Thanks to striking progress in both the understanding of anti-tumor immune response and the characterization of several tumor associated antigens (TAA), a more rational design and more sophisticated strategies for anti-tumor vaccination have been possible. However, the effectiveness of cancer vaccines in clinical trial is still partial, indicating that additional studies are needed to optimize their design and their pre-clinical testing. Indeed, anti-tumor vaccination success relies on the choice of the best TAA to be targeted and on the translational power of the pre-clinical model used to assess its efficacy. The chondroitin sulfate proteoglycan-4 (CSPG4) is a cell surface proteoglycan overexpressed in a huge range of human and canine neoplastic lesions by tumor cells, tumor microenvironment and cancer initiating cells. CSPG4 plays a central role in the oncogenic pathways required for malignant progression and metastatization. Thanks to these features and to its poor expression in adult healthy tissues, CSPG4 represents an ideal oncoantigen and thus an attractive target for anti-tumor immunotherapy. In this review we explore the potential of CSPG4 immune-targeting. Moreover, since it has been clearly demonstrated that spontaneous canine tumors mimic the progression of human malignancies better than any other pre-clinical model available so far, we reported also our results indicating that CSPG4 DNA vaccination is safe and effective in significantly increasing the survival of canine melanoma patients. Therefore, anti-CSPG4 vaccination strategy could have a substantial impact for the treatment of the wider population of spontaneous CSPG4-positive tumor affected dogs with a priceless translational value and a revolutionary implication for human oncological patients. It is when oncology meets immunology that cancer immunotherapy begins. Strengthen the patient’s own immune response against cancer cells represents one of the most challenging and exciting concept of active cancer immunotherapy .Neoplastic initiation and progression are accompanied by the accumulation of several genetic modifications in somatic cells. The transcriptional and mutational landscape of tumors indicates that there is a clear opportunity for the immune system to distinguish tumor cells from healthy tissue and trigger a specific attack against both conserved and mutated tumor-associated antigens (TAA) , 3. IndeFor these reasons, the clear need for more refined and predictive pre-clinical models becomes apparent. Therefore, in 2003 the National Cancer Institute’s Center for Cancer Research introduced the Comparative Oncology Program in order to foster the study of naturally occurring cancer in pet animals as models of human tumors , 9. A EuThis review will highlight the role of chondroitin sulfate proteoglycan-4 (CSPG4) as one of the most attracting TAA identified so far for translational immunotherapy studies in both human and veterinary field.Displaying a low level of expression in healthy tissues and a high level of expression in tumors, in addition to own a “driving” role in the promotion of cancer development—pushing the progression of a neoplastic lesion from one stage to the next—are the features of “ideal” immunotherapeutic targets.The term “oncoantigen” was coined to describe those TAA that display the above-mentioned characteristics , 12. TheThe expression level in neoplastic and/or cancer initiating cells (CIC), the cellular localization and the role in oncogenic pathways, together with the percentage of patients with tumors expressing a given TAA, are characteristics also used by the National Cancer Institute of the U.S. to prioritize cancer antigens for the development of focused immunotherapeutic vaccines . The CSPCSPG4, high molecular weight-melanoma associated antigen (HMW-MAA) and melanoma chondroitin sulfate proteoglycan (MCSP) are different names to label a cell surface molecule with unique structural characteristics. CSPG4 was first characterized on human melanoma cells more than 30 years ago ; in the The extracellular portion of the CSPG4 is followed by a 25-aa transmembrane sequence (2222–2246 aa) joined to a 75-aa cytoplasmatic domain 2247–2322 aa) encompassing sites critical for CSPG4 function: (a) the PDZ binding domain on the carboxyl terminal portion that is the site for the attachment of scaffold proteins such as MUPP1, syntenin and GRIP1 ; (b) two2 aa encoAs a membrane-spanning molecule, CSPG4 plays an important role in the communication between the outside and the inside compartments of the cell. Notably, CSPG4 is devoid of a catalytic activity ; howeverCSPG4 is involved mainly in tissue development or homeostasis and retains a limited expression in adult healthy tissues . Its expMarked by a restricted distribution in normal tissues, CSPG4 is over-expressed in several haematological and solid neoplastic conditions besides melanomas, including oligodendrocytomas, gliomas, childhood acute lymphoblastic leukemia and acute myeloid leukemia, renal cell carcinomas, chondrosarcomas, pancreatic and triple-negative breast carcinomas. In neoplastic lesions CSPG4 is highly expressed on both malignant cells and activated pericytes within the tumor microenvironment , 43. TheV600E mutation determines a constitutive activation of the downstream MAPK pathway positively impacting on cell proliferation, the expression of CSPG4 is required to maximize the tumorigenic effect [cas, an adaptor protein involved in the linkage of actin cytoskeleton to the extracellular matrix during cell migration, invasion and transformation [As a consequence of its ability to coordinate several intracellular pathways regulating different cell functions, CSPG4 becomes involved in tumorigenesis at multiple levels , 46. Spec effect . CSPG4 oc effect –54. By bc effect . Throughc effect . Activatormation , 57, conormation , 59. Finormation  that areormation –64.In recent years, the study of naturally occurring tumors in pet animals as models of human cancer, i.e. the comparative oncology, has been on the rise. Indeed, the difficulties in translating into the clinics several immunotherapeutic strategies found to be effective in rodents , 7 prompAt the light of the comparative oncology concepts and considering the biological role of the CSPG4 oncoantigen in human tumors and its high conservation in structural and functional properties through phylogenetic evolution, the identification of CSPG4 in canine tumors could represent an unprecedented opportunity to pre-clinically test anti-CSPG4 immunotherapies in the veterinary field, highly predictive of their clinical efficacy in the human oncology. With this in mind and considering the well-known link between CSPG4 expression and melanoma progression in humans, other than the urgent need of effective strategies for the treatment of both human and canine melanomas –73, we wMoreover, we have evaluated the presence of CSPG4 on different cell lines generated from three canine melanoma patients: one named OLGA, generated starting from the bioptic material obtained from a metastatic lymph node, and two lines named CMM9 and CMM10, derived from primary oral melanomas. CSPG4 expression on tumor cell membrane has been confirmed by flow cytometry analysis in all the canine melanoma cell lines analyzed  positive and 6 (21%) negative cases. Particularly, the positivity score assigned as previously described  among thAll together, these findings highlight the great value of CSPG4 as a translational immunotherapeutic target in veterinary clinical practice, not only for melanoma but also for other tumor types.The development of anti-cancer therapies directed against CSPG4 represents an unprecedented opportunity to simultaneously target tumor cells, CIC and pericytes on tumor vasculature. Moreover, since CSPG4 orchestrates multiple intracellular signaling pathways , its tarOne explored strategy in the immunotherapy field, consisting in the stimulation of autologous tumor-specific lymphocytes ex vivo to improve cell mediated immunity before adoptively transferring them back to the patient, were first described almost four decades ago. Since then, several attempts to treat metastatic melanoma were carried out exploiting the adoptive cell transfer (ACT) of autologous lymphocytes derived from the tumor or from the blood of patients –80. Desp+ T cells reactivity against specific peptides located in the extracellular domain of CSPG4 antigen in the peripheral blood of both healthy donors and melanoma patients, in the absence of clinical signs of autoimmunity. Importantly, these peptide-specific CD4+ T cells could strongly recognize CSPG4 expressing melanoma cells, suggesting that the identified peptides are naturally processed by tumor cells. These findings supported the idea that the activation and the expansion of CSPG4-reactive CD4+ T cells circulating in the blood could be considered for further development of anti-CSPG4 immunotherapeutic strategies [The importance of CSPG4 as a promising target for T cell-based immunotherapy has been also supported by Erfurt and colleagues, which demonstrated the presence of a CD4rategies , 92.However, since in several malignancies tumor cells have been reported to escape T-cell recognition , 94, morAn alternative approach to mAb administration is obviously active immunization, which has the potential to bring about effective and long-lasting anti-tumor responses without significant side effects and the risk of resistance development. First evidences of the effectiveness of active immunization against CSPG4 in melanoma patients were achieved through vaccination with the anti-idiotypic antibody MK2-23, which bears the internal image of the mAb 763.74 against a defined CSPG4 epitope. Interestingly, the induction of CSPG4-specific antibodies in immunized patients was associated with significantly longer survival and metastasis regression , 105. HoOverall, these are encouraging data providing a strong rationale for the development of new strategies of active immunization against CSPG4.Amongst the various possible approaches for active immunization, DNA vaccination has some advantages over other strategies. Among them there are the high stability of DNA and the possibility to generate the vaccine in large amounts in a cheaper and faster manner than other vaccines . DNA vacA large body of studies assessed the efficacy and the safety of the DNA vaccination technique for cancer treatment and prevention in a variety of animal models and even in humans with plasmids encoding different TAA –120. Howen bloc surgically resected stage II and III CSPG4-positive spontaneous oral malignant melanoma [The ability of xenogeneic DNA vaccines to break the immune tolerance and to induce an effective immune response against a self-oncoantigen has been extensively demonstrated also by us , 128 andmelanoma , 121. Thmelanoma , 121.Our results indicate that CSPG4 xenogeneic DNA vaccination associated to electroporation in dogs affected by MM is safe and able to overcome host unresponsiveness to the self-antigen, resulting in significantly increased overall and disease-free survival, thanks to the induction of anti-CSPG4 antibodies. These findings corroborate the clinical impact of CSPG4 immune-targeting for the management of canine melanoma patients. Moreover, the recognition of CSPG4 expression in other aggressive and incurable tumor histotypes may promote the spread of anti-CSPG4 vaccination strategy for the treatment of the wider population of CSPG4-positive tumor affected dogs. Finally, the recognized value of spontaneous tumors in dogs as a priceless model for predicting tumor behavior and response to immunotherapy in humans, highlights the translational potential of active CSPG4 immune-targeting that might be revolutionary for the management of both canine and human CSPG4-positive cancer patients.


FKBP5 and early adversity, it is delineated how this interaction could contribute to transdiagnostic disease risk via a combined genetic and epigenetic disinhibition of FKBP5 transcription. This knowledge may now allow to develop biomarkers for a transdiagnostic subset of psychiatric patients and to personalize treatment.Epidemiological studies indicate a combined contribution of genetic and environmental factors, mainly exposure to adverse life events, in the risk for psychiatric disease. Understanding how adverse life events interact with genetic predisposition on the molecular level to shape risk and resilience to psychiatric disorders may yield important insight into disease mechanism. Using the example of the molecular mechanisms of interaction of functional genetic variants within the stress-regulating gene  Epidemiological studies indicate a combined contribution of genetic and environmental factors, mainly exposure to adverse life events, in the risk for psychiatric disease  secretion from the adrenal gland . More condensed chromatin would thus be more prohibitive for binding of GRs and transcriptional regulation of target genes. A series of studies have shown that environmental exposure, especially adversity early in life, can lead to lasting changes in gene function by inducing such epigenetic changes  by stress or glucocorticoids in a number of tissues, including the brain  is associated with decreased stress coping behaviour, increased anxiety and impaired extinction learning  in the FKBP5 locus (Binder et al., FKBP5 following GR activation; 35% carry the allele that leads to a more exaggerated mRNA response. These genetic effects on the molecular stress regulation are reflected on the endocrine, behavioural and imaging level. First, the changes in GR sensitivity during the feedback regulation of the HPA axis lead to prolonged stress-related cortisol release in individuals carrying the variant that is associated with higher FKBP5 mRNA induction (Buchmann et al., On the genetic level, this stress-related induction of FKBP5 are necessary to unveil the stress interaction effect.This genetic change in the endocrine, circuit level and behavioural stress response is associated with an altered risk for stress-related psychiatric disorders. Interactions between FKBP5 and stressful life events, especially those occurring in childhood, were found to be associated with a variety of psychiatric disorders, including PTSD, depression, aggression, suicidality and psychosis in a large number of studies including well over 18,000 individuals (for reviews, see Halldorsdottir & Binder, FKBP5, genetic and epigenetic changes need to come together. Specifically, changes in DNA methylation of FKBP5 locus glucocorticoid response elements have been implicated that lead to an enhancement of the transcription response of FKBP5 to GC (Klengel & Binder, FKBP5 expression following GR activation, delaying the negative feedback phase and resulting in prolonged cortisol response to stress and trauma. This in turn would then precipitate GR-induced DNA demethylation at GREs in FKBP5 and a transcriptional disinhibition (Klengel & Binder, FKBP5 (Klengel et al., In the case of FKBP5 following stress, caused by both a genetic and an epigenetic disinhibition, could result in long-lasting changes in the neural circuits involved in stress and anxiety regulation. This could be driven by changes in GR tone but also by downstream effects of FKBP5 on additional pathways that are relevant for neuronal function and synaptic plasticity. As mentioned above, early trauma x FKBP5 genotype interactions predict risk for a range of psychiatric disorders. It could be possible that patients suffering from such an FKBP5 disinhibition, but presenting with distinct psychiatric symptoms, could benefit from blocking FKBP5 activity. A selective high-affinity antagonist for FKBP5 has been developed, and first experiments in experimental animals show that is has anxiolytic effects and increases stress coping (Gaali et al., This disruption in regulatory homeostasis of FKBP5 has identified a combined genetic and epigenetic disinhibition of stress-induced FKBP5 transcription as a possible underlying cause of disease risk. This knowledge may now allow the development of biomarkers for this transdiagnostic subset of psychiatric patients and to personalize treatment. This could include selective inhibition of FKBP5 but also targeted psychotherapy approaches for the underlying risk behaviours such as an increased bias towards threat or increased intrusions observed with increased FKBP5 function (see FKBP5 genotype moderates long-term effectiveness of exposure-based psychotherapy for posttraumatic stress disorder, with patients carrying the genotype associated with higher FKBP5 function showing increase relapse rates at 10 months (Wilker et al., FKBP5 status could thus help guide the type of treatment as well as the frequencies of follow-up visits. The next step will be to expand this set of biomarkers and mechanistic insights to genome-wide approaches.Dissecting the molecular mechanisms underlying the statistical gene x early trauma association of tion see . A study


ECs have also been shown to regulate the maintenance and regeneration of organ‐specific stem cells in mammals. In the hematopoietic system, hematopoietic stem cells (HSCs) are dependent on signals from the bone marrow (BM) vascular niche for their maintenance and regeneration after myelosuppressive injury. Recent studies have demonstrated the essential functions of BM ECs and perivascular stromal cells in regulating these processes. In the present study, we summarize the current understanding of the role of BM ECs and perivascular cells in regulating HSC maintenance and regeneration and highlight the contribution of newly discovered EC‐derived paracrine factors that regulate HSC fate. S Recent studies have shown that blood stem cells require signals from the bone marrow microenvironment or niche for their survival and regeneration. In the present study, the current understanding of the interactions between blood stem cells and niche cells is summarized and the potential for niche‐derived secreted factors as therapeutic agents for regenerative medicine is highlighted.Vascular endothelial cells (ECs) constitute the intimal lining of arteries, capillaries, and veins within the human vasculature and serve critical roles in the delivery of oxygen and nutrients, in the regulation of coagulation, and as the gatekeepers to inflammatory cells for entry into tissues Within the embryo, development of the hematopoietic system occurs in multiple locations at various stages, including the extraembryonic yolk sac, fetal liver, spleen, and, finally, adult BM. Evidence exists for two discrete anatomic origins of hematopoietic activity, one extraembryonic and one intraembryonic Before 1975, it was widely accepted that definitive HSCs originated from the yolk sac blood islands. To evaluate this dogma, Françoise Dieterlen‐Lièvre took advantage of the ability to distinguish chick and quail cell nuclei and grafted 2‐day‐old quail embryos on to chick yolk sacs of comparable developmental stages, before or shortly after the establishment of vascularization The first definitive HSC known to be able to fully reconstitute the hematopoietic system on transplantation was identified in the aorto‐gonad‐mesonephros region (AGM) in mice and humans + pericytes associate with portal vessels, forming a fetal perivascular niche that promotes HSC expansion + pericytes, also occurs, because the portal vessels undergo a transition from neuropilin‐1+ephrin‐B2+ arteries to an EphB4+ vein phenotype. This suggests that the changes in postnatal circulation and hemodynamics alter the liver vasculature and are associated with the loss of HSC‐supporting cells. The HSCs then migrate to the BM wherein long‐term hematopoiesis persists postnatally.Later in development, HSCs reside in the fetal liver. Many questions regarding the fetal liver vascular niche remain, especially regarding the process of migration of HSCs from the fetal liver to the BM following birth. A recent study showed that nestin‐positive NG2In 1968, Wolf and Trentin directly implanted BM stroma into the spleens of irradiated recipients and showed that the proportions of outgrowing erythroid and myeloid colonies could be biased by their local environment, providing some evidence for microenvironmental influence of immature hematopoietic cell fate op/op, Fos‐deficient, and Rankl‐deficient mice, which lack osteoclasts In addition to HSCs and their progeny, the BM is composed of a diverse array of cells with specialized functions. These include vascular ECs, perivascular cells, osteoblasts, sympathetic nerves, adipocytes, macrophages, and many subsets of stromal cells + , nestinbright pericytes, which are distinct from sinusoid‐associated leptin receptor (LepR)+ or Nestindim perivascular cells + cells Most mammalian hematopoiesis occurs in the axial skeleton  in the flat bones, such as the pelvis, sternum, skull, ribs, vertebrae, and the metaphyseal and epiphyseal ends of long bones. Other BM, composed of higher fat content (“yellow marrow”), can be found in the hollow interior of the diaphyseal portion (shaft) of the long bones. Arteries enter through the bone cortex, terminate in the endosteum (the connective tissue lining the inner surface of compact bone), and branch in the metaphysis or diaphysis of long bones +CD244−CD48−) on HSCs catalyzed the ability of researchers to localize HSCs in the niche. Most SLAM+ HSCs were found in that study to be associated with sinusoidal endothelium Defining the anatomic location of HSCs in the adult BM as it relates to distinct niche cells has proved to be challenging owing to the difficulty in retaining histological integrity when sectioning bone, the number of markers necessary to identify HSCs, the limitations of microscopy, and the close proximity of bone and vascular elements throughout the BM. The discovery of a pattern of SLAM family receptor expression  required the recovery of an intact vasculature CXCL12 in BM ECs also impaired HSC maintenance in mice. In the same studies, Ding and Morrison demonstrated that deletion of CXCL12 from LepR+ perivascular stromal cells depleted HSCs CXCL12 from Prx1+ mesenchymal progenitor cells markedly decreased HSC content Ding et al. established the essential role of BM ECs in regulating the maintenance of the HSC pool via cell‐specific deletion of SCF Human ECs can promote and maintain HSCs in culture VEGFR2 or antibody‐mediated blockade of VE‐cadherin‐mediated vasculogenesis was shown to disrupt BM vascular regeneration in irradiated mice and result in prolonged hematopoietic toxicity and delayed HSC regeneration −/−;Baxfl/−Tie2Cre;Bak1 mice, which bear deletion of the intrinsic mediators of apoptosis, BAK and BAX, in Tie2+ ECs, were shown to have radioprotection of the BM vasculature and the hematopoietic system compared with mice that retained BAX expression in Tie2+ ECs + stromal cells were also shown to have a necessary role in maintaining splenic HSCs via CXCL12 secretion in the setting of extramedullary hematopoiesis in the same study Several lines of evidence suggest that BM ECs have a necessary role in HSC regeneration Although recent studies have confirmed the essential role of BM ECs in maintaining the HSC pool in steady state and regeneration, the precise mechanisms through which BM ECs regulate HSC maintenance and regeneration remain incompletely understood. Although cell‐cell interactions are clearly important, our laboratory has focused on discovering and characterizing the paracrine factors that are produced by BM ECs that regulate HSC fate −/−;Baxfl/−Tie2Cre;Bak1 mice secreted significantly increased concentrations of epidermal growth factor (EGF) and amphiregulin, another EGF receptor (EGFR) agonist, in the BM at steady state and early after myelosuppressive TBI + BM ECs and that EGFR signaling might be functionally relevant to HSC regeneration. Subsequently, Doan et al. showed that systemic administration of EGF promoted HSC regeneration and improved survival in mice after lethal irradiation PUMA‐mediated apoptosis in HSCs, leading to increased hematopoietic recovery and improved survival Doan et al. reported that BM ECs in radioprotected Jagged‐1 in BM ECs led to hematopoietic exhaustion and HSC depletion over time Poulos et al. reported that BM ECs also secrete Jagged‐1, a Notch ligand, and conditional deletion of + perivascular stromal cells surround BM sinusoidal vessels As noted, several recent studies have highlighted the importance of perivascular cells in regulating HSC fate. Anatomically, BM vessels are ensheathed by pericytes or adventitial reticular cells, including CXCL12‐abundant reticular cells (CAR cells) + cells led to BM hypocellularity and anemia SCF or CXCL12 from nestin‐positive MSCs had no effect on HSC maintenance CXCL12 from LepR+ perivascular stromal cells caused a significant depletion of HSCs. Greenbaum et al. also demonstrated that deletion of CXCL12 from nestin‐negative, Prx1‐positive mesenchymal cells yielded HSC depletion The functional role of mesenchymal stromal cells (MSCs) in the HSC niche was suggested by the observation that HSCs reside near nestin‐expressing MSCs and that deletion of nestin‐positive cells depleted BM HSC content Ext1, in Mx1+ BM stromal cells was shown to cause a reduction in heparan sulfate production σ (PTPσ), a receptor for heparan sulfate and chondroitin sulfate, was shown to substantially increase the BM repopulating capacity The production of extracellular matrix proteins by BM stromal cells and the interaction of these molecules with HSCs have also recently come into focus. Deletion of the glycosyltransferase gene, + HSCs + perivascular stromal cells was shown to accelerate hematopoietic and vascular recovery in mice while increasing vascular permeability In the present review, we focused on the role of BM vascular and perivascular cells in regulating HSC fate. All the important HSC regulatory mechanisms could not be covered comprehensively in the present review, including the Wnt signaling pathway, the Notch pathway and the Tie2/angiopoietin pathway. The function of Wnt ligands in the hematopoietic niche has been previously reviewed in detail Although it was not the focus of the present review, recent studies have suggested that corruption of BM niche cells might contribute to or promote the transformation of hematopoietic progenitor cells to malignancy + stromal cells in the spleen supported EMH via elaboration of SCF or CXCL12 + pericytes and that such fetal liver hematopoiesis ceases with closure of the umbilical vein and the loss of nestin‐positive NG2+ pericytes as the portal vessels transition from the arterial to the venous phenotype In the setting of myelofibrosis, chronic myelogenous leukemia, and other myelophthisic processes and stress conditions, extramedullary hematopoiesis can occur in humans in various organs, including the liver, spleen, lymph nodes, retroperitoneum, lungs, genitourinary system, skin, muscle, and rarely, the central nervous system Elucidation of the mechanisms through which BM ECs, perivascular cells, and osteolineage cells regulate HSC self‐renewal, regeneration, and malignant transformation will increase the potential for development of targeted therapeutic agents for the human hematopoietic system. Novel vascular niche‐derived paracrine factors that regulate HSC fate are particularly exciting in this regard because the systemic administration of such factors could be readily developed for clinical testing. Several BM EC‐derived soluble proteins described in the present study, such as PTN and EGF, and derivatives of these proteins, are in advanced preclinical testing for investigational new drug evaluation. The ongoing discovery of novel mechanisms through which vascular, perivascular, and osteolineage cells regulate hematopoiesis, coupled with the explication of the cross‐talk signals among BM ECs, perivascular cells, osteolineage cells, and other niche elements, will certainly yield many exciting tools for regenerative medicine.J.P.S., K.T.Y., and J.P.C.: manuscript writing, final approval of manuscript.The authors indicated no potential conflicts of interest.


Tertiary lymphoid organs are found at sites of chronic inflammation in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. These organized accumulations of T and B cells resemble secondary lymphoid organs and generate autoreactive effector cells. However, whether they contribute to disease pathogenesis or have protective functions is unclear. Here, we discuss how tertiary lymphoid organs can generate potentially pathogenic cells but may also limit the extent of the response and damage in autoimmune disease. TLOs are also known as tertiary lymphoid tissues, ectopic lymphoid follicles, or ectopic lymphoid structures and are accumulations of lymphocytes and stromal cells in an organized structure that occur outside of secondary lymphoid organs (SLOs). TLOs share many features with SLOs, such as the presence of T and B cell compartmentalization into T cell zones and B cell follicles, chemokines that mediate the compartmentalization, antigen-presenting cells, lymphatic sinuses, high endothelial venules, follicular dendritic cells, and fibroblastic reticular cells (FRCs)11. In SLE, inflammation in the kidney interstitial tissue is associated with greater risk for kidney failure12. Up to almost half of patients have well-circumscribed aggregates of B cells, plasma cells, and T cells and a small fraction can have well-organized germinal centers with follicular dendritic cells2. In rheumatoid arthritis, TLOs ranging from B and T cell aggregates to germinal centers are found in the inflamed synovium of about half of biopsied patients and are associated with more severe joint and systemic inflammation13. TLOs are also found in other organs in other autoimmune diseases or models, such as in the salivary and lacrimal glands in Sjögren’s syndrome16, the central nervous system in multiple sclerosis22, the pancreas in diabetes25, the thymus in myasthenia gravis27, and the intestines in inflammatory bowel disease29. While findings in recent years have begun to delineate the mechanisms that regulate the formation of TLOs (recently reviewed inAutoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis are marked by chronic inflammation in end organs that can be associated with the development of tertiary lymphoid organs (TLOs)32 that can maintain and reactivate memory CD8+ T cells33 and produce plasma cells and antiviral serum immunoglobulins31. Remarkably, although there is a delay in anti-viral immunity development, TLOs are sufficient for protection when the hosts are deficient in SLOs33, underscoring the idea that TLOs can generate effector cells that provide effective host defense. Innate lymphoid cells (ILCs) in the lung are induced after influenza infection and have been shown to maintain lung function, epithelial integrity, and airway remodeling34. Although it is unknown whether TLOs play a role in maintaining or stimulating lung ILCs after influenza infection, ILCs have been shown to be associated with TLOs and have even been associated with decreased disease progression in lung cancer37. It is possible that influenza-induced lung TLOs also provide a suitable environment for ILCs to populate and function in a protective manner. Similar to influenza virus infection, TLOs form with pulmonaryMycobacterium tuberculosis (MTB) infection40. Latent tuberculosis is associated with more frequent, well-organized TLOs while TLOs are less frequent and less well-formed in active tuberculosis40, suggesting a protective role for TLOs in controlling disease. The TLOs contribute to the formation of granulomas, which function to promote immunity and limit tissue damage41. Additionally, CXCL13 expression that organizes the B cell follicles serves to recruit CXCR5-expressing T helper (Th) cells into granulomas to activate macrophages that are essential to infection control40. These studies on TLOs in infection models highlight the ability of TLOs to support immune responses that are capable of protecting the host.In the setting of infections, TLOs have been generally considered to be protective, adopting SLO-like functions and acting as “outposts” of SLOs that are directly positioned at the site of inflammation. TLOs form in the lung of influenza-infected mice2, demonstrating well-developed effector responses. The TLOs correlate strongly with the presence of immune complexes, suggesting that the locally generated antibodies are autoantibodies to renal antigens that can fix complement and thus cause tissue inflammation and damage2. Similarly, the B cell responses associated with TLOs in the rheumatoid arthritis synovium42, the salivary glands in Sjögren’s syndrome43, and other target tissues show autoimmunity10. SLE kidneys and rheumatoid synovium are also characterized by the accumulation of Th17 cells, which can have proinflammatory roles48. While IL-17-expressing cells could help to induce TLO formation, as has been shown in the central nervous system and in neonatal lung49, the TLOs could potentially also help to support Th17 cell maintenance or acquisition of additional proinflammatory properties51. Indeed, B cells are necessary for the accumulation of activated T cells, likely by presenting antigen to the T cells54, and B cells in TLOs may be pathogenic in part by stimulating autoreactive T cells, which then can contribute to the inflammatory milieu in the affected end organs. TLOs in autoimmune diseases, then, can be a source of potentially pathogenic lymphocytes.Similar to immune responses generated in SLOs, immune responses targeted to self may be harmful to the host. The TLOs in SLE kidneys contain germinal centers that show clonal expansion and somatic hypermutation characteristic of germinal center responses in SLOs12, but failure to sequester lymphocytes within the interstitial tissue could potentially result in the migration of lymphocytes to the glomeruli and worsened glomerular damage. Alternatively, in the absence of TLOs, the lymphocytes could enter the circulation to home to and potentially damage additional organs outside the kidneys. That inflammatory cells are able to find alternative niches despite the absence of TLOs is seen in MTB infection, where antigen-specific T cells still accumulate, showing an altered, perivascular location, in the absence of TLOs40. Also, B cell selection in the pancreas is unaltered by follicular disruption of TLO in the pancreas of non-obese diabetic mice25. Interestingly, TLOs within tumors but not at the tumor periphery are correlated with good outcomes in a study of pancreatic carcinoma patients55, raising the possibility that the TLOs at the tumor periphery prevent potential anti-tumor lymphocytes from accessing the tumor parenchyma. The concept that TLOs might have a sequestration function is analogous to the sequestration of lymphocytes within SLOs with the S1P agonist fingolimod, which is used to treat multiple sclerosis56. Fingolimod downregulates SIP receptor 1, preventing lymphocyte egress from the SLOs and subsequent migration to end organs57. Sequestration of potentially pathogenic cells, then, may help limit the extent of disease.Despite the generation of autoreactive and proinflammatory cells, TLOs could also have a protective role by sequestering pathogenic lymphocytes and preventing them from leaving the specific tissue or tissue compartment to cause further damage. For example, in SLE, glomerular damage is unrelated to the extent of interstitial inflammation58. These results suggested that the TLOs were protective, perhaps by the generation of the Tregs. Here, the TLO stroma may be critical for the generation of regulatory cells. Lymph node FRCs have been implicated in Treg generation by presenting self-antigen on MHCII and by guiding T cells into a tolerance-inducing environment60. FRCs, along with endothelial cells, can additionally promote tolerance by MHCI presentation of autoantigen62 and regulate the magnitude of T cell activation by expressing inducible nitric oxide synthase65. Tregs have also been shown to mediate tissue repair via amphiregulin in lung with influenza infection66 and in muscle after injury67, and thus TLO generation of Tregs can have protective effects independent of their immunosuppressive functions. Similarly, ILCs are another source of amphiregulin that is important for repair34, and TLOs may potentially support their development68. Interestingly, both SLE patients and lupus-prone mice possess decreased numbers and abnormal function of Tregs72 while exhibiting increased calcium/calmodulin-dependent protein kinase IV (CamK4)74, which is responsible for an imbalance in Th17 cells and Tregs with a shift towards more Th17 cells. Inhibition of CamK4 corrected this imbalance in lupus-prone mice, decreasing Th17 cells and increasing Tregs in the kidney in association with reduced organ damage75. It is tempting to speculate that the TLOs (and perhaps the SLOs) in SLE do not function correctly to foster optimal Treg generation. TLOs, then, may not only sequester potentially pathogenic cells but also provide an environment that limits the magnitude or severity of the response.TLOs can also be protective if they provide a microenvironment that generates regulatory or reparative cells that reduce the pathogenicity of inflammatory cells. In the apoE-/- model of atherosclerosis, TLOs that form on the outer aspects of the atherosclerotic vessel wall generate regulatory T cells (Tregs). These TLOs are dependent on lymphotoxin β receptor (LTβR) stimulation of presumably local vascular smooth muscle, and preventing TLO formation by deleting LTβR from smooth muscle cells resulted in more and enlarged plaquesIn conclusion, in autoimmune diseases, TLOs can generate and harbor autoreactive and proinflammatory, potentially pathogenic lymphocytes but could potentially serve to limit pathogenic responses by sequestering these cells or by reducing the magnitude of the response. Therapeutically, targeting TLOs may offer opportunities to ameliorate disease, and more understanding of the potential pathogenic and protective functions is needed. For example, can we identify TLOs that generate more pathogenic cells versus those that have more regulatory functions? Do these different functions in part reflect the evolution of TLO development and maturation? What are the vascular, stromal, and hematopoietic elements that contribute to the different microenvironments, and can we modulate them to generate a more immunoregulatory environment? Furthermore, understanding how the affected tissue outside the TLOs may be similar or distinct in supporting the generation and maintenance of autoreactive lymphocytes would enrich our understanding of the distinct nature of TLOs and also allow us to prevent the lymphocytes from potentially accumulating elsewhere upon TLO disruption. Continued improved understanding of TLO biology will help us better understand how to treat autoimmune disease.


Eating disorders and some forms of obesity are characterized by addictive-like, compulsive eating behavior which contains numerous similarities with compulsive drug use. Food intake is in part mediated by reward and reinforcement processes that can become dysregulated in these disorders. Additionally, impairments in inhibitory control regulation of reward-related responding can cause or further exacerbate binge and compulsive eating. Dysfunctions in two neurotransmitter systems in the mesocorticolimbic pathway, dopamine and glutamate, are thought to contribute to maladaptive eating behaviors. The trace amine associated receptor 1 (TAAR1) system is a promising therapeutic target for compulsive eating behavior due to the role of TAAR1 in synaptic transmission and in the modulation of dopaminergic and glutamatergic signaling. In support of this notion, the TAAR1 agonist RO5256390 was found to decrease the reinforcing effects of palatable food-cues and to reduce binge-like and compulsive-like eating of palatable food. Additionally, prolonged, intermittent access to palatable food was shown to downregulate TAAR1 in the prefrontal cortex, suggesting a potential role for TAAR1 signaling in inhibitory control processes. Research into the role of TAAR1 in addiction, including TAAR1’s ability to modulate psychostimulant reward and reinforcement, bolsters support for TAAR1 agonism as a pharmacological treatment for compulsive eating and other addictive behaviors. This review summarizes the evidence for TAAR1 agonism as a promising therapeutic for compulsive eating behavior as well as the hypothesized mechanism responsible for these effects. Eating disorders, including binge eating disorder (BED), bulimia nervosa (BN), anorexia nervosa (AN), ‘food addiction,’ and some forms of obesity are associated with significant economic and social costs  and limiBinge eating is defined as consuming large quantities of food in a short period of time accompanied by a loss of control over food intake . In a tyIn a different study, RO5256390 was also able to decrease, as well as slow down, responding for chocolate flavored pellets in rats . In thisAnother study, performed in mice, reported similar effects of TAAR1 agonism. R5166017 (0.3 mg/kg) was able to reduce high fat diet intake in a model of diet-induced obesity . Followi+ channels .Interestingly, partial agonism at TAAR1 (by RO5203648 or RO5263397) has been shown to have no effects on food self-administration  or to enchannels . Howeverchannels  as well channels  have oppEnvironmental food-associated stimuli exert a powerful control over behavior and can robustly enhance the desire to binge, even in satiated humans  and animA procedure used in the preclinical laboratory setting to assess the magnitude of influence of a conditioned stimulus (CS) over food or drug-seeking behavior is the second-order schedule of reinforcement . In thisAnother paradigm used to test the rewarding properties of food or drugs and the ability of paired contextual cues to drive behavior is the conditioned place preference (CPP) test. In animals with a history of binge-eating a highly palatable, sugary diet, following conditioning, tactile and visual cues associated with the highly palatable food are able to induce a strong place preference, unlike stimuli associated to the standard chow diet . When giThese experiments show that TAAR1 agonism greatly reduced the strength of palatable-food associated stimuli in driving behavior in rats with a history of binge eating. Though exact mechanisms have not yet been examined specifically for palatable food-cues, studies have found that the ability of TAAR1 to decrease the ability of drug-paired cues to elicit cocaine-seeking behavior can be pinpointed the ventral tegmental area and the prelimbic region of the medial prefrontal cortex (mPFC) as critical sites of action for TAAR1 in reducing cue-induced seeking behavior and drug-related memory expression , 2017.Compulsivity is a behavioral construct observed in different disorders, including drug addiction and disorders of pathological eating, where there is a loss of control over behavior that results in continued use despite incurring negative consequences  . CompulsTo test continued overeating despite consequences in animals, a commonly used paradigm is the light/dark conflict test, where feeding is typically suppressed in control rats when they face the aversive bright compartment of the light/dark box. However, rats with a history of intermittent access to palatable food will compulsively consume the palatable diet in face of the risky environment . TAAR1 aFigure 1).Compulsivity shares some commonalities with impulsivity in that both constructs involve a dysfunction in inhibitory control, mediated by frontal cortex projections to subcortical regions . DysfuncInhibitory control processes are largely governed by prefrontocortical circuits, and dysfunctions in this pathway are thought to underlie the loss of control and continued intake despite aversive consequences ,b,c. DecFigure 1). In line with this hypothesis, TAAR1 KO mice display behaviors consistent with prefrontal dysfunctions, such as perseverative behavior and impulsivity, suggesting that a deficiency in TAAR1 may result in deficits in cognitive function and behavioral flexibility .Importantly, TAAR1 effects on compulsive, binge eating are being driven by direct actions on food reward mechanisms, rather than homeostatic needs. Evidence for this is twofold. The first is that no food restriction or deprivation was used in the above-summarized studies showing its efficacy . Second,Though TAAR1 agonism effects on binge, compulsive eating are not being mediated by homeostatic mechanisms, it is interesting to note that TAAR1 activation has been studied for its metabolic effects in models of obesity and diabetes . In the The TAAR1 full agonist RO5256390 was not found to have any effects on anxiety-like  or depreAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To obtain satisfactory welds in positional laser beam welding, it is necessary to know how process parameters will influence the quality of welds in different welding positions. In this study, the titanium alloy Ti6Al4V sheets were laser welded in two vertical welding positions , and the appearance, porosity, strength, and ductility of the laser joints were evaluated. Results show that undercuts of the vertical up welds were greater than that of vertical down welds, while the porosity contents were much higher in vertical down welds than that in vertical up welds. When welding with a higher heat input, the vertical up welding position resulted in poor weld profiles (undercuts and burn-through holes), whereas the vertical down welding position led to excessive porosity contents in welds. Both severe undercut and excessive porosity were detrimental to the tensile properties of the welds. Weld appearance was improved and porosity contents were reduced by using a lower heat input, achieving better weld quality. Therefore, it is suggested that process parameter settings with relatively high laser powers and welding speeds, which can result in lower heat inputs, are used when laser welding the Ti6Al4V titanium alloys vertically. Nonferrous alloys, such as titanium alloys and aluminium alloys have been increasingly used in the aerospace and automobile industries because of their high specific strengths and, therefore, high fuel efficiency . MeanwhiStudies on the laser welding of high-strength steel sheets showed that using a horizontal welding position was helpful in mitigating the defects of undercut and sagging, which are commonly encountered when welding in a flat welding position ; moreove2 laser, the Nd:YAG laser [Laser welding of titanium alloys has been studied ever since the birth of the laser in 1960s. The laser beams used included both the early stage COAG laser ,11,12, aAG laser ,14,15. EAG laser ,17. SignConsidering that the laser welding of titanium alloys in vertical positions is sometimes inevitable, this paper aims to investigate the laser welding of a titanium alloy (Ti6Al4V) in two vertical welding positions , and to reveal the influences of welding positions and laser welding parameters on the laser weld quality. This study used titanium alloy sheets of Ti6Al4V, which had a thickness of 3 mm. Because titanium alloys are very sensitive to oxidation at high temperature, special measures were taken to protect the workpieces. The shielding scheme adopted in this study is shown in 3 + 88 mL water), based on which the weld bead appearance, undercuts, and microstructures were examined under an optical microscope. Afterwards, three mechanical testing specimens were prepared for each welding parameter setting, which were then tested under static tensile conditions. Fracture surfaces after tests were analyzed using a scanning electron microscope. With all the results obtained, the correlations between the bead appearance, inner porosity, and tensile behaviour were analysed comprehensively.After welding, the bead appearance, inner porosity, and mechanical properties of welds were examined to evaluate the quality of the laser joints obtained. X-ray radiography was firstly used to detect the pores in the welds. Then, metallographic samples were prepared by grinding, polishing, and etching in a solution , a few small pores existed in the vertical up position weld c, indicaThe total lengths of pores in the welds were measured for the four welding cases, and are shown in For both vertical up and vertical down positions, the butt weld joints of the titanium alloy fractured through the base metal when the laser heat input was lower (2500 W and 1.2 m/min) in tensile testing, while the fracture occurred in the joint zone, which consisted of weld metal and the heat affected zone (HAZ), for those joints produced with higher energy input (2200 W and 0.5 m/min). The yield strengths and ductility (in terms of specific elongation) of the laser welds are shown in Examination over the bead appearances showed that burn-through holes existed in the vertical up position welds if the higher heat input of 275 kJ/m (2200 W laser beam power at 0.5 m/min welding speed) was used, which was absent when welding in the vertical down welding position. Furthermore, laser welding with a lower heat input of 125J/s (2500 W laser beam power at 1.2 m/min) could avoid the formation of burn-through holes.For vertical up welding position shown in a, the doThe intermittent formation of burn-through holes during vertical up laser welding process may render the weld pool very dynamic and unstable. This accounts for the inconsistent bead profiles (undercuts and widths) along the length of vertical up welds, which were absent in vertical down welds. X-ray examination results of porosity showed that the vertical up welding position led to less pores in laser welds than the vertical down welding position. During laser welding, gas bubbles or voids may form as a result of collapsing keyhole or turbulent fluid flow of molten metal in the weld pool. If there are routes available for theses bubbles and voids to escape, and the molten metal remains there long enough, the bubbles and voids will be able to leave the weld pool and won’t form pores in welds. Otherwise, if the escape routes are not available and/or the solidifying time is too short, the bubbles and voids will be retained in welds and form pores. For vertical up and vertical down welding positions, although the dimensions and solidifying time of weld pools can be similar when laser welding with the same welding parameters, there exist totally different escape routes. For vertical up welding position a, the voBesides the welding positions, the final amount of porosity in laser welds can also be influenced by laser welding parameters in two basic ways. First, the laser parameters used will affect the stability of the fluid flow and keyhole, and then the amount of voids and bubbles formed in laser welding. As indicated by CFD modeling, the molten metal in weld pool is more unstable when laser welding with lower power and corresponding lower welding speed, which may then cause a larger number of pores . By contExperiment results show that, for both vertical up and vertical down positions, all welds produced with lower heat input (2500 W at 1.2 m/min) fractured through base metal, and had higher yield strengths and better ductility than those produced with higher heat input (2200 W at 0.5 m/min), for which the fracture occurred not through the base metal but through the joint zone consisting of the weld metal and heat affected zone (HAZ). Such a deterioration of mechanical properties can partially be attributed to the coarse martensite microstructure in the joint zone, as shown in For vertical up position welds fabricated with 2200 W laser power and 0.5 m/min welding speed (the higher heat input condition), the crack initiated from and propagated through the boundary between the base metal and weld metal during the static tensile tests, as shown in (1)The undercuts of vertical up welds were greater than those of vertical down welds for the same welding parameters in this study. Burn-through holes and inconsistent weld bead appearance formed when vertical up welding using the higher laser energy input.(2)The vertical down position could lead to more pores in laser welds than the vertical up position. Welding with the higher laser power at higher speed could reduce the porosity in vertical down welds.(3)Both undercut and porosity were detrimental to the tensile mechanical properties of welds when the higher heat input was used. The vertical up weld fractured through the greatest undercut, while the vertical down weld fractured through the most severe porosity.(4)Irrespective of welding positions studied, all laser joints welded using the lower energy input failed from the base metal, and had higher yield strengths and better ductility than those welded using the higher energy input, which failed from the weld metal.(5)For both vertical up and vertical down welding, it is suggested that relatively high laser powers in combination with high travelling speeds are employed for better welds.In this paper, titanium alloy Ti6Al4V was laser welded in vertical positions, and the effects of gravity were studied on the weld bead profile and inner porosity, in addition to their correlations with the mechanical properties of the laser welds that resulted. The conclusions of the work can be summarized as follows:


In vitro and in vivo tau models demonstrated that FKBP51 synergizes with Hsp90 to increase neurotoxic tau oligomer production. Inversely, protein phosphatase 5 (PP5), which dephosphorylates tau to restore microtubule-binding function, is repressed with aging and activity is further repressed in AD. Similarly, levels of cyclophilin 40 (CyP40) are reduced in the aged brain and further repressed in AD. Interestingly, CyP40 was shown to breakup tau aggregates in vitro and prevent tau-induced neurotoxicity in vivo. Moreover, the only known stimulator of Hsp90 ATPase activity, Aha1, increases tau aggregation and toxicity. While the levels of Aha1 are not significantly altered with aging, increased levels have been found in AD brains. Overall, these changes in the Hsp90 heterocomplex could drive tau deposition and neurotoxicity. While the relationship of tau and Hsp90 in coordination with these co-chaperones is still under investigation, it is clear that imbalances in these proteins with aging can contribute to disease onset and progression. This review highlights the current understanding of how the Hsp90 family of molecular chaperones regulates tau or other misfolded proteins in neurodegenerative diseases with a particular emphasis on the impact of aging.The ATP-dependent 90 kDa heat shock protein, Hsp90, is a major regulator of protein triage, from assisting in nascent protein folding to refolding or degrading aberrant proteins. Tau, a microtubule associated protein, aberrantly accumulates in Alzheimer's disease (AD) and other neurodegenerative diseases, deemed tauopathies. Hsp90 binds to and regulates tau fate in coordination with a diverse group of co-chaperones. Imbalances in chaperone levels and activity, as found in the aging brain, can contribute to disease onset and progression. For example, the levels of the Hsp90 co-chaperone, FK506-binding protein 51 kDa (FKBP51), progressively increase with age.  Aging is the biggest risk factor for developing a neurodegenerative disease, but the specific factors which cause these predominantly sporadic diseases are still under investigation  that are divided based on their molecular mass. The small Hsps regulate general protein aggregation, Hsp40s regulate Hsp70 ATP hydrolysis, Hsp70 folds proteins during translation, and Hsp90 maintains and triages a subset of clients  activity through TPR domains including cyclophilin 40 (CyP40) and five FK506-binding proteins: FKBP51, FKBP52, FKBP36, FKBP38, FKBPL/WISp39  have been shown to stimulate toxic tau aggregation , however their relationship to tau, if any, is still unknown at this point and so they will not be discussed in detail.XAP2, otherwise known as FKBP37 or Aryl hydrocarbon receptor interacting protein (AIP), contains a PPIase homologous domain. While a direct role of XAP2 in tau pathogenesis has not been described, studies have shown that XAP2 is activated by histone deacetylase (HDAC) 6, which has been linked to pathogenic tau , is repressed in aging. PP5 is contains a PPIase homology domain, but does not display classical PPIase activity, since it can bind FK506 , is involved in helping transfer client proteins from the early stages of protein maturation involving Hsp70 and Hsp40 to the later stages of the cycle involving Hsp90  is highly involved in the Hsp70-Hsp90 machinery acting not only as a co-chaperone, but also as an E3 ubiquitin ligase responsible for ubiquitin-dependent proteasomal degradation Edkins, . CHIP haes Table . OverexpDnaJC7, also known as Tpr2, simultaneously binds Hsp70 and Hsp90 via its two TPR domains  slows the ATPase activity of Hsp90 allowing a prolonged interaction between Hsp90 and its client proteins  works as a co-chaperone to stimulate the ATPase function of Hsp90 to regulate the folding and activation of client proteins. Aha1 interacts with Hsp90 independent of its nucleotide status and allows the Hsp90 ATPase cycle to skip the I1 phase, thus accelerating the progression of the ATPase cycle dramatically  interacts with Hsp90. One study used siRNA to screen several Hsp90 co-chaperones to investigate the effect on tau. This study found that reductions in S100A1 also led to massive reductions in both phospho- and total tau levels in cells  is able to interact with Hsp90 as a co-chaperone in order to inhibit its ATPase activity. One study found that FNIP1, in complex with FNIP2 and Hsp90, was able to stabilize the tumor suppressor folliculin  and found that in aged human samples there was an increase in Hsp90 under normal physiological conditions when compared to young samples (Njemini et al., In addition to the differences in expression levels of Hsp90, there are also changes in expression levels of co-chaperone proteins during the aging process. Almost all of the Hsp90 co-chaperones are repressed in aging, suggesting that these proteins could play important roles in maintaining homeostasis within the cell (Brehme et al., Hsp90 is able to form many unique heterocomplexes with different co-chaperones in order to regulate protein triage. Hsp90 heterocomplexes are unique in that there is usually a specific progression of co-chaperones that interact with Hsp90 (Schopf et al., In addition to altering Hsp90 heterocomplex composition and client selection, altered Hsp90 co-chaperone expression can interfere with degradation of aberrant proteins via the proteasome or autophagy. As mentioned above, aged cells are often inundated with misfolded and aggregated proteins, which can overload the Hsp90 chaperone network causing a negative spiral where there are not enough healthy chaperone molecules to refold or degrade aberrant proteins. In addition to the problems faced with an overwhelmed chaperone network, the proteolytic activity of the proteasome also declines with aging, and in fact Hsp90 has been shown to protect the proteasome from age-related, oxidative-dependent decline (Conconi and Friguet, There are many different PTMs that can affect Hsp90 including phosphorylation, acetylation, S-nitrosylation, oxidation, and ubiquitination; and all of these PTMs can impact the chaperoning function of Hsp90. Phosphorylation of Hsp90 leads to reduced chaperoning ability and phosphorylation of specific tyrosine residues can affect the ability of Hsp90 to interact with distinct client proteins (Zhao et al., Inhibition of the ATPase activity of Hsp90 has been shown to have positive outcomes in cell culture and animal models of tauopathy. Hsp90 ATPase inhibitors have been developed to target each of the three domains; with the majority of Hsp90 inhibitors targeting the N-terminal domain (Bhat et al., in vivo. There is still much to be done to develop compounds which target the Hsp90 chaperone network, but there are a lot of promising leads which can be targeted to develop disease-modifying therapeutics.In addition to offering more potential therapeutic targets, small molecules which modify Hsp90/co-chaperone interactions may also show more specificity for a specific pool of Hsp90 which may reduce the number of on-target side effects. There are very few Hsp90 co-chaperone targeting small molecules, and of these, only a handful of these have been investigated for their role in effecting tau. There is a Hop/Hsp90 complex specific inhibitor, C9, however, there is no available data on how chemical inhibition of this complex affects tau accumulation (Pimienta et al., in vitro and PP5 dephosphorylates tau restoring microtubule binding, suggesting that increasing the levels or activity of these co-chaperones could have a beneficial, neuroprotective role in diseases such as AD. These are just a few examples of how important maintaining the balance of Hsp90 co-chaperones is to homeostasis, and what can happen when they are altered in aging and disease. Hsp90 co-chaperones offer a unique target for potential therapeutics due to their specific roles within the Hsp90 machinery. Overall, more work needs to be done to develop BBB-permeable therapeutics to target discrete Hsp90 co-chaperones for the treatment of AD and other tauopathies.The Hsp90 chaperone machinery plays a huge role in both aging and neurodegenerative diseases. Hsp90 is one of the most highly expressed proteins in the cell and is involved in a myriad of cellular processes. Previous work has focused on inhibition of Hsp90 to triage misfolded proteins. There are also many co-chaperones that associate with Hsp90 and play their own roles in aging and neurodegeneration. As these Hsp90 co-chaperones change with age they can significantly impact the propensity for certain neurodegenerative diseases. FKBP51 steadily increases with age and both FKBP51 and Aha1 are induced in the AD brain suggesting that these two co-chaperones negatively affect tau pathology. On the other hand, both CyP40 and PP5 are repressed in aged and AD brains. CyP40 disaggregates tau fibrils LS wrote the manuscript. JK created the figures and edited the manuscript. LB critiqued and edited the manuscript drafts.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hepatitis C virus (HCV) still represents a major public health threat, with a dramatic burden from both epidemiological and clinical points of view. New generation of direct-acting antiviral agents (DAAs) has been recently introduced in clinical practice promising to cure HCV and to overcome the issues related to the interferon-based therapies. However, the emergence of drug resistance and the suboptimal activity of DAAs therapies against diverse HCV genotypes have been observed, determining treatment failure and hampering an effective control of HCV spread worldwide. Moreover, these treatments remain poorly accessible, particularly in low-income countries. Finally, effective screening strategy is crucial to early identifying and treating all HCV chronically infected patients. For all these reasons, even though new drugs may contribute to impacting HCV spread worldwide a preventive HCV vaccine remains a cornerstone in the road to significantly reduce the HCV spread globally, with the ultimate goal of its eradication. Advances in molecular vaccinology, together with a strong financial, political, and societal support, will enable reaching this fundamental success in the coming years. In this comprehensive review, the state of the art about these major topics in the fight against HCV and the future of research in these fields are discussed. Among infectious diseases, hepatitis C virus (HCV) still represents a major public health threat, with a dramatic burden from both epidemiological and clinical points of view. Chronically infected individuals are estimated to reach 150–170 million worldwide and estimates of incidence, performed in the United States by the Center for disease control and prevention (CDC), reported nearly 30,000 new HCV infections in 2013 [Although HCV infection is characterized by a global diffusion, its prevalence greatly differs according to geographic area , 4. CentAfter acute infection, 75% of infected subjects become chronically infected and approximately 20% of this population develops liver cirrhosis during the two decades after infection if left untreated . HoweverIn recent years, substantial advances have been made to understand HCV biology and to develop a new generation of effective direct-acting antiviral agents (DAAs) able to cure HCV. However, several challenges hamper an effective control of HCV spread worldwide. In fact, the emergence of drug resistance and the suboptimal activity of these therapies against diverse HCV genotypes have been observed and have been associated with treatment failure. Moreover, the high costs of these drugs and the high prevalence of HCV-infected individuals, especially in low-income countries, jeopardized the affordability for the healthcare system to treat all infected patients in developed countries and, even more, in developing countries , 9. FinaThis comprehensive review summarized the state of the art about three major unresolved issues in the fight against HCV: which are the perspectives for the universal screening of HCV? Do we need DAAs resistance testing in the future? How close is an effective preventive HCV vaccine?The rate of underdetection of HCV infection is still relevant because of clinical, educational, technical, organizational, and economic issues. In fact, recent estimates suggest that most of people with HCV remain undiagnosed or unaware of their HCV infection , 13.Another criticism is represented by the difficulty in early diagnosing HCV infection. Indeed, few people are diagnosed during the acute phase because it is usually asymptomatic . FurtherTherefore, it is crucial to implement the most sensitive and specific approaches to diagnose chronic HCV infection before the development of liver damage and to assure the linkage to care of infected patients .A testing strategy for HCV infections characterized by high sensitivity and specificity should be established.The WHO recommends offering the HCV serology test to individuals belonging to population with high HCV prevalence or who have a history of HCV risk exposure/behavior . Given tAmong serologic screening test, the enzyme immunoassays (EIAs) are generally used . EIAs arRecently, the possibility of a single serologic assay for the detection of HCV active infection has become available. This test is based on the identification of the HCV core antigen (HCVAg) that is detectable in acute infection almost at the same time as HCV RNA  and persIn consideration of improving the testing access, some countries such as US  and FranThe variability of the risk factors and history of behaviors linked to HCV infection, depending on the geographical setting and population studied, make the identification of the target population for screening policies challenging worldwide.In many high-income countries HCV screening recommendations are targeted to subjects with a history of HCV risk exposure or behavior and certain medical conditions and who belong to a population of known high HCV prevalence , 34. In In 2012, the Center of Disease Control and Prevention (CDC) and the US Preventive Services Task Force (USPSTF) extended the HCV screening recommendation to all persons born from 1945 through 1965, without prior ascertainment of HCV risk factors  –37. The In Japan, where the overall prevalence of HCV infection is comparable with the USA, but different transmission patterns determined different age-specific prevalence, the national screening for HCV in both the high-risk group and the general population from ages 40 to 70 started at 2002 and has demonstrated to be cost-effective in containing the epidemic , 47.In Europe, accurate estimates of HCV incidence and prevalence are not available; however prevalence estimates vary from 0.4%  to 1.5%  in Western Europe and from 0.7% (Czech Republic) to 4.5% (Moldova) in Central and Eastern Europe . HCV scrDeveloping nations, where the population-based prevalence of anti-HCV may reach as high as 11% (Mongolia) to 15% (Egypt), are less likely to be able to afford screening . Many ofConsidering the existing barriers that limit the implementation of screening strategies such as low provider knowledge about HCV and its related risk factors , 51–54, CDC and the US Preventive Services Task Force (USPSTF) recommend a one-time HCV test in asymptomatic persons belonging to the 1945 to 1965 birth cohort and subjects with exposures, behaviors, and conditions that increase risk for HCV infection.As regards the correct periodicity of testing persons at risk for ongoing exposure to HCV, evidence is lacking. Thus, physicians should determine the frequency of testing on the basis of the risk of reinfection. Owing to the high incidence of HCV infection among HIV-infected men who have unprotected sex with men and subjects who inject drugs –60, the In order to enter the patients in the cascade of care and thus provide them with the so-called “continuum of care,” the detection of HCV-infected patients should be followed by linkage to care via a physician referral with expertise in evaluation and treatment HCV infection , 61. HowThese gaps may be both patient- and practitioner-related. The most common patient-related barriers to treatment initiation include contraindications because of the presence comorbidities, lack of acceptance, and access of treatment, due to often asymptomatic course of the infection, long treatment duration and possible adverse reactions of the treatment, high cost, and distance to specialist –66. In pMoreover, the linkage to care is critical among patients coinfected with HIV , 69, thoGeneral practitioner-related barriers include lack of expertise in HCV treatment, lack of specialty referral resources, resistance to treating persons currently using illicit drugs or alcohol, and concern about cost of HCV treatment –73.Evidence-based interventions to address linkage to care have been summarized by Meyer JP and colleagues  and by tThese interventions include the integration of HCV screening, evaluation, and treatment center with other medical or social services, such as correctional facilities and programs providing needle exchange, substance abuse treatment, and methadone maintenance –76. MultIn order to address lack of access to specialists, models involving close collaboration between primary care practitioners and subspecialists also through telemedicine and multidisciplinary networks of specialists have been implemented , 78, 79.Additional strategies for improving linkage to and retention in care could be the use of patient navigators or care coordinators , 81; howThe extension of best and new strategies for linkage to HCV care is essential to optimize the impact of HCV treatment.Treatment of chronic hepatitis C has rapidly evolved from PEG-INF/RBV to more potent highly effective DAAs combination therapies. In particular, DAAs act on key stages of the HCV lifecycle and are classified on the basis of their molecular target and mechanism of action . Four caThe efficacy of treatment containing DAAs is very high in terms of sustained virological response (SVR); nonetheless 10–15% of therapeutic failure is observed in clinical practice, mainly associated with the selection of DAAs-resistant viral variants, resulting from mutations produced by amino acid substitutions in the target virus protein that reduce viral sensitivity to DAAs . ResistaSince DAAs are widely used for treatment of HCV, the role of resistance-associated variants (RAVs) is becoming clearer.Natural polymorphisms, before treatment, associated with resistance to NS3/4A, NS5A, and NS5B inhibitors have a considerable prevalence in DAAs naïve patients that is variable and depends on HCV genotype and subtype . These vIn the IFN-free regimen the presence of Q80K was associated with treatment failure only in a small sample size of patients with genotype 1a and cirrhosis: a phase 3 study (OPTIMIST-2) evaluated the efficacy and safety of 12 weeks of simeprevir plus sofosbuvir in HCV genotype 1 infected treatment-naïve or treatment-experienced patients with cirrhosis. Of the 72 patients with chronic HCV genotype 1a, SVR rate was lower for the 34 with Q80K at baseline than for the 38 without Q80K (74% versus 92%) . For thiReduced sensitivity to the first generation protease inhibitors (PIs) (boceprevir and telaprevir) and to the more recent PIs was associated with other NS3/4A mutations, such as those involving the amino acid positions V36, T54, R155, A156, and D168. The list of the major NS3/4A RAVs is reported in The RAVs conferring resistance to NS5A inhibitors more frequently occurred as natural variants in HCV G1 infected patients naïve to DAAs  and seemRecently, the FDA recommended testing for the presence of virus with NS5A resistance-associated polymorphisms  in genotype 1a, regardless previous treatment history and cirrhosis status, before the combination therapy with grazoprevir and elbasvir to determine dosage regimen and duration (for 12 or 16 weeks and if ribavirin should be added) .It is noteworthy that the persistence of NS5A RAVs has been observed in about 85% of patients with treatment failure over 1-2 years after the start of therapy , 105–107Regarding the NS5B RAVs, no cross resistance is observed across currently approved nucleotide and nonnucleoside polymerase inhibitors. Nucleos(t)ide inhibitors demonstrate activity against different HCV genotypes and have a high resistance barrier . The priResistance testing at baseline and following treatment failure is not yet indicated, as more data are needed to demonstrate its role in clinical practice, but the unresolved issue is if it may be useful to individualize the best treatment option for each patient in the future. With the exception of Q80K and NS5A RAVs detection in G1a patients before starting simeprevir plus PEG-INF/RBV and grazoprevir plus elbasvir, respectively, whether resistance mutation testing should be done in treatment-naïve patients remains controversial.Some authors highlight the importance of resistance testing in patients who fail multiple DAAs for deciding retreatment and selecting salvage therapy , 109. MoThe development of an effective HCV vaccine is hampered by several factors. First, HCV is characterized by an extraordinary genetic variability resulting from the lack of proofreading activity of the NS5B RNA-dependent polymerase . This deIn addition to evading antibody and CTL recognition by passively mutating its genome in response to immune pressure, HCV exploits several further strategies to escape adaptive immune response and achieve a high rate of chronic infection. In fact, HCV, through a yet-unknown mechanism, fails to properly initiate the CD4+ T cell response at the beginning of infection and determines a rapid immune exhaustion and depletion of CD8+ T cells –117. MorThe weaker and ineffective adaptive immune response to HCV mainly contributes to the infection progressing into a chronic state. On the other hand, these immune evasion mechanisms have relevant implications in the development of the HCV vaccine. In fact, an effective vaccine should be able to induce strong neutralizing antibodies as well as powerful cellular immune responses during the very first stages of the HCV infection, before the virus has the chance to activate its many immune escape mechanisms .A further barrier that has challenged the research on HCV vaccine is the lack of convenient experimental model systems for the study of HCV pathogenesis and vaccine design. To date, the only suitable infectious animal model is the chimpanzee. However, some ethical issues, the high cost of acquiring and maintaining the chimpanzees, and their limited supply restricted the use of these animals and the statistical power of studies , 120. FuSeveral strategies have been adopted to develop an effective preventive HCV vaccine. They ranged from the traditional strategy of producing recombinant envelope proteins combining them with adjuvant substances to complex manufacturing of viral vectors directing the expression of multiple viral antigens. All strategies have been targeted to enhance the T cell response and, in particular, to determine a long-lasting cellular immune responses involving helper CD4+ and CD8+ T cells rather than only improving humoral responses.Despite two decades of research efforts, few HCV candidate vaccines have reached the clinical trials phase and the evidences on both the efficacy and safety of these vaccines in humans remain limited .The first prophylactic HCV vaccine tested in human beings was a C-terminally shortened recombinant E1 protein adjuvant with aluminum hydroxide, called T2S-918/InnoVac-C, demonstrating good tolerability and good antibody response against E1. Moreover, a robust specific cellular immune response towards E1 was stimulated in all vaccinees . HoweverFurther HCV prophylactic vaccines based on recombinant proteins combined with adjuvant substances have been developed and evaluated in clinical trials.A recombinant E1/E2 heterodimer vaccine (derived from HCV 1a) adjuvant with MF59C was tested in phase I clinical trial involving 60 healthy subjects. Vaccine demonstrated inducing neutralizing antibodies and T cell responses to E1/E2 in all subjects. Although the vaccine was safe and well-tolerated, its usage was prevented by manufacturing difficulties .Another approach, based on recombinant protein, consisted of combining recombinant HCV core protein with an adjuvant substance, called ISCOMATRIX™. In a phase I clinical trial, conducted on 30 healthy volunteers, all subjects except one demonstrated antibodies against HCV core protein, without indication of a dose response. However, T cell responses were detected in only two subjects who received the highest dose of vaccine. The candidate vaccine was generally well-tolerated .Also the development strategies based on HCV peptides vaccine that were able to induce HCV specific T cell responses by presenting vaccine peptide to the T cell receptor via HLA molecules have obtained some encouraging findings.In particular, a peptide vaccine, known as IC41 and consisting of five synthetic peptides derived from conserved regions of core, NS3 and NS4 proteins of HCV genotypes 1 and 2 with a poly-L-arginine adjuvant, was evaluated in 128 healthy volunteers in a phase I clinical trial where it resulted in being safe and well-tolerated . On the More innovative strategies to develop an effective HCV preventive vaccine include the manufacturing of DNA vaccine and viral vectors expressing HCV genes. DNA vaccines showed the capability of inducing cytotoxic lymphocyte responses in animal models; however, the induced immunity is often brief, weak, and unlikely to be effective in infection prevention. Viral vectors able to express foreign antigens represent an effective tool to induce a broader CD4+ and CD8+ T cell responses, compared to peptide vaccines.Two preventive vaccines based on human adenovirus rare serotype 6 (HADV6) and chimpanzee Ad 3 (ChAd3) expressing the HCV nonstructural proteins were tested in a phase I clinical trial conducted on 36 healthy volunteers. The study demonstrated that both vaccines induced by specific T cell responses against multiple HCV proteins and T cell were capable of recognizing heterologous HCV strains (genotypes 1a and 3a). These data suggested that an adenoviral vector strategy may induce sustained T cell responses of a magnitude and quality associated with protective immunity, thus encouraging studies of novel prophylactic vaccines for HCV .On the basis of some promising data obtained in phase I clinical trials, different types of prophylactic HCV vaccine are approaching phase II and phase III clinical trials . In the Research efforts for the development of alternative therapeutic options have been made contextually to the development of DAAs and the research on preventive vaccines. To date, several approaches have been adopted in the development and production of HCV therapeutic vaccines including peptide vaccines, recombinant protein vaccines, DNA vaccines with different carriers, and virally vectored vaccines.γ responses and SVR rates within patients treated with interferon [Some studies were conducted among chronic hepatitis C patients, obtaining promising results in terms of T cell proliferation and IFN-terferon , 131.Clinical trials, conducted by administering candidate HCV therapeutic vaccines in HCV chronic infected patients, have demonstrated HCV specific immune responses and transiently reduction of viral RNA, but the vaccines were not able to completely clear HCV infection or consistently reduce viral titers , 133.Some authors discussed the possibility to improve the efficacy of these vaccines by prior treatment with DAAs to first suppress HCV viremia , but theA further therapeutic option to cure HCV is represented by neutralizing monoclonal antibodies (mAbs) directed against HCV envelope glycoproteins E1 and E2, which are involved in the HCV entry into host cells or against phospholipids expressed on infected host cells , 135. WiTwo studies involved anti-HCV/2 human mAb HCV-AB68 demonstrating only a modest and short-lasting reduction in viremia , 137. MoAs regards mAbs against host receptors, a phase II unpublished clinical trial involving bavituximab was conducted to study the safety profile and early virological response in HCV genotype 1 infected patients treated with this mAb in combination with ribavirin in comparison with patients treated with PEG-INF/RBV .In recent years, novel mAbs with immunomodulatory effects have been explored. Among these, mAbs that target programmed death 1 (PD-1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and CD3 receptors were studied in phase II clinical trials with encouraging results in terms of safety, tolerability, and viral response –141.The most likely future for clinical use of mAbs is represented by regimen for HCV treatment consisting of mAb cocktails or combination of mAbs with other available drugs with the main goal of avoiding viral escape and the development of resistance.Substantial advances have been made in HCV research and treatment in recent years. Although highly effective anti-HCV drugs are now available, these treatments remain poorly accessible because of their high costs; moreover, their efficacy is challenged by the high frequency of resistance-associated mutations. A further major obstacle for the control of the disease is represented by the lack of availability of an effective screening strategy to identify all people in need of treatment. Therefore, even though new drugs may contribute to impacting HCV spread worldwide, substantially modifying the natural history of the disease, eradication will be reached only through the development of an effective prophylactic vaccine. Future research directions should bridge this gap and progress in the comprehension of biological and immunological mechanisms of the disease and advances in molecular vaccinology, together with a strong financial, political, and societal support, will enable reaching this fundamental success in the coming years.