Diagnostic Utility of Cytomegalovirus Nucleic Acid Testing during Antigenemia-Guided Cytomegalovirus Monitoring after Hematopoietic Stem Cell Transplantation or Liver Transplantation.

Cytomegalovirus (CMV) is an opportunistic pathogen, and careful monitoring of CMV is important for immunocompromised patients. Antigenemia-based CMV monitoring is a standard test used for managing CMV infection in transplant recipients; however, in Japan, there are no reports of CMV monitoring using the standardized test. The utility of a standardized CMV nucleic acid test (NAT) was evaluated during antigenemia-based CMV monitoring after hematopoietic stem cell transplantation (HSCT) or liver transplantation. Blood collection for CMV monitoring was performed under the physician's instructions depending on the condition of the patient, and CMV NAT and antigenemia was evaluated. For HSCT recipients, blood collection only for NAT was additionally performed during the pre-engraftment phase. The results of the NAT were blinded to those evaluating the results. A total of 34 patients were enrolled (11 HSCT recipients and 23 liver transplant recipients). NAT detected the first CMV episode no later than antigenemia in 2 (18.2%) HSCT recipients and 3 (13.0%) liver transplant recipients, earlier than antigenemia in 3 (27.3%) HSCT recipients and 7 (30.4%) liver transplant recipients, and later than antigenemia in 1 (9.1%) HSCT recipient and 1 (4.3%) liver transplant recipient. In 5 HSCT recipients, NAT was positive during the pre-engraftment phase. Among the 468 blood samples which were evaluated by both NAT and antigenemia, 124 (26.7%) were positive in NAT and 51 (10.9%) were positive in antigenemia. The standardized CMV NAT is useful for accurately diagnosing CMV infection and determining appropriate therapeutic interventions for HSCT recipients and liver transplant recipients.

Coexistence of dementia with smear-positive pulmonary tuberculosis is associated with patient in-hospital mortality.

BACKGROUND: The impact of dementia on the survival of patients with pulmonary tuberculosis (TB) remains unclear. This study sought to describe the risk factors influencing in-hospital mortality in patients with pulmonary TB and comorbid dementia. METHODS: A 9-y, medical record-based retrospective study of hospitalized adult patients with newly diagnosed, smear-positive, non-multidrug-resistant pulmonary TB without human immunodeficiency virus infection was performed. Clinical presentations, biochemical tests, radiographic findings, and clinical outcomes were collected. Variables were compared between groups. Statistically significant (p-value < 0.05) variables were entered into a multivariate stepwise logistic regression model. Survival analysis was performed using the Kaplan-Meier method, and groups were compared by log-rank test. RESULTS: Of the 279 enrolled patients (178 men; median age, 76 y), the mortality rate was 12.2% (34/279). Univariate analysis showed a higher frequency of dementia in patients who died in hospital than that in surviving patients. Multivariate stepwise logistic analysis showed that dementia was significantly associated with higher rates of in-hospital mortality (odds ratio, 3.20; 95% confidence interval, 1.15-8.88, p = 0.026). In addition, subgroup survival curves showed that dementia was associated with reduced survival rates, even after adjusting for age (log-rank test, p = 0.0007). CONCLUSIONS: The comorbidity of dementia with pulmonary TB was associated with patient in-hospital mortality. Medical practitioners should be aware of dementia in patients with smear-positive pulmonary TB to identify high-mortality groups.

Strong influence of human leukocyte antigen-DP variants on response to hepatitis B vaccine in a Japanese population.

Genome-wide association studies (GWASs) have reported that human leukocyte antigen (HLA) variants are associated with chronic hepatitis B, spontaneous hepatitis B virus (HBV) clearance, and response to hepatitis B vaccine. Single nucleotide polymorphisms (SNPs) in HLA-DP (rs9277535 and rs3077) and HLA-DQ (rs2856718 and rs7453920) have been repeatedly associated with chronic hepatitis B and spontaneous HBV clearance. However, the data on the SNPs associated with response to hepatitis B vaccine are inconclusive. The objective of this study was to determine whether these four HLA SNPs that have been identified as risk loci for chronic HBV infection are associated with response to hepatitis B vaccine in a Japanese population. We enrolled 278 medical students who received hepatitis B vaccination and measured anti-hepatitis B surface (HBs) antibody titers 1month after a three-dose vaccination series. We found that rs9277535 and rs3077 in HLA-DP were strongly associated with response to hepatitis B vaccine (odds ratio [OR]=0.31 and 0.32, P=0.004 and 0.010, respectively). These two SNPs were significantly associated with anti-HBs titers in an allele-dependent manner. On the other hand, rs2856718 and rs7453920 in HLA-DQ were not associated with response to hepatitis B vaccine. These results indicate that rs9277535 and rs3077 in HLA-DP are the major determinants of response to hepatitis B vaccine, whereas rs2856718 and rs7453920 in HLA-DQ have little effect on the immune response to hepatitis B vaccine.

Deep Phylogeographic Breaks in Magpie Pica pica Across the Holarctic: Concordance with Bioacoustics and Phenotypes.

We examined sequences of the mitochondrial control region in magpies (Pica pica) from the entire distribution range and found deep genetic splits into four major lineages: (1) group West (Europe-Siberia), (2) group East (southern Far East), (3) P. p. mauritanica (North Africa), and (4) P. p. hudsonia (North America). These lineages show a geographic pattern corresponding to known subspecies or subspecies groups. Genetic variation within the widely-distributed group West is low and neutrality tests supported a recent expansion scenario. The haplotypes from Kamchatka, representing a separated sublineage with clear affinity to the European-Siberian group, are almost identical, implying a recent bottleneck. Group East contained two subclades without clear geographic pattern, presumably due to admixing of populations that had diverged in Pleistocene refuges. The homogeneity of the Kyushu population supports historical reports of introduction of the species from Korea. In contrast, the high variation in the recently established Hokkaido population may reflect an ongoing invasion from several populations of the Far Eastern mainland. Bioacoustic data based on chatter call differentiate groups of subspecies and reflect phylogeographic patterns, i.e., mitochondrial lineages. Furthermore, we report the fast spreading of P. p. jankowskii towards the west along the upper Amur River, and a slower shifting of P. p. leucoptera in the opposite direction thus yielding a new contact zone. Overall, our data support a scenario of divergence in geographic isolation, but the ongoing expansion of distribution ranges may lead to major changes in phylogeographic patterns.

Simultaneous screening for JAK2 and calreticulin gene mutations in myeloproliferative neoplasms with high resolution melting.

INTRODUCTION: Recently, novel calreticulin (CALR) mutations were discovered in Janus kinase 2 (JAK2) non-mutated myelofibrosis (PMF) and essential thrombocythemia (ET) cases, with a frequency of 60-80%. We examined clinical correlations and CALR mutation frequency in our myeloproliferative neoplasms (MPN) cases, and introduce an effective test method for use in clinical practice. METHODS: We examined 177 samples previously investigated for the JAK2 mutation for differential diagnosis of MPN. JAK2 and CALR mutations were analyzed using melting curve analysis and microchip electrophoresis, respectively. Next, we constructed a test for simultaneous screening of the JAK2 and CALR mutations utilizing high resolution melting (HRM). RESULTS: Among 99 MPN cases, 60 possessed the JAK2 mutation alone. Of the 39 MPN cases without the JAK2 mutation, 14 were positive for the CALR mutation, all of which were ET. Using our novel screening test for the JAK2 and CALR mutations by HRM, the concordance rate of conventional analysis with HRM was 96% for the JAK2 mutation and 95% for the CALR mutation. CONCLUSION: Our novel simultaneous screening test for the JAK2 and CALR gene mutations with HRM is useful for diagnosis of MPN.

[Diagnostic Utilities of an Automated and Standardized DNA Quantification during Cytomegalovirus Monitoring].

The accurate and standardized diagnosis of cytomegalovirus (CMV) infection is important for immunocom- promised patients. We prospectively evaluated the performance of an automated and standardized real-time polymerase chain reaction (PCR) -based DNA quantification for the detection of CMV. The results of PCR- based analysis were also compared with pp65 antigenemia (Ag) assay in the clinical records. The PCR- based analysis of 144 plasma samples from 26 patients with hematologic diseases detected CMV in 69 (48.0%) samples (range, <150-1.28 X 104 copies/mL) while Ag detected CMV in 32(22.2%) samples (range, 1-37/50,000 cells). The number of concordant samples between the two tests was 95(66.0%). There were nine patients who had an Ag-positive period sandwiched by Ag-negative periods and, in all these patients, the Ag-positive period was completely covered by PCR-positive period. These results suggest that PCR can detect CMV more sensitively than Ag. The automated and standardized PCR for detection of CMV can support the appropriate management in patients with risks of CMV infection. [Original].

The level of bone marrow WT1 message is a useful marker to differentiate myelodysplastic syndromes with low blast percentage from cytopenia due to other reasons.

OBJECTIVE: Myelodysplastic syndromes (MDS) are a group of hematological neoplasms associated with ineffective hematopoiesis and that transform to acute leukemia. Distinguishing MDS from other cytopenias is sometimes difficult even for trained hematologists. WT1, the gene mutated in Wilms' tumor, was found expressed in acute myeloid leukemia and MDS. The amount of WT1 in peripheral blood and bone marrow (BM) is low in low-risk MDS subtypes, and is high in high-risk MDS subtypes. However, the role of WT1 in the differential diagnosis between MDS and other diseases showing cytopenia has not been fully addressed. The present study evaluated whether WT1 expression level can assist in the differential diagnosis of MDS from other cytopenias. METHODS: The amount of WT1 message was evaluated among 56 MDS patients and 47 patients with cytopenia for various other reasons (cytopenia VR) at the Nagasaki University Hospital. RESULTS: The level of WT1 was significantly related to the percentage of blasts in BM among MDS cases, and the type of French-American-British classification of MDS; refractory anemia (RA) cases showed significantly lower WT1 level than patients with RA with excess blasts. WT1 level was significantly related to the prognostic risk categories of MDS by the International Prognostic Scoring System (IPSS) and the revised IPSS. Although the blast percentage in the BM of RA and cytopenia VR were both less than 5%, there was a significant difference in the level of WT1 between MDS and cytopenia VR. CONCLUSION: WT1 might be a good marker to differentiate low blast percentage MDS and cytopenia VR.

Molecular analysis of the BCR-ABL1 kinase domain in chronic-phase chronic myelogenous leukemia treated with tyrosine kinase inhibitors in practice: study by the Nagasaki CML Study Group.

An appropriate trigger for BCR-ABL1 mutation analysis has not yet been established in unselected cohorts of chronic-phase chronic myelogenous leukemia patients. We examined 92 patients after 12 months of tyrosine kinase inhibitor (TKI) treatment in Nagasaki Prefecture, Japan. Univariate analysis revealed that significant factors associated with not attaining a major molecular response (MMR) were the presence of the minor BCR-ABL1 fusion gene, a low daily dose of TKI, and the emergence of BCR-ABL1 kinase domain mutations conferring resistance to imatinib. Factors associated with the loss of sustained MMR were a low daily dose of TKI and the emergence of alternatively spliced BCR-ABL1 mRNA with a 35-nucleotide insertion. Taken together, our results suggest that the search for BCR-ABL1 mutations should be initiated if patients have not achieved MMR following 12 months of TKI treatment.

Human T-cell leukemia virus type 1 infection worsens prognosis of hepatitis C virus-related living donor liver transplantation.

Severe and life-threatening donor-transmitted human T-cell leukemia virus type 1 (HTLV-1) infections after solid organ transplantation have been reported. However, in HTLV-1-infected recipients, graft and patient survival were not fully evaluated. A total of 140 patients underwent living donor liver transplantation (LDLT). Of these, 47 of 126 adult recipients showed indications of hepatitis C virus (HCV)-related liver disease. The HTLV-1 prevalence rate was 10 of 140 recipients (7.14%) and three of 140 donors (0.02%). In HCV-related LDLT, graft and patient survival was worsened by HTLV-1 infection in recipients (seven cases). The 1-, 3-, and 5-year survival rates in the HCV/HTLV-1-co-infected group were 67%, 32%, and 15%, respectively, and the corresponding rates in the HCV-mono-infected group were 80%, 67%, and 67%, respectively. Only the 5-year survival rates were statistically significant (P=0.04, log-rank method). HTLV-1 infection in recipients is also an important factor in predicting survival in HTLV-1 endemic areas.

Heterogeneity in clonal nature in the smoldering subtype of adult T-cell leukemia: continuity from carrier status to smoldering ATL.

To better understand indeterminate HTLV-1 carriers and smoldering (SM) subtype of adult T-cell leukemia (ATL), HTLV-1 proviral integrated status, proviral load (PVL) and ATL-related biomarkers were examined in 57 smoldering cases, including unusual carriers with a percentage of ATL-like cells. We found that according to Southern blot hybridization analytic features, 28 patients with SM ATL could be divided into 3 groups consisting of 16 (57.4%) patients with a monoclonal band, 6 (21.4%) with oligoclonal bands and the remaining 6 with smears. Although no clinical differences were observed among the 3 SM subtypes, HTLV-1-infected CD4 T-cell counts increased in order of poly-, oligo- and monoclonal subtypes. This trend began in the carrier stage and also was observed in PVL, CD25 and CCR4, indicating that a clone consisting of leukemic phenotypic cells was continuously growing. Moreover, the antigen modulation rates of CD26 and CD7 and the increasing rate of CD25 and CCR4 cells were closely correlated to growing clonal size, indicating that these markers had the possibility to predict a monoclonal band. In particular, CD26 or the ratio of CD26/CD25 had a validity differential for leukemic nature and predictive detection of clonal band. Conclusively, the present study shows that smoldering ATL is heterogeneous in the leukemogenic process, and the behavior of CD26 plays a central role in the evolution from early occult to overt smoldering ATL.

Paradoxical expression of IL-28B mRNA in peripheral blood in human T-cell leukemia virus type-1 mono-infection and co-infection with hepatitis C virus.

BACKGROUND: Human T-cell leukemia virus type-1 (HTLV-1) carriers co-infected with and hepatitis C virus (HCV) have been known to be at higher risk of their related diseases than mono-infected individuals. The recent studies clarified that IL-28B polymorphism rs8099917 is associated with not only the HCV therapeutic response by IFN, but also innate immunity and antiviral activity. The aim of our research was to clarify study whether IL-28B gene polymorphism (rs8099917) is associated with HTLV-1/HCV co-infection. RESULTS: The genotyping and viral-serological analysis for 340 individuals showed that IL-28B genotype distribution of rs8099917 SNP did not differ significantly by respective viral infection status. However, the IL-28B mRNA expression level was 3.8 fold higher in HTLV-1 mono-infection than HTLV-1/HCV co-infection. The high expression level was associated with TT (OR, 6.25), whiles the low expression was associated with co-infection of the two viruses (OR, 9.5). However, there was no association between down-regulation and ATL development (OR, 0.8). CONCLUSION: HTLV-1 mono-infection up-regulates the expression of IL-28B transcripts in genotype-dependent manner, whiles HTLV-1/HCV co-infection down-regulates regardless of ATL development.

Successful treatment of a chronic-phase T-315I-mutated chronic myelogenous leukemia patient with a combination of imatinib and interferon-alfa.

The T315I BCR-ABL mutation in chronic myelogenous leukemia (CML) patients is responsible for up to 20% of all clinically observed resistance. This mutation confers resistance not only to imatinib, but also to second-generation BCR-ABL tyrosine kinases, such as nilotinib and dasatinib. A number of strategies have been implemented to overcome this resistance, but allogeneic stem cell transplantation remains the only established therapeutic option for a cure. A 61-year-old male was diagnosed with Philadelphia chromosome-positive chronic-phase CML in 2002. He was initially treated with imatinib and complete cytogenetic response (CCyR) was achieved 12 months later. However, after 18 months, a loss of CCyR was observed and a molecular study at 24 months revealed a T315I mutation of the BCR-ABL gene. At 30 months, imatinib/interferon-alfa (IFNα) combination therapy was initiated in an effort to overcome the resistance. Thirty months later, he re-achieved CCyR, and the T315I BCR-ABL mutation disappeared at 51 months. To our knowledge, this is the first case report showing the effectiveness of imatinib/IFNα combination therapy for CML patients bearing the T315I BCR-ABL mutation.

Suppressor of cytokine signal 3 and IL28 genetic variation predict the viral response to peginterferon and ribavirin.

AIM: The aim of this study was to investigate the relationship among the expression of suppressor of cytokine signaling 3 (SOCS 3) in the liver, the SNPs in the IL28B locus, and the outcome of interferon therapy. METHODS: Prior to interferon treatment, we immunostained 67 liver specimens from chronic hepatitis C (CHC) patients who were receiving peginterferon alpha-2b/ribavirin therapy for suppressor of cytokine signaling 3 (SOCS3), and compared the expression of SOCS3, IL28 polymorphisms and other clinical factors between the patients and compared their eventual outcomes. RESULTS: Significant differences between the low SOCS3 group and high SOCS3 group were found in age, as well as in the platelet, transaminase, gamma-glutamyl transpeptidase levels. The incidence of high SOCS3 was not significantly different between subjects with the TT genotype and the TG genotype (TT : TG = 71%:29%, P = 0.250). In a multivariate analysis, age (≥65 years old) (odds ratio 0.221 [0.120-0.966], P = 0.045), IL28B gene (genotype TT) (odds ratio 5.422 [1.254-23.617], P = 0.024) and SOCS3 (high) (odds ratio 0.308 [0.104-0.948], P = 0.040) were significant predictors of the interferon response. In patients with the TT genotype, those with low SOCS3 immunostaining showed a high sustained virological response (69%), while the sustained virological rate was low (27%) in the patients with high SOCS3 immunostaining. CONCLUSIONS: Using a combination of the SOCS3 immunostained area in the liver and the expression of IL28B single nucleotide polymorphisms might be a useful predictor of hepatitis C virus clearance by interferon therapy.

An instructive case suggesting warfarin resistance which is independent on the regulation of the CYP2C9 and VKORC1 genotype.

To improve the safety and effectiveness of warfarin (WF) therapy, the initial dose trends to be practically decided based on single nucleotide polymorphism (SNP) genotyping of two genes, cytochrome P450 (CYP) 2C9 and vitamin K epoxide reductase complex1 (VKORC1). We encountered a 43-year-old female who was hospitalized for investigation and treatment because of intermittent convulsive seizures. Right brain cortical vein thrombi were confirmed by magnetic resonance imaging (MRI) scan; therefore, a 3 mg dose of WF was empirically initiated. The prothrombin time (PT), expressed as the international normalized ratio (INR), did not change at all, even when WF was increased to a dose of 11 mg/day. Direct sequence analysis revealed *3 in CYP2C9 and 3673 GA, 6484 CT, 6853 GC and 9041 GA in VKORC1, indicating that the genotypic pattern of the two genes is the responsible SNP for the moderate phenotype on WF sensitivity. Conclusively, our case may present an unknown mechanism other than the concern mentioned above.

Hoxb4 transduction down-regulates Geminin protein, providing hematopoietic stem and progenitor cells with proliferation potential.

Retrovirus-mediated transduction of Hoxb4 enhances hematopoietic stem cell (HSC) activity and enforced expression of Hoxb4 induces in vitro development of HSCs from differentiating mouse embryonic stem cells, but the underlying molecular mechanism remains unclear. We previously showed that the HSC activity was abrogated by accumulated Geminin, an inhibitor for the DNA replication licensing factor Cdt1 in mice deficient in Rae28 (also known as Phc1), which encodes a member of Polycomb-group complex 1. In this study we found that Hoxb4 transduction reduced accumulated Geminin in Rae28-deficient mice, despite increasing the mRNA, and restored the impaired HSC activity. Supertransduction of Geminin suppressed the HSC activity induced by Hoxb4 transduction, whereas knockdown of Geminin promoted the clonogenic and replating activities, indicating the importance of Geminin regulation in the molecular mechanism underlying Hoxb4 transduction-mediated enhancement of the HSC activity. This facilitated our investigation of how transduced Hoxb4 reduced Geminin. We showed in vitro and in vivo that Hoxb4 and the Roc1 (also known as Rbx1)-Ddb1-Cul4a ubiquitin ligase core component formed a complex designated as RDCOXB4, which acted as an E3 ubiquitin ligase for Geminin and down-regulated Geminin through the ubiquitin-proteasome system. Down-regulated Geminin and the resultant E2F activation may provide cells with proliferation potential by increasing a DNA prereplicative complex loaded onto chromatin. Here we suggest that transduced Hoxb4 down-regulates Geminin protein probably by constituting the E3 ubiquitin ligase for Geminin to provide hematopoietic stem and progenitor cells with proliferation potential.

High-resolution melting analysis for a reliable and two-step scanning of mutations in the tyrosine kinase domain of the chimerical bcr-abl gene.

For relevant imatinib therapy against Philadelphia (Ph)-positive leukemias, it is essential to monitor mutations in the chimerical bcr-abl tyrosine kinase domain (TKD). However, there is no universally acceptable consensus on how to efficiently identify mutations in the target TKD. Recently, high-resolution melting (HRM) technology was developed, which allows gene scanning using an inexpensive generic heteroduplex-detecting dsDNA-binding dye. This study aimed to validate the introduction of HRM in a practical clinical setting for screening of mutations in sporadic sites of the chimerical bcr-abl TKD. All chimerical and wild-type abl TKD regions selectively amplified were used for HRM assays and direct sequencing. The HRM test had approximately 5-90% detection sensitivity for mutations. In contrast to mixture samples with mutant and wild-type cells, all mutant cell samples had indeterminate melting curves equivalent to those of the wild-type due to formation of only a homodulex. This issue was improved by the addition of exogenous wild-type DNA after PCR. Subsequently, HRM results gave a high accordance rate of 97.8% (44/45 samples) compared to the sequencing data. The discordant results in one appear to be due to unsuccessful amplification. Thus, HRM may be considered to be suitable for reliable scanning of mutations in the chimerical abl TKD in a clinical setting.

Screening for genetic heterogeneity in the interferon sensitivity determining region of the hepatitis C virus genome by polymerase chain reaction with melting curve analysis.

BACKGROUND: Although mutations in the interferon (IFN) sensitivity determining region (ISDR) of hepatitis C virus (HCV) have been reported to be useful as a predictive viral factor for IFN therapy in patients infected with HCV-1b, such laboratory research has not been favorably translated into the clinic. To promote such translation, we attempted the establishment of a rapid and simple polymerase chain reaction (PCR) combined with melting curve analysis (MCA) to screen for mutations in the ISDR and for the monitoring of HCV quasispecies. METHODS: A PCR-MCA protocol was established using in-house primers and hybridization probes designed according to the results of direct sequencing of 34 HCV-1b samples. Then, the performance of PCR-MCA was verified by comparing with mutation profiles obtained by direct sequencing and sequencing after cloning. RESULTS: The MCA assay revealed that melting temperature (Tm) was inversely correlated with the number of nucleotide (nt) and amino acid substitutions in the ISDR deduced on the basis of the results of direct sequencing. A boundary Tm of 58.0 degrees C allowed us to discriminate HCV genomes into two groups: one with a Tm >58.0 degrees C had no or a low number of nt substitutions, while the other genomes with a Tm <58.0 degrees C had a high number of nt substitutions, corresponding to wild-type in the former and mutant-type in the latter in respect of a clinical setting for IFN therapy. Moreover, this MCA assay provided precise discrimination of Tm between clones, reflecting the degree of the genetic complexity of HCV genomes. CONCLUSIONS: This study indicates that the MCA assay is useful to rapidly and simply screen the mutational status of the ISDR of HCV, as well as in using the ISDR as one of the targets for discriminating the genetic complexity of HCV genomes. The MCA assay could also be applicable as a convenient and useful screen of the genetic heterogeneity of clones relating to HCV quasispecies.

The possibility of a valuable resource of circulating DNA for single nucleotide polymorphisms genotyping: the application of a rapid and simple polymerase chain reaction with melting curve analysis for methyltetrahydrofolate reductase polymorphisms.

Circulating DNA from plasma is easily stored and is a valuable resource to access genetic information indispensable to modern hematology. The aim of the present project was to evaluate the integrity of circulating DNA and to investigate whether such DNA is practically tolerable for genotyping single nucleotide polymorphisms (SNP) of methyltetrahydrofolate reductase (MTHFR). We first established a protocol combined with polymerase chain reaction (PCR) and melting curve analysis (MCA) based on the different melting temperatures of heteroduplex amplicons. This method was simple and rapid, requiring 3 hours without any complex manipulation, and allowed for a reliable test and diagnostic validity. The median of the circulating DNA density in 240 donors was 33.5 ng/mL. The DNA consisted of fragments with approxiately 100 to 500 base pairs. Such DNA fragments were acceptable for quantifying the housekeeping genes of - globin using a real-time PCR method and also for genotyping the MTHFR SNP using the method of PCR with MCA. Circulating DNA from storage plasma is acceptable for genetic tests, but it is necessary to note the integrity of DNA.

Expression of glycolytic enzymes is increased in pancreatic cancerous tissues as evidenced by proteomic profiling by two-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry.

The prognosis of patients with pancreatic cancer is very poor because of late diagnosis and the lack of response to various therapies. We tried to identify proteins that might be available for early diagnosis and effective therapies by proteomic profiling of pancreatic cancer tissues. Pancreatic cancerous and paired non-cancerous tissues obtained from surgical resections or autopsies of 10 patients were analyzed by two-dimensional gel electrophoresis. The differential display showed 11 spots whose expression was increased in cancerous tissues compared with the paired non-cancerous tissues. The liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) system identified the spots as alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triosephosphate isomerase, transgelin, calmodulin, superoxide dismutase(Mn) mitochondrial precursor, glutathione S-transferase P, cyclophilin A, protein disulfide isomerase A3 precursor, and apolipoprotein A-I precursor. Two of the 11 spots were detected as GAPDH. We noticed that 4 of 11 spots were enzymes involved in glycolytic pathway. Increased glycolysis in cancer cells has been regarded as the effect of intratumoral hypoxia and is possibly associated with tumor invasion, metastasis or resistance to therapies. These glycolytic proteins and transgelin, were confirmed by Western blotting and immunohistochemistry.

Usefulness of a comprehensive PCR-based assay for human herpes viral DNA in blood mononuclear cell samples.

Human herpes virus (HHV) is well known to reactivate in immunocompromised situations and plays an immunomodulatory role leading to indirect effects through viral replication to the host. The aim of this study was to determine the laboratory and clinical relevance of a comprehensive PCR-based assay for detecting eight HHVs, which are lymphotropic and cause disease in humans. Using 176 samples collected from 146 specimens of peripheral blood, 12 skin nodules, 11 lymph nodes and 7 others of patients who were suspected to have adult T cell leukemia (ATL), the PCR-based assay was validated to simultaneously detect one or more herpes viral DNA with two consensus primer sets. Although most samples were seropositive for either of the HHVs, only 50% of them were positive for either herpes viral DNA with EBV in 76%, HHV-6 in 14% and VZV in 9%. Furthermore, such a herpes viral DNA positive status was not always associated with clinical symptoms relating to the virus, implying active replication in the blood cells, but being asymptomatic. HHV-8 viral DNA, although Kaposi's sarcoma has been reported to be complicated with ATL, was not demonstrable. HHV-6B was detected only in HTLV-1 healthy carriers and ATL patients, and may imply a co-factor role with HTLV-1. This PCR-based assay provides a herpes viral infectious status compensatory for virus-serology and is a clinically relevant laboratory test, serving as a screening marker for active infection.