Evaluation of next-generation sequencing performance for in vitro detection of viruses in biological products.

Next-Generation Sequencing (NGS) can detect nucleic acid sequences in a massively parallel sequencing. This technology is expected to be widely applied for the detection of viral contamination in biologics. The recently published ICH-Q5A (R2) draft indicates that NGS could be an alternative or supplement to in vitro viral tests. To examine the performance of NGS for the in vitro detection of viruses, adenovirus type 5 (Ad5), a model virus, was inoculated into Vero cells, which are the most popular indicator cells for the detection of adventitious viruses in the in vitro test. Total RNA extracted from the Vero cells infected with Ad5 was serially diluted with that from non-infected Vero cells, and each sample was analyzed using short- or long-read NGSs. The limits of detection of both NGS methods were almost the same and both methods were sensitive enough to detect viral sequences as long as there was at least one copy in one assay. Although the multiplexing in NGS carries the risk of cross-contamination among the samples, which could lead to false positives, this technology has the potential to become a rapid and sensitive method for detecting adventitious agents in biologics.

Evaluation of the reproducibility and positive controls of cellular immortality test for the detection of immortalized cellular impurities in human cell-processed therapeutic products.

Introduction: Contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic/immortalized cellular impurities is a major concern in the manufacturing and quality control of hCTPs. The cellular immortality test based on cell growth analysis is a method for detecting tumorigenic/immortalized cellular impurities in hCTPs. However, the performance of the cellular immortality test has not yet been well characterized. In this study, we examined the reproducibility of the cellular immortality test in detecting HeLa cells as a model of tumorigenic cellular impurities, as well as the applicability of other models of cellular impurities with different tumorigenicity to the cellular immortality test. Methods: Using HeLa cells as a model for cellular impurities, we measured the growth rate of human mesenchymal stem cells (hMSCs) supplemented with HeLa cells at concentrations ranging from 0.01 to 0.0001% at each passage in three laboratories and evaluated the reproducibility of the detection of immortalized cellular impurities. In addition, HEK293 cells (another immortalized cell line) and MRC-5 cells (a non-immortalized cell line) were employed as cellular impurity models that exhibit different growth characteristics from HeLa cells, and the ability of the cellular immortality test to detect these different impurities when mixed with hMSCs was examined. Results: In the multisite study, the growth rate of hMSCs supplemented with 1 and 10 HeLa cells (0.0001% and 0.001%) significantly increased and reached a plateau in all three laboratories, whereas those of hMSCs alone eventually decreased. Moreover, when hMSCs were supplemented with 10 and 100 HEK293 and MRC-5 cells (0.001% and 0.01%), the growth rate significantly increased. The growth rate of hMSCs supplemented with HEK293 cells increased with passage and remained high, whereas that of hMSCs supplemented with MRC-5 cells eventually decreased, as in the case of hMSCs alone. Conclusions: These results indicate that the cellular immortality test is reproducible and can detect immortalized (i.e., potentially tumorigenic) cells such as HEK293 cells with a lower growth rate than HeLa cells by discriminating against normal cells, which could contribute to ensuring the safety and quality of hCTPs.

A selective cytotoxic adenovirus vector for concentration of pluripotent stem cells in human pluripotent stem cell-derived neural progenitor cells.

Highly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.

Infectivity assessment of porcine endogenous retrovirus using high-throughput sequencing technologies.

Xenogenic cell-based therapeutic products are expected to alleviate the chronic shortage of human donor organs. For example, porcine islet cell products are currently under development for the treatment of human diabetes. As porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the case of products that use living pig cells as raw materials. Although several PERV sequences exist in the porcine genome, not all have the ability to infect human cells. Therefore, polymerase chain reaction analysis, which amplifies a portion of the target gene, may not accurately assess the infection risk. Here, we determined porcine genome sequences and evaluated the infectivity of PERVs using high-throughput sequencing technologies. RNA sequencing was performed on both PERV-infected human cells and porcine cells, and reads mapped to PERV sequences were examined. The normalized number of the reads mapped to PERV regions was able to predict the infectivity of PERVs, indicating that it would be useful for evaluation of the PERV infection risk prior to transplantation of porcine products.

Validation of novel identification algorithms for major adverse cardiovascular events in a Japanese claims database.

Predicting clinical outcomes can be difficult, particularly for life-threatening events with a low incidence that require numerous clinical cases. Our aim was to develop and validate novel algorithms to identify major adverse cardiovascular events (MACEs) from claims databases. We developed algorithms based on the data available in the claims database International Classification of Diseases, Tenth Revision (ICD-10), drug prescriptions, and medical procedures. We also employed data from the claims database of Jichi Medical University Hospital, Japan, for the period between October 2012 and September 2014. In total, we randomly extracted 100 potential acute myocardial infarction cases and 200 potential stroke cases (ischemic and hemorrhagic stroke were analyzed separately) based on ICD-10 diagnosis. An independent committee reviewed the corresponding clinical data to provide definitive diagnoses for the extracted cases. We then assessed the algorithms' accuracy using positive predictive values (PPVs) and apparent sensitivities. The PPVs of acute myocardial infarction, ischemic stroke, and hemorrhagic stroke were low only by diagnosis (81.6% [95% CI 72.5-88.7]; 31.0% [95% CI 22.8-40.3]; and 45.5% [95% CI 34.1-57.2], respectively); however, the PPVs were elevated after adding the prescription and procedure data (87.0% [95% CI 78.3-93.1]; 44.4% [95% CI 32.7-56.6]; and 46.1% [95% CI 34.5-57.9], respectively). When we added event-specific prescription and procedure data to the algorithms, the PPVs for each event increased to 70%-98%, with apparent sensitivities exceeding 50%. Algorithms that rely on ICD-10 diagnosis in combination with data on specific drugs and medical procedures appear to be valid for identifying MACEs in Japanese claims databases.

A highly sensitive method for the detection of recombinant PERV-A/C env RNA using next generation sequencing technologies.

Several xenogenic cell-based therapeutic products are currently under development around the world for the treatment of human diseases. Porcine islet cell products for treating human diabetes are a typical example. Since porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the development of these products. Four subgroups of infectious PERV have been identified, namely PERV-A, -B, -C, and recombinant PERV-A/C. Among them, PERV-A/C shows a high titre and there was a paper reported that an incidence of PERV-A/C viremia was increased in diseased pigs; thus, it would be important to monitor the emergence of PERV-A/C after transplantation of porcine products. In this study, we developed a highly sensitive method for the detection of PERV-A/C using next generation sequencing (NGS) technologies. A model PERV-C spiked with various doses of PERV-A/C were amplified by RT-PCR and the amplicons were analysed by NGS. We found that the NGS analysis allowed the detection of PERV-A/C at the abundance ratios of 1% and 0.1% with true positive rates of 100% and 57%, respectively, indicating that it would be useful for the rapid detection of PERV-A/C emergence after transplantation of porcine products.

Ethnic differences in association of outcomes with trimethylamine N-oxide in acute heart failure patients.

AIMS: The aim of this study was to investigate whether ethnicity influences the associations between trimethylamine N-oxide (TMAO) levels and heart failure (HF) outcomes. METHODS AND RESULTS: Trimethylamine N-oxide levels were measured in two cohorts with acute HF at two sites. The UK Leicester cohort consisted mainly of Caucasian (n = 842, 77%) and South Asian (n = 129, 12%) patients, whereas patients in the Japanese cohort (n = 116, 11%) were all Japanese. The primary endpoint was the measurement of all-cause mortality and/or HF rehospitalization within 1 year post-admission. Association of TMAO levels with outcome was compared in the entire population and between ethnic groups after adjustment for clinical parameters. TMAO levels were significantly higher in Japanese patients [median (interquartile range): 9.9 µM (5.2-22.8)] than in Caucasian [5.9 µM (3.6-10.8)] and South Asian [4.5 µM (3.1-8.4)] (P < 0.001) patients. There were no differences in the rate of mortality and/or HF rehospitalization between the ethnic groups (P = 0.096). Overall, higher TMAO levels showed associations with mortality and/or rehospitalization after adjustment for confounders ( P = 0.002). Despite no differences between ethnicity and association with mortality/HF after adjustment (P = 0.311), only in Caucasian patients were TMAO levels able to stratify for a mortality/HF event (P < 0.001). CONCLUSIONS: Differences were observed in the association of mortality and/or rehospitalization based on circulating TMAO levels. Elevated TMAO levels in Caucasian patients showed increased association with adverse outcomes, but not in non-Caucasian patients.

Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells.

Cell-processed therapeutic products (CTPs) derived from human pluripotent stem cells (hPSCs) have innovative applications in regenerative medicine. However, undifferentiated hPSCs possess tumorigenic potential; thus, sensitive methods for the detection of residual undifferentiated hPSCs are essential for the clinical use of hPSC-derived CTPs. The detection limit of the methods currently available is 1/105 (0.001%, undifferentiated hPSCs/differentiated cells) or more, which could be insufficient for the detection of residual hPSCs when CTPs contain more than 1 × 105 cells. In this study, we developed a novel approach to overcome this challenge, using adenovirus and adeno-associated virus (AdV and AAV)-based selective cytotoxic vectors. We constructed AdV and AAV vectors that possess a suicide gene, iCaspase 9 (iCasp9), regulated by the CMV promoter, which is dormant in hPSCs, for the selective expression of iCasp9 in differentiated cells. As expected, AdV/CMV-iCasp9 and AAV/CMV-iCasp9 exhibited cytotoxicity in cardiomyocytes but not in human induced pluripotent stem cells (hiPSCs). The vectors also induced apoptosis in hiPSC-derived cardiomyocytes, and the surviving cells exhibited higher levels of hPSC marker expression. These results indicate that the AdV- and AAV-based cytotoxic vectors concentrate cells expressing the undifferentiated cell markers in hiPSC-derived products and are promising biological tools for verifying the quality of CTPs.

Correlation between B-Type Natriuretic Peptide and N-Terminal pro-B-Type Natriuretic Peptide in a Large Japanese Population at Risk of Stage A Heart Failure.

BACKGROUND: The measurements of B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) are useful for ruling out heart failure and as prognostic markers in not only heart failure populations but also general populations. It is not clear whether these two biomarkers are elevated in parallel or associated with demographic characteristics in large populations at risk of stage A heart failure. Here we investigated the relationship between BNP and NT-proBNP and extended the evaluation of this association to known demographic disparities in stage A heart failure. METHODS: Of 4,310 ambulatory patients, we analyzed the cases of the 3,643 (mean age 65 ± 11 years, 46$ male, and 79$ on antihypertensive medication) patients whose serum BNP and NT-proBNP levels were both measured and who had a history of and/or risk factors for cardiovascular disease from the Japan Morning Surge-Home Blood Pressure (J-HOP) Study dataset. RESULTS: The median (25th-75th percentiles) BNP and NT-proBNP values were 18.7 (9.3-38.5) pg/mL and 50.3 (25.5-97.4) pg/mL. There was a significant association between log-transformed BNP and log-transformed NT-proBNP (r = 818, p < 0.001). A multiple linear regression analysis showed that log-transformed NT-proBNP was significantly associated with log-transformed BNP (beta coefficient = 0.774, p < 0.001). When stratified by demographic characteristics, these associations remained (all p < 0.001). CONCLUSION: In a large Japanese population at risk of stage A heart failure, there was a significant association between BNP and NT-proBNP after adjustment and stratification by demographics.

Sex Differences in the Prognostic Power of Brain Natriuretic Peptide and N-Terminal Pro-Brain Natriuretic Peptide for Cardiovascular Events　- The Japan Morning Surge-Home Blood Pressure Study.

BACKGROUND: Brain natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are prognostic biomarkers. Although these 2 peptides differ with regard to biological characteristics, there are few reports on the differences between BNP and NT-proBNP with regard to cardiovascular events or according to sex.Methods and Results:Between 2005 and 2012, this study analyzed 3,610 of 4,310 Japanese outpatients (mean age, 65 years; men, n=1,664; women, n=1,947) with a history of at least one cardiovascular event who were recruited to the Japan Morning Surge-Home Blood Pressure Study. During an average 4-year follow-up, there were 129 cardiovascular events. Both median BNP (21.1 pg/mL; IQR, 10.9-40.6 pg/mL vs. 16.2 pg/mL, IQR, 7.2-36.2 pg/mL, P<0.001) and median NT-proBNP (54.7 pg/mL; IQR, 30.2-102.6 pg/mL vs. 44.9 pg/mL, IQR, 20.7-92.6 pg/mL, P<0.001) were significantly higher in women than in men. A 1-SD increment in log-transformed BNP (hazard ratio [HR], 2.18; 95% CI: 1.53-3.10) and NT-proBNP (HR, 2.39; 95% CI: 1.73-3.31) was associated with a significant increase in cardiovascular events in women; in men, only NT-proBNP showed this association. There was an interaction between log-transformed BNP (P=0.007) or NT-proBNP (P=0.001) and cardiovascular events according to sex. CONCLUSIONS: Both BNP and NT-proBNP predicted cardiovascular outcomes in a large Japanese clinical population. BNP and NT-proBNP were significantly stronger predictors in women than in men.

In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

Application of cell growth analysis to the quality assessment of human cell-processed therapeutic products as a testing method for immortalized cellular impurities.

In human cell-processed therapeutic products (hCTPs) for clinical application, tumorigenic cellular impurities in the manufacturing process are a major concern. Because cellular immortalization is one of the prerequisite steps in tumorigenesis, we tested whether cell growth analysis can be employed to check for immortalized (and potentially tumorigenic) cellular impurities in hCTPs. We monitored the growth of human bone marrow-derived mesenchymal stem cells (BMSCs) mixed with HeLa cells at a ratio of 1/106 or more and compared their growth rates with that of BMSCs alone. The cell growth analysis detected a significant increase in the growth rate of the BMSCs spiked with 0.0001% HeLa within 30 days at a probability of 47%. When human adipose-derived stem cells (ADSCs) were spiked with ASC52telo cells, a human telomerase reverse transcriptase (hTERT)-immortalized adipose-derived mesenchymal stem cell line, at a ratio of 0.001% or more, their growth rates were significantly increased within 15 passages, compared with that of ADSCs alone. These results indicate that cell growth analysis for the detection of immortalized cellular impurities in human somatic stem cells is simple and can be useful for the quality assessment of hCTPs in the manufacturing process.

Fluorescent image analysis of HIV-1 and HIV-2 uncoating kinetics in the presence of old world monkey TRIM5α.

Uncoating of Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2) conical cores is an important early step for establishment of infection. In Old World Monkey (OWM) cells, the TRIM5α cellular factor potently suppresses an early step of infection by HIV-1. Previously, biochemical studies using whole cell lysates of infected cells revealed that OWM TRIM5α accelerates the uncoating of HIV-1, leading to premature reverse transcription. In the present study, we re-evaluated uncoating kinetics of HIV-1 in the presence of OWM TRIM5α by using an in situ uncoating assay, which allowed us to differentiate productive HIV-1 entry from simple (non-productive) endocytosis. Results showed that the uncoating kinetics of HIV-1 was indeed accelerated in the presence of OWM TRIM5α. Furthermore, we adapted an in situ uncoating assay to HIV-2, which showed wide variations in TRIM5α sensitivity among different isolates. HIV-2 isolate GH123, whose infectivity was suppressed by cynomolgus monkey (CM) TRIM5α, showed accelerated uncoating in the presence of CM TRIM5α. In contrast, mutant HIV-2 ASA, whose infectivity was unaltered by CM TRIM5α, showed no change in uncoating kinetics in the presence of CM TRIM5α. These results confirmed and further extended the previous notion that accelerated uncoating is associated with restriction activity of TRIM5α against lentiviruses.

Characterization of the cell growth analysis for detection of immortal cellular impurities in human mesenchymal stem cells.

The analysis of in vitro cell senescence/growth after serial passaging can be one of ways to show the absence of immortalized cells, which are frequently tumorigenic, in human cell-processed therapeutic products (hCTPs). However, the performance of the cell growth analysis for detection of the immortalized cellular impurities has never been evaluated. In the present study, we examined the growth rates of human mesenchymal stem cells (hMSCs, passage 5 (P = 5)) contaminated with various doses of HeLa cells, and compared with that of hMSCs alone. The growth rates of the contaminated hMSCs were comparable to that of hMSCs alone at P = 5, but significantly increased at P = 6 (0.1% and 0.01% HeLa) or P = 7 (0.001% HeLa) within 30 days. These findings suggest that the cell growth analysis is a simple and sensitive method to detect immortalized cellular impurities in hCTPs derived from human somatic cells.

Therapeutic strategy using extracorporeal life support, including appropriate indication, management, limitation and timing of switch to ventricular assist device in patients with acute myocardial infarction.

The appropriate indication for, management of and limitations to extracorporeal life support (ECLS) and the timing of a switch to a ventricular assist device (VAD) remain controversial issues in patients with acute myocardial infarction (AMI) complicated with cardiogenic shock or cardiopulmonary arrest. To evaluate and discuss these issues, we studied patients with AMI treated with ECLS and compared deceased and discharged patients. Thirty-eight patients with AMI who needed ECLS [35 men (92.1 %), aged 59.9 ± 13.5 years] were enrolled in this study. Of these 38 patients, 34 subsequently underwent percutaneous coronary intervention (PCI), and four subsequently received coronary artery bypass grafting (CABG). Fourteen patients (36.8 %) were discharged from the hospital. The outcome was not favorable for those patients with deteriorating low output syndrome (LOS) and the development of leg ischemia, hemolysis and multiple organ failure during ECLS. Levels of creatine kinase, creatine kinase-MB (CK-MB), lactate dehydrogenase, serum creatinine (Cr) and amylase after the patient had been put on ECLS and fluctuation of the cardiac index, blood pressure, arterial blood gas analysis and CK-MB and Cr levels during ECLS were indicators to switch from the ECLS to VAD. In the case of patients with no complication associated with ECLS, 4.6-5.6 days after initiation of ECLS was assumed to be the threshold to decide whether to switch from ECLS to VAD. Patients with AMI who suddenly developed refractory pulseless ventricular tachycardia or ventricular fibrillation without deteriorating LOS and who underwent successful PCI or CABG, and who prevented the complications associated with ECLS, showed a high probability of recovering with ECLS.

Slower uncoating is associated with impaired replicative capability of simian-tropic HIV-1.

Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously generated a simian-tropic HIV-1 that replicates efficiently in CM cells. This virus encodes a capsid protein (CA) with SIVmac239-derived loops between α-helices 4 and 5 (L4/5) and between α-helices 6 and 7 (L6/7), along with the entire vif from SIVmac239 (NL-4/5S6/7SvifS). These SIVmac239-derived sequences were expected to protect the virus from HIV-1 restriction factors in monkey cells. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the impaired replicative capability of the virus in human cells. This adapted virus encoded a G-to-E substitution at the 116(th) position of the CA (NL-4/5SG116E6/7SvifS). In the work described here, we explored the mechanism by which the replicative capability of NL-4/5S6/7SvifS was impaired in human cells. Quantitative analysis (by real-time PCR) of viral DNA synthesis from infected cells revealed that NL-4/5S6/7SvifS had a major defect in nuclear entry. Mutations in CA are known to affect viral core stability and result in deleterious effects in HIV-1 infection; therefore, we measured the kinetics of uncoating of these viruses. The uncoating of NL-4/5S6/7SvifS was significantly slower than that of wild type HIV-1 (WT), whereas the uncoating of NL-4/5SG116E6/7SvifS was similar to that of WT. Our results suggested that the lower replicative capability of NL-4/5S6/7SvifS in human cells was, at least in part, due to the slower uncoating of this virus.

Generation of rhesus macaque-tropic HIV-1 clones that are resistant to major anti-HIV-1 restriction factors.

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.

TRIM5 genotypes in cynomolgus monkeys primarily influence inter-individual diversity in susceptibility to monkey-tropic human immunodeficiency virus type 1.

TRIM5α restricts human immunodeficiency virus type 1 (HIV-1) infection in cynomolgus monkey (CM) cells. We previously reported that a TRIMCyp allele expressing TRIM5-cyclophilin A fusion protein was frequently found in CMs. Here, we examined the influence of TRIM5 gene variation on the susceptibility of CMs to a monkey-tropic HIV-1 derivative (HIV-1mt) and found that TRIMCyp homozygotes were highly susceptible to HIV-1mt not only in vitro but also in vivo. These results provide important insights into the inter-individual differences in susceptibility of macaques to HIV-1mt.

Calcium-incorporated titanium surfaces influence the osteogenic differentiation of human mesenchymal stem cells.

In this study, a titanium surface was chemically modified with calcium ions and assessed for its influence on osteogenic differentiation and molecular responses of human mesenchymal stem cells (hMSCs). Titanium disks were treated with NaOH (NaOH treatment), NaOH + CaCl2 (CaCl2 treatment), or NaOH + Ca(OH)2 (Ca(OH)2 treatment). Ca(OH)2 treatment caused significantly greater calcium incorporation onto the titanium surface and apatite formation than CaCl2 treatment. The morphology of hMSCs differed on CaCl2- and Ca(OH)2-treated disks. The osteopontin (OPN) expression in hMSCs cultured on CaCl2-treated titanium was significantly higher than that in cells cultured on NaOH-treated disks; OPN expression was significantly higher in cells cultured on Ca(OH)2-treated disks than on un-, NaOH-, and CaCl2-treated disks. Osteocalcin (OCN) protein expression in hMSCs cultured on Ca(OH)2-treated disks was significantly higher than that on all the other disks. Comparative expression profiling by DNA microarray and pathway analyses revealed that calcium modification of the titanium surface induced integrin ß3 after OPN upregulation and promoted Wnt/ß-catenin signaling in hMSCs. In addition, Ca(OH)2 treatment upregulated the expression of bone morphogenetic protein 2, cyclooxygenase 2, and parathyroid hormone-like hormone in comparison to CaCl2 treatment. These observations suggest that calcium-modified titanium surfaces affect osteogenic differentiation in hMSCs and that Ca(OH)2 treatment induced osteogenic differentiation in hMSCs, whereas CaCl2 treatment had a limited effect.