A recombinant vesicular stomatitis virus encoding CCR5-tropic HIV-1 receptors targets HIV-1-infected cells and controls HIV-1 infection.

Anti-retroviral therapy is useful to treat human immunodeficiency virus type 1 (HIV-1)-infected individuals, but has some major problems, such as the generation of multidrug-resistant viruses. To develop a novel supplemental or alternative therapeutic for CCR5-tropic (R5) HIV-1 infection, we generated a recombinant vesicular stomatitis virus (rVSV) in which the gene encoding its envelope glycoprotein (G) was replaced with the genes encoding R5 HIV-1 receptors (human CD4 and CCR5), designated VSVΔG-CC5. Our present data demonstrate that this rVSV specifically infects cells that are transiently expressing R5 HIV-1 envelope glycoproteins, but does not infect those expressing CXCR4-tropic HIV-1 envelope glycoproteins. Notably, after a CD4+CCR5+ T cell line or primary cells initially infected with R5 HIV-1 were inoculated with G-complemented VSVΔG-CC5, the rVSV significantly reduced the number of HIV-1-infected cells, probably through direct targeting of the rVSV and VSV-mediated cytolysis and/or through syncytium formation- or cell-cell fusion-dependent killing, and markedly inhibited HIV-1 production. Furthermore, G-complemented VSVΔG-CC5 also efficiently inhibited HIV-1 infection in R5 HIV-1-infected humanized immunodeficient mice. Taken together, our findings indicate that a cytolytic rVSV that targets and eliminates R5 HIV-1-infected cells potentially has therapeutic value for controlling R5 HIV-1 infection.

Development of an infectious surrogate hepatitis C virus based on a recombinant vesicular stomatitis virus expressing hepatitis C virus envelope glycoproteins and green fluorescent protein.

To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies.

Highly sensitive detection of hepatitis B virus surface antigen by use of a semiautomated immune complex transfer chemiluminescence enzyme immunoassay.

The performance of hepatitis B surface antigen (HBsAg) screening assays is continuously improved to reduce the risk of transfusion-associated hepatitis B. In this study, a semiautomated immune complex transfer chemiluminescence enzyme immunoassay (ICT-CLEIA) for the detection of HBsAg, which is as sensitive as hepatitis B virus (HBV) DNA PCR, was developed; the ICT-CLEIA assay performance was compared with the performance of the Architect HBsAg QT assay and HBV DNA PCR. The specificities in the initial assay and after retesting were 99.50% (1,988/1,998 samples) and 99.95% (1,997/1,998 samples), respectively. The analytical detection limit was determined to be 0.2 mIU/ml using the 2nd International WHO HBsAg standard, and the cutoff value (0.5 mIU/ml) of the ICT-CLEIA assay was 8.0 standard deviations (SD) above the mean of the HBsAg-negative specimens. The ICT-CLEIA assay could detect HBsAg even in the presence of anti-HBs antibodies and demonstrated a 23.6-day-shorter window period using commercially available HBsAg seroconversion panels than the Architect HBsAg QT assay. Furthermore, the monitoring of the viral kinetics by the ICT-CLEIA assay and the HBV DNA PCR produced very similarly shaped curves during both the HBsAg seroconversion and reverse seroconversion periods. Therefore, the ICT-CLEIA assay may be useful not only for an earlier detection of HBV reactivation but also for the monitoring of hepatitis B patients.

Serum hepcidin levels and reticulocyte hemoglobin concentrations as indicators of the iron status of peritoneal dialysis patients.

Hepcidin is the key mediator of renal anemia, and reliable measurement of serum hepcidin levels has been made possible by the ProteinChip system. We therefore investigated the iron status and serum hepcidin levels of peritoneal dialysis (PD) patients who had not received frequent doses of an erythrocytosis-stimulating agent (ESA) and had not received iron therapy. In addition to the usual iron parameters, the iron status of erythrocytes can be determined by measuring reticulocyte hemoglobin (RET-He). The mean serum hepcidin level of the PD patients (n = 52) was 80.7 ng/mL. Their serum hepcidin levels were significantly positively correlated with their serum ferritin levels and transferrin saturation (TSAT) levels, but no correlations were found between their serum hepcidin levels and RET-He levels, thereby suggesting that hepcidin has no effect on the iron dynamics of reticulocytes. Since low serum levels of CRP and IL-6, biomarkers of inflammation, were not correlated with the serum hepcidin levels, there is likely to be a threshold for induction of hepcidin expression by inflammation.

HMGA1a is involved in specific splice site regulation of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) utilizes a highly complex splice site regulation system, taking advantage of host proteins, to express its own viral protein in an orderly way. We show here that one of the host proteins, high mobility group A protein 1a (HMGA1a), is involved in splice site regulation of 3' splice site 2 (A2) and 5'splice site 3 (D3) of HIV-1 genomic RNA. shRNA knockdown of HMGA1 in HeLa cells resulting in a decrease of HMGA1 showed a significant decrease of Vpr mRNA. RNA electrophoretic mobility shift assays showed HMGA1a specifically binds to a sequence adjacently upstream D3. In vitro splicing using heterologous pre-mRNA with A2 and D3, showed HMGA1a induced a splicing intermediate which decreased when an RNA decoy of the HMGA1a binding site was added. RT-PCR of in vitro splicing products revealed that HMGA1a induced an incomplete splicing product resulting from usage of A2 but inhibition of D3, which is reminiscent of the splicing pattern necessary for Vpr mRNA formation. HMGA1a interacted with hnRNPA1 shown by coimmunoprecipitation and supershifted U1 snRNP in an RNA electrophoretic mobility shift assay. We conclude that HMGA1a anchors U1 snRNP to inhibit D3 function, and that HMGA1a inhibits hnRNPA1 function on exon splicing silencer of Vpr (ESSV) to activate A2 function. We show here for the first time that HMGA1a is involved in specific splice site regulation of HIV-1.

A recombinant vesicular stomatitis virus encoding HIV-1 receptors and human OX40 ligand efficiently eliminates HIV-1-infected CD4-positive T cells expressing OX40.

OX40 protein is highly expressed on activated CD4-positive T cells that are susceptible for human immunodeficiency virus type 1 (HIV-1) infection. To target and kill HIV-1-infected OX40(+) T cells, we used a recombinant vesicular stomatitis virus (rVSV) lacking its envelope glycoprotein (ΔG) and instead expressing HIV-1 receptors CD4/CXCR4 and OX40 ligand (OX40L). Expression of OX40L as well as HIV-1 receptors on the VSV particles led to specific infection of OX40(+) T cells, including primary cells, either acutely or chronically infected with X4 HIV-1. Consequently, the rVSV rapidly eliminated these infected cells and caused a marked reduction of HIV-1 viral load in culture. Inclusion of the OX40L gene in the VSV recombinant led to significantly better infection and HIV-1 elimination compared with an rVSVΔG expressing only HIV-1 receptors. A novel rVSVΔG encoding both HIV-1 receptors and OX40L has a potentially greater therapeutic value than an rVSVΔG expressing only HIV-1 receptors.

Development of rapid diagnostic reagents for measles.

Measles diagnosis has thus far been based on clinical symptoms in daily clinical practice. However, atypical cases are not uncommon; a simple and rapid method of measles diagnosis is clinically required. Lateral flow-based rapid diagnosis reagents are widely used for point-of-care testing in Japanese clinics, because it does not require special skills and facilities. We have developed a rapid diagnostic reagent for measles that employs the lateral flow method. The lower limit of detection of this reagent was almost the same for recombinant proteins of wild-type and vaccine strain, at 5.1 x 10(3) copies/test. This lower limit of detection and the specificity of this reagent suggest its efficacy for the diagnosis of measles infection.

Automated enumeration of immature granulocytes.

The performance characteristics of the XE-2100 (Sysmex, Kobe, Japan) automated immature granulocyte (IG) count were studied. The automated IG count was compared with the manual morphology count and with a proposed reference flow cytometric count. The comparison data were analyzed by both least-squares and Passing-Bablok regression analysis. Long-term imprecision using preserved blood quality control specimens at different levels showed a range from 2.59% to 3.57% coefficient of variation (CV) for within-run imprecision and 3.57% to 6.85% CV for total imprecision. The within-run reproducibility performed using fresh blood on 3 different specimens showed a range from 5.55% to 8.24% CV. The counts were stable at both room temperature and after refrigeration for 24 hours.Passing-Bablok regression analysis showed excellent agreement between the proposed reference flow cytometric IG count and the XE-2100 IG count, while there was less agreement with the manual morphology count. Our results indicate that the automated IG count can replace the manual morphology count for IG counting in the clinical laboratory. The results also confirm that the flow cytometric IG count is superior to and can replace the manual morphology count as a reference method for IG counting.

Enumeration of bacterial cell numbers and detection of significant bacteriuria by use of a new flow cytometry-based device.

A new, automated flow cytometry-based urine bacterium analyzer (UBA) was developed. We assessed the UBA for linearity of measurement, reproducibility of results, carryover rate, and correlation of measured results with those determined by urine culture. We also evaluated its ability to screen urine samples for significant bacteriuria. The UBA showed excellent linearity and a minor carryover rate. Results from the UBA were highly reproducible, and in between-run precision assays, the coefficients of variation for the UBA results were smaller than those for the urine culture results. Two hundred seventy-three urine specimens from patients attending the outpatient clinics of two university-based hospitals were examined. The results for the UBA were compared with those for urine culture. The UBA detected significant bacteriuria with a sensitivity of 96.6%, a specificity of 79.9%, a positive predictive value of 57.0%, a negative predictive value of 98.8%, a false-positive rate of 15.8%, a false-negative rate of 0.7%, and an accuracy of 83.5%. These results were comparable to or better than those obtained with previously reported screening procedures. The UBA can perform accurate enumeration of bacterial cells automatically in 90 seconds and can be used for the screening of significant bacteriuria.

Feasibility of flow cytometry for the detection of bacteria from body fluid samples.

The detection of microorganisms in body fluids is indispensable for identifying the source of infection and is one of the important examinations that influence subsequent treatment. In order to quickly detect bacteria in body fluid samples, a flow cytometry-based experimental automated bacteria counter (BF-FCM), was tested to determine its clinical value. The results for detectability obtained with the BF-FCM were compared with those obtained by conventional culture and Gram-staining techniques. We evaluated a total of 318 body fluid samples, excluding bile samples from which fungus alone was isolated. The samples consisted of 176 bile, 64 ascites, 42 pleural fluid, and 36 cerebrospinal fluid samples. Among the 318 samples, 154 (48.4%) were culture-positive. Of these 154, the BF-FCM identified 130 as positive (sensitivity, 84.4%). Of the 164 samples that were culture-negative, 141 were negative by the BF-FCM (specificity, 86.0%). Based on the culture results, the BF-FCM detected bacteria with a positive predictive value of 85.0% (130 of 153 samples), a negative predictive value of 85.5% (141 of 165 samples), and percent agreement of 85.2%. Although there were 23/164 (14.0%) false-positive samples, we consider that the BF-FCM, in combination with Gram staining and conventional cultures, would be helpful in the diagnosis and management of patients with diseases such as bacterial meningitis that present emergently.

Usefulness of measurement of reticulated platelets for diagnosis of idiopathic thrombocytopenic purpura.

Reticulated platelets (RP) and large platelets (LP) were measured by an automated hematology analyzer (modified R-2000) in 287 healthy volunteers and 131 patients with thrombocytopenia or thrombocytosis. RP was significantly higher in patients with idiopathic thrombocytopenic purpura (ITP), especially in active phase, while RP was markedly lower in patients with essential thrombocytosis (ET) or chronic myelocytic leukemia (CML). LP was significantly higher in patients with ITP, especially in active phase, while LP was markedly lower in patients with aplastic anemia (AA), ET, or CML. In ITP, RP and LP were significantly higher in patients positive for anti-glycoprotein (Gp) IIb/IIIa antibody. RP and LP were poorly correlated with platelet-associated IgG (PAIgG). RP and LP were poorly correlated with plasma thrombopoietin levels, and negatively correlated with platelet count. These results show that RP reflects the pathology of thrombocytopenic disorders, and that measurement of RP is useful for the differential diagnosis and analysis of platelet kinetics.

Usefulness of fully automated measurement of reticulated platelets using whole blood.

Reticulated platelets (RP) were measured with an automated hematology analyzer (modified R-2000) in 287 healthy volunteers and in 212 patients with thrombocytopenia. In healthy volunteers, the RP was 0.48 +/- 0.26% in men and 0.48 +/- 0.32% in women. No significant difference in the RP values due to gender or age (21-60 years) was observed. Furthermore, the reverse correlation was observed between platelet counts and RP. The RP was high in patients with idiopathic thrombocytopenic purpura (ITP), those with high fibrinogen and fibrin degradation products (FDP), and those with high C-reactive protein (CRP), but low in patients after chemotherapy. The RP was highest in active phase of ITP, and relatively high in the partial remission phase of aplastic anemia. In patients after chemotherapy, the patients had a minimum phase of RP and then a maximum phase of RP before platelet counts increased. RP was significantly high in the maximum phase and significantly low in the minimum phase. The relationships between platelet count and RP were negatively correlated in patients with ITP, high FDP, or high CRP, but were not correlated in patients with aplastic anemia, liver disease, or after chemotherapy. These results show that RP reflects the pathology of thrombocytopenic disorders and the measurement of RP is useful for the differential diagnoses and analysis of platelet kinetics.

Improved detection of minimal acute myeloid leukemia cells by the use of the combined parameters of XE-2100 hematology analyzer.

BACKGROUND: For the diagnosis and therapy of acute leukemia, it is important to detect a small number of leukemic cells precisely. Although several automated hematology analyzers that carry blast-detecting programs have been developed, they do not exert sufficient detection sensitivity to exceed the sensitivity of manual eye counting method. METHODS: We constructed a new blast-detecting program by combining the numerical information acquired from five cytometric parameters presented by XE-2100. The sensitivity and specificity of this blast multi-scoring program were assessed in comparison with the Blasts flag program equipped originally in XE-2100. RESULTS: The blast-detecting sensitivity was found to be highly improved in the blast multi-scoring program as compared with the Blasts flag program without much decreasing the specificity. A small number of leukemic myeloblasts was detected at the better sensitivity than the eye counting method in the clinical course of the patients with acute myeloid leukemia. CONCLUSIONS: The daily practical use of this blast multi-scoring program will surely contribute to sensitive, objective, and real-time evaluation of the control of acute myeloid leukemia with a low cost.

Clinical and oncologic implications in epigenetic down-regulation of CD26/dipeptidyl peptidase IV in adult T-cell leukemia cells.

CD26/dipeptidyl peptidase IV (DPPIV), a T-cell-activation antigen, is a 110-kD type II surface glycoprotein expressed on various types of normal cells. CD26/DPPIV is considered a multifunction housekeeping protein. Malignant cells often show altered CD26/DPPIV expression or no CD26/DPPIV expression, thus suggesting a useful marker for assessing some T-cell malignancies. In this study, cell surface protein and messenger RNA expression profiles for CD26/DPPIV were examined in 49 patients with adult T-cell leukemia (ATL), 10 carriers of human T-lymphotropic virus I (HTLV-I), and 4 HTLV-I-infected cell lines to assess the utility of CD26/DPPIV expression as a useful molecular marker for ATL pathology. In contrast to normal lymphocytes, ATL cells and HTLV-I-infected cell lines apparently down-regulated or completely lost the CD26/DPPIV antigen. Furthermore, the positive rate and antigen density for CD26/DPPIV in ATL cells gradually declined along with the advancement of ATL stage. Analysis of genomic DNA and the CD26/DPPIV transcript showed that CD26- ATL cells possessed faintly detected transcripts of the gene that were aberrantly methylated at the CpG islands within the promoter region in parallel with the advancement of ATL, a finding supported by a rescue experiment for transcript reexpression using 5-azacytidine as demethylation agent. Moreover, there was no relationship between loss of CD26/DPPIV and HTLV-I tax expression. These results indicate that ATL cells down-regulate CD26 antigens by means of epigenetic machinery and that this antigen abnormality is a useful molecular marker for the pathology of ATL.

Automation of bone marrow aspirate examination using the XE-2100 automated hematology analyzer.

BACKGROUND: Attempts to analyze bone marrow aspirates have been reported with the use of several automated blood cell counters, but sufficient accuracy in examination is not acquired yet. Major problems have included difficulties in correctly differentiating various immature cells and interference by lipid in bone marrow aspirates. The goal of this study was to solve these problems to attain more accurate assessment of bone marrow aspirates with automated blood cell counters. METHODS: We modified the XE-2100 Automated Hematology Analyzer (Sysmex Corporation, Kobe, Japan) to fit it for bone marrow aspirate measurement and evaluated its performance. Measurements were performed with the modified XE-2100 on 81 patient samples of bone marrow aspirates; as a reference, the manual visual method was used and flow cytometric analysis were carried out. RESULTS: Good correlations between results with the modified XE-2100 and the manual visual method were obtained for total nucleated cell count (TNCC; r = 0.99), erythroblast/TNCC ratio (r = 0.93), and myeloid cell/TNCC ratio (r = 0.75). CONCLUSIONS: When this device is used, bone marrow aspirate differentials can be determined quickly and easily. This device will be useful for preliminary examination to obtain a summary of various blood cell ratios in bone marrow aspirates before performance of microscopic examination.

Evaluation of immature granulocyte counts by the XE-IG master: upgraded software for the XE-2100 automated hematology analyzer.

We evaluated an automated immature granulocyte (IG) count in the peripheral blood with the XE-IG Master (Sysmex Corporation). The XE-IG Master is the upgraded software package for the XE-2100 automated hematology analyzer. Reproducibility tests demonstrated a mean coefficient of variation of 7.02% for the IG percentage (IG%) and 6.93% for the absolute IG count. Correlations of the IG counts were assessed in two ways. A flow cytometric IG count using CD11b, CD16, and CD45 monoclonal antibodies and a manual differential count were used as reference methods. The regression equation and the correlation coefficient of the IG% for the flow cytometric reference count versus results with the XE-IG Master were: y = 0.91x + 0.10; r = 0.96. For the comparison with the manual differential count of promyelocytes, myelocytes, and metamyelocytes, the regression equation and correlation coefficient were: y = 0.81x + 1.27; r = 0.78. Samples were found to be stable up to 48 hours both at room temperature and when refrigerated. We investigated the clinical significance of the IG count as a new marker of acute inflammation. In this preliminary study, most samples with a high IG count had positive values for C-reactive protein and the erythrocyte sedimentation rate (positive sample rates were 84.0% and 95.0%, respectively) despite neutrophil counts within the normal range. Elevated IG counts correlated most closely with CD64 expression on polymorphonuclear cells and less so with the concentration of interleukin 6. Compared with other available inflammation markers, the IG count is rapidly generated with each full blood count at no extra cost and with no delay in sample analysis.

Automated analysis of differentiation-induced leukemic cells during all-trans retinoic Acid therapy of acute promyelocytic leukemia.

During differentiation-induction therapy of acute promyelocytic leukemia (APL) patients with all-trans retinoic acid (ATRA), a variety of APL-derived bizarre granulocytic cells appear in the peripheral blood. To evaluate the differentiation induction of leukemic cells, we have developed a new scattergram analyzing program with an automated hematology analyzer and compared the data with the flow cytometry measuring the expression of differentiation-associated cell surface antigens, CD11b and CD16. We used the fluorescence intensity and side scatter as parameters of granulocytic maturation in the analysis with the automated hematology analyzer. The analysis of 2 ATRA-treated APL patients and in vitro study using HL-60 cells demonstrated that the levels of fluorescence intensity and side scatter decreased as accompanied with granulocytic maturation, and these changes were parallel with the results of flow cytometry. Our automated scattergram analysis of cell differentiation will contribute to general, objective, and real-time evaluation of differentiation-induction therapy of APL with ATRA.

Immature granulocyte count after liver transplantation.

We evaluated the significance of immature granulocyte (IG) count during the clinical course after liver transplantation. We counted IG using the flow cytometric method with CD16, CD11b, and CD45 antibodies. Samples were obtained from 31 patients in the Department of Transplantation and Immunology, and we determined (i) the distribution of IG peak value, (ii) the distribution of IG peak time-points, (iii) the clinical background of patients with high IG, and (iv) the clinical course of high IG cases. We observed the appearance of IG (100/microl or higher) in the majority of the patients (23 out of 31 patients; 74.2%). The IG peak was detected on the 19th day after transplantation. We observed serious complications, such as melena, rejection, or severe infection, in high IG (500/microl or higher) cases. We observed instances of inflammation with low C-reactive protein (CRP) value in the presence of IG. We believe that IG is a useful marker to monitor inflammation.

Automated sperm concentration analysis with a new flow cytometry-based device, S-FCM.

The S-FCM uses flow cytometry technology to measure sperm concentrations. Semen samples from 104 men attending a male infertility clinic were used to evaluate the reproducibility of results and the carryover rate with the S-FCM by performing between- and within-run imprecision analyses. In addition, sperm concentrations measured with the S-FCM were compared with those obtained by manual analyses with the Makler chamber and the improved Neubauer hemacytometer. The results showed that automated analyses with the S-FCM were highly reproducible and the carryover rate was 0.17% or less. In within-run imprecision assays, the coefficients of variation for the S-FCM were less than 5% at all sperm concentrations, while those for the Makler chamber were between 17.7% and 28.7% at lower sperm concentrations. The overall correlation between values measured with the S-FCM and those measured with the Makler chamber and improved Neubauer hemacytometer was excellent, but at lower sperm concentrations the correlation was lower. The S-FCM performed sperm concentration analyses in 110 seconds compared with 5 minutes for the Makler chamber and 10 minutes for the improved Neubauer hemacytometer. The S-FCM is suitable for quantitative measurement of lower sperm concentrations.