Human populations at high altitude exhibit both unique physiological responses and strong genetic signatures of selection thought to compensate for the decreased availability of oxygen in each breath of air. With the increased availability of genomic information from Tibetans, Andeans, and Ethiopians, much progress has been made to elucidate genetic adaptations to chronic hypoxia that have occurred throughout hundreds of generations in these populations. In this perspectives piece, we discuss specific hypoxia-pathway variants that have been identified in high-altitude populations and methods for functional investigation, which may be used to determine the underlying causal factors that afford adaptation to high altitude. Many of humankind’s smallest but greatest secrets are packaged within the human genome. Recent advancements have accelerated our ability to unravel these mysteries and discover how the genome contributes to shared and distinct variations in human traits, including those that have been essential for survival. Some of the most striking examples of adaptation within our species occurred in populations that migrated to the Tibetan, Andean, and Ethiopian highlands in the past several millennia. Physiologists first noted distinct characteristics among highland populations centuries ago, postulating that specific traits were helpful or harmful to challenges imposed by environmental hypoxia due to decreased oxygen availability at high altitudes . Given tWithin the past decade, it has become increasingly feasible to obtain insight into the evolutionary past of our species through genome-wide scans in search of adaptive signatures that highlight outlier patterns within the genome . While mBioinformatic screens of human genetic variation are increasingly used by geneticists and physiologists to better understand features of the human genome, including variable sites, chromosomal arrangements, and population-level variation. Genome-wide tests of selection have been successfully employed across continental populations, yielding candidate genes that are putatively associated with adaptation to high altitude . With reEGLN1 and EPAS1. Both genes are among the top targets of adaptation in Himalayan populations  were identified in Tibetans: p.Asp4Glu (p.D4E) and p.Cys127Ser (p.C127S)  . While tp.C127S) , effectsfunction . Due to termined .EPAS1 variant p.H194R is reported as a target of selection in Argentinian highlanders  in isogenic cell lines, multiple SNVs may be examined simultaneously  . This neOther publicly available tools may be used to assist in understanding the relationship between genetic variation and tissue-specific expression, including the Genotype-Tissue Expression (GTEx) database . This resource, in tandem with epigenetic information gathered from resources such as The Encyclopedia of DNA Elements (ENCODE) Consortium, provide the potential to uncover complex gene regulation networks within a wide array of human tissues . Such inin vivo and in vitro model systems. The development of multiplexed base-editing systems, which introduce point mutations in aggregate , will allow for functional investigation of not only epistatic interactions under selection, but also the functional impact of the inheritance of entire haplotype blocks in model systems , which has been associated with lower systolic pulmonary arterial pressure in Tibetans , also known as copy number variation (CNVs), include identification of insertions, deletions, inversions, and translocations that may help explain some unaccounted heritability . Early aulations . Newly dulations  allow foaptation . OuzhuluTibetans .de novo assemblies may also be compared to archaic hominid assemblies  to illuminate population-specific evolutionary histories. Recent comparisons of high-altitude adapted populations from the Himalayas and archaic hominids suggest a higher frequency of genome-wide archaic hominid introgression in Himalayan populations relative to East Asian genomes  was identified in the SCUBE2 gene, which has been linked with lung function  in Tibetans  has the potential to offer key insights into epigenetic and transgenerational inheritance. Previous research indicates that altitude-induced epigenetic changes occur , and DNA in Peru . Another in Peru .in vivo/in vitro epigenome editing systems will provide an opportunity to tease apart direct causal/mechanistic relationships.Many genomic studies suggest regulatory variation underlies responses to hypoxia . Multiplin vitro cellular systems have a number of limitations. Three dimensional (3D) cellular models  offer the opportunity to functionally investigate the effects of cellular development, self-organization, and tissue-specific, organ-specific function .While isogenic cellular models offer a relevant system to functionally evaluate mutations that are putatively under selection, two dimensional (2D) function . 3D cellEPAS1 or EGLN1) in pluripotent stem cell tissue systems that may be differentiation into physiologically relevant organoid systems. For example, human blood vessel organoids engineered as models of diabetic vasculopathy could also be genome-edited to create isogenic cellular models to functionally investigate the role of specific mutations in the development and arborization of vascular structures/networks putatively implicated in high-altitude adaptation  at high altitude . During The potential to engineer new tools to functionally investigate genetic variation is an exciting prospect as it establishes greater accountability in research (e.g., in population genetics, reducing the tendency toward evolutionary adoptionism narratives . While sEGLN1 and EPAS1, would not be made possible without partnerships in Indigenous communities thriving at high altitude. Understanding the function of specific variants in these groups contributes to a more complete picture of the human story and some of the most notable examples of adaptation within our species.With the generation of data presented thus far and the ability to manipulate and test functional relevance, the scientific community will continue to gain meaningful insights into hypoxia adaptation. The mechanistic characterization of these observations, through the identification of adaptive gene targets like JH, KF, and TS designed the article. JH and KF designed the figures. JH, EL, TS, and KF drafted and contributed comments to the final manuscript.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.


Successful clinical application requires innovative future investigations enhancing vascularization.Bone tissue engineering techniques are a promising alternative for the use of autologous bone grafts to reconstruct bone defects in the oral and maxillofacial region. However, for successful bone regeneration, adequate vascularization is a prerequisite. This review presents and discusses the application of stem cells and new strategies to improve vascularization, which may lead to feasible clinical applications. Multiple sources of stem cells have been investigated for bone tissue engineering. The stromal vascular fraction (SVF) of human adipose tissue is considered a promising single source for a heterogeneous population of essential cells with, amongst others, osteogenic and angiogenic potential. Enhanced vascularization of tissue-engineered grafts can be achieved by different mechanisms: vascular ingrowth directed from the surrounding host tissue to the implanted graft, vice versa, or concomitantly. Vascular ingrowth into the implanted graft can be enhanced by (i) optimizing the material properties of scaffolds and (ii) their bioactivation by incorporation of growth factors or cell seeding. Vascular ingrowth directed from the implanted graft towards the host tissue can be achieved by incorporating the graft with either (i) preformed microvascular networks or (ii) microvascular fragments (MF). The latter may have stimulating actions on both vascular ingrowth and outgrowth, since they contain angiogenic stem cells like SVF, as well as vascularized matrix fragments. Both adipose tissue-derived SVF and MF are cell sources with clinical feasibility due to their large quantities that can be harvested and applied in a one-step surgical procedure. During the past years, important advancements of stem cell application and vascularization in bone tissue regeneration have been made. The development of engineered  To rehabilitate patients with critical-sized bone defects, surgical reconstructions are required. A critical-sized defect will not heal spontaneously or regenerate more than 10% of the lost bone during patients' lifetime , generalBone grafting procedures are carried out to reconstruct a bone defect . In thesex vivo seeded cells on the scaffold play a key role and orchestrate the mechanism of bone formation at the target site. Multiple techniques have been investigated, applying a variety of stem cell sources and cell processing protocols [Unfortunately, the ideal bone regeneration technique and material have not yet been developed. However, recent developments in tissue engineering have led to new and better treatment options called “cellular bone tissue engineering.” In this approach, a scaffold with mesenchymal stem cells (MSCs) and/or osteoprogenitor cells of an external source is implanted into the bone defect site. The rotocols . Furtherrotocols .The rationale behind the application of MSCs and/or osteoprogenitor cells is their key role in bone formation. Natural bone formation in the pre- and postnatal development of the oral and maxillofacial area is performed intramembranously by recruiting mesenchymal bone marrow cells. These cells undergo osteoblastic differentiation and initiate new bone formation in the defect site. In other words, this method is aimed at inducing bone regeneration by mimicking biologic processes that occur during embryogenesis , 14.α (TNF-α), platelet-derived growth factor (PDGF), interleukin-1 (IL-1), and IL-6. These secreted factors may recruit resident MSCs to the regenerated site [The mechanism by which MSCs promote bone regeneration can be directed by engraftment of the transplanted cells into the newly regenerated tissue, differentiating into osteoblasts that eventually will secrete osteoid and initiate mineralization –17. In ated site , 19.in situ tissue engineering [ex vivo, seeded on adequate three-dimensional (3D) scaffolds , and proliferated and/or predifferentiated in controlled culture conditions [In cellular bone tissue engineering, MSCs are applied using two different approaches. The first approach is to directly transplant MSCs and/or osteoprogenitor cells combined with a scaffold  into the bone-defected site, which is a kind of an ineering , 21. Autineering . The secnditions . Such a nditions .μm from a supply blood vessel, cells lying beyond this physiological border suffer from hypoxia [μm exceeds the capacity of nutrient supply and waste removal from the tissue and, therefore, requires an intimate supply of vascular networks [A major challenge in bone tissue engineering is the vascularization of the implanted graft. Graft survival requires rapid and sufficient vascularization. Since the amount of oxygen is limited to a diffusion distance of only ~150-200  hypoxia . Under t hypoxia . New insnetworks . NeovascThe aim of this review is to present and discuss the advancement of stem cell application, vascularization, and bone regeneration in the oral and maxillofacial region, with emphasis on the human jaw. Moreover, we propose new strategies to improve the current techniques, which may lead to feasible clinical applications.In vitro and in vivo animal studies reported on the application of embryonic stem cells (ESCs) [Undifferentiated: multipotent adult MSCs, pluripotent ESCs, or IPSCsEarly differentiated: MSCs differentiated towards specific lineage, such as osteogenic lineageDifferentiated: specialized cell, such as osteoblastSomatic stem cells, mainly mesenchymal stem cells (MSCs), that are applied in bone tissue engineering are isolated from various tissues. The clinically applied sources of stem cells in the oral and maxillofacial region originate from bone marrow, adipose tissue , and dens (ESCs) –32 and is (ESCs)  in bone s (ESCs) . In Figuex vivo relative to more highly proliferative stem/progenitor cells. The use of stem cells is also intended to achieve a complete physiological repair process that involves the MSC-mediated activation of not only bone formation but also neovascularization. Nevertheless, it is of pivotal importance to prohibit unwanted side effects such as teratoma formation which may occur by ESCs and IPSCs.Clinically applicable tissue engineering involving stem cells is focused on the use of patient-derived (adult) stem cells that are undifferentiated, given that terminally differentiated cells are difficult to expand in vivo applied stem cell sources is given. Besides, In the following, an overview of the currently vivo have been reported in the oral and maxillofacial region (in vitro cultivated BMSCs (expanded with or without differentiation factors) (in vivo [Bone marrow was the first source reported to contain MSCs . Until tl region . There afactors) . Concent(in vivo . An overin vitro) after maxillary sinus floor elevation.Several studies showed promising results applying BMSCs in surgical procedures in the oral and maxillofacial region. Some maxillary sinus floor elevation studies presented histomorphometrical data that showed increased new bone formation after 3 to 4 months compared to traditional methods using bone substitutes alone , 39. Kaiin vitro culture expansion is required to obtain sufficient numbers of cells for clinical application [Unfortunately, BMSC application comes with limitations: bone marrow aspiration is an invasive and painful procedure for the donor, and cell retrieval is scarce, since the frequency of BMSCs in human bone marrow is rather low (0.001%–0.01%) . Consequlication . This celication , 48 and lication , 50. Laslication .Adipose tissue-derived mesenchymal stem cells (ASCs) have opened appealing new possibilities in adult stem cell therapies. ASCs show many similarities with BMSCs with regard to surface marker profiles, multilineage potential, and growth properties . Howeverin vitro culturing steps can be avoided.Helder and colleagues formulated the concept of the one-step surgical procedure (OSP) to apply ASCs in the regeneration of bone tissues . After hin vitro studies made important advancements in the application of ASCs in bone tissue engineering [β-tricalcium phosphate or biphasic calcium phosphate  [in vitro cultivated, and seeded on demineralized bovine bone mineral (DBBM) and autologous bone (AB), in alveolar cleft reconstruction. Cone beam-computed tomography 6 months after the treatment showed more bone formation in the test group with supplementation of ASCs. Castillo-Cardiel et al. [in vitro cultivated and injected at the fracture site. After 12 weeks of the surgical treatment, the test group with the supplemented ASCs had a 37% higher ossification rate compared to the traditional treatment (control group). A disadvantage of SVF harvesting so far is that it is performed under general anesthesia and requires (short) hospitalization. Also, postoperative care and complaints are to be regarded. However, clinical studies using local anesthesia are currently being undertaken, which may widen the applicability of this intraoperative approach.Multiple ineering . Recentlineering . Prins eosphate) . Khojastosphate)  used ASCl et al.  treated Dental tissues provide several populations of stem cells, including the pulp of both exfoliated and adult teeth, periodontal ligament, and dental follicle . Dental However, published clinical studies with successful results are scarce . D' Aquihttp://www.clinicaltrials.gov).The question remains open whether in spite of the low numbers of cells, DSCs might become an attractive source of autologous SCs for bone regeneration. This source is being investigated with at least more than ten new trials underway  and/or regulate preexisting ECs via paracrine or juxtacrine signals .Angiogenesis is defined as new blood vessel sprouting from preexisting vessels. The first step in this process is the activation of the host microvasculature at the implantation site by angiogenic growth factors, such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor . These fThe endothelial cells, which are lining blood vessels, allow the formation of new blood capillaries by the sprouting of an existing small vessel , 76. UpoAccordingly, successful vascularization of an implanted graft via vasculogenesis and angiogenesis is dependent on the coordinated sequence of various humoral and cellular mechanisms and, in particular, the close interaction between the host tissue and the implanted graft. This process allows tissue growth and repair by extending and remodeling the network of blood vessels , 80.μm/h [Several approaches to improve vascularization, through enhanced vasculogenesis and angiogenesis, of the implanted grafts are currently investigated. The classical vascularization strategies focus on the stimulation of vascular ingrowth into the implanted grafts from the surrounding host tissue by (i) optimizing the material properties of scaffolds and (ii) their bioactivation by incorporation of growth factor delivery systems or by cell seeding. However, endothelial cell migration and physiological growth of new blood vessels has been demonstrated not to be faster than ~5 μm/h . TherefoTo overcome this problem, vascular ingrowth directed from the implanted graft towards the host tissue has been proposed to complement vascular ingrowth from the host tissue into the implanted graft. This can be achieved by incorporating the graft with either (i) preformed microvascular networks which can directly be perfused with blood by developing interconnections (inosculation) to the host microvasculature or (ii) microvascular fragments which rapidly develops into microvascular networks after transfer into the host tissue . In the in vivo and in vitro, e.g., polymers, bioactive ceramics, and hybrids (composites) [The characteristics of the scaffold material play an important role in angiogenesis of the graft. Many different scaffold materials for bone tissue engineering have been investigated posites) .in vivo biocompatibility, have been shown to be characterized by a poor vascularization [The chemical composition of scaffold materials has been shown to influence the angiogenic process at the implantation site. For instance, poly(lactic-co-glycolic acid) (PLGA), hydroxyapatite (HA), and dentin scaffolds show a slight inflammatory response after implantation, inducing marked angiogenic response and a good vascularization of the grafts after 14 days , 82. In rization . These fβ-TCP allows to overcome the problems due to PLGA's acidic degradation products that may induce tissue necrosis and negatively affect neoangiogenesis, since HA and β-TCP neutralize the acidic degradation products of PLGA [Combinations of biomaterials have been investigated to improve the scaffold properties. Composites consist of a combination of two or more materials with different properties, each displaying only some advantages and specific drawbacks. Polymer-ceramic composites have been successful in bone regeneration, exceeding the results obtained when these materials are used separately, showing improved mechanical and biological results . The com of PLGA .μm [μm [Not only the chemical composition but also the architecture of scaffolds is an important determinant for adequate vascularization . It shouμm  based onμm , but subμm [μm , 92. Relμm [μm , 93.A common strategy to improve scaffold vascularization is the stimulation of the angiogenic host tissue response at the implantation site by incorporation of angiogenic growth factors. For this purpose, VEGF , 95, basin vivo more strongly depends on the liberation of VEGF by the seeded cells than the differentiation potential of the BMSCs [in vivo implantation of PGLA scaffolds seeded with osteoblast-like cells. This indicated that this method alone is not sufficient to accelerate the vascularization to ensure the survival of seeded cells. Qu et al. [Another important aspect to consider is that many angiogenic growth factors are known to be released spontaneously by cells under stress-related conditions, including hypoxia. Due to hypoxia, bone-derived osteoblast-like cells as well as bone marrow mesenchymal stem cells (BMSCs) are known to liberate growth factors such as VEGF. Based on this cellular mechanism, an accelerated vascularization of scaffolds is also achieved by seeding the scaffolds with differentiated tissue-specific cells , 100 or he BMSCs . Even thu et al.  showed tu et al. .in vitro by seeding of vessel-forming cells onto scaffolds are being investigated. After seeding onto the scaffold, these cells rapidly assemble into immature microvessels. In contrast to the above-mentioned approaches that focus on the stimulation of vascular ingrowth into the implanted graft, prevascularization is aimed at generating preformed microvascular networks inside the graft prior to their implantation. After implantation, these networks can be rapidly perfused with blood by inosculation with the surrounding host microvasculature [Different approaches to prevascularize the graft culature .Proangiogenic cells, such as endothelial cells, endothelial progenitor cells, and mural cells (pericytes and smooth muscle cells), are widely used as cell source. Other cell sources including adult stem cells, such as pluripotent mesenchymal stem cells from bone marrow , 106 or Originally, endothelial and endothelial progenitor cells were used for the formation of blood vessels, but this resulted in blood vessels with suboptimal lifespan . Due to in vivo, through cocultivation of human umbilical vein endothelial cells (HUVECs) with mural precursor cells. This is in contrast to microvessels engineered with HUVECs alone, which rapidly regressed after 60 days [A better alternative being investigated seems to be the cocultivation of endothelial cells with mural cells. These cells are crucial for the stabilization, maturation, and long-term survival of newly formed microvessels. Koike et al.  demonstr 60 days . However 60 days , 116. Thin vitro preformed microvascular networks, the enzymatic digestion period for the isolation of microvascular fragments is much shorter (5-10 min) than that of single source cells and does not require complex and time-consuming in vitro incubation periods. Moreover, MF can also be obtained from patients in a one-step surgical procedure with a liposuction technique under local anesthesia [Prevascularization methods by cell seeding using cellular isolates may result in uncertain outcomes. Moreover, the correct ratio of cells to be used is difficult to determine. This led to a novel concept exploiting the use of microvascular fragments (MF) isolated from adipose tissue by short (5-10 min) digestion –119. MF esthesia .However, the MF procurement does not avoid the regulatory burden of using stem cell preparations obtained by enzymatic digestion, which are considered “more than minimally manipulated” by the FDA and the European counterpart the EMA. Therefore, recently, much effort was put in the development of mechanical disruption of the tissue creating microfragmented adipose tissue/nanofat (MFAT/NFAT) .Strikingly, it was found that the microfragmentation of the adipose tissue, which kept the microarchitecture  of the fat intact but disrupts most mature adipocytes, showed a remarkable enrichment of blood vessel-stabilizing pericytes and release of many more growth factors and cytokines involved in tissue repair and regeneration, noticeably via angiogenesis, compared to enzymatically obtained SVF . Moreoveex vivo, in addition to donor-dependent variability regarding the bone forming potency, further investigations are needed to standardize the production and quality of stem cells for therapeutic applications.Future investigations in cellular bone tissue engineering applications should be focused on enhancing vascularization, since adequate vascularization is a prerequisite for successful clinical bone regeneration. Moreover, due to existing discrepancies in the way human MSC are harvested and whether they are either directly applied without cultivation or isolated and cultured A promising future direction for cellular tissue engineering in jaw bone reconstruction with feasible clinical application is the use of the stromal vascular fraction (SVF) of human adipose tissue. SVF is considered a “single source” for cellular tissue engineering due to its heterogeneous population of essential cells, i.e., multipotent stem cells and progenitor cells, including endothelial cells, stroma cells, pericytes, preadipocytes, and hematopoietic cells. SVF also contains macrophages, which secrete a multitude of vascular growth factors and cytokines .in vivo [in vitro observations that ASCs in SVF secrete a variety of angiogenic and antiapoptotic growth factors [in vivo [The adipose stem cells (ASCs) in SVF have been shown to attach, proliferate, and osteogenically differentiate on calcium phosphate scaffolds  and secrin vivo  but alsoin vivo . This is factors  and that factors . Moreove[in vivo .in vitro [A first clinical trial confirmed that SVF/ASCs are capable to enhance bone and blood vessel formation , 138. Thin vitro .In vitro results of short (minutes) incubation of ASCs with a low dose of bone morphogenetic protein-2 (BMP-2) before seeding the cells on the scaffold (β-TCP and BCP) showed promising results; i.e., proliferation and osteogenic differentiation were enhanced by BMP-2 pretreatment, with concomitant downregulation of adipogenic gene expression. Stimulated gene expression of the osteogenic markers core binding factor alpha 1, collagen-1, osteonectin, and osteocalcin in the seeded ASCs was observed [Further investigations should also address the possibilities to enhance the osteogenic capacity of the ASCs within the treatment time of the “one-step surgery.” observed .in vitro and in vivo research needs to confirm these findings. The MF and MFAT/NFAT variants of adipose tissue may spur future developments in particular for homologous applications since the regulatory burden can be avoided and the angiogenic, anti-inflammatory, and regenerative growth factor secretion properties appear at least equal but likely even higher than collagenase-digested SVF [Recently, several studies suggested that adipose tissue-derived microvascular fragments (MF) show higher vascularization potential than SVF , 126. Hosted SVF .The major clinical benefit of applying adipose tissue-derived SVF, MF, or MFAT/NFAT compared to other single-cell sources is that a native mixture of essential cells can be harvested in large quantities in a one-step surgical procedure. This makes clinical application of adipose tissue-derived SVF or MF feasible, due to its lower morbidity rate and shorter treatment duration compared to the traditional treatment options, such as autologous bone harvesting, bone marrow-derived stem cells, and endothelial cells.in vitro 3D models of bone defects to investigate cellular bone tissue engineering techniques, and specifically vascularization, are lacking. Such models would enhance the understanding of the interaction of cells with the host environment for osteogenesis and angiogenesis. Moreover, it would facilitate new possibilities for vascularization strategies. Currently exploited 2D-models and in vivo animal models have several limitations, including controllability, reproducibility, and flexibility of design. Recently, novel strategies in 3D-models are investigated to mimic human physiology in vitro, including bone niche-on-a-chip and bone bioreactors [Appropriate reactors .in vitro models to study bone tissue engineering are lacking. Engineered in vitro 3D models mimicking the bone defect environment are crucial to facilitate new bone regeneration strategies. Successful bone reconstruction in the oral and maxillofacial region, using bone tissue engineering techniques, requires innovative future investigations focusing on the enhancement of vascularization.Important advancements have been made regarding the application of stem cells and the development of new strategies to improve vascularization in bone tissue engineering. However, adequate graft vascularization, which is a prerequisite to successful bone regeneration, is still considered a major challenge. The use of SVF of human adipose tissue seems to be a promising source for bone tissue engineering due to its heterogeneous population of essential cells for osteogenesis and angiogenesis. Besides, adipose tissue-derived MF is suggested as a promising cell source, due to its correct native cell ratios, for vascularization strategies. SVF, MF, and MFAT/NFAT are treatment options with clinical feasibility due to their large quantities that can be harvested and applied in a one-step surgical procedure. Appropriate


Women experience adverse drug reactions, ADRs, nearly twice as often as men, yet the role of sex as a biological factor in the generation of ADRs is poorly understood. Most drugs currently in use were approved based on clinical trials conducted on men, so women may be overmedicated. We determined whether sex differences in drug pharmacokinetics, PKs, predict sex differences in ADRs.Searches of the ISI Web of Science and PubMed databases were conducted with combinations of the terms: drugs, sex or gender, pharmacokinetics, pharmacodynamics, drug safety, drug dose, and adverse drug reaction, which yielded over 5000 articles with considerable overlap. We obtained information from each relevant article on significant sex differences in PK measures, predominantly area under the curve, peak/maximum concentrations, and clearance/elimination rates. ADRs were identified from every relevant article and recorded categorically as female-biased, male-biased, or not sex-biased.For most of the FDA-approved drugs examined, elevated blood concentrations and longer elimination times were manifested by women, and these PKs were strongly linked to sex differences in ADRs. Of the 86 drugs evaluated, 76 had higher PK values in women; for 59 drugs with clinically identifiable ADRs, sex-biased PKs predicted the direction of sex-biased ADRs in 88% of cases. Ninety-six percent of drugs with female-biased PK values were associated with a higher incidence of ADRs in women than men, but only 29% of male-biased PKs predicted male-biased ADRs. Accessible PK information is available for only a small fraction of all drugsSex differences in pharmacokinetics strongly predict sex-specific ADRs for women but not men. This sex difference was not explained by sex differences in body weight. The absence of sex-stratified PK information in public records for hundreds of drugs raises the concern that sex differences in PK values are widespread and of clinical significance. The common practice of prescribing equal drug doses to women and men neglects sex differences in pharmacokinetics and dimorphisms in body weight, risks overmedication of women, and contributes to female-biased adverse drug reactions. We recommend evidence-based dose reductions for women to counteract this sex bias. Women historically were excluded from clinical pharmaceutical trials because of potential risks to individuals of childbearing potential. The now discredited belief that studies of men apply without modification to women also contributed to this oversight. Remediation by the US National Institute of Health (NIH) Revitalization Act of 1993 mandated enrollment of women in federally supported phase III clinical trials; a European Union initiative provided practical tools regarding sex differences in drug study design , most stA consequence of this sex inequality hides in plain sight today: most drugs are prescribed to women and men at the same dose. Many currently prescribed drugs were approved by the US Food and Drug Administration (FDA) prior to 1993, with inadequate enrollment of female animals in preclinical research and of women in clinical trials . An illuVigiBase ; less frequently, data were available for drug half-life [t1/2], clearance/elimination measures conducted over fixed time intervals, systemic exposure, Vd, and Tmax.Each article was examined for relevance with additional searches conducted in PubMed and Web of Science for specific drugs; relevant publications identified in target articles were also inspected. Many drugs do not have a sex bias in pharmacokinetics. In an unknown number of instances, this reflects a true absence of sex differences based on empirical findings. For many drugs, however, the available PK data are lacking because female subjects were either not included in study design, or data were not reported by sex. Only studies of drugs that have a sex bias in pharmacokinetics were considered. Risk bias was examined in all articles to determine whether both sexes were included, whether sample size was adequate, and if subjects enrolled were adults or children. Sample size was considered adequate if there were 10–12 subjects/group. Several articles reporting statistically significant results with smaller sample sizes were reported but flagged with a disclaimer. From each relevant article, data on sex differences in PK measures was extracted; the majority of these measures were in units of either area under the curve [AUC] or peak/maximum concentration [CVigiBase, were searched. ADR data from this site are listed in instances where we were unable to find primary reports of ADRs for a specific drug. The ratio of women:men reporting ADRs was computed, and a value that deviated by more than 20% from 1.0 in either direction indicated that VigiBase reports were sex biased: values > 1.2 and < 0.80 indicated female and male sex biases, respectively. Values between 0.80–1.20 (inclusive) were interpreted as indicating no sex bias in ADRs. As discussed above, a major limitation of VigiBase is that it does not account for differences in the frequency with which women and men are treated with a specific drug. To directly examine this limitation in the context of sex differences in ADRs, we separately compared the prevalence of clinical ADRs (from data in the literature) with sex-specific reporting rates in VigiBase.ADRs were identified from every relevant article and recorded categorically as female-biased or male-biased. The EU database of ADR reports and the WHO’s ADR monitoring website, also known as Drugs@FDA database [Label information and PK and PD data, where available, were obtained from the database . Only daGenerally, sex differences refer to differences between women and men in the gonads, genitals, hormones, chromosome, and gene complements. Gender differences are more difficult to define and usually encompass gender roles and gender identity. With a few exceptions, we use the term sex differences to describe differences in pharmacokinetics, pharmacodynamics, and adverse drug reactions.Results are provided in the form of a descriptive analyses of whether studies with sex-specific patterns of PK bias predict sex-specific patterns of ADR bias. Only studies with clear sex differences in pharmacokinetics were included in the data synthesis. Chi-square analyses also evaluated whether PK biases predicted VigiBase data.VigiBase existed; as a result, tirilizad is discussed below but not included in final tabular calculations. Another drug, theophylline (R03DA04), exhibited conflicting PK data , no experimentally identified ADRs, but clear sex-biased adverse responses in post marketing reports and is therefore included in both the discussion and tabular calculations below. Lastly, for 4 drugs , ADRs were not uniformly present in one sex. For mirabegron, a number of publications indicated a female bias in ADRs, and for aspirin and sertraline, the overall number of ADRs reported was greater for women; these drugs were classified as having female-biased ADRs. For clozapine, empirical reports and meta-analyses identified over a dozen sexually differentiated ADRs, but overall, more ADRs occurred in men than in women, so clozapine was classified as having a male-biased ADR profile.Among the 86 drugs that were identified as having sex-specific PK data, one compound, tirilazad (ATC code N07XX01), exhibited clear sex-biased PK values (greater exposure in men), but neither clinical ADRs nor sufficient entries in Data for all drugs are contained in Additional file max and AUC values or drug distribution and elimination rates. Many drugs with striking sex differences in ADRs did not make it onto this list because there were no available PK data, almost always because drugs were approved prior to the year 2000, and thus the relevant information is not available in the online FDA database.We identified 86 drugs with significant sex differences in PKs, based on CThe correspondence of sex differences in PKs with sex differences in ADRs was striking. In 88% of instances, a sex difference in PKs was linked to a similar sex difference in ADRs , among these drugs, sex-biased PKs predicted sex-biased VigiBase ADR reporting ratios  in 74% of instances  for 15 of 26 (58%) ADR–VB ratio discordant drugs, and a sex-biased VB ratio in a direction opposite to that of the actual ADR bias  in 11 of 26 (42%) discordant drugs .Some have argued that what we ascribe to sex differences in the genesis of ADRs is causally related to differences in body weight. In reference to the sex difference in ADRs among patients taking zolpidem, Richardson et al.  stated “F statistics or p values, and were further complicated by small sample sizes and inflated type II error. Estimates of treatment effect size were also largely absent.Lastly, while we appreciate the difficulties in conducting clinical research, the standards of evidence encountered in many reports cited herein were disappointing, especially so in the clinical trials submitted to, and approved by, the FDA: reports of no statistical differences between the sexes were routinely not supported by For all drugs currently in use, the FDA should post, on their website, PK data that were submitted as part of the drug approval process  and associated peer reviewed publications. Generally, this information is unavailable for drugs approved prior to 2000. Once this information becomes accessible, scores if not hundreds of drugs with substantial PK sex differences will be revealed, which can facilitate corrective measures that will reduce female ADRs.WebMD, the list of adverse effects should include known sex differences.Credible evidence of sex differences in PKs should be made available in drug labels. In popular websites directed at the public either by drug companies, or by sites such as Almost all ADRs emerge from post-marketing reports, sometimes years after a drug appears on the market. The high correlation between elevated PKs in women and increased ADRs suggests that for drugs with higher female PKs, the initial dose should be lower for women than men and increased only if the lower dose fails to achieve the desired therapeutic effect. We anticipate this would eliminate or ameliorate many sex differences in adverse drug reactions. For orally administered drugs, pill splitting can begin to approximate a correction to the current practice where “one dose fits all.” Wherever feasible, drugs should be administered on a body weight adjusted basis (mg/kg) for men as well as women.P values, corrections deployed to control type I and type II error, and post hoc estimates of treatment effect magnitude. Such information is essential if clinicians are to develop informed decisions regarding sex differences in drug effects.The FDA should require all empirical NDA data to be accompanied by documentation of statistical analyses which should meet requirements for publication in a peer-reviewed journal. Experiments reported in NDAs are less likely to be subjected to replication attempts by the scientific community, and therefore methodological or interpretive errors are less likely to be rapidly corrected by the iterative scientific process. Standards of evidence should be accordingly maintained high. At a minimum, this should include mandatory reporting of information on: a priori power analyses, measures of central tendency, variance, treatment of outlier data, statistical tests used to support statements regarding drug effects and lack thereof, specific An appropriate understanding of the clinical relevance of sex-differences in drug treatment should be part of the board certification process for healthcare providers and reinforced in continuing medical education courses.Establishing sex parity in the drug approval process should be explicitly identified as a long-term goal of the Department of Health and Human Services. The decades-long pattern of neglect of female animals in preclinical research and underrepresentation of women in clinical trials and research must be corrected, and the recent NIH oversight and vigilance must be maintained.Pharmaceutical companies should pay more attention, beginning in the early drug development phases to sex-appropriate dosing . Drug deThe substantially elevated incidence of adverse drug reactions in women is undisputed, but the biological basis for this sex difference is poorly understood. The present study tested the hypothesis that sex differences in pharmacokinetics contributes to the female sex bias in adverse drug reactions. The analyses indicate that for many drugs administration of the same standard drug dose to women and men results in higher blood concentrations and/or longer elimination times in women and that these female-biased pharmacokinetics are a striking predictor of adverse drug reactions in women. Pharmaceutical pipelines that optimize drugs using males in vitro  and in vivo  may contribute to the prevalence of these PK-ADR relations. Pervasive unintended and adverse drug responses may reflect routine overmedication of women for many drugs. Measures to ameliorate the sex difference in adverse drug reactions include reducing drug doses for women.Additional file 1:. Supplementary Results.


The role of palliative surgery in the management of acute complications in patients with disseminated malignancy remains controversial given the complexity of assessing acute surgical risk and long-term oncologic outcome. With the emergence of checkpoint blockade immunotherapy, there appears to be an increasing role for historically palliative procedures as a bridge to systemic immunotherapy. This is especially evident in advanced microsatellite instability-high (MSI-H) colorectal cancer where malignant obstruction and fistula formation are more common and where immunotherapy with checkpoint blockade  has a high response rate with potential for favorable oncologic outcomes. We present a series of three patients with MSI-H metastatic colorectal cancer complicated by malignant bowel obstruction and fistula formation, who having progressed on standard chemotherapy, underwent palliative intervention as a bridge to immune checkpoint blockade with durable and clinically meaningful anti-cancer responses. These cases highlight the need to re-evaluate the role of historically palliative operations in the setting of disease progression for immunotherapy-responsive tumors. Surgery as a component of palliative care in the setting of metastatic colorectal cancer (mCRC) has been, and remains, controversial due to a narrow risk/benefit ratio with the potential for significant postoperative morbidity and mortality impacting quality of life, performance status, and tolerability of additional anti-cancer therapies . The decision to intervene surgically for patients with disseminated malignancy continues to revolve around various challenging medical and ethical considerations including disease burden, trajectory of disease progression with and without systemic therapy, availability of efficacious systemic treatment options, and patients' goals of care . Our preOne subset of colorectal cancer patients where this risk/benefit ratio is changing is in those with tumors exhibiting microsatellite instability-high (MSI-H) genotypes—a marker of dysfunctional DNA repair proteins, which can represent either somatic or germline  mutations. Given the high rate of response and durability of response with immune checkpoint blockade in MSI-H  when determined by IHC) colorectal cancer patients , palliative interventions may alleviate acute surgical processes, and rather than strictly improving quality of live, can act as a bridge to immunotherapy with the potential for prolonged survival.The most common palliative surgical intervention in advanced colorectal cancer remains stoma creation for relieving malignant bowel obstruction and its associated symptoms. Ostomy creation does not provide a cancer-related survival benefit, but does provide symptom relief and can improve quality of life . The rolP = 0.035) as well as OS from recurrence to death  compared to MSS disease  imaging revealed a 3.9 × 3.7 cm heterogenous soft tissue mass in the ascending colon near the hepatic flexure with invasion of the right anterior pararenal fascia and the second/third portion of the duodenum. There were associated enlarged and necrotic portacaval and mesenteric lymph nodes and a 1 cm hypodense lesion in the right hepatic lobe. Colonoscopy with biopsy confirmed a diagnosis of adenocarcinoma, and subsequent immunohistochemistry (IHC) showed loss of MLH1 and PMS2 gene expression consistent with MSI-H/dMMR. The patient was started on 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX), but continued experiencing symptoms of partial bowel obstruction including severe nausea, emesis, abdominal pain and dizziness from hypovolemia. CT imaging at this time is shown in A 62 year-old man with a history of liver transplantation for alcoholic cirrhosis underwent screening colonoscopy and was found to have moderately differentiated adenocarcinoma of the ascending colon. Molecular analysis identified loss of MLH1 and PMS2 genes, consistent with MSI-H/dMMR disease. The patient initially underwent a right hemicolectomy, and surgical pathology revealed AJCC stage IIIc (pT3N2b) disease with 7 of 27 lymph nodes positive for adenocarcinoma. He was treated with adjuvant FOLFOX, and in the following 6 months developed hepatic metastases along with diffuse omental and mesenteric disease consistent with failure of first-line therapy . AdditioIn the setting of concurrent liver dysfunction, prior liver transplantation, and new extensive liver metastases, discussions regarding surgical intervention vs. a less invasive percutaneous option were debated. With the potential to receive immunotherapy and given the increased risk associated with this unique patient, an interventional radiology-guided percutaneous gastrostomy tube was placed to palliate his obstructive symptoms, and he initiated TPN for caloric support. After extensive discussions regarding the unique risks of immunotherapy in the setting of liver transplantation, the patient was started on pembrolizumab. On-treatment follow-up imaging showed a partial response of both the primary and metastatic lesions  and the The final case is a 37 year-old man who initially experienced persistent alternating diarrhea and constipation. Within 5 months, these symptoms progressed to urinary retention, and imaging revealed a 5.5 cm sigmoid mass with invasion into the bladder and metastatic liver lesions . Image-gThe three cases presented here highlight the changing landscape of oncology care in the era of immunotherapy for colorectal cancer patients with MSI-H disease and a reasonable expectation of response to immune checkpoint blockade. For those who respond to treatment, advanced MSI-H disease now carries a much different prognosis compared to the pre-checkpoint blockade era. This is dramatically changing the decision-making and management of medical and surgical specialists who provide care to these advanced cancer patients.In colorectal cancer, checkpoint inhibitors such as pembrolizumab, nivolumab, and ipilimumab have been approved for MSI-H/dMMR metastatic colorectal cancer that has failed standard therapies –17. WhilSince checkpoint blockade immunotherapeutics are not yet approved for first-line therapy, there is often a significant period on traditional chemotherapy before starting immunotherapy. In the CheckMate-142 trial, 54% of patients had received 3 or more chemotherapy regimens before starting immunotherapy on trial . As for Our recent experience suggests that interventions that are historically viewed as “palliative” should be re-evaluated and thoughtful consideration be given for surgical interventions as a bridge to immunotherapy in select metastatic colorectal cancer patients with MSI-H disease. Because MSI-H disease is frequently less responsive to standard chemotherapy, intervening for complications such as bowel obstruction and fistulae formation may be more appropriate as this allows for palliation of symptoms and for patients to transition to checkpoint blockade therapy, where response rates are more pronounced. Our experience described in this study is limited by a small sample size and has potential for selection bias. Therefore, the criteria for patient selection, route and timing of intervention requires investigation and should be discussed in a multidisciplinary setting. Further investigation is warranted in this subset of MSI-H mCRC patients to validate the role of palliative interventions that may provide a window for immunotherapy, potentially leading to prolonged and durable responses in this unique patient population.Written informed consent was obtained from the patients for the publication of any potentially identifiable images or data included in this article.SJ: manuscript, planning, writing, and editing. JJ: case write-up, figure creation, references, manuscript editing, and submission. RC: case submission, surgical expertise, and manuscript editing. JL and MK: manuscript editing and adding references. EK and KT: patient care/submission and manuscript editing. JG and AG: expert opinion, references, and manuscript editing. AK, SG, and RB: surgical expertise, patient care/submission, and manuscript editing. MF: expert opinion and manuscript editing. MC: principle investigator.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.


Biological heterogeneity and low inherent immunogenicity are two features that greatly impact therapeutic management and outcome in colorectal cancer. Despite high local control rates, systemic tumor dissemination remains the main cause of treatment failure and stresses the need for new developments in combined-modality approaches. While the role of adaptive immune responses in a small subgroup of colorectal tumors with inherent immunogenicity is indisputable, the challenge remains in identifying the optimal synergy between conventional treatment modalities and immune therapy for the majority of the less immunogenic cases. In this context, cytotoxic agents such as radiation and certain chemotherapeutics can be utilized to enhance the immunogenicity of an otherwise immunologically silent disease and enable responsiveness to immune therapy. In this review, we explore the immunological characteristics of colorectal cancer, the effects that standard-of-care treatments have on the immune system, and the opportunities arising from combining immune checkpoint-blocking therapy with immune-modulating conventional treatments. Colorectal cancer (CRC) is the third-most commonly diagnosed cancer worldwide with 1.4 million new cases annually . Age is Even though the majority of CRC cases are of a sporadic nature, a subgroup of patients presents with hereditary forms of CRC, such as the common manifestation of Lynch syndrome or the rare familial adenomatous polyposis. In individuals with the former, inherited mutations or epigenetic inactivations of DNA mismatch-repair (MMR) genes result in a deficient MMR (dMMR) function. For those developing CRC, MMR deficiency usually causes a high tumor mutational burden or microsatellite-instable (MSI) status . Apart fA quarter of all CRC patients present with metastatic disease at the time of diagnosis, whereas approximately half of the total patient population will eventually have developed metastases. The most frequently affected distant organ is the liver, followed by the lungs and peritoneum . The preGenerally, no curative treatment exists for multi-organ metastasis. In the past two decades, a number of new systemic therapies of cytotoxic and biologically targeted agents have been taken into routine use for advanced CRC, extending patients’ lives and most importantly, alleviating their symptoms. Nevertheless, unresectable metastases, particularly in abdominal cavity organs, remain the cause of severe morbidity and poor survival . New insWithin this frame of reference, the potential of using the immune system to fight progressing cancer has opened paradigm-changing therapeutic avenues. Tumor-defeating immunity involves both tumor-antigen recognition and the action of cytotoxic T-lymphocytes. However, as the tumor is also “self”, protective mechanisms against auto-immunity impede immune surveillance. This counterbalance between the cancer and the immune system creates a state of equilibrium, or immune tolerance, which can be edited therapeutically. Therefore, immune therapies are aimed at overcoming tumor-induced immune-suppressive mechanisms and invoking antitumor immune responses. So far, this concept has proven successful in the treatment of a limited number of immunogenic tumors, but for less immunogenic cancers such as the majority of CRC cases, additional stimulation is required to breach the immune tolerance and for patients to achieve beneficial and durable treatment responses.Hypoxia (low tumor oxygenation), a major TME (tumor microenvironment) hallmark, continuously selects for tumor cell clones with aggressive phenotypes and stimulates epithelial-to-mesenchymal transition, a process during which tumor cells lose the ability to adhere to each other in favor of increased migration within the extracellular matrix . HypoxicStudies have demonstrated major responses and long-term survival to ICB in early-stage, locally advanced, and metastatic dMMR CRC, characterized by high tumor mutational burden and positive MSI status and therefore high densities of tumor-associated antigens ,17,18,19The heterogeneous nature of CRC at the molecular and clinical level has warranted a more comprehensive method of classification that combines molecular signatures of tumor cells with TME features  . The conIt has been over a decade since Galon and colleagues elegantly demonstrated that the tumor’s immune–cell contexture, as assessed in large cohorts of CRC patients with early-stage disease, was better at predicting patient survival than well-established histopathological methods . This coHowever, it has been argued that the Immunoscore fails to include other immune-cell populations hosted by the TME such as macrophages, dendritic cells, and natural killer cells, all of which may also be important for predicting prognosis . These cThis class of agents constitutes the mainstay of most CRC treatment regimens. Their mechanism of action relies on disruption of DNA synthesis by the incorporation into DNA and RNA structures and by inhibition of the DNA synthesis enzyme thymidylate synthase . The mosThis agent is a third-generation platinum analog. The mechanism behind its cytotoxicity relies on the adduct formation with DNA, resulting in the blocking of DNA replication and transcription. Reduction of thymidylate synthase synthesis is considered as a secondary effect of oxaliplatin that contributes to the synergy observed when oxaliplatin is combined with fluorouracil .Oxaliplatin perfectly illustrates the interplay between cytotoxic and immune-modulating effects of chemotherapy, the latter in terms of immunogenic cell death (ICD) ,50 Figu. In the The ability of oxaliplatin to initiate ICD may play a critical role with respect to its rational combination with ICB therapy. Preclinical studies demonstrated that oxaliplatin provoked TME infiltration of cytotoxic T-cells and sensitization of CRC and other adenocarcinomas to ICB therapy ,52,53. AAnother platinum analog, cisplatin, was unable to sensitize CRC models to ICB therapy . InteresApproximately 50% of cancer patients receive radiotherapy during the disease course . IonizinHowever, not all radiation effects are in favor of the antitumor response, and radiation has been shown to also enhance TME immune-suppressive features in experimental models. For example, dendritic cells co-cultured with irradiated murine mammary carcinoma cells had significantly reduced surface expression of both antigen-presenting and co-stimulatory markers . TransfoIn principle, three scenarios can be set for “on-target” cytotoxicity combined with “off-target” tumor-targeting immune responses in the standard-of-care CRC management. They are: neoadjuvant treatment of locally advanced disease in order to impede metastatic progression, neoadjuvant treatment of oligometastatic disease in order to achieve systemic tumor clearance, and early-line therapy in unresectable metastatic disease for durable systemic control.In locally advanced rectal cancer (LARC), neoadjuvant chemoradiotherapy (CRT) with a fluoropyrimidine in a non-cytotoxic radiosensitizing dose and resection of the residual tumor result in low local recurrence rates , but metIn liver-confined metastatic CRC with a therapeutic intent for surgical resection or radiofrequency ablation, oxaliplatin-based NACT can be used for patient selection with respect to tumor aggressiveness, disease down-sizing, or conversion of initially unresectable disease , three sHepatic artery infusion (HAI) chemotherapy is attractive as it limits systemic toxicity and allows combination with systemic therapy. A number of early-phase trials have investigated the concept, using oxaliplatin as the HAI compound for unresectable CLM with treatment toxicity and response as endpoints, but only one study has been carried on with the aim of transforming to resectable disease by using oxaliplatin, irinotecan, and fluorouracil . The invIt is clear that immune responses arise from the ”off-target” effects of these various treatment approaches. However, their involvement towards the eradication of occult tumor at other sites  has been essentially unexamined. Thus, we studied antitumor immunity invoked by oxaliplatin-HAI and the long-term outcome of primarily unresectable CLM in patients who had their first recurrence of oxaliplatin-naïve isolated CLM that was considered technically unresectable . Those wPatients with disseminated CRC make up an extremely heterogeneous group, with survival rates depending on the metastatic sites, tumor load, and severity of systemic inflammation; however, median OS has improved to approximately 30 months in clinical trials . The powAs conveyed in the above sections, the effects that cytotoxic therapies have on the immune system are complex and often hanging on a balance between activating and suppressive responses. Despite the accumulating preclinical evidence displaying synergies between cytotoxic and immune therapies, the heterogeneous nature of cancer as it progresses and the individual differences of the hosts’ immune systems present practical barriers that need to be overcome for this partnership to be clinically successful. A better biological insight into the individual patient’s systemic responses to conventional treatment is an important step towards a rational integration of immune therapy in this context. Conceptually, detection of circulating proteins that may reflect the changing TME and its systemic manifestations, as well as the constitutional and acquired physiology of the patient, is a promising path to treatment-related biomarker discovery. We employed an antibody array technology to monitor serial serum samples collected throughout an intensified neoadjuvant treatment course in LARC study patients, in order to dissect the contribution of the individual modalities to treatment outcome and tolerance . The comThe particular study (NCT00278694) prospectively enrolled 97 patients with T2-4N0-2M0 rectal cancer between 2005 and 2010, who were given oxaliplatin-/fluorouracil-containing induction NACT and sequential CRT with concomitant oxaliplatin weekly and capecitabine daily on the days of radiotherapy before pelvic surgery ,121. TheThe study biobank consists of serum and plasma samples collected at the time of diagnosis, immediately following NACT and then CRT completion, and at the time of treatment evaluation four weeks after the neoadjuvant treatment. Following the initial screening with the antibody array , we systThe almost exclusively tumor-specific CA9, whose gene is induced by the hypoxia-inducible factor-1α, is a transmembrane enzyme aimed at controlling intracellular pH changes that result from the accumulation of acidic metabolites under hypoxic conditions. Specifically, CA9 catalyzes the reversible hydration of carbon dioxide to bicarbonate and protons, which restores physiological intracellular pH . HoweverWe found that patients who from their individual baseline level showed a strong increase in circulating CA9 after completion of the short-course induction NACT, had significantly better survival without metastatic progression than patients with low post-NACT versus baseline alteration. Strikingly, this strong CA9 increase was significantly correlated with tumor down-staging and node sterilization, but not with the degree of surviving tumor cells relative to fibrosis  of the surgical specimen . The hisThis metallopeptidase belongs to a family of proteases with critical roles in extracellular matrix degradation and remodeling as well as angiogenesis . LipocalWe found that circulating MMP9, together with lipocalin-2, had significantly declined from the baseline values at completion of the induction NACT and gradually reverted over the remaining neoadjuvant course. Moreover, the greater the fall in post-NACT and post-CRT MMP9 levels, the more favorable survival without metastatic progression . These oThis soluble tumor necrosis factor decoy receptor is a glycoprotein that binds to the ligand of the receptor activator of nuclear factor-kappaB (RANK), RANKL . The OPGWe found that circulating OPG appeared to show contradictory biological behavior. On the one hand, high baseline levels were associated with metastatic progression after the pelvic surgery . It mighThis is a hematopoietic factor that can activate functionally mature dendritic cells ,146,147 We showed that circulating FLT3LG increased following NACT and the sequential CRT and that the post-NACT increase was associated with favorable survival outcome. We further observed that patients who had an oxaliplatin dose intensity under CRT that maintained compliance to the radiotherapy protocol, had low probability of metastatic progression after the definitive surgery . Our intIn this LARC study, the initial modality of the oxaliplatin-containing neoadjuvant therapy course, consisting of two well-tolerated cycles of the Nordic FLOX regimen , led to Conventional chemotherapy and radiotherapy are particularly appealing now that more is known about their effects on the immune system, and about the fact that dose and timing of the administration play a decisive role. From this, a next step in CRC management will be the rational combination of cytotoxic agents with ICB therapy in patients with locally advanced or advanced pMMR CRC , as for In our own study, METIMMOX: Colorectal Cancer Metastasis—Shaping Antitumor Immunity by Oxaliplatin, patients with unresectable and previously untreated metastatic pMMR CRC are randomized to either the standard-of-care Nordic FLOX regimen or repeat sequential two FLOX cycles and two cycles of the PD-1 inhibitor nivolumab . The priAs discussed in this review, there is clear evidence that the effects of cytotoxic therapies extend beyond the shrinking of the tumor masses and involve factors with critical roles in tumor-targeting immunity. Investigations of these factors, as they appear systemically and intratumorally over the course of treatment, will lead to a better understanding of the immune-modulating effects of conventional cancer therapies and match the accumulating knowledge on the immunologic characteristics of individual tumors. In CRC, with a complex TME consisting of hypoxic regions with highly invasive propensity and immune tolerance that pose therapeutic challenges, the next therapeutic milestone is likely found here.


The use of inertial measurement units (IMUs) has gained popularity for the estimation of lower limb kinematics. However, implementations in clinical practice are still lacking. The aim of this review is twofold—to evaluate the methodological requirements for IMU-based joint kinematic estimation to be applicable in a clinical setting, and to suggest future research directions. Studies within the PubMed, Web Of Science and EMBASE databases were screened for eligibility, based on the following inclusion criteria: (1) studies must include a methodological description of how kinematic variables were obtained for the lower limb, (2) kinematic data must have been acquired by means of IMUs, (3) studies must have validated the implemented method against a golden standard reference system. Information on study characteristics, signal processing characteristics and study results was assessed and discussed. This review shows that methods for lower limb joint kinematics are inherently application dependent. Sensor restrictions are generally compensated with biomechanically inspired assumptions and prior information. Awareness of the possible adaptations in the IMU-based kinematic estimates by incorporating such prior information and assumptions is necessary, before drawing clinical decisions. Future research should focus on alternative validation methods, subject-specific IMU-based biomechanical joint models and disturbed movement patterns in real-world settings. Evaluating kinematical characteristics is crucial for a correct clinical understanding of complex functional movements such as gait , a forwaA 3D optical motion capture system is currently the gold standard and the most commonly used technique to study lower limb movement ,5 and soTo overcome these restrictions, the use of wearable devices to monitor human movements has been studied extensively ,7. RecenHowever, when applying IMU-based joint kinematics to specific applications in a clinical setting, a good understanding of the methodological requirements is still lacking. The aim of this review is twofold—to evaluate the methodological requirements for IMU-based, lower limb joint kinematic estimation to be applicable in a clinical setting, and to suggest future research directions.This review focused on peer-reviewed articles and conference papers published in English which included a description of the methodology used to obtain kinematic variables of the lower limb by means of IMUs. To be included in the review, a validation against a reference system e.g., an optical motion capture system, should be reported. Also, studies of which the methodology was extended with measurement modalities other than these available within IMUs, i.e., a pressure sensor, were included. A journal paper was preferred over a conference paper when similar content was covered.Review papers and book chapters were excluded. Studies related to upper quadrant movements were not considered for this review. Studies of which the methodology was not applicable for outside-laboratory usage were left out. Articles lacking a description of a reproducible algorithm to obtain joint kinematics were also excluded.The available literature was searched in a systematic way through a personalized four step PRISMA method . These four steps are summarized in Data from all selected articles were extracted and are structured in The results of our systematic literature search initially identified 4290 articles of which thirty-one articles were ultimately included. Extracted information of included studies on study characteristics , signal The study characteristics summarized in Among the thirty-one studies included in this review, gait was the most commonly evaluated activity, which was assessed either on a treadmill ,17,23,24Most articles did not study the full 3D kinematics of the entire lower quadrant. Sagittal knee joint movements were most extensively studied ,40,43,44Nearly all the studies made use of sensors placed on adjacent segments surrounding the joint of interest. Four studies opted for a more distal placement on the segments ,22,35,37Signal processing characteristics  were splEight studies compensated for gyroscope bias ,34,37,42The analysis of human motion yields biomechanically related prior information such as joint range of motion (RoM) ,39,41, sBesides prior knowledge, specific assumptions were also exploited to ease the estimation of joint kinematics. Both a single description of joint acceleration in a common reference frame (up to a time-variant rotation matrix) and segment rigidity were often used as assumptions ,36,38,42A set of common approaches for information fusion were identified in the thirty-one studies considered in this review. Strap-down integration was used ,35,42,45Different from conventional information fusion methods, other methods were proposed for the application of joint kinematic estimation. For example, three studies used regression techniques to map raw sensor data to known kinematic parameters ,31,43. FIntegration drift originates as an artifact by integration of noise- and bias-affected gyroscope measures. In this article, global and relative drift are defined as drift with respect to a global or an adjacent segment reference frame, respectively. Drift in the vertical plane was compensated by tilt estimates from accelerometer readings, which are dominated by gravity during characteristic samples ,34,35,42The information fusion techniques from To overcome misalignment between sensor and segments, different approaches were identified. Two studies aligned the sensor and segment with active  or passiStudy validity with respect to a reference system is reported in This review systematically evaluated the methodological requirements for IMU-based lower limb joint kinematic estimation. Human motion analysis with inertial sensors has the potential to increase understanding in movement patterns in trusted well-known environments . HoweverIn summary, lower limb kinematic estimation from inertial sensors requires a well-defined application and study characteristics. The study characteristics define which sensor modalities will be measured and processed to compensate for the following sensor restrictions: (1) due to their microelectromechanical architecture, raw sensor measurements are prone to noise and non-zero biases; (2) an integration step of measurements is typically necessary to obtain joint kinematics, resulting in drifting estimates of sensor orientations and joint kinematics; (3) inertial sensors are usually not aligned with the bone, which implies that misalignment with respect to anatomical coordinate frames needs to be identified; (4) initial sensor orientations need to be determined.In order to overcome these sensor restrictions, all of the included articles were required to rely in some way on application-specific prior information and assumptions. However, by including this additional information in the methodology, the resulting kinematic estimates need to be interpreted carefully, taking into consideration a number of factors, before drawing any clinical decision.First, the biomechanical system yields usable prior information, but this information can be violated in practical applications. For example, assumptions on the range of motion can restrict the kinematic solution to be within a given interval of normal physical ability. However, RoM boundaries are not generalizable across patient populations who might be hypermobile or hypomobile, exceeding or not reaching normal RoM respectively. Also, segment lengths are relevant priors that can be obtained, as described by Crabolu et al. . MultiplSecond, assuming periodicity in motion dates back to Morris et al. , to solvMoreover, calibration movements are typically required to obtain a misalignment matrix between the sensor and anatomical reference frames. However, predefined calibration movement with a fully extended leg can be difficult within certain patient populations or during post-op periods ,42. As aThe incorporation of additional information such as assumptions and prior information can easily be done in an optimization-based smoothing approach for applications that demand high accuracy ,39. SolvThis review highlights the application dependency and inherent connection of methodological characteristics for lower limb IMU-based kinematic estimation. Assumptions and prior information are typically used to compensate for sensor limitations and to enhance the quality of kinematic estimates. Because of this, IMU-based kinematic estimates have to be interpreted carefully, before drawing any clinical decision. We identified a number of directions and pieces of advice for future research for the estimation of clinically relevant lower limb kinematics.For the clinical interpretability of the joint kinematics, the general reporting standards from the International Society of Biomechanics (ISB) ,70 need Gait predominantly occurs in the sagittal plane, and therefore the knee is often modeled as a hinge joint. A hinge joint axis can be estimated from IMU readings ,66 to coFurthermore, a recent trend is visible towards the inclusion of multiple joints and segments, rather than estimating kinematics for separate joints ,38,42. WOptical motion capture systems are the most commonly used technique to study lower limb movement. They are therefore also most often used as a reference to evaluate IMU-based joint kinematic estimation methods. However, due to manual marker placement errors  and softWhilst IMUs are proposed for long term observations, there are still few studies tackling measurements beyond 30 s. With respect to long, in-the-wild studies, measurement duration must be increased ,82,83. RMost of the published work recruited young, healthy participants. However, in clinics, most attention must go to the investigation of different patient populations with disturbed daily functional movement such as gait, sit-to-stand, stand-to-sit or climbing stairs . One of Where are we now? Despite the ongoing research regarding the computation of joint kinematics by means of IMUs, there still appear to be difficulties which prevent their use in daily clinical practice. It is reasonable to assume that the complexity in obtaining meaningful kinematic measures from noisy and biased measured sensor data and sensor restrictions regarding integration drift, sensor-to-segment alignment and initial sensor orientation explain these study restrictions. What can already be measured with sufficient accuracy? Most often, biomechanically inspired assumptions and prior information are used to compensate for sensor limitations. Both clinicians and engineers have to be aware of the possible adaptations in the IMU-based kinematic estimates by incorporating such prior information and assumptions, before drawing clinical decisions. What needs to be tackled with high priority? Investigating the appropriate validation methods that might be superior in tracking underlying bone movement and can overcome the restrictions of optical motion capture systems as a reference. What might yield novel results? Subject-specific IMU-based biomechanical joint models applied to populations with disturbed movement patterns in real-world settings. Combined efforts of engineers and clinical experts can result in application- and patient-specific implementations that will be valuable to clinicians.This review systematically evaluated the methodological requirements for IMU-based lower limb joint kinematic estimation.


Seem DL, Lee I, Umscheid CA, Kuehnert MJ. PHS guidelinefor reducing human immunodeficiency virus, hepatitis B virus, and hepatitisC virus transmission through organ transplantation. Public Health Rep2013;128:247–343), hereafter referred to as the 2013 PHSguideline. PHS evaluated and revised the 2013 PHS guideline because of severaladvances in solid organ transplantation, including universal implementation ofnucleic acid testing of solid organ donors for HIV, HBV, and HCV; improvedunderstanding of risk factors for undetected organ donor infection with theseviruses; and the availability of highly effective treatments for infection withthese viruses. PHS solicited feedback from its relevant agencies, subject-matterexperts, additional stakeholders, and the public to develop revised guidelinerecommendations for identification of risk factors for these infections amongsolid organ donors, implementation of laboratory screening of solid organdonors, and monitoring of solid organ transplant recipients. Recommendationsthat have changed since the 2013 PHS guideline include updated criteria foridentifying donors at risk for undetected donor HIV, HBV, or HCV infection; theremoval of any specific term to characterize donors with HIV, HBV, or HCVinfection risk factors; universal organ donor HIV, HBV, and HCV nucleic acidtesting; and universal posttransplant monitoring of transplant recipients forHIV, HBV, and HCV infections. The recommendations are to be used by organprocurement organization and transplant programs and are intended to apply onlyto solid organ donors and recipients and not to donors or recipients of othermedical products of human origin . The recommendations pertain to transplantation of solid organsprocured from donors without laboratory evidence of HIV, HBV, or HCV infection.Additional considerations when transplanting solid organs procured from donorswith laboratory evidence of HCV infection are included but are not required tobe incorporated into Organ Procurement and Transplantation Network policy.Transplant centers that transplant organs from HCV-positive donors shoulddevelop protocols for obtaining informed consent, testing and treatingrecipients for HCV, ensuring reimbursement, and reporting new infections topublic health authorities.The recommendations in this report supersede the U.S Public Health Service (PHS)guideline recommendations for reducing transmission of human immunodeficiencyvirus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) through organtransplantation ( Since the emergence of human immunodeficiency virus (HIV) in the United States,the U.S. Public Health Service (PHS) has made recommendations to minimize therisk for potential HIV transmission to organ transplant recipients  have universally implemented screening of organdonors for HIV, HBV, and HCV infections by NAT since 2017, the 12-monthtime frame should be shortened , which was initially approved in 1984and subsequently amended . Therecommendations apply to OPOs, transplant centers, and other transplantrecipient health care providers. The recommendations pertain to use of solidorgans procured from donors without laboratory evidence of HIV, HBV, or HCVinfection. Additional considerations when using solid organs procured fromdonors with laboratory evidence of HIV, HBV, or HCV infection are included. As aresult of the opioid epidemic, an increasing number of deceased donors havedetectable HCV RNA  regardingblood, organ, and tissue policies; andInput and feedback from the public solicited by posting draft recommendationsin the Federal Register . All titles and abstractswere evaluated, and 86 articles that described transmission to recipients werereviewed in detail to ascertain donor risk factors for HIV, HBV, and HCVinfections. In addition, the results of a systematic review performed to informdevelopment of the 2013 PHS guideline were reviewed to identify pertinenttransmission events  to assess the risk for recent HIV, HBV, or HCVinfection among donors. These criteria were evaluated to determine whether theirpresence among organ donors was associated with a substantive risk for HIV, HBV,or HCV transmission to recipients despite negative donor serologic and nucleicacid screening test results because of an acute, undetected donor infection. For all criteria, two methods were used toassess this risk. First, CDC conducted a review of OPTN data on alldonor-derived HIV, HBV, and HCV infections investigated, reviewed, andadjudicated by the OPTN ad hoc Disease Transmission Advisory Committee during2008–2018 to identify reported donor risk criteria . Second,Four criteria were not described as donor risk factors in HIV, HBV, or HCVtransmission events reported to the OPTN or identified through the literaturesearch; two additional criteria had only been reported as donor risk factorsonce . Of theshttps://stacks.cdc.gov/view/cdc/88644).Hemodialysis was included as a risk criterion in the 2013 PHS guideline becauseof reports of an association between HCV infection and outpatient dialysis born to a mother at increased riskfor HIV, HBV, or HCV infectionChild breastfed by a mother at increased risk for HIVinfectionContinue use of the following criteria that would result in augmenteddonor risk designation if present in the 3 months before organprocurement:Sex with a person known or suspected to have HIV, HBV, or HCVinfectionMan who has had sex with another manSex in exchange for money or drugsSex with a person who had sex in exchange for money or drugsDrug injection for nonmedical reasonsSex with a person who injected drugs for nonmedical reasonsIncarceration  for ≥72 consecutive hoursUnknown medical or social historyChild born to a mother with HIV, HBV, or HCV infectionChild breastfed by a mother with HIV infectionSupport the development and use of tools and processes to educatetransplant providers and enhance the process of transplant candidatecounseling to increase organ use.The diversity of opinions expressed during the deliberations of the AdvisoryCommittee for Blood and Tissue Safety and Availability were considered,including the use of a term to designate organ donors with HIV, HBV, or HCVinfection risk factors. Proposed revisions to the 2013 PHS guidelinerecommendations were published in the Federal Register .Attendees included relevant representatives from federal agencies, transplantcenters, and professional societies. Attendees gave presentations and discussedthe epidemiology of HCV infections, ethics of transplanting organs fromHCV-viremic donors to HCV-negative recipients, the use of direct actingantiviral (DAA) therapy as prophylaxis or treatment, and guiding principles fortransplant centers to consider when transplanting organs from HCV-viremic donorsto HCV-negative recipients. Considerations included in this guideline are basedon this workshop and are not required to be incorporated into OPTN policy.The guideline recommendations are categorized into the following six topic areas:Risk assessment of living and deceased donorsLiving and deceased solid organ donor testingTransplant candidate informed consentRecipient testing and vaccinationCollection and storage of donor and recipient specimensTracking and reporting of donor-derived disease transmission eventshttps://stacks.cdc.gov/view/cdc/88644).Steps of the organ transplantation process related to the recommendations , differeBOX1. Initial transplant candidate informed consent discussion and vaccination2. Risk assessment of living and deceased donors3. Living and deceased solid organ donor testing4. Transplant candidate informed consent discussion, including donor riskfactors5. Recipient testing6. Collection and storage of donor and recipient specimens7. Tracking and reporting of donor-derived disease transmission eventshttps://stacks.cdc.gov/view/cdc/88644). No further rationale isincluded here for these recommendations. Recommendations in this guideline are included in the bulleted lists that follow. Thelists are followed by the rationale for new or modified recommendations. Certainrecommendations remain unchanged from the 2013 PHS guideline except for minorwording changes or reorganization  should be informed of the donor evaluationprocess, including the review of medical and social history, physicalexamination, and laboratory tests to identify the presence of infectiousagents or medical conditions that could be transmitted by organtransplantation.OPOs should ascertain, confidentially, whether any of the followingcriteria that would put organ recipients at risk for acquiring HIV, HBV,and HCV infections were applicable to potential organ donors within 30days before organ procurement:Sex  with a person known or suspected to have HIV, HBV, orHCV infectionMan who has had sex with another manSex in exchange for money or drugsSex with a person who had sex in exchange for money or drugsDrug injection for nonmedical reasonsSex with a person who injected drugs for nonmedical reasonsIncarceration  for ≥72 consecutive hoursChild breastfed by a mother with HIV infectionChild born to a mother with HIV, HBV, or HCV infectionUnknown medical or social historyLiving potential donors who have past or ongoing risk for acquiring HIV,HBV, or HCV infection should receive individualized counseling onspecific strategies to prevent exposure to these viruses during theperiod before surgery.Remove any specific term  todescribe donors with risk factors for acute HIV, HBV, or HCVinfection.Donors who meet one or more of the listed criteria might be at risk for having anacute HIV, HBV, or HCV infection , eight were repeatedly reported asdonor criteria associated with donor-derived disease transmission events fromreports to the OPTN ad hoc Disease Transmission Advisory Committee, including 1)sex with a person known or suspected to have HIV, HBV, or HCV infection; 2) manwho has had sex with another man; 3) sex in exchange for money or drugs; 4) sexwith a person who has had sex in exchange for money or drugs; 5) drug injectionfor nonmedical reasons; 6) sex with a person who injected drugs for nonmedicalreasons; 7) incarceration for ≥72 consecutive hours; and 8) unknownmedical or social history . FourriAlthough OPTN does not routinely collect data on specific criteria resulting inIRD designations, a recent analysis was conducted to determine the criteriaresulting in IRD designation among a random sample of IRDs during 2018  in addition to NAT for all three viruses, regardless of therisk criteria identified during screening using assays licensed,approved, or cleared by the Food and Drug Administration (FDA) for donorscreening.For living potential donors, testing should continue to beperformed as close as possible to the surgery but at leastwithin the 28 days before organ procurement.For deceased potential donors, the donor specimen should becollected within 96 hours before organ procurement, with resultsof these screening tests available at the time of organprocurement.Because next-of-kin interviews used to identify risk factors might be unreliable may be used, and the blood vesseldonor meets risk criteria listed under Risk Assessment of Living andDeceased Donors, then the transplant center team should include thisrisk information in the informed consent discussion.Regardless of a donor’s risk profile for HIV, HBV, or HCVinfections, transplant programs should test all organ recipients usingassays licensed, approved, or cleared by FDA for diagnosis.Before transplantation, HIV testing should be performed using aCDC-recommended laboratory HIV testing algorithm  after transplantationshould be tested for viral hepatitis, even if previous hepatitisB or hepatitis C testing was negative.All organ transplant candidates should be vaccinated against HBVinfection.All recipients should be screened for HIV, HBV, and HCV infections aftertransplantation because next-of-kin interviews do not accurately identify alldonors with risk factors (Use of organs from donors who are positive for anti-HCV or HCV RNA (μg) hepatitis B vaccineseries  has recommended hepatitis Bvaccination for adults at risk for HBV infection since 1982, for all infantssince 1991, and for children aged ≤18 years since 1999 (OPOs and living donor recovery centers should archive donor bloodspecimens for at least 10 years. These specimens should be collectedwithin 24 hours before organ procurement. Two blood specimens should becollected for archiving: an ethylenediaminetetraacetic acid (EDTA)plasma specimen or serum specimen for serologic assays and a separateEDTA plasma specimen for NAT. If only feasible to collect one specimen,a plasma specimen collected in EDTA, rather than a serum specimen, isoptimal.For deceased donors, living donors, transplant candidates, andrecipients, two blood specimens should be collected when HIV, HBV, orHCV infection testing is planned: an EDTA plasma specimen or serumspecimen for serologic assays and a separate EDTA plasma specimen forNAT.All stored blood vessel conduits from a donor found to be infected withHIV, HBV, or HCV should be quarantined immediately and not released forclinical use unless the HIV-, HBV-, or HCV-infected vessel conduits areneeded for the initial transplant procedure in the recipient. Aftercompleting the initial transplant procedure, any remaining vesselconduits should be disposed of in accordance with hospital policy toprevent inadvertent release from quarantine and unintentional use inother patients.https://stacks.cdc.gov/view/cdc/88644).The recommendations for Collection and Storage of Donor and Recipient Specimenshave not been changed from the 2013 PHS guideline, with the exception that thisguideline includes a recommendation on the timing of blood donor specimencollection. The remaining recommendations in this section have been reorganizedto increase clarity  an organ or bloodvessel conduit donor meets one or more of the criteria as specifiedunder Risk Assessment of Living and Deceased Donors; 2) the donor isinfected with HIV, HBV, or HCV; or 3) an organ recipient infection withHIV, HBV, or HCV is suspected to be donor derived, they should contactother organizations involved with organs or tissue procured from thedonor, including 1) OPTN, 2) the OPO or living donor recovery center, 3)transplant centers, and 4) any institutions considering tissue and eyerecovery. Living donors who test positive for HIV, HBV, or HCVinfections should be notified of the results.An OPO, living donor recovery center, or transplant center also shouldnotify the appropriate public health authorities if the deceased donor,living donor, or transplant recipient is infected with HIV, HBV, orHCV.OPOs, in coordination with OPTN, should have a system in place allowingtracking between a common deceased donor and 1) recovered organs, 2)recovered associated blood vessel conduits, and 3) recovered tissues andeyes to facilitate notification when a donor-derived diseasetransmission is suspected. This system should include accurate recordsof the distribution and disposition of each organ and initialdistribution of associated blood vessel conduits, along with proceduresto facilitate the timely notification of transplant centers and tissueand eye recovery establishments when a donor-derived diseasetransmission is suspected. To facilitate notification by OPO, transplantcenters should keep accurate records of all organs and associated bloodvessel conduits received and the disposition of each.https://stacks.cdc.gov/view/cdc/88644).The recommendations for Tracking and Reporting of Donor-Derived DiseaseTransmission Events have not been changed from the 2013 PHS guideline. They haveonly been reorganized to increase clarity  Act (Historically, HCV transmission through organ transplantation has been associated withpoor clinical outcomes (Transplant centers should develop and maintain a plan for education andinformed consent of HCV-negative transplant candidates who are consideringan organ from an HCV-viremic donor.Transplant centers should ensure development of a testing and treatmentprotocol for their transplant program for HCV-negative transplant candidateswho receive an organ from an HCV-viremic donor.Transplant centers should ensure payment- and reimbursement-related barrierswill not result in a delay of HCV diagnosis or treatment of recipients whoreceive organs from HCV-viremic donors.Clinicians caring for recipients of organs from donors with HCV infection orrecent drug injection for nonmedical reasons should maintain awareness ofmultiple rapidly changing infectious disease risks associated with druginjection for nonmedical reasons, including but not limited to hepatitis Avirus (In accordance with state requirements for reporting notifiable infectiousdiseases, if an organ recipient becomes newly infected with HCV, thetransplant center should notify public health authorities in therecipient’s residence jurisdiction.The recommendations presented in this guideline reflect recentorgan-transplant–specific evidence and are intended to increase the use oforgans while continuing to maintain transplant recipient safety. PHS will continueto review evidence on donor-derived transmission events of HIV, HBV, and HCV;development and use of new screening and diagnostic technologies; treatment andoutcomes of donor-derived infections; the impact of this guideline on organ safetyand use; and other related topics. As technology and scientific evidence progress,PHS will consider future revisions to this guideline as warranted.