Application of dsRNA in cancer immunotherapy: current status and future trends.

Cancer cells create a microenvironment that prevents tumor rejection by the host's immune system. The activation of pattern recognition receptors (PRRs) can elicit an innate immune response and guide the adaptive immune response to overcome this. dsRNA analogs can trigger TLR3, RIG-I, MDA5, NLRP3 and several other PRRs to induce not only robust immune response against cancer but also programmed cell death. This review focuses on the signal pathways activated by dsRNA and examines examples of their clinical application in cancer treatment.

The six-minute walk test is an excellent predictor of functional ambulation after total knee arthroplasty.

BACKGROUND: The Six-minute walk (6 MW) and Timed-Up-and-Go (TUG) are short walk tests commonly used to evaluate functional recovery after total knee arthroplasty (TKA). However, little is known about walking capacity of TKA recipients over extended periods typical of everyday living and whether these short walk tests actually predict longer, more functional distances. Further, short walk tests only correlate moderately with patient-reported outcomes. The overarching aims of this study were to compare the performance of TKA recipients in an extended walk test to healthy age-matched controls and to determine the utility of this extended walk test as a research tool to evaluate longer term functional mobility in TKA recipients. METHODS: The mobility of 32 TKA recipients one year post-surgery and 43 healthy age-matched controls were assessed using the TUG, 6 MW and 30-minute walk (30 MW) tests. The latter test was repeated one week later. Self-reported function was measured using the WOMAC Index and a physical activity questionnaire. RESULTS: 30 MW distance was significantly shorter amongst TKA recipients (mean 2108 m [95% CI 1837 to 2381 m]; Controls 3086 m [2981 to 3191 m], P < 0.001). Test-retest repeatability was high (ICC = 0.97, TKA; 0.96, Controls). Amongst TKA recipients, the 30 MW distance correlated strongly with the shorter tests (6 MW, r = 0.97, P < 0.001; TUG, r = -0.82, P < 0.001). Multiple regression modeling found 6 MW distance to be the only significant predictor (P < 0.001) of 30 MW distance, explaining 96% of the variability. The TUG test models were moderate predictors of WOMAC function (55%) and physical activity (36%) and were stronger predictors than 6 MW and 30 MW tests. CONCLUSIONS: Though TKA recipients are able to walk for 30 minutes one year post-surgery, their performance falls significantly short of age-matched norms. The 30 MW test is strongly predicted by 6 MW test performance, thus providing strong construct validity for the use of the 6 MW test in the TKA population. Neither a short nor long walk test is a strong predictor of patient-reported function after TKA.

The effects of TLR activation on T-cell development and differentiation.

Invading pathogens have unique molecular signatures that are recognized by Toll-like receptors (TLRs) resulting in either activation of antigen-presenting cells (APCs) and/or costimulation of T cells inducing both innate and adaptive immunity. TLRs are also involved in T-cell development and can reprogram Treg cells to become helper cells. T cells consist of various subsets, that is, Th1, Th2, Th17, T follicular helper (Tfh), cytotoxic T lymphocytes (CTLs), regulatory T cells (Treg) and these originate from thymic progenitor thymocytes. T-cell receptor (TCR) activation in distinct T-cell subsets with different TLRs results in differing outcomes, for example, activation of TLR4 expressed in T cells promotes suppressive function of regulatory T cells (Treg), while activation of TLR6 expressed in T cells abrogates Treg function. The current state of knowledge of regarding TLR-mediated T-cell development and differentiation is reviewed.

Changes in left atrial volume in diabetes mellitus: more than diastolic dysfunction?

AIM: To evaluate left atrial (LA) volume and function as assessed by strain and strain rate derived from 2D speckle tracking and their association with diastolic dysfunction (DD) in patients with diabetes mellitus (DM). METHODS AND RESULTS: Seventy three patients with DM were compared with age- and gender-matched normal controls; 30 patients with DM alone were compared to those with hypertension (HT) alone. The maximum LA volume, traditional measures of atrial function, 2D strain and strain rate were analysed. The LA indexed volume (LAVI) was larger in DM group than that in normal controls (38.2 ± 9.9 vs. 20.5 ± 4.8 ml/m(2), P< 0.0001), as well as in DM alone compared with hypertensive patients (33.9 ± 10 vs. 25.7 ± 8 ml/m(2), P< 0.0001). Global strain was significantly reduced in the DM group compared with that in normal controls (22.5 ± 8.67 vs. 30.6 ± 8.27%; P< 0.0001) but was similar with HT. There was a weak correlation between LAVI and global strain with increasing grades of DD (r= 0.439, P< 0.0001 and r= - 0.316, P< 0.0001, respectively) in the diabetic group. However, there was no significant difference in LAVI between these groups. A logistic regression analysis for predictors of LAVI demonstrated that only diabetes was a determinant of LAVI. Patients with diabetes showed a significant reduction in global strain compared with normal controls but no difference with increasing grades of diastolic function. CONCLUSIONS: LA enlargement in DM is independent of associated HT and diastolic function. LA enlargement is associated with LA dysfunction as evaluated by 2D strain. It is likely that a combination of DD and a diabetic atrial myopathy contribute to LA enlargement in patients with DM.

Improvements in knee range and symptomatic and functional behavior after knee arthroplasty based on preoperative restriction in range.

Recovery of knee range and Oxford Knee Score post knee arthroplasty based on preoperative knee range is described. A total of 191 patients recruited across 5 hospitals were assessed preoperatively, at 8 weeks postoperatively and 1 year. Preoperative knee range was categorized into "low" (≤ 109), "moderate" (> 109 to ≤ 120), and "high" (> 120°) flexion and "normal" (± -5) and "restricted" (> +5°) terminal extension. Recovery was analyzed using MIXED modeling procedures. The low-flexion group gained flexion across time. The moderate-flexion and high-flexion groups lost flexion initially then recovered, but 1-year flexion remained lower than preoperative values. The restricted terminal extension group gained extension across time. The normal terminal extension group lost extension initially then recovered to preoperative values at 1 year. Recovery in Oxford score was independent of preoperative knee range limitation. Improvement in knee range postoperatively, but not self-reported behavior, is highly dependent on the initial restriction in range.

Is discharge knee range of motion a useful and relevant clinical indicator after total knee replacement? Part 1.

OBJECTIVES: Knee range of motion (ROM) at the point of discharge from acute care is used as a clinical indicator to benchmark performance between hospital services after total knee replacement (TKR). The utility of the current benchmark, including whether discharge ROM varies between hospitals, is unknown. This study aimed to determine whether the benchmark [≥80 degrees flexion and ≤5 degrees fixed flexion (extension)] is realistic and whether the service provider is a predictor of knee ROM. METHODS: A prospective, observational cohort study was conducted involving 176 TKR patients from four hospitals. Knee ROM was photographically assessed preoperatively and at discharge. 'Hospital', typical patient demographic data and preoperative ROM were identified a priori as potential predictors of knee ROM. RESULTS: Overall, 2% [95% CI (confidence interval) 1-6] of patients attained the ROM benchmark. Individual hospital attainment of the benchmark ranged 0-7% with a significant difference (P = 0.047) evident between the best performer and the remaining hospitals. The overall rates of attainment of the individual flexion (25%, 95% CI 19-32) and extension (15%, 95% CI 10-21) components were similarly low, although the scatter between hospitals was large [flexion (2-47%); extension (8-44%)]. Preoperative flexion and hospital were significant (P = 0.002) predictors of discharge flexion, explaining 21% of the variance. Similarly, hospital and preoperative extension together with gender were significant (P < 0.001) predictors of discharge extension, explaining 26% of the variance. CONCLUSIONS: A small minority of patients attained the knee ROM benchmark, indicating the existing standard is unrealistic. Nevertheless, that 'hospital' is an important predictor confirms the potential of ROM for benchmarking purposes. Differences in physiotherapy practices may contribute to inter-hospital variation in discharge knee ROM together with other undefined factors. The causal relationships explaining the variation and the relationship between discharge ROM and longer-term outcome are avenues for future exploration which will help define the clinical relevance of the indicator.

Is discharge knee range of motion a useful and relevant clinical indicator after total knee replacement? Part 2.

OBJECTIVE: Knee range of motion (ROM) at discharge from acute care is used as a clinical indicator following total knee replacement (TKR) surgery. This study aimed to assess the clinical relevance of this indicator by determining whether discharge knee ROM predicts longer-term knee ROM and patient-reported knee pain and function. METHODS: A total of 176 TKR recipients were prospectively followed after discharge from acute care. Outcomes assessed included knee ROM and Oxford knee score post rehabilitation and 1 year post surgery. Discharge ROM and other patient factors were identified a priori as potential predictors in multiple linear regression modelling. RESULTS: A total of 133 (76%) and 141 (80%) patients were available for follow-up post rehabilitation [mean postoperative week 8.1 (SD 2.7)] and at 1 year [mean postoperative month 12.1 (SD 1.4)], respectively. Greater discharge knee flexion was a significant (P < 0.001) predictor of greater post-rehabilitation flexion but not 1-year knee flexion (P < 0.083). Better discharge knee extension was a significant predictor of better post-rehabilitation (P = 0.001) and 1-year knee extension (P = 0.013). Preoperative Oxford score and post-rehabilitation knee flexion independently predicted post-rehabilitation Oxford score, and gender predicted 1-year Oxford score. Discharge ROM did not significantly predict Oxford score in either model. CONCLUSION: The finding that early knee range predicts longer-term range provides clinical evidence favouring the relevance of discharge knee ROM as a clinical indicator. Although longer-term patient-reported knee pain and function were not directly associated with discharge knee ROM, they were associated with ROM when measured concurrently in the sub-acute phase. No causal effect has been demonstrated, but the findings suggest it may be important for physiotherapists to maximize range in the early and sub-acute periods.

Mimotope ELISA for detection of broad spectrum antibody against avian H5N1 influenza virus.

BACKGROUND: We have raised a panel of broad spectrum neutralizing monoclonal antibodies against the highly pathogenic H5N1 avian influenza virus, which neutralize the infectivity of, and afford protection against infection by, most of the major genetic groups of the virus evolved since 1997. Peptide mimics reactive with one of these broad spectrum H5N1 neutralizing antibodies, 8H5, were identified from random phage display libraries. METHOD: The amino acid residues of the most reactive 12mer peptide, p125 (DTPLTTAALRLV), were randomly substituted to improve its mimicry of the natural 8H5 epitope. RESULT: 133 reactive peptides with unique amino acid sequences were identified from 5 sub-libraries of p125. Four residues (2,4,5.9) of the parental peptide were preserved among all the derived peptides and probably essential for 8H5 binding. These are interspersed among four other residues (1,3,8,10), which exhibit restricted substitution and probably could contribute to binding, and another four (6,7,11,12) which could be randomly substituted and probably are not essential for binding. One peptide, V-1b, derived by substituting 5 of the latter residues is the most reactive and has a binding constant of 3.16×10(-9) M, which is 38 fold higher than the affinity of the parental p125. Immunoassay produced with this peptide is specifically reactive with 8H5 but not also the other related broad spectrum H5N1 avian influenza virus neutralizing antibodies. Serum samples from 29 chickens infected with H5N1 avian influenza virus gave a positive result by this assay and those from 12 uninfected animals gave a negative test result. CONCLUSION: The immunoassay produced with the 12 mer peptide,V1-b, is specific for the natural 8H5 epitope and can be used for detection of antibody against the broad spectrum neutralization site of H5N1 avian influenza virus.

RNA Interference inhibits hepatitis B virus of different genotypes in vitro and in vivo.

BACKGROUND: Hepatitis B virus (HBV) infection increases the risk of liver disease and hepatocellular carcinoma. Small interfering RNA (siRNA) can be a potential new tool for HBV therapy. Given the high heterogeneity of HBV strains and the sensitivity towards sequences changes of siRNA, finding a potent siRNA inhibitor against the conservative site on the HBV genome is essential to ensure a therapeutic application. RESULTS: Forty short hairpin RNA (shRNA) expression plasmids were constructed to target conserved regions among nine HBV genotypes. HBV 1.3-fold genome plasmids carrying various genotypes were co-transfected with shRNA plasmids into either Huh7 cells or mice. The levels of various viral markers were examined to assess the anti-HBV efficacy of siRNA. Four (B245, B376, B1581 and B1789) were found with the ability to potently inhibit HBV RNA, DNA, surface antigen (HBsAg), e antigen (HBeAg) and core antigen (HBcAg) expression in HBV genotypes A, B, C, D and I (a newly identified genotype) in Huh7 cells and in mice. No unusual cytotoxicity or off-target effects were noted. CONCLUSIONS: Such siRNA suggests an alternate way of inhibiting various HBV genotypes in vitro and in vivo, promising advances in the treatment of HBV.

Immunomodulatory effects of dsRNA and its potential as vaccine adjuvant.

dsRNA can be detected by pattern recognition receptors, for example, TLR3, MDA-5, NLRP3 to induce proinflammatory cytokines responsible for innate/adaptive immunity. Recognized by endosomal TLR3 in myeloid DCs (mDCs), dsRNA can activate mDCs into mature antigen presenting cells (mAPCs) which in turn present antigen epitopes with MHC-I molecules to naïve T cells. Coadministration of protein and synthetic dsRNA analogues can elicit an antigen-specific Th1-polarized immune response which stimulates the CD8+ CTL response and possibly dampen Th17 response. Synthetic dsRNA analogues have been tested as vaccine adjuvant against viral infections in animal models. However, a dsRNA receptor, TLR3 can be expressed in tumor cells while other members of TLR family, for example, TLR4 and TLR2 have been shown to promote tumor progression, metastasis, and chemoresistance. Thus, the promising potential of dsRNA analogues as a tumor therapeutic vaccine adjuvant should be evaluated cautiously.

Low incidence of complications from endoscopic gastric variceal obturation with butyl cyanoacrylate.

BACKGROUND & AIMS: Endoscopic variceal obturation with tissue adhesive is used to control gastric variceal bleeding. We investigated the prevalence of serious complications from this therapy. METHODS: We performed a retrospective analysis of complications that occurred in 753 patients with gastric variceal hemorrhages who were hospitalized in 2 tertiary referral hospitals. All patients received N-butyl-2-cyanoacrylate as therapy for endoscopic variceal obturation. RESULTS: Complications occurred in 51 patients. Thirty-three patients experienced rebleeding because of early-onset (within 3 months) extrusion of the N-butyl-2-cyanoacrylate glue cast (4.4%), 10 patients developed sepsis (1.3%), and 5 patients developed distant embolisms (0.7%; 1 pulmonary, 1 brain, and 3 splenic). One patient had major gastric variceal bleeding after endoscopic variceal obturation (0.1%), 1 developed a large gastric ulcer (0.1%), and 1 had mesentery hematoma, hemoperitoneum, and infection in the abdominal cavity (0.1%). The complication-related mortality was 0.53% (3 deaths from sepsis and 1 death from rebleeding after early-onset glue cast extrusion). CONCLUSIONS: The occurrence of complications after endoscopic variceal obturation with N-butyl-2-cyanoacrylate in gastric varices treatment is rare.

Performance of detecting IgM antibodies against enterovirus 71 for early diagnosis.

Enterovirus 71 (EV71) infection is more likely to induce severe complications and mortality than other enteroviruses. Methods for detection of IgM antibody against EV71 had been established for years, however, the performance of the methods in the very early diagnosis of EV71 infection had not been fully evaluated, which is especially meaningful because of the short incubation period of EV71 infection. In this report, the performance of an IgM anti-EV71 assay was evaluated using acute sera collected from 165 EV71 infected patients, 165 patients infected with other enteroviruses, and more than 2,000 sera from healthy children or children with other infected diseases. The results showed a 90% sensitivity in 20 patients who were in their first illness day, and similar sensitivity remained till 4 days after onset. After then the sensitivity increased to 95% to 100% for more than one month. The specificity of the assay in non-HFMD children is 99.1% (95% CI: 98.6-99.4), similar as the 99.9% specificity in healthy adults. The cross-reaction rate in patients infected with other non-EV71 enteroviruses was 11.4%. In conclusion, the data here presented show that the detection of IgM anti-EV71 by ELISA affords a reliable, convenient, and prompt diagnosis of EV71 infection.

Role of heat-shock protein 90 in hepatitis E virus capsid trafficking.

p239 is a virus-like particle constituted from hepatitis E virus (HEV) recombinant proteins. It can be used as a surrogate for HEV and as an investigative tool to study cellular interactions because of its ability to adsorb to and penetrate HepG2 cellular membranes. Our objective was to use p239 to define the role of HEV capsid proteins during the early stages of infection. Pull-down and MALDI-TOF MS experiments identified three host-cell proteins, Grp 78/Bip, alpha-tubulin and heat-shock protein 90 (HSP90), and the latter was investigated further. Antibodies to p239 alone or HSP90 alone could detect p239 or HSP90, suggesting the formation of a complex between p239 and HSP90. In the HepG2 cell, geldanamycin (GA), an HSP90-specific inhibitor, blocked intracellular transportation of p239, but had no effect on the binding and cellular entry of p239, suggesting that HSP90 was important for HEV capsid intracellular transportation. RT-PCR results showed that the efficiency of wild-type HEV infection was inhibited significantly by GA treatment, suggesting the importance of HSP90 in virus infectivity. It was concluded that HSP90 plays a crucial role in the intracellular transportation of viral capsids in the early stage of HEV infection.

A rapid test for the detection of influenza A virus including pandemic influenza A/H1N1 2009.

A new rapid diagnostic test for detection of influenza A virus was evaluated with four sets of experiments: first, a comparison with a commercial diagnostic kit against a panel of virus strains was conducted; second, the kit was tested against a collection of 40 strains of influenza A virus isolated from five different host species and 26 strains of other respiratory viruses used as controls; third, the kit was tested against specimens collected in the field obtained from human and chicken; and fourth, the kit was tested against the novel pandemic influenza A/H1N1 2009 clinical specimens obtained from admitted to hospital patients. The test kit displayed a sensitivity of 88% for both human specimens and avian specimens. The corresponding specificity was 99.3% for human specimens and 96.5% for avian specimens. This test kit may be useful for rapid diagnosis of influenza A virus.

Novel double-antigen sandwich immunoassay for human hepatitis B core antibody.

A novel diagnostic immunoassay testing procedure for hepatitis B virus core antibody (anti-HBc) using homogeneous purified full-length hepatitis B virus core antigen (HBcAg) capsids obtained from Escherichia coli was compared with Abbott Architect anti-HBc chemiluminescent microparticle immunoassay (CMIA; indirect method) against a library of specimens. A monoclonal anti-HBc neutralization confirmatory assay was then used to determine the degree of discordance between specimens. The new assay was found to be superior in both sensitivity and specificity.

Molecular characteristics of occult hepatitis B virus from blood donors in southeast China.

The characteristics of 30 carriers with occult hepatitis B virus (HBV) infection (OBI) were compared with those of 30 individuals diagnosed as being HBV carriers at the time of blood donation, 60 asymptomatic carriers, and 60 chronic hepatitis patients. The prevalence of genotype C was significantly higher in carriers with OBIs than in any other HBsAg-positive (HBsAg(+)) group (P < 0.001). Specific amino acid substitutions in the regions from amino acids 117 to 121 and amino acids 144 to 147 located in the major hydrophilic region of the S gene were associated with carriers with OBIs (P < 0.01 for carriers with OBIs versus HBsAg(+) donors, carriers with OBIs versus HBsAg(+) asymptomatic carriers, and carriers with OBIs versus HBsAg(+) chronic hepatitis patients). G145R was the major variation in the HBV isolates responsible for local occult HBV infections.

Decreased NK cell frequency in chronic hepatitis C does not affect ex vivo cytolytic killing.

Prior studies have suggested that natural killer (NK) cell function might be impaired in chronic hepatitis C virus (HCV) infection. Circulating NK cell frequency and cytolytic activity were examined freshly ex vivo in HCV-infected and uninfected subjects. Surprisingly, the intrinsic cytolytic activity of peripheral blood NK-enriched cells was similar between HCV-infected and uninfected groups (P = .91). Although the percentage of circulating CD3- CD16/56+ NK cells was 30% lower in HCV-infected compared with uninfected subjects (P = .02) paralleled by a decrease of CD56(dim) cytolytic NK cells (P = .02), overall K562 cytolysis by unfractionated peripheral blood mononuclear cells was not affected (P = .29). Analysis of the relationships between NK cytolytic activity and other clinical information revealed an inverse association with liver fibrosis stage (P = .035). In conclusion, NK cell cytolytic function does not appear to be impaired in chronic hepatitis C, but higher levels of NK cell cytolysis are associated with less liver fibrosis.

Prognostic indicators of breakthrough hepatitis during lamivudine monotherapy for chronic hepatitis B virus infection.

BACKGROUND: Breakthrough hepatitis (BTH), defined as a flare of transaminases alanine aminotransferase [ALT]) can occur during lamivudine monotherapy for hepatitis B virus (HBV) infection. There have been many reports of lamivudine-resistant mutations within the C domain of the viral reverse transcriptase; however, the appearance of these mutants is not necessarily correlated with BTH during lamivudine therapy. METHODS AND RESULTS: Entire serial HBV genomic sequences before and during lamivudine therapy for 4 patients with BTH and 1 patient without BTH were analyzed and showed changes in the pre-S region. These changes may be associated with ALT flares. Further investigation in a cohort of 36 patients with a median treatment period of 25 months showed that 21 patients had a rise in HBV-DNA titer, of whom 18 had BTH. Univariate statistical analyses showed that possible prognostic indicators for the occurrence of BTH were pre-S deletions ( P = 0.03) and L180M/M204L mutations ( P = 0.04). By multivariate Cox regression analyses, significant variables were pre-S deletions (hazard ratio, 0.17; 95% confidence internal (CI), 0.044-0.66) and precore mutations (hazard ratio, 5.70; 95% CI, 1.74-18.71) prior to the commencement of lamivudine monotherapy. Interestingly, BTH occurred after the selection of the wild-type species in the pre-S region during lamivudine monotherapy. CONCLUSIONS: These results suggest that patients with HBV pre-S deletion mutants should be monitored carefully during lamivudine therapy.