Number of musculoskeletal pain sites leads to increased long-term healthcare contacts and healthcare related costs - a Danish population-based cohort study.

BACKGROUND: People with musculoskeletal pain seek more healthcare than the general population, however little is known about the long-term effect on healthcare use. The aim of this study was to examine the consequences of number of musculoskeletal pain sites on long-term care-seeking and healthcare-related costs and explore how health anxiety influences this relationship. METHODS: We conducted a Danish population-based longitudinal cohort study of 4883 participants combining self-reported survey data from 2008 with ten-year follow-up data from national health registers. Using a causal inference framework, we examined associations between number of pain sites (range 0-7)/level of health anxiety (high/low level) and face-to-face healthcare contacts/healthcare-related costs. Data were analyzed using negative binomial regression with generalized estimating equations. Regression models were adjusted for sex, age, duration of pain, level of education, comorbidity, personality traits, risk of depression, marital status, physical job exposure, and previous healthcare utilization. RESULTS: For each additional pain site general healthcare contacts (Incidence Rate Ratio (IRR): 1.04 (95% CI: 1.03-1.05)), healthcare-related costs (IRR: 1.06 (95% CI: 1.03-1.08) and musculoskeletal healthcare contacts (IRR: 1.11 (95% CI:1.09-1.14) increased. Those with high levels of health anxiety at baseline had a slightly higher number of general healthcare contacts (IRR 1.06 (1.01-1.11), independent of number of pain sites. However, level of anxiety did not influence the effect of number of pain sites on any healthcare use or cost outcomes. CONCLUSIONS: We found evidence for a causal association between increasing number of pain sites and greater healthcare use and cost, and high levels of health anxiety did not increase the strength of this association. This suggests that number of pain sites could be a potential target for biopsychosocial interventions in order to reduce the need for future care-seeking.

Complement component 5 does not interfere with physiological hemostasis but is essential for Escherichia coli-induced coagulation accompanied by Toll-like receptor 4.

There is a close cross-talk between complement, Toll-like receptors (TLRs) and coagulation. The role of the central complement component 5 (C5) in physiological and pathophysiological hemostasis has not, however, been fully elucidated. This study examined the effects of C5 in normal hemostasis and in Escherichia coli-induced coagulation and tissue factor (TF) up-regulation. Fresh whole blood obtained from six healthy donors and one C5-deficient individual (C5D) was anti-coagulated with the thrombin inhibitor lepirudin. Blood was incubated with or without E. coli in the presence of the C5 inhibitor eculizumab, a blocking anti-CD14 monoclonal antibody (anti-CD14) or the TLR-4 inhibitor eritoran. C5D blood was reconstituted with purified human C5. TF mRNA was measured by quantitative polymerase chain reaction (qPCR) and monocyte TF and CD11b surface expression by flow cytometry. Prothrombin fragment 1+2 (PTF1·2) in plasma and microparticles exposing TF (TF-MP) was measured by enzyme-linked immunosorbent assay (ELISA). Coagulation kinetics were analyzed by rotational thromboelastometry and platelet function by PFA-200. Normal blood with eculizumab as well as C5D blood with or without reconstitution with C5 displayed completely normal biochemical hemostatic patterns. In contrast, E. coli-induced TF mRNA and TF-MP were significantly reduced by C5 inhibition. C5 inhibition combined with anti-CD14 or eritoran completely inhibited the E. coli-induced monocyte TF, TF-MP and plasma PTF1·2. Addition of C5a alone did not induce TF expression on monocytes. In conclusion, C5 showed no impact on physiological hemostasis, but substantially contributed to E. coli-induced procoagulant events, which were abolished by the combined inhibition of C5 and CD14 or TLR-4.

Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

The key roles of complement and tissue factor in Escherichia coli-induced coagulation in human whole blood.

The complement system and the Toll-like (TLR) co-receptor CD14 play important roles in innate immunity and sepsis. Tissue factor (TF) is a key initiating component in intravascular coagulation in sepsis, and long pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)-induced transcription of TF. The aim of this study was to study the mechanism by which complement and CD14 affects LPS- and Escherichia coli (E. coli)-induced coagulation in human blood. Fresh whole blood was anti-coagulated with lepirudin, and incubated with ultra-purified LPS (100 ng/ml) or with E. coli (1 × 10(7) /ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti-CD14 F(ab')2 antibody and a control F(ab')2 . TF mRNA was measured using quantitative polymerase chain reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF functional activity in plasma microparticles was measured using an amidolytic assay. Prothrombin fragment F 1+2 (PTF1.2) and PTX3 were measured by enzyme-linked immunosorbent assay (ELISA). The effect of TF was examined using an anti-TF blocking antibody. E. coli increased plasma PTF1.2 and PTX3 levels markedly. This increase was reduced by 84->99% with compstatin, 55-97% with anti-CD14 and > 99% with combined inhibition (P < 0·05 for all). The combined inhibition was significantly (P < 0·05) more efficient than compstatin and anti-CD14 alone. The LPS- and E. coli-induced TF mRNA levels, monocyte TF surface expression and TF functional activity were reduced by > 99% (P < 0·05) with combined C3 and CD14 inhibition. LPS- and E. coli-induced PTF1.2 was reduced by 76-81% (P < 0·05) with anti-TF antibody. LPS and E. coli activated the coagulation system by a complement- and CD14-dependent up-regulation of TF, leading subsequently to prothrombin activation.

Immersion challenge with low and highly virulent infectious salmon anaemia virus reveals different pathogenesis in Atlantic salmon, Salmo salar L.

The salmonid orthomyxovirus infectious salmon anaemia virus (ISAV) causes disease of varying severity in farmed Atlantic salmon, Salmo salar L. Field observations suggest that host factors, the environment and differences between ISAV strains attribute to the large variation in disease progression. Variation in host mortality and dissemination of ISAV isolates with high and low virulence (based on a previously published injection challenge) were investigated using immersion challenge. Virus dissemination was determined using real-time PCR and immunohistochemistry in several organs, including blood. Surprisingly, the low virulent virus (LVI) replicated and produced nucleoprotein at earlier time points post-infection compared to the virus of high virulence (HVI). This was particularly noticeable in the gills as indicated by different viral load profiles. However, the HVI reached a higher maximum viral load in all tested organs and full blood. This was associated with a higher mortality of 100% as compared to 20% in the LVI group by day 23 post-infection. Immersion challenge represented a more natural infection method and suggested that specific entry routes into the fish may be of key importance between ISAV strains. The results suggest that a difference in virulence is important for variations in virus dissemination and pathogenesis (disease development).

C1-inhibitor efficiently inhibits Escherichia coli-induced tissue factor mRNA up-regulation, monocyte tissue factor expression and coagulation activation in human whole blood.

Both the complement system and tissue factor (TF), a key initiating component of coagulation, are activated in sepsis, and cross-talk occurs between the complement and coagulation systems. C1-inhibitor (C1-INH) can act as a regulator in both systems. Our aim in this study was to examine this cross-talk by investigating the effects of C1-INH on Escherichia coli-induced haemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultrapurified E. coli lipopolysaccharide (LPS) in the absence or presence of C1-INH or protease-inactivated C1-INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1·2 (PTF1·2) and long pentraxin 3 (PTX3) were measured by enzyme-linked immunosorbent assay (ELISA). Cytokines were analysed using a multiplex kit. C1-INH (1·25-5 mg/ml) reduced both LPS- and E. coli-induced coagulation, measured as a reduction of PTF1·2 in plasma, efficiently and dose-dependently (P < 0·05). Both LPS and E. coli induced marked up-regulation of TF mRNA levels and surface expression on whole blood monocytes. This up-regulation was reduced efficiently by treatment with C1-INH (P < 0·05). C1-INH reduced the release of PTX3 (P < 0·05) and virtually all cytokines measured (P < 0·05). Complement activation was inhibited more efficiently with compstatin than with C1-INH. C1-INH inhibited most of the other readouts more efficiently, consistent with additional non-complement-dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1-INH is a broad-spectrum attenuator of the inflammatory and haemostatic responses.

The effects of selective complement and CD14 inhibition on the E. coli-induced tissue factor mRNA upregulation, monocyte tissue factor expression, and tissue factor functional activity in human whole blood.

BACKGROUND: The complement pathway and CD14 play essential roles in inflammation, but little is known about the relative roles of complement and CD14 in E. coli-induced tissue factor (TF) mRNA upregulation, expression by monocytes, and functional activity in human whole blood. METHODS: Whole E. coli bacteria were incubated for up to 4 h in human whole blood containing the anticoagulant lepirudin, which does not affect complement activation. TF mRNA levels were analyzed using reverse transcription, quantitative real-time PCR (RT-qPCR), and the expression of TF on the cell surface was analyzed using flow cytometry. Complement was selectively inhibited using the C3 convertase inhibitor compstatin or a C5a receptor antagonist (C5aRa), while CD14 was blocked by an anti-CD14 F(ab')2 monoclonal antibody. RESULTS: The E. coli-induced TF mRNA upregulation was reduced to virtually background levels by compstatin, whereas anti-CD14 had no effect. Monocyte TF expression and TF activity in plasma microparticles were significantly reduced by C5aRa. Anti-CD14 alone only slightly reduced E. coli-induced monocyte TF expression but showed a modest additive effect when combined with the complement inhibitors. Inhibiting complement and CD14 efficiently reduced the expression of the E. coli-induced cytokines IL-1beta, IL-6, IL-8, and platelet-derived growth factor bb. CONCLUSION: Our results indicate that E. coli-induced TF mRNA upregulation is mainly dependent on complement activation, while CDI4 plays a modest role in monocyte TF expression and the plasma TF activity in human whole blood.

Differential effect of inhibiting MD-2 and CD14 on LPS- versus whole E. coli bacteria-induced cytokine responses in human blood.

BACKGROUND: Sepsis is a major world-wide medical problem with high morbidity and mortality. Gram-negative bacteria are among the most important pathogens of sepsis and their LPS content is regarded to be important for the systemic inflammatory reaction. The CD14/myeloid differentiation factor 2 (MD-2)/TLR4 complex plays a major role in the immune response to LPS . The aim of this study was to compare the effects of inhibiting MD-2 and CD14 on ultra-pure LPS - versus whole E. coli bacteria-induced responses. METHODS: Fresh human whole blood was incubated with upLPS or whole E. coli bacteria in the presence of MD-2 or CD14 neutralizing monoclonal antibodies, or their respective controls, and/or the specific complement-inhibitor compstatin. Cytokines were measured by a multiplex (n = 27) assay. NFκB activity was examined in cells transfected with CD14, MD-2 and/or Toll-like receptors. RESULTS: LPS-induced cytokine response was efficiently and equally abolished by MD-2 and CD14 neutralization. In contrast, the response induced by whole E. coli bacteria was only modestly reduced by MD-2 neutralization, whereas CD14 neutralization was more efficient. Combination with compstatin enhanced the effect of MD-2 neutralization slightly. When compstatin was combined with CD14 neutralization, however, the response was virtually abolished for all cytokines, including IL-17, which was only inhibited by this combination. The MD-2-independent effect observed for CD14 could not be explained by TLR2 signaling. CONCLUSION: Inhibition of CD14 is more efficient than inhibition of MD-2 on whole E. coli-induced cytokine response, suggesting CD14 to be a better target for intervention in Gram-negative sepsis, in particular when combined with complement inhibition.

Effects of geographic distance, sea barriers and habitat on the genetic structure and diversity of all-hybrid water frog populations.

The history of population size and migration patterns leaves its mark in the genetics of populations. We investigate the genetic structure of the edible frog, Pelophylax esculentus in the Danish archipelago and adjacent countries. This frog is of particular interest because it is a hybrid that, in this area, forms all-hybrid populations of diploid (LR) and triploid (LLR and LRR) genomotypes with no (or very few) adults of the parental species (LL and RR). This study is the first to cover the entire geographic range of Danish, Swedish and German all-hybrid populations, documenting their extent and providing a broad picture of their diversity of neutral genetic markers and genomotype proportions. With 18 microsatellite markers, we found that genetic diversity declines northwards in agreement with the glacial refuge and central-marginal hypotheses; however, populations on small and medium-sized islands are no less diverse than those on large islands and continental peninsulas. Isolation by distance exists across the archipelago with limited influence of fragmentation by brackish seawater. The extremely low genetic diversity in all-hybrid populations, compared with adjacent populations, may be responsible for the maintenance of their special breeding system. We also show large variation among ponds in proportions of LLR, LR and LRR genomotypes, but little geographic pattern in their distribution. Instead, we found relationships between the genomotype proportions and some of 15 habitat parameters monitored. Body size differences among LLR, LR and LRR further suggest ecological differences.

The role of age and mode of delivery in the STEPS intervention: a longitudinal pilot-study in treatment of posttraumatic stress symptoms in Danish survivors of sexual assault.

Background: The STEPS programme has been succesfully implemented as a group-based treatment of trauma symptoms after rape for adolescents. The STEPS intervention was translated from Dutch to Danish and offered to adults in addition to adolescents as well as an individual intervention in addition to a group-based intervention at a Danish Centre for Rape Victims through 2011 to 2014. The programme was translated from Dutch to Danish and expanded to adults in addition to adolescents as well as to an individual intervention in addition to a group-based intervention at a Danish Centre for Rape Victims through 2011 to 2014. Objective: The present study observes  development in trauma symptoms and ICD-11 diagnostic status during an adapted version of the intervention programme 'STEPS' for survivors of sexual assault. Methods: A prospective uncontrolled study was conducted, monitoring symptoms of posttraumatic stress and other trauma-related symptomatology before treatment, after treatment and at 6 and 12 months' follow up for 103 referrals receiving individual or group-based STEPS. Tentative diagnoses of posttraumatic stress disorder (PTSD) and complex PTSD were assigned to participants according to the ICD-11 to observe the development in diagnostic status across time, and multilevel modelling was used to assess the development of symptom severity and to assess the moderating effect of age-group and mode of delivery. Results: A loglinear function representing large and statistically significant decline in symptomatology over time provided the best fit for all measures of trauma-related symptomatology. The decline was not moderated by age-group or mode of intervention. Dropout rates were independent of mode of intervention and age. Conclusion: The adaption of the STEPS programme to adults and as an individual intervention is feasible and maintains effect sizes comparable to those observed in the original intervention. Further research using randomized controlled trials is needed to ascribe the observed effect to the STEPS programme.

Antecedentes: El programa STEPS se ha implementado con éxito como un tratamiento grupal de síntomas de trauma después de una violación en adolescentes. La intervención STEPS se tradujo del holandés al danés y se ofreció a adultos además de adolescentes, así como una intervención individual además de una intervención grupal en un Centro danés para víctimas de violación hasta el 2011 y el 2014.Objetivo: El presente estudio observa el desarrollo de síntomas de trauma y el estado de diagnóstico de la CIE-11 durante una versión adaptada del programa de intervención "PASOS" para los sobrevivientes de agresión sexual.Métodos: Se realizó un estudio prospectivo no controlado, monitorizando los síntomas de estrés postraumático y otras sintomatologías relacionadas con trauma antes del tratamiento, después del tratamiento y a los 6 y 12 meses de seguimiento para 103 derivaciones. Se asignaron a los participantes los diagnósticos tentativos de trastorno de estrés postraumático (TEPT) y TEPT complejo según la CIE-11 para observar el desarrollo en el estado de diagnóstico a través del tiempo, y se usó un modelado multinivel para evaluar el desarrollo de la severidad de los síntomas y evaluar el efecto moderador del grupo de edad y modo de entrega.Resultados: Un función lineal logarítmica que representa una disminución grande y estadísticamente significativa de la sintomatología a lo largo del tiempo proporcionó el mejor ajuste para todas las medidas de la sintomatología relacionada con trauma. El efecto no fue moderado por grupo de edad o tipo de intervención. Las tasas de abandono fueron independientes del tipo de intervención y edad.Conclusión:: La adaptación del programa STEPS para adultos y como una intervención individual es factible y mantiene el tamaño del efecto comparable con aquellos observados en la intervención original. Se necesita más investigación usando estudios controlados aleatorizados para atribuir el efecto observado al programa STEPS.

Phagocytosis of live and dead Escherichia coli and Staphylococcus aureus in human whole blood is markedly reduced by combined inhibition of C5aR1 and CD14.

BACKGROUND: Sepsis is a dysregulated host response to infection. The aim of this study was to investigate the effects of complement- and CD14 inhibition on phagocytosis of live and dead Gram-negative and Gram-positive bacteria in human whole blood. METHODS: Lepirudin-anticoagulated blood was incubated with live or dead E. coli or S. aureus at 37 °C for 120 min with or without the C5aR1 antagonist PMX53 and/or anti-CD14. Granulocyte and monocyte phagocytosis were measured by flow cytometry, and five plasma cytokines by multiplex, yielding a total of 28 mediators of inflammation tested for. RESULTS: 16/28 conditions were reduced by PMX53, 7/28 by anti-CD14, and 24/28 by combined PMX53 and CD14 inhibition. The effect of complement inhibition was quantitatively more pronounced, in particular for the responses to S. aureus. The effect of anti-CD14 was modest, except for a marked reduction in INF-ß. The responses to live and dead S. aureus were equally inhibited, whereas the responses to live E. coli were inhibited less than those to dead E. coli. CONCLUSION: C5aR1 inhibited phagocytosis-induced inflammation by live and dead E. coli and S. aureus. CD14 blockade potentiated the effect of C5aR1 blockade, thus attenuating inflammation.

Pre-clinical evaluation of a quadrivalent HCV VLP vaccine in pigs following microneedle delivery.

The introduction of directly acting antiviral agents (DAAs) has produced significant improvements in the ability to cure chronic hepatitis C infection. However, with over 2% of the world's population infected with HCV, complications arising from the development of cirrhosis of the liver, chronic hepatitis C infection remains the leading indication for liver transplantation. Several modelling studies have indicated that DAAs alone will not be sufficient to eliminate HCV, but if combined with an effective vaccine this regimen would provide a significant advance towards achieving this critical World Health Organisation goal. We have previously generated a genotype 1a, 1b, 2a, 3a HCV virus like particle (VLP) quadrivalent vaccine. The HCV VLPs contain the core and envelope proteins (E1 and E2) of HCV and the vaccine has been shown to produce broad humoral and T cell immune responses following vaccination of mice. In this report we further advanced this work by investigating vaccine responses in a large animal model. We demonstrate that intradermal microneedle vaccination of pigs with our quadrivalent HCV VLP based vaccine produces long-lived multi-genotype specific and neutralizing antibody (NAb) responses together with strong T cell and granzyme B responses and normal Th1 and Th2 cytokine responses. These responses were achieved without the addition of adjuvant. Our study demonstrates that our vaccine is able to produce broad immune responses in a large animal that, next to primates, is the closest animal model to humans. Our results are important as they show that the vaccine can produce robust immune responses in a large animal model before progressing the vaccine to human trials.

Increased FXYD1 and PGC-1α mRNA after blood flow-restricted running is related to fibre type-specific AMPK signalling and oxidative stress in human muscle.

AIM: This study explored the effects of blood flow restriction (BFR) on mRNA responses of PGC-1α (total, 1α1, and 1α4) and Na+ ,K+ -ATPase isoforms (NKA; α1-3 , ß1-3 , and FXYD1) to an interval running session and determined whether these effects were related to increased oxidative stress, hypoxia, and fibre type-specific AMPK and CaMKII signalling, in human skeletal muscle. METHODS: In a randomized, crossover fashion, 8 healthy men (26 ± 5 year and 57.4 ± 6.3 mL kg-1  min-1 ) completed 3 exercise sessions: without (CON) or with blood flow restriction (BFR), or in systemic hypoxia (HYP, ~3250 m). A muscle sample was collected before (Pre) and after exercise (+0 hour, +3 hours) to quantify mRNA, indicators of oxidative stress (HSP27 protein in type I and II fibres, and catalase and HSP70 mRNA), metabolites, and α-AMPK Thr172 /α-AMPK, ACC Ser221 /ACC, CaMKII Thr287 /CaMKII, and PLBSer16 /PLB ratios in type I and II fibres. RESULTS: Muscle hypoxia (assessed by near-infrared spectroscopy) was matched between BFR and HYP, which was higher than CON (~90% vs ~70%; P < .05). The mRNA levels of FXYD1 and PGC-1α isoforms (1α1 and 1α4) increased in BFR only (P < .05) and were associated with increases in indicators of oxidative stress and type I fibre ACC Ser221 /ACC ratio, but dissociated from muscle hypoxia, lactate, and CaMKII signalling. CONCLUSION: Blood flow restriction augmented exercise-induced increases in muscle FXYD1 and PGC-1α mRNA in men. This effect was related to increased oxidative stress and fibre type-dependent AMPK signalling, but unrelated to the severity of muscle hypoxia, lactate accumulation, and modulation of fibre type-specific CaMKII signalling.

Staphylococcus aureus-induced complement activation promotes tissue factor-mediated coagulation.

Essentials Complement, Toll-like receptors and coagulation cross-talk in the process of thromboinflammation. This is explored in a unique human whole-blood model of S. aureus bacteremia. Coagulation is here shown as a downstream event of C5a-induced tissue factor (TF) production. Combined inhibition of C5 and CD14 efficiently attenuated TF and coagulation. SUMMARY: Background There is extensive cross-talk between the complement system, the Toll-like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression. Objectives To study the relative roles of complement, TLRs and TF in Staphylococcus aureus-induced coagulation. Methods Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF1 + 2 ) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively. Results All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF1 + 2 , and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF1 + 2 levels to baseline levels. Conclusions S. aureus-induced coagulation in human whole blood was mainly attributable to C5a-induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation.

Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine.

The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.

Alternative genetic pathways and cooperating genetic abnormalities in the pathogenesis of therapy-related myelodysplasia and acute myeloid leukemia.

Alternative genetic pathways were previously outlined in the pathogenesis of therapy-related myelodysplasia (t-MDS) and acute myeloid leukemia (t-AML) based on cytogenetic characteristics. Some of the chromosome aberrations, the recurrent balanced translocations or inversions, directly result in chimeric rearrangement of genes for hematopoietic transcription factors (class II mutations) which disturb cellular differentiation. Other genetic abnormalities in t-MDS and t-AML comprise activating point mutations or internal tandem duplications of genes involved in signal transduction as tyrosine kinase receptors or genes more downstream in the RAS-BRAF pathway (class I mutations). The alternative genetic pathways of t-MDS and t-AML can now be further characterized by a different clustering of six individual class I mutations and mutations of AML1 and p53 in the various pathways. In addition, there is a significant association between class I and class II mutations possibly indicating cooperation in leukemogenesis, and between mutations of AML1 and RAS related to subsequent progression from t-MDS to t-AML. Therapy-related and de novo myelodysplasia and acute myeloid leukemia seem to share genetic pathways, and surprisingly gene mutations were in general not more frequent in patients with t-MDS or t-AML as compared to similar cases of de novo MDS and AML studied previously.

Amplification or duplication of chromosome band 21q22 with multiple copies of the AML1 gene and mutation of the TP53 gene in therapy-related MDS and AML.

Amplification or duplication of the AML1 gene at chromosome band 21q22 was detected by FISH using a locus-specific probe in three out of 171 unselected patients with therapy-related myelodysplasia (t-MDS) or t-AML (1.7%). In two patients AML1 signals were located tandemly on derivative chromosomes, in one patient on a dic(9;21) and in the the other patient on a derivative chromosome 18 made up of interchanging layers of material from chromosomes 9, 14, 18, and 21. In the third patient three single supernumerary copies of AML1 were located on derivatives of chromosomes 19 and 21. All three patients were older, had previously received therapy with alkylating agents without topoisomerase II inhibitors, had complex karyotypes including abnormalities of chromosomes 5 or 7, and presented acquired point mutations of the TP53 gene. No point mutations of the AML1 gene were observed. The results support a pivotal role of impaired TP53 function in the development of gene amplification or duplication in t-MDS and t-AML.

Mutations of genes in the receptor tyrosine kinase (RTK)/RAS-BRAF signal transduction pathway in therapy-related myelodysplasia and acute myeloid leukemia.

Mutations of the FLT3, c-KIT, c-FMS, KRAS, NRAS, BRAF and CEBPA genes in the receptor tyrosine kinase (RTK)/RAS-BRAF signal-transduction pathway are frequent in acute myeloid leukemia (AML). We examined 140 patients with therapy-related myelodysplasia or AML (t-MDS/t-AML) for point mutations of these seven genes. In all, 11 FLT3, two c-KIT, seven KRAS, eight NRAS and three BRAF mutations were identified in 29 patients (21%). All but one patient with a FLT3 mutation presented with t-AML (P=0.0002). Furthermore, FLT3 mutations were significantly associated with previous radiotherapy without chemotherapy (P=0.03), and with a normal karyotype (P=0.004), but inversely associated with previous therapy with alkylating agents (P=0.003) and with -7/7q- (P=0.001). RAS mutations were associated with AML1 point mutations (P=0.046) and with progression from t-MDS to t-AML (P=0.008). Noteworthy, all three patients with BRAF mutations presented as t-AML of M5 subtype with t(9;11)(p22;q23) and MLL-rearrangement (P=0.01). In t-AML RAS/BRAF mutations were significantly associated with a very short survival (P=0.017). Half of the patients with a mutation in the RTK/RAS-BRAF signal-transduction pathway (denoted 'class-I' mutations) simultaneously disclosed mutation of a hematopoietic transcription factor (denoted 'class-II' mutations) (P=0.046) suggesting their cooperation in leukemogenesis.

Large scale production of a mammalian cell derived quadrivalent hepatitis C virus like particle vaccine.

A method for the large-scale production of a quadrivalent mammalian cell derived hepatitis C virus-like particles (HCV VLPs) is described. The HCV core E1 and E2 coding sequences of genotype 1a, 1b, 2a or 3a were co-expressed in Huh7 cell factories using a recombinant adenoviral expression system. The structural proteins self-assembled into VLPs that were purified from Huh7 cell lysates by iodixanol ultracentrifugation and Stirred cell ultrafiltration. Electron microscopy, revealed VLPs of the different genotypes that are morphologically similar. Our results show that it is possible to produce large quantities of individual HCV genotype VLPs with relative ease thus making this approach an alternative for the manufacture of a quadrivalent mammalian cell derived HCV VLP vaccine.

Mutations with loss of heterozygosity of p53 are common in therapy-related myelodysplasia and acute myeloid leukemia after exposure to alkylating agents and significantly associated with deletion or loss of 5q, a complex karyotype, and a poor prognosis.

PURPOSE: To study mutations and loss of heterozygosity (LOH) of p53 in therapy-related myelodysplasia (t-MDS) and acute myeloid leukemia (t-AML). PATIENTS AND METHODS: Fifty-two unselected patients with t-MDS and 25 patients with t-AML were studied by polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) at the DNA level and by reverse transcriptase (RT)-PCR-SSCP at the mRNA level, and cases with aberrant SSCP patterns were sequenced. RESULTS: Somatically acquired mutations of p53 were observed in 21 of 77 cases of t-MDS or t-AML, and 19 of these 21 patients had received alkylating agents. Single-base substitutions at A:T pairs were more common in t-MDS and t-AML, whereas single-base substitutions at G:C pairs are most common in MDS and AML de novo and in solid tumors. Six patients demonstrated a cytogenetic loss of 17p13, and these six and an additional nine patients with p53 mutations demonstrated LOH of p53 at the DNA or mRNA level. This suggests a cytogenetic loss of the normal p53 allele in these nine cases combined with duplication of the homologous chromosome 17 carrying the mutated p53 allele. Mutations of p53 were significantly associated with deletion or loss of 5q (P <.0001) and a complex karyotype (P =.0001), but surprisingly were not associated with deletion or loss of 7q (P =.73), and were infrequent in patients with balanced chromosome translocations (P =.03). Mutations of p53 were more common in older patients (P =.036) and were associated with an extremely poor prognosis (P =.014), apparently restricted to the 15 cases with LOH of p53 ( P =.046). CONCLUSION: Mutations with loss of function of p53 are significantly associated with deletion or loss of 5q in t-MDS and t-AML after previous treatment with alkylating agents and are associated with genetic instability.