Toll-like receptors (TLRs) are key pathogen sensing receptors that respond to diverse microbial ligands, and trigger both innate and adaptive immune responses to infection. Since their discovery, a growing body of evidence has pointed to an important role for TLRs in retroviral infection and pathogenesis. These data suggest that multiple TLRs contribute to the anti-retroviral response, and that TLR engagement by retroviruses can have complex and divergent outcomes for infection. Despite this progress, numerous questions remain about the role of TLRs in retroviral infection. In this review, I summarize existing evidence for TLR-retrovirus interactions and the functional roles these receptors play in immunity and pathogenesis, with particular focus on human immunodeficiency virus (HIV). The innate immune system represents the front line of defense against invading microbes, mediating rapid responses at mucosal surfaces that can prevent the establishment of disseminated infection. This system is comprised of a diverse set of molecules and pathways that serve to sense the presence of invading pathogens, and to inhibit replication of these pathogens. A remarkable feature of the innate immune system is its ability to detect infection by a wide range of pathogens with different characteristics—ranging from viruses to bacteria and fungi, and to initiate rapid expression of anti-microbial genes within hours of infection. These anti-microbial responses can take the form of proteins that target specific steps in viral replication (often referred to as “restriction factors”) or pro-inflammatory signaling pathways and cytokine networks that recruit and activate cell types such as natural killer cells and macrophages that clear pathogens. The signaling pathways activated by the innate immune system in response to infection are complex, and vary depending on the microbe and on the sensing pathway, but typically involve activation of both proinflammatory transcription factors such as NF-κB, and antiviral cytokines such as type 1 interferons (IFNs) . In someDespite the vital importance of the innate immune system, strict regulation of innate signaling pathways is also essential. Inappropriate or unregulated activation of innate immunity can be highly detrimental to an organism’s health, resulting in chronic inflammatory diseases and autoimmunity . A critiOver the past 25 years, enormous progress has been made in discovering and characterizing PRRs in the vertebrate immune system. The overall diversity of PRRs is beyond the scope of this review, and has been reviewed elsewhere ,12. BrieIntracellular signaling by TLRs is initiated by ligand-induced dimerization and/or ligand-induced conformational changes of the Toll-Interleukin-1 receptor homology region (TIR) domains located within the cytoplasmic tail of the TLRs ,25. ThesThe retrovirus family has been present in vertebrate species for at least 450 million years  and retrThe past 15 years have seen dramatic advances in our understanding of how retroviruses interact with host innate sensing pathways. For retroviruses, however, the problem of pathogen sensing is uniquely complicated, due the presence of thousands of retroviral sequences within the human genome. Although human endogenous retroviruses (ERVs) are not actively replicating, many ERVs carry out parts of the retroviral replication cycle and can generate potential intracellular ligands for retroviral PRRs . This crSeveral genes have been identified as encoding potential PRRs for retroviruses, and the overall diversity of retrovirus PRRs has been reviewed elsewhere ,40,41. TOver the past decade, considerable evidence has accumulated that TLRs play a key role in the immune response to retroviruses. This evidence has come from experiments using a variety of approaches and model systems–biochemical studies of TLR binding, reverse genetic approaches to manipulate TLR expression in cell culture models of infection, and from the analysis of infections in TLR knockout mice. More recently, the analysis of genetic variants in TLRs in humans has provided intriguing clues about the possible roles of TLRs in clinical HIV infection. When considering the evidence for the activity of TLRs in anti-retroviral responses, it is important to distinguish between direct and indirect roles for TLRs in infection. A direct role would consist of cell-intrinsic TLR engagement by viral ligands in a cell type naturally infected by the virus, during the course of an infection cycle. However, cells that are not fully permissive for retroviral infection can still participate in TLR-dependent immunity, and viral entry or internalization is likely sufficient to trigger TLR-dependent immunity, resulting in the release of cytokines or cell-mediated responses that have an indirect or “bystander’ effect on virus infection . DefectiTLR2 forms heterodimeric complexes with TLR1 and TLR6 to detect bacterial lipoproteins . NeverthTLR3 is a nucleic acid sensing PRR that is triggered by binding to dsRNA . Some viTLR4 localizes to the plasma membrane and senses the presence of lipopolysaccharide (LPS), an outer wall component of Gram-negative bacteria. As such, it is unclear if it can act as a direct sensor of retroviral particles. Nevertheless, a handful of studies have indicated that viral components may have intrinsic TLR4 stimulating ability. Two studies demonstrated that the HIV transcription factor Tat could trigger TLR4 and induce IDO-1, TNFα and IL-10 expression in monocytes via an NF-κB dependent pathway ,61. HIV The majority of natural retroviral infections are initiated at mucosal surfaces, at which commensal bacteria could regulate infection through TLR-dependent pathways. Important evidence to support this hypothesis comes from a study demonstrating that mucosal transmission of mouse mammary tumor virus (MMTV) depended on LPS binding to virions, leading to TLR4-dependent IL-6 and IL-10 secretion . RemarkaTLR7 binds and responds to ssRNA within endosomal compartments and, given that retroviruses have ssRNA genomes, the role of TLR7 in retroviral infection has received considerable attention. In particular, TLR7 responds to uridine-rich ssRNA sequences , and botIt is important to exercise caution in interpreting studies using isolated TLR ligands from viral genomes, since these molecules may exhibit different properties than intact genomes. Indeed, compared to other RNA viruses such as influenza, the HIV genome has a relatively low abundance of polyuridine tracts, raising the possibility that HIV has been selected for a genome that has a low intrinsic ability to activate TLR7. Consistent with this notion, studies have found that HIV particles are a comparatively poor inducer of cell intrinsic innate immunity   or with the gammaretrovirus Friend virus (FV) have demonstrated that these mice exhibit a profound deficit in their ability to generate anti-retroviral antibodies, highlighting an important connection between innate signaling and the adaptive immune response ,81. For Some evidence from mice also indicates that retroviruses can co-opt TLR signaling to enhance viral replication. Innate-like B1 cells represent a key early target for Friend murine leukemia virus (FrMLV), and mice that are deficient in B1 cells exhibit reduced susceptibility to FrMLV infection . RemarkaRecently, genetic studies in humans have also pointed to a role for TLR7 in HIV infection. A polymorphism in TLR7 (rs179008) is associated with increased viral loads and altered CD4 T cell counts during infection ,86 and iDespite mediating an overlapping functional role with TLR7 (ssRNA sensing), TLR8 has received somewhat less attention. Nevertheless, a handful of studies point to an important role for TLR8 in the innate response to HIV infection. Meas et al. showed that endosomal HIV can stimulate cytokine secretion from CD4 T cells in a TLR8-dependent manner , while BTLR9 senses the presence of unmethylated CpG dsDNA and localizes to the endosome . As suchTLR10 represents an unusual member of the TLR family. Like TLR8, no functional murine homolog exists, so evidence for its role in vivo is limited. TLR10 is not expressed in T cells, but it is expressed on B cells, monocytes, and neutrophils. Also, no natural ligand for TLR10 is yet known. Interestingly, antibody-mediated engagement/activation of TLR10 suppresses pro-inflammatory cytokine secretion, suggesting an anti-inflammatory role . HoweverUntreated HIV infection is characterized by profound perturbations in the host immune system, leading to severe immune deficiency and increased susceptibility to opportunistic infections . Loss ofThe impact of increased TLR expression on HIV infection is unknown. Increased expression may sensitize cells to TLR ligands and boost activation of both innate and adaptive immune cells, leading to improved anti-viral immunity. On the other hand, overactivation of TLRs might contribute to non-productive immune activation, or help create a pool of highly activated CD4 T cells for HIV to infect. Some insights regarding this question have come from non-human primate studies. Analysis of SIV-infected Rhesus macaques found that rapidly progressing infection was associated with elevated TLR expression in CD8 T cells . FurtherA number of studies have also examined the responsiveness of different immune subsets to exogenous TLR agonists in samples from HIV infected individuals. Many of these studies are difficult to compare, since they use a wide variety of TLR agonists at differing concentrations. Also, it is likely that different results could be obtained, depending on whether the studies examined isolated CD4 cells or heterogeneous cell mixtures such as PBMCs. Results may also differ depending on the stage of infection and on whether the individual is receiving ART or not. Overall, studies examining TLR signaling in the context of untreated HIV infection have indicated mixed results, with some studies reporting increased TLR sensitivity, while others reporting decreased responsiveness. Increased TLR sensitivity could potentially be explained by increased expression of TLRs themselves, or of intermediate signaling proteins during HIV infection. For example, in addition to increased TLR expression, Lester et al. observed increased responsiveness to exogenous TLR ligands in samples from individuals with viremic HIV infection . SimilarBy contrast, studies of whole blood from HIV infected individuals on ART demonstrated reduced IFNα and IL-12 secretion in response to TLR7/8 agonists but that responsiveness was restored during treatment interruption ,114. DhaThe primary function of the innate immune system is to provide rapid upregulation of genes that inhibit microbial replication. As such, it is of considerable interest whether agonists for TLRs have antiviral activity that might be therapeutically beneficial during retroviral infection. In addition to the rapid expression of antiviral genes such as interferon-stimulated genes (ISGs), activating the innate immune system through TLRs could promote B or T cell responses to HIV, thereby enhancing adaptive immune-mediated clearance. Counterbalancing this hypothesis, retroviruses, including HIV, often exhibit enhanced replication in an inflammatory environment , and actBy contrast, studies using more complex experimental systems have highlighted ways in which TLR stimulation could potentially promote viral replication or transmission. Dendritic cells (DCs) can enhance HIV infection of CD4 T cells through capture and internalization of HIV at mucosal surfaces via DC-SIGN, and subsequent formation of a transmission synapse with the T cell–known as “trans-enhancement” of HIV Infection ,128. Stu6 CD4 T cells), making their direct observation and analysis difficult, yet, sporadic reactivation of these cells reseeds infection, leading to rebound during treatment interruption [Despite the success of anti-retroviral therapy in reducing viral loads and restoring lifespan in people infected with HIV, treatment interruption leads to viral rebound within weeks ,133. Thurruption ,136. As rruption . The clerruption ,139. Givrruption . FurtherSeveral reports have examined the potential of TLR agonists to serve as both LRAs and immune boosting agents by ex vivo stimulation of samples from patients on ART. Most TLRs are not expressed at physiological levels in CD4 T cells, and as such, the most encouraging results from this approach have come from stimulation of mixed cultures such as PBMCs, suggesting that indirect mechanisms likely play a key role in the LRA activity of TLR agonists. Novis et al. screened a panel of TLR agonists for latency reversing activity and identified Pam3CSK4 (TLR1/TLR2 agonist) as being able to increase HIV transcription and p24 expression in a primary cell latency model . Further3 copies of vRNA per mL of plasma, after four doses [Some recent studies have begun to examine the potential of TLR agonists as curative tools for SIV infection in non-human primates. Notably, Lim et al. found that the TLR7 agonists GS986 and GS9620 were able to induce “blips” of viremia up to 10ur doses . This stur doses . Howeverur doses . A limitur doses . Howeverur doses . Howeverur doses . Overallur doses . AlthougSince the discovery of mammalian TLRs 24 years ago, a substantial body of evidence has accumulated that demonstrates that this family of PRRs plays key roles in triggering and shaping the immune response to retroviral infection. These studies have revealed a complex network of interactions and outcomes that can inhibit and, in some cases, enhance infection. TLRs regulate both innate and adaptive immune responses to retroviral infection, and it is likely that both viral and non-viral ligands engage TLRs during infection. Although only a small number of TLRs have been confirmed to directly regulate immunity to retroviruses by analysis of infection in TLR-deficient mice, a combination of biochemical studies, in vitro manipulation, and genetic studies of TLR variants in humans, suggest that a broader set of TLRs contribute to retroviral immunity and pathogenesis. A critical question that remains is whether TLR sensing of HIV during clinical infection is suboptimal for host immunity, either due to the low intrinsic TLR-agonist properties of the virus, or due to viral interference with TLR activity or signaling. If this hypothesis is correct, then this raises the possibility that TLRs could be targeted to further enhance immunity to HIV, and/or that disruption of TLR function by infection contributes to HIV-induced immune dysfunction. Although the limited attempts so far to pursue pharmacological targeting of TLRs in HIV infection have produced mixed results, a more thorough effort to test the potential of TLRs for HIV therapy will be required. A deeper understanding of TLR-retrovirus interactions will likely lead to improved strategies to modulate TLR activity in vivo and improve clearance of HIV. In particular, determining the precise molecular mechanisms by which genetic variants of TLRs in humans impact the outcome of HIV infection or regulate HIV transmission will be of the highest importance. Due to the technical challenges of functional studies for genetic variants in humans, the application of genetic tools to small animal models of HIV infection, such as humanized mice (huMice) may permit a more mechanistic understanding of the role of TLRs in HIV infection. The mechanisms of TLR dysfunction during HIV infection will also need to be elucidated before tools to reverse this effect can be developed. Furthermore, optimal regimens of TLR agonists will need to be identified and fully tested for therapeutic benefit during active and ART-treated HIV infection. Cross-talk and interactions between TLRs and other innate retrovirus sensing pathways will also need to be examined. In summary, although TLRs are now clearly established as key regulators of the anti-retroviral immune response, much remains to be learned about how TLRs impact diverse aspects of retroviral infection and the host immune response.


The spread of infection from reservoir host populations is a key mechanism for disease emergence and extinction risk and is a management concern for salmon aquaculture and fisheries. Using a quantitative environmental DNA methodology, we assessed pathogen environmental DNA in relation to salmon farms in coastal British Columbia, Canada, by testing for 39 species of salmon pathogens  in 134 marine environmental samples at 58 salmon farm sites (both active and inactive) over 3 years. Environmental DNA from 22 pathogen species was detected 496 times and species varied in their occurrence among years and sites, likely reflecting variation in environmental factors, other native host species, and strength of association with domesticated Atlantic salmon. Overall, we found that the probability of detecting pathogen environmental DNA (eDNA) was 2.72  times higher at active versus inactive salmon farm sites and 1.76  times higher per standard deviation increase in domesticated Atlantic salmon eDNA concentration at a site. If the distribution of pathogen eDNA accurately reflects the distribution of viable pathogens, our findings suggest that salmon farms serve as a potential reservoir for a number of infectious agents; thereby elevating the risk of exposure for wild salmon and other fish species that share the marine environment. TransmiLepioptheirus salmonis and Caligus spp.), from domesticated salmon can elevate infection rates of wild juvenile salmon [Oncorhynchus kisutch), Chinook (Oncorhynchus tshawytscha), and sockeye (Oncorhynchus nerka) as well as wild populations of Atlantic salmon and sea trout in other regions where salmonid aquaculture encroaches on wild habitat [Tracking the dynamics of multi-host parasite transmission has been a key challenge for marine aquaculture where domesticated fish may function as a reservoir host for parasites in coastal environments ,9. In ree salmon ,13 and ie salmon ,15 as wee salmon –18. Despe salmon –21, there salmon ,23). Pat habitat –28. The Salmo salar) via their shared marine environment. Salmonids can also contract pathogen infections from wild non-salmonid hosts. Our study area contains 58 salmon farm tenures, each of which can be stocked with over one million domesticated Atlantic salmon that spend approximately 18 months in marine net pens before being harvested , Canada. In this system, transmission among salmonids can occur between five species of wild Pacific salmon and net-pen farmed Atlantic salmon We collected samples of seawater at active and inactive salmon farm locations between Vancouver Island and the BC mainland over three years To isolate the community of cellular microbes and viruses in seawater collections, 10–12 l samples were filtered in the field immediately after collection and the viral fraction was stabilized via addition of an iron flocculant. Subsequently, in the laboratory, nucleic acids were extracted and DNA (or complementary DNA (cDNA) synthesized from RNA viruses) was analysed via quantitative polymerase chain reaction (PCR) targeting 39 species of pathogens as well as eDNA of Atlantic salmon .(c)For our statistical analyses, we included data from only those pathogen species that were encountered at least once in a survey . We modelled the presence of pathogen DNA as a binary variable (present or absent) per site using a generalized linear mixed-effects model (GLMM) with a binomial error distribution, that included fixed effects for farm activity (active versus empty) or Atlantic salmon eDNA (continuous), and random effects for site (intercept) and species (intercept and slope). The model was fitted for all sampling years combined by adding a fixed effect of sampling year on the intercept. To quantify the Atlantic salmon eDNA covariate, we calculated the inverse of Atlantic salmon eDNA cycle threshold (Ct) values before centring values on zero and dividing by their standard deviation. To evaluate whether small variations in water volumes influenced the results, the volumes processed were also included as a covariate for both pathogen eDNA detection, and Atlantic salmon eDNA levels. We fitted the GLMMs using the ‘glmmTMB' package in R ,30 and wWe quantified the strength and direction of the association between pathogen eDNA detections and salmon farm status (active versus inactive) and relative Atlantic salmon eDNA concentration based on the odds ratios derived from the GLMM model fits. In both cases, an odds ratio equal to one suggests no association; whereas, an odds ratio less than one indicates a negative association between the predictor and pathogen detections and greater than one indicates a positive association. For binary predictors , odds ratios represent the proportional change in the likelihood of detecting a pathogen associated with one level of the predictor relative to the alternative level of the predictor. For continuous predictor variables , odds ratios represent the proportional change in the likelihood of detecting a pathogen per standard-deviation increase in Atlantic salmon eDNA concentration.3.(a)Ichthyophonus hoferi) was only assessed in 2016. The majority of virus positive detections were observed in the cell-associated (greater than 0.22 µm) sample fraction; however, three viruses including cutthroat trout virus (CTv-2), Atlantic salmon Calicivirus (ASCV), and viral encephalopathy and retinopathy (VER) exhibited the opposite pattern; whereby, they were more commonly detected in the sub-cellular (less than 0.22 µm) sample fraction .Over the three years of surveys, we detected eDNA from 22 of the 39 pathogen species screened in our samples  times higher at active salmon farms relative to inactive sites and 1.76  times higher per standard deviation increase in Atlantic salmon eDNA variation, respectively , Piscirickettsia salmonis, and Erythrocytic necrosis virus, were the most commonly detected  risk of, nearby host populations ,3. Our sFarmed salmon have previously been shown to function as a reservoir host for ectoparasitic sea lice, elevating infection risk in the marine environment over a radius of approximately 15 km per farm . Our resWe found that the probability of detecting pathogens was on average 2.72 times greater in the environment near active salmon farms when compared with control (inactive) sites, with consistent estimation accuracy among years but lower precision in 2017 when the sample size was smaller . These results also indicate that the detection of pathogens at a site increased with the relative concentration of Atlantic salmon eDNA measured at that site . If pathogen eDNA is a reliable indicator of the presence of viable pathogens, these results would suggest there is an increased infection risk for wild salmon at locations of active salmon farms. However, pathogens vary in their infectivity and progression to cause disease in wild salmon depending on species, host condition, and environmental factors; therefore, further studies will be required to quantify the potential impacts of this environmental pathogen exposure on wild populations.We found that Atlantic salmon eDNA was a better predictor of pathogen presence than farm status  are one of the most commonly reported species of bycatch on salmon farms, and are competent hosts for a number of pathogens that infect farmed and wild salmon [D. lepeophtherii), which appear to exhibit relatively strong associations with active salmon farms that are not supported by Atlantic salmon eDNA model estimates ; [Wild fish species also likely contribute to the distribution of pathogen eDNA in the environment because many miocroparasites are generalists. For example, Erythrocytic necrosis virus (ENv) and 24.004); ).Moritella viscosa is capable of infecting diverse fishes at temperatures below 8°C [M. viscosa eDNA were higher in 2016 spring surveys , the sign of estimates differed between the activity status of a farm and Atlantic salmon eDNA , bacterial , and viral pathogens , Atlantic salmon calicivirus, salmon pescarenavirus (SPAv), Piscine orthoreovirus (PRv-1)) were detected at three or fewer sites in a given season , or empty; which ignores the history of the site and age of the fish. We speculate that this may have decreased observable differences between active and inactive sites, as some inactive sites were recently fallowed and active sites with recently stocked young farmed fish may not have yet accumulated some microparasitic infections. Many pathogens are capable of persisting in the environment and on farm equipment for extended periods in the absence of their hosts , and thea priori expectations, given the suite of factors, both natural and methodological, which can influence the distribution and subsequent recovery of genetic material in environmental samples [Our results relied on qPCR detection of gene fragments to explore variation in the abundances of hosts and pathogens. It is likely that a fraction of the genetic material that we detected in seawater samples was associated with non-viable pathogens; although, we suspect that such variation should be spatially correlated with the abundance of viable organisms for a given pathogen species. The environmental stability of pathogens, the efficacy of nucleic acid recovery and purification, and the relative instability of RNA compared to DNA could all contribute to detection biases among pathogens. For example, we collected samples near the surface, which might reduce the chance of detecting viruses that are more sensitive to UV degradation. We focused our analysis of the directly filtered cellular (greater than 0.22 µm) fraction on DNA for bacterial and eukaryotic agents, and our analysis of the chemically flocculated ‘free virus' (less than 0.22 µm) sample fraction on viruses (RNA and DNA). In both cases, we did not enzymatically remove RNA or DNA. Hence, for the free virus fraction, cDNA was synthesized from a portion of the RNA extract, and the remainder was used to assess DNA viruses. For 2017 and 2018 samples, we included cell-associated RNA viruses in our analysis by synthesizing cDNA from both the cellular (greater than 0.22 µm) and subcellular (less than 0.22 µm) sample fractions, and found that most viral detections were present in the cell-associated fraction . Without explicitly evaluating these and other potential sources of pathogen detection bias, we cannot conclude that species which we failed to observe were not present. This uncertainty is inherent in many eDNA studies when eDNA distributions cannot be evaluated against  samples ,57. Alth−1, it is also possible that faecal matter was not well represented in samples at 2 m depth [One pathogen, PRv-1, has been reported at high prevalence in farmed Atlantic and Pacific salmon populations , with g m depth . However m depth , their cet al. [D. lepeophtherii as the most prevalent agent in farmed Atlantic salmon, with 88% of fish testing positive for this agent during their 2011–2013 surveys BC surveys. This is consistent with our assessment of D. lepeophtherii. This species was the second most frequently detected pathogen in our surveys (detected at 63% of sampled sites) and the estimated species effect from the multi-year model indicated a positive correlation with active salmon farms  reported in [Despite the fact that we did not have paired pathogen prevalence data in farmed fish for comparison, we can compare our results to a previous assessment  of pathoet al.  identifion farms . Additio  in Atlantic salmon farms in  were notacterium , Flavobaerved in  was alsoalmon in  but not almon in ; due to almon in .et al. [The shedding of pathogens from infected hosts depends upon the host tissue in which pathogens localize and often also on the progression of infection, both of which may have contributed to the differences between pathogen prevalence from  and thoset al.  (collectThe prevalence of infection and disease depends not only on the presence of the parasite but also on characteristics of the host individuals, community, and the environment. A number of laboratory studies have explored the relationship between host infection and pathogen release, as well as how pathogen exposure translates to host infection ,68. HoweOur study adds to a growing body of empirical work testing predictions from community epidemiology to better understand domestic-wildlife interactions ,2,72,73.


Clarke and Patani review the involvement of microglia in ALS, focusing specifically on evidence for spatiotemporally regulated neurotoxic and neuroprotective roles. Understanding how microglia contribute to the ‘cellular phase’ of ALS promises to advance the development of therapies with a mechanistic rationale. Microglia are the primary immune cells of the CNS, carrying out key homeostatic roles and undergoing context-dependent and temporally regulated changes in response to injury and neurodegenerative diseases. Microglia have been implicated in playing a role in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by extensive motor neuron loss leading to paralysis and premature death. However, as the pathomechansims of ALS are increasingly recognized to involve a multitude of different cell types, it has been difficult to delineate the specific contribution of microglia to disease. Here, we review the literature of microglial involvement in ALS and discuss the evidence for the neurotoxic and neuroprotective pathways that have been attributed to microglia in this disease. We also discuss accumulating evidence for spatiotemporal regulation of microglial activation in this context. A deeper understanding of the role of microglia in the ‘cellular phase’ of ALS is crucial in the development of mechanistically rationalized therapies. C9orf72, SOD1, TARDBP and FUS are the most common genetic forms, accounting for more than half of familial ALS cases. Although motor neurons are primarily affected in ALS, non-neuronal cells play a key role in disease progression  is a rapidly progressing neurodegenerative disease involving the degeneration of both upper and lower motor neurons in the motor cortex, brainstem and spinal cord   and Chil3 (Ym1) and decreased genes associated with neurotoxicity including Tnfa, Ifng and Il1b , a molecule that is able to cause motor neuron death in vitro (Inducible nitric oxide synthase (iNOS) is an enzyme that is upregulated in microglia following activation of NF-κB and is increased in the spinal cord of symptomatic mSOD1 mice  have been implicated in microglial-mediated motor neuron death in ALS. NOX2 is a ROS-producing catalytic subunit of the NADPH oxidase that has been implicated in neurotoxicity in ALS. Reduced NOX2 activity has been correlated with increased survival in ALS patients , the UK Medical Research Council (FC010110), and the WellcomeTrust (FC010110). R.P. holds an MRC Senior Clinical Fellowship [MR/S006591/1].The authors report no competing interests.


Recent years have witnessed much progress in medical device manufacturing and the needs of the medical industry urges modern nanomaterials science to develop novel approaches for improving the properties of existing biomaterials. One of the ways to enhance the material properties is their nanostructuring by using severe plastic deformation (SPD) techniques. For medical devices, such properties include increased strength and fatigue life, and this determines nanostructured Ti and Ti alloys to be an excellent choice for the engineering of implants with improved design for orthopedics and dentistry. Various reported studies conducted in this field enable the fabrication of medical devices with enhanced functionality. This paper reviews recent development in the field of nanostructured Ti-based materials and provides examples of the use of ultra-fine grained Ti alloys in medicine.  Presently, Ti and its alloys represent the top choice when a combination of high strength, light weight, and affordable cost are required, such as in the area of medical device manufacturing. However, the clinical demands for implantable medical devices are growing rapidly, and nowadays new Ti alloys are being investigated in terms of their chemical composition optimization, manufacturing processes and modification of surface to meet the appropriate medical standards and comply with regulation ,2. One pDifferent SPD techniques are applied to refine grains in metallic materials to below micrometer range or even to the nanosized range. SPD techniques are also efficient for the formation of nanoclusters and nanoprecipitates of secondary phases, enhancing the mechanical and functional properties of the materials ,5. A whoNanostructuring of metallic materials increases material strength due to work hardening and grain refinement ,14, consRecently, materials scientists have been exploring possibilities of improved interaction of nanostructured materials with body tissues, for instance bones. In this respect, surface modifications of bulk nanomaterials demonstrate encouraging results ,18,20,21The first studies devoted to Ti-based materials potentially applicable in medicine were applied to commercial purity titanium (CP Ti) due to its high biocompatibility with living tissues . UnparalThe first results on nanostructured CP Ti Grade 4  titanium alloys such as Ti-6Al-4V and Ti-6Al-7Nb continue to be the most important metallic materials in the dental and orthopedic fields due to their excellent mechanical properties and satisfactory biocompatibility ,22,30,31Several recent studies reported improved mechanical and functional properties of nanostructured titanium alloys.Microstructure and mechanical properties of Ti-6Al-4V ELI  prepared by SPD are reported in ,32,33. RTransmission electron microscopy (TEM) studies showed that SPD leads to a complex UFG structure containing refined grains and subgrains with a mean size of about 300 nm.Stress–strain curves for the initial coarse-grained and UFG material shown in In accordance with , enhanceFine tuning of mechanical properties by annealing after the SPD processing is limited mainly by grain growth occurring at elevated temperatures. Thermal stability of UFG structure of commercially pure Ti follows classical grain growth depending on temperature via Arrhenius equation  and limi7 cycles in comparison to 600 MPa in the initial coarse-grained condition  after SPD processing and subsequent annealing at 500 °C; resulted in an enhancement of fatigue limit to 740 MPa after 10ondition  32]. . 7 cycleThe fatigue limit of the Ti-6Al-4V alloy in UFG condition reported in  tested bAchieved results show that high strength can be achieved in UFG Ti-6Al-4V ELI alloy by processing by ECAP and subsequent thermo-mechanical treatment. Selection of SPD regimes and adjustment of processing parameters of SPD processing such as temperature, strain rate and strain allow us to manipulate the grain boundary structure and phase morphology in the two-phase UFG alloy. As the result, the best combination of strength and ductility can be achieved along with the improved fatigue endurance limit. Enhancement of strength and ductility of the biomedical Ti-6Al-7Nb alloy was reported in another comprehensive study . In compConsidering that vanadium and partly also aluminum are rather toxic elements and, simultaneously, that reducing of the Young’s modulus is required for avoiding so-called stress-shielding , the devGiven the above mentioned requirements, the interest is drawn to titanium alloys containing high content of the β phase, because this phase is characterized by lower Young’s modulus in the range of 55–90 GPa, and thus exhibit lower stress shielding ,41,42,43Advancements in the areas of orthopedics and dentistry called for new strategies for development of new generation of β-Ti alloys with reduced Young’s modulus and high strength, which would be more suitable for such applications. Recently, SPD processing has been proposed to fabricate nanocrystalline β-Ti alloys with high strength, low modulus of elasticity and excellent biocompatibility ,49,50,51Microstructural refinement in β-Ti alloys can be also enhanced by multiple twinning and/or martensitic transformation β → α’’ . The nanNiTi alloys exhibit unique mechanical behavior—shape memory effect (SME) and superelasticity, which arise from a transformation between martensite and austenite phases ,62. NiTiDuring the past two decades, there has been interest in the application of SPD methods to NiTi alloys because the formation of nanocrystalline and UFG structures allows enhancing mechanical and functional properties in comparison to coarse grained materials ,64. HPT processing of NiTi alloys leads to a transformation from crystalline to amorphous phase. Microstructural changes in deformed NiTi during thermal treatment are of key interest as they are responsible for the shape memory effect ,66. DuriEqual channel angular pressing is another SPD processing technique applied for producing uniform UFG structure in bulk NiTi alloys. The ECAP processing of NiTi at 400–450 °C results in formation of UFG structure with grain size of about 200 nm . rmax increases from 6% (in CG state) to 9% after ECAP and the maximum recovery stress σrmax reaches 1120 MPa, which is twice more than the level of CG alloys (about 500 MPa) [UFG structure formation leads to significant improvement of mechanical and functional properties of NiTi-based alloys ,68,69,70500 MPa) .The high-strength NC and UFG NiTi alloys with improved functional characteristics are very promising for medical applications in particular for manufacturing of stents, embolic protection filters, guide wires, and other peripheral vascular devices see . Enhanced mechanical properties of nanostructured metals allow development of medical implants with better design, for instance with a more subtle design which is less harmful for human body .Application of stronger nanostructured CP Ti instead of common CG Ti, allows for altering the design of devices. Recently, detailed computations were conducted to analyze the possible geometries of miniplates for maxillofacial surgery manufactured from nanostructured Ti .CP Ti miniplate specified by ASTM F 67, was considered by Conmet Company  as the benchmark for redesigning the product dimensions of mini-plates manufactured from nanostructured CP Ti. The mechanical properties in a cross-section of a newly designed plate were calculated with the use of estimates of the fatigue performance limit for coarse-grained Grade 4 CP Ti and nanostructured Grade 4 CP Ti. In practical use, the mini-plates are subjected to bending loads, therefore bending strength of mini-plates from conventional and nanostructured CP Ti was compared. The result indicates that the plate from nanostructured Ti has significantly improved bending strength and therefore, it is clearly advantageous over the standard device currently manufactured from CG Ti.Recently, three-dimensional finite element models (FEM) were developed using CAE software  and then imported into ANSYS Workbench 18.2   for geomThe following procedure was used to assess possible ways to miniaturize the implants. The device with a standard geometry was assumed to be made from the conventional coarse-grained CP Ti. The model was designed in a way to obtain nearly critical stress state both in terms of static and fatigue failure. Afterwards, the same loading was applied to a model with reduced dimensions but with the properties of nano CP Ti.A one-stage dental implant with generic geometry was considered in the study. The shape of the implant is similar to the implant geometry produced from nano CP Ti by company Timplant s.r.o.  . Figure The applied loading scheme was inspired by the testing procedures used in the ISO 14801 standard. The performed calculations revealed that application of the nanoTi allows reduction of the diameter of implant by at least 10%, while 20% diameter reduction leads to an unacceptable decrease of the device’s fatigue strength. Maximal principal stress zone for the implant with the diameter reduced by 10% loaded with a 67.75 N force is shown in Recently, manufacturing and successful testing of several medical implants fabricated from nanostructured Ti have been considered in detail . AnotherAnother interesting example of the innovative application is the removable clipping device for blood vessels, tubular structures, and soft tissues fabricated from UFG NiTi with enhanced shape-memory effect and designed for bleeding control during laparoscopic operations. This device has been created and tested in collaboration between Ufa State Aviation Technical University (USATU) and National University of Science and Technology “MISIS” .The conducted tests demonstrated that the removable clipping devices produced from UFG NiTi alloy obtain several advantages when compared to the standard counterpart. Higher completely recoverable strain allows for a more convenient shape of the clipping device to be used for laparoscopy and manipulation; it also helps to reduce the diameter of a laparoscope tube, i.e., to create more comfortable surgery conditions for a doctor and a patient. Due to higher deformation with a reversible shape memory effect, it is possible to improve non-invasiveness when removing the clipping device. Increased maximum recovery stress provides high force that develops at triggering the clipping device, high reliability of tissue crimping and fixation, and also makes it possible to reduce the weight of the clipping device.Recent studies have proven that nanostructuring of titanium materials by means of severe plastic deformation (SPD) techniques achieving grain refinement, increase of dislocation density, dissolution, and formation of secondary phase precipitations allows for considerable improvement of the strength and fatigue properties. In the present paper the advantages of nanostructuring were demonstrated for CP Ti, Ti alloys including new β-Ti alloys as well as the NiTi alloy with shape memory effect. The approaches to computer design of a number of miniaturized medical implants made from high-strength nanomaterials have been suggested. In addition, the paper includes the examples of manufacturing and tests of selected advanced medical devices for traumatology and surgery from Ti nanobiomaterials. Taking into account the results of recent studies on surface modification, including chemical etching of nanometals and deposition of bioactive coatings, it is assumed that the developments of Ti-based nanomaterials opens new possibilities for advanced medical implants and devices with improved design and functionality.


Background: ISGylation is the conjugation of ISG15 with target proteins. ISGylation occurs through an enzymatic cascade, which is similar to that of ubiquitination. Through ISGylation, ISG15 can bind to proteins involved in cell proliferation and differentiation, thus promoting genesis and progression of malignancies. The present study aims to investigate expression of genes involved in ISGylation and ubiquitination in patients with hepatocellular carcinoma and to correlate gene expression with clinical laboratory parameters of these patients.Methods: mRNA expression of genes encoding enzymes involved in the ISGylation process  was evaluated by quantitative real-time PCR in 38 pairs of tumour and adjacent non-tumour tissues from patients with hepatocellular carcinoma and correlated with distinct clinical laboratory parameters.Results: Relative mRNA expression of EFP, HERC5, UBA1 and USP18 was significantly higher in tumour tissues compared to adjacent non-tumour tissues . The correlation pattern of mRNA expression between genes in the tumours differed from the pattern in adjacent non-tumour tissues. Relative expression of EFP, HERC5 and UBA1 in adjacent non-tumour tissues was positively associated with direct bilirubin levels  and relative expression of USP18 in adjacent non-tumour tissues correlated negatively with ALT levels .Conclusions: EFP, HERC5, UBA1, and USP18 genes are upregulated in tumour tissues of patients with HCC and, thus, may be associated with the pathogenesis of hepatocellular carcinoma. Hepatitis B virus (HBV) infection is responsible for up to 90% of hepatocellular carcinoma (HCC) cases, especially in regions with a high prevalence of HBV infection including Vietnam In response to HBV infection, immune cells secrete interferon (IFN) and other inflammatory cytokines. Subsequently, cellular signalling pathways are activated and IFN-stimulated genes (ISGs) are induced ISGylation has multiple functions, including inhibition of release of exosomes, downregulation of protein translation and regulation of distinct protein functions EFP, HERC5 and USP18 genes contribute to tumourigenesis through ISG15/ISGylation or other mechanisms. In addition, the exact role of ISG15 and other key enzymes involved in ISGylation in the formation and development of HBV-related HCC is still unclear. In the present study, we aim to investigate the expression profiles of genes encoding enzymes involved in the ISGylation process  in patients with HCC and their association with clinical outcomes.It remains, however, unclear so far whether the 0C until further use. Patients were further grouped as Child-A, Child-B or Child-C according to Child-Pugh scores The study included 38 patients with HCC who underwent surgery at the 108 Military Central Hospital in Hanoi, Vietnam. The patients were diagnosed according to the guidelines of the American Association for the Study of Liver Diseases (AASLD) for management of HCC All details of the study were explained to the participants and informed written consent was obtained before taking samples. All clinical procedures and experiments were performed in accordance with applying guidelines and regulations. The study was approved by the Institutional Review Board of the 108 Central Hospital, Hanoi, Vietnam.GAPDH  used as reference gene. Sequences of primers used in this study are presented in Table ® 480 real-time PCR system . Relative expression of target genes was normalized to the expression of GAPDH based on the ΔCt method.Total RNA was extracted from the 38 tumour and adjacent non-tumour tissue pairs by Trizol reagent  according to the protocol recommended by the manufacturer. Extracted total RNA samples were reverse transcribed into cDNA by using the QuantiTect Reverse Transcription Kit . cDNA was quantified by real-time PCR with P<0.05.Quantitative parameters including laboratory tests and relative gene expression values are given as means with standard deviation or medians with ranges where appropriate. The relative gene expression between tumour and adjacent non-tumour tissues was compared using the Wilcoxon signed ranks test. Spearman's rank correlation coefficient was used to analyze the correlation of relative gene expression between two genes or between expression of the genes with clinical parameters. The SPSS software version 22.0  was used for all statistical analyses; the significance level was set at EFP, HERC5, UBA1, UBC and USP18, which are related to ISGylation, was quantified and compared between HCC tumour and adjacent non-tumour tissues. Expression of EFP, HERC5, UBA1, and USP18 was significantly higher in HCC tumour tissues compared to the adjacent non-tumour tissues . No significant difference of UBC expression was observed between HCC tumour tissues and non-tumour tissues (P=0.91) . BCLC stages C and D did not occur among the study participants.We then examined whether gene expression was associated with progression of HCC by comparing their expression between different BCLC stages. However, expression of the genes did not differ between stage A and B HCC tissues as well as between the corresponding non-tumour tissues (P<0.0001). The strongest correlation was observed between expression of EFP and HERC5 , followed by expression of HERC5 and USP18  and of EFP and UBA1  . A similar result was observed when the correlation of HERC5 expression with that of UBA1 and USP18 was analysed  . A similar correlation was seen between UBA1 and USP18 . These results indicate that the expression pattern of the genes under study differed between HCC tumour and adjacent non-tumour tissues.We analysed the correlations of mRNA expression in both HCC tumour and non-tumour tissues. In HCC tissues, gene expression was strongly correlated with each other  Table . In non-1) Table . The corEFP, HERC5 and UBA1 in non-tumour tissues were positively correlated with direct bilirubin levels  , but positively correlated with albumin levels   , UBA1 and USP18 is significantly upregulated in HCC tumours compared to adjacent non-tumour liver tissues. In addition, the expression levels of the genes in non-tumour tissues is associated with several clinical parameters. Our results indicate that E3 ligases, UBA1 and USP18 are associated with HCC development and may possibly be considered as targets in the treatment of HCC.ISGylation plays a key role both in immunity against infections and in the formation and progression of tumours by affecting a wide range of target proteins EFP, HERC5, UBA1 and USP18 in HCC tissues and the different patterns of correlation of the genes in HCC tumour and in adjacent non-tumour tissues suggest that these genes may play a role in the pathogenesis of HCC. Studies have shown that EFP is closely associated with breast cancer EFP regulates metastasis of breast cancer through its E3 ubiquitin ligase activity rather than through estrogen signalling EFP is upregulated in breast and gastric cancers but downregulated in endometrial cancer EFP expression is associated with a poor prognosis of breast cancer EFP expression is a prognostic factor in prostate cancer EFP increases proliferation and survival of prostate cancer cells by affecting p53 signals EFP is also associated with HCC progression through degrading the metastasis-associated 1 protein (MTA-1) EFP is significantly upregulated in HCC tissue compared to non-tumour liver tissues and EFP expression in non-tumour tissues is correlated with the levels of direct bilirubin.The upregulation of in vitroHERC5 in HCC tumour tissues and a correlation of HERC5 expression with direct bilirubin levels in non-tumour tissues. Although UBA1 has been considered as a potential target gene for cancer therapy, especially for acute myeloid leukemia UBA1 expression in HCC tumour and adjacent non-tumour tissues and points towards its correlation with direct bilirubin levels.HERC5 is the dominant E3 enzyme for ISGylation and has been shown to be highly expressed in HCC tissues and USP18 is significantly upregulated in HCC tissues compared to corresponding non-tumour tissues. USP18 is an important factor in the immune response directed against HBV replication and its overexpression promotes growth of HCC cells USP18 mRNA in peripheral blood mononuclear cells (PBMCs) has been proposed as a predictive factor for monitoring treatment of HBV patients with interferon-alpha In agreement with a previous report UBC expression is upregulated in lung cancer UBC does not differ significantly between HCC tumour and non-tumour tissues, UBC expression in non-tumour tissues is correlated with AST levels, total bilirubin and albumin, indicating that UBC may not significantly contribute to the pathogenesis of HCC, but rather plays a role in liver functions. Limitations of this study apply to the rather small number of HCC patients, particularly the small number of HCC patients in BCLC stages C and D, implying the impossibility to perform association analyses of gene expression with HCC progression.EFP, HERC5, UBA1 and USP18 genes, upregulated in tumour tissues of HCC patients, is correlated with liver function parameters and, thus, may be associated with the pathogenesis of liver cancer.In conclusion, our study shows that expression of


Pain is a percept of critical importance to our daily survival. In most cases, it serves both an adaptive function by helping us respond appropriately in a potentially hostile environment and also a protective role by alerting us to tissue damage. Normally, it is evoked by the activation of peripheral nociceptive nerve endings and the subsequent relay of information to distinct cortical and sub-cortical regions, but under pathological conditions that result in chronic pain, it can become spontaneous. Given that one in three chronic pain patients do not respond to the treatments currently available, the need for more effective analgesics is evident. Two principal obstacles to the development of novel analgesic therapies are our limited understanding of how neuronal circuits that comprise these pain pathways transmit and modulate sensory information under normal circumstances and how these circuits change under pathological conditions leading to chronic pain states. In this review, we focus on the role of inhibitory interneurons in setting pain thresholds and, in particular, how disinhibition in the spinal dorsal horn can lead to aberrant sensory processing associated with chronic pain states.The online version of this article (10.1007/s13311-020-00936-0) contains supplementary material, which is available to authorized users. The dorsal horn of the spinal cord is the principal termination site of primary afferents that innervate the skin and deeper tissues of the trunk and limbs and is composed of several distinct classes of neurons. These afferent fibers engage discrete, modality-specific circuits comprised of spinal interneurons that play important roles in modulating and gating afferent input, and projection neurons that relay the processed information to higher brain centers . NocicepGABA acts as the major inhibitory neurotransmitter throughout most regions of the central nervous system, although glycinergic neurotransmission predominates in parts of the spinal cord and brainstem, and in the retina. Immunohistochemical studies in the rat and mouse show that GABA-immunoreactive (GABA-IR) cells in the spinal dorsal horn are concentrated in laminae I–III, whereas glycine-IR cells are rarely seen in laminae I and II, but are common in laminae III and IV , 19–22. All dorsal horn inhibitory interneurons are believed to express the developmental transcription factor Pax2 –34. InhiMorphological heterogeneity among dorsal horn neuron populations has been a consistent finding, from early studies using Golgi labelling –39, to mSimilar morphological populations of lamina II neurons have been described in various species, including rat , 48, 49 Given that a variety of markers commonly used to define inhibitory interneurons in the dorsal horn can also be expressed in glutamatergic interneuron populations , 12, 45,A and glycine receptors, respectively). Presynaptic inhibition is a GABA-mediated event resulting principally from the release of transmitter from axons (or presynaptic dendrites) that synapse with primary afferent terminals to act on GABAA receptors primarily [B receptors have also been implicated in this form of inhibition on both group Ia muscle afferents and Aβ cutaneous afferents [A receptors at synapses [A receptor and gephyrin colocalize extensively at synapses formed by axon terminals containing both GABA and glycine [A receptor subunits but not for gephyrin [Inhibitory interneurons in the spinal cord can generate two distinct forms of synaptic inhibition mediated through the release of GABA and/or glycine and activation of ligand-gated ion channels  receptors on the central terminals of primary afferents [At the ultrastructural level, the central terminals of both non-peptidergic C-fibers and Aδ down hair afferents display distinctive anatomical features where they form the central elements of Type I and Type II glomeruli, respectively –87. Thesfferents , 63, 89,fferents , 84, 90.fferents –95, and fferents , 69–71, A receptor antagonists, respectively) were administered via intrathecal routes in rats [The importance of spinal inhibition in somatosensory processing was demonstrated in studies where strychnine and bicuculline  a loss of GABA levels in the spinal dorsal horn remain topics of debate, other alternative mechanisms have been proposed to explain heightened excitability of dorsal horn circuits in conditions of neuropathic pain. One hypothesis implicates the downregulation of the chloride ion transporter KCC2 following peripheral nerve injury –115, broAlthough the loss of spinal inhibition has been shown to allow A-LTMR afferent input to activate pain circuits in lamina I , 122, thCre; Ai9 mouse line, no change in excitatory drive to PV cells was seen, but distinct differences in the excitability and action potential firing patterns of these interneurons were reported [Precisely how peripheral nerve injury leads to a loss of PV cell-mediated inhibition has yet to be established. There is no apparent loss of PV interneurons , 123, bureported . These fin vitro electrophysiological studies where the properties of interneuron populations were recorded under normal or chronic pain conditions [in vivo to determine [One approach in helping to develop new therapies to tackle chronic pain states is to establish the functional significance of discrete neuronal populations in the spinal dorsal horn and then determine precisely how their associated anatomical features or electrophysiological properties change under pathological conditions. Experiments involving topical application of specific neurotransmitter receptor antagonists in freely moving animals were the first to establish the importance of spinal inhibition in somatosensory processing , 98–100.nditions , 119, anetermine , 137. Gietermine , 36, 138in vivo has been instrumental in establishing their role in setting mechanical thresholds [For example, manipulating the activity of PV interneurons specifically in both naïve and nerve-injured mice resholds . By deteresholds , 60. Wheresholds –143, andresholds  and liesA receptors in primary afferents and spinal dorsal horn neurons [h subthreshold currents and are enriched in both HCN1 and HCN4 subunits [To achieve this selectively with pharmacological approaches may be challenging given the widespread distribution and heterogeneity of GABA neurons , 144, bu neurons –147. HCNsubunits , 77. It subunits . Althougsubunits , 148. ShESM 1(PDF 1224 kb)ESM 2(PDF 1224 kb)


Sickle cell anemia (SCA) is the most common inherited hemolytic anemia worldwide. Here, we performed an exploratory study to investigate the systemic oxidative stress in children and adolescents with SCA. Additionally, we evaluated the potential impact of hydroxyurea therapy on the status of oxidative stress in a case–control study from Brazil. To do so, a panel containing 9 oxidative stress markers was measured in plasma samples from a cohort of 47 SCA cases and 40 healthy children and adolescents. Among the SCA patients, 42.5% were undertaking hydroxyurea. Multidimensional analysis was employed to describe disease phenotypes. Our results demonstrated that SCA is associated with increased levels of oxidative stress markers, suggesting the existence of an unbalanced inflammatory response in peripheral blood. Subsequent analyses revealed that hydroxyurea therapy was associated with diminished oxidative imbalance in SCA patients. Our findings reinforce the idea that SCA is associated with a substantial dysregulation of oxidative responses which may be dampened by treatment with hydroxyurea. If validated by larger prospective studies, our observations argue that reduction of oxidative stress may be a main mechanism through which hydroxyurea therapy attenuates the tissue damage and can contribute to improved clinical outcomes in SCA. This disease is characterized by altered hemoglobin synthesis (sickle hemoglobin [HbS]), which leads to several pathological effects, including hemolysis, vaso-occlusive crises, progressive organic damage, and eventual early death3. Notably, such hereditary hemolytic anemia exhibits a high prevalence in Brazil, especially in the state of Bahia4. Due to the scarcity of an effective pharmacological treatment, understanding of fundamental mechanisms underlying SCA may lead to development of novel therapies and optimized patient care6.Sickle cell anemia (SCA) is the most common monogenic hemoglobinopathy disease in the world8. Notably, a large number of studies investigating oxidative stress in SCA patients have indicated that several types of reactive oxygen species (ROS) affect red blood cells (RBCs), resulting in metabolic dysfunction of these cells and alteration of their biochemical properties10. There is also evidence supporting the idea that oxidative damage mediates cytoskeleton remodeling in RBCs, leading to morphological deformities, and consequently affecting traffic through smaller capillaries. Many consequences of this process have been described, such as inflammatory endothelial injury, platelet adhesion, vaso-occlusion, nitric oxide depletion, ischemia and pain12.Numerous aspects are long proposed to influence the pathogenesis of SCA14. Among the treatment options, the use of hydroxyurea has emerged as an important pharmacological intervention16. Nevertheless, although hydroxyurea is now largely used in SCA to prevent acute vaso-occlusive crises, the specific effects of hydroxyurea therapy on the oxidative response remains uncertain. Here, we performed an exploratory case–control study in Brazilian children and adolescents using multidimensional analysis to evaluate the expression of biomarkers related to oxidative stress, measuring the global oxidative perturbation in SCA and comparing to that detected in non-SCA healthy controls. In addition, we evaluated whether hydroxyurea treatment is associated with changes in such oxidative perturbation in SCA patients.The clinical management of SCA patients is a largely explored field with several lines of investigationsAll clinical investigations were conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from each participant or legal guardian at the study enrollment. This study was approved by the Ethics Committee of the Bahiana School of Medicine and Public Health, . Access to the registry data was authorized by the boards of the participating institutions.In the present study, plasma samples were collected from 47 children and adolescents from 6 to 18 years old, diagnosed with SS Hemoglobinopathy, enrolled and followed up at the Hematology Outpatient Clinic of the Magalhães Neto Ambulatory—Professor Edgard Santos University Hospital Complex (HUPES) of the Bahia School of Medicine—UFBA and at the Foundation for Hematology and Hemotherapy of Bahia (HEMOBA). The aforementioned institutions are reference centers in the care and treatment of hematological diseases in the state of Bahia, Brazil. Inclusion criteria for sickle cell patient recruitment included HbSS diagnosis via electrophorese or high-performance liquid chromatography (HPLC), and absence of recent infectious or SCA-related acute events. The current study was a case–control analytical investigation with cases (SCA patients) and control group , performed between March 2013 and June 2015. Additional comparisons within the SCA group were performed between participants stratified based on use of hydroxyurea. Plasma sample collection was performed from June 1, 2014 to March 31, 2015. Healthy participants consisted of children and adolescents of the same age group, whose inclusion criteria included absence of sickle cell disease determined by Hb electrophoresis and/or high-performance liquid chromatography, or any other acute or chronic diseases, enrolled at the general pediatric teaching-care ambulatory of Roberto Santos General Hospital (HGRS) / Bahiana School of Medicine and Public Health (EBMS) and at the outpatient clinic of Hebiatra of the Magalhães Neto Ambulatory—HUPES Complex of the Bahia Medical School—Federal University of Bahia (UFBA).18. The following parameters were assessed in a reference laboratory: hematocrit (%), hemoglobin (g/dL), mean corpuscular volume (MCV) (fL), corpuscular hemoglobin concentration (MCHC) (pg), total leukocyte count (× 109/L), platelet count (× 109/L), reticulocyte count (%), Glutamic oxaloacetic transaminase (GOT) (U/L), glutamate-pyruvate transaminase (GPT) (U/L), direct bilirubin (DB) (mg/dL), indirect bilirubin (IB) (mg/dL), total cholesterol (mg/dL), low density lipoprotein-cholesterol (LDL-c) (mg/dL), high density lipoprotein-cholesterol (HDL-c) (mg/dL), triglycerides (mg/dL), and high sensitivity C-reactive protein (CRP) (mg/L), as previously described18. Details of the assays described below, which used to assess oxidative stress, have been formerly published19. Total oxidant status was assessed using a commercially available kit  and results are described as micro molar hydrogen peroxide equivalent per liter (μmol H2O2 Equiv./L). Total antioxidant status was measured using the Antioxidant Assay kit from Cayman Chemical , where the capacity of the antioxidants to counteract ABTS  oxidation is compared with that of Trolox, and is calculated as molar Trolox equivalents19. Lipid peroxidation in plasma was quantified using a kit from Cayman Chemical, which measures the formation of malondialdehyde (MDA)19. Levels of lactate dehydrogenase (LDH), glutathione (GSH) and of superoxide dismutase protein level (SOD) were measured using kits from Cayman Chemical following the manufacturer’s protocol. ELISA commercial kits from R&D Systems  were used to quantify concentrations of soluble CD14 (sCD14), heme oxygenase-1 (HO-1) and of vascular endothelial growth factor (VEGF).Blood samples were obtained after a fasting period of at least 8 hU test (when 2 groups were compared) or the Kruskal–Wallis test with the Dunn’s multiple-comparison (when more than 2 groups were compared) were used to evaluate continuous variables. For variables which values displayed a Gaussian distribution in the study population, Student’s t test (for 2 groups) or One-Way ANOVA with the Tukey’s ad hoc were employed to compare the groups. The Fisher’s exact test was used to compare variables displayed as frequencies (%). We used principal component analysis (PCA) to test which combination of oxidative stress related markers could distinguish individuals with or without SCA. The PCA integrated with vector analysis was chosen due to its capacity to perform dimension reduction of multiple biomarker measurements and display the overall distribution of the global data variation. This approach also allowed us to visualize the impact of each parameter inputted on the distribution of the data and individuals from the distinct clinical groups. A hierarchical cluster analysis (Ward’s method) of log10 transformed and z-score normalized data was employed to depict the overall expression profile of indicated biomarkers in the study subgroups, as described previously22. All comparisons were pre-specified and two-tailed. Statistically significant differences were defined as displaying p-values < 0.05 after Holm-Bonferroni’s adjustment for multiple comparisons. Profiles of correlation between oxidative stress parameters were examined using network analysis of the Spearman correlation matrices as previously described24. Correlations with p-value < 0.05 were included in the network visualization.Descriptive statistics were performed to characterize the study population. Continuous variables were tested for Gaussian distribution using the D’Agostino-Pearson test. For parameters which values exhibited a non-Gaussian distribution, the median values with interquartile ranges (IQR) were used as measures of central tendency and dispersion. The Mann–Whitney 26. In the present study, “healthy control” (non-SCA) was defined as the “reference” group and the average level and standard deviation of this reference group was calculated for the plasma concentrations of each plasma marker26. The DOP score of an individual marker in a given sample “s” was defined by taking the difference in concentration level in sample “s” from the average of the marker in reference group divided by the corresponding standard deviation26. Thus, the DOP score represents the number of standard deviations from the reference26.The molecular degree of perturbation was calculated using values of the markers related to oxidative stress to infer the degree of oxidative perturbation (DOP) associated with SCA and impacted by hydroxyurea therapy. This method has been used and detailed previouslyA total of 87 children and adolescents were enrolled, 47 SCA patients (HbSS confirmed), from whom 20 were undertaking hydroxyurea, and 40 healthy controls Table . ParticiU test revealed that only two parameters (MCV and direct bilirubin) were statistically significant, with higher values being detected in participants treated with hydroxyurea , cell lysis/death (LDH) and sCD14 were found in SCA patients compared to those detected in non-SCA controls Table . Next, wThe vector analysis integrated with a PCA model showed that heightened levels of total oxidant status, MDA, HO-1, LDH, SOD and GSH were more associated with SCA participants, whereas the values of total antioxidant status were associated with the healthy control group. This finding highlights a potential role of oxidative stress in the SCA pathophysiology, with a higher expression of oxidant factors being able to distinguish SCA patients from non-SCA controls.Given that results reported above indicated that SCA is hallmarked by increased oxidative stress, we further investigated the possible impact of the hydroxyurea therapy in this oxidative milieu. To do so, we compared the expression of oxidative markers between subgroups of SCA patients stratified according to use of hydroxyurea Fig. .Figure 2An unsupervised two-way hierarchical clustering analysis of the log10-transformed and z-score normalized values/concentrations of each parameter uncovered a clear distinction of oxidative profiles between the SCA and controls when all markers were examined simultaneously, revealing a lower expression of the all oxidative markers in healthy controls, except for the total antioxidant status Fig. A. This f27. We also evaluated the correlation between DOP values and hemoglobin levels or reticulocyte count, which are markes directed associated with degree of SCA disease activity   of statistical interactions between values of the oxidative stress related markers  total oxidant status, (ii) HO-1, (iii) MDA and (iv) LDH Fig. . Of noteThe network analysis highlighted that in healthy controls, LDH was the most highly connected marker, followed by GSH, SOD and HO-1 Fig. . In SCA 30. The identification of oxidative stress related biomarkers is of utmost importance to help understanding the mechanisms that drive the physiopathology and clinical outcomes in SCA32. Additionally, the paucity of approved pharmacological treatments for SCA hinders the achievement of a highly successful therapeutic intervention as well as appropriate patient care, culminating in poor outcome in carriers of this genetic condition. In the present investigation, we performed a detailed analysis of plasma biomarkers related to redox status and inflammation in SCA children and adolescents undertaking or not hydroxyurea treatment in order to better understand the nuances of the association between oxidative stress and inflammation in this disease. We also tested association between the hydroxyurea therapy and alteration of the oxidative responses in the patients evaluated.It has been long proposed that oxidative stress plays a critical role in the pathophysiology of SCA35. The results presented here expand the disease repertoire linked to oxidative stress as they demonstrated a marked increased oxidative milieu in patients with SCA, which is characterized by augmented total oxidant status as well as increased lipid peroxidation  and cell lysis/necrosis (read by LDH levels). In addition, higher concentrations of sCD14 and HO-1 were observed in SCA patients. All of those markers have been previously reported individually, but not together, to be associated with SCA and vascular injury37. Importantly, the hierarchical cluster analysis inputting values of all the biomarkers measured here indicated that the homeostasis is so dramatically altered by SCA that the combination of such markers could accurately distinguish the patients from the non-SCA control group. The same analysis failed to reliably discriminate the groups of SCA patients undertaking or not hydroxyurea. These findings demonstrate that hyperexpression of oxidative factors in response to SCA hallmark this condition, regardless of the hydroxyurea treatment. This result increments the current knowledge in the field, and identifies parameters involved in the pathophysiology of SCA that may be targeted in future studies exploring individualized therapeutic strategies, as previously suggested10.Oxidative stress is a direct consequence of the imbalance between production of free radicals versus total antioxidant factors. It is already known that excessive oxidative responses can culminate in tissue damage and consequently poor outcomes in several pathological conditions. This process has been largely described by our group in a large number of diseases38, vessel occlusive syndrome39. The same pathological process results in complications related to unfavorable outcomes, for example endothelial dysfunction40, chronic inflammation41, acute chest syndrome42 and others45. Despite the recent advances in studies depicting pathophysiologic aspects of SCA, establishment of clinical interventions to avoid SCA-associated complications remains elusive, and tools to infer disease activity are needed. Here, we found an inverse correlation between the DOP levels and hemoglobin, suggesting that the intensity of the oxidative milieu is associated with increased red blood cell degradation and its consequences, or that an intense red blood cell destruction and the consequent tissue damage is associated with increases in the oxidative status. Regardless of the direction of the association, the degree of oxidative perturbation based on the markers explored here hallmarks SCA. Quantifying the oxidative disturbance through DOP could emerge as a useful tool to follow up SCA carriers and guide onset of early interventions. Future prospective studies evaluating more markers and performing mechanistic investigations ex vivo are warranted to directly test the utility of this approach.An important contribution of our study was the assessment of the degree of oxidative perturbation (DOP). To our knowledge, no previous study has estimated the global oxidative disturbance in SCA patients. It is known that the oxidative milieu with production of free radicals would favor hemoglobin polymerization and RBCs sickling, leading to onset of SCA-related clinical manifestations, such as intravascular hemolysis46, and its effects on chronic complications of SCA remain unclear47. Nevertheless, hydroxyurea therapy has been associated with decreases in frequency of painful crises48, and other complications such as acute chest syndrome, dactylitis, hospitalization and transfusions49. It has been recently suggested that hydroxyurea therapy could modulate oxidative stress through its effect on the hemoglobin50. Here, using the DOP analysis, we found that the hydroxyurea therapy leads to consistent decreases in the overall degree of oxidative perturbation, which values were indistinguishable from non-SCA healthy controls. It is reasonable to hypothesize that hydroxyurea therapy attenuates the oxidative stress status in SCA patients, contributing to clinical improvement and a tendency towards a milder disease. Importantly, the networks of correlation matrices demonstrated an increase in the number of connections and an apparent balance in the oxidative responses, reflected by the similar number of correlations between the markers, in hydroxyurea treated SCA patients. This observation suggest that hydroxyurea therapy may reduce the activation of mechanisms leading to production of free radicals in a coordinated manner, reflected by increased number of statistically significant correlations between concentrations of parameters associated with regulation of oxidative and inflammatory responses. Recently, in vitro analyses using human peripheral blood cell and human umbilical vein endothelial cells treated with hemin demonstrated that hydroxyurea therapy scavenges free radicals and induces the expression of antioxidants genes in these cells51, reinforcing our hypothesis. Additionally, we found higher levels of mean corpuscular volume in SCA patients who were being treated with hydroxyurea, which may suggest attenuated polymerization of hemoglobin. Altogether, these observations support the idea that hydroxyurea therapy may mitigate the systemic oxidative stress, directly scavenging free radicals and indirectly inducing transcription of antioxidant genes, contributing to reduce the hemoglobin polymerization and, consequently, all unfavorable clinical events linked to the cell sickling, such as painful crises, acute chest syndrome, inflammatory activation and tissue damage. Although this is an interesting idea at first glance, a large prospective study is needed to direct test our hypothesis.A relevant aspect shown here was the decrease of the degree of oxidative perturbation in SCA patients undertaking hydroxyurea. The risks and benefits of this pharmacological intervention are discussed elsewhereOur study has some limitations. We had a limited sample size, and limited number of oxidative markers to establish a more detailed profile of oxidative disorder in SCA. The present investigation was exploratory and was focused on a particular population composed by children and adolescents. Regardless of such limitations, the robust multidimensional analysis adapted to relative limited sample size was able to reveal distinct profiles that could characterize the disease groups. In summary, our findings extend the current knowledge about SCA, highlighting more nuances of the systemic oxidative stress that characterizes this disease. We also propose a strategy to measure DOP to track disease activity, which could be useful in the clinical management of patients. Additionally, our observations suggest that hydroxyurea therapy may mitigate the degree of oxidative perturbation by activating coordinated mechanisms. A larger, prospective interventional study is warranted to findings on hydroxyurea described here.Supplementary Information.


The steadily growing elderly population calls for efficient, reliable and preferably ambulant health supervision. Since cardiovascular risk factors interact with psychosocial strain , we investigated the potential contribution of psychosocial factors in discriminating generally healthy elderly from those with a cardiovascular condition, on and above routinely applied physiological assessments. Fifteen elderly (aged 60 to 88) with a cardiovascular diagnosis were compared to fifteen age and gender matched healthy peers. Six sequential standardized lab assessments were conducted (one every two weeks), including an autonomic test battery, a 6-min step test and questionnaires covering perceived psychological state and experiences over the previous two weeks. Specific combinations of physiological and psychological factors (most prominently symptoms of depression) effectively predicted  cardiovascular markers. Additionally, a highly significant prognostic model was found, including depressive symptoms, recently experienced negative events and social isolation. It appeared slightly superior in identifying elderly with or without a cardiovascular condition compared to a model that only included physiological parameters. Adding psychosocial parameters to cardiovascular assessments in elderly may consequently provide protocols that are significantly more efficient, relatively comfortable and technologically feasible in ambulant settings, without necessarily compromising prognostic accuracy. The steady increase of the elderly population (65 and older) encourages scientific efforts focused on healthy aging and elderly well-being . InsightCommon diagnostic and prognostic methods primarily focus on physiological factors , typically assessed using extensive protocols executed in hospitals, whilst their accuracy and efficiency might potentially be strengthened by including psychosocial factors. This might enhance “efficiency”, as assessing psychosocial factors is generally cheaper, less intrusive and faster.Previous scientific work clearly showed the potential relevance of prognostic models that include multisystem dynamics to predict health outcomes ,11,12,13In addition to the mentioned findings of the reciprocal effects of psychophysiological factors on health, evidence exists for social relationships to be directly related to physical and mental health and even mortality. Long-term detrimental health effects of negative social relations and social tensions appear to be comparable to those of smoking, obesity and lack of physical activity . Other sConsequently, in the present study we set out to investigate the potential of prognostic models combining physiological and psychosocial factors to discriminate between elderly with or without CVD. We hypothesize that including psychosocial factors  provides potentially simpler, thus more efficient, assessment methods, without necessarily compromising their prognostic accuracy.Subjects included in this study were non-smoking, independently living elderly, aged between 60 and 88 years and able to provide informed consent. Subjects were assigned into one of two groups: 1) The CVD subjects: all diagnosed with chronic heart disease  with either ischemic or non-ischemic cardiomyopathy for > 3 months, and no or slight functional limitations in physical activity  ; 2) age/The procedure consisted of two sequential stages: an intake and a series of 6 standardized lab assessments including an autonomic test battery, a 6-min step test and a selection of (self-report) psychological questionnaires. The assessments were conducted by two experienced staff members in a dedicated lab inside the independently living center. The intake questionnaires and the lab assessments were handed out and performed by trained lab technicians. Each lab visit lasted up to 75 min. There were no time limits set for the subjects during the lab assessments. Climate control remained constant at a comfortable level for the elderly.Following the intake, covering an intake interview including medical status and (signing of) informed consent (copy available with the editor and/or the corresponding author), participants were asked to come to the lab every other week for six sequential assessments. Among other assessments, the biweekly set of questionnaires included the 12-item Short-Form version-2 (SF12v2) : 12 items scored on a 5-point Likert scale ; the PANAS Short Form (assessing positive and negative affect in the previous two weeks): 10 items scored on a 5-point Likert scale ; a listing of all positive and negative life events of the last two weeks; the Interpersonal Support Evaluation List : 12 items scored on a 5-point Likert scale ; a social integration questionnaire : 4 items scored on a 4-point Likert scale (ranging from “hardly ever or never” to “often”); and the Geriatric Depression Short Form : 15 items, scored with “yes” or “no” ,23,24,25Finishing the questionnaires and connecting the ECG leads typically required at least 20 min. This time was used as a resting period to physiologically recover from the walk to the laboratory, and to set a baseline for the physiological measures. As part of an autonomic test battery, subjects performed 5 min of paced breathing . During the autonomic testing, a three lead ECG was recorded continuously using a Nexfin device . The ECGFollowing, the 6-min step test was conducted, which consists of 2 min resting, 1 min stepping at 30 steps per minute (SPM), 1 min stepping at 60 SPM, 1 min stepping at 90 SPM and 1 min recovery. Following this exercise, the continued ECG recordings were used to determine Heart Rate Recovery (HRRec) . During 2); (2) Oxygen Uptake Efficiency Slope (OUES); (3) Oxygen Saturation (SaO2); (4) resting end-tidal carbon dioxide partial pressure (resting PetCO2); (5) PetCO2 change in response to exercise (ΔPetCO2); and (6) a standardized index for pulmonary capacitance  .,36.32,362Slope and PO2P). These analyses provided multiple significant models for MVI and PO2P. Depression scores appeared to be a prominent factor providing slightly superior statistics over models without psychosocial factors. However, the preselected set of independent variables did not provide satisfying models for VEVCO2Slope, a variable potentially more closely related to physiological and cardiovascular disturbances. Interestingly, in the healthy elderly, HRRec appeared to be a dominant factor in both QoL and in relation to clinical indicators. This finding is in accordance with previous findings identifying HRRec as a representative parameter of fitness in generally healthy individuals [Based on the previous steps, regression analyses were applied to identify the most accurate combinations of factors to predict clinical indicators of CVD  appeared to be significantly inferior. As a consequence, we may conclude that adding psychosocial factors to models predicting CVD risk in elderly may very likely be a useful contribution, not least to increase predictive power. Specifically, adding depression scores appears to provide a statistical surplus in modeling cardiovascular risk outcomes (chronic heart failure/CVD) on and above well-known cardiovascular parameters. When both CVD and reference samples were analyzed separately, in the CVD group depression scores gained even more statistical prominence. This suggests that in individuals experiencing symptoms of chronic heart disease, even if they are mild, depressive symptoms may become an increasingly relevant indicator of the presently used health outcomes. However, more research in more robust and heterogeneous samples  is necessary to further unravel the  contribution of symptoms of depression in the prognosis of cardiovascular risk in the elderly population.Nevertheless, using stepwise backward regression analyses, most presented models combine physiological and psychosocial factors. The few models that only comprised physiological factors , which appeared to be superior to most models including psychological factors in discriminating between high and low risk elderly. However, a psychophysiological model including a wide variety of psychosocial and cardiovascular parameters appeared to be the most accurate see . This laUnfortunately, two elderly with significant initial depression scores and a notable negative outlook on life prematurely resigned from the study. Given their state of mind, this was not a surprise, although this significantly decreased the distribution within the sample specifically in the negative direction. Consequently, stronger, and potentially more valid and representative effects may have been observed than those reported here.Secondly, the present sample was rather small. Consequently, some methodological concessions had to be made, primarily with respect to the applied statistical strategy and methods. For example, statistical power was insufficient to engage in more sophisticated repeated measurement analytical designs. This, of course, limits the interpretation and generalizability of the presented results. Additionally, all participants were non-smokers, possibly biasing the found results to some extent as compared to the general population. On the other hand, the participating elderly, both from the participating institute and those coming from other locations appeared to be remarkably compliant and motivated to participate in six consecutive assessment rounds in a period of 3 months. This provided a considerable body of data, resulting in outcomes that are generally in line with previously reported findings  .In relation to that last point, it must be noted that several parameters of HRV applied here may actually reflect overlapping sources of variance. The appropriate interpretation of (short time series of) SDNN, for example, is yet under debate . In addiLastly, although the present findings underline the prognostic potency of combining cardiovascular, physiological and psychosocial factors in adequately identifying elderly at risk for CVD, to effectively implement such prognostic strategies in clinical practice might yet be a challenge in itself. It requires the combination of expertise, skills and technologies from quite specific domains in, more or less standardized, assessment batteries. Nevertheless, in the context of personalized medicine, and supported by developments in sensor and analytical technologies, models like the ones outlined here might be of significant importance for clinical decision making in elderly in the near future.The use of Artificial Intelligence to process ECG tracings into valid and reliable health risk indicators (for example in relation to potassium changes or ejection fraction) progresses steadily into clinical practice and routine. This development is paralleled by the unpreceded current developments in sensory technology. An important example of the latter is the assessment of cardiovascular and psychophysiological parameters via smartphones, a development that seems to quickly mature into reliable and valid assessment methods . The comIn the present study, we set out to explore the potential additional contribution of psychosocial factors in identifying elderly at risk for cardiovascular disease on-and-above routinely used cardiovascular variables and clinical parameters. With this aim, the present sample of elderly provided prognostic models with comparable and sometimes even superior accuracy over models incorporating established cardiovascular or clinical markers only. Apart from the found contribution in prognostic accuracy, an additional advantage might be that assessing the relevant psychosocial factors  can be done rather efficiently, for example by means of mobile technology. Consequently, extending diagnostic routines with assessments aimed at these factors might be considered useful. Nevertheless, future studies are necessary to shed more light on the presently reported relations; to further develop the here proposed prognostic strategies; and to tackle associated technological assessment challenges.