Tumor microenvironments are the result of cellular alterations in cancer that support unrestricted growth and proliferation and result in further modifications in cell behavior, which are critical for tumor progression. Angiogenesis and therapeutic resistance are known to be modulated by hypoxia and other tumor microenvironments, such as acidic stress, both of which are core features of the glioblastoma microenvironment. Hypoxia has also been shown to promote a stem-like state in both non-neoplastic and tumor cells. In glial tumors, glioma stem cells (GSCs) are central in tumor growth, angiogenesis, and therapeutic resistance, and further investigation of the interplay between tumor microenvironments and GSCs is critical to the search for better treatment options for glioblastoma. Accordingly, we summarize the impact of hypoxia and acidic stress on GSC signaling and biologic phenotypes, and potential methods to inhibit these pathways. Glioblastoma (GBM), also known as a World Health Organization grade IV astrocytoma, is the most common and aggressive primary brain tumor in adults. From 2012-2016, the average incidence of malignant brain tumors in the U.S. was 7.08 per 100,000, and GBM accounted for about 15% of all central nervous system tumors, and close to half of all malignant brain tumors diagnosed. GBMs have a disproportionate incidence rate by sex and race, occurring 1.58 times more often in males than females, 1.95 times more often in whites than blacks, and 2.39 times more often in whites than Asian or Pacific Islanders in vitro via neurosphere formation assays and the expression of molecular markers  in symmetric and asymmetric division studies A characteristic that tumor cells share with non-neoplastic stem cells is their ability to proliferate indefinitely. Accordingly, many tumors appear to be maintained via a hierarchical organization that consists of slowly-dividing stem cells, precursor cells, and differentiated cells Through genetic and epigenetic characterization, a number of adult GBM subtypes have been defined GSCs assist in establishing the tumor microenvironment through complex crosstalk within their niche. The two most commonly described niches in which GSCs have been characterized are the perivascular and the perinecrotic niches Other GSC-associated niches, the peri-hypoxic, peri-immune, and extracellular matrix niche are reviewed in-depth in Aderetti et al. 2) of 5-9 mm Hg and an acidic pH of 6.8 or lower 2, and ratio imaging to measure pH, have demonstrated these microenvironments occur independently Figure 1. As hypoxia and/or low pH correlate with many aspects of tumorigenicity, including therapeutic resistance, patient survival, and tumor invasion, understanding tumor microenvironmental effects on GBM growth and recurrence is critical A pathologic hallmark of GBMs, pseudopalisading necrosis, often occurs near a collapsed blood vessel, and tends to be surrounded by cells surviving in a hypoxic and often, acidic zone 2) and extracellular pH compared to non-neoplastic tissue, as well as the development of regions of extreme hypoxia and low pH in vitro experiments continue to be performed in atmospheric oxygen (~21% O2 or 159 mmHg) using buffered media to minimize pH changes  in vitro approaches, a shift towards improved modeling of physiologic microenvironments is desperately needed.The presence of hypoxia promotes the use of anaerobic glycolysis to generate energy and essential precursors (nucleic and amino acids as well as lipids) required for cell growth Hypoxia-responsive signaling in both tumors and nonmalignant tissue is mediated through transcription factors called hypoxia-inducible factors (HIFs). HIFs exist as heterodimers, consisting of an alpha and a beta subunit. The beta subunit is constitutively expressed in all cells, while the alpha subunit exists in three isoforms, which are unstable and rapidly degraded in the presence of oxygen Two well-characterized HIF alpha isoforms are HIF1α and HIF2α. Suggesting that these two isoforms program distinct responses to changes in the microenvironment, HIF2α expression can be stabilized at higher oxygen levels (approximately 5%) than the more severely hypoxic conditions , where HIF1α is induced in vivo-like multicellular tumor spheroids derived from GBM short-term culture with tumor stem cell properties, indicating that tumor cell phenotypes associated with stemness and chemoresistance may depend on the oxygen tension surrounding that particular tumor cell as well as cellular interactions GSCs are identified using cell surface markers or cell selection techniques that take advantage of phenotypes increased in this cellular subset, with sorting or validation using GSC markers that can include CD133, OCT4, and SOX2 among others in vivoFigure HIF1α and HIF2α are both critical for GSC function and can be expressed in different patterns dependent upon the level of hypoxia as mentioned above. The stabilization of HIF1α leads to the expansion of the GSC population within the bulk of the tumor, which is, in part, mediated by the extracellular signaling related kinase (ERK) and the PI3K/AKT pathways in vitro and decreased tumor growth in vivoUnder less extreme hypoxia, when HIF2α is preferentially expressed in GSC populations, HIF2α levels were regulated not only via post-translational modification but also through increased transcription in vivo, relatively few studies focus on the biological effects of acidic stress in GBM or other solid tumor cells. The extracellular pH of solid tumors including GBMs has been measured as low as 5.9 with an average pH of 6.8, whereas the normal brain pH is approximately pH 7.1 Extracellular< pHIntracellular), drugs altering proton export or production, and buffer therapies are being explored as novel treatments in vivo and improved the efficacy of immunotherapy Although low pH is recognized to be an important component of the tumor microenvironment and has been shown to exist in the absence of hypoxia Applying acidic stress to GBM cells upregulates VEGFA mRNA via the ERK1/2 and MAPK pathway, which enhances AP-1 binding to the VEGF promoter Acidic stress also promotes glioma stem cell phenotypes independently of hypoxia, but these may still involve HIF2α expression. Exposure of GBM cells to acidic stress increased GSC marker expression as well as self-renewal and tumor growth CAIX and CAXII contribute to cellular growth by maintaining extracellular acidification and an alkaline intracellular pH in response to tumor acidosis in vivo, suggesting that this enzyme is active in the necrotic zones of GBM tumors Figure Acidic stress-induced changes in metabolism occur in GBM, and GSCs can adapt to different tumor microenvironments via shifts between glycolysis and oxidative respiration One of the most well-defined biological effects of hypoxia and HIFs in solid tumors including GBMs is the promotion of new blood vessel formation, or angiogenesis. While gliomas first support their growth by taking advantage of existing brain blood vessels in a process called vascular co-option, hypoxia will still ultimately occur as the tumor grows. A hypoxic microenvironment has also been linked to defective neovasculature, resulting in significantly poorer prognosis in vitro. Experiments with fluorescently labeled GSCs demonstrated that GSCs can incorporate into the vasculature of GBM xenografts. However, the lack of endothelial cells with common driver mutations present in GSCs suggests that transdifferentiation is very rare in human GBMs GSCs express VEGF receptor 2 (VEGFR2), permitting an autocrine loop of VEGF/VEGFR signaling that can become further activated with increased VEGF under hypoxia VEGF production may also be regulated by the pro-angiogenic chemokine CXCL12, also known as stromal cell-derived factor 1, in GSCs. CXCL12 is highly expressed in GSCs Solid tumor cells in a hypoxic niche contribute to tumor aggressiveness and metastases, with specific roles for cancer stem cells that can have epithelial-to-mesenchymal transition (EMT) phenotypes 3 Adenosine Receptor was demonstrated to promote the migration/invasion of GSCs via a HIF-2-dependent mechanism Hypoxia is an important modulator of GSCs in the context of epithelial to mesenchymal transition, which often leads to greater migration and invasion and ultimately, tumor recurrence The increased invasiveness of GBMs in hypoxic condition has also been linked to their enhanced hyaluronic acid production in vivo in abundance in areas of pseudopalisades, where glioma cells are actively migrating A recent study focused on the importance of recombination signal binding protein for immunoglobulin kappa J . CBF1 is a master transcriptional regulator of the notch signaling pathway and contributor of GSC maintenance as well as an important regulator of EMT in GSCs Chromatin remodeling regulates the GSC state as well as therapeutic resistance, and a rapidly developing area of interest is regulation of the epigenetic landscape by the hypoxic tumor microenvironment. While it is known that there are many epigenetic alterations that are involved in the initiation and progression of brain tumors, much less is known about how hypoxia contributes to these mechanisms. The discovery that expression of mixed-lineage leukemia 1 (MLL1), a histone methyltransferase specific for the lysine 4 methylation of histone H3, is increased in GSCs by hypoxic conditions has driven interest in this field. MLL1 enhances hypoxia response gene expression, including VEGF, by enforcing expression of HIFs, mainly HIF2α, in hypoxia In GSCs, there are also established roles for some Jumonji Domain-Containing proteins that modify histones and are known to be oxygen dependent. GSCs that survived kinase inhibitor treatment had differential H3K27me3 profiles, and the Jumonji Domain-Containing Protein 3 (JMJD3)/Lysine Demethylase 6B (KDM6B) was implicated in cellular maintenance Hypoxia and acidic stress also repress the epigenetic modifier chromodomain-helicase-DNA-binding protein 7 (CHD7) Although we have thus far focused on direct effects of hypoxia on GBM cells to regulate tumor growth, the tumor microenvironment also impacts GBM cells indirectly via paracrine effects mediated through nearby non-neoplastic cells. Under a hypoxic microenvironment, macrophages undergo phenotypic changes that activate the expression of mitogenic and proangiogenic cytokines and enzymes Macrophage migration inhibitory factor (MIF) is a type of cytokine released by leukocytes. MIF levels have been associated with immunosuppression as well as angiogenesis, cell differentiation, and cell proliferation in tumor cell lines Gliomas have been characterized as immunologically “cold” tumors that have a highly immunosuppressive microenvironment, particularly in terms of adaptive immunity. Using immunogenomic characterization data compiled by The Cancer Genome Atlas (TCGA), Thorsson et al. characterized these tumors as immunologically quiet and lymphocyte depleted, as the current understanding of immune activity in these tumors is that it is largely driven by monocytes and innate immunity The hypoxic microenvironment of GBMs has many effects that result in tumor cell resistance to chemotherapy and radiation. These include effects on DNA repair, DNA stability, ABC transporter expression, cell cycle checkpoint protein expression, and vasculature function. As a broad indicator of GSC survival in the hypoxic microenvironment, the CD133-positive GSC fraction are more resistant to apoptosis under hypoxia Changes in the ability to repair DNA in the hypoxia microenvironment lead to therapeutic resistance Cell surface transporter proteins are considered important mechanisms of drug resistance and are affected by hypoxia in GSCs. The ATP-binding cassette (ABC) transporters are a class of proteins that have a wide range of biological effects. These transporters promote therapeutic resistance by removing chemotherapy from the tumor cells, minimizing the time that the drugs have to be effective Hypoxic regions form when tumor cells lack proximity to a functional blood vessel leading to low oxygen tensions. Cells located at least 70 µm away from the nearest functional blood vessel do not receive adequate amounts of oxygen, leading to a conversion to a hypoxic cell state Table 1 and Figure 4) focused on improved imaging modalities that identify tumors by regions of hypoxia or acidosis. An innovative study synergized radiomic features in MRI scans and RNA expression data from hypoxia markers to generate a hypoxia enrichment score (HES) that was highly predictive of the survival of GBM patients in vivo have proven challenging to develop but have recently gained traction. Cutting-edge MRI techniques that highlight regions of acidosis are being developed as novel imaging tools to improve patient care and explore the pathophysiology of brain tumors Diagnostic monitoring of tumor progression post-resection can be complicated by ischemia, and there are a number of clinical trials  is an intracellular kinase that regulates cell growth and cell cycle progression via signaling from nutrients and growth factors Mitogen-activated protein kinase (MAPK) can be activated by stress signaling such as that resulting from hypoxia Nitro compounds, N-oxides, and quinones are bioreductive prodrugs that target the hypoxic tumor cells by being reduced by intracellular oxidoreductases in an oxygen-sensitive manner to form cytotoxins in vivoThe quinone mitomycin C (MMC) is activated under hypoxic conditions in tumor cells in vitro or that we still do not fully understand. It is of our opinion that more accurately modeling the tumor microenvironment in vitro and consideration of its links to differentiation state and therapeutic resistance will allow for more efficient transfer of novel therapeutics from in vitro studies to the clinic. In addition, added emphasis on targeting these microenvironments during therapeutic design may lead to desirable improvements in standard of care. Considering this, hypoxia and acidic stress are major micro-environmental stresses that occur commonly in GBM solid tumors. This is especially important when considering the GSC niche that can be found within hypoxic and acidic zones. Establishing efficient models of these microenvironments in vitro and targeting these in vivo to exploit resistant cell populations, i.e. GSCs, is of substantial importance when identifying novel treatment strategies to synergize with standard of care.Glioblastoma remains a formidable tumor that is notoriously difficult to prevent from recurrence, which contributes to abysmal survival in patients. Compared to solid tumors outside the brain, glioblastoma treatment is also more complex due to the presence of the blood-brain-barrier: development of certain types of treatment modalities is precluded without novel delivery methods that are able to circumvent the restrictions of the blood-brain-barrier. For example, many conventional chemotherapies and antibodies that target antigens in and on the surface of tumor cells have difficulty crossing an intact blood-brain-barrier, although there are antibodies that work well in the bloodstream without having to cross into the tumor . While the tumor associated blood-brain-barrier is remarkably fenestrated and is constantly being remodeled, the blood-brain-barrier in regions where invading tumor cells reside may be intact preventing eradication of the disease. Continual failures with novel treatment strategies suggest that there are major characteristics of these tumors that are not adequately being modeled for drug testing


Pattern recognition receptors (PRRs) are a pivotal part of the immune system. They are distributed in almost every site of higher organisms, able to recognize foreign pathogens or unwanted remnants of metabolism and mount innate immune response. Moreover, PRRs create bridging signaling to initiate adaptive immunity. The liver being the largest organ of the body, exposed to myriads of foreign substances often being immunogenic, is well equipped with PRRs. They act as sentinels of the organ, both in health and disease. In viral hepatitis C at least two of them, RIG-1 and TLR3 sense HCV, induce protective interferon production and create proinflammatory status. The hepatitis B virus is apparently invisible to PRRs, which has recently been denied. Besides, they are active in the course of infection. In liver injury and hepatic fibrogenesis Toll-like receptors (TLRs), predominantly TLR4, TLR3 and TLR9 are associated with gut microflora-related products and DNA from dying hepatocytes, lead to the activation of hepatic stellate cells. The latter initiate production of fibrillar collagens, the main agents forming hepatic fibrosis. Tumor cells of primary liver cancer also express PRRs, mainly TLRs. In concert with non-resolving liver inflammation, they are considered pivotal factors leading to carcinogenesis. Eukariota but absent in Prokariota, such as unicellular bacteria, protozoa, viruses and fungi  are evolutionally very old, germ line encoded receptors expressed on cell membranes and also in the cytoplasm of several cells but predominantly on those of the immune system. They are present in most of +T cell infiltration and liver injury in mice.Apart from the significance of PRRs in various pathological conditions of the liver described below, there is a growing interest in the role of these receptors in a normal organ. For example, Lang et al.  demonstrExpression of PRRs in hepatitis B and C was noticed by several authors, including ourselves  and C virus-infected hepatocytes. It was shown that type I IFN production is only made via TLR7 signaling, when both cell types are in direct contact but pDCs do not become infected, in spite of apparent infusion of viral RNA. Moreover, IFN production requires the concordance of cell species (human/human) and highly differentiated hepatocytes. It was concluded by the authors that this phenomenon constitutes a novel cell–cell viral RNA transfer process, which might have an important role in the course and treatment of HCV-infected patients  therapy on regulatory T cells (Tregs) and T helper (Th)17 cell response to TLR2 agonist Pam3Csk4 in treated HCV patients were researched . It was shown that each of the three adaptors markedly reduced the levels of HBV mRNA and DNA in both cell types. Studies on TLRs polymorphisms in HBV infection also did not exclude the role of interactions of viral molecules with TLRs in disease. The TT genotype of the missense variant TLR5 was found to be significantly more frequent in the control group than in HBV patients, indicating the protective effect of this abnormality against infection . The virus is apparently “invisible” to the innate immune system, which was proven in both animals and human  formation and development has been found to be TLR9 pathway activation dependent. Plasma from individuals with NASH contains high amounts of highly oxidized mitochondrial DNA, mostly in microparticles from hepatocytes. This DNA, relatively CpG rich, is able to activate TLR9. Mitochondrial DNA acts as a ligand of TLR9, which results in the stimulation of proinflammatory cytokines in hepatic microenvironment. Total body TLR9 deletion results in the disappearance of NASH in high fat diet (HFD) fed mice. TLR9 antagonist such as IRS954 prevents NASH formation in HFD fed mice  may lead to autoimmune hepatitis by releasing the ligands for TLR2/TLR4. Upregulated cytokines and chemokines in the liver activate STAT3 and STAT4 in hepatic CD4+CD25+ Treg cells suppressing FOXp3 gene expression resulting in the reduction of suppressive activity of Tregs.Malaria parasites infestation—there is a growing opinion that PRRs, mainly TLRs recognize Plasmodium components and shape innate, albeit inadequate immune response (Kalantari Plasmodium yoelli that grows in mouse liver. The drug was also able to clear parasites from the liver after establishment of infection.Gut–liver axis—bile acid metabolism connects the liver with the intestine. Dysregulation of bile acids leads to intestinal dysbiosis that promotes pathogenic microorganisms entrance in the liver via portal vein and subsequent hepatic inflammation. Dysbiosis is also due to faulty bidirectional liver-microbiome crosstalk (Adolph et al. Liver transplantation—in liver transplant recipients, the damage of cells during ischemia–reperfusion injury poses a significant clinical problem. Sosa et al. (.a et al.  found TLPattern recognition receptors are still not fully appreciated by clinical hepatologists as an important factor in the management of liver diseases. The data provided in this article suggest that the complex nature of their function may hamper direct transfer of this knowledge to clinics. Nevertheless, the growing number of PRR synthetic ligands, showing therapeutic effect in various liver pathologies appears to be a promising agent for future liver therapies.


The trigeminal ganglion with its three trigeminal nerve tracts consists mainly of clusters of sensory neurons with their peripheral and central processes. Most neurons are surrounded by satellite glial cells and the axons are wrapped by myelinating and non-myelinating Schwann cells. Trigeminal neurons express various neuropeptides, most notably, calcitonin gene-related peptide (CGRP), substance P, and pituitary adenylate cyclase-activating polypeptide (PACAP). Two types of CGRP receptors are expressed in neurons and satellite glia. A variety of other signal molecules like ATP, nitric oxide, cytokines, and neurotrophic factors are released from trigeminal ganglion neurons and signal to neighboring neurons or satellite glial cells, which can signal back to neurons with same or other mediators. This potential cross-talk of signals involves intracellular mechanisms, including gene expression, that can modulate mediators of sensory information, such as neuropeptides, receptors, and neurotrophic factors. From the ganglia cell bodies, which are outside the blood–brain barrier, the mediators are further distributed to peripheral sites and/or to the spinal trigeminal nucleus in the brainstem, where they can affect neural transmission. A major question is how the sensory neurons in the trigeminal ganglion differ from those in the dorsal root ganglion. Despite their functional overlap, there are distinct differences in their ontogeny, gene expression, signaling pathways, and responses to anti-migraine drugs. Consequently, drugs that modulate cross-talk in the trigeminal ganglion can modulate both peripheral and central sensitization, which may potentially be distinct from sensitization mediated in the dorsal root ganglion. The trigeminal ganglion is outside the blood–brain barrier  and substance P were first immunohistochemically described in dorsal root ganglion neurons, with broad overlap in shared secretory vesicles but double the number of vesicles with CGRP immunoreactivity  channels or their isolectin B4 (IB4) binding. The first group containing the peptidergic neurons is sensitive to nerve growth factor (NGF), while the second group is sensitive to glial cell line-derived neurotrophic factor (GDNF) during its development , a seven transmembrane spanning protein, the receptor activity-modifying protein 1 (RAMP1), a single membrane-spanning protein, and the receptor component protein (RCP) an intracellular component Fig. . CLR comIn the trigeminal ganglion of several species, neurons of mainly medium sizes and glial cells (Schwann cells and SGCs) have been found to be immunopositive for both CLR and RAMP1  and RAMP1, is now established  receptors, receptors for substance P, has indirectly been shown by functional studies in the rat trigeminal ganglion. The activity of spinal trigeminal neurons with afferent input from inflamed temporomandibular joint and facial skin was decreased by injection of an NK1 receptor antagonist into the trigeminal ganglion   mRNA has been detected in rat trigeminal ganglion neurons and inhibition of both receptors reduced the expression of downstream proteins associated with sensitization of peripheral nociception in a model of temporomandibular joint inflammation  and metabotropic glutamate receptors (mGluR), have been localized in rat trigeminal ganglion neurons  and muscarinic acetylcholine receptors (mAchR) has been found on the mRNA level and with immunohistochemistry in rat trigeminal ganglion  and Cl− currents recorded in whole cell patch clamping showed that the subunits form functional GABA receptors. The authors discussed a possible GABA-driven inhibition of neurons within the trigeminal ganglion. Glycine receptors were also found in rat trigeminal ganglia using immunohistochemistry  binds to several types of G-protein-coupled 5-HT receptors, only the 5-HT3 receptor is a cation channel. Three subtypes of the Gi-protein-coupled serotonin 1 receptors are targets of antimigraine triptans (5HT1B/1D) or “ditans” like lasmitidan (5HT1F) and have been found by immunohistochemistry in the rat trigeminal ganglion  or form cation channels (P2X). Expression of different subtypes of P2X receptors, predominantly P2X2 and P2X3, was described in rat trigeminal ganglion neurons of small and medium size, frequently co-expressed with neuropeptides  family. TRP receptors form transduction channels in peripheral sensory endings and may also be involved in synaptic transmission at the central afferent terminals  controlled by calcium binding proteins usually involved in vesicular exocytosis , predominantly the ASIC3 subtype, which is suggested to contribute to headaches under acidic or inflammatory conditions , a TRPA1 receptor activating sibling of NO, may play a similar role in the ganglia by increasing the release of CGRP  and cytokines in SGCs ? Both ganglia serve similar roles of encoding somatosensory modalities such as pain, touch, temperature and proprioception. However, despite their functional overlap, there are distinct differences in their ontogeny, gene expression, including distribution of CGRP receptors, and responses to antimigraine drugs.The trigeminal ganglion and the dorsal root ganglia arise from different embryological origins. Sensory neurons in the trigeminal ganglion are from a mixed origin of both cranial neural crest cells and the trigeminal ophthalmic and maxillomandibular placodes (Baker and Bronner-Fraser In the adult trigeminal and dorsal root ganglion neurons, there are unique patterns of expression that mirror the embryological differences. Using transgenic mice with GFP expression in only sensory cells, neurons in the DRG and trigeminal ganglion were separated from non-neuronal cell types by FACS and analyzed by RNAseq (Lopes et al. With respect to CGRP signaling in dorsal root and trigeminal ganglion, there are some potential differences in receptor expression, distribution and site of action. Using immunohistochemistry, in adult trigeminal ganglion neurons the CGRP receptor components RAMP1 and CLR were predominantly found in medium-sized cell bodies, presumably with Aδ fibers, whereas CGRP expression was predominantly seen in small neurons with unmyelinated C-fibers (Lennerz et al. Likewise, CGRP may act at different presynaptic and postsynaptic sites when released from the trigeminal and dorsal root ganglion neurons, although there are conflicting reports. In the dorsal horn, CLR expression was initially found predominantly on cell bodies and dendrites of second-order neurons (Ye et al. B/D receptors for triptans are present on both trigeminal and dorsal root ganglion neurons, yet to date it appears that triptans are efficacious for only relief of pain initiated in the trigeminal pathway (Ahn and Basbaum Responses to antimigraine medication may also be a point of difference between the two neuron populations. The 5HT1Overall, while dorsal root and trigeminal ganglion neurons are similar sensors of peripheral stimuli, they have different embryonic origins, transcription patterns, signaling pathways, and responses to antimigraine medications. Differences between the two groups of sensory neurons may provide clues for selective targeting of CGRP and other targets that modulate peripheral and central sensitization in pain states.


With conventional treatments for primary immunodeficiency diseases (PIDs), such as allogeneic stem cell transplantation or autologous gene therapy, still facing important challenges, the rapid development of genome editing technologies to more accurately correct the mutations underlying the onset of genetic disorders has provided a new alternative, yet promising platform for the treatment of such diseases. The prospect of a more efficient and specific therapeutic tool has pushed many researchers to apply these editing tools to correct genetic, phenotypic, and functional defects of numerous devastating PIDs with extremely promising results to date. Despite these achievements, lingering concerns about the safety and efficacy of genome editing are currently being addressed in preclinical studies. This review summarizes the progress made toward the development of gene editing technologies to treat PIDs and the optimizations that still need to be implemented to turn genome editing into a next-generation treatment for rare monogenic life-threatening disorders. Although the majority of PIDs are inherited, with an incidence rate approximately between 1 in 5,000 to 1 × 106 live birth, acquired forms of the disease have been also described.3Primary Immunodeficiency Diseases (PIDs) constitute a heterogeneous group of rare genetic disorders impairing the development, regulation, and function of the immune system. Increased susceptibility toward infections, autoimmunity, and inflammatory complications as well as risk of developing malignancies are major clinical manifestations observed in individuals affected by PIDs.4 While the above treatments may provide adequate temporary support, allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation has proven a definitive cure for many PIDs with a hematopoietic cell background. Unfortunately, the limited availability of Human Leukocyte Antigen (HLA)-matched donors poses a constraint for many patients, and although transplantation using HLA-mismatched donors is increasingly successful, it comes with significant risks, including graft versus host disease and graft rejection leading to incomplete immune cell reconstitution and higher risks of mortality and long-term morbidity. Autologous gene therapy provides an attractive option by genetically correcting the patient's own HSPCs through the use of viral vectors.5 By improving safety features of viral vectors, current phase I/II gene therapy trials have been applied to the treatment of various PIDs, including adenosine deaminase-severe combined immunodeficiency (ADA-SCID), X linked-SCID (SCID-X1), chronic granulomatous disease (CGD) and Wiskott–Aldrich syndrome (WAS) with largely successful clinical outcomes.6–10 Despite this, long-term safety/efficacy follow-up studies are still required for most of these gene therapy products to be approved as medicinal drugs,11 as they come with some limitations, including the semirandom integration pattern of viral vectors and often unregulated transgene expression in transduced cells.12The management of many PIDs is based on preventing infection through prophylaxis and where necessary anti-inflammatory medication.in situ ensures that a physiologically regulated expression of the correct gene is more likely to be preserved in edited cells. In this review, we will illustrate the different types of gene editing platforms that have been developed so far and how these technologies have been utilized as therapeutic applications to treat severe forms of PID before, focusing on key challenges and mandatory requirements that gene editing needs to fulfill before translation into a cell therapeutic medicine for routine clinical application.Precise targeting by means of gene editing has recently emerged as an alternative technology to overcome the limitations of conventional gene therapy. Engineered endonucleases that introduce double-strand breaks (DSBs) at specific sequences in the genome offer much more control over viral vector integration. Moreover, the site-specific correction of the disease-causing mutant DNA FokI endonuclease domain.13 The CRISPR/Cas9 platform relies on a DNA-binding guide RNA (gRNA), which is complementary to the target DNA sequence, and a Cas9 endonuclease.14 As a simple modification of the gRNA sequence is required to target different genetic loci, without the need of complex nuclease engineering as in the case of ZFNs and TALENs, CRISPR Cas9 has become a cost-effective, easy-to-use, and popular choice for genome editing among the scientific community.Over the past decade, there has been a global upsurge in the identification and discovery of novel gene editing tools. To date, in the context of PID research, three main platforms have been extensively utilized: Zinc finger nucleases (ZFNs), transcription activator effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats–associated Cas9 nuclease (CRISPR/Cas9) . These eAlthough they differ in terms of target recognition, activity, and specificity, these platforms have been designed to create DSBs upon binding to the target site, triggering the activation of two main endogenous repair pathways: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Each of these pathways could be exploited for therapeutic purposes. The efficient but error-prone NHEJ pathway repairs the DSBs by ligating the two DNA strands directly without the need of a template, thus leading to the random insertion and/or deletion (indels) of nucleotides. The generated indels can abolish the expression of a protein or the function of a regulatory region and thus this repair pathway could be utilized to treat pathological dominantly active genetic elements. In contrast, HDR mediates the accurate repair of the cut by utilizing a DNA sequence homologous to the region flanking the DSB as a template. As this process results in the insertion of a correct DNA sequence, this pathway could be harnessed when treating those diseases, for which correcting or adding a genetic element may lead to a therapeutic benefit.15 or into its own genomic locus, allowing the preservation of its physiological expression. Although the latter strategy is specific for each single gene, it could be particularly amenable for diseases in which endogenous gene regulation is essential.In the context of PIDs, HDR-mediated editing is the most frequently used repair pathway to correct loss-of-function mutations underlying the disease, by either site-specific insertion of a gene or correction of patient-specific mutations . Site-spex vivo modification of HSPCs. Since the isolation and autologous transplantation of HSPCs to patients is now relatively easily accomplished, PID clinical trials involving gene editing of HSPCs will likely follow the ex vivo route for the foreseeable future. Overall, the application of genome editing to HSPCs has shown substantial benefits toward functional gene correction for different types of PIDs. When applying genome editing with the aim to repair the functional and phenotypic defects of a disease, researchers not only need to choose which specific tool to utilize, but also the delivery route, dose, and timing of editing reagents. This is to ensure that high HDR efficiency, and negligible cytotoxic and off-target effects are achieved in the cell type of interest. Depending on the type and location of the mutation, researchers have shown that it is possible to fully recapitulate a physiological gene expression pattern and restore a functional immune response by HDR-mediated editing of the desired genomic locus in HSPCs. Evidence has also shown the ability of patient-derived edited cells to engraft the bone marrow and differentiate into various immune cells in vivo. Large-scale preclinical studies are underway to assess the long-term safety and efficacy of the gene editing products and to define the minimum level of targeted correction required to achieve a therapeutic benefit for each type of PID without significantly affecting lifelong haematopoiesis. In this study, we will review the current progresses in the genome editing field by highlighting the most promising strategies that have been put in place to correct PIDs.From the very first PID gene therapy clinical trial, successful treatment outcomes have been generated from the et al. were among the first groups to demonstrate functional correction of a mutated IL2RG gene, which is responsible for SCID-X1, an X-linked recessive monogenic PID characterized by a complete block in the development of T and NK lymphocytes with dysfunctional B cells.17 To edit IL2RG, Urnov et al. transfected K562 and T cells with ZFNs together with a donor plasmid carrying an exon 5 fragment of IL2RG, achieving a frequency of HDR-mediated repair of up to 5% in primary T cells.17 These experiments highlighted the limitations associated to the delivery of editing reagents through plasmid transfection in clinically relevant primary cells, where elevated cell toxicity and a negligible HDR frequency could be observed. To address these issues, Lombardo et al. utilized an integrase-defective lentiviral vector (IDLV) to package and deliver ZFN dimers and the donor template to target IL2RG, resulting in 0.2% of GFP-expressing HSPCs derived from healthy donors with minimum toxicity.18 To improve the overall IL2RG-targeting rates in HSPCs, Genovese et al. optimized the ex vivo culture conditions as well as timing and delivery route of the editing reagents.19 Through electroporation of ZFN mRNA 2 days after cell thawing, followed by IDLV transduction of an IL2RG-GFP donor template, an increase to up of 20% GFP-positive HSPCs was achieved, with a marked increase in the frequency and yield of GFP-positive targeted cells in primitive, long-term repopulating hematopoietic stem cells (HSCs). Recently, two additional studies have demonstrated a tremendous advancement in the correction of SCID-X1 by gene editing, using either the ZNF or CRISPR/Cas9 system coupled to the delivery of the donor template to HSPCs using an AAV6 vector.21 Both studies demonstrated that higher HDR-mediated editing can be achieved in HSPCs and HSCs population both in vitro and in vivo when cells are electroporated with a gRNA–Cas9 ribonucleoprotein (RNP) complex followed by transduction with an AAV6 donor template. Indeed, this strategy facilitated up to 40% of targeted integration in SCID-X1 HSPCs, fully restoring physiological gene expression and successfully reconstituting all hematopoietic lineages in immunodeficient mice transplanted with gene-corrected cells.21 SCID-X1 represents an ideal target for proof-of-principle gene editing studies, as the selective advantage that functionally corrected cells have over mutated ones in an SCID setting can compensate for the relatively low rate of HDR-mediated correction in HSPCs.20 Additional protocol optimizations may be required to increase the rates of gene correction in HSPCs and more primitive HSCs to revert the disease phenotype in PIDs, where such a strong selective advantage is missing.In 2005, Urnov phox, p22phox, p40phox, p47phox, and p67phox) of the nicotinamide adenine dinucleotide phosphate oxidase (NADPH) enzyme complex. Apart from gp91phox, which is encoded by the CYBB gene located in the X chromosome and associated to the X-linked form of the disease, the remaining subunits are responsible for autosomal recessive CGD. Since the NADPH oxidase is essential for the production of reactive oxygen species and nuclear extracellular trap in phagocytes, CGD patients with nonfunctional NADPH oxidase are susceptible to severe infection and inflammation, including pneumonia and blood sepsis.22CGD is a group of life-threatening PID caused by mutations in any of the five protein subunits  as donor templates. By electroporating ZFN mRNA targeting the “safe harbor” AAVS1 locus, followed by transduction with an AAV6 containing a NCF1 gene, which interestingly, is also found in two NCF1 pseudogenes, NCF1A and NCF1C. By electroporating ZFNs targeting NCF1 exon 2 together with a AAV6 vector containing an exon 2 repair template for HDR-mediated correction, Merling et al. reported the replacement of the mutated exon in the NCF1 locus as well as in both pseudogenes, restoring oxidase function in 6% of myeloid cells differentiated from p47-CGD-induced pluripotent stem cells (iPSCs).25 A similar approach was recently pursued by Klatt et al., who employed a plasmid-based CRISPR/Cas9 system to correct the ΔGT mutation in the NCF1 locus of p47-CGD iPSCs, resulting in the complete reconstitution of phagocyte function without altering any sequences in the pseudogenes.26 Optimization of this mutation-specific approach and its application to clinically relevant HSPCs may provide a valid therapeutic alternative for patients suffering from p47-CGD.Contrary to X-CGD, where multiple mutations are responsible for the onset of the disease, p47-CGD could be tackled with a gene correction, rather than gene insertion approach, as >80% of p47-CGD patients are homozygous for a two-nucleotide deletion (ΔGT) exon 2 of the WAS gene encoding the WAS protein (WASp), an actin cytoskeleton regulator. Clinical manifestation of WAS includes thrombocytopenia, recurrent infection, eczema, autoimmunity, and cancer.27 The first proof-of-concept study for the use of gene editing to tackle WAS was reported by Laskowski et al. in patient-derived iPSCs using a plasmid-based ZNF platform. Despite very low rates of targeted integration, protein expression level in edited iPSC-derived hematopoietic cells were comparable to wild-type controls, and functional correction of NK cells was demonstrated.28 By using ZFN and CRISPR/Cas9 plasmid-based systems, a later study compared the efficiency of integration of a GFP cassette in WAS intron 1 in K562 cells, achieving an HDR frequency of 5%.29 Despite encouraging early results, such reduced levels of targeted integration achieved in cell lines are unlikely to lead to a therapeutic correction of the defects associated with WAS when applied to patient-derived HSPCs. Our group has recently overcome this limitation by developing a highly specific and efficient CRISPR/Cas9 coupled to AAV6 editing system to insert a codon-optimized WAS cDNA in frame with its endogenous translation start codon in wild-type and WAS HSPCs.30 This strategy not only resulted in up to 60% of targeted integration in WAS HSPCs derived from various patients, but also significantly restored the functional and phenotypic defects in macrophages, platelets, T and B cells derived from corrected HSPCs, associated with relatively physiological expression of WASp.30 Primary and secondary transplantation of edited HSPCs into immunodeficient mice revealed the preservation of their differentiation potential in vivo, with evidence of corrected WASp expression being preserved in long-term repopulating cells and lack of any noticeable toxicity. This proof-of-concept study provides a demonstration of the efficacy and the safety of a CRISPR/Cas9-based gene editing approach to treat WAS, setting the ground for an alternative, yet highly efficient, safe, and precise treatment for this disease.Compared with other PIDs, such as SCID-X1 and CGD, where the immune defect is restricted to either lymphocytes or granulocytes, WAS patients suffer from a complex functional defect in all mature hematopoietic cells, with the exception of red blood cells. This X-linked disease is caused by mutation in the CD40L) gene. Expression of CD40L is finely tuned so that its protein is only upregulated on the surface of activated T cells, where it induces the immunoglobulin class switch upon binding to CD40 on B cells. Without effective antibody production, XHIM male infants are prone to recurrent infection and autoimmunity with reduced chance of survival.31 Although previous gene therapy attempts in an XHIM mouse model reconstituted adaptive immunity,32 the constitutive ectopic expression of CD40L from the viral promoter resulted in the abnormal proliferation of transduced T cells leading to the development of lymphomas in the experimental mice . Hubbard et al. tried to overcome this bottleneck by targeting a correct CD40L gene under the control of its endogenous promoter in primary T cells from three different XHIM donors using TALENs coupled with AAV6 as a donor template, reaching up to 30% of HDR-mediated targeted integration.33 Importantly, gene-edited cells recapitulated the wild-type T cell activation–resting kinetics both in vitro and in vivo, which is indicative of normal CD40L physiological restoration, and restored IgG class switching. Although autologous transplantation of CD40L-edited T cells is feasible for treating XHIM and could be used as a bridge therapy before curative HSC transplant, a sustained life-long clinical benefit probably requires correction of the mutated gene at the HSPCs level. Kohn's group developed TALEN and CRISPR/Cas9-based platforms to correct the functional defects of XHIM in healthy donor HSPCs, reporting a relatively high gene integration rate in CRISPR/Cas9-edited cells and no aberrant multilineage differentiation both in vitro and in vivo.34 An average of 4% of edited cells persisted for more than 5 months after transplantation in immunodeficient mice, demonstrating correction of long-term repopulating HSCs, and more than 60% of the mice showed thymic reconstitution. The results obtained in primary T cells and HSPCs suggest that a small number of gene-corrected T cells may be sufficient to restore IgG class switching and ameliorate the disease phenotype. These promising data may provide a backbone for further work to address whether edited patient-derived XHIM-HSPCs can be differentiated into corrected T cells without the occurrence of any adverse event.For some PID, especially those caused by mutations in genes that require a tightly regulated expression, introduction of the correct gene in its own locus by targeted gene editing could represent the strategy of choice. X-linked hyper-IgM syndrome (XHIM) is caused by mutations in the CD40 ligand  and transiently upregulated in activated effector T cells (Teff). Impairment of both Treg and Teff functions underlies the onset of IPEX and patients present with many different autoimmune manifestations, such as type 1 diabetes, life-threatening enteropathy, eczema, and cytopenia.35 For the development of a lifelong FOXP3 gene therapy using HSPCs, it is necessary to achieve constitutive expression of FOXP3 in the Treg compartment without having FOXP3 overexpression perturb the proliferation and function of HSPCs or Teff cells. To achieve such cell-type-specific expression, a recent study has described a one-size-fits-all CRISPR/Cas9 and AAV6-based gene editing strategy to deliver FOXP3 to its endogenous genomic locus.36 Initial protocol optimizations showed 15% and 25% of targeted integration in healthy donor Treg and Teff, respectively. Aside from emulating the wild-type activation/resting kinetics, edited IPEX Treg was able to suppress the proliferation of 40% of wild-type responder Teff compared with nonedited controls. Such promising findings were complemented with in vivo experiments in FOXP3 humanized mice, where gene-corrected IPEX HSPCs persisted in the bone marrow for up to 3 months and were able to differentiate into Treg in the spleen. Further investigation on whether this approach can rescue the scurfy mice phenotype, which is an equivalent model for human IPEX, while stabilizing the tight regulation of FOXP3 during T cell development in the thymus, remains to be sought.Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is another example of an X-linked monogenic PID, where the application of gene editing is desirable to preserve the cell-type-specific expression of the gene upon targeted integration. The disease is caused by mutations in the Encouraging proof-of-concept data for the precise treatment of PID continue to stem from the research field of genome editing and its innovative therapeutic power. To translate this platform from bench to bedside in a safe and effective way, major challenges need to be addressed during basic and preclinical experiments. Some of these hurdles include:ex vivo cell engineering is the delivery of the nuclease reagents to the target cells. Apart from the Cas9 nuclease, which is commercially manufactured as purified protein, ZFNs and TALENs must be delivered as either plasmid DNA or mRNA. Electroporation of plasmid DNA is the least used technique due to high toxicity in human primary cells and off-target insertion.37 Careful consideration must also be taken when delivering mRNA forms of ZFNs, TALENs, and Cas9 to ensure that premature degradation due to impurities and improper handling, as well as an antiviral response in HSPCs, do not occur.38 These unwanted effects are minimal when delivering the Cas9 coupled to the gRNA in a RNP complex, leading to high editing rates.39 Due to their nonintegrative property and high tropism toward HSPCs, viral vectors, such as AAVs and IDLVs, have been extensively adopted not only for nuclease delivery but also to transfer the donor templates for HDR-mediated editing. One of the drawbacks of AAVs is their 4.5 kb cargo capacity, which limits the delivery of large transgenes. In contrast, IDLVs can handle >10 kb transgene cassettes but suffer from low titer, which has a costly implication for large-scale vector production, and non-negligible rates of integration into the genome in a semirandom fashion.40 Similarly, lipid and gold-based nanoparticles are considered safer in terms of overall toxicity and immunogenicity profile, but the rate of targeted integration achieved so far in HSPCs is not sufficiently high to fully correct most PIDs.41 Currently, AAV6 is considered the genetic tool of choice to deliver the donor template for efficient HSPC gene editing, given its overall low toxicity, minimal rates of random integration and high recombination frequencies even when small homology regions are provided.39One of the crucial processes during ex vivo delivery of the editing reagents, NHEJ is the preferential pathway utilized to correct the DSB in nondividing cells, such as stem cells. To overcome this issue, various groups have implemented strategies to either inhibit NHEJ,42 or increase the frequency of HDR,43 and have optimized timing and dosage of the editing reagents44 to enhance knockin efficiency, resulting in targeted integration rates of >60% in vitro. Selection of gene-targeted HSCs and/or HSPCs after editing and before transplantation is another potential route to compensate for the low frequency of HDR in stem cells, as it would guarantee the infusion of a virtually pure population of corrected cells. However, further improvements in the selection protocol and cell culture techniques are required before applying this strategy in a clinical setting, since despite an increase in the HDR rate, the yield and the engraftment ability of edited HSCs after cell sorting are dramatically decreased.39Achieving sustained and higher levels of targeted integration is one of the ultimate goals to completely cure PID by gene editing. However, following the e.g., exon 1 vs. exon 2) and which genetic elements must be included in the donor template. In this regard, the choice of suitable transcriptional  and post-transcriptional  regulatory sequences in the donor cassette may significantly affect the expression of the corrective protein.33To ensure a therapeutic benefit from the gene editing approach, it is necessary to mediate expression of the corrected gene at curative levels and in the desired cell type upon targeted integration. Site-specific correction or insertion of the donor cassette in its own locus, under the control of its endogenous regulatory regions, secures a regulated protein expression that follows the kinetics observed in wild-type cells and healthy individuals . Howeverin vivo while preserving their long-term repopulating potential. While good engraftment rates of up to 60% have been observed following transplant of HSPCs electroporated with Cas9:gRNA RNP into immunodeficient mice, the persistence of edited cells in the hematopoietic tissues decreases significantly within 8–16 weeks after transplant and in serial transplantation experiments.44One of the critical aspects of HSPC gene editing that requires thorough evaluation is the capability of corrected stem cells to engraft into the bone marrow in vivo could be due to the inefficient HDR-mediated editing in quiescent long-term repopulating HSCs, or their inability to self-renew upon their manipulation in vitro, including exposure to the editing reagents and culture conditions. Different strategies have been put in place in recent years to maintain and expand the primitive pool of self-renewing HSCs, such as the use of small molecules ,44 as well as the optimization of culture conditions and timing of delivery of the editing reagents to HSPCs to preserve their engraftment potential. Some studies have suggested that p53-mediated damage may alter HSC stemness as well as lead to cell cycle arrest, which decreases the frequency of DSB repair by HDR.45 Although transient p53 inhibition has been sought to eliminate its detrimental effects on stem cells and increase HDR,45 the use of such inhibitors in preclinical and clinical settings must be carefully evaluated, considering the essential tumor suppressor function of the p53 pathway. In addition, other groups have uncovered cellular factors, such as the interferon-inducible antiviral factor, which may be responsible for an innate immune response to viruses in cells where the gene editing reagents are delivered by lentivectors; a recent study has shown that this phenomenon could be counteracted by the use of Cyclosporin H.46The decline in the frequency of corrected cells 47 Such in silico approaches are fast and easy to use, but often fail to give a comprehensive picture of the genome-wide activity of the editing machineries, therefore they should be complemented with unbiased genome-wide detection techniques, including IDLV capture, CIRCLE-seq, GUIDE-seq, DIGEOME-seq, and UDiTaS™.47 However, these methods do not always identify all potential off-targets, often show limited sensitivity, and/or are applicable only to nonclinically relevant cell lines, which may exhibit huge variability in terms of nuclease off-target activity compared with the therapeutic target cell type. Recent findings to quantify off-targets in vivo are encouraging,48 however, efforts are still warranted to develop protocols and reagents suitable for assessing off-target fluctuation in HSPCs before, during, and after transplantation in preclinical and clinical settings. Moreover, the prediction of the impact that unintended genetic modifications may have on cell fitness is not trivial and requires the development of functional readouts of safety that must be tailored to the therapeutic cell type of interest and the editing platform used. In a parallel effort, researchers have tried to improve the specificity of the editing tools to limit their off-target activity. For instance, a key modification for ZFNs and TALENs has been the substitution of amino acid residues in the DNA-binding and cleaving domains, which resulted in enhanced on-target to off-target ratio.49 On the other side, the development of chemically modified and shorter gRNAs combined with the use of mutants and high-fidelity Cas9 variants have demonstrated significant improvement in the specificity of the CRISPR/Cas9 gene editing platforms.50 With these modifications, therapeutic gene editing for PID is reaching a crucial milestone, exhibiting improved efficacy and robust safety profiles; the research community must continue designing and improving the editing platforms and apply them in disease-relevant settings, rigorously identifying the frequency, location, and consequence of any potential off-targets.Despite their ability to induce targeted modifications, all the gene editing platforms used so far do not possess perfect specificity and may introduce unintended DSBs at random genomic loci. Off-target modifications introduce permanent genetic mutations that may ultimately lead to cancer. To prevent such risks, comprehensive detection and analysis of the widespread activity of genome editors are vital. Several types of methods to identify off-target indels and chromosomal aberration have been described, each with certain benefits and limitations. Although a gold standard has not been laid out yet to determine which method to apply, initial screening to select the ideal ZFN and TALEN pair or gRNA sequence for a given target and their potential off-target sites are provided through numerous computational algorithms.During the last few years, tremendous advances have been made in the field of gene editing applied to monogenic disorders. Many proof-of-principle studies applying editing platforms to modify HSPCs, including CRISPR/Cas9 and TALENs, have demonstrated the feasibility of such technologies in correcting the genetic defects underlying the onset of PID. Despite the exciting prospects, no PID clinical trials are currently underway that deploy gene editing technologies. Many research groups are now conducting large-scale preclinical work to assess the long-term safety and efficacy of gene-edited products and to define thresholds of genetic and functional correction needed to reverse the phenotype of each set of PIDs. Fundamental biological concerns remain to be addressed to ensure that significant and unexpected adverse events do not occur. The design of preclinical studies that unequivocally mimics the human clinical trials for a particular PID is therefore desirable, to minimize the translational distance between preclinical and clinical results and ensure that the potential risks for trial participants are negligible. Additionally, direct comparison between gene editing and existing gene therapy platforms or any other treatment option available for a specific PID should be carefully evaluated. This is of paramount importance to assess the applicability and feasibility of gene editing as a curative treatment for such ultra-rare patient populations, especially when taking in account the costs incurred from Good Manufacturing Practice-compliant editing reagents, viral vector production, and the setup of the manufacturing facilities and logistics. Correction of patient HSPCs by means of gene editing remains cumbersome and there are still many challenges facing the field, including the specificity and efficiency of the editing system. Continuous efforts are required to overcome such hurdles, with the final aim of translating gene editing into the next generation of therapeutic tools for severe PID and other blood disorders.


Neuropathic pain is a frequent condition caused by a lesion or disease of the central or peripheral somatosensory nervous system. A frequent cause of peripheral neuropathic pain is diabetic neuropathy. Its complex pathophysiology is not yet fully elucidated, which contributes to underassessment and undertreatment. A mechanism-based treatment of painful diabetic neuropathy is challenging but phenotype-based stratification might be a way to develop individualized therapeutic concepts. Our goal is to review current knowledge of the pathophysiology of peripheral neuropathic pain, particularly painful diabetic neuropathy. We discuss state-of-the-art clinical assessment, validity of diagnostic and screening tools, and recommendations for the management of diabetic neuropathic pain including approaches towards personalized pain management. We also propose a research agenda for translational research including patient stratification for clinical trials and improved preclinical models in relation to current knowledge of underlying mechanisms. Numerous reviews have been written about neuropathic pain (NP) in general  of peripheral origin , painful polyneuropathy (third-level diagnosis)  worldwide, dPNP is and will be one of the most important and common causes of NP. In 2000, 171 million (2.8% of the world population) people suffered from DM  can be divided into central or peripheral syndromes, depending on the site of lesion or underlying disease. This section focuses on conditions that are considered consequences of a peripheral insult. Central NP conditions are less well understood and might differ in their underlying mechanisms, so they need separate consideration  pain and hypersensitivity, including allodynia and hyperalgesia (evoked pain) , chronic constriction injury (CCI), and spinal nerve ligation (SNL). Additionally, pathogenesis of NP has also been studied in rodent models of diabetes, chemotherapy, herpes zoster and HIV–peripheral neuropathy  at the injury site can generate ectopic activity and so pain originates from an area with reduced sensitivity to thermal and mechanical stimuli. Microneurographic recordings of single C-fibers have demonstrated spontaneous activity in human studies investigating several NP syndromes  in primary afferents  and thinly myelinated (Aδ) primary afferent neurons, is sensitization. Sensitization, which typically develops as a consequence of tissue injury and inflammation, is defined as a reduction in the threshold, an increase in the magnitude of response to noxious stimulation and spontaneous activity. The inflammatory processes in Wallerian degeneration may hence render the remaining intact fibers after nerve injury hyperexcitable  family led to a better understanding of how nociceptors detect external stimuli and how they can be sensitized , an activity-dependent persistent synaptic strengthening, intensively studied in the hippocampus, appears to play a role in spinal sensitization after noxious input  activity contributes to central sensitization after nerve damage. Activation of intracellular pathways by protein kinases leads to phosphorylation of NMDARs. Afterwards, NMDARs respond stronger to agonists. Under normal circumstances, NMDA receptor channels are blocked by Mg2+ ions. Phosphorylation by protein kinase C increases the opening probability and decreases the affinity of NMDARs for extracellular Mg2+   as well as low levels of the anti-inflammatory mediator IL-10 supporting the idea of central neuroinflammation in NP patients  gives insight into endogenous descending inhibition and facilitation  describes a group of syndromes with many different causes and varying clinical manifestations. Diagnostic algorithms differ depending on whether the underlying lesion or disease is in the peripheral or central nervous system. Hence, a first subdivision of NP is peripheral versus central NP  of the International Association for the Study of Pain (IASP) issued diagnostic criteria for NP, the Neuropathic Pain Grading System, developed to determine the level of certainty that a patient’s pain is neuropathic in nature or has a neuropathic component in mixed pain syndromes . A detailed clinical examination should then be performed to find confirmatory evidence for the pain distribution and the underlying lesion or disease. Step 3: Since there is no confirmatory test for the spatial extent of perceived ongoing pain, the spatial extent of sensory signs is used as a surrogate. If this condition is also met, the neuropathic nature of the pain is considered to be likely (probable NP). Step 4: Depending on the suspected lesion or disease, appropriate confirmatory tests are performed. When positive, they lead to the diagnosis of “definite NP”. The level “probable NP” is considered sufficient to initiate treatment. The level “definite NP” indicates that a physician is able to confirm that the patient has a neurological lesion or disease that might explain his/her pain . When these two conditions are met, the possibility of NP is considered possible   (Krause and Backonja Bedside examination (BSE) in patients with DM is essential when suspecting diabetic polyneuropathy (dPNP) and/or pDN. Most guidelines advise yearly screening for dPNP  to examine vibration perception. It is a valid and reliable tool for screening purposes, manageable in daily clinical practice  tests that confirm the sensory changes and (b) tests that confirm the specific underlying lesion or disease of the somatosensory nervous system explaining the symptoms of the patient , multi-center reference data are available for different body regions in both sexes and a broad age range  through skin biopsy is validated for diagnostics of small fiber neuropathies, including dPNP , vitamin deficiencies  or intoxications . Thus, diabetic neuropathy is a diagnosis of exclusion  is a common disorder marked by persistent hyperglycemia and other metabolic disturbances, such as dyslipidemia. Increased glucose levels affect primarily cells that have a limited capacity to regulate their glucose intake, including vascular cells, Schwann cells, and neurons of the peripheral and central nervous systems. Consequently, hyperglycemia leads to largely intractable complications such as retinopathy, nephropathy, hypertension, and neuropathy. However, not all patients with DM develop diabetic polyneuropathy (dPNP). In prospective studies with diabetic patients, environmental risk factors for the development of neuropathy in type 1 (T1DM)  and less so of T2DM using diet or genetically induced DM  where they are not protected by a blood–nerve barrier. Although dPNP is not considered primarily a demyelinating neuropathy, Schwann cells are targeted by chronic hyperglycemia and more severe cases of dPNP in patients include features of demyelination   for developing NP in dPNP  in pDN. In rodent models of pDN, MGO induced signs of hypersensitivity via activation of the sodium channel Nav1.8 and transient receptor potential channel ankyrin 1 (TRPA1)  is defined as “re Table  consistiDistribution of both pain and sensory changes should be mapped  is much less frequent in pDN (< 10%) than in postherpetic neuralgia, where it was originally described , while deep tissue mechanical pain (assessed by pressure pain threshold) is relatively intact , pain sensations to normally non-painful stimuli  and spontaneous pain. pDN patients often describe a prickling, burning, deep aching, sharp, stabbing, and/or electric pain. Signs of sensory gain were traditionally thought to be rare in pDN, but recent studies using QST demonstrated a high prevalence of mechanical hyperalgesia in pDN : glycemic control, foot care, and symptomatic treatment, and predominantly pain therapy.While diabetes is defined by increased glucose levels and tight glycemic control has a high value in its treatment, it is currently recommended to individualize glycemic control in patients with T2DM considering the benefit/risk ratio  in general and painful diabetic neuropathy (pDN) in particular is a challenge. A number of clinical practice guidelines, e.g., by the International Association for the Study of Pain (IASP) , serotonin–norepinephrine reuptake inhibitors  and calcium channel alpha-2-delta ligands gabapentin and pregabalin.Second-line recommendations by most guidelines  is tramadol (weak opioid + SNRI).Third- and fourth-line treatments commonly include strong opioids, anti-convulsants (other than gabapentinoids), and cannabinoids.Between these guidelines, there is broad agreement regarding pharmacological management of NP  are based on a large meta-analysis, including unpublished trials   and neuropathic pain (NP) in general, treatment options to eliminate the root causes are still lacking. The research agenda for mechanism-based treatment of NP proposed in 1990 is still valid , by modulating synaptic transmission  or by mimicking and enhancing endogenous inhibitory mechanisms . However, there is still a large gap between theoretically effective target mechanisms and actual efficacy in clinical studies.After the discovery of complete insensitivity to pain in patients with null mutations in the sodium channel Nav1.7  has been promising in preclinical animal models  the wide variety of potential underlying lesions or diseases of NP suggests that a variety of models should be studied. (2) Multiple endpoints should be assessed including measures of both evoked and ongoing pain. (3) Comorbidities and quality of life should be assessed. (4) Animals with genetic variability should be used, e.g., outbred strains. (5) Studies should be done in male and female animals.Another key factor for a better clinical translation of preclinical studies is an improvement in clinical trial design. Compared with fields like oncology or infectious diseases, clinical trials on treating pain conditions can be quite primitive in assuming that all pain is one and the same. In the presence of evidence for multiple distinct mechanisms of pain generation, it is necessary to shift paradigms towards more specific mechanism-based indications. Clinical practice is moving towards individualized pain management, so it is advisable for the pharmaceutical industry to move in the same direction and supply the necessary specific analgesics. The European Medicines Agency published guidelines on how to conduct trials for the development of pharmaceutics for pain therapy. This guideline encourages stratification according to the phenotype  of the patients as a first step to identify possible responder populations for existing or future medication , so significant progress is expected to be made over the coming years.


Diabetes is a worldwide disease also affecting the sports field. The two main forms of diabetes, namely type 1 diabetes (T1D) and type 2 diabetes (T2D), differ in both their pathological and pharmacological characteristics and thus require a distinct nutritional treatment. Diet plays an important role in the management of athletes with diabetes and is crucial to achieving their best performance. This review aims to investigate the objectives of nutritional therapy before, during and after training, in order to improve the best composition of macronutrients during meals. In this review, we provide a brief overview of recent studies about nutritional approaches to people with diabetes for performance optimization and for the control of diabetes-related complications. Thereafter, we discuss the differences between macronutrients and dietary intake before, during and after training. It can be concluded that each sport has particular characteristics in terms of endurance and power, hence demanding a specific energy expenditure and consequent nutritional adjustments. Therefore, the management of athletes with diabetes must be personalized and supported by medical professionals, including a diabetologist, physiologist and a nutritionist. Many professional athletes, practicing different sports, suffer from diabetes. Currently, there are no specific dietary indications to be applied in the sporting field. Athletes with diabetes should adjust their diet according to the guidelines suggested to the general population ,2, possiDiabetes mellitus is a chronic disease that includes several physiological dysfunctions  with difNutritional treatment in athletes with diabetes requires an in-depth assessment that passes through several critical points: 1) early evaluation based on the sport practiced, including the type of activity  ,16; (2)  early evThe purpose of this review is to investigate the objectives of nutritional therapy before, during and after training, in order to improve the composition of macronutrients during meals for performance optimization and for the control of diabetes-related complications.As mentioned above, pharmacological treatment of T1D requires insulin supplementation, therefore athletes affected by T1D must pay attention to the ratio between the prandial insulin dosage  and the CHO monitoring is crucial (1) to maintain a constant content of CHO in each meal ; (2) to T1D subjects usually present with a morphotype incline to normal weight, thus not needing caloric restrictions but with a high susceptibility to hypoglycemia compared to individuals with T2D . The objThe American Diabetes Association (ADA) suggested a diet based on a flexible percentage of CHO, which can vary from 45 to 55% of total calorie intake ,29,30,31Sugar should not be banned , althougThe diet composition of athletes with diabetes must contain other essential macronutrients such as proteins and fats  which, i2 max)) and mainly rely on aerobic metabolism by utilizing muscle glycogen stores and, to a minor extent, circulating free fatty acids (FFAs). When muscle glycogen is depleted, hepatic glycogenolysis and glucose uptake in the skeletal muscle increase upon reduction of insulin secretion and upregulation of released catecholamines. However, athletes with T1D may experience hypoglycemia due to increased insulin absorption, reduced glucagon secretion and impaired catecholaminergic response. Similarly, high-intensity activities (85 to 100% VO2 max) are primarily sustained by aerobic metabolism while brief intermittent bouts  activate anaerobic metabolism. Moreover, increased catecholamine release following intermittent sprints may likely result in hyperglycemia during and/or after the exercise [However, nutritional recommendations may vary upon the type of exercise practiced by the individual athlete with T1D. Endurance sports  are considered moderate-intensity activities  is detected, extra CHO should be taken before beginning the activity . If traiEating during exercise depends on training length and intensity. Generally, CHO supplements should be assumed only when the length of training exceeds 1 h or during competitive sports events . This isThe main goal after training is to provide for adequate liquids, electrolytes, energy, and CHO to replace muscle glycogen and guarantee a rapid recovery .A CHO intake of approximately 1–1.5 g/kg of body weight during the first 30 min after physical activity and every 2 h for 4–6 h will be enough to reconstitute liver and muscle glycogen sources . SimilarThe nutritional approach in the diet of T1D and T2D athletes should strictly adhere to the principles recommended for diabetes treatment, providing the energy and nutritional needs and water loss related to physical activity . The MedAthletes with diabetes need to follow a diet that is similar to the one recommended for non-athletic diabetic individuals: caloric and energetic requirements must always be satisfied, with a cautious reintegration of hydric and nutrients loss after exercise. It is important to detect early and recognize signs of nutritional deficiency with intensive control of glycemia and body weight, in order to practice physical activity in a safe and efficient way. The continuous assistance of professional experts is required to optimize the management of athletes with diabetes for long-term safe and effective dietary therapy.


Aging is associated with immunosenescence which leads to severe consequences of infection as well as inefficient protection after vaccination. This process is defined by immune remodeling of the innate and adaptive branches of the immune system. One effect of this change is that current vaccine strategies do not sufficiently protect older adults hence the development of efficient approaches could substantially improve morbidity and mortality rates for these under protected people. Aging affects the quality and quantity of antibody responses, including specificity and class of antibody produced. High-affinity antibodies are produced in the germinal centers (GC) of secondary lymphoid tissues where processes like affinity maturation and somatic hypermutation (SHM) take place. The decreased ability of older adults to produce a protective antibody response is due, in part, to defects in T cells. In addition to impairments at the haematopoietic stem cell and lymphoid progenitor level in the bone marrow and thymus, the reduction in clonal diversity of naïve T cells and the skewing of their function to a Th1 pro-inflammatory role . In humaAnother large factor in the progression to a fulfilled adaptive immunity is the innate immune response. Initiation of the innate response is triggered when pathogen-associated molecular patterns (PAMPS) engage pattern recognition receptors (PRRs) in cells including monocytes, macrophages, epithelial cells, and dendritic cells (DCs). Activation of these key innate cells leads to induction of type I interferon (IFN) responses that mediate anti-viral immunity and initiate and support the adaptive immune system through their functions, such as phagocytosis and antigen presentation. Alterations resulting from aging in these pathways have a crucial role in what is called ‘inflammaging’ or the chronic basal production of pro-inflammatory cytokines like IL-6, TNF-α, and type I IFNs . This deGCs, through virtue of their function, are critical for potent immune responses against foreign antigen. The production of high-affinity antibody results from complex interactions of DCs and naïve T cells, followed by B and Tfh cells in the GC reaction. Any deviation from this complex dance, results in aberrant humoral immunity as we see in aging individuals in response to vaccine or infection. Along with restructuring of the microarchitecture of the spleen and lymph nodes, GCs from older nonhuman primates (NHP) have recently been shown to have altered baseline cell composition including decreased follicular Tfh, proliferating GC B cells and follicular monocytes, granulocytes, and increased T-follicular regulatory (Tfr) cells ,7 Figur. Underst


Br. J. Cancer 122:1425–1427, 2020), the National Institute for Health and Care Excellence completed its consultation process on larotrectinib. With an improved pricing agreement, it was recommended in April 2020 for use within the Cancer Drugs Fund (CDF) for neurotrophic tyrosine receptor kinase (NTRK) fusion-positive solid tumours in adults and children with locally advanced or metastatic disease or where surgery could cause severe health problems and no satisfactory treatment options exist. It was recognised during the consultation that patients and clinicians may wish to access the drug earlier in treatment and that further information is required to determine optimal positioning within the patient pathway . The conditions for use associated with the CDF will allow further real-world data collection to inform future marketing authorisation.Following our Comment on Tumour-agnostic drugs in paediatric cancers  with a canonical ETV6-NTRK3 fusion who failed conventional chemotherapy, developed rapid resistance to larotrectinib and subsequently showed resistance to the second-generation TRK inhibitor selectrectinib (Loxo-195). They elegantly map the molecular evolution of the resistance. They conclude that comparative clinical trials beyond basket studies are needed to learn when and how to deploy drugs like larotrectinib and that until such evidence is available, cautious use of TRK inhibitors in children should be advocated.In our view, TRK inhibitors should only be used under direct supervision by oncologists experienced in the use of such targeted agents. While agreeing that the place of larotrectinib in treatment of tumours with NTRK mutations remains to be defined, the unusually aggressive and ultimately fatal case with incompletely resectable disease Oliver et al. present highlights the potential role for tumour-agnostic drugs like larotrectinib to facilitate surgery or prolong life, emphasises the need to address emergent resistance mechanisms and compels us to urgently explore whether earlier use of these agents may lead to better outcomes for patients.The level of evidence required to introduce tumour-agnostic agents such as larotrectinib into frontline treatment requires careful consideration. The tension is to ensure equipoise in trying to design a randomised/comparative registration level trial for a heterogeneous group of tumours with a known but rare target, when a new class of drugs with high efficacy, favourable short-term toxicity profile and an oral formulation suitable for the youngest of infants becomes available. This may be even more important for patients likely to survive their disease, where long-term chemotherapy side effects or morbid surgical approaches may impact significantly on quality of life.1 of which 19 (38%) were resected as primary treatment and 31 (62%) were Group III . Overall, 30/31 (97%) Group III patients received chemotherapy , 2/31 patients (6%) had mutilating surgery (amputations), 1/31 (3%) was irradiated and 3/31 (10%) died . This study demonstrates that the potential population for performing prospective trials in newly diagnosed IFS is extremely small. Moreover, fatal toxicity, potential late effects of chemotherapy, such as nephrotoxicity, reduced fertility or cardiotoxicity and the need for mutilating surgery are a real risk for these patients.IFS, a tumour of young children and the most common paediatric malignancy harbouring an NTRK fusion, might present a more uniform population for comparative or randomised paediatric trials of TRK inhibitors. In the experience of the European paediatric Soft tissue Sarcoma Group, only 50 cases of non-metastatic IFS were registered in 14 countries in 7 years,The ongoing phase II study in newly diagnosed NTRK fusion-positive IFS, solid and brain tumours and relapsed leukaemia in patients <30 years (NCT03834961) is addressing objective response rate to first-line treatment with larotrectinib, with secondary endpoints of toxicity, event-free survival and overall survival and, for IFS, duration of response. It will provide further information on the use of the drug in frontline and results are eagerly awaited. Meanwhile, the role of the second-generation TRK inhibitor seletrectinib is also under investigation in a phase I/II study (NCT03215511) and an expanded access programme (NCT03206931). Additional TRK inhibitors such as entrectinib (Roche) and repotrectinib (Turning Point Therapeutics) are in development. While acknowledging the excellent points raised by Dr. Oliver and colleagues, it remains our view that given the favourable toxicity profile compared to conventional options and the demonstrated high response rates, TRK inhibitors constitute a highly attractive frontline option in IFS that for many patients may allow definitive, curative surgery before development of drug resistance.