Distinguishing functional exosomes and other extracellular vesicles as a nucleic acid cargo by the anion-exchange method.

The development of a new large-scale purification protocol is required for research on the reliable bioactivity and drug discovery of extracellular vesicles (EVs). To address this issue, herein, we propose an effective method for preparing high-performance exosomes (EXOs) by using an anion-exchange method. Cytotoxic T-lymphocyte (CTL) EVs from 4 L of culture supernatant through a 220 nm cut-off filter are divided into two populations at a deproteinization rate of over 99.97%, which are eluted at low (0.15 M-0.3 M) and high (0.3 M-0.5 M) NaCl concentrations (approximately 2 × 1012 and 1.5 × 1012 particles, respectively) through the anion-exchange column chromatography. The former are abundant in EXO proteins, including late endosome-associated proteins and rab-family and integrin-family proteins, and functional micro (mi) RNAs, and have bioactivity for preventing tumour metastasis by depleting mesenchymal cell populations in the primary tumour lesions. By contrast, the latter is microvesicle (MV)-like particles including DNA, core histone and ribosomal proteins, and GC-rich miRNAs with unknown function, and are easily phagocytosed by mannose receptor+ Kupffer cells. Thus, the anion-exchange method is suitable for the large-scale separation of bioactive EXOs and MV-like EVs as a cargo for dangerous nucleic acids at high-purity.

Cellular HIV-1 DNA levels in patients receiving antiretroviral therapy strongly correlate with therapy initiation timing but not with therapy duration.

BACKGROUND: Viral reservoir size refers to cellular human immunodeficiency virus-1 (HIV-1) DNA levels in CD4(+) T lymphocytes of peripheral blood obtained from patients with plasma HIV-1-RNA levels (viral load, VL) maintained below the detection limit by antiretroviral therapy (ART). We measured HIV-1 DNA levels in CD4(+) lymphocytes in such patients to investigate their clinical significance. METHODS: CD4(+) T lymphocytes were isolated from the peripheral blood of 61 patients with a VL maintained at less than 50 copies/ml for at least 4 months by ART and total DNA was purified. HIV-1 DNA was quantified by nested PCR to calculate the copy number per 1 million CD4(+) lymphocytes (relative amount) and the copy number in 1 ml of blood (absolute amount). For statistical analysis, the Spearman rank or Wilcoxon signed-rank test was used, with a significance level of 5%. RESULTS: CD4 cell counts at the time of sampling negatively correlated with the relative amount of HIV-1 DNA (median = 33 copies/million CD4(+) lymphocytes; interquartile range [IQR] = 7-123 copies/million CD4(+) lymphocytes), but were not correlated with the absolute amounts (median = 17 copies/ml; IQR = 5-67 copies/ml). Both absolute and relative amounts of HIV-1 DNA were significantly lower in six patients in whom ART was initiated before positive seroconversion than in 55 patients in whom ART was initiated in the chronic phase, as shown by Western blotting. CD4 cell counts before ART introduction were also negatively correlated with both the relative and absolute amounts of HIV-1 DNA. Only the relative amounts of HIV-1 DNA negatively correlated with the duration of VL maintenance below the detection limit, while the absolute amounts were not significantly correlated with this period. CONCLUSIONS: The amounts of cellular HIV-1 DNA in patients with VLs maintained below the detection limit by the introduction of ART correlated with the timing of ART initiation but not with the duration of ART. In addition, CD4(+) T lymphocytes, which were newly generated by ART, diluted latently infected cells, indicating that measurements of the relative amounts of cellular HIV-1 DNA might be underestimated.

Development and application of a simple LC-MS method for the determination of plasma maraviroc concentrations.

Maraviroc is an orally available antagonist of the CCR5 chemokine receptor, which acts as a human immunodeficiency virus type 1 (HIV-1) coreceptor. Binding of maraviroc to this receptor blocks HIV-1 attachment to the coreceptor and prevents HIV-1 from entering host cells. Maraviroc does not require intracellular processing to exert this activity. Drug interaction studies have shown changes in maraviroc exposure when given with other anti-HIV medications, and thus quantification of maraviroc in human plasma is important to manage drug interactions and to evaluate the relationship between plasma concentrations and treatment response. We developed a conventional LC-MS method for determining plasma maraviroc concentrations, validated by estimating precision and accuracy for inter- and intraday analysis in the concentration range of 0.011-2.188 µg/ml. The calibration curve was linear within this range. The average accuracy ranged from 92.7% to 99.7%, while the relative standard deviations of both inter- and intraday assays were less than 7.1%. Recovery of maraviroc exceeded 86.7%. Our LC-MS method provides a conventional, accurate and precise way to determine the maraviroc concentration in human plasma. This method enables dose adjustment based on monitoring plasma maraviroc concentrations and permits management of drug interactions and toxicity.

An 11-Year Surveillance of HIV Type 1 Subtypes in Nagoya, Japan.

Abstract To monitor active HIV-1 transmission in Nagoya, Japan, we have been determining the subtypes of HIV-1 infecting therapy-naive individuals who have newly visited the Nagoya Medical Center since 1997. The subtypes were determined by phylogenetic analyses using the base sequences in three regions of the HIV-1 genes including gag p17, pol protease (PR) and reverse transcriptase (RT), and env C2V3. Almost all HIV-1 subtypes from 1997 to 2007 and 93% of all HIV-1 isolates in 2007 were subtype B. HIV-1 subtypes A, C, D, and F have been detected sporadically since 1997, almost all in Africans and South Americans. The first detected circulating recombinant form (CRF ) was CRF01_AE (11-year average annual detection rate, 7.7%). Only two cases of CRF02_AG were detected in 2006. A unique recombinant form (URF ) was first detected in 1998 and the total number of URFs reached 25 by year 2007 (average annual detection rate, 4.7%). Eleven of these 25 were detected from 2000 to 2005 and had subtypes AE/B/AE as determined by base sequencing of the gag p17, pol PR and RT, and env C2V3 genes (average annual detection rate, 3.7%). Unique subtype B has been detected in six cases since 2006. All 17 of these patients were Japanese. Other recombinant HIV-1s have been detected intermittently in eight cases since 1998. During the 11-year surveillance, most HIV-1s in Nagoya, Japan were of subtype B. We expect that subtype B HIV-1 will continue to predominate for the next several years. Active recombination between subtype B and CRF01_AE HIV-1 and its transmission were also shown.

A conventional LC-MS method developed for the determination of plasma raltegravir concentrations.

Raltegravir belongs to a new class of antiretrovirals acting for a human immunodeficiency virus (HIV)-1 integrase inhibition. Clinical trials of this drug have demonstrated potent antiviral activity in both therapy naïve and experienced patients. Thus, raltegravir has become an important component of combination treatment regimens used to treat patients with multidrug-resistant HIV-1. The quantification of raltegravir in human plasma is important to support clinical studies and determine pharmacokinetic parameters of raltegravir in HIV-1 infected patients. Recently, the LC-MS/MS superfine system was developed to determine plasma concentration of raltegravir; however, the system needs to be delicately set and the equipment is very expensive. Therefore, we developed a conventional LC-MS method to overcome these difficulties. Subsequently the method was validated by estimating the precision and accuracy for inter- and intraday analysis in the concentration range of 0.010-7.680 microg/ml. The calibration curve was linear in this range. Average accuracy ranged from 97.2 to 103.4%. Relative standard deviations of both inter- and intraday assays were less than 10.4%. Recovery of raltegravir was more than 80.6%. This novel LC-MS method is accurate and precise enough to determine raltegravir levels in human plasma samples.

Analysis of near full-length genomic sequences of drug-resistant HIV-1 spreading among therapy-naïve individuals in Nagoya, Japan: amino acid mutations associated with viral replication activity.

We analyzed a total of 12 near full-length genomes of drug-resistant HIV-1 spreading among therapy-naïve individuals in Nagoya, Japan. Genomes comprised seven protease inhibitor (PI)-resistant viruses possessing an M46I (n = 6) or L90M mutation (n = 1) and five non-nucleoside reverse transcriptase inhibitor-resistant viruses possessing a K103N mutation. All 12 viruses conserved both an H87Q mutation in the cyclophilin A-binding site of Gag p24 (capsid) and a T23N mutation in the cysteine-rich domain of Tat protein. PI-resistant viruses commonly possessed two cleavage site mutations in the p6(Pol)/protease of Pol polyprotein (F48L in p6(Pol)) and the anchor/core domains of Nef protein (L57V). These amino acid mutations represent candidates for enhancing replication activity of drug-resistant viruses and supporting expansion of such viruses in therapy-naïve individuals.

Trend of drug-resistant HIV type 1 emergence among therapy-naive patients in Nagoya, Japan: an 8-year surveillance from 1999 to 2006.

We studied the emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) with major amino acid mutations in 402 therapy-naive patients at Nagoya Medical Center, Japan, between 1999 and 2006. The mean prevalence of drug-resistant HIV-1 was 6.7% (range, 2.3-10.0%; n = 27). HIV-1 variants with protease inhibitor (PI)-resistant mutations alone were most frequently found (3.5%, n = 14), followed by those with nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutations alone (1.7%, n = 7). Variants with nucleoside reverse transcriptase inhibitor (NRTI)-resistant mutations alone were sporadically found (1.0%, n = 4). A variant possessing both NRTI- and PI-resistant mutations was detected in one patient (0.2%) and a variant possessing both NNRTI- and PI-resistant mutations was identified in another patient (0.2%). In addition, another 17 variants (4.2%, n = 17) with only 215-revertant mutations (T215C/D/G/L/S) that can easily reconvert to the nucleoside analogue-associated mutation of T215Y/F were found. The 402 viruses were phylogenetically analyzed, revealing three independent clusters comprising PI-resistant variants with the M46I or L90M mutation, NNRTI-resistant variants with the K103N mutation, and 215-revertant variants. The PI-resistant and 215-revertant strains have been spreading since 2000, and the NNRTI-resistant strain has started spreading since 2003. The nature of the epidemic and information for successfully blocking the spread of drug-resistant HIV-1 were clarified in this study.

The validation of plasma darunavir concentrations determined by the HPLC method for protease inhibitors.

Darunavir (DRV) is a new protease inhibitor used to treat human immunodeficiency virus (HIV) type-1. The aim of this study was to validate the determination of plasma DRV concentrations using the HPLC method, a simple procedure for simultaneous determination of seven HIV protease inhibitors and efavirenz. The calibration curve was linear (range of 0.13 to 10.36 microg/ml). The average accuracy ranged from 100.7 to 105.6%. Both the interday and intraday coefficients of variation were less than 6.7%, which was similar to or much lower than previously reported values by the LC/MS/MS method. It is concluded that HPLC can be used to determine plasma DRV concentrations and routinely in the clinical setting; thus, this HPLC method enables further study of DRV pharmacokinetics in conventional hospital laboratories.

Determination of plasma tenofovir concentrations using a conventional LC-MS method.

The quantification of tenofovir, a nucleoside reverse transcriptase inhibitor prescribed once daily, in human plasma is important due to a recent increase in its use. HPLC, however, can not easily detect and quantify tenofovir because of interfering peaks. Therefore, we developed a rapid and conventional LC-MS method, validated by estimating the precision and accuracy for inter- and intraday analysis in the concentration range of 0.019-1.567 microg/ml. The calibration curve was linear in the described concentration range. Average accuracy ranged from 95.9 to 100.7%. Relative standard deviations of both inter- and intraday assays were less than 11.6%. Recovery of tenofovir was more than 80.2%. This novel method provides a conventional, accurate and precise way to determine tenofovir in human plasma samples.

No observable correlation between central nervous system side effects and EFV plasma concentrations in Japanese HIV type 1-infected patients treated with EFV containing HAART.

The present study assessed the relationship between central nervous system (CNS) side effects and plasma concentrations of efavirenz (EFV) in Japanese HIV-1-infected patients. Subjects consisted of 69 HIV-1-infected patients (57 therapy-naive and 12 therapy-experienced patients) being treated using EFV in combination with other antiretroviral agents at the outpatient HIV clinic. Successful virological treatment was achieved in 61 patients. Eight patients discontinued EFV containing therapy because CNS symptoms did not resolve (four patients), EFV-specific mutations were detected (two patients), or skin rash was observed (two patients). Mean EFV plasma concentration for 61 effectively treated patients, measured at 15 h postdosing, was 2.42 microg/ml (range: 0.78-6.82 microg/ml). This EFV concentration range contributed to suppressed viral load in these Japanese patients. Adverse CNS effects were observed in 19 patients soon after therapy onset. These effects disappeared within 1 month except for four patients who suffered severe CNS side effects. Mean EFV plasma concentrations were not significantly different between subjects with (2.45 +/- 1.08 microg/ml) and without (2.42 +/- 1.40 microg/ml) CNS side effects. We concluded no correlation existed between the plasma EFV concentration and the emergence of CNS side effects in Japanese HIV-1-infected patients. Further investigations, enforced with the drug concentration measurement at earlier time points and more appropriate assessment of CNS symptoms, are required.

Performance and quality assurance of genotypic drug-resistance testing for human immunodeficiency virus type 1 in Japan.

Highly active antiretroviral therapy (HAART) can suppress human immunodeficiency virus type 1 (HIV-1) replication and plasma HIV-1 to below detectable levels. However, HAART becomes ineffective when drug-resistant viruses emerge during HAART. Monitoring drug-resistance mutations in viruses is necessary for selecting new drugs or therapies effective at inhibiting such HIV-1 variants. Most laboratories in Japan perform the tests using in-house protocols. However, the quality of these tests has never been assessed. Our study assessing the accuracy and reliability of HIV-1 genotypic drug-resistance testing in 15 laboratories in Japan revealed that the quality was very high (97.3% accurate). The errors, though rare, were caused by human errors, poor electropherograms, and the use of inadequate primers. Here, we propose troubleshooting procedures to improve testing accuracy and reliability in Japan.

Beneficial effect of GB virus C co-infection in Human Immunodeficiency Virus type 1-infected individuals.

Several reports have documented a better prognosis for HIV-1-infected patients co-infected with GBV-C, while other reports have contradicted such findings with the result that this issue remains controversial. We attempted to clarify the complicated status of the effect of GBV-C co-infection on HIV-1-infected patients. GBV-C RNA was detected in 37 samples in 182 HIV-1-infected patients (20.3%) using RT/nested PCR. Of these, 3 were determined to be GBV-C genotype 1, 12 were genotype 2, and the remaining 22 were genotype 3. The GBV-C viral load quantified by real-time PCR ranged from 7.8x10(3) to 3.3x10(6) copies/ml. Weakly negative correlation was observed between GBV-C viral load and HIV-1 viral load in 19 HAART-naïve patients, indicating that a higher GBV-C viral load is associated with a greater suppression of HIV-1 replication. A previously published in vitro study suggested that GBV-C infection would induce up-regulation of RANTES, leading to suppression of HIV-1 replication. However, in our present study, the blood RANTES level was significantly lower in the GBV-C co-infected group than in the uninfected group (190-9,959 vs. 264-31,038 pg/ml, P=0.004). Our results suggested that a suppression of HIV-1 replication by GBV-C co-infection is not mediated by up-regulated RANTES, and thus call for another as yet unknown factor.

Identification and expression of a sialyltransferase responsible for the synthesis of disialylgalactosylgloboside in normal and malignant kidney cells: downregulation of ST6GalNAc VI in renal cancers.

Although disialyl glycosphingolipids such as GD3 and GD2 have been considered to be associated with malignant tumours, whether branched-type disialyl glycosphingolipids show such an association is not well understood. We investigated the sialyltransferases responsible for the biosynthesis of DSGG (disialylgalactosylgloboside) from MSGG (monosialylgalactosylgloboside). Among six GalNAc:alpha2,6-sialyltransferases cloned to date, we focused on ST6GalNAc III, V and VI, which utilize sialylglycolipids as substrates. In vitro enzyme analyses revealed that ST6GalNAc III and VI generated DSGG from MSGG with V(max)/K(m) values of 1.91 and 4.16 respectively. Transfection of the cDNA expression vectors for these enzymes resulted in DSGG expression in a renal cancer cell line. Although both ST6GalNAc III and VI genes were expressed in normal kidney cells, the expression profiles of ST6GalNAc VI among 20 renal cancer cell lines correlated clearly with those of DSGG, suggesting that the sialyltransferase involved in the synthesis of DSGG in the kidney is ST6GalNAc-VI. ST6GalNAc-VI and DSGG were found in proximal tubule epithelial cells in normal kidney tissues, while they were downregulated in renal cancer cell lines and cancer tissues. All these findings indicated that DSGG was suppressed during the malignant transformation of the proximal tubules as a maturation arrest of glycosylation.

Quantitative SNP-detection method for estimating HIV-1 replicative fitness: application to protease inhibitor-resistant viruses.

We have improved the methods for the standard competitive growth assay of human immunodeficiency virus type 1 (HIV-1). The cloning step for the mixed viral population and subsequent genotype analysis for arbitrary numbers of clones were excluded from procedures. Instead, a single nucleotide polymorphism (SNP)-detection step was devised for the determination of viral populations. The quantitative SNP-detection method can rapidly estimate the proportion of wild-type and mutant populations with high reproducibility. Consequently, this method allows manipulation of many samples within a short period. Using this new competitive growth assay, replicative fitness of drug-resistant HIV-1 containing an M46I amino acid mutation in the protease was assessed in the presence or absence of indinavir. Without indinavir, replicative fitness of wild-type HIV-1 surpassed that of M46I-mutated HIV-1, and the fraction of mutated virus was reduced to about 10% at passage #9. In contrast, the fraction of M46I-mutated virus increased to >90% at passage #5 in the presence of 26.4 nM indinavir. Almost identical results were obtained for L90M-mutated HIV-1 with or without saquinavir. HIV-1 can survive under indinavir pressure by acquiring M46I mutation, as with acquisition of the L90M mutation under saquinavir pressure. However, these mutations damage viral replicative fitness under natural conditions without any drugs. Subtle differences between wild-type and mutant viruses are thus easily detected using the improved method.

PNA-in situ hybridization method for detection of HIV-1 DNA in virus-infected cells and subsequent detection of cellular and viral proteins.

We describe in situ hybridization protocols using peptide nucleic acid (PNA) as a probe for detecting HIV-1 DNA in virus-infected cells and the subsequent detection of cellular and/or viral proteins. Because a PNA probe of approx 20 bases was sufficiently long to detect a specific target sequence, a conserved sequence of such a short length was easily identified. Therefore, this probe is valuable even to identify quasi-species of HIV-1. In addition, we adopted a catalyzed signal amplification method to amplify weak viral DNA signals; thus, stringent washing was crucial for eliminating false-positive signals. Our double-staining method using PNA-in situ hybridization and subsequent immunostaining enabled the active and inactive proviruses to be distinguished.

Conventional HPLC method used for simultaneous determination of the seven HIV protease inhibitors and nonnucleoside reverse transcription inhibitor efavirenz in human plasma.

We developed a simple HPLC method for the simultaneous quantitative determination of seven HIV protease inhibitors: amprenavir (APV), atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), and a nonnucleoside reverse transcription inhibitor, efavirenz (EFV). This method involves a rapid liquid-liquid drug extraction from plasma, the use of an isocratic elution on a reversed-phase C18 column, and an ultraviolet detection at a single wavelength (205 nm). The mobile phase consisted of 39% 50 mM phosphate buffer (pH 5.9), 22% methanol and 39% acetonitrile. Forty-eight samples could be measured in one day since the runtime of one sample is 30 min. The assay has been validated over a concentration range of 0.05 to 12.20 microg/ml for APV, 0.09 to 12.05 microg/ml for ATV, 0.05 to 12.01 microg/ml for IDV, 0.12 to 12.36 microg/ml for LPV, 0.18 to 12.20 microg/ml for NFV, 0.12 to 12.33 microg/ml for RTV, 0.12 to 12.06 microg/ml for SQV, and 0.05 to 12.17 microg/ml for EFV. Calibration curves were linear in the described concentration ranges. The average accuracy ranged from 97.2 to 106.8%. Both the interday and intraday coefficients of variation for all drugs tested were less than 8.5%. This method provides a simple, accurate, and precise method for the therapeutic drug monitoring of the seven protease inhibitors and EFV in clinical routine use.

New estimation method for highly sensitive quantitation of human immunodeficiency virus type 1 DNA and its application.

A new estimation method for quantitation of HIV-1 DNA was established by introducing a pre-quantitation polymerase chain reaction (PCR) before conventional real-time PCR. Two alternative methods for estimating the copy number can be used: the first method utilizes the rate of beta2-microglobulin (beta2M) gene amplification during the pre-quantitation PCR, and the second utilizes a calibration curve of the crossing point of real-time PCR versus the standard HIV-1-plasmid concentration. These methods could be used to reproducibly and accurately detect a provirus density down to five copies/10(6) cells (for methods 1 and 2, inter-assay CV=17 and 16% and accuracy=81 and 92%, respectively). The levels of HIV-1 DNA could be measurable using as little as 100 microl of whole blood or buffy coat cells. Using a combination of a conventional and highly sensitive methods, we found that the amount of HIV-1 DNA ranged from 2 to 5960 copies/10(6) cells (median of 830 copies/10(6) cells) in CD4-positive T lymphocytes isolated from 30 patients responding well to highly active antiretroviral therapy (HAART). Thus, the highly sensitive method developed in this study allows estimation of the HIV-1 reservoirs in peripheral CD4-positive T lymphocytes of patients responding well to HAART.

Delayed HIV-1 infection of CD4+ T lymphocytes from therapy-naïve patients demonstrated by quantification of HIV-1 DNA copy numbers.

Measuring the amount of HIV-1 DNA in infected cells is important to estimate the size of the viral reservoir in patients. However, the clinical impact of the intracellular viral DNA level remains unclear. The present study examines the clinical significance of the HIV-1 DNA level in peripheral CD4+ T lymphocytes from 21 therapy-naïve patients. HIV-1 DNA levels in purified peripheral CD4+ T lymphocytes were measured by the real-time PCR method using the Roche LightCycler system that can detect 200 copies/10(6) cells. We detected intracellular HIV-1 DNA in 15 (71.4%) of 21 patients at levels ranging from 270 to 98,120 copies/10(6) CD4+ cells, with a median of 2,220 copies/10(6) cells. We also found HIV-1 DNA that was below the detection limit in the remaining 6 patients, although 8,800-150,000 copies/ml of HIV-1 RNA were detected in plasma. Circular HIV-1 DNA was not detected in 5 of 6 cases, suggesting that reverse transcription in CD4+ T lymphocytes of these cases was not active. Thus, delayed HIV-1 infection of CD4+ T lymphocytes was demonstrated in these patients. The level of HIV-1 DNA in peripheral CD4+ T lymphocytes indicates the clinical status of therapy-naïve patients.

Pharmacokinetics of lopinavir after administration of Kaletra in healthy Japanese volunteers.

The pharmacokinetic parameters of lopinavir (LPV) were examined by administering Kaletra (LPV+ritonavir) to 8 healthy Japanese volunteers both in the fasting and postprandial conditions. LPV showed a biphasic decline, which was slower in the initial phase and became more rapid in the later phase. The behavior of LPV in the initial phase could be modeled using a one-compartment model with first-order absorption. In the fasting study, calculations based on the pharmacokinetic model revealed that the time to reach the maximum concentration (T(max)), maximum concentration (C(max)), half-life (T(1/2)), lag time, apparent volume of distribution (Vd/F) and oral clearance (Cl/F) were 3.2+/-1.0 h, 6.9+/-1.9 microg/ml, 10.0+/-3.7 h, 0.71+/-0.32 h, 51.0+/-12.4 l and 4.2+/-2.6 l/h, respectively. On the other hand, in the postprandial study, the calculated T(max), C(max), T(1/2), lag time, Vd/F and Cl/F were 5.6+/-2.0 h, 7.6+/-1.8 microg/ml, 16.7+/-7.0 h, 2.35+/-0.78 h, 48.0+/-15.9 l and 2.1+/-0.6 l/h, respectively. The values for the area under the curve for data collected over a 24-h period (AUC(24 h)) in the fasting and postprandial studies were 86.0+/-27.7 and 102.1+/-31.0 microg.h/ml, respectively. The T(1/2) had a tendency to be prolonged after food intake, but there were 2 cases with shortened T(1/2). Food intake prolonged the lag time 3-fold and as a result, the postprandial T(max) was 2 times longer.