Origanum majorana L. protects against neuroinflammation-mediated cognitive impairment: a phyto-pharmacological study.

BACKGROUND: Neuroinflammation and oxidative stress are critical players in the pathogenesis of numerous neurodegenerative diseases, such as Alzheimer's disease (AD) which is responsible for most cases of dementia in the elderly. With the lack of curative treatments, natural phenolics are potential candidates to delay the onset and progression of such age-related disorders due to their potent antioxidant and anti-inflammatory effects. This study aims at assessing the phytochemical characteristics of Origanum majorana L. (OM) hydroalcohol extract and its neuroprotective activities in a murine neuroinflammatory model. METHODS: OM phytochemical analysis was done by HPLC/PDA/ESI-MSn. Oxidative stress was induced in vitro by hydrogen peroxide and cell viability was measured using WST-1 assay. Swiss albino mice were injected intraperitoneally with OM extract at a dose of 100 mg/kg for 12 days and with 250 µg/kg LPS daily starting from day 6 to induce neuroinflammation. Cognitive functions were assessed by novel object recognition and Y-maze behavioral tests. Hematoxylin and eosin staining was used to assess the degree of neurodegeneration in the brain. Reactive astrogliosis and inflammation were assessed by immunohistochemistry using GFAP and COX-2 antibodies, respectively. RESULTS: OM is rich in phenolics, with rosmarinic acid and its derivatives being major constituents. OM extract and rosmarinic acid significantly protected microglial cells against oxidative stress-induced cell death (p < 0.001). OM protected against the LPS-induced alteration of recognition and spatial memory in mice (p < 0.001) and (p < 0.05), respectively. Mice that received OM extract prior to the induction of neuroinflammation showed comparable histology to control brains, with no overt neurodegeneration. Furthermore, OM pre-treatment decreased the immunohistochemistry profiler score of GFAP from positive to low positive and COX-2 from low positive to negative in the brain tissue, compared to the LPS group. CONCLUSION: These findings highlight the potential preventive effects of OM phenolics against neuroinflammation and pave the way toward drug discovery and development for neurodegenerative disorders.

Egyptian adaptation and validation of the Edinburgh Cognitive and Behavioral Amyotrophic Lateral Sclerosis Screen (ECAS-EG).

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is the most common, fatal adult neuromuscular disease. It is a multi-system disorder characterized primarily by motor manifestations, but there is established evidence for cognitive and behavioral impairment, which is associated with poor prognosis, hence, the importance of tools for its assessment. The Edinburgh Cognitive and Behavioral Assessment Screen (ECAS) is an invaluable assessment tool for cognition in ALS-front temporal spectrum dementia (FTSD), as it accommodates physical challenges that usually confound traditional neuropsychological testing in those patients. OBJECTIVE AND METHODS: To validate the Egyptian Arabic version of ECAS (ECAS-EG) based on the original English scale. This is a prospective study. The ECAS was adapted and administered to 62 Egyptian ALS patients and 60 healthy controls. Patients were recruited from the Neuromuscular Unit, Ain Shams University Hospital. The ECAS was adapted to Egyptian Arabic after being translated using the back translation method. Internal consistency of the test, inter-rater reliability, and construct validity were assessed. RESULTS: The Egyptian Arabic version of ECAS (ECAS-EG) showed good internal consistency using Cronbach's alpha of 0.84. Inter-rater reliability was tested, values for all variables were compared, and no statistically significant differences were found (ICC = .997). ECAS-EG discriminated significantly between the patients from the control subjects (p-value of 0.001). There was a strong positive correlation between the ECAS-EG total score and the MoCA total score with a p-value of 0.001, thus indicating convergent validity. The test showed that 63% of Egyptian ALS patients were cognitively affected; most affected domains were executive functions and verbal fluency. CONCLUSION: The current study proves that the Egyptian version of the ECAS (ECAS-EG) is valid and reliable among Egyptian ALS patients and it would be applicable to the general Arabic-speaking population.

A novel homozygous p.Ser69Pro SOD1 mutation causes severe young-onset ALS with decreased enzyme activity.

BACKGROUND: The dose-effect of various SOD1 mutations on SOD1 enzymatic activity offers valuable insights into ALS pathogenesis with possible therapeutic implications. Homozygous SOD1 mutations, yet scarce, are of special interest. We report a novel homozygous SOD1 mutation with decreased enzymatic activity and severe early onset ALS phenotype. METHODS: Whole exome sequencing and targeted screening of commonly implicated genes were conducted. Repeat-primed PCR and fragment length analysis were used for C9orf72. Bi-directional Sanger sequencing was used for SOD1 and other genes. SOD1 activity was measured by direct spectrophotometry. Serum neurofilament light chain level was measured by the ELLA immunoassay system. RESULTS: The homozygous patient for a novel SOD1 variant p.Ser69Pro showed poor SOD1 enzymatic activity (16% of controls) and an early onset ALS phenotype predominantly affecting lower motor neurons with rapid involvement of the trunk, upper limbs and bulbar muscles. The asymptomatic heterozygous relatives had at least 68% of normal enzyme activity. Level of serum neurofilament light chain was much higher (148 pg/ml) in the patient than the relatives who had normal levels (6-10 pg/ml). CONCLUSION: This novel mutation adds knowledge to the ALS genotype-phenotype spectrum and supports the strong dose-effect of SOD1 mutations associated with severely decreased enzymatic activity.

Assessment of diagnostic potential of some circulating microRNAs in Amyotrophic Lateral Sclerosis Patients, an Egyptian study.

OBJECTIVE: Numerous studies have been carried out to identify the role of microRNA (miRNA) as potential biomarkers for many diseases including amyotrophic lateral sclerosis (ALS). The aim of this study was to explore the circulating levels of some miRNAs in cohort of Egyptian ALS patients in an attempt to correlate the selected miRNA profiles with disease progression. METHODS: Thirty ALS patients and 20 age and sex matched healthy controls were enrolled. Circulating miRNA levels were determined in venous blood samples, collected on EDTA, from all the study subjects. The selection of miRNA species (miR-206, miR-142-3p, miR-143-3p, miR-181a-5p, miR-106b-3p, miR-4516 and Let7f-5p) was based on their involvement in the pathophysiology of ALS and was further confirmed by data mining of specific miRNA databases (miRBase and miRDB). RESULTS: As compared to the control group, significant consistent upregulation was found in the levels of miR-206, miR-143-3p and to a lesser extent in miR-142-3p. An elevation trend, although not significant, was also found in the levels of miR-181a-5p, miR-106b-3p, and miR-4516. Interestingly, we found that the levels of miR-142-3p were elevated in familial cases, while that of miR-4516 were significantly increased in sporadic cases. Furthermore, the levels of Let7f-5p, although were generally lowered in ALS patients but were also decreased in familial cases as well as in spinal onset ALS as compared to bulbar onset. CONCLUSION: This is the first study investigating miRNA profiles in Egyptian ALS patients. We found that some miRNAs are significantly altered in ALS patients, and some may be used to distinguish familial and sporadic cases and bulbar and spinal onset. Larger study is needed, in which we will conduct a correlation of miRNA levels against variations in disease onset, progression as well as systemic inflammatory responses and the extent of neuromuscular involvement in Egyptian ALS patients in an attempt to identify environmental/occupational risk factors.

Magnetic localization and control of helical robots for clearing superficial blood clots.

This work presents an approach for the localization and control of helical robots during removal of superficial blood clots inside in vitro and ex vivo models. The position of the helical robot is estimated using an array of Hall-effect sensors and precalculated magnetic field map of two synchronized rotating dipole fields. The estimated position is used to implement closed-loop motion control of the helical robot using the rotating dipole fields. We validate the localization accuracy by visual feedback and feature tracking inside the in vitro model. The experimental results show that the magnetic localization of a helical robot with diameter of 1 mm can achieve a mean absolute position error of 2.35 ± 0.4 mm (n = 20). The simultaneous localization and motion control of the helical robot enables propulsion toward a blood clot and clearing at an average removal rate of 0.67 ± 0.47 mm3/min. This method is used to localize the helical robot inside a rabbit aorta (ex vivo model), and the localization accuracy is validated using ultrasound feedback with a mean absolute position error of 2.6 mm.

Neuromodulatory Activity of Dietary Phenolics Derived from Corchorus olitorius L.

Dietary phenolics are known for their potent antioxidant and anti-inflammatory activities, making them promising candidates for protection against neuroinflammation and neurodegeneration. Hydroalcohol extract of Egyptian species of Corchorus olitorius L. (Co) leaves was investigated for its neuroprotective effects in a lipopolysaccharide-induced neuroinflammatory mouse model. Twenty five metabolites were characterized from the bioactive extract using high-performance liquid chromatography HPLC/PDA/HRESI/MSn , revealing 1,5-dicaffeoylquinic acid (Co11) as one of the major constituents (5.7%), which was isolated and its identity was confirmed by spectral data as first report. Co significantly protected microglia against H2 O2 -induced cytotoxicity and immunohistochemistry showed reduced expression of the astrocytic marker, glial fibrillary acidic protein, and the inflammatory marker, cyclooxygenase-2. These findings correlated with significant improvement of cognitive functions and reduction of LPS-induced neurodegeneration in Co-treated mice as revealed by histopathology. The current study shows promising effects of Co in limiting neurodegeneration and cognitive impairment caused by neuroinflammation and glial cell activation. PRACTICAL APPLICATION: Information presented here shed light on the promising effects of Corchorus olitorius (Co) for the modulation of neuroinflammatory pathways improving the neuroinflammation-related neurodegeneration and cognitive decline. This makes Co a promising candidate as a nutraceutical supplement to be used against neuroinflammation-related disorders.

Characterization of Helical Propulsion Inside In Vitro and Ex Vivo Models of a Rabbit Aorta.

In this work, the propulsion of a helical robot is experimentally characterized inside whole blood (in vitro model) and against the flowing streams of phosphate buffered saline (PBS) inside rabbit aorta (ex vivo model). The helical robot is magnetically actuated inside these models under the influence of rotating magnetic fields. The frequency response of the helical robot is characterized. Averaged speed is measured at actuation frequency of 8 Hz as 11.3 ± 0.52 (n = 5) and 7.45 ± 1.2 mm/s (n = 3) inside rabbit aorta and whole blood, respectively. The Speed of the robot inside rabbit aorta is characterized against flowing streams of PBS at flow rate of 90 ml/hr.

The Influence of Mechanical Rubbing on the Dissolution of Blood Clots.

Mechanical rubbing of blood clots is a potential minimally-invasive method for clearing clogged blood vessels. In this work, we investigate the influence of the interaction of the tip of a helical robot with blood clots. This interaction enables the dissolution of the blood clot and the release of the entrapped red blood cells and platelets from its three-dimensional fibrin fiber network. We analyze the pre- and post-conditions of the blood clots following 40 minutes of mechanical rubbing, under the influence of a rotating magnetic field in the frequency range of 20 Hz to 45 Hz. Our measurements show that the weight of the blood clots is decreased by 22.5 ± 11.1% at frequency of 25 Hz. We also validate the influence of mechanical rubbing using cell count and spectrophotometric analysis on phosphate buffered saline samples past the robot and the clot. The maximum cell count is measured as 654 ± 108 × 104 cells/m1 and 54 ± 12 × 104 cells/m1, whereas the absorbance is measured as 4. 35 × 10-6 mol and 1. 05 × 10-6 mol under the influence of mechanical rubbing and without mechanical rubbing, respectively.

Methylene Blue Improves Brain Mitochondrial ABAD Functions and Decreases Aß in a Neuroinflammatory Alzheimer's Disease Mouse Model.

Methylene blue (MB) phase II clinical trials reported improvements in cognitive functions of Alzheimer's disease (AD) patients. Regarding MB mechanism of action, its antioxidant and mitochondrial protective effects have been previously described. In relation to AD, it has been recently reported that MB reduced amyloid beta (Aß) levels in AD models. The mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) has been shown to bind Aß inducing mitochondrial dysfunction, providing a direct relation between Aß toxicity and mitochondrial dysfunction occurring in AD. Since it has been reported that inhibiting ABAD protects mitochondrial functions and prevents Aß-induced toxicity, the aim of the current study was to investigate if the protective effects of MB could be associated with an effect on ABAD levels and functions. The current study shows that MB is able to enhance cell viability, reduce both reactive oxygen species levels and importantly Aß oligomers in a lipopolysaccharide (LPS) mouse model. Interestingly, ABAD levels were increased in the brains of the LPS mouse model and MB treatment was able to reduce its levels. Given that regulation of the estradiol level is a well-established function of ABAD, brain estradiol level was compared in LPS mouse model and in MB-treated mice. The results of the current study show that MB treatment is able to improve significantly the LPS-induced decrease of estradiol levels in mice brains, indicating its ability to modulate both levels and function of ABAD. These results give a new insight to possible mechanisms of MB in AD.

Minocycline attenuates Aß oligomers-induced pro-inflammatory phenotype in primary microglia while enhancing Aß fibrils phagocytosis.

Microglia, the brain innate immune cells, are activated in response to amyloid beta (Aß) resulting in neuroinflammation in AD brains. Recently, two phenotypes have been described for microglia: the pro-inflammatory classical and the anti-inflammatory alternative. Changes in microglia phenotype that control their phagocytic function are yet to be determined. The highly neurotoxic Aß oligomers (oAß) formed at an early disease stage induce pro-inflammatory microglia activation releasing neurotoxic mediators and contributing to neurodegeneration. A novel strategy for AD treatment is to attenuate microglia-induced inflammation while maintaining efficient Aß clearance. Minocycline effectively crosses the blood-brain barrier and has widely reported neuroprotective effects. Yet, its exact mechanism of neuroprotection and its effects on microglia are still unknown. The aim of this study is to investigate the effect of minocycline on the phagocytic uptake of fAß by primary microglia in relation to their activation state in an inflammatory milieu generated by oAß or LPS. The study shows that minocycline is able to attenuate oAß-induced neuroinflammatory response of microglia by inhibiting their pro-inflammatory phenotype activation. In addition, a significant enhancement of fAß phagocytosis by minocycline- treated microglia is reported for the first time, providing novel insight into its neuroprotective role in AD.

Predictive value of interferon-lambda gene polymorphisms for treatment response in chronic hepatitis C.

BACKGROUND: IL28B gene polymorphism is the best baseline predictor of response to interferon alfa-based antiviral therapies in chronic hepatitis C. Recently, a new IFN-L4 polymorphism was identified as first potential functional variant for induction of IL28B expression. Individualization of interferon alfa-based therapies based on a combination of IL28B/IFN-L4 polymorphisms may help to optimize virologic outcome and economic resources. METHODS: Optimization of treatment outcome prediction was assessed by combination of different IL28B and IFN-L4 polymorphisms in patients with chronic HCV genotype 1 (n = 385), 2/3 (n = 267), and 4 (n = 220) infection treated with pegylated interferon alfa (PEG-IFN) and ribavirin with (n = 79) or without telaprevir. Healthy people from Germany (n = 283) and Egypt (n = 96) served as controls. RESULTS: Frequencies of beneficial IL28B rs12979860 C/C genotypes were lower in HCV genotype 1/4 infected patients in comparison to controls (20-35% vs. 46-47%) this was also true for ss469415590 TT/TT (20-35% vs. 45-47%). Single interferon-lambda SNPs (rs12979860, rs8099917, ss469415590) correlated with sustained virologic response (SVR) in genotype 1, 3, and 4 infected patients while no association was observed for genotype 2. Interestingly, in genotype 3 infected patients, best SVR prediction was based on IFN-L4 genotype. Prediction of SVR with high accuracy (71-96%) was possible in genotype 1, 2, 3 and 4 infected patients who received PEG-IFN/ribavirin combination therapy by selection of beneficial IL28B rs12979860 C/C and/or ss469415590 TT/TT genotypes (p<0.001). For triple therapy with first generation protease inhibitors (PIs) (boceprevir, telaprevir) prediction of high SVR (90%) rates was based on the presence of at least one beneficial genotype of the 3 IFN-lambda SNPs. CONCLUSION: IFN-L4 seems to be the best single predictor of SVR in genotype 3 infected patients. For optimized prediction of SVR by treatment with dual combination or first generation PI triple therapies, grouping of interferon-lambda haplotypes may be helpful with positive predictive values of 71-96%.

Tamoxifen downregulates MxA expression by suppressing TLR7 expression in PBMCs of males infected with HCV.

Gender discrepancies in immune response to HCV infections and during HCV therapy exist and previous findings including those from this research team indicate the female sex hormone, 17ß-estradiol (E2), to be one probable cause of such inconsistencies. Also, it was recently demonstrated that estrogen receptor modulator Tamoxifen (TAM) exerts an upmodulating/enhancing effect on the TLR7 and JAK-STAT pathways in PBMCs of premenopausal females infected with HCV. Pursuing this work, a discrepancy was noticed in the results from male patients, therefore this study aimed to determine whether the effects of TAM previously observed in the PBMCs of women would hold true in PBMCs from males infected with HCV. Isolated PBMCs were pooled and relative expression of the TLR7 was quantified using RTqPCR. Sets of PBMCs were treated with exogenous interferon alpha (IFNα) or the TLR7 ligand, Imiquimod; these stimulations were performed with and without E2 and TAM pretreatment and the relative gene expressions of TLR7 and MxA were measured. Pretreatment with E2 and IFNα downregulated TLR7 (**P = 0.0080) and TAM further decreased this expression significantly (*P = 0.0284). TAM pretreatment also caused a significant downregulation in MxA expression in Imiquimod-stimulated PBMCs (*P = 0.0218). In conclusion, TAM displays several paradoxical effects in PBMCs of males infected with HCV compared to those of females. Contrary to the previous study involving premenopausal females, in PBMCs of infected males TAM may decrease IFNα release as indicated by reduced MxA expression possibly via the suppression of TLR7 expression.

Progesterone suppresses interferon signaling by repressing TLR-7 and MxA expression in peripheral blood mononuclear cells of patients infected with hepatitis C virus.

This study aimed at investigating the effect of progesterone on interferon signaling pathways in peripheral blood mononuclear cells (PBMCs) of patients infected with hepatitis C virus (HCV). PBMCs were isolated from peripheral blood of 38 treatment-naïve HCV-infected patients, pooled, and stimulated with progesterone in the presence and absence of its receptor antagonist, mifepristone, along with interferon alpha (IFN-α) or imiquimod. Toll-like receptor (TLR) 7 and myxovirus resistance protein A (MxA) were quantified in PBMCs using RT-qPCR. Imiquimod alone or combined with progesterone did not change MxA expression in HCV-infected PBMCs. Progesterone decreased the inducing effect of IFN-α on TLR-7 expression in both males and females. Moreover, progesterone stimulation prior to IFN-α treatment attenuated the Jak/STAT pathway, which was reflected by decreased expression of MxA in females. Progesterone showed a negative impact on the IFN signaling pathway in HCV-infected PBMCs as it decreased the expression of TLR-7 in both genders, while MxA expression was decreased only in females.

Hepatocytes that express variants of cyclophilin A are resistant to HCV infection and replication.

BACKGROUND & AIMS: Hepatitis C virus (HCV) uses several host factors to infect and replicate in human hepatocytes. Cyclophilin A (CypA) is required for viral replication, and CypA inhibitors are in development. We investigated the effects of nonsynonymous single nucleotide polymorphisms (SNPs) in the region of peptidyl-prolyl isomerase A (PPIA) that encodes CypA on HCV infection and replication of human hepatocytes. METHODS: We used a combination of virologic, biochemical, and genetic approaches to investigate the effects of PPIA variants on HCV replication in cultured Huh-7.5 cells. We reduced levels of CypA in these cells using small hairpin RNAs (shRNAs). RESULTS: Using shRNAs, we showed that CypA was required for replication of HCV in Huh-7.5 cells and identified 3 SNPs in PPIA that protected cells from HCV entry or replication. Levels of HCV RNA were reduced 3-4 log in cells homozygous for the variant alleles; release of new particles was also reduced, but viral entry was not affected. The effects of the variant alleles were recessive and stronger for preventing replication of full-length HCV genomes than subgenomes. CypA inhibitors prevented replication of residual HCV in hepatocytes. The variants appeared to destabilize the CypA protein; the single amino acid changes led to rapid degradation of the protein. CONCLUSIONS: We identified variants in PPIA that destabilize its product, CypA, and prevent HCV infection and replication. These findings indicate mechanisms by which some cells might be resistant to HCV infection and that CypA is a good therapeutic target.

Estrogen-related MxA transcriptional variation in hepatitis C virus-infected patients.

Sex has been reported to influence the rates of viral clearance in hepatitis C virus (HCV)-infected patients. However, little is known regarding the influence of sex on the host genetic response to HCV, which is mediated by the expression of interferon (IFN)-stimulated genes (ISGs) after the activation of janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway by IFN. Thus, we investigated gender differences in MxA genetic profile, which is a downstream reliable marker for JAK/STAT pathway activation. In all, 40 untreated HCV-infected patients were subclassified into premenopausal, postmenopausal, and male patients. The peripheral blood mononuclear cells (PBMCs) from premenopausal women showed the highest MxA gene expression compared to both postmenopausal females and males before and after IFN stimulation. The prestimulation of PBMCs with 17beta-estradiol prior to IFN treatment resulted in a decrease of MxA expression in all groups of patients. That was confirmed by the reversal of this effect using estrogen antagonist ICI182/780. This study demonstrates for the first time the presence of gender variations in the genetic response to chronic HCV infection and to interferon treatment. It also clarifies that estrogen is not the key player in enhancing the JAK/STAT pathway.

Repressed induction of interferon-related microRNAs miR-146a and miR-155 in peripheral blood mononuclear cells infected with HCV genotype 4.

MicroRNAs regulate the expression of many genes and subsequently control various cellular processes, such as the immune response to viral infections mediated by type I interferon (IFN). In this study, the expression pattern of two interferon-related microRNAs, miR-146a and miR-155, was examined in healthy and HCV-genotype-4-infected peripheral blood mononuclear cells (PBMCs) using qRT-PCR. In contrast to other viral infections, the expression pattern was similar in both healthy and infected PBMCs. This could be attributed to attenuation of IFN pathway by HCV, which was assessed by investigating the expression of MxA, an interferon-stimulated gene, that showed lower expression in HCV-infected PBMCs. To determine the site of interference of HCV in the IFN pathway, expression of both microRNAs was examined following stimulation of PBMCs with IFN-α2a, an activator of the JAK/STAT pathway as well as with imiquimod, a toll-like receptor-7 (TLR-7) agonist that promotes interferon release. IFN stimulation induced the expression of miR-146a and miR-155 in HCV-infected and healthy PBMCs. Stimulation with imiquimod led to a down-regulation of both microRNAs in infected PBMCs, while it increased their expression in healthy PBMCs, indicating that HCV might interfere with miR-146a and miR-155 expression at sites upstream of interferon release, specifically in the TLR-7 pathway. The pattern of expression of both miR-146a and miR-155 was very similar with a strong positive correlation, but showed no correlation to the patients' clinical or histopathological parameters or response to treatment. In conclusion, HCV infection might repress the induction of miR-146a and miR-155 by interfering with TLR-7 signaling.

Development of a second version of the Cobas AmpliPrep/Cobas TaqMan hepatitis C virus quantitative test with improved genotype inclusivity.

Hepatitis C virus (HCV) RNA measurement has been facilitated by the introduction of real-time PCR-based assays with low limits of detection and broad dynamic ranges for quantification. In the present study, the performance of two second-version prototypes of the Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test (CAP/CTM v2) with decreased sample input volume and improved genotype inclusivity was investigated. A total of 232 serum and plasma samples derived from patients with chronic hepatitis C (genotype 1 [GT1], n = 108; GT2, n = 8; GT3, n = 24; GT4, n = 87; GT5, n = 3; and GT6, n = 2) were processed in parallel with the Cobas AmpliPrep/Cobas TaqMan HCV Test (CAP/CTM), Cobas Amplicor HCV Monitor Test v2.0 (CAM), and two second-version prototype formulations of CAP/CTM, Mastermix 1 (MMx1) and MMx2. In addition, three GT4 transcripts containing rare variant sequences were tested. The mean log(10) HCV RNA differences for the best-performing CAP/CTM v2/MMx2 formulation in comparison to CAM were -0.05, 0.05, -0.12, -0.10, -0.44, and -0.29 for patients with GT1, GT2, GT3, GT4, GT5, and GT6 infections, respectively. GT1, GT2, and GT4 samples including isolates with known variants within the 5' untranslated region (G145A, A165T) that were underquantified with CAP/CTM were correctly quantified with the second-version prototype. In addition, CAP/CTM v2 was able to accurately quantify the three transcripts with rare variant sequences. In conclusion, CAP/CTM v2 accurately quantifies HCV RNA across all HCV genotypes, including specimens with rare polymorphisms previously associated with underquantification.

Impact of intra- and interspecies variation of occludin on its function as coreceptor for authentic hepatitis C virus particles.

Hepatitis C virus (HCV) is characterized by a narrow host range and high interindividual variability in the clinical course of infection. Both of these traits are thought to be largely due to genetic variation between species and between individual hosts. The tight junction component occludin (OCLN) is essential for HCV entry into host cells, and the differences between human and murine OCLN are thought to account in part for the inability of HCV to infect mice and hence preclude their use as a convenient small-animal model. This study assesses the impact of genetic variation in OCLN on cell culture-grown HCV (HCVcc) using a newly generated and characterized OCLN(low) subclone of the Huh-7.5 cell line (Huh-7.5 subclone in which endogenous OCLN expression has been downregulated by a short hairpin RNA). We report the frequency of coding nonsynonymous single nucleotide polymorphisms, i.e., polymorphisms resulting in amino acid exchanges, present in the human population and determine their ability to function as HCV (co)receptors. Moreover, we show that murine OCLN can sustain HCVcc entry, albeit with about 5-fold reduced efficiency compared to that of human OCLN. This reduction in efficiency is due solely to two amino acid residues previously identified by others using an HCV pseudoparticle approach. Finally, we use the Huh-7.5/OCLN(low) cell line to show that HCV spread between neighboring cells is strictly dependent on OCLN.