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Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal meningoencephalitis complicated with neurogenic pulmonary oedema. Its pathogenesis, especially the CNS involvement, is not clearly understood. The aim of this study was to set up a mouse EV71 infection model with CNS involvement. EV71 virus was administrated orally to neonatal mice. The EV71-infected mice manifested a skin rash at an early stage and hind limb paralysis or death at a later stage. Immunohistochemical staining and virus isolation demonstrated that EV71 replicated in the small intestine, induced viraemia and spread to various organs. Kinetic studies showed that EV71 antigen was first detected in the intestine at 6 h, in the thoracic spinal cord at 24 h, in the cervical spinal cord at 50 h and in the brain stem at 78 h post-infection. Leukocyte infiltration was evident in the spinal cord and brain stem. Furthermore, EV71 virus could be transmitted to littermates within the same cage.

Fourteen cytokines or chemokines were analyzed on 88 RT-PCR-confirmed severe acute respiratory syndrome (SARS) patients. IFN-gamma, IL-18, TGF-beta, IL-6, IP-10, MCP-1, MIG, and IL-8, but not of TNF-alpha, IL-2, IL-4, IL-10, IL-13, or TNFRI, were highly elevated in the acute phase sera of Taiwan SARS patients. IFN-gamma was significantly higher in the Ab(+) group than in the Ab(-) group. IFN-gamma, IL-18, MCP-1, MIG, and IP-10 were already elevated at early days post fever onset. Furthermore, levels of IL-18, IP-10, MIG, and MCP-1 were significantly higher in the death group than in the survival group. For the survival group, IFN-gamma and MCP-1 were inversely associated with circulating lymphocytes count and monocytes count, but positively associated with circulating neutrophils count. It is concluded that an interferon-gamma-related cytokine storm was induced post SARS coronavirus infection, and this cytokine storm might be involved in the immunopathological damage in SARS patients.

Severe acute respiratory syndrome (SARS), a new disease with symptoms similar to those of atypical pneumonia, raised a global alert in March 2003. Because of its relatively high transmissibility and mortality upon infection, probable SARS patients were quarantined and treated with special and intensive care. Therefore, instant and accurate laboratory confirmation of SARS-associated coronavirus (SARS-CoV) infection has become a worldwide interest. For this need, we purified recombinant proteins including the nucleocapsid (N), envelope (E), membrane (M), and truncated forms of the spike protein (S1-S7) of SARS-CoV in Escherichia coli. The six proteins N, E, M, S2, S5, and S6 were used for Western blotting (WB) to detect various immunoglobulin classes in 90 serum samples from 54 probable SARS patients. The results indicated that N was recognized in most of the sera. In some cases, S6 could be recognized as early as 2 or 3 days after illness onset, while S5 was recognized at a later stage. Furthermore, the result of recombinant-protein-based WB showed a 90% agreement with that of the whole-virus-based immunofluorescence assay. Combining WB with existing RT-PCR, the laboratory confirmation for SARS-CoV infection was greatly enhanced by 24.1%, from 48.1% (RT-PCR alone) to 72.2%. Finally, our results show that IgA antibodies against SARS-CoV can be detected within 1 week after illness onset in a few SARS patients.

Severe acute respiratory syndrome (SARS) has raised a global alert since March 2003. After its causative agent, SARS-associated coronavirus (SARS-CoV), was confirmed, laboratory methods, including virus isolation, reverse transcriptase–polymerase chain reaction (RT-PCR), and serologic methods, have been quickly developed. In this study, we evaluated four serologic tests ( neutralization test, enzyme-linked immunosorbent assay [ELISA], immunofluorescent assay [IFA], and immunochromatographic test [ICT]) for detecting antibodies to SARS-CoV in sera of 537 probable SARS case-patients with correlation to the RT-PCR . With the neutralization test as a reference method, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.2%, 98.7%, 98.7%, and 98.4% for ELISA; 99.1%, 87.8%, 88.1% and 99.1% for IFA; 33.6%, 98.2%, 95.7%, and 56.1% for ICT, respectively. We also compared the recombinant-based western blot with the whole virus–based IFA and ELISA; the data showed a high correlation between these methods, with an overall agreement of >90%. Our results provide a systematic analysis of serologic and molecular methods for evaluating SARS-CoV infection.

Background/Aims : Inducible nitric oxide synthase (iNOS) is found to have antiviral activity. Its role is evaluated using a murine acute hepatitis B virus (HBV) infection model. Methods : pHBV3.6 plasmid containing HBV genome was injected into mice by hydrodynamics-based in vivo transfection. HBV antigenemia and serum HBV-DNA were detected by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction, respectively. HBV replication in liver was analyzed by Northern and Southern blot. Intrahepatic leukocytes were isolated and analyzed with flow cytometry. Results : After hydrodynamics injection of pHBV3.6, HBV genome was synthesized in the liver and HBV-DNA, as well as hepatitis B surface antigen and hepatitis B e antigen were secreted into the blood. Anti-HBV antibody responses developed afterward. A murine acute HBV infection model was established with hydrodynamics injection of non-transponase based HBV-DNA. Using this protocol in iNOS deficient or wild type B6 mice, the level of HBV transcript, replicative intermediate, and antigenemia were higher in iNOS −/− than in B6 mice. The intrahepatic leukocytes in iNOS −/− mice were also affected after transfection. Conclusions : Our data suggests that the iNOS expression not only affects the HBV clearance, but also modulates the infiltrating leukocytes response to HBV antigens.

Summary. Natural killer (NK)/T-cell lymphoma is a special subtype of rare malignant lymphoma that is more prevalent in Asia than in America and Europe. This newly characterized haemato-lymphoid malignancy is highly aggressive and frequently present in nasal and upper aerodigestive sites. Several studies have reported the commonly deleted region of chromosome 6q21–25 in this particular type of lymphoma. To refine the smallest region of overlapping (SRO) deletion for localization of potential tumour suppressor (TS) genes, we performed loss of heterozygosity (LOH) and homozygosity mapping of deletion (HOMOD) analyses on 37 nasal and nasal-type NK/T-cell lymphoma patients using a panel of 25 microsatellite markers, covering the 6q21–q25 region. In all patients studied, LOH was detected in eight (89%) paired-sample patients, while hemizygous deletion was detected in three (11%) single-sample patients. Combination of the LOH and HOMOD results defined a distinct 3 Mb SRO on chromosome 6q25. Quantitative multiplex polymerase chain reaction analysis of 10 sequence-tagged sites further refined the putative TS-gene-containing region to a 2·6 Mb interval between TIAM2 and SNX9. Eighteen known genes/Unigene clusters and 25 hypothetical genes are located within this 2·6 Mb region, but none are previously identified TS genes. These results provide a framework for future positional cloning of novel TS gene(s) at 6q25.2–q25.3.

Reactivation of hepatitis is one of the most serious complications of chemotherapy in lymphoma patients who are carriers of the hepatitis B virus (HBV). Glucocorticoids are linked to increased risk of HBV reactivation. This study seeks to clarify whether removal of glucocorticoids from chemotherapy regimens may decrease the risk of HBV reactivation. Eligible patients were seropositive for hepatitis B surface antigen (HBsAg) and had histologically proven non-Hodgkin's lymphomas for which intensive chemotherapy was indicated. Patients were randomized to receive either ACE (epirubicin, cyclophosphamide, and etoposide) or PACE (prednisolone + ACE). A total of 50 patients were enrolled, 25 each for the ACE and PACE arms. The cumulative incidence of HBV reactivation at 9 months after starting chemotherapy was 38% and 73% for ACE and PACE arm, respectively (P =.03). The degree of clinical hepatitis was significantly more severe in the PACE arm: 11 patients (44%) in the PACE and 3 patients (13%) in the ACE arm had ALT elevation more than 10-fold of normal (P =.025), and 7 patients (28%) in the PACE and 1 patient (4%) in the ACE arm had icteric hepatitis (P =.049). Complete remission of tumors occurred in 11 (46%) patients in the PACE and 8 (35%) patients in the ACE arm (P =.556). The estimated overall survival rate at 46 months was 68% in the PACE arm and 36% in the ACE arm, respectively (P =.18). In conclusion, steroid-free chemotherapy decreases the incidence and severity of HBV reactivation in HBsAg-positive lymphoma patients. However, further research is needed to evaluate whether steroid-free chemotherapy may confer a less satisfactory control of lymphoma.

EBV infects most of the global population, but only a small percentage of infected individuals develop EBV-associated malignancies. Host and viral factors may play a role in determining the clinical outcome. Because EBNA-1 functions as an oriP binding protein and links the episomal viral DNA to metaphase chromosomes, variation of EBNA-1 has been suggested to contribute to determining the tissue tropism of EBV and the development of various EBV-associated diseases. Five subtypes have been described, according to the amino acid at residue 487: P-ala (B95-8 prototype), P-thr (aa 487 ala to thr), V-val, V-pro and V-leu. Other studies, however, concluded that EBNA-1 sequence variation simply reflects the geographical distribution of EBV. To clarify these possibilities, we collected DNA samples from healthy individuals and patients with various EBV-associated diseases in Taiwan for PCR amplification and DNA sequencing. The results indicate that: 1) V-val EBNA-1 was detected in patients with nasopharyngeal carcinoma (NPC) and other EBV-associated malignant diseases; 2) the prototype P-ala strain was detected only in peripheral blood lymphocytes; 3) mixed populations of different subtypes of N-terminal and C-terminal sequences were observed in samples from one patient with nasopharyngeal carcinoma, one with T lymphoma and one with infectious mononucleosis sample; 4) intermediate variations between P-ala and V-val were observed in T-lymphoma, Hodgkin disease and infectious mononucleosis samples; and 5) in comparison with the major sequences identified in healthy carriers, the EBNA-1 sequences in peripheral lymphocytes from nasopharyngeal carcinoma were mixed types in 4 of 5 patients, implying increasing frequency of V-val might correlate with the progression of nasopharyngeal carcinoma.

The infection of human T cells by Epstein-Barr virus (EBV) may result in a fatal hemophagocytic syndrome (HS). We have previously shown that EBV can selectively upregulate the tumor necrosis factor-alpha (TNFalpha) gene and lead to activation of macrophages in a manner similar to the pathobiology of HS in EBV-infected T lymphoproliferative disorders (LPDs). This study was designed to further clarify the specific EBV gene product(s) responsible for TNFalpha upregulation. RT-PCR analysis of EBV gene expression was performed on 2 CR2-transfected EBV-infected T lymphoma lines and 2 EBV-infected B cell lines. To identify the EBV gene responsible for upregulation of TNFalpha, 2 reporter recombinant plasmids, pTNF-CAT and pTNFalpha-Luc, were then constructed and cotransfected with the expression plasmids of the EBV latent and lytic genes (EBNA-1, EBNA-2, LMP-1, LMP-2A, and BZLF-1) in both T and B cell lines. Analyses using ELISA and Western blotting were further performed to detect the secreted TNFalpha. The results revealed that EBNA-1 and LMP-1 were consistently expressed in EBV-infected T cell lines (type II latency), while a type III latency with expression of EBNA-1, EBNA-2, LMP-1, and lytic BZLF transcripts was detected in EBV-infected B cell lines. LMP-1 was demonstrated to be the only EBV gene product to transactivate the TNFalpha gene, and this phenomenon was observed only in T, not in B, cells. Enhanced secretion of TNF-alpha protein was also detected in LMP1-transfected T cell lines. We concluded that LMP1 is the candidate protein in the upregulation of the TNFalpha gene in T cells and is probably responsible for the pathogenesis of HS in EBV-infected T lymphoproliferative disorders.

Metabolic syndrome has closely linked to the development of human hepatocellular carcinoma (HCC). By using the hepatitis B virus (HBV) X (HBx) transgenic mouse model, we studied the dynamic evolution of serum and liver profiles of lipids and global cDNA expression at different stages of HBx tumorigenesis. We observed that the lipid (triglycerides, cholesterol, and fatty acids) profiles revealed a biphasic response pattern during the progression of HBx tumorigenesis: a small peak at early phase and a large peak or terminal switch at the tumor phase. By analyzing cDNA microarray data, the early peak correlated to the oxidative stress and pro-inflammatory response, which then resolved at the middle phase and were followed by the terminal metabolic switch in the tumor tissues. Five lipid metabolism-related genes, the arachidonate 5-lipoxygenase, lipoprotein lipase, fatty acid binding protein 4, 1-acylglycerol-3-phosphate O-acyltransferase 9, and apolipoprotein A-IV were identified to be significantly activated in HBx transgenic HCCs and further validated in human HBV-related HCCs. Inhibition of these lipid genes could reverse the effect of HBx on lipid biosynthesis and suppress HBx-induced cell proliferation in vitro. Our results support the concept that metabolic syndrome plays an important role in HBV tumorigenesis. The dysregulation of lipid metabolic genes may predict the disease progression to HCC in chronic hepatitis B patients.

BACKGROUND We present the first comprehensive analysis of Mycobacterium tuberculosis (MTB) isolates circulating in southern Taiwan. In this 9-year population-based study, the TB situation in the Kaohsiung region was characterized by genotypic analysis of 421 MTB isolates. METHODS All 421 isolates of MTB were analyzed by spoligotyping and MIRU-VNTR typing. Drug-resistance patterns were also analyzed. RESULTS The percentage of EAI (East African-Indian) strains increased across sampling years (2000-2008) in southern Taiwan, whereas the proportion of Beijing lineages remained unchanged. Clustering was more frequent with EAI genotype infections (odds ratio = 3.6, p

Neoplasms of histiocytes and dendritic cells are rare, and their phenotypic and biological definition is incomplete. Seeking to identify antigens detectable in paraffin-embedded sections that might allow a more complete, rational immunophenotypic classification of histiocytic/dendritic cell neoplasms, the International Lymphoma Study Group (ILSG) stained 61 tumours of suspected histiocytic/dendritic cell type with a panel of 15 antibodies including those reactive with histiocytes (CD68, lysozyme (LYS)), Langerhans cells (CD1a), follicular dendritic cells (FDC: CD21, CD35) and S100 protein. This analysis revealed that 57 cases (93%) fit into four major immunophenotypic groups (one histiocytic and three dendritic cell types) utilizing six markers: CD68, LYS, CD1a, S100, CD21, and CD35. The four (7%) unclassified cases were further classifiable into the above four groups using additional morphological and ultrastructural features. The four groups then included: (i) histiocytic sarcoma (n =18) with the following phenotype: CD68 (100%), LYS (94%), CD1a (0%), S100 (33%), CD21/35 (0%). The median age was 46 years. Presentation was predominantly extranodal (72%) with high mortality (58% dead of disease (DOD)). Three had systemic involvement consistent with `malignant histiocytosis'; (ii) Langerhans cell tumour (LCT) (n =26) which expressed: CD68 (96%), LYS (42%), CD1a (100%), S100 (100%), CD21/35 (0%). There were two morphological variants: cytologically typical (n =17) designated LCT; and cytologically malignant (n =9) designated Langerhans cell sarcoma (LCS). The LCS were often not easily recognized morphologically as LC-derived, but were diagnosed based on CD1a staining. LCT and LCS differed in median age (33 versus 41 years), male:female ratio (3.7:1 versus 1:2), and death rate (31% versus 50% DOD). Four LCT patients had systemic involvement typical of Letterer-Siwe disease; (iii) follicular dendritic cell tumour/sarcoma (FDCT) (n =13) which expressed: CD68 (54%), LYS (8%), CD1a (0%), S100 (16%), FDC markers CD21/35 (100%), EMA (40%). These patients were adults (median age 65 years) with predominantly localized nodal disease (75%) and low mortality (9% DOD); (iv) interdigitating dendritic cell tumour/sarcoma (IDCT) (n =4) which expressed: CD68 (50%), LYS (25%), CD1a (0%), S100 (100%), CD21/35 (0%). The patients were adults (median 71 years) with localized nodal disease (75%) without mortality (0% DOD). In conclusion, definitive immunophenotypic classification of histiocytic and accessory cell neoplasms into four categories was possible in 93% of the cases using six antigens detected in paraffin-embedded sections. Exceptional cases (7%) were resolvable when added morphological and ultrastructural features were considered. We propose a classification combining immunophenotype and morphology with five categories, including Langerhans cell sarcoma. This simplified scheme is practical for everyday diagnostic use and should provide a framework for additional investigation of these unusual neoplasms.

Background : Human herpesvirus 8 (HHV-8), a γ-herpesvirus, is associated with Kaposi's sarcoma (KS). A variety of assays for the diagnosis HHV-8 infection have been developed. However, the accuracy of current HHV-8 antibody testing is uncertain, mainly in patients with asymptomatic HHV-8 infection. Methods : We utilized a new immunoblot assay, comprised of three recombinant HHV-8 encoded proteins (ORF16, ORF57, and ORF71) to determine the seroprevalence of HHV-8 in Taiwan. ORF16 encodes a Bcl-2 homologue and is expressed only in the lytic cycle. ORF57 is an immediate–early gene product and can activate gene expression by a post-transcriptional mechanism. ORF71 encodes a viral FLICE inhibitory protein and is a latently expressed protein. We collected 6 KS(+)/HIV(−) patients, 10 KS(−)/HIV(+) individuals, 174 blood donors, and 62 non-KS cancer patients in this study. Results : Among the different populations, ORF57 showed highest reactivities. The frequency of positive results for KS(+)/HIV(−), KS(−)/HIV(+), blood donors, and non-KS cancer patients were 83.3%, 40%, 23%, and 12.9%, respectively. Compared to the widely used lytic immunofluorescence assay, our immunoblotting assay using ORF57 showed higher reactivities among the blood donors. Conclusions : ORF57 is useful for the detection of HHV-8 infection, especially during the asyptomatic infections.

Induction of apoptosis of virus-infected cells is an important host cell defense mechanism. It is well documented that T cells may undergo apoptosis due to interactions between Fas and Fas ligand (FasL). In addition, signals that induce apoptosis in T cells can result from interaction of tumor necrosis factor (TNF)-alpha with TNF receptors (TNFRs). It has been shown that human T cell lines expressing HTLV-I have decreased sensitivity to Fas-mediated apoptosis. The susceptibility of HTLV-I-infected cells to TNF-alpha-induced apoptosis remains to be elucidated. In the present study, we examined the expression of TNFRs on HTLV-I-infected T cell lines that expressed T-cell activation markers and thus phenotypically resemble activated T cells. Different from primary activated T cells that expressed both TNFRs, none of the five HTLV-I-infected T cell lines studied had detectable TNFR1 and only three had TNFR2 on their cell surfaces, although, the RNA transcripts of both TNFR genes could be detected via reverse transcription-polymerase chain reaction in these cell lines. The T cell blasts, which we activated in vitro, were sensitive to apoptosis induced by TNF-alpha and by antibodies to TNFR1 and/or TNFR2. However, all of the HTLV-I-infected cell lines expressing TNFR2 were resistant to TNF-alpha-mediated apoptosis. These findings suggest that HTLV-I infection may interfere with the autonomous suicide programs of T cells, not only Fas/FasL but also TNFRs/TNF-alpha pathways, to prolong the life of the infected cells. This may contribute to viral persistence and favor survival and subsequent expansion of dysregulated infected T cells with the potential to produce HTLV-I-associated autoimmune-like diseases or malignancies.

Betel quid (BQ) chewing has been a well-documented cause of oral epithelial lesions (OEL). Evolution from early hyperplastic lesions to the late or carcinomatous stage has been recognized. The pathobiological and molecular mechanism, however, remains to be elucidated. In this study, a total of 232 samples obtained from 153 cases of BQ-related OEL were retrospectively evaluated for the expression of p53 and bcl-2 in comparison with 26 cases of BQ-unrelated lesions (n = 29). The possible role of human papillomavirus (HPV) was also investigated. These BQ-related OELs included verrucous hyperplasia (VIH, n = 57, 24.6%), epithelial dysplasia (n = 23, 9.9%), verrucous carcinoma (VC, n = 5, 2.1%) and squamous cell carcinoma (SCC, n = 106, 45.7%). Fifty-four cases (35.3%) had multiple lesions. In comparison with the BQ-unrelated OELs, the characteristics of BQ-related OELs were a younger age, male predilection and multicentricity. In contrast to the tongue in BQ-unrelated OELs, the most common site for all types of BQ-related lesions was the buccal mucosa. Immunohistochemical studies of BQ-related lesions showed p53 staining in 30% of dysplasia and 38% of SCC, but a consistent absence in VH and VC. The cases with p53-positive SCC had a higher recurrence rate than p53-negative ones. Bcl-2 expression was negligible for all types of lesions. HPV-6/11 was detectable in 10% of dysplasia and 13% of SCC, but in neither VH nor VC. HPV-16/18, however, was consistently negative for all types of lesions. Our data suggest that p53, but not bcl-2, may play a role in tumor progression of BQ-related OELs, and that VH and VC are distinct and closely related histological lesions. The consistent absence of the malignant-type HPV in all BQ-related lesions suggests that HPV plays an insignificant role in the tumorigenesis of BQ-related oral cancers, although a cooperative role may exist between the benign-type HPV and BQ chewing.

Two outbreaks of hand-foot-and-mouth disease (HFMD) occurred in Taiwan between 1998 and 2000. Enteroviruses were isolated from a total of 1,892 patients in this laboratory during this period. Of the virus isolates, enterovirus 71 (EV71) was diagnosed in 44.4% of the patients (132 of 297) in 1998, 2% (13 of 646) in 1999, and 20.5% (195 of 949) in 2000. Genetic analyses of the 5′-untranslated and VP1 regions of EV71 isolates by reverse transcription-PCR and sequencing were performed to understand the diversity of EV71 in these outbreaks of HFMD. Most EV71 isolates from the 1998 epidemic belonged to genotype C, while only one-tenth of the isolates were genotype B. Interestingly, all EV71 isolates tested from 1999 to 2000 belonged to genotype B. This study indicated that two genogroups of EV71 capable of inducing severe clinical illness have been circulating in Taiwan. Furthermore, the predominant EV71 genotypes responsible for each of the two major HFMD outbreaks within the 3-year period in Taiwan were different.

We previously reported the therapeutic efficacy of 13-cis retinoic acid (13-cRA) in some subtypes of peripheral T-cell lymphoma (PTCL). This study sought to clarify if the addition of interferon-alpha2a (IFN-alpha2a), an agent with synergistic cytotoxicity with 13-cRA in many types of malignant cells, may be more effective in the treatment of PTCL. Eligible patients has histologically proven PTCL, which was recurrent after or refractory to anthracycline-containing systemic chemotherapy. The treatment included oral administration of 13-cRA 1 mg/kg/day, divided into three doses, and intramuscular injection of IFN-alpha2a 4.5 MU/M2, three times per week. From March 1995 to July 2000, a total of 17 patients, 10 men and 7 women, with a median age of 47 years (range, 18-77 years), were recruited. The histologic diagnosis included 7 cases of unspecified PTCL, 6 cases of Ki-1 anaplastic large cell lymphoma (ALCL), 1 case of angioimmunoblastic T-cell lymphoma, and 3 cases of angiocentric nasal NK/T cell lymphoma. They received a median of 1.7 months of treatment (range, 0.4-13.3 months). One patient refused further treatment due to toxicity. The doses of 13-cRA and IFN-alpha2a had to be decreased in 7 and 7 patients, respectively. Grade III/IV hematologic and non-hematologic toxicity developed in 2 and 5 patients, respectively. There were 5 partial responses (Ki-1, 4; unspecified PTCL, 1), with a total response rate of 31.3% (95% CI, 5.7-56.8%). The median duration of response for the responders was 2.5 months (range, 0.8-7.2 months). The median overall survival for the entire group of patients was 3.6 months. In conclusion, a combination of 13-cRA and IFN-alpha2a is a useful salvage treatment for selected patients with recurrent or refractory PTCL, particularly those with the Ki-1 subtype. However, the data does not support that addition of IFN-alpha2a is superior to 13-cRA alone.

Although hepatitis B virus (HBV) has been established to cause hepatocellular carcinoma (HCC), the exact mechanism remains to be clarified. Type II ground glass hepatocytes (GGHs) harbouring the HBV pre-S2 mutant large surface protein (LHBS) have been recognized as a morphologically distinct hallmark of HCC in the advanced stages of chronic HBV infection. Considering its preneoplastic nature, we hypothesized that type II GGH may exhibit high genomic instability, which is important for the carcinogenic process in chronic HBV carriers. In this study we found that pre-S2 mutant LHBS directly interacted with importin α1, the key factor that recognizes cargos undergoing nuclear transportation mediated by the importin α/β-associated nuclear pore complex (NPC). By interacting with importin α1, which inhibits its function as an NPC factor, pre-S2 mutant LHBS blocked nuclear transport of an essential DNA repair and recombination factor, Nijmegen breakage syndrome 1 (NBS1), upon DNA damage, thereby delaying the formation of nuclear foci at the sites of DNA double-strand breaks (DSBs). Pre-S2 mutant LHBS was also found to block NBS1-mediated homologous recombination repair and induce multi-nucleation of cells. In addition, pre-S2 mutant LHBS transgenic mice showed genomic instability, indicated by increased global gene copy number variations (CNVs), which were significantly higher than those in hepatitis B virus X mice, indicating that pre-S2 mutant LHBS is the major viral oncoprotein inducing genomic instability in HBV-infected hepatocytes. Consistently, the human type II GGHs in HCC patients exhibited increased DNA DSBs representing significant genomic instability. In conclusion, type II GGHs harbouring HBV pre-S2 mutant oncoprotein represent a high-risk marker for the loss of genome integrity in chronic HBV carriers and explain the complex chromosome changes in HCCs. Mouse array CGH raw data: GEO Accession No. GSE61378 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61378).

The recurrence rate of Mycobacterium tuberculosis in Taiwan is 3%. Here, we present the draft genome sequences of M. tuberculosis strains A2 and A4 from a relapse patient. The draft genome sequences comprise 4,443,031 bp and 4,487,096 bp, revealing 4,220 and 4,143 coding sequences for A2 and A4, respectively, as well as 49 tRNA genes for the both isolates.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Previous studies have noted the induction of endoplasmic reticulum stress or apurinic endonuclease 1 (APE1) expression in many tumors. Therefore, the aim of this study was to investigate the relationship between endoplasmic reticulum (ER stress) and APE1 in hepatocellular carcinoma. Here we investigate the expression of APE1 during ER stress in HepG2 and Huh-7 cell lines. Tunicamycin or brefeldin A, two ER stress inducers, increased APE1 and GRP78, an ER stress marker, expression in HepG2 and Huh-7 cells. Induction of APE1 expression was observed through transcription level in response to ER stress. APE1 nuclear localization during ER stress was determined using immunofluorescence assays in HepG2 cells. Furthermore, expression of Hepatitis B virus pre-S2∆ large mutant surface protein (pre-S2∆), an ER stress-induced protein, also increased GRP78 and APE1 expression in the normal hepatocyte NeHepLxHT cell line. Similarly, tumor samples showed higher expression of APE1 in ER stress-correlated liver cancer tissue in vivo. Our results demonstrate that ER stress and HBV pre-S2∆ increased APE1 expression, which may play an important role in resistance to chemotherapeutic agents or tumor development. Therefore, these data provide an important chemotherapeutic strategy in ER stress and HBV pre-S2∆-associated tumors.

Purpose A phase II trial was conducted to evaluate the therapeutic efficacy and safety profiles of frontline concurrent chemoradiotherapy (CCRT) plus consolidation chemotherapy for patients with stage I/II nasal natural killer/T-cell lymphoma (NKTCL). Patients and methods Patients with newly diagnosed, measurable stage I/II nasal NKTCL were eligible. The CCRT included two cycles of the DEP regimen (dexamethasone, etoposide, and cisplatin) every 4 wk with concurrent 5040 cGy radiation in 28 fractions for 5 wk. Patients without disease progression after CCRT were subjected to two cycles of DVIP consisted of dexamethasone, etoposide, ifosphamide, mesna, and cisplatin every 4 wk. The primary endpoint was tumor response rate, and secondary endpoints were survival and toxicities. This phase II study has been registered in the ClinicalTrials.gov (NCT00292695). Results Thirty-three patients received CCRT, and 29 patients received two cycles of consolidation DVIP after CCRT. Among the 32 evaluable patients, 20 achieved complete response and 6 achieved partial response. The overall and complete response rate was 81% (95% CI, 68–95%) and 63% (95% CI, 46–79%), respectively. The 2-yr and 5-yr progression-free survival rate for intention-to-treat population was 64% (95% CI, 47–80%) and 60% (95% CI, 39–73%), respectively; while the corresponding overall survival rate was 73% (95% CI, 57–88%) and 66% (95% CI, 50–83%), respectively. The most common treatment-related grade 3/4 adverse event was leukopenia (85%). Conclusion Frontline CCRT plus consolidation chemotherapy is feasible and effective for treating localized nasal NKTCL.

Tuberculosis remains a major infectious disease in Taiwan. Here we present the draft genome sequence of the Mycobacterium tuberculosis C2 strain, belonging to the Latin American–Mediterranean lineage. The draft genome sequence comprises 4,453,307 bp with a G+C content of 65.6%, revealing 4,390 coding genes and 45 tRNA genes.

Aims Follicular dendritic cell tumours are very rare neoplasms that often occur in lymph nodes. We report here a case in the colon, a hitherto unreported site, in a 37-year-old female. The differentiation from gastrointestinal stromal tumour is emphasized. Methods and results The tumour was tan, elastic and solid with surface ulceration. Microscopically, it was composed of oval to spindle tumour cells with syncytial cytoplasm arranged in fascicular and whorled patterns. There were many infiltrating lymphocytes. The histological appearance resembled gastrointestinal stromal tumour, thymoma or meningioma. Distinct from the stromal tumour, the lymph node was also involved by the tumour. Immunohistochemically, the tumour cells were positive for CD21, CD35 and CD68, but negative for cytokeratin, CD34, smooth muscle actin, desmin, S100 protein, epithelial membrane antigen, leukocyte common antigen, HMB-45 and c-kit. In-situ hybridization study was negative for Epstein-Barr virus RNA sequences. Ultrastructurally, the tumour cells possessed cytoplasmic processes joined by desmosomes. Conclusions This entity should be considered in the list of differential diagnoses for gastrointestinal stromal tumour. The lymph node metastasis and immunohistochemical features are of value for identification of this rare neoplasm.

Retinoic acid (RA) has been used to induce the regression of refractory T-cell lymphoma. In vitro and in vivo studies have shown that RA exerts this effect through the induction of apoptosis. This study was designed to investigate the molecular pathway of RA-induced apoptosis in T-lymphoma cell lines.RA-induced apoptosis was verified by morphology, flow cytometry, and DNA ladder analysis. Differential display method using a combination of 12 poly(A)-anchored primers and 20 arbitrary primers was adopted for gene cloning. Total RNAs were extracted from H9 cell line at 0, 6, 12, and 24 hours after All-trans RA (ATRA) treatment and the serial expression patterns of the candidate fragments were recognized. The cloned gene fragments were then analyzed and confirmed by Northern blot analysis on H9 and SR786 cell lines.ATRA-induced apoptosis of T-cell lymphoma was protein synthesis-dependent. The execution or irreversible phase of apoptosis appeared to occur at 6-12 hours of RA treatment. Among the 60,000 arbitrarily displayed bands, 25 of 250 candidate fragments were selected for further cloning and sequencing. A total of 14 clones could be matched to known genes and were categorized into four groups: A) transcription factors: prothymosin, CA150, p78 serine/threonine kinase, IL-1beta-stimulating gene, glucocorticoid receptor, MLN64/CAB1, gastrin-binding protein, and polypeptide from glioblastoma; B) chaperone: 90 kDa heat shock protein; C) ion channel: chloride channel protein 3; and D) cytoskeleton: cytovillin2/ezrin and vimentin. Another two clones of genes were of unrecognized functions. The remaining 11 clones belonged to unmatched or novel genes. The expression of these genes varied, either upregulated or downregulated, in response to ATRA treatment.RA-induced apoptosis may involve a cascade of genes that are related to transcription regulation, stress response, housekeeping, and the execution of apoptosis. The clarification of the RA-induced apoptotic pathway will help us to understand the molecular mechanism of cancer differentiation agents.

Experimental infection with enterovirus type 71 (EV71) induced death in neonatal mice in an age- and dose-dependent manner. The mortality rate was 100% following intraperitoneal inoculation 1-day-old ICR mice and this gradually decreased as the age at the time of inoculation increased (60% in 3-day-old mice and no deaths occurred in mice older than 6 days of age). A lethal dose greater than 10(8) PFU was necessary. Lethargy, failure to gain weight, rear limb tremors and paralysis were observed in the infected mice before death. EV71 was isolated from various tissues of the dead mice. Using a reverse transcription polymerase chain reaction technique with a specific primer pair, a 332-bp product was detected in the tissues that produced a culture positive for EV71. Protection against EV71 challenge in neonatal mice was demonstrated following passive transfer of serum from actively immunized adult mice 1 day after inoculation with the virus. Pups from hyperimmune dams were resistant to EV71 challenge. Additionally, maternal immunization with a formalin-inactivated whole-virus vaccine prolonged the survival of pups after EV71 lethal challenge.

Background: An epidemic of enterovirus 71 (EV71) occurred in Taiwan from April to December of 1998, with two peaks, one in June and the other in October. Many enteroviruses were isolated in our laboratory from 258 cases during this outbreak. Approximately half of the enteroviruses isolated were EV71 and one fifth were coxsackievirus A16. Objectives: To analyze laboratory findings in the EV71 epidemic of 1998 in Taiwan, various EV71 specimens in different cell lines were examined. In addition, genetic analysis of 5′ non-coding region (NCR) was performed to analyze the strain variation in this outbreak. Results: The cytopathic effect induced by EV71 was observed 2–13 (mean of 4.5) days post-inoculation in Vero cells and 4–15 (mean of 6.6) days in green monkey kidney (GMK) cells inoculated with throat swabs. Of the total positive EV71 cases, virus was most frequently obtained from throat swabs (91.7%), less from stools (64.8%), and none from cerebral spinal fluid (CSF). Molecular analyses of EV71 by sequencing the 5′ NCR of 34 strains obtained from different clinical categories and various geographic areas showed that their sequences differed (0–13 bp in 681 bp sequenced) by ∼0–2%. The sequences of these isolates differed from EV71 prototype BrCr or MS strain by 17.5–19%, with the exception of two samples which exhibited nucleotide variation by only 8.9 and 8.2%, when compared to the MS strain. Conclusion: EV71 was most frequently isolated from throat swab specimens in Vero cells. The molecular analyses of the 5′ NCR of EV71 revealed that most isolates from this epidemic belonged to a group of closely related clones and only two were in a different group which was clustered with the EV71 MS strain.

Background: In a recent enterovirus outbreak in Taiwan, serotype 71 was the culprit of encephalitis causing rapid clinical deterioration and death among young children. Objectives: Since knowledge of enterovirus 71 (EV71) infection in the central nervous system is still limited, the purpose of the present case study was attempted to uncover the pathogenesis of the virus. Study design: We performed a detailed pathological examination, virological and molecular studies on a case of EV71 infection with a rapidly fatal outcome. In addition, the whole genome of the virus was sequenced to determine the genetic relationships to other enteroviruses and two other EV71 strains (a prototype BrCr and a neurovirulent MS strain), and to provide the genetic basis of its neurovirulence of the new isolate, NCKU9822 strain. Results: Characteristic features of acute encephalomyelitis were observed, with most prominent lesions in the spinal cord and brain stem. Mild myocarditis and pancreatitis were also noticed. EV71 antigen was localized to neurons on immunohistochemical staining. EV71 was recovered from all organs with inflammatory reaction. Sequence analysis showed that overall NCKU9822 and the two EV71 strains shared 80% nucleotide identity and 95% amino acid identity. It had only 45% amino acid and 52% nucleotide identities with polioviral P1 capsid region. Conclusion: The spinal cord and brain stem were the main targets of EV71 in the fatal cases in this outbreak, however, heart and pancreas might also be involved. Since the amino acid sequences in the P1 region are conserved (97% identity) among the three EV71 strains as compared to other enteroviruses and polioviruses, these EV71 neurovirulent strains might share the same mechanisms of neurovirulence, and the mechanisms might be different from those in polioviruses.

Background and Aims: The expression of hepatitis B viral (HBV) antigens in liver tissue reflects the replicative status of chronic HBV infection. We have previously recognized a novel marginal pattern of hepatitis B surface antigen (HBsAg) in hepatocytes, which usually clusters in groups and emerges at the late non-replicative phase. This study was designed to investigate whether the marginal-type HBsAg represented the gene product of a specific HBV-surface mutant. Methods: Microdissection of cirrhotic nodules homogeneously expressing marginal HBsAg was performed on two of 12 resected livers from HBsAg-seropositive patients with hepatocellular carcinoma. The gene presumably encoding marginal HBsAg was polymerase chain reaction (PCR)-cloned, sequenced and analysed. In vitro transfection and expression of the cloned surface mutant plasmids were performed on the Huh7 cell line to illustrate intrahepatic HBsAg expression. Results: Immunohistochemical staining revealed that the marginal HBsAg was positive for pre-S1 and thus contained large surface proteins.The PCR cloning and sequencing of the genes presumably encoding marginal-type HBsAg in both cases revealed the same deletion at the 5¢ terminus (nt 2‐55) of preS2. A point mutation on the small-surface (S) antigen was also found in one case. The pre-S2 deletion sequence and the mutation sites of the S gene coincide with human lymphocyte antigen-restricted Tand/or B-cell epitopes. In vitro transfection of the mutant plasmid revealed a blot-like retention or accumulation of HBsAg in the cytoplasm or at the periphery of hepatocytes, accompanied by a decreased secretion of HBsAg in the culture supernatant, mimicking intrahepatic expression. Conclusion: A natural pre-S2 deletion mutant was identified in hepatocytes expressing a novel marginal pattern of HBsAg, which probably contains mutant, large, surface proteins. The biological significance of the pre-S2 deletion mutant should be interesting in view of the clustering proliferation of hepatocytes expressing marginal HBsAg. © 2000 Blackwell Science Asia Pty Ltd

Nasopharyngeal carcinoma (NPC) is an epithelial cancer with a high incidence in Southeast Asia. How it escapes attack from the host immune system is not fully understood. Recently, pieces of evidence show that Fas-ligand (Fas-L)-mediated apoptosis may be involved in immune privilege of tumours. To determine whether a similar mechanism may exist in NPC, the expression of Fas-L was analysed. Biopsy specimens of the nasopharynx were taken from 27 NPC patients. Histologically, they were either non-keratinizing or undifferentiated carcinomas. Nasopharyngeal biopsies of 11 other patients that proved to have no tumour served as control. The transcripts of Fas-L were detected by reverse transcription-polymerase chain reaction. Localization of Fas-L protein was performed with immunohistostaining using an antibody recognizing human Fas-L. All nasopharyngeal tissues have a similar amount of transcripts of Fas-L. However, the Fas-L protein was detected exclusively on the cell surface of malignant epithelial cells of NPC. The present findings suggest that Fas-L protein may be involved in evading immune attack of NPC.

Intravascular lymphomatosis (IL) is an unusual neoplasm characterized by multifocal proliferation of lymphoma cells exclusively within the blood vessels. We report here a patient with acquired immunodeficiency syndrome (AIDS) and disseminated Kaposi's sarcoma. A 233-bp amplification product of HHV-8 was detected in the DNA extracted from specimens of Kaposi's sarcoma at different sites by polymerase chain reaction (PCR). At autopsy, the vessels within the Kaposi's sarcoma were dilated and filled with atypical large mononuclear cells. No such feature was seen in the vessels of non-Kaposi's sarcomatous regions. Immunohistochemically, the spindle cells of Kaposi's sarcoma were positive for CD31 (endothelial cell marker). The intravascular tumor cells were positive for CD45 (leukocyte common antigen) but negative for others, including chloroacetate esterase, CD45-RO (UCHL-1, Pan-T), CD3, CD43, CD20 (L26, Pan-B), CD30 (Ki-1), immunoglobulin heavy chains and light chains, CD56 (natural killer cell antigen), and CD31. Monoclonal rearrangement of immunoglobulin heavy chain gene was detected in the DNA extracts from fresh tissue of Kaposi's sarcoma by PCR, which indicated that the lymphoma cells within the Kaposi's sarcoma were of monoclonal B cell origin. In situ hybridization revealed that EBER-1 transcripts were present in the lymphoma cells of IL but not in the spindle cells of Kaposi's sarcoma. To the authors' best knowledge, this is the first instance of IL in an AIDS patient with direct evidence of EBV association.

Aim BRCA1, a nuclear phosphoprotein, normally functions as a negative regulator of the cell cycle and may be an active inhibitor of neoplastic progression. Mutation of the BRCA1 gene has been demonstrated in 80% of familial breast cancer. Decreased mRNA levels or aberrant subcellular locations of BRCA1 have been identified in breast cancer lines and in sporadic cases of breast cancer tissues. The expression of BRCA1 in large series of variously differentiated breast carcinomas with correlation with other biological parameters has not been clarified. Methods and results The BRCA1 expression in normal breast tissue (n = 15) and in sporadic cases of invasive ductal carcinoma (n = 108) was determined using immunohistochemistry. BRCA1 expression was correlated with other prognostic parameters including p53, c-erbB-2, bcl-2, oestrogen receptor (ER), histological grade, tumour size, axillary lymph node status and age. BRCA1 was exclusively (100%) localized in the nuclei of normal ductal and lobular epithelia. However, this nuclear expression pattern was variable in breast carcinoma (76.8%). Loss of nuclear BRCA1 expression (22 of 108 cases, 20.4%) correlated well with high histological grade (P

We aimed to develop an H. pylori-infected mousemodel using clinically stored strains in Taiwan and totest whether development of H. pylori infection in an invivo animal model is related to the status of the cagA gene. A total of 100 male BALB/cmice, 6-8 weeks old, including 80 in the experimentalgroup and 20 in the control group, were used. Twoclinically stored H. pylori isolates, a cagA-positive and a cagA-negative strain, were selected toinduce the H. pylori infection in half (N = 40) of themice in the experimental group. Bacterial isolates of0.8 × 109 CFU/ml were orally inoculatedin each mouse of the experimental group for threeconsecutive days. Ten mice in the control group weresacrificed to confirm the initial absence of H. pylori.Eight weeks after inoculation of the experimental group and no inoculation of the remaining 10mice of the control group, each mouse was killed.Gastrectomy was then performed for rapid urease test(CLOtest) and histology. In the control group, none of 20 mice had positive results from the CLOtestor histology. In contrast, excluding eight of 80 micethat died before the eighth week, 90.3% (65/72) of themice challenged with H. pylori showed persistent presence of H. pylori by histology. Theseverity of gastritis at the eighth week was moreevident in H. pylori-infected mice than in control andnoninfected mice (P 0.05). In summary, storedstrains of H. pylori can be applied to induce aninfection model in BALB/c mice. The less virulentcagA-negative strain can induce H. pylori infection inmice as effectively as the cagA-positive strain. Thehigh prevalence of cagA-positive strains in Taiwanesepatients may be related to factors other than only thecagA gene of the bacteria.

Lymphoproliferative diseases of large granular lymphocytes (LDGL) may arise from either CD3+ T cells or CD3− natural killer (NK) cells. LDGL with clonal proliferation of large granular lymphoeytes (LGL) is defined as LGL leukaemia. The number of patients with NK-LGL leukaemia reported is limited and the pathogenesis of the disease is not yet clear. From 1991 to 1998 six patients with cytogenetically proved clonal disease of NK-LGL were identified in our institute. All were seropositive for Epstein-Barr virus (EBV). EBV RNA or DNA could be detected in LGL from four patients by EBV in situ hybridization or Southern blot analysis. Most patients ran an aggressive clinical course and five died of the disease. Nonrandom clonal chromosomal abnormalities, including duplication of 1q, rearrangement at 3q and loss of chromosomes Y, 13 or 10, were noted in the six patients from this study and in eight from the literature. The implications of these recurrent cytogenetic aberrations in the development and progression of the disease deserve further studies.

The long-term outcome of 22 children treated with etoposide-containing regimens for haemophagocytic syndrome (HS) were longitudinally studied; none of them had a family history of the disease. All patients received etoposide-containing (150 mg/m2/d) regimens, combined, in 16 cases, with intravenous immunoglobulin (IVIG) and prednisolone. Complete remission (CR) was achieved in 12 patients, partial remission in seven, and early mortality occurred in three. Of the 12 CR patients, only four remain alive and disease-free, with a median follow-up of 47.4 months; one CR patient died due to infection and the remaining seven had relapsed diseases. Three patients with a partial response or with relapsed disease progressed to T-cell lymphoma, characterized, in the two cases tested, by clonal chromosomal abnormalities. Epstein-Barr virus (EBV) infection was implicated in disease pathogenesis in 15/22 patients. The overall survival was 45.5%, 40.9% and 40.9% at 1, 3 and 5 years, respectively, and disease-free survival for CR patients at these same times was 45.5%, 36.4% and 36.4%. The etoposide-containing regimen would appear to be an effective initial therapeutic option for childhood HS. However, in view of the frequency of partial remissions and relapsed disease, a more intensive chemotherapy or bone marrow transplantation should be applied. The progression to EBV-containing T-cell lymphoma in three patients is consistent with the previous observation that EBV-associated HS is a potentially malignant disease.

BACKGROUND Epstein-Barr virus (EBV) infection in nonsmall cell lung carcinoma (NSCLC) has been demonstrated in some ethnic groups. The pathobiology and the role of EBV and oncoprotein expression in these tumors have not been studied extensively. In this study, the authors investigated EBV-encoded RNA-1 (EBER1) transcripts by in situ hybridization and the expression of latent membrane protein-1 (LMP-1) and bcl-2 protein by immunohistochemistry in NSCLC patients from Taiwan, where nasopharyngeal carcinoma is endemic. METHODS A total of 127 cases of NSCLC (43 cases of squamous cell carcinoma [SCC], 67 cases of adenocarcinoma [AD], 12 cases of large cell carcinoma [LCC], and 5 cases of lymphoepithelioma-like carcinoma [LE]) were included. A sensitive polymerase chain reaction-derived, digoxigenin-labeled DNA probe for in situ detection of EBER1 transcripts was performed for the detection of EBV. Immunohistochemistry using the avidin-biotin-immunoperoxidase method was also performed to evaluate the expression of bcl-2 and LMP-1. RESULTS EBER1 was detected in 11 of the 127 NSCLC cases (8.7%; 6 SCC cases and 5 LE cases). All 5 LE cases were EBV-positive, whereas only 6 of the 43 SCC cases (14%), 0 of 67 AD cases, and 12 LCC cases were EBV-positive (P 0.05). All LE cases had bcl-2 oncoprotein expression (100%). This frequency was significantly different from other histologic types (P < 0.05). The LMP-1 detection rate was low and demonstrated no correlation with bcl-2 expression. CONCLUSIONS In this study, the authors found that the primary LE of the lung is associated with young age, a history of not smoking, high bcl-2 immunoreactivity, and better survival rate. These characteristics demonstrate that EBV-associated LE of the lung is a unique entity. The findings of the current study suggest that EBV infection may play a different role in the tumorigenesis of primary LE of the lung than it does in other EBER1-positive NSCLCs. Cancer 1998;82:2334-2342. © 1998 American Cancer Society.

Without culture, differentiation of bacille Calmette-Guerin-induced lymphadenitis (BCG-LA) from tuberculosis (TB) is sometimes difficult by histology, but is important because of different treatment schemes. The purpose of this study was to investigate the feasibility of differentiating BCG-LA from TB in lymph nodes (LNs) by molecular analyses of two recently identified genes, pncA and oxyR. In both genes, a single nucleotide difference exists between Mycobacterium bovis and M. tuberculosis. M. tuberculosis complex (MTC) DNA was first detected in nine of ten formalin-fixed, paraffin-embedded LNs from patients aged under 20 years with suspected mycobacterial infections, using polymerase chain reaction (PCR) for IS6110, an insertion sequence specific for MTC species. PCR, together with direct DNA sequencing and PCR–restriction fragment length polymorphism (RFLP) assay, was then performed to identify polymorphic nucleotide in pncA and oxyR, respectively. For comparison, 37 adult cases of tuberculous lymphadenitis were also analysed by PCR–single strand conformation polymorphism (SSCP) assay for pncA and by PCR–RFLP for oxyR. The results revealed that five of the nine IS6110-positive child cases had a G residue at nucleotide 169 in pncA, and also had a three-band pattern after digesting the amplified oxyR segment with AluI, suggesting BCG-LA. The remaining four child cases, as well as all adult cases with detectable IS6110, showed no motility shift in pncA PCR–SSCP and had the same one-band pattern as M. tuberculosis in oxyR PCR–RFLP, suggesting TB lymphadenitis. The data from molecular analyses showed a good correlation with the vaccination history and clinicopathological findings, except for one case. This study indicates that molecular assay of either oxyR or pncA could be a rapid and useful tool to distinguish BCG-LA from TB. © 1998 John Wiley & Sons, Ltd.

The class I tyrosine kinase growth-factor receptors include epidermal growth factor receptor (EGFR), ErbB2 (c-erbB-2, HER-2/neu), ErbB3 and ErbB4. To elucidate their role in the regulation of homeostasis and carcinogenesis, we examined the expression of the receptors in normal urothelium and in urothelial carcinoma by immunohistochemistry. EGFR was expressed in the basal cells of normal urothelium, while ErbB2, ErbB3 and ErbB4 were present mainly in the superficial layer. A distinct reciprocal distribution was observed between the EGFR and the remaining members of the subclass (P = 0.0001). Both BCL-2 protein and Ki-67 antigen (MIB-1) showed a strong positive association with EGFR (P = 0.002) and an inverse correlation with ErbB2, ErbB3 or ErbB4 (P = 0.0004, 0.0000, and 0.001, respectively). With regard to carcinoma, there was no important relationship between receptor overexpression and tumour grading (P > 0.1), while only EGFR overexpression was correlated with muscular invasion (P = 0.02). Coexpression of EGFR-ErbB3 and ErbB3-ErbB4 was more often detected in high-grade tumours and correlated with the extent of tumour invasion. Our data indicate that class I receptors are differentially expressed in normal urothelium in vivo, but an orchestrated expression pattern does not exist during tumorigenesis.

Angiogenesis is of vital importance during the development and progression of solid tumors. To examine the role of vascular endothelial growth factor (VEGF) in hepatocarcinogenesis, we evaluated the expression of peptide in normal human liver (n = 6) and in 36 cases of hepatocellular carcinoma (HCC). Immunoreactivity for VEGF was present in the extracellular matrix of the portal tracts in the normal and nontumor part of liver, but not in hepatocytes and bile duct epithelium. For HCC, variable amounts of VEGF were expressed in 13 cases (36.1%) of tumor cells. Using a logistic regression model, expression of VEGF was significantly associated with a higher proliferative index (P = .01) and sonographic portal vein thrombosis (P = .05). However, VEGF expression did not correlate with a biochemical liver profile, alpha-fetoprotein levels, histological grading, gender, or clinical stage of cirrhosis (P0.1, respectively). Log-rank test showed that evaluation of VEGF did not provide more prognostic information (P.5) than that from tumor volume and portal vein thrombosis (P.01, respectively). In addition, VEGF was always present in the fibrovascular stroma or pericellular matrix of HCC, although no strong relationship was observed with the expression of VEGF in tumor cells (P.5). Our data suggested that expression of VEGF may characterize a progression toward higher proliferation in hepatocarcinogenesis in vivo. The relevance of VEGF existing in the extracellular matrix of the normal liver and HCC remains to be clarified.

Nasal T/natural killer (NK) cell lymphoma is a distinct clinicopathologic entity which is more prevalent in Asia than in America and Europe. The clonal nature of the infiltrating lymphoid cells is difficult to demonstrate because of the lack of immunologic markers for clonality and the absence of clonal T-cell receptor gene rearrangement in most cases. In this study, clonal chromosomal abnormalities were detected in the tumour cells from four patients with nasal T/NK cell lymphoma. This finding provided direct evidence for clonality of the disease. Moreover, nonrandom cytogenetic abnormalities, including isochromosome for the short arm (p) of chromosome 6, isochromosome for the long arm (q) of chromosome 1, partial deletion of 6q, and aberrations at 11q, were disclosed. Isochromosome 6p was the sole structural abnormality in one patient, which may be a pathognomonic change in nasal lymphoma.

Chronic suppurative otitis media, averaging 20 or more years of duration, has been associated with cancer in this region in 40%-80% of cases. Although human papillomaviruses (HPV) have been implicated in many human squamous-cell neoplasms, their role in the pathogenesis of middle-ear malignancies remains unexplored. In this study, we investigated the presence and subtypes of HPV in middle-ear carcinomas. Formalin-fixed and paraffin-embedded tumor tissues were sampled for DNA extraction. PCR was done with consensus primers, capable of detecting HPV 16, 18, 31, 33, 52b and 58. Typing of the products generated by consensus primers was performed with restriction enzyme digestion. It was found that a resulting 89% (8/9) of the middle-ear carcinomas contained HPV DNA. Coexistence of HPV 16 and 18 was detected in 3 squamous-cell carcinomas. HPV 16 was detected in 4 squamous-cell carcinomas and 1 adenocarcinoma. The high prevalence of high-risk-type HPV in carcinomas of the middle ear suggests that viral infection may be an important etiologic component in the carcinogenic process.

Recently, a new human herpesvirus (KSHV/HHV-8) has been identified in classic, transplant, endemic, and AIDS Kaposi's sarcoma that may be involved in the pathogenesis of Kaposi's sarcoma. The purpose of this study was to evaluate oral AIDS-Kaposi's sarcoma for detection of KSHV/HHV-8 DNA. DNA extracted from 54 oral AIDS-Kaposi's sarcoma lesions (47 initial, 7 postvinblastine treated), 5 non-Kaposi's sarcoma HIV-positive lesions, and 3 non-Kaposi's sarcoma HIV-negative lesions was evaluated by polymerase chain reaction (KS330 233bp amplicon) for KSHV/HHV-8. The AIDS-Kaposi's sarcoma study population consisted of 52 patients (51:1, men:woman; 92% men having sex with men, 8% heterosexual; mean age, 38 years; mean, CD4 59/mm 3 ) Opportunistic infections occurred in 88% (candidiasis, 65%; Pneumocystis carinii pneumonia, 31%; nonoral Kaposi's sarcoma, 25%, mycobacterium avium-intracellulare (MAI), 16%; cytomegalovirus, 14%; herpes simplex virus, 14%). Sexually transmitted diseases occurred in 73% (gonorrhea, 37%; syphilis, 23%; condyloma, 22%; HSV, 16%). Most frequent lesion sites were palate (74%) and gingiva (17%). Most common lesion types were purple nodular (48%) and macular (42%). Histopathologic subtypes were nodular (71%), plaque (27%), and patch (2%). Polymerase chain reaction analysis detected KSHV/HHV-8 DNA in 53 of 54 AIDS-Kaposi's sarcoma lesions (47 of 47 initial, 6 of 7 postvinblastine treatment). KSHV/HHV-8 DNA was not detected in non-Kaposi's sarcoma lesions in HIV-positive or HIV-negative persons. KSHV/HHV-8 DNA sequence is present in a high proportion of oral AIDS-Kaposi's sarcoma lesions. Whether KSHV/HHV-8 is an etiologic agent or a cofactor in the development of this vascular neoplasm is uncertain and remains to be proven. Polymerase chain reaction analysis for KSHV/HHV-8 DNA sequence detection may be helpful in identifying Kaposi's sarcoma in early vascular proliferations, when the characteristic histopathologic features are not present.

It has been suggested that angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is closely related to peripheral T cell lymphoma (PTCL). However, the clinical course of AILD-like PTCL is notoriously unpredictable. A minor portion of patients enjoyed prolonged remission with steroid-only treatments (indolent AILD) while most others died rapidly despite the use of intensive chemotherapy (aggressive AILD). Recently, it has been suggested that histological features such as the presence or absence of clear cells and convoluted cells are of high prognostic value. The validity of this observation was addressed in this study. Eighteen patients who presented between 1977 and 1994 at the National Taiwan University Hospital were retrospectively studied. There were 11 men and 7 women, with a median age of 47 years. Twelve patients had received various regimens of systemic chemotherapy, and the other 3 patients had been treated with steroids alone. Eight patients had indolent AILD and 6 aggressive AILD. The follow-up period in 4 patients was too short to be analyzed. The histopathology of these cases was divided, according to the criteria of Aozasa et al., into group I (neither cells), 4 patients; group II (only convoluted cells), 1 patient, and group III (clear cells with or without convoluted cells), 13 patients. Contrary to others, our data revealed that group III patients were doing better than group I patients. Univariate analysis of other pertinent clinical features, including sex, age, lymphadenopathy, B symptoms, hepatosplenomegaly, hypergammaglobulinemia, elevated serum lactate dehydrogenase, and treatment regimens, revealed none of them to be prognostically relevant. However, patients who had achieved complete remission by steroids or other systemic chemotherapy had a significantly better prognosis than those who had not. Together, these preliminary data suggested that (1) the presence or absence of clear cells and convoluted cells failed to predict the clinical behavior, and (2) induction of complete remission by steroids or other chemotherapeutic agents is an important prognostic index.

This preliminary study attempted to test whetherpretreatment gastric histology of H. pylori infectionmay affect the success of dual therapy and to identifywhich parameter of gastric histology could be improved after dual therapy. One hundredforty-five dyspeptic patients with H. pylori infectionreceived a two-week course of dual therapy (Amoxicillin500 mg every 6 hr plus omeprazole 20 mg twice a day). In each patient, three pairs of gastricbiopsies, sampled from the antrum, lower body, and upperbody near the cardia, were collected before treatmentand four weeks after completion of dual therapy. The density of H. pylori (score 1-5) and parametersof the modified Sydney system were applied to test theseverity of H. pylori-related gastric histology in eachspecimen. The total bacterial load (score 1-15) was a sum of the density of H. pylorisampled from three biopsies. The overall rate of H.pylori eradication rate by dual therapy is 73.1%(106/145). Univariate analysis of parameters inpretreatment histology disclosed that the presence ofmucosal atrophy (P < 0.01), lymphoid follicles (P< 0.005), and higher-density H. pylori (P < 0.001)predisposed to dual therapy failure. Multivariateanalysis by stepwise logistic regression further confirmed that boththe density of bacteria and the presence of lymphoidfollicles are the two major factors related to theoutcome of dual therapy (P < 0.001). Four weeks after dual therapy was completed, only patients withsuccessful eradication significantly improved in thesegastric histology parameters: acute activity, chronicinflammation, eosinophil infiltration, and mucosal atrophy. However, the lymphoid follicle andintestinal metaplasia were not significantly improvedduring the study period. The eradication rates amongthree subgroups with different total bacterial loads (group I: 1-5; II: 6-10; III: 11-15) discloseda downward trend (I: 89.1%; II: 73%; III: 52.7%). It isconcluded that dual therapy could improve gastrichistology especially among patients with successful eradication of H. pylori. Evaluatingpretreatment histologic parameters, including thedensity of H. pylori and the presence of lymphoidfollicles, is valuable in predicting the success of dualtherapy.

Hepatitis B virus (HBV) X protein (HBx) and pre-S2 deletion mutant large surface antigens are oncoproteins that induce hepatocellular carcinoma (HCC). The interaction of these two oncoproteins in hepatocytes and its significance in tumorigenesis remain to be elucidated. In this study, we observed the co-expression of HBx with surface antigens in ground-glass hepatocytes in 5 of 20 hepatitis B surface antigen-positive livers. In vitro, hepatocytes co-expressing HBx and a pre-S2 mutant showed enhanced expression of vascular endothelial growth factor-A, phosphorylated Akt 1/2/3, phosphorylated extracellular signal-regulated kinase 1/2, and phosphorylated mammalian target of rapamycin signals. Transgenic mice harboring both HBx and pre-S2 mutant construct plasmids developed HCCs at an average of 15.1 months, earlier than animals carrying either HBx (16.9 months) or pre-S2 mutant (24.5 months) alone. The oncogenic signals of vascular endothelial growth factor-A, phosphorylated Akt 1/2/3, phosphorylated extracellular signal-regulated kinase 1/2, and phosphorylated mammalian target of rapamycin were sequentially and differentially activated at different stages in tumorigenesis. Phosphorylated mTOR was consistently activated in transgenic and human HCCs. We conclude that ground-glass hepatocytes co-expressing HBx and surface antigens exhibit enhanced oncogenic effects and tumorigenesis in chronic HBV infections. The mTOR signal cascade may be the key regulator in HBV tumorigenesis and may be useful targets in the design of HCC therapy.

Arginine biosynthesis and nitric oxide (NO) production are important for cancer homeostasis. Degradation of arginine may be used to inhibit liver tumors with low argininosuccinate synthetase (ASS) expression. In this report, we investigated an alternative therapeutic approach by targeting argininosuccinate lyase (ASL). ASL is transcriptionally induced by endoplasmic reticulum stress and is overexpressed in some human liver tumors. Knockdown of ASL expression by short hairpin RNA (shRNA) in three liver cancer cell lines, ML-1, HuH-7, and HepG2, decreased colony formation in vitro and tumor growth in vivo. Furthermore, lentiviral infection of ASL shRNA inhibited tumor growth in a therapeutic animal tumor model. Analysis of ASL shRNA on the cell-cycle progression revealed a G2–M delay. Among cell-cycle regulatory molecules, cyclin A2 expression was reduced. Reintroduction of exogenous cyclin A2 restored the cell growth in ASL-knockdown cells. Autophagy was observed in the cells treated with ASL shRNA, as shown by an increase in LC3-II levels and autophagosome formation. The total cellular arginine level was not altered significantly. Inhibition of autophagy further attenuated cell growth, suggesting that autophagy induced by ASL shRNA plays a feedback prosurvival function. Knockdown of ASL reduced NO content, and addition of NO donor partially recovered the growth inhibition by ASL shRNA. In summary, downregulation of ASL attenuated tumor growth and the inhibition was mainly mediated by a decrease of cyclin A2 and NO. Mol Cancer Ther; 12(11); 2505–16. ©2013 AACR.

Background To control tuberculosis (TB), it is critical to identify at risk populations. Schizophrenia is recognized as an important risk factor for TB. However, previous studies have been confounded by comorbidities, and reports of TB infection outcomes are rare. Therefore, the current nation-wide study aimed to compare the adjusted incidence and outcome of TB diseases in schizophrenics and the general population. Method Using the National Health Insurance Research Database from 1998 to 2009, this retrospective longitudinal study included 60,409 schizophrenics and general population matched for age, Charlson’s score, and comorbidities. Diagnosis of TB was based on the international classification of disease, ninth revision and use of anti-TB drugs. Unfavorable outcome for TB was defined as death, loss to follow-up, or use of anti-TB treatment for more than 9 months. Results The adjusted incidence of TB in schizophrenics was significantly higher than in the general population [hazard ratio, 1.52; 95% confidence interval (CI), 1.29-1.79; p < 0.001; Kaplan-Meier log-rank test, p < 0.001]. Cox regression revealed age and male gender as risk factors for newly-diagnosed TB. The outcome of TB was comparable in schizophrenics and the general population [odds ratio (OR), 0.78; 95% CI, 0.55-1.09; p =0.144]. Logistic regression revealed a statistical trend for diabetes mellitus to predict poor outcome in schizophrenics with TB (OR, 2.30; 95% CI, 0.96-5.74; p = 0.062). Conclusions Schizophrenics are at increased risk for TB, and screening may be warranted for those living in areas with high prevalence of TB.