Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study.

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.

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.

Meeting report on the possible proposal of an extranodal primary cutaneous variant in the lymphoma type of adult T-cell leukemia-lymphoma.

Based on the advances in research on the clinicopathophysiology of adult T-cell leukemia-lymphoma (ATL), Japanese researchers collected and evaluated cases of smoldering ATL exhibiting primary cutaneous manifestation but showing poor prognosis. Macroscopic findings of skin eruptions were categorized into the patch, plaque, multipapular, nodulotumoral, erythrodermic and purpuric types, as previously reported. Pathological findings were divided into low or high grade based on epidermotropism, tumor cell size and perivascular infiltration. Eight eligible cases were evaluated among 14 collected cases. Macroscopic findings were nodulotumoral in six cases, a subcutaneous tumor in one case and plaque in one case, and the number and size were heterogeneous in each case. Pathological findings of all eight cases were T-cell lymphoma, high-grade type (pleomorphic, medium or large size), with prominent perivascular infiltration and scant epidermotropism. To diagnose such cases as the "lymphoma type of ATL, extranodal primary cutaneous variant", it is essential to examine each case carefully, including cutaneous lesions at onset, lymph nodes and other organ involvement using computed tomography (CT) and/or positron emission tomography/CT, as well as the percentage of abnormal lymphocytes in peripheral blood. Based on the results of an ongoing nationwide survey on ATL, ATL with cutaneous lesions will be analyzed to investigate the incidence and prognosis of the so-called "lymphoma type of ATL, extranodal primary cutaneous variant".

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.

Relevance of molecular tests for HTLV-1 infection as confirmatory tests after the first sero-screening.

The diagnosis of human T-cell leukemia virus type-1 (HTLV-1) infection has been widely examined by serologics. In the first screening tests, serological false negative and positive samples have been reduced thanks to advances in assay techniques that apply new emission agents and sensors. On the other hand, western blot (WB) remains problematic. For example, WB analysis yields many samples equivalent to antibody positive ones. To reduce the need for WB, an alternative testing strategy is required to detect HTLV-1 infection. Polymerase chain reaction (PCR) for the HTLV-1 provirus has recently been recommended for a final diagnosis of infection. However, although PCR is thought to be one element, the validation of detection performance for HTLV-1 infection between serological and molecular testing is not always clear. Thus, this study aimed to evaluate the accuracy and test the validity of an improved methodology for serological detection of HTLV-infection, as well as that of PCR. In conclusion, the high values of kappa-statistics are expected to deliver high quality in chemiluminescent enzyme immunoassay (or chemiluminescent immunoassay), while the problems with WB assays remain to be elucidated. As an alternative to WB, a combination of real-time qPCR and nested PCR is proposed as a suitable confirmatory test.

The tyrosine kinase inhibitor dasatinib suppresses cytokine production by plasmacytoid dendritic cells by targeting endosomal transport of CpG DNA.

Plasmacytoid dendritic cells (pDCs) produce a vast amount of interferon (IFN)-α in response to nucleic acids from viruses and damaged self-cells through Toll-like receptor (TLR)7 and TLR9. Pharmaceutical agents that suppress IFN-α production by pDCs are instrumental in elucidating the mechanisms behind IFN-α production, and in developing novel therapies for inflammatory disorders that involve pDCs. Here, we show that a tyrosine kinase inhibitor for chronic myeloid leukemia with multiple targets, dasatinib, strongly suppresses production of IFN-α and proinflammatory cytokines by human pDCs stimulated with multimeric CpG oligodeoxynucleotides (CpG-A) without reducing viability. In contrast, other tyrosine kinase inhibitors, imatinib, and nilotinib, did not suppress the cytokine production at clinically relevant concentrations. Inhibitors of SRC family kinases (SFKs), which are prominent targets of dasatinib, also suppressed the cytokine production. Notably, however, dasatinib, but not SFK inhibitors, abrogated prolonged localization of CpG-A in early endosomes, which is a critical step for pDCs to produce a large amount of IFN-α. This study suggests that dasatinib suppresses IFN-α production by pDCs by inhibiting SFK-dependent pathways and SFK-independent endosomal retention of CpG DNA. Kinases controlling the distinctive endosomal trafficking in pDCs may be exploited as targets to develop novel therapies for pDC-related inflammatory disorders.

Relationships of diverse apoptotic death process patterns to mitochondrial membrane potential (Δψ(m)) evaluated by three-parameter flow cytometric analysis.

Recently, it has been proposed that novel methodologies are needed to re-evaluate apoptotic cell death, as studies of apoptosis have shown it to be a complex process. Since mitochondria are key regulators in cell death pathways, we developed a simultaneous 3-parameter flow cytometric analysis that incorporates the change in mitochondrial membrane potential (Δψ(m)) in an Annexin-V [for phosphatidyl-serine (PS)] and propidium iodide (PI) assay system (3 parameters with 4 colours), and evaluated the apoptotic process using various haematological malignant cell lines and death triggers. The present method enabled visualization of cell composition during apoptosis and captured complicated molecular events. For example, apoptotic cells that lost Δψ(m) did not always externalize PS, while some late apoptotic cells had polarized Δψ(m). The findings of unchanged PS-externalization and aberrant cell death suggest that there is no relationship of PS externalization and apoptosis with an unknown apoptotic mechanism. Based on PS-externalization, sensitivity to staurosporine, and the combination of cell lines and triggers, the apoptotic process was classified into 2 types. Importantly, most of our findings could not be observed by PS-PI and Δψ(m) assays when independently performed. Our method may be useful for examining mitochondrial-related apoptosis and death signalling pathways, as well as screening novel apoptosis-inducing cancer drugs.

Molecular characterization of quinolone-insensitive Streptococcus pneumoniae isolates from Japanese patients.

Fluoroquinolone resistance in Streptococcus pneumoniae has become a growing concern. Using S. pneumoniae isolates (n = 61) for which the minimum inhibitory concentration (MIC) of levofloxacin was not less than 1 µg/ml, we investigated the susceptibility of S. pneumoniae isolates to other fluoroquinolones (ciprofloxacin, pazufloxacin, moxifloxacin, garenoxacin, and sitafloxacin) and sequenced the quinolone resistance-determining regions of two topoisomerase genes, parC and gyrA, in these isolates to identify mutations. As the number of missense mutations increased, the MIC values for each drug increased. However, moxifloxacin, garenoxacin, and sitafloxacin showed potent activities against the isolates, while the MICs of ciprofloxacin and pazufloxacin were higher than the MICs of levofloxacin.

Attenuation of an adult T-cell leukemia skin lesion after treatment of a concomitant herpes simplex infection: a case study.

We report the development and treatment of eczema herpeticum in a 51-year-old male suffering from adult T-cell leukemia (ATL). Lesions of eczema herpeticum coexisted with the skin lesions of ATL. Treatment of eczema herpeticum resulted in a concomitant improvement in the symptoms of ATL, including a reduction in the size of the ATL plaques, for over 2 months before relapse.

Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene.

Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from positive blood culture samples. One hundred and two positive blood culture bottles from 68 patients were randomly selected and the bacteria were identified by phenotyping and pyrosequencing. The results of pyrosequencing identification displayed 84.3 and 64.7 % concordance with the results of phenotypic identification at the genus and species levels, respectively. In the monomicrobial samples, the concordance between the results of pyrosequencing and phenotypic identification at the genus level was 87.0 %. Pyrosequencing identified one isolate in 60 % of polymicrobial samples, which were confirmed by culture analysis. Of the samples identified by pyrosequencing, 55.7 % showed consistent results in V1 and V3 targeted sequencing; other samples were identified based on the results of V1 (12.5 %) or V3 (31.8 %) sequencing alone. One isolate was erroneously identified by pyrosequencing due to high sequence similarity with another isolate. Pyrosequencing identified one isolate that was not detected by phenotypic identification. The process of pyrosequencing identification can be completed within ~4 h. The information provided by DNA-pyrosequencing for the identification of micro-organisms in positive blood culture bottles is accurate and could prove to be a rapid and useful tool in standard laboratory practice.

Impact of miR-155 and miR-126 as novel biomarkers on the assessment of disease progression and prognosis in adult T-cell leukemia.

OBJECTIVE: Micro RNAs (miRNAs) provide new insight in the development of cancer, but little is known about their clinical relevance as biomarkers in the assessment of diagnosis, classification, progression and prognosis of various cancers. To explore a potential novel biomarker, we examined the cellular and plasma miRNA profiles in adult T-cell leukemia (ATL) characterized by diverse clinical features. METHODS AND RESULTS: Using CD4-positive cells isolated from 2 non-infected healthy individuals, 3 chronic ATL patients and 3 acute ATL patients, cellular miRNAs were profiled by microarray. The microarray screened 5 miRNAs namely miR-155, let-7g, miR-126, miR-130a and let-7b because of the large difference in their expression in diseased vs. that of healthy controls. The expression levels of before 5 miRNAs re-quantified by reverse transcription quantifiable polymerase chain reaction (RT-qPCR) were not always accordant in cells and plasma. The high and low plasma levels of miR-155 and miR-126 changed with ATL stage. CONCLUSION: The present study revealed that there is a quantitative discrepancy between cellular and plasma miRNAs. The elevation of plasma miR-155 and the reduction in miR-126 correlated with poor prognosis, indicating their usefulness as a novel biomarker for the assessment of disease stage.

[Diagnosis and management of antibiotic-resistant bacteria].

Antibiotic-resistant infections acquired in hospitals are of great concern, and have become a serious public issue. Antibiotic-resistant infections can be associated with a variety of bacteria, such as methicillin resistant Staphylococcus aureus(MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRP). Since clinical laboratories are responsible for detecting information regarding antibiotic-resistant bacteria, they are required to perform analysis and dissemination of the information. Currently, rapid methods for detecting antibiotic-resistant bacteria using molecular techniques are being developed in response to the problem of the conventional methods for bacteriological testing, which require a few days to obtain results. This article presents the diagnosis and management of antibiotic-resistant bacteria, which comprise a serious health care issue.

The transcription factor Spi-B regulates human plasmacytoid dendritic cell survival through direct induction of the antiapoptotic gene BCL2-A1.

Plasmacytoid dendritic cells (pDCs) selectively express Toll-like receptor (TLR)-7 and TLR-9, which allow them to rapidly secrete massive amounts of type I interferons after sensing nucleic acids derived from viruses or bacteria. It is not completely understood how development and function of pDCs are controlled at the transcriptional level. One of the main factors driving pDC development is the ETS factor Spi-B, but little is known about its target genes. Here we demonstrate that Spi-B is crucial for the differentiation of hematopoietic progenitor cells into pDCs by controlling survival of pDCs and its progenitors. In search for Spi-B target genes, we identified the antiapoptotic gene Bcl2-A1 as a specific and direct target gene, thereby consolidating the critical role of Spi-B in cell survival.

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.

Live Legionella pneumophila induces MUC5AC production by airway epithelial cells independently of intracellular invasion.

The airway epithelium is the initial barrier against airborne pathogens, and it plays many roles in host airway defense. Legionella pneumophila is an intracellular pathogen that causes rapidly advancing pneumonia and is sometimes life-threatening. Here, we evaluated the role of the airway epithelial cells in the defense against L. pneumophila by examining mucus production in vitro. The production of MUC5AC, a major mucin protein, was not induced by formalin- or ultraviolet-killed L. pneumophila, but it was induced by live L. pneumophila. Similarly, nuclear factor-kappaB (NF-κB) was activated only by live L. pneumophila. Inhibitors of ERK and JNK, but not p38, dose-dependently inhibited the induction of MUC5AC by live L. pneumophila. Inhibition of intracellular invasion by cytochalasin D did not affect MUC5AC production. Taken together, the results suggest that live L. pneumophila induces MUC5AC production via the ERK-JNK and NF-κB pathways without internalization of bacteria and that the airway epithelium produces mucin as part of the immune response against L. pneumophila.

Clinical characteristics of tertiary hospital patients from whom Acinetobacter calcoaceticus-Acinetobacter baumannii complex strains were isolated.

OBJECTIVE: Acinetobacter baumannii is a worldwide nosocomial pathogen that has become increasingly common over the past few decades, and strains of multidrug-resistant A. baumannii have been increasing. The aim of this study was to assess the clinical characteristics of A. calcoaceticus-A. baumannii complex (Acb complex) strains and to determine the risk factors of this infection. METHODS: The medical records of 121 patients at Nagasaki University Hospital from whom Acb complex had been isolated between January 2007 and December 2009 were retrospectively reviewed. Patient backgrounds, sensitivity to antibiotics, risk factors for infection, and prognosis were evaluated. RESULTS: Lower respiratory isolates accounted for 73% (147 strains) of all 201 isolates. Most of the isolates were sensitive to carbapenems. Of the 121 patients (74 males and 47 females; mean age: 62.1 years), 48 (39.7%) had malignancy and 75 (62.0%) were treated with antibiotics prior to isolation. Thirty-seven of the patients in this study (30.6%) were infected by Acb complex and the most frequent clinical manifestation was pneumonia (18 cases; 48.6%). Approximately 60% of infected patients were treated with ß-lactam agent in combination with ß-lactamase inhibitors or carbapenems. The mortality rate of infected patients was significantly higher than that of colonized patients (infected: 24.3%, colonized: 6.0%, p<0.05). Risk factors for Acb complex infection include being over 60 years of age, chronic liver disease, and the use of first-generation cephalosporins prior to isolation. CONCLUSION: Acb complex was relatively sensitive to antibiotics. The appropriate usage of antibiotics should be continued for the prevention of drug resistance in Acb complex.

In vivo efficacy and pharmacokinetics of biapenem in a murine model of ventilator-associated pneumonia with Pseudomonas aeruginosa.

Biapenem (BIPM) has high bactericidal activity against Pseudomonas aeruginosa and similar activity in vitro as meropenem (MEPM). We used a murine model to examine the efficacy of biapenem against ventilator-associated pneumonia (VAP) caused by P. aeruginosa. Mice were treated by intraperitoneal injection with 100 mg/kg BIPM or MEPM every 12 h beginning 12 h after inoculation with P. aeruginosa. Survival was evaluated for 7 days, and 24 h after infection, lung histopathology was analyzed and the number of viable bacteria in the lungs and blood was counted. In addition, the pharmacokinetics of BIPM and MEPM were analyzed after the initial treatment. BIPM and MEPM significantly prolonged survival compared to control (P < 0.05). The lungs of mice treated with BIPM or MEPM had significantly fewer viable bacteria (3.54 ± 0.28 vs. 3.77 ± 0.14 log(10) CFU/ml) than in the lungs of control mice (6.65 ± 0.57 log(10) CFU/ml) (P < 0.05). Furthermore, viable bacteria were not detected in the blood of mice treated with BIPM or MEPM (control 2.85 ± 0.85 log(10) CFU/ml) (P < 0.05). Histopathological examination of lung specimens indicated that BIPM and MEPM prevent the progression of lung inflammation, including alveolar neutrophil infiltration and hemorrhage. The % time above MIC for BIPM and MEPM was 15.4% and 18.3% in plasma and 19.8% and 19.8% in lungs, respectively. These results show that BIPM and MEPM significantly prolongs survival and reduces the number of viable bacteria in a murine model of VAP caused by P. aeruginosa. Therefore, BIPM might be a potent and effective treatment for VAP caused by this bacterium.

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.