. We conclude that EBV DNA levels generally reflect EBER status, and a panel of at least two Q-PCR assays is recommended for sensitive identification of infected cancers.

Epstein-Barr virus genetic variation in Vietnamese patients with nasopharyngeal carcinoma: full-length analysis of LMP1

Genetic variation in tumor virus genes and its impact on function might contribute to the understanding of geographic differences in risks for virus-associated tumors. This is particularly true for the genes known to contribute to the biology of the tumor. It is has been proposed that Epstein-Barr virus (EBV) gene variation has a role in the high risk of nasopharyngeal carcinoma (NPC) in South-East Asia. NPC is among the five most common cancers in Vietnam. EBV-NPC cells always express EBV nuclear antigen 1 (EBNA1) and also frequently latent membrane protein 1 and 2 (LMP1 & LMP2). To investigate EBV gene variation in Vietnamese NPC patients we analyzed the full length of LMP1 gene including its promoter region, and the N-termini of both EBNA1 and LMP2A genes from five NPC biopsies. We detected two EBV variants V1 and V2 based on the LMP1 nucleotide sequence pattern compared with the prototype B95-8 and some available sequences including Chinese variants. The V1 variant shows strong similarity to a variant dominant in Southern China (China 1), while the V2 variant is similar to a Thai variant SEA 2 and partly identity with GD1 in the C-terminus. The promoter region and transmembrane domain of the SEA 2-like samples contained some specific differences compared with previously published variants. In contrast, analysis of EBNA1 N- and LMP2A N-termini only revealed minor changes. Our findings reinforces that the polymorphisms of whole LMP1 sequence should be considered in future EBV molecular epidemiology studies in different geographic populations.

LMP1 gene variants of Russian origin from patients with EBV- associated pathologies and healthy individuals

LMP1 gene variants of Russian origin from patients with EBV- associated pathologies and healthy individuals

Differential Gene Regulation by Epstein-Barr Virus Type 1 and Type 2 EBNA2

A transfection assay with a lymphoblastoid cell line infected with Epstein-Barr virus was used to compare the abilities of type 1 and type 2 EBNA2 to sustain cell proliferation. The reduced proliferation in cells expressing type 2 EBNA2 correlated with loss of expression of some cell genes that are known to be targets of type 1 EBNA2. Microarray analysis of EBNA2 target genes identified a small number of genes that are more strongly induced by type 1 than by type 2 EBNA2, and one of these genes (CXCR7) was shown to be required for proliferation of lymphoblastoid cell lines. The Epstein-Barr virus LMP1 gene was also more strongly induced by type 1 EBNA2 than by type 2, but this effect was transient. Type 1 and type 2 EBNA2 were equally effective at arresting cell proliferation of Burkitt's lymphoma cell lines lacking Epstein-Barr virus and were also shown to cause apoptosis in these cells. The results indicate that differential gene regulation by Epstein-Barr virus type 1 and type 2 EBNA2 may be the basis for the much weaker B-cell transformation activity of type 2 Epstein-Barr virus strains compared to type 1 strains.

Follicular dendritic cell tumor of the spleen associated with diffuse large B-cell lymphoma

We report the case of a 50-year-old woman with an isolated large mass of the lower pole of the spleen. Splenectomy was performed and revealed a follicular dendritic cell (FDC) tumor associated with diffuse large cell lymphoma. Dendritic cells were CD21(+), CD35(+), CNA42(+), CD20(-), and Epstein-Barr virus (EBV)(+). They contained a clonal EBV genome as shown by polymerase chain reaction analysis of the LMP-1 gene polymorphism. Interestingly, the lymphoma cells were intermingled with the neoplastic FDCs and displayed a germinal center cell phenotype (CD20(+), CD10(+), Bcl2(+), and EBV(-)). Double staining confirmed that EBV was restricted to the FDCs. Clinical, radiologic, and pathologic staging showed no other lymphoma localization. To the best of our knowledge, this association has never been reported. Based on the well-established role of FDCs in B-cell survival and proliferation, this observation suggests that the FDC tumor represented a favorable microenvironment for lymphoma cells with germinal center phenotype.

Analysis of Epstein-Barr virus strains and variants in classical Hodgkin's lymphoma by laser microdissection.

Epstein-Barr virus (EBV) seems to have an etiological role in the pathogenesis of classical Hodgkin's lymphoma (cHL). Studies of whole tissue DNA by polymerase-chain reaction (PCR) have shown a considerable number of cHL cases with co-infections by different EBV strains and variants, which apparently contradict the clonality of EBV in cHL previously demonstrated by Southern blot analysis. Due to the paucity of HRS cells in HL tissues, studies on single cell DNA are necessary to identify the specific cellular location (HRS cells and/or bystander B lymphocytes) of the EBV strains and variants present in tissue specimens. In the current study, the presence of EBV was determined by PCR of the 3' end of the LMP-1 gene and EBNA-3C gene in whole tissue and, consecutively, in isolated cells from 26 cases of cHL: 10 HIV-positive and 16 sporadic cHL cases. EBV EBERs were present in all but 2 sporadic cHL cases, which were used as negative controls. At isolated cell level, EBNA-3C gene PCR was more sensitive. Indeed, from the cHL cases in which dual-infection was present, it was observed that, in most of them, HRS cells were infected by type 1 virus, and B lymphocytes were co-infected by both types, which points towards EBV infection occurring early in cHL development. Moreover, the finding of 2 cases with dual-infection in HRS may suggest that, in a small percentage of cHL cases, HRS cells derive from different neoplastic clones, or that HRS cells are superinfected by other viral types after the establishment of the neoplastic clone.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F-->I, 21/21) and 212 (G-->S, 19/21) or (G-->N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G-->D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L-->F) and 129 (M-->I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W-->C), and 5/21 specimens showed another novel change at codon 115 (G-->A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.

Molecular Mechanisms of Uterine Leiomyosarcomas: Involvement of Defect in LMP2 Expression

Patients with uterine leiomyosarcoma (LMS) typically present with vaginal bleeding, pain, and a pelvic mass. Typical presentations with hypercalcemia or eosinophilia have been reported. Radiographic evaluation with combined positron emission tomography/computed tomography may assist in the diagnosis and surveillance of women with uterine LMS. A recently developed risk-assessment index is highly predictive of disease-specific survival. Ovarian preservation does not appear to negatively impact outcome, and the addition of adjuvant therapy after surgical treatment does not seem to improve survival. It is noteworthy that LMP2-deficient mice exhibit spontaneous development of uterine LMS with a disease prevalence of approximately 37% by 12 months of age. The LMP2 gene is transcribed from a promoter containing an interferon (IFN)-gamma-response factor element; thus, the IFN-gamma-signal strongly induces LMP2 expression. Furthermore, a recent report demonstrated the loss of ability to induce LMP2 expression, which is an interferon (IFN)-gamma-inducible factor, in human uterine LMS tissues and cell lines. Analysis of human uterine LMS shows somatic mutations in the IFN gamma signalling pathway, thus the loss of LMP2 induction is attributable to a defect in the earliest steps of the IFN-gamma signalling pathway. The discovery of an impaired key cell-signalling pathway may provide new targets for diagnostic approaches and therapeutic intervention.

Mammalian Proteasome Subpopulations with Distinct Molecular Compositions and Proteolytic Activities

The proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far have yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multiprotein complexes in a laminar flow that support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26 with additional subpopulations focusing in the range from pH 5.10 to 5.33. In contrast, the majority of hepatic 20 S proteasomes had a pI of 5.05 and focused from pH 5.01 to 5.29. Importantly proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities because proteasomes with pI 5.21 contained 58% less of the inducible subunit beta 2i compared with those with pI 5.28. In addition, dephosphorylation of 20 S proteasomes demonstrated that besides molecular composition posttranslational modifications largely contribute to their pI values. These data suggest the possibility of mixed 20 S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery. Thus, proteasome subpopulations may serve as novel therapeutic targets in the myocardium.

Modulation of LMP1 protein expression by EBV-encoded microRNAs

Epstein-Barr virus (EBV) was the first human virus found to encode microRNAs (miRNAs), but the function of these miRNAs has been obscure. Nasopharyngeal carcinoma (NPC) is associated with EBV infection, and the EBV-encoded LMP1 is believed to be a key factor in NPC development. However, detection of LMP1 protein in NPC is variable. Here, we report that EBV-encoded BART miRNAs target the 3' UTR of the LMP1 gene and negatively regulate LMP1 protein expression. These miRNAs also modulate LMP1-induced NF-kappaB signaling and alleviate the cisplatin sensitivity of LMP1-expressing NPC cells. Consistent with a previous study on the NPC C666-1 cell line and C15 xenograft, we found abundant expression of BART miRNAs in NPC tissues. Furthermore, DNA sequencing revealed that the 3' UTR of LMP1 is highly conserved in NPC-derived EBV isolates. The data provide insight into the discrepancy between LMP1 transcript and protein detection in NPC and highlight the role of the EBV miRNAs in regulating LMP1 downstream signaling to promote cancer development.

Induction of matrix metalloproteinases by Epstein–Barr virus latent membrane protein 1 isolated from nasopharyngeal carcinoma

Epstein–Barr virus latent infection is associated with human malignancies including Burkitt's lymphoma, gastric carcinoma and the highly invasive nasopharyngeal carcinoma (NPC). Increased expression of EBV latent membrane protein 1, LMP1, is correlated with tumor progression and metastasis in NPC. LMP1 induces cellular proteins including cytokines and matrix metalloproteinases (e.g., MMP1, MMP2 and MMP9). MMPs are endopeptidases involved in the degradation of extracellular matrix proteins; and their upregulation in cancer implicates their potential role in tumor metastasis. In light of the role of LMP1 in cytokine dysregulation and the fact that MMPs are regulated by cytokines, we examined whether LMP1 promotes NPC metastasis via the induction of MMPs. To delineate the oncogenic role of LMP1 in NPC, we first investigated the induction of MMP1, MMP2, MMP3 and MMP9 in LMP1-positive NPC tumor samples ( n = 15) by quantitative RT-PCR. We showed a significant induction of MMP1 and MMP3 transcripts in the EBV LMP1-positive NPC tissues, compared with biopsies obtained from the adjacent non-tumor tissues. To investigate the role of LMP1 in MMP expression in NPC, we cloned the LMP1 gene from NPC samples and transiently expressed it in MRC5 cells (human lung fibroblasts). Following transfection, a time-dependent elevation of endogenous MMP3 expression was found in the LMP1-transfectants by quantitative RT-PCR and Western analysis. Taken together, we observed that MMP3 is upregulated in LMP1-positive NPC tumors and LMP1-expression in fibroblasts is associated with MMP3 and cytokine expression. Our results suggest that LMP1 may contribute to invasiveness of NPC cells via the expression of MMP3 in fibroblasts.

The low rate of HLA class I molecules on the human embryonic stem cell line HS293 is associated with the APM components' expression level

Human embryonic stem cells (hESCs) represent a promise for future strategies of tissue replacement. However, there are different issues that should be resolved before these cells can be used in cellular therapies; among others, the rejection of transplantable hESCs as a result of HLA incompatibility between donor cells and recipients. The hESCs exhibit a weak HLA class I expression on the cell surface, but today the responsible mechanisms are unknown. We have analyzed the level expression of HLA class I heavy chain, beta2-microglobulin (beta2-m), and antigen-processing machinery (APM) components (TAP1, TAP2, LMP2, LMP7, and Tapasin) using the HS293 hESC line by real-time quantitative RT-PCR. This analysis has revealed a low expression of beta2-m, HLA-B, and Tapasin, and an absence of expression of: TAP1, TAP2, LMP2, and LMP7 genes in the HS293 hESC line respect to the embryoid bodies (EBs) and the induced stem cells with IFNgamma (with significant differences, p<0.05). The lack or loss of HLA class I molecules due to the down-regulation of the APM components has been frequently found in tumors of different histology as specific mechanisms of immune-evasion. We described for the first time in this report that the hESCs shared similar mechanisms with respect to tumor cells responsible for the weak HLA class I expression on the cell surface.

Role of Antigen Processing and Presentation Genes in Development of Acute and Chronic Hepatitic Viral Infections

Purpose: Progression of viral infection to either acute or chronic state depends on the efficacy of the host immune response. Whereas acute viral hepatitis invites a vigorous, multispecific T-cell response, chronic viral hepatitis is associated with a weaker, narrowly focused response. Specific T-cell hyporesponsiveness in the later could be due to inefficient delivery of antigens to MHC class I molecules. Antigen processing and transport to MHC class I primarily involves, Low molecular weight protein (LMP);2 and7 and Transporter Associated with Antigen Processing (TAP)1 and2 genes. Immune system modifies this complex by addition of these subunits induced by IFN-γ. We hypothesize that proteasome subunits shape the specific T-cell response and it may be dysfunctionally regulated in peripheral blood lymphocytes in chronic infections. Aim: To investigate the expression of antigen processing and presentation genes in acute hepatitis B (AHB), acute hepatitis E (AHE), chronic hepatitis B (CHB) and chronic hepatitis C (CHC) patients. Methods: Fifty subjects were studied:10 of each category, AHB (age 33.3 ± 19.8 yr., M = 7), AHE (age31.7 ± 16.5 yr., M = 7), CHB (age 29.4 ± 13.2 yr., M = 8), CHC (age 45.5 ± 18.1 yr., M = 8) and controls (age 27.9 ± 4.6 yr., M = 7). Total RNA was isolated from PBMC's and cDNA was prepared and mRNA expression of TAP1, TAP2, LMP2 and LMP7 genes was quantified by Real-Time PCR. Results: No significant difference in expression patterns between AHB and AHE was found. LMP2 was upregulated in AHB (P= 0.051) and AHE (0.059) compared to controls. In CHC, LMP2 was significantly downregulated and TAP2 was upregulated in comparison to AHB and AHE. In CHB, TAP 1 and LMP 7 were significantly downregulated compared to controls and AH groups (P= 0.034, 0.006). Conclusion: i) Downregulation of LMP7 and TAP1 in CHBV and LMP2 in CHC suggests that the catalytic subunits of proteasome complex and antigen presentation genes play significant role in HBV and HCV persistence, ii) Similar profile in AHB and AHE suggests that while viral strains differ, acute hepatitis invites similar antigen processing pathways.[figure1]Figure