High DNA Load Research Articles

HHV-6 encephalitis may complicate the early phase after allogeneic hematopoietic stem cell transplantation: Detection by qualitative multiplex PCR and subsequent quantitative real-time PCR.

Viral reactivation following hematopoietic stem cell transplantation (HSCT) can cause various complications especially viral encephalitis. In this prospective study, we investigated the correlation of post-HSCT viral reactivation in blood with CNS dysfunction. We employed a multiplex PCR that detects 13 kinds of viruses as a first-line screening test and real-time PCR for subsequent quantitative evaluation. Five hundred ninety-one whole blood samples were collected from 105 patients from before until 42 days after HSCT. Seven patients developed CNS dysfunction such as altered consciousness. In six of the seven, the multiplex PCR test detected HHV-6 DNA in at least one sample. In contrast, DNA from other viruses, such as CMV, EBV, HHV-7, adenovirus, and HBV was never detected in any of the seven patients throughout the study period. Quantitative measurement of whole blood HHV-6 DNA levels demonstrated four of the six HHV-6 DNA loads were elevated at successive time points during the CNS dysfunction. In addition, the virus DNA peaks were temporally associated with the development of CNS dysfunction. CSF was tested in two of the four patients and high HHV-6 DNA levels comparable to those in whole blood were confirmed in both. These four patients were, thus, suspected to have developed HHV-6 encephalitis, a rate of 3.8% in the study population. Our results suggest that early diagnosis of probable HHV-6 encephalitis can be improved by confirming high HHV-6 DNA load in blood.

A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA

Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression.Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes.Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism (“energetic stress”).The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control.To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses.

Study of human cytomegalovirus replication in body fluids, placental infection, and miscarriage during the first trimester of pregnancy.

Intrauterine infection caused by human cytomegalovirus (HCMV) can lead to embryo, fetal, and neonatal damage. The prevalence of HCMV replication in body fluids (blood, urine, and cervicovaginal secretion) was investigated, and its effects on HCMV vertical transmission and miscarriages in early pregnant women were evaluated. HCMV DNA in body fluids was detected in 1,064 early pregnant women (624 normal pregnancies and 440 miscarriages). There were 101 cases who were HCMV DNA positive in cervicovaginal secretion and the rates were 10.9% (48/440 cases) and 8.5% (53/624 cases) in miscarriages and normal pregnancies, respectively (P > 0.05). A total of 101 cases (63 and 38 cases with and without HCMV DNA in cervicovaginal secretion, respectively) were given HCMV DNA detection in placental villi/deciduas. There were five cases (7.9%; two normal pregnancies and three miscarriages) with HCMV DNA in placental villi/deciduas among the 63 cases with HCMV DNA in cervicovaginal secretion, whereas none of the other 38 cases were detected HCMV DNA positive in their placental villi/deciduas. The percentage of HCMV DNA in placental villi/deciduas was higher in miscarriage group (9.1% [3/33]) than that in the normal pregnancy group (6.7% [2/30]), but there was no statistical significance (P > 0.05). Two cases with a higher HCMV loads in cervicovaginal secretion and placental villi/deciduas had miscarriages. These findings suggest that HCMV replication in cervicovaginal secretion can involve in placental HCMV infection, and high HCMV DNA loads in cervicovaginal secretion and placental villi/deciduas are associated with miscarriage.

Expression of BKV and JCV encoded microRNA in human cerebrospinal fluid, plasma and urine

BackgroundBK and JC polyomaviruses encode microRNAs which may facilitate the establishment of persistent infection. MicroRNAs contribute to disease pathogenesis, and may provide useful tools in laboratory diagnostics and patient management. ObjectivesIn this pilot work we studied whether viral and cellular microRNAs can be extracted and detected from body fluids to provide added value in a diagnostic laboratory. Study designAltogether 120 human plasma, urine, and cerebrospinal fluid samples from individuals diagnosed with, or suspected of, a severe polyomavirus associated disease, were included in the study. The samples were spiked with unrelated synthetic microRNA to control for sample quality and inhibition. BKV specific bkv-miR-B1-5p, JCV specific jcv-miR-J1-5p, and bkv-miR-B1-3p/jcv-miR-J1-3p, sharing identical sequences between the two viruses, were amplified from human samples using specific TaqMan assays. Expression of 84 circulating human microRNAs was studied in four selected plasma samples in microarray. Resultsjcv-miR-J1-5p and bkv-miR-B1-3p/jcv-miR-J1-3p were frequently amplified from human plasma, urine, and cerebrospinal fluid samples. bkv-miR-B1-5p was amplified from one-third of the samples, which often contained high viral DNA loads. A microarray screen of human microRNAs in plasma samples suggested regulation of several human microRNA expression in BKV positive vs negative samples. ConclusionsViral and cellular microRNAs can be processed and detected from human body fluids. They may prove useful in the diagnosis and management of severe polyomavirus associated diseases, calling for further clinical evaluation.

Co-dependence of HTLV-1 p12 and p8 functions in virus persistence.

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.

Presence of human polyomavirus 6 in mutation-specific BRAF inhibitor-induced epithelial proliferations.

A frequent adverse effect of mutation-specific BRAF inhibitor therapy is the induction of epithelial proliferations including cutaneous squamous cell carcinomas. To date, the only factor identified contributing to their development is the activation of the mitogen-activated signal transduction cascade by mutations in the RAS genes. However, these mutations explain only 60% of the tumors; hence, it is important to identify what is causing the remaining tumors. To test for the presence of human papillomaviruses (HPVs) and the recently identified human polyomaviruses (HPyVs), Merkel cell polyomavirus (MCPyV), and trichodysplasia spinulosa-associated polyomavirus (TSPyV), as well as HPyV-6, HPyV-7, HPyV-9, and HPyV-10, in epithelial proliferations occurring after BRAF inhibitor therapy to determine whether these oncogenic viruses may contribute to BRAF inhibitor-induced skin tumors. Retrospective study at a university hospital in Austria of epithelial proliferations that developed in patients with melanoma after initiation of treatment with the BRAF inhibitor vemurafenib. Samples were analyzed for (1) presence of the most frequently observed RAS mutations by SNaPshot technology, (2) detection of the viruses by real-time polymerase chain reaction, and (3) presence of capsid proteins of the most abundantly detected virus by immunohistochemical analysis. RAS mutational status, as well as HPV and HPyV presence, in BRAF inhibitor-induced epithelial proliferations. Eighteen biopsy samples from 6 patients were retrieved from our hospital's archive. We identified RAS mutations in 10 (62%) of the 16 samples with clear results. DNA of HPyV-9, HPyV-10, and TSPyV were virtually absent in the samples. MCPyV DNA was present in 13 of 18 samples, and HPV, HPyV-6, and HPyV-7 DNA were present in all samples. In general, the amount of DNA encoding the latter viruses was rather low, with the exception of HPyV-6 in several samples of 1 individual patient. Notably, the relevance of the presence of HPyV-6 in the epithelial proliferation was underlined by immunohistochemical detection of the core protein VP1 of HPyV-6. The presence of both high HPyV-6 DNA load and VP1 protein suggests that polyomaviruses may contribute to the epithelial proliferations observed in patients receiving BRAF inhibitor therapy, albeit the relative impact as compared with that of RAS mutations appears circumstantial.

Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR.

IntroductionIn HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples.Materials and MethodsWe compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid.ResultsGenomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland–Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10).Conclusions2-LTR circles quantification in HIV-infected patients proved to be more accurate with a modified plasmid DNA isolation procedure compared to total genomic DNA isolation. This method enables the processing of more blood cells, thus enhancing quantification accuracy and sensitivity. An improved quantification of 2-LTR circles will contribute to the better understanding of ongoing replication in the HIV reservoir of patients on cART.

Untreated highly viraemic pregnant women from Asia or sub‐Saharan Africa often transmit hepatitis B virus despite serovaccination to newborns

Mother-to-child (MTC) hepatitis B virus (HBV) transmission has been mainly studied in Asia. The geographical origins of women and HBV genotypes differ in Europe. The aims were to determine the rate and risk factors of MTC HBV transmission from women with high HBV DNA loads in a maternity hospital in Paris, France. Retrospective study of HIV-negative, HBs Ag-positive pregnant women with HBV DNA loads above 5 Log10 I.U/ml who were not given lamivudine or tenofovirDF during pregnancy between 2004 and 2011. Among 11 417 pregnant women, 437 (4%) showed a positive HBs Ag. Among these women, 52 had HBV DNA loads above 5 Log10 I.U/ml: 41, 10 and 1 born in Asia, sub-Saharan Africa and Europe respectively. Among the 52 women, 40 were eligible for the analysis: no antiviral therapy during pregnancy; children over 9 months old. Twenty-eight (70%) women were assessed, corresponding to 41 childbirths. Eleven children (27%) had positive HBs Ag, 14 (34%) had positive HBc and HBs Ab, 16 (39%) had positive HBs Ab only. The risk of having positive HBs Ag, according to maternal HBV DNA loads, was 14% for HBV DNA loads less or equal to 8 Log10 I.U/ml, 42% for HBV DNA loads over 8 Log10 I.U/ml, P = 0.04, but not related to the women's origin, HBV genotype. This study confirms that serovaccination does not fully protect newborns from MTC HBV transmission, when maternal HBV DNA loads exceed 5 Log10 I.U/ml, regardless of the women's origin or HBV genotype.

Tumor prevention in HPV8 transgenic mice by HPV8-E6 DNA vaccination

The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.

Quantification of two viral transcripts by real time PCR to investigate human herpesvirus type 6 active infection

BackgroundHuman herpesvirus 6 (HHV-6) causes exanthema subitum and is associated with symptomatic reactivations in immunocompromised patients, particularly after hematopoietic stem cell transplantation. The detection of viral mRNA can help to make the difference between latent, chromosomally integrated and true replicating virus. It can also be a useful tool to investigate viral multiplication in different cell types. ObjectivesTo develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting. Study-designTwo one-step reverse-transcriptase quantitative real-time PCR (RT-qPCR) were developed for the quantification of the immediate early U90 and the late U100 mRNAs. Viral mRNA loads were compared to viral DNA loads during infection in vitro and in blood samples collected from stem cell transplanted patients. ResultsAnalytical performances of the two quantitative real-time PCR were good. In vitro, kinetics of both transcripts was well correlated with DNA levels. Sixty blood samples from patients with active HHV-6 infection were analyzed. Overall agreement of qualitative results for HHV-6 DNA, U90 RNA and U100 RNA was good. HHV-6 DNA loads were significantly higher than mRNA loads. In clinical samples, the amounts of U100 and U90 mRNAs were low and their detection was mainly associated to viral DNA loads upper than 1000copies/ml of blood. ConclusionThe new assays are sensitive and reliable methods for the monitoring of viral transcription in vitro and in vivo. As their detection is associated to high DNA loads in vivo, they can be helpful tools for the diagnosis of active infection.

Looking for biological factors to predict the risk of active cytomegalovirus infection in non-immunosuppressed critically ill patients

The identification of non-immunosuppressed critically ill patients most at risk for developing cytomegalovirus (CMV) reactivation is potentially of great clinical relevance. The current study was aimed at determining (i) whether single nucleotide polymorphisms in the genes coding for chemokine receptor 5 (CCR5), interleukin-10 IL-10), and monocyte chemoattractant protein-1 (MCP-1) have an impact on the incidence rate of active CMV infection, (ii) whether serum levels of CMV-specific IgGs are associated with the risk of CMV reactivation, and (iii) whether detection of CMV DNA in saliva precedes that in the lower respiratory tract or the blood compartment. A total of 36 out of 78 patients (46%) developed an episode of active CMV infection. The incidence rate of active CMV infection was not significantly associated with any single nucleotide polymorphisms. A trend towards a lower incidence of active CMV infection (P = 0.06) was noted in patients harboring the IL10 C/C genotype. Patients carrying the CCR5 A/A genotype had high CMV DNA loads in tracheal aspirates. The serum levels of CMV IgGs did not differ significantly between patients with a subsequent episode of active CMV infection (median, 217 IU/mL) or without one (median, 494 IU/mL). Detection of CMV DNA in saliva did not usually precede that in plasma and/or tracheal aspirates. In summary, the analysis of single nucleotide polymorphisms in the IL10 and CCR5 genes might help to determine the risk of active CMV infection or the level of CMV replication within episodes, respectively, in non-immunosuppressed critically ill patients.

Visual optical biosensors based on DNA-functionalized polyacrylamide hydrogels

Biosensors are devices that can provide quantitative or semi-quantitative analytical information about target molecules, where molecular recognition is based on biomolecular interactions. In recent years, DNA has emerged as a useful molecule for biosensor development since DNA can not only recognize its complementary strand, but also metal ions, small molecules, proteins and cells utilizing DNA aptamer technology. Converting DNA binding events into useful biosensors often require sensor immobilization. Among the various materials for sensor immobilization, hydrogels are particularly attractive. Hydrogels are crosslinked hydrophilic polymer networks that undergo swelling in water. In a gel, DNA immobilization can take place in 3D, allowing for high DNA loading capacity. Hydrogels are transparent, offering low optical background. The gel volume is affected by many environmental parameters such as temperature, pH, ionic strength, and solvent composition. In this paper, we present a concise summary of recent developments in DNA-functionalized hydrogel biosensors for visual detection. Detailed methods for immobilizing DNA biosensors in monolithic polyacrylamide gels and gel microparticles are supplied.

Enhanced Nasal Mucosal Delivery and Immunogenicity of Anti-Caries DNA Vaccine through Incorporation of Anionic Liposomes in Chitosan/DNA Complexes

The design of optimized nanoparticles offers a promising strategy to enable DNA vaccines to cross various physiological barriers for eliciting a specific and protective mucosal immunity via intranasal administration. Here, we reported a new designed nanoparticle system through incorporating anionic liposomes (AL) into chitosan/DNA (CS/DNA) complexes. With enhanced cellular uptake, the constructed AL/CS/DNA nanoparticles can deliver the anti-caries DNA vaccine pGJA-P/VAX into nasal mucosa. TEM results showed the AL/CS/DNA had a spherical structure. High DNA loading ability and effective DNA protection against nuclease were proved by gel electrophoresis. The surface charge of the AL/CS/DNA depended strongly on pH environment, enabling the intracellular release of loaded DNA via a pH-mediated manner. In comparison to the traditional CS/DNA system, our new design rendered a higher transfection efficiency and longer residence time of the AL/CS/DNA at nasal mucosal surface. These outstanding features enable the AL/CS/DNA to induce a significantly (p<0.01) higher level of secretory IgA (SIgA) than the CS/DNA in animal study, and a longer-term mucosal immunity. On the other hand, the AL/CS/DNA exhibited minimal cytotoxicity. These results suggest that the developed nanoparticles offer a potential platform for DNA vaccine packaging and delivery for more efficient elicitation of mucosal immunity.

Calcium Phosphate/Octadecyl-Quatemized Carboxymethyl Chitosan Nanoparticles: An Efficient and Promising Carrier for Gene Transfection

Calcium phosphate (CaP) has been widely used as the vector for gene transfection in the past three decades. However, clinical application is still not popular due to the poor-controlling of DNA/CaP complexes preparation, cytotoxicity and its low transfection efficiency. In this study, a novel amphipathic octadecyl-quatemized carboxymethyl chitosan (OQCMC) derivative from chitosan was combined with calcium phosphate to synthesize CaP/OQCMC nanoparticles (CaP/OQCMC NPs). The nanoparticles were 122-177 nm in diameter exhibited neutral zeta potential (from -0.115 mV to 0.216 mV), and they were applied as DNA vectors for DNA loading and in vitro transfection. The results showed that CaP/OQCMC NPs displayed high DNA loading capacity and enhanced transfection efficiency with extremely low cytotoxicity. In addition, both CaP and OQCMC are biocompatible and biodegradable, thus the as-prepared CaP/OQCMC NPs are promising in gene delivery.

High Coxiella burnetii DNA Load in Serum during Acute Q Fever Is Associated with Progression to a Serologic Profile Indicative of Chronic Q Fever

PCR is very effective in diagnosing acute Q fever in the early stages of infection, when bacterial DNA is present in the bloodstream but antibodies have not yet developed. The objective of this study was to further analyze the diagnostic value of semiquantitative real-time PCR (qPCR) in diagnosing acute Q fever in an outbreak situation. At the Jeroen Bosch Hospital, in 2009, qPCR testing for Coxiella burnetii DNA was performed for 2,715 patients suspected of having acute Q fever (positive, n = 385; negative, n = 2,330). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the qPCR assay were calculated for patients with negative qPCR results with a follow-up sample obtained within 14 days (n = 305) and qPCR-positive patients with at least one follow-up sample (n = 369). The correctness of the qPCR result was based on immunofluorescence assay results for samples submitted for qPCR and follow-up testing. The sensitivity of the Q fever qPCR assay was 92.2%, specificity 98.9%, PPV 99.2%, and NPV 89.8%. Patients who later developed serologic profiles indicative of chronic Q fever infection had significantly higher C. burnetii DNA loads during the acute phase than did patients who did not (P < 0.001). qPCR testing is a valuable tool for the diagnosis of acute Q fever and should be used in outbreak situations when the onset of symptoms is <15 days earlier. Special attention is needed in the follow-up monitoring of patients with high C. burnetii DNA loads during the acute phase, as this might be an indicator for the development of a serologic profile indicative of chronic infection.

FCGR2A and FCGR3A polymorphisms in classical Hodgkin lymphoma by Epstein–Barr virus status

FCGR2A and FCGR3A polymorphisms in classical Hodgkin lymphoma by Epstein–Barr virus status

Chitosan–plasmid DNA nanoparticles encoding small hairpin RNA targeting MMP‐3 and ‐13 to inhibit the expression of dedifferentiation related genes in expanded chondrocytes

Overexpression of matrix metalloproteinase (MMP)-3 and -13 can lead to the dedifferentiation of expanded chondrocytes. After implanting dedifferentiated cells for cartilage defect repair, graft failure may occur. Short hairpin RNA (shRNA) is a powerful genetic tool to reduce the expression of target genes. This study investigated the effects of chitosan-plasmid DNA (pDNA) nanoparticles encoding shRNA targeting MMP-3 and -13 on the dedifferentiation of expanded chondrocytes. The objective was to optimize the parameters of chitosan-pDNA formulation for achieving higher efficiency of pDNA delivery and gene silencing. The chitosan-pDNA nanoparticles were prepared using a complex coacervation process. Then the characteristics including size, shape, stability, and transfection efficiency were compared in different groups. The results indicated that chitosan of 800 kDa at N/P ratio of 4 and pH 7.0 was optimal to prepare chitosan-pDNA nanoparticles. These nanoparticles showed high DNA loading efficiency (95.8 ± 1.5%) and high gene transfection efficiency (24.5 ± 1.6%). After the expanded chondrocytes were transfected by chitosan-pDNA nanoparticles, MMP-3-610 and MMP-13-2024 groups showed greater suppression in mRNA and protein levels. The results indicated that chitosan-pDNA nanoparticles encoding shRNA targeting MMP-3 and -13 had great potential in silencing the dedifferentiation-related genes for regenerating prolonged and endurable cartilage.

Quantification and genotyping of hepatitis B virus in a single reaction based on dual molecular beacon real-time PCR

Objective To develop a new method for simultaneous quantifying and genotyping of HBV in a single reaction based on dual molecular beacon real-time PCR. Methods Genotype B and C recombinant plasmids were constructed as the standards and genotype-specific primers and molecular beacons were designed for each genotype.The molecular beacons of genotype B and C were labeled with FAM and Hex respectively.In this way,a simultaneous qualification and genotyping method for HBV DNA in a single real-time PCR reaction system was developed.Firstly,10-fold gradient dilution of genotype B and C standard plasmids (103-1011 kIU/L) were utilized to evaluate the linear ranges and sensitivity of this approach.The clinical specificity was tested with twenty different serum specimens (5 cases with hepatitis C virus,5 cases with herpes simplex virus and 5 cases with human papilloma virus as well as 5 healthy volunteers) ; the reproducibility was assessed by intra-assay and inter-assay coefficient of variation (CV) of cycle threshold (Ct) value through 10 repeated detections within a batch and between batches of the B,C standard plasmids (108,106and 104kIU/L).Then the accuracy of qualifying and genotyping of the self-built method was evaluated by a parallel examination with 132 HBV infected patients by use of two commercial kits as the references.Finally,these HBVpositive patients were divided into 4 groups:asymptomatic carrier (n=21),chronic hepatitis (n=77),liver cirrhosis (n=25) and hepatocellular carcinoma (n=9) to investigate the relationship of genotypes,stages of disease progression and HBV DNA load. Results A simultaneous qualification and genotyping assay was successfully built and its genotyping sensitivity was 103kIU/L and the linear range was 103-1011k IU/L.The intra-assay CV of B genotyping was 1.51% to 1.80% and the interassay CV was 2.11% to 3.03%,while the intra-assay CV of C genotyping was 1.79% to 1.95% and the inter-assay CV was 2.53% to 2.91%.The results of non HBV infected cases and healthy volunteers showed negative.In the test of 132 HBV infected patients,the general coincident rate of genotyping results comparing our assay and HBV DNA genotyping kit was 90.9% (120/132,Kappa=0.832,P<0.05).The HBV DNA quatitive results between the assay[5.07 (3.89-6.33)]and HBV DNA quatitive kit[5.19 (4.15-6.32) lg kIU/L] were well correlative (R2=0.8477,P<0.05).69 genotype B cases,51 genotype C cases and 12 B/C mixed-genotype cases were detected by dual molecular beacon real-time PCR method and their HBV DNA load were 4.54 (3.83-6.17),5.53 (4.02-6.55),4.58 (3.68-4.98) lg kIU/L respectively.Where the patients with genotype C had higher DNA load than the patients with other two genotypes (Z=-2.195 and-2.162,P<0.05).The HBV DNA load of asymptomatic group,chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group were 7.02 (6.35-7.84),4.94 (4.16-6.25),4.37 (3.50-5.17) and 3.45 (3.25-4.92) lg kIU/L, respectively.Among them,the asymptomatic group was significantly higher than those of other three groups (Z=-4.244,-4.568 and-3.489,P<0.001) and DNA load comparing with the chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group also showed statistically different (Z=-2.894 and-2.413,P<0.05).However,compared with the liver cirrhosis group and hepatocellular carcinoma group there was no significant difference (Z=-0.995,P=0.335). Conclusion A dual molecular beacon real-time PCR assay which can simultaneously quantifying and genotyping HBV DNA with highly accuracy,sensitive and specificity is successfully developed.(Chin J Lab Med,2013,36:333-338) Key words: Hepatitis B virus; DNA; viral; Genotype; Polymerase chain reaction

Decationized crosslinked polyplexes for redox-triggered gene delivery

The clinical applicability of polymers as gene delivery systems depends not only on their efficiency, but also on their safety. The cytotoxicity of these systems remains a major issue, mainly due to their cationic nature. Therefore, it is highly preferable to have a system based on biocompatible neutral polymers, lacking polycations, without compromising the DNA condensing and protecting capacities. Here, we introduce a concept to obtain a neutral polymeric gene delivery system, through a 3-step process (charge-driven condensation; stabilization through disulfide crosslinking; polyplex decationization) to generate polyplexes with a core of disulfide crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) in which plasmid DNA (pDNA) is entrapped and a shell of poly(ethylene glycol) (PEG). The resulting polyplexes combine beneficial features of high and stable DNA loading capacity, stealth behavior and reduced toxicity. The nanoparticles are designed to release the pDNA after cellular uptake through cleavage of disulfide crosslinks within the intracellular reducing environment. This was shown by forced introduction of the polyplexes into the cytosol of HeLa cells by electroporation, which resulted in a high level of expression of the reporter gene. Additionally, the decationized polyplexes showed no interference on the cellular cell viability or metabolic activity (even at high dose) and no complex-induced membrane destabilization. Furthermore, decationized polyplexes showed a low degree of non-specific uptake, which is a highly favorable property for targeted therapy. Summarizing, the stabilized, decationized polyplexes presented here contribute to solve the high toxicity, low stability and lack of cellular/tissue specificity of cationic polymer based gene delivery systems.

Prospective Study of HPV16 Viral Load and Risk of In Situ and Invasive Squamous Cervical Cancer

A strong association has been shown between high viral DNA load (VL) of human papillomavirus (HPV) type 16 and risk for cervical cancer in situ (CIS). However, little data is available for the significance of VL in invasive squamous cell carcinoma (SCC). In 2 nested case-control studies among women participating in cervical screening, with a cytologically normal first smear, we collected 5,665 smears from 621 women with CIS, 457 with SCC, and individually matched controls. All smears were tested for HPV, and VLs of HPV16 positive smears were quantified using real time-PCR. The median follow-up until diagnosis of CIS or SCC was 6.1 to 7.7 years. Low VL's were common among both CIS and SCC case women, until 1 to 2 years before diagnosis when a surge in VL occurred. The relative risk (RR) associated with low viral load of HPV16 was around 10 for CIS, and 10 to 20 for SCC throughout 10 years before diagnosis, compared with HPV16-negative women. For women with medium to high VL, the risk for CIS was greatly increased from 5 years before diagnosis [RR, 19; 95% confidence interval (CI), 7-48]. In SCC, a high VL conferred an increased risk, but only from 3 years before diagnosis [RR, 60; 95% CI, 6-580]. We show differing risk functions associated with HPV16 viral load in CIS and SCC, respectively. We further show that viral loads were unexpectedly low early in the SCC disease process. HPV16 viral load appears highly complex which may limit its use in cervical screening.