Survival rates after heart transplant have significantly improved over the last decade. Nevertheless, long-term allograft viability after 10 years remains poor and the sequelae of transplant-associated immunosuppression increases morbidity. Although several studies have implicated roles for lymphocyte-mediated rejection, less is understood with respect to non-major histocompatibility, and innate immune reactivity, which influence graft viability. As immature and mature dendritic cells (DCs) engage in both Major Histocompatibility Complex (MHC)-dependent and MHC-independent immune responses, these cells are at the crossroads of therapeutic strategies that seek to achieve both allograft tolerance and suppression of innate immunity to the allograft. Here we review emerging roles of DC subsets and their molecular protagonists during allograft tolerance and allograft rejection, with a focus on cardiac transplant. New insight into emerging DC subsets in transplant will inform novel strategies for operational tolerance and amelioration of cardiac vasculopathy. The volume of heart transplants performed worldwide has continued to rise as surgical transplantation remains a standard of care for patients with advanced heart failure . AccordiChronic allograft vasculopathy (CAV), an accelerated version of atherosclerosis characterized by diffuse thickening of arterial walls, remains a noteworthy cause of long-term graft attrition with 29.3% of transplant recipients experiencing CAV 5 years post-transplant and an astounding 47.4% of patients within 10 years , and macrophages, following solid organ transplantation and their involvement in this chronic inflammatory state. While these cells are classically thought of as first responders, emerging evidence encourages us to reevaluate this “surface level” thinking and consider a deeper investigation as events occurring early after transplant have the potential to contribute to or even perpetuate long term damage. While much could be said about each of the innate immune cell types, our discussion will focus specifically on the role of DCs in heart transplantation.DCs are canonically recognized as professional antigen presenting cells (APCs) that serve as a key linking cell between the non-specific innate immune response and the memory producing, antigen specific adaptive immune response. As such, DCs are a remarkably heterogenous population of cells, existing in a variety of subtypes that differ in surface phenotype, function, and location in the body . AttemptDCs develop from a common progenitor cell in the bone marrow into classical DCs (cDCs) with potent antigen presenting abilities, or plasmacytoid DCs (pDCs) implicated in the production of type I interferons (IFNs) and subsequent innate immunity against viral infection  Figure . Recent in vitro  which are routinely generated in vitro  but are in vitro . Howeverin vitro . Below w+ Cadm1+ CD172a− cDC1s and XCR1− Cadm1− CD172a+ cDC2s (+ cDC1 and CD4+ cDC2 or lung CD103+ cDC1 and CD11b+ cDC2.Following migration of a committed precursor cell (pre-cDC) from the bone marrow to peripheral lymphoid and non-lymphoid tissues , cDCs wi+ CD172− cDC1s present to and subsequently stimulate a CD8+ T cell response , CD45R (B220), Sca-1, Siglec-H, Bst2, and CCR9 in addition to markers that are thought to be related to maturation state such as Ly6C, CD4, and CD8 .Functionally, activated pDCs are able to perform the canonically associated antigen presenting role of a DC, however they do so much less efficiently than cDCs , 29. pDCWith these somewhat “weak” antigen presenting capabilities and ability to prime T cells, pDCs are more recognized for their role in production of type I Interferon in response to viral stimulation . This su+CD25+ regulatory T cells (Tregs) as demonstrated following co-culture of CD4+CD25− naïve T cells with pDCs enriched from human peripheral blood mononuclear cells (PBMCs) + pDC dependent manner  . Relatedt manner .As we begin to assess the innate response of the aforementioned DC subsets in cardiac transplant, it is important to consider the environment these cells currently or will soon occupy. Organ transplantation induces rapid activation of the innate immune system as damaged vascular and parenchymal tissue from organ procurement, organ storage, and engraftment yield numerous inflammatory stimuli derived from dead or dying graft cells. These released damage-associated molecular patterns (DAMPs) are then recognized by toll-like receptors (TLRs), a type of pattern-recognition receptor (PRR), which initiate a signaling cascade that results in production of multiple cytokines and cellular responses to further enhance this inflammatory milieu .The onslaught of immune cell infiltration has long been assumed to be damaging to the graft with cell-specific depletion studies further confirming. Depletion of macrophages in a mouse model of heart transplantation revealed markedly reduced development of CAV lesions  and a reThe innate immune response is composed of physical, cellular, and chemical components that function as a first line defense to protect the body from invading pathogens and/or foreign antigens. As such, the innate response can be thought to consist of elements that respond directly to this invader and elements responsible for signaling to members of the adaptive response, allowing for formation of immunological memory . DCs havImmature DCs specialize in the capture and processing of antigens, after which they mature and lose much of their endocytic capacity while upregulating the expression of MHCII and other costimulatory molecules such as CD40, CD80, and CD86 . EngagemContinuing with the concept of DCs as cellular director of the innate response, elegant experiments from the lab of Florent Ginhoux have identified skin cDC1s (distinct from epidermis associated antigen presenting Langerhans cells) as producers of the cytokine VEGF-α, recognized to be important in the recruitment of neutrophils . DepletiHaving discussed the direct responses of DCs to foreign antigen, the second category of elements in the innate response to consider is methods of signaling to members of the adaptive immune system to initiate a primary immune response. While other innate immune cells are recognized for their phagocytic properties (namely macrophages), the responsibility of uptake and subsequent presentation of foreign bodies to lymphocytes to trigger an adaptive response fall on DCs. The efficiency by which different DC subsets present antigen to T cells has already been mentioned, however it is important to recognize the complexity which underlies antigen presentation and allorecognition in the setting of donor and recipient immune cells.A series of potential allorecognition pathways amongst DC and T cells exist including direct, indirect, and semi-direct allorecognition. Direct allorecognition refers to the recognition of MHC-peptide complexes on donor APCs directly by recipient T cells  which liA worthy goal in the realm of organ transplantation is the induction of operational tolerance in which there is long-term survival of an allograft without need for immunosuppressive therapies . This isIt has also been noted that the relative composition of DC subsets found in the peripheral blood following heart transplantation is mutable, dependent upon factors such as choice of pharmacologic immunosuppressant and length of time since transplant . In a stA number of studies have begun to further probe this interesting observation by adoptively transferring pDCs into rodent models of heart or lung transplant and observing graft outcome [reviewed in Rogers et al. ]. Remark+ cytotoxic T cells through programmed death 1 ligand (PD-L1) signaling  was shown to be required for thymic allo-tolerance in a murine model of GVHD following BMT. Implicating this pathway further, a blinded analysis of peripheral blood of patients following BMT revealed a correlation between decreased CD36 expression and CD141+ DCs  with increased frequency of GVHD development, despite no significant differences in prevalence of other cell types, demographic, or clinical characteristics. While the thymic environment and processes associated with central tolerance are admittedly removed from tolerance induction following solid organ transplant, evaluating the mechanisms by which tolerance in various settings is successfully achieved in parallel with the involved cellular players may help to facilitate the generation of new tolerogenic therapies.cDC1s have also been found to play a vital role in central tolerance by presentation of cell-surface antigens from apoptotic medullary thymic epithelial cells yielding development of a diverse repertoire of regulatory T cell receptors . InteresA variety of protocols have been developed and refined to commandeer and direct the interactions of immune cells in a manner that would promote a tolerogenic environment following organ transplantation leading to graft acceptance. Of special interest to the authors is the use of donor cells, with or without additional combinatorial therapy, as a means to harness the body's ability to clear naturally occurring apoptotic cells via phagocytosis without damaging healthy neighboring cells or initiating an inflammatory milieu . We willTreatment of donor splenocytes with the chemical crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (ECDI-SP) induces apoptosis, allowing for the processing and presentation of donor antigens in a non-immunogenic manner . Donor E+ DCs via administration of diphtheria toxin to CD11c+ DTR mice was able to inhibit islet allograft survival following ECDI-SP treatment , a complex pathway of downstream signaling is initiated that alters DC phenotype and functionality in order to promote an effective T cell response. This includes increasing MHC and costimulatory molecule expression, increasing production of inflammatory cytokines (When CD40 (expressed on DCs) binds to CD40L  as far as DCs are concerned. Continuing to further delineate the cellular and molecular mechanisms by which tolerance is induced and maintained may allow for use of such therapies in combination and improved pharmacological targeting .Over the past 5 years there has been a developing interest in the relationship between cellular metabolism and its influence on cell function. Recent studies have shown alterations to metabolic pathways, including glycolysis, the Krebs cycle, and fatty acid metabolism, are able to profoundly influence the function of macrophages and DCs in notably specific ways . While m+ and succinate, are able to provide signals to immune cells that regulate their function  do indeed differentially express genes associated with metabolic pathways. Pathways of oxidative phosphorylation, lipid, and sugar metabolism were shown to be two-fold higher in these tolerogenic DCs compared to mature inflammatory DCs . Such fiThis basic understanding of the powerful role DCs play at the intersection of innate and adaptive immunity in combination with strategies already showing therapeutic potential are an encouragement to the pursuit of achieving operational tolerance. Additional strategies and future research directions should consider complementary or supplemental interrogation of regulatory DC receptors that have yet to be evaluated.in vitro bone marrow-derived DCs as having greater levels of inflammation associated Axl in comparison to Mer, a finding further supported by similar levels of expression on CD11c+ DCs isolated from murine spleen. Characterization of these receptors within specific DC subsets has not performed and use of gene and cell specific knockout models or receptor specific antibodies could aid in elucidating targetable pathways amongst the TAM receptors for use in transplant.The TAM family of receptor tyrosine kinases—TYRO3, Axl, and Mer—are expressed among cells of the immune system including macrophages, resting and activated DCs, and natural killer cells. These receptors are recognized to play essential roles in innate immunity including inhibition of the inflammatory response, phagocytosis of apoptotic cells, and maturation of natural killer cells . HoweverCurrently identified ligands of the TAM receptors include growth-arrest specific six protein (GAS6) and Protein S . These pA potential strategy toward harnessing the selective activation of the aforementioned receptors may be through the use of nanobiologics that selectively target DCs, such as dendritic cell-targeted polymersomes . This idIn this review, we have sought to highlight what is currently known as it relates to DCs in the setting of cardiac transplantation. We highlight this unique cell as a prominent director of the innate immune response with the ability to activate and recruit additional cell types, such as NK cells and neutrophils, and participate in allogenic recognition and signaling to the adaptive system. We describe DC subset classification and what has been found regarding subset specific roles in transplantation tolerance where both pDCs and cDC1s could serve as important cellular mediators of tolerance, although this has yet to be fully elucidated. We assess the impact of tolerization therapies, such as delivery of apoptotic cells or costimulation blockade, acting on DCs in related albeit dissimilar mechanisms through upregulation of inhibitory molecules vs. reduced cytokine secretion, respectively. Finally, we address areas in which research has only just begun including the implications of immunometabolic modulation and the interrogation of regulatory DC receptors such as the TAM family with nanobiologics.As our ability to probe deeper into specific immune cell populations continues to advance, we find ourselves at a unique time in immunology to be able to ask questions and address pathways and cellular functions in ways like never before. There has been a drastic increase in understanding of the innate immune system over the past few years, yet gaps in knowledge certainly remain within the realm of transplantation and DCs. Future directions of research require careful consideration of subset specific receptors and subsequent responses in order to better delineate DC roles in tolerance and maximize potential therapeutic targets. We must also deepen our awareness of the mechanisms by which DCs mediate both the adaptive and innate response in transplant, evaluating DC signaling utilized for cellular recruitment, innate sensing of self vs. non-self, and receptors necessary for tolerogenic DC programming. With a growing and evolving knowledge of this complex cell and the innate immune system, true transplant tolerance, considered to be the “holy grail” of transplant research, may become within reach.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sirtuins compose a unique collection of histone deacetylase enzymes that have a wide variety of enzymatic activities and regulate diverse cell functions such as cellular metabolism, longevity and energy homeostasis, mitochondrial function, and biogenesis. Impaired sirtuin functions or alterations of their expression levels may result in several pathological conditions and contribute to the altered metabolic phenotype of malignantly transformed cells in a significant manner. In the twenty-first century, principles of personalized anticancer treatment need to involve not only the evaluation of changes of the genetic material, but also the mapping of epigenetic and metabolic alterations, to both of which the contribution of sirtuin enzymes is fundamental. Since sirtuins are central players in the maintenance of cellular energy and metabolic homeostasis, they are key elements in the development of metabolic transformation of cancer cells referred to as the Warburg effect. Although its most well-known features are enhanced glycolysis and excessive lactate production, Warburg effect has several aspects involving both carbohydrate, lipid, and amino acid metabolism, among which different tumor types have different preferences. Therefore, energy supply of cancer cells can be impaired by a growing number of antimetabolite agents, for which appropriate vectors are strongly needed. However, data are controversial about their tumor suppressor or oncogenic properties, the biological effects of sirtuin enzymes strongly depend on the tissue microenvironment (TME) in which they are expressed. Immune cells are regarded as key players of TME. Sirtuins regulate the survival, activation, metabolism, and mitochondrial function of these cells, therefore, they are not only single elements, but key regulators of the network that determines anticancer immunity. Altered metabolism of tumor cells induces changes in the gene expression pattern of cells in TME, due to altered concentrations of metabolite cofactors of epigenetic modifiers including sirtuins. In summary, epigenetic and metabolic alterations in malignant diseases are influenced by sirtuins in a significant manner, and should be treated in a personalized approach. Since they often develop in early stages of cancer, broad examination of these alterations is required at time of the diagnosis in order to provide a personalized combination of distinct therapeutic agents. First definition of epigenetics is derived from Conrad Hal Waddington 1942), who proposed it as “the causal interactions between genes and their products, which bring the phenotype into being” 942, who . These fFirst epigenetic alteration to be linked to cancer was global DNA hypomethylation described by Feinberg and Vogelstein in 1983  are divided into four groups, among which sirtuin enzymes, that are homologs to the yeast SIR2 protein (Silent Information Regulator 2), comprise group III, requiring NADIn cancer, sirtuins have both oncogenic and tumor suppressor properties, however, data are controversial at several points. As an example for emphasizing the importance of tissue microenvironment, SIRT1 has been proposed both as a tumor suppressor and as an oncogene in different types of malignancies . SIRT4 aAltered expression levels of sirtuins contribute to chemoresistance and metastasis formation, and in some cases, they are associated with clinical outcome. In endometria carcinoma cell lines, SIRT1 overexpression enhanced resistance to paclitaxel and cisplatin . SIRT1 aSirtuins are also involved in the pathogenesis of hematological malignancies. SIRT1 is overexpressed in human leukemia stem cells (LSC), and its inhibition suppressed proliferation of primitive progenitor cells and increased apoptosis in LSC . Due to The central role of sirtuin enzymes in the metabolism of cancer cells is confirmed by a growing number of evidences about their role in both promoting and inhibiting the Warburg effect (see below) in several tumor types. This strong impact on metabolism is highly associated with the numerous interactions of sirtuins with oncogenic and tumor suppressor proteins, microRNAs that regulate metabolism, and proteins involved in signal transduction pathways as well.18FDG PET . TIGAR is responsible for the cleavage of fructose-2,6-bisphosphate, the allosteric activator of phosphofructokinase I (PFK I) .de novo synthesis of amino acids, phospholipids, and ribonucleotides, respectively , tumor cells primarily express the M2 isoform that is responsible for the regulation of the final and rate-limiting reaction of the glycolytic pathway . PKM2 acBesides the tumor specific isoforms, the mutations of some metabolic enzymes also contribute to the metabolic switch featuring cancer cells. Gain of function mutations of isocitrate dehydrogenase (IDH) result in novel enzymatic activity and the production of the oncometabolite 2-hydroxyglutarate (2-HG) . MutatioCancer stem cells, the maintenance and self-renewal properties of which are tightly regulated by sirtuin enzymes, demonstrate unique metabolic flexibility. In general, they are characterized by even higher glycolytic rate , howeverDevelopment of Warburg-like metabolic phenotype of cancer cells is strongly regulated by epigenetic mechanisms where sirtuin enzymes play a key role. Various enzymatic activities, subcellular localizations and an enormous interactome with metabolic enzymes, epigenetic modifiers, and proteins of signal transduction pathways enable sirtuins to be central players in this field. In this section, we summarize data about the involvement of distinct sirtuins in the regulation of metabolism of cancer cells.SIRT1 is a key metabolic sensor and regulator of mitochondrial biogenesis as well. SIRT1 and AMP-activated protein kinase (AMPK) directly activate the nuclear receptor Peroxisome Proliferator Activated Receptor Gamma Coactivator 1 alpha (PGC1α) through deacetylation and phosphorylation, respectively, resulting in increased mitochondrial biogenesis . In glioIn acute myeloid leukemia (AML) cell models, SIRT2 promoted the Warburg effect by deacetylating and activating glucose-6-phosphate dehydrogenase (G6PD) enzyme, increasing the production of NADPH, and supporting the biosynthesis of macromolecules that are essential for rapid cell proliferation . HoweverSIRT3 is the best characterized mitochondrial sirtuin enzyme, its expression level is increased by caloric restriction, fasting, and exercise training . The firSIRT4 also has an impact on metabolism of cancer cells by promoting the Warburg effect. Besides the inhibition of glutamate dehydrogenase (GDH) enzyme , SIRT4 wSIRT5 regulates metabolic enzymes by deacetlyation, desuccinylation, deglutarylation, and demalonylation as well. Carbamoyl phosphate synthetase 1 (CPS1) is the only known protein that is deacetylated by SIRT5 . ElevateSIRT6, which is one of the major epigenetic regulator of the glucose homeostasis of cells, exerts anti-Warburg effect by the inhibition of increased glucose uptake and overexpression of glycolytic enzymes as well. The latter results from the deacetylase activity of the enzyme, because H3K9 and H3K56 deacetylation of glycolytic genes inhibits their transcription by HIF1α and MYC, respectively . SIRT6 aSIRT7 has recently been described as an enzyme with ADP-ribosyl transferase activity . SIRT7 c* contributes to the down-regulation of enzymes involved in TCA  in colorectal cancer cells, which inhibits the conversion of pyruvate to acetyl-CoA in the tricarboxylic acid cycle (TCA) . Both tud in TCA .MicroRNAs and sirtuins cannot be regarded as independent regulators of the metabolism of cancer cells, since numerous interactions have been revealed between them. For example, miR-31 targets SIRT3 enzyme to increase oxidative stress in oral carcinoma .It has been clear for more than a half century that one of the most important functions of the immune system is to identify and eliminate transformed cell clones (Macfarlane Burnet−1950) . On the Cancer cells have been proved to induce decreased levels of nutrients and a hypoxic, acidic condition in the tumor microenvironment . TherefoT cells are the major components for antitumor immunity , howeverM2 polarized tumor-associated macrophages (TAMs) support tumor growth and provide a barrier against the natural killer (NK) cells and cytotoxic T lymphocytes . They exSimilarly to M2 polarized TAMs, cytotoxic T lymphocytes are also featured by a decreased rate of glycolysis and enhanced oxidative phosphorylation .Central role of mitochondria in the maintenance of metabolic and redox homeostasis also has a strong impact on antitumor immunity. Mitochondrial oxidative metabolism was described as a critical suppressor of metastasis , while h+ are signals that regulate innate immunity, by acting via deacetylases such as SIRT1 and SIRT2 and regulating HIF1α, respectively (Activation of the transcription factor aryl hydrocarbon receptor (AHR) induces the differentiation of CD4+ naive T cells into immunosuppressive regulatory T cells , 89. Besectively .in vivo (In the twenty-first century, principles of personalized anticancer treatment need to involve the mapping of epigenetic and metabolic alterations, to both of which the contribution of sirtuin enzymes is fundamental. In parallel with this aim, growing number of antimetabolites and sirtuin inhibitors are administered. As an example for the latter, inhibition of SIRT1 by the up-regulation of miR-211-5p was associated with the induction of apoptosis in breast cancer cells . Inhibitin vivo .Manipulating metabolism is also a tool to enhance antitumor immunity, since metabolic pathways have been proved to shape both function and survival of antitumor T cells . HoweverIt should also be considered, that inhibitors of sirtuin enzymes can induce different changes in distinct cell types of anticancer immunity. In myeloid cells, SIRT1 inhibition leads to increased transcription of proinflammatory cytokines, but on the other hand, targeting SIRT1 results in net immunosuppressive effect in case of T cells .Sirtuins are key hubs in the regulatory network of etiological factors of tumors, having strong impact on the growth, survival, and metabolism of cancer cells, that also influences antitumor immunity in a significant manner. One of the major challenges of modern oncology is to establish novel elements of this regulatory-etiological network in order to provide personalized treatment for patients. Investigation of the exciting world of sirtuin enzymes is definitely one of the most effective tools for this. Further examinations are required to elucidate cell-specific metabolic and immunological effects of sirtuin inhibitors and activators. Appropriate vectors are also needed to deliver these small molecules and antimetabolites to their definitive target cells. Together with widespread genetic, epigenetic, and metabolic mapping at the time of the diagnosis, these efforts could improve therapeutic results, leading to longer disease-free and overall survival, with improved life quality as well.Text of the manuscript was written by ZG with the supervision and instructions of LC.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.


While there are constraints on the number or function of Tregs which can be exploited to evoke an effective anti-tumor response, sufficient expansion of Tregs is essential for successful organ transplantation and for promoting tolerance of self and foreign antigens. The immune-suppressive property of Tregs equips this T lymphocyte subpopulation with a pivotal role in the establishment and maintenance of maternal tolerance to fetal alloantigens, which is necessary for successful pregnancy. Elevation in the level of pregnancy-related hormones including estrogen, progesterone and human chorionic gonadotropin promotes the recruitment and expansion of Tregs, directly implicating these cells in the regulation of fetal-maternal immune tolerance. Current studies have provided evidence that a defect in the number or function of Tregs contributes to the etiology of several reproductive diseases, such as recurrent spontaneous abortion, endometriosis, and pre-eclampsia. In this review, we provide insight into the underlying mechanism through which Tregs contribute to pregnancy-related immune tolerance and demonstrate the association between deficiencies in Tregs and the development of reproductive diseases.Regulatory T cells (Tregs) are a specialized subset of T lymphocytes that function as suppressive immune cells and inhibit various elements of immune response  Regulatory T cells (Tregs), a key subset of T lymphocytes, play a critical role in regulating the immune response and maintaining immune tolerance both in physiological and pathological processes. Many studies have shown that Tregs are compromised in patients with autoimmune diseases as well as in patients with graft-versus-host disease after receiving transplanted organs , however+ Tregs has been shown to cause implantation failure in allogeneic mated mice  and inducible regulatory T cells (iTregs). NTregs originate from the thymus in response to self-antigens, whereas iTregs are peripherally induced from T cells responsible for restraining immune responses to foreign antigens, such as commensal bacteria, food antigens and allergens , 17. Theficiency . An incrficiency –21.+CD25− T cells with the capacity to suppress the proliferation of CD4+ T cells  and CTLA4 are the most important molecules involved in cell-contact dependent mechanism.Most Tregs abundantly express high-affinity IL-2 receptor α (CD25) and IL-2/IL-2R signaling provides indispensable signaling during the development and maturation of Tregs both in the thymus and in the periphery. Furthermore, the lack of the IL-2R cannot be compensated by other cytokine receptors . IL-2, ICTLA4, a key molecule constitutively expressed in Tregs, is crucial for maintaining T cells homeostasis and tolerance induction, and its expression is in part controlled by Foxp3 , 40. MicIn addition to the cell-contact dependent mechanism, Tregs also exert suppressive activity in a cell-contact independent manner, mainly through the secretion of inhibitory cytokines. Unlike other T cells, Tregs fail to produce exclusive cytokines. However, certain cytokines, such as TGF-β and IL-10, secreted by Tregs are essential for the expansion and suppressive activity of Tregs.−/− mice and addition of TGF-β results in increased Foxp3 expression  and intrauterine insemination (IUI), has enabled an analysis of earlier gestational stages from oocyte fertilization to implantation in humans. Adequate endometrial receptivity is considered a pivotal precondition for successful embryo implantation. Endometrial scratching before embryo transfer has been proposed as a clinical treatment to increase uterine receptivity, and some studies have demonstrated that endometrial scratching improves the pregnancy outcome by triggering an inflammatory response and enhancing angiogenesis at the implantation site, providing indirect evidence for the role played by inflammation during implantation  increase dramatically during pregnancy. These hormones play an essential role in maintaining immune tolerance and in supporting successful pregnancy. Currently, there is increasing evidence that the mechanisms through which hormones contribute to immune homeostasis during pregnancy are in part due to the expansion of Tregs and their suppressive activity .in vivo and in vitro experiments, estrogen treatment triggers the expansion of Tregs. Furthermore, the addition of estrogen in combination with TCR stimulation enhances Foxp3 mRNA expression in CD4+CD25−T cells in vitro, suggesting that estrogen may potentially induce the conversion of CD4+CD25−T cells to Tregs  and a significant shift toward a Th1/Th17 response with the onset of labor , defined as the loss of three or more consecutive pregnancies, affects ~1% of women attempting to conceive , 128. RSThe mechanisms through which Tregs contribute to RSA primarily involve an imbalance of the Th1/Th2/Th17/Treg cells paradigm and the abnormal proportion and activity of Tregs. Dysregulation of T lymphocyte homeostasis is also involved in the etiology of RSA. In peripheral blood from patients with RSA, the balance between Th1 and Th2 cells is disrupted in favor of Th1 cells, and the ratio of Th17/Treg cells is skewed toward Th17 cells , 133. It+CD25−T cells  and paternal or third-party lymphocyte immunization therapy have been proposed for the treatment of patients with RSA due to the potential immunomodulatory effects. Although the benefit for these immunotherapies is controversial, a growing body of evidence suggests that they may increase rates of live birth and decrease rates of miscarriage –188. A vTregs-enhancing drugs are another type of Tregs-based target therapy. Rapamycin (Sirolimus) is an mTOR inhibitor, which acts as an immunosuppressive drug by selectively promoting the expansion of Tregs and inducing differentiation of T helper cells into Tregs. Royster et al. established a murine model with conditional knockdown of Tregs induced by diphtheria toxin. They found the deletion of Tregs decreased litter sizes and triggered embryo implantation failure, effects that were reversed after the treatment with rapamycin . A multiTaken together, some studies have demonstrated the effectiveness of Tregs-based therapy in treating several autoimmune diseases and cases of organ transplantation. However, the methods cannot be directly applied for pregnancy-related diseases because the dynamic change in the immune state during pregnancy and the possibility of fetal drug toxicity must be taken into account. Most of the current treatments for pregnancy-related diseases focus on a reduction in an overactive immune response with the use of non-specific immunosuppressive therapy. This triggers the simultaneous activation of numerous immune cells and makes it difficult to control the dose and to evaluate the curative effect because of individual heterogeneity. Therefore, more studies should be conducted to further explore the effectiveness and safety of Tregs-based target therapies for the treatment of pregnancy-related diseases.Tregs are generally viewed as arising from a specific T cell lineage generated in the thymus or induced in peripheral organs. Being the most predominant immune-suppressive cells, a tremendous amount of research has focused on determining the molecular mechanisms responsible for inducing the expansion of Tregs and their activity in the periphery and in specific organs. This effort will provide new insights that will guide the improvement of Tregs-based targeted immune therapy. In recent years, increasing data has shown that the expansion of Tregs is triggered after exposure to the fetal alloantigens and changes dynamically over the course of pregnancy. Hormones such as estradiol and progesterone as well as HCG are significantly increased during pregnancy, and regulate the number and function of Tregs to sustain a proper pregnancy-related immune tolerance. Furthermore, various reproductive diseases such as recurrent miscarriage, endometriosis and preeclampsia result in part from the deficiency in the number and activity of Tregs. Therefore, modulating the immune response by boosting the number of Tregs and enhancing their activity may be a potential therapeutic strategy for managing these pregnancy-related complications.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, these basic science discoveries have yet to translate into new therapies for human patients. This review addresses both the promise and barriers to developing translational therapies that target CaMKII signaling to abrogate pathologic remodeling in the setting of chronic disease. Efforts in small molecule design are discussed, as well as alternative targeting approaches that exploit novel avenues for compound delivery and/or genetic approaches to affect cardiac CaMKII signaling. These alternative strategies provide hope for overcoming some of the challenges that have limited the development of new therapies.Heart failure remains a major health burden around the world. Despite great progress in delineation of molecular mechanisms underlying development of disease, standard therapy has not advanced at the same pace. The multifunctional signaling molecule Ca Despite the wealth of basic research supporting CaMKII as a master regulator of pathways important for cardiac remodeling and arrhythmia, the field has yet to witness translation of these findings to the clinic. The gap between advances at the bench and new treatments in human patients has not gone unnoticed, and efforts are underway that will hopefully fix the pipeline for development of effective therapy. In this review, we address a brief history of progress in small molecule drug design, followed by consideration of alternative approaches that take advantage of recent advances in novel delivery mechanisms as well as genetic approaches for manipulating CaMKII signaling in the heart. These alternative strategies address the ability to circumvent the challenges and limitations of small molecule drug design while highlighting next-generation therapeutic paradigms.For the past 50 years, therapy based on antagonism of beta adrenergic receptors, angiotensin converting enzyme (ACE), and/or AT2 receptors has been the staple for heart failure (HF) treatment . However2+/calmodulin  into adaptations in cell function through direct phosphorylation of a large number of target proteins  the C-terminal association domain responsible for the oligomerization of individual CaMKII molecules to create a mature dodecameric-holoenzyme  the N-terminal catalytic domain responsible for ATP binding and kinase function; (2) the regulatory domain responsible for binding CaIn addition to the autophosphorylation at Thr287 maintaining the active conformation, other posttranslational modifications have been identified on the regulatory domain, which maintain an active state and have been associated with cardiac disease states. These include the oxidation of Met281/282 in response to elevated reactive oxygen species , O-linke2+ cycling to mitochondrial function. CaMKII has been implicated in pathologic phosphorylation of a number of Ca2+ handling proteins including phospholamban, leading to activation of the sarcoplasmic reticulum (SR) ATP-driven Ca2+ pump SERCA2a (2+ release channel (RyR2)  , promotitivation . Collecttivation . CaMKII-izations . Mitochopoptosis .Beyond ion channels, CaMKII has been found to target chromatin remodeling protein class 2 histone deacetylases 4 and 5 (HDAC4/5). The phosphorylation of these proteins induces their nuclear export, freeing the transcription factor MEF2 from its repressed state, contributing to hypertrophic remodeling . RelevanThe approach of using pharmacologic inhibitors to target CaMKII activity has been used extensively in basic research with less progress in translational medicine, which is somewhat surprising given the widespread use of protein kinase inhibitors in cancer therapeutics for targeting tumor proliferation and cell survival . In fact2+ channels  and isoproterenol (100 nM) and did so in a dose-dependent manner, completely eliminating PACs at 100 and 300 nM concentrations, which occurred in 50% of nontreated samples. In parallel, GS-680 was found to reduce Ca2+ sparks and SR Ca2+ leak and attenuated triggered activity, as well as increased action potential amplitude and maximal upstroke velocity within isolated human atrial myocytes. However, the drug was found to impair systolic atrial contractions, which was negated after pretreating the tissue with isoproterenol, suggesting that CaMKII inhibition at baseline had a negative ionotropic impact that could be compensated by PKA stimulation. Consistent with this idea, human HF ventricular trabeculae preparations, which are characterized by increased PKA activity and negative force–frequency relationships, experienced improved contractility and increased Ca2+ transients at higher pacing frequencies when treated with GS-680, showing that this compound holds promise for the potential treatment of both atrial arrhythmias and HF ventricular remodeling.Another pyrimidine-based, ATP-competitive molecule, GS-680, has been developed by Gilead Sciences . GS-680 v4.3, and Nav1.5 over 30 and 14 µM for Cav1.2. The therapeutic potential of this compound was tested in a transgenic mouse model of dilated cardiomyopathy, but unique to these studies was the delivery of the drug as an orally bioavailable agent, giving it significant advantage for potential clinical investigation. The drug was found to improve ejection fraction in a genetic mouse model of dilated cardiomyopathy, which associated with reduced levels of phosphorylated phospholamban (Thr17), a direct target of CaMKIIδ. Critically, a five-fold lower exposure for the drug was found in the brain compared to the heart, limiting the potential for off-target impacts of CaMKII inhibition in the brain.Like the compounds just discussed, the most recently designed compound named RA306 developed by Sanofi R&D was derived from a chemical optimization program based on the pyrimidine ATP-competitive class of CaMKII inhibitors . The druThe last several years has seen a strong push in the development of translational pharmacologic agents to inhibit CaMKII for the treatment of pathologic heart conditions. While only one compound (RA306) has been shown to be orally bioavailable, several new agents are available with favorable selectivity and potency profiles. Therefore, based on these efforts, it seems reasonable to remain optimistic about the development of a translationally relevant and novel HF pharmacologic.in vitro and in vivo, mitigating HF, increasing cardiac function, and preventing lethal cardiac arrhythmias , double-stranded siRNAs, and miRNAs . ASOs arB, in comparison to the other cardiac variant CaMKIIδC , there are options that are unique to RNA therapeutics for potentially overcoming this limitation, including gene therapy which could utilize cardiac specific promoters (limited to miRNAs for polymerase compatibility) or local delivery/injection directly into cardiac tissue. There is also exciting opportunity to combine emerging nanoparticle therapy (discussed in previous section) to carry RNA-based drugs instead of peptide cargos through the same inhalation delivery to deliver directly to the heart.2+ handling proteins) that may serve as attractive targets for electrical and hypertrophic remodeling. Perhaps the best known example of therapy aimed at a downstream target in the CaMKII pathway comes from ranolazine, an approved antianginal agent that inhibits late Na+ current among other actions  is one such compound that has been shown to improve Ca2+ leak and left ventricular function in a canine model of HF  and type-2 phosphatases  , which cand PR59 , while sand PR59 . Future and PR59 , but cleA unique opportunity for further discerning the potential impact of phosphatase activation may lie in the field of cancer therapeutics. Currently, cancer treatment strategies are beginning to consider phosphatase activators in earnest for tumor disruption . It is wIV-spectrin has been identified in targeting CaMKII to the cardiomyocyte intercalated disc to facilitate regulation of Nav1.5  and the American Heart Association .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.


Human papillomavirus (HPV) infection is associated with a range of malignancies that affect anogenital and oropharyngeal sites. α-HPVs dominantly infect basal epithelial cells of mucosal tissues, where they dysregulate cell division and local immunity. The cervix is one of the mucosal sites most susceptible to HPV infections. It consists of anatomically diverse regions, and the majority of cervical intraepithelial neoplasia and cancers arise within the cervical squamo-columnar junction where undifferentiated basal progenitor cells with stem cell properties are found. The cancer stem cell theory particularly associates tumorigenesis, invasion, dissemination, and metastasis with cancer cells exhibiting stem cell properties. In this perspective, we discuss evidence of a cervical cancer stem cell niche and explore the association of stemness related genes with 5-year survival using a publicly available transcriptomic dataset of a cervical cancer cohort. We report that poor prognosis in this cohort correlates with overexpression of a subset of stemness pathway genes, a majority of which regulate the central Focal Adhesion pathway, and are also found to be enriched in the HPV infection pathway. These observations support therapeutic targeting of stemness genes overexpressed by mucosal cells infected with high-risk HPVs. Oncogenic human papillomavirus (HPV) infections are responsible for 5% of the global human cancer burden, resulting in over 634,000 new cancers annually, of which ~90% are in women  within the transformation zone  in the cervical SCJ and in HPV associated neoplastic lesions is decreased, and this might be related to the lack of HD5, which acts as a chemoattractant. Furthermore, elevated TGF-β levels inhibit E-cadherin expression, which disrupts APC adhesion and subsequently alters APC maturation and regulator T cell polarization , allowing indefinite proliferation, and understanding how these self-renewal pathways are maintained in cancer has therapeutic implications. Cancer stem cells (CSCs) are found in many malignant diseases and are associated with poor survival , of which 93% have previously been determined to be HPV positive , including one study in patients with head and neck cancer. Integrins have been classified as metastasis-promoting genes due to their function in regulating adhesion, proliferation, and migration through cell-cell and cell-matrix interactions , as well as upstream integrin subunits ITGB1, ITGB3, and ITGA5, paxillin (PXN), and thrombospondin 1 (THBS1) represented hub nodes next to the common cancer associates VEGFA and EGFR, and all these genes are part of the Focal Adhesion pathway. A range of PTK2 inhibitors are being tested in clinical cancer trials, with 40 studies currently registered (Expression of some of the genes identified above has impact on the HPV viral life cycle. According to our current understanding of α-HPVs, viral gene expression can modulate cellular pathways to either impart basal stem-cell like characteristics to infected cells in the epithelium or infect specialized epithelial stem-cell populations. In any event, the virus interacts with and affects genes and proteins involved in stemness regulation and maintenance. HPV viral gene expression is highly regulated with limited expression in the infected basal epithelial layer where early protein E2 facilitates viral episomal genome partitioning during the division of infected cells. E2 also regulates cell differentiation and has been shown to interact with focal adhesion kinase PTK2B and FN1 (Muller and Demeret, During the phase of low HPV viral gene expression, E6 also plays an important role by helping delay the migration of infected epithelial basal cells to the suprabasal layers. Viral genome amplification occurs in the middle epithelial layers, where deregulated expression of E6 and E7 is seen in association with the manifestation of high-grade neoplasia. In addition to suppressing host immune responses, and associating with key cell cycle proteins p53 and Rb, the early HPV viral proteins also bind to other cellular proteins, enabling suprabasal cell cycle entry. As observed in the epithelial basal layers, levels of FN1 and other adhesion proteins are also increased by E6 and E7 expression in suprabasal layers (Hellner et al., HPV early proteins also influence cell proliferation. Among the candidate genes identified in E5, E6, and E7 have previously been shown to co-operate to drive tumorigenesis. Co-expression of E5 with either E6 or E7 promotes transformation to a greater extent than with either oncoprotein alone (Ilahi and Bhatti, The biggest challenges in the treatment of HPV-mediated cervical malignancy are associated with acquired local immune-suppression, and disruption of cellular homeostatic processes leading to impaired focal adhesion and break-down of extracellular matrix, promoting cancer cell invasion, dissemination, and metastasis. The cancer stem cell theory suggests that these processes are more likely to occur when pluripotent cells with active stemness signaling pathways undergo transformation. HPV infects mucosal epithelial cells at many different anatomical sites, including distinct sub-anatomic sections of the cervix, but malignant transformation occurs predominantly at the SCJ, which contains unique basal cells with stem-like properties, whereas the productive HPV life cycle occurs mainly in the squamous section of the cervix where no undifferentiated cells are seen. The cervical SCJ/transformation zone microanatomy is unique compared to those of other HPV-infected anogenital regions including vagina, vulva, and anus, and this specialized undifferentiated cervical SCJ is associated with a higher risk for malignant transformation. In our analysis of stemness-pathway genes in association with cervical cancer prognosis, we confirmed 18 genes known to be dysregulated in cervical cancer cells. We also identified 27 novel genes whose overexpression significantly associated with poor survival and a majority of these play a role in the central Focal Adhesion pathway. Furthermore, some of these genes were also associated with the HPV infection pathway, and viral protein interactions with stemness proteins have been demonstrated previously.Importantly, the data presented here indicates but does not prove a direct contribution of the cervical stem cell niche to cervical cancer survival outcomes, as it was collected from bulk RNA sequencing data of primary tumor biopsies. An alternative interpretation would be that cervical cancers which overexpress genes associated with the Focal Adhesion pathway have further progressed on a trajectory towards invasion and dissemination, leading to poorer survival outcomes. Nevertheless, the pathway overlap with HPV infection confirms importance of stemness associated genes in HPV driven cervical cancer progression and might prove important for cancer therapy target selection. While all drivers of HPV infection and malignant transformation are still to be determined, treatment of cervical, and other HPV-associated cancers may be improved by a holistic approach targeting multiple cancerogenic pathways, as well as by initiating a strong HPV-specific immune response through immunization-based immunotherapy that enhances virus-specific immunity (Frazer and Chandra, https://xenabrowser.net/.Publicly available TCGA-CESC datasets were analyzed in this study. This data can be found here: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MB and JC conceived the work, analyzed data, and wrote the first draft of the manuscript. SL supervised data analysis and revised the manuscript. SP and IF wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.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.


It also exerts minor inhibitory action against PDE6, which is present exclusively in rod and cone photoreceptors. The effects of sildenafil on the visual system have been investigated in a wide variety of clinical and preclinical studies due to the fact that a high dose of sildenafil may cause mild and transient visual symptoms in some patients. A literature review was performed using PubMed, Cochrane Library and Clinical Trials databases from 1990 up to 2020, focusing on the pathophysiology of visual disorders induced by sildenafil. The aim of this review was not only to gather and summarize the information available on sildenafil clinical trials (CTs), but also to spot subpopulations with increased risk of developing undesirable visual side effects. This PDE inhibitor has been associated with transient and reversible ocular side effects, including changes in color vision and light perception, blurred vision, photophobia, conjunctival hyperemia and keratitis, and alterations in the electroretinogram (ERG). Sildenafil may induce a reversible increase in intraocular pressure (IOP) and a few case reports suggest it is involved in the development of nonarteritic ischemic optic neuropathy (NAION). Reversible idiopathic serous macular detachment, central serous retinopathy and ERG disturbances have been related to the significant impact of sildenafil on retinal perfusion. So far, sildenafil does not seem to cause permanent toxic effects on chorioretinal tissue and photoreceptors as long as the therapeutic dose is not exceeded and is taken under a physician’s direction to treat a medical condition. However, the recreational use of sildenafil can lead to harmful side effects, including vision changes.Phosphodiesterase type 5 (PDE5) inhibitors such as Viagra To maintain Ca2+ homeostasis within the photoreceptor, K+ and Ca2+ are, in parallel, continuously extruded via the potassium-dependent sodium-calcium exchanger (NCKX). This constant inward current, referred to as the dark current, causes photoreceptor depolarization and glutamate release at the synaptic terminal, inhibiting postsynaptic second-order neurons (bipolar cells). Absorption of photons by rhodopsin leads to the sequential activation of G-protein transducin and phosphodiesterase 6 (PDE6), responsible for the hydrolysis of cGMP and the consequent closure of CNG channels. This interrupts the dark current, resulting in the hyperpolarization of outer segments due to the continued activity of NCKX. As a result, the generation of this electro-chemical signal halts the release of neurotransmitters at the photoreceptor axon terminal and the visual signal is propagated to postsynaptic cells [The phototransduction process is a G-protein mediated signaling cascade where rod or cone opsins couple photon absorption to current flow at the photoreceptor outer segment plasma membrane . In the ic cells .The role of cGMP as a second messenger is key in the regulation of phototransduction since the whole signaling cascade depends on the balance between its synthesis by retinal guanylyl cyclase (GC) and its hydrolysis by PDE6. Thus, it seems obvious that the disruption of cGMP metabolism implies serious consequences for visual functioning, including photoreceptor toxicity and cell death (reviewed in ). Procesrd1 and rd10, which carry loss-of-function mutations in the beta subunit of rod PDE6 [2+ homeostasis, impairing the function of Ca2+-dependent phototransduction proteins such as recoverin and guanylyl cyclase-activating proteins (GCAPs), and even triggering photoreceptor death via the calpain signaling pathway [It is known that the NO/GC/cGMP/PKG signaling pathway is functional and widely distributed in specific cell types of both the internal and external retina of mice . Studiesrod PDE6 , have shrod PDE6 . Althougrod PDE6 , Paquet-rod PDE6 . On the ®), vardenafil (Levitra®), and tadalafil  are highly selective for PDE5 and represent the first successful application of PDE inhibition therapy to an individual isoenzyme. Nonetheless, despite this high selectivity, each of these drugs inhibits other PDE isoenzymes to some extent. Sildenafil and vardenafil, for example, have shown only 10 and 15 times lower specificity for PDE6 than for PDE5, respectively , skeletal muscles, and many other tissues including kidney, pancreas, heart, lung, liver, brain, placenta and various gastrointestinal tissues . By contiewed in ), which iewed in ,17.®) became internationally available in 2013. Avanafil exhibited 100 times lower specificity for PDE6 than for PDE5, presumably reducing the potential side effects derived from the nonselective inhibition of PDE6 by sildenafil and vardenafil (reviewed in [PDE5 inhibitors have been used for the treatment of erectile dysfunction (ED), which is a form of peripheral vascular disease that impairs men’s abilities to achieve and maintain an erection, and have become some of the best-selling medications worldwide. Sildenafil and its analogues operate by increasing the cGMP levels because they occupy the active site of PDE5 and prevent cGMP catalysis. cGMP acts as a powerful smooth muscle relaxant that promotes blood flow to the corpus cavernosum, facilitating penis erection . Apart fiewed in ). Other iewed in . Severaliewed in ,23,24.The occurrence of side effects increases with both serum levels and exposure time to the PDE5 inhibitor . To overOf the above mentioned drugs, sildenafil is the one that has exhibited a higher incidence of visual side effects, given that it is only 10-fold more potent on PDE5 than on PDE6 ,31. As aOverall, the most common adverse effects of sildenafil are strongly associated with its pharmacological nature as an inhibitor of PDE5  and as a weak inhibitor of PDE6 , being dose-dependent and observed in 6–18% of men taking sildenafil . In thisThe purpose of this literature review was to gather and summarize the information available on sildenafil clinical trials (CTs), focusing on the possible adverse effects related to different aspects of visual health.www.cochranelibrary.com, accessed on 20 December 2020) we retrieved 2001 entries including the term “sildenafil” in the title, abstract or keywords, of which we curated contents and selected all those results related with diverse features of the eye’s anatomy and physiology. The first CT on this topic “The effects of sildenafil citrate (Viagra®) on color discrimination in volunteers and patients with erectile dysfunction” (CN-00675062) was published in 1999 and over the following decade (2000–2009) a total of twenty-six CTs were conducted to assess adverse effects of sildenafil regarding visual health. Later on, the number of CTs declined and in the 2010–2019 decade only seven trials were registered  and ClinicalTrials , and in the Cochrane Library, twenty-six performed ophthalmic examinations, of which seven assessed ocular anatomy , thirteen evaluated IOP, and six measured blood flow. Regarding visual function and perception, eleven CTs evaluated visual acuity (Snellen test), seven assessed contrast sensitivity (Pelli–Robson test), seven measured static perimetry , four performed electroretinogram (ERG), and nine assayed color perception. In some of the trials (five), the most common transient and subjective visual adverse events, derived from sildenafil consumption, were also evaluated. These included color perception distortion , changes in sensitivity to light, and blurred vision. In two of the CTs , the main outcomes were not publicly available  using a slit-lamp and/or a fundoscope . Most CTs were carried out on patients with previous pathologies such as ED ,58,87,91Sildenafil inhibition of PDE5 leads to an increase in the concentration of cGMP, which, in animals, is known to lower IOP . HoweverSildenafil has a strong systemic vasodilating effect and it is known to decrease systemic blood pressure, which could lead to a decrease in choroidal blood flow . HoweverMost studies suggest an increase in choroidal blood flow velocity, with a lesser effect on the retinal vasculature in healthy subjects. This may be due to the production of NO, which plays a key role in the local regulation of ocular blood flow, and probably as a consequence of PDE5 inhibition in smooth muscle cells in a time-dependent manner . It is wEleven CTs evaluated the best-corrected visual acuity (BCVA) following sildenafil administration in healthy people ,57,76,91Nine CTs evaluated color discrimination using different tests. Five studies were carried out on healthy volunteers ,62,79,91Seven CTs evaluated contrast sensitivity in healthy people ,80,82 anThe 30-2 program on the Humphrey visual field (HVF) analyzer was used to assess the visual field in seven CTs. Three of them were carried out in healthy volunteers ,74,91, oFive CTs evaluated the presence or absence of the most common visual adverse effects such as light sensitivity changes, blurred vision and blue-tinted vision in healthy subjects ,79, in ASildenafil intake can be expected to inhibit the phototransduction process, thus inducing changes in the ERG. However, only minimal changes were observed at sildenafil doses ranging from 50 to 200 mg compared with placebo . Indeed,2+ homeostasis can be detrimental to photoreceptor survival. PDE5 inhibitors such as sildenafil are often used for the treatment of ED and PAH. Although sildenafil exhibits a high selectivity for PDE5, in high doses it is also capable of binding and inhibiting PDE6 nonselectively. Inhibition of both isoenzymes, PDE5 and PDE6, is the main cause of sildenafil visual side effects. PDE5 is expressed in some ocular tissues such as the endothelial cells of retinal and choroidal vessels. PDE6 is exclusively expressed in photoreceptors and its inhibition directly alters the phototransduction cascade due to an increase in cGMP levels. This idea has prompted many scientists and researchers to conduct CT to evaluate the safety and the visual side effects of sildenafil. From 1999, the moment when the first CT assessing visual parameters after sildenafil uptake was published, many other studies have been released and, although visual disturbances have been extensively reported, all of them seem to be transient and mild. However, many case reports regarding ocular side effects linked to sildenafil consumption have recently arisen in the medical literature  did not resolve until two weeks postadministration of the drug [+/tm1Pde6g), another murine model of autosomal recessive retinitis pigmentosa [Pde6a) over a 4-month period. Despite the low number of animals used in their experiment, the results were statistically significant, showing that sildenafil-treated Pde6a+/− dogs exhibited thinner outer nuclear layers and lower photoreceptor counts than untreated Pde6a+/− dogs [Additionally, evidence from preclinical studies carried out in animal models of human retinal diseases suggests that sildenafil consumption can be detrimental in some cases ,120,121.-subunit . The rd1the drug . Behn etgmentosa . Likewis+/− dogs . These dwww.fda.gov, accessed on 20 December 2020) and the European Medicines Agency , and associations of eye physicians and surgeons such as the American Academy of Ophthalmology  warn about the lack of controlled clinical data on the safety of sildenafil in patients with retinitis pigmentosa or with a family history of the disease. Thereby, it is essential that general practitioners supervise the treatment of a medical condition such as ED and guarantee a safe use of PDE5 inhibitors. The possibility of illegally purchasing online sildenafil and their analogues brings up relevant issues such as the risks linked to the irrational use of medicines. This is one of the factors that has prompted some countries to consider the reclassification of sildenafil from prescription-only medicine to a pharmacy medicine. Among the countries whose regulatory authorities have already taken that step are New Zealand in 2014 ; the United Kingdom in 2017 ; Norway in 2019 ; Ireland in 2020 . Although in these countries sildenafil can be sold without prescription, pharmacists receive specific training so they can provide proper guidance and request patients who answer some of the questions in the affirmative to contact their general practitioner for further assessment [Important regulatory agencies such as the U.S. Food and Drug Administration (FDA, sessment . The aimFinally, alternatives that minimize unwanted side effects should be pursued by scientists in general and by the pharmaceutical industry in particular. These may include, among others, the design, screening and development of drugs highly selective for PDE5 with no inhibitory effects on other PDE isoenzymes ; the invIn conclusion, from the literature review we can affirm that visual side effects derived from the consumption of sildenafil are generally mild and transient, but the cessation of sildenafil therapy is advised if certain rare conditions such as central serous chorioretinopathy or NAION appear. Moreover, caution should be taken in patients with a family history of retinal dystrophy because available evidence in animal research supports the hypothesis that carriers of some recessive alleles are more sensitive to sildenafil toxicity.


Large-scale, sustainable production of lignocellulosic bioenergy from biomass will depend on a variety of dedicated bioenergy crops. Despite their great genetic diversity, prospective bioenergy crops share many similarities in the polysaccharide composition of their cell walls, and the changes needed to optimize them for conversion are largely universal. Therefore, biomass modification strategies that do not depend on genetic background or require mutant varieties are extremely valuable. Due to their preferential fermentation and conversion by microorganisms downstream, the ideal bioenergy crop should contain a high proportion of C6-sugars in polysaccharides like cellulose, callose, galactan, and mixed-linkage glucans. In addition, the biomass should be reduced in inhibitors of fermentation like pentoses and acetate. Finally, the overall complexity of the plant cell wall should be modified to reduce its recalcitrance to enzymatic deconstruction in ways that do no compromise plant health or come at a yield penalty. This review will focus on progress in the use of a variety of genetically dominant strategies to reach these ideals. Due to the breadth and volume of research in the field of lignin bioengineering, this review will instead focus on approaches to improve polysaccharide component plant biomass. Carbohydrate content can be dramatically increased by transgenic overexpression of enzymes involved in cell wall polysaccharide biosynthesis. Additionally, the recalcitrance of the cell wall can be reduced via the overexpression of native or non-native carbohydrate active enzymes like glycosyl hydrolases or carbohydrate esterases. Some research in this area has focused on engineering plants that accumulate cell wall-degrading enzymes that are sequestered to organelles or only active at very high temperatures. The rationale being that, in order to avoid potential negative effects of cell wall modification during plant growth, the enzymes could be activated post-harvest, and post-maturation of the cell wall. A potentially significant limitation of this approach is that at harvest, the cell wall is heavily lignified, making the substrates for these enzymes inaccessible and their activity ineffective. Therefore, this review will only include research employing enzymes that are at least partially active under the ambient conditions of plant growth and cell wall development. Lignocellulosic plant biomass represents the largest renewable source of organic carbon on earth. Organic carbon that can be converted into a wide variety of compounds, including high-energy liquid fuel, thereby curbing our dependence on non-renewable sources and limiting the net production of carbon dioxide. The bulk of plant biomass is contained in the cell wall, specifically the thick secondary cell walls (SCW) of the vasculature and fiber tissues. The cell wall has evolved a highly complex and rigid structure to resist the mechanical forces of growth and protect the plant from various stresses. The most valuable component of the cell wall, from a bioenergy perspective, is the 6-carbon sugar glucose comprising the linear polysaccharide cellulose. Produced and extruded into the developing cell wall by cellulose synthase (CesA) complexes at the plasma membrane, individual cellulose chains coalesce through hydrogen bonding to form crystalline cellulose microfibrils. In all vascular plants, the SCW is composed of cellulose microfibrils embedded in a matrix of the aromatic polymer lignin. Hemicellulose like xylan coat the cellulose microfibrils and can form covalent linkages with cell wall proteins, lignin, and other hemicelluloses . This naPennisetum purpureum) produces more biomass per hectare annually than any other vegetation. Grasses like Miscanthus × giganteus, sorghum, and switchgrass (Panicum virgatum), and tree species like poplar, aspen, and willow are capable of producing large amounts of biomass in temperate regions like Europe and the United States   .in planta concurrent with cell wall development. The engineering of plants to accumulate hyperthermophilic or inactive cell wall-degrading enzymes has been well reviewed in other works  by overexpression of either of the primary cell wall (PCW) CesAs, CesA2, CesA5 and CesA6 . While plants, many bacteria and some fungi can produce cellulose, only one group of animals is known to do so: the marine invertebrates of subphylum Tunicata, otherwise known as sea squirts (Ciona savignyi (CsCesA) was used to create transgenic sugarcane overexpressing a functional form of the protein  for cellulose production. It could be that this depletion acts as a signal to source organs to increase production and/or transport of sucrose to sink organs. It has previously been demonstrated that induced depletion of sucrose from sugarcane stems increases photosynthetic productivity and phloem loading from leaves  cellulose, which accounts for the bulk of cellulosic biomass in bioenergy-relevant crops, is synthesized at the plasma membrane by three, non-redundant cellulose synthase (CesA) proteins CesA4, CesA7, and CesA8 . Overexpnd CesA6 . CesA2,  squirts . A cellu protein . Use of m leaves . In the m leaves . While sGossypium hirsutum) SuSy in hybrid poplar  resulted in small (2–6%) increases in cellulose and increased cellulose crystallinity (CrI) compared to controls (Populus simonii × Populus nigra) gene PsnSuSy2 in tobacco led to 25% thicker cell walls, containing up to 18% more cellulose and decrease in lignin of up to 28%, when compared to controls. Additionally, the degree of cellulose crystallinity (CrI) was reduced by 9–11%  lines overexpressing OsSuSy3 under a SCW-specific or a constitutive promoter resulted in dramatic improvements in multiple bioenergy-relevant characteristics  proteins catalyze the cleavage of sucrose to fructose and UDP-Glc, which is the sole substrate for the biosynthesis of glucans like cellulose, callose and mixed-linkage glucans. Evidence suggests that some SuSy isoforms interact directly with the CesA complex, channeling UDP-Glc directly to cellulose biosynthesis . Overexpcontrols . CrI refcontrols . Overexpby 9–11% . Overexpecreased . Celluloeristics , 2019. Tynthesis . The β-, in poplar (Populus deltoides) and Arabidopsis  is the only previously characterized gene encoding a DUF266-containing protein. OsBC10 encodes a Type II transmembrane Golgi protein and loss-of-function mutant plants were dwarfed with reductions in cellulose, and increases in xylose and lignin, indicating a role in cell wall biosynthesis , GhCOBL9A, was overexpressed in Arabidopsis, leading to dramatic increases in total biomass and cellulose content  that preferentially binds to crystalline cellulose. COBLs are required for cellulose production, believed to direct the orderly deposition of nascent cellulose microfibrils but are not components of the cellulose synthase complex  with the gene encoding an enzyme that provides its substrate, UDP-Glc/UDP-Gal-4-Epimerase2 (UGE2), in Arabidopsis increased galactose content in the cell wall by up to 80% , responsible for transport of UDP-Gal from the cytoplasm to the Golgi, in combination with GalS1 and UGE2 further boosted galactose content in the stems to four times the levels in wild-type plants (Cellulose synthase-like F6 from rice (OsCslF6) , a promoter active only during senescence.Although it is by far the most abundant, cellulose is not the only C6-sugar polysaccharide in the cell wall. Co-overexpression of p to 80% . GalS1 te plants . In thesOsCslF6) . The celt growth . The sucIrregular Xylem 10 (IRX10) and its partially redundant homolog Irregular Xylem 10-like (IRX10-L) encode GT47 enzymes that unambiguously exhibit xylan β--xylosyltransferase activity in recombinant systems (IRX10 (dnIRX10) genes in wild-type Arabidopsis, the mutant isoforms (IRX10G283D and IRX10E293Q) out-competed the native IRX10 for its place in the proposed xylan synthase complex. The stems of dnIRX10-overexpressing plants had reductions in xylose content of up to 55% compared to wild type. While xylan has detrimental effects on recalcitrance and biomass conversion, it is critical to the strength of vessel and fiber SCWs, and mutants in xylan biosynthesis exhibit severe growth defects due primarily to collapsed xylem vessels and impaired water and nutrient transport  dramatically improved biomass quality without significant growth or developmental defects  for building and remodeling the wall in many different tissues and stages of development . The GH9 defects . The celose ends .Acidothermus cellulolyticus AcCel5A suggests that even minimal cuts in cellulose chains can have dramatic effects on microfibril crystallinity, increasing enzymatic digestibility and sugar yield  have been cloned and used to engineer overexpression lines in tobacco (Expansin-A1 and Expansin-A2 (ClEXPA1 and ClEXPA2) overexpressing lines grew taller and had thicker stems than wild type. The cell walls of xylem cells were 1.13 to 1.45 times thicker in transgenic plants and when the composition of stem cell wall material was analyzed, they contained 30–50% more cellulose. It is also interesting to explore the effects that CBMs alone can have on cell wall architecture. In Arabidopsis, the coding sequence of the CBM of Starch Synthase III (SSIII) was overexpressed and targeted to the cell wall  and, in grasses, arabinose (Ara) side chains -linked xylose residues, many of which can be mono- or di-acetylated, and is decorated to varying degrees with glucuronic acid (GlcA), 4-e chains . ArabinoAspergillus niger (AnAXE1) was overexpressed in Arabidopsis (Populus tremula L. × tremuloides Michx)  had beneficial effects on biomass composition, despite increasing xylan acetylation by 8%  . The traon by 8% . It is lon by 8% , than toelopment . TherefoOsARAF1 and OsARAF3), were used to engineer rice lines accumulating high levels of the protein in the cell wall (In planta expression of a variety of ferulic acid esterases (FAEs) has demonstrated beneficial effects to reduce recalcitrance and increase biomass digestibility, but in many cases there are trade-offs to be considered. Expression of Aspergillus nidulans FAE in Arabidopsis and Brachypodium distachyon enhanced saccharification but increased susceptibility to fungal pathogens (Medicago sativa), expression of A. niger FAE did reduce arabinoxylan feruloylation, but unexpectedly led to increased lignification and decreased digestibility , an important animal forage crop  in poplar  led to increased growth and cellulose deposition and up to 81% more glucose released after enzymatic hydrolysis (Populus trichocarpa (PtPL1) in hybrid aspen (Populus tremula × tremuloides) resulted in improved saccharification  was used to engineer overexpressors in Arabidopsis and tobacco (Phaseolus vulgaris) has been identified and well described to eliminate PGA2 activity (Pectobacterium carotovorum (PcPL1) and the A. niger polygalacturonase (AnPGA2)  . The β-eAtPMEI-2) and kiwi  were overexpressed in Arabidopsis and wheat (Triticum durum), respectively (AtPMEI-2 overexpressing Arabidopsis stem increased by 50%, while wheat plants overexpressing AcPMEI had 2.5-fold greater saccharification efficiency than controls. In contrast to polygalacturonase-overexpressing plants, the total biomass of Arabidopsis plants overexpressing AtPMEI-2 was greater than wild type by 68%. This increase was not observed in AcPMEI overexpressing wheat plants, suggesting differing impacts of homogalacturonan methylesterification between monocots and dicots. The role of minor matrix polysaccharides in the development and structure of SCWs is poorly understood, but the successful results suggest that this would be a fertile ground for further research.Homogalacturonan is extensively methylesterified as it is synthesized and secreted. Once it reaches the cell wall, pectin methylesterases remove many of these methyl groups, revealing carboxylic acid moieties that subsequently form linkages with various components of the cell wall. Free carboxyl groups of de-methylesterified homogalacturonan can additionally form rigid, calcium-mediated “egg box” structures with adjacent stretches of homogalacturonan . Therefoectively . SaccharPlant biotechnological research can make a major contribution to meeting perhaps humanities’ most common and critical goals. That is, mitigating the effects of climate change by reducing our net production of carbon dioxide and developing an alternative to the fundamentally unsustainable fossil resources on which we have become dependent. While traditional breeding techniques will be important in the development of dedicated bioenergy crops, genetic engineering allows for significant modifications to biomass to be made much more quickly. As our fundamental knowledge of plant cell biology and the regulation of cell wall development continues to grow, so too will our ability to approach the perfect balance of maximizing yield and quality while minimizing recalcitrance and deleterious side effects. Here we have described a number of dominant genetic engineering strategies to improve plant biomass for bioenergy conversion. Though they may have only been demonstrated in one or two species, they have potential for broad usability in the wide variety of dedicated bioenergy species currently being researched. In several of the studies we have reviewed here, the results observed in different species were somewhat contradictory. For example, overexpression of CesAs or Susy in different species resulted in both increased or decreased cellulose and lignin depending on the particular study. This suggests that direct translation of an engineering strategy from a model plant or from one bioenergy crop to another is not entirely straightforward. The reasons for these contradictory results are not clear. They could be due to differences in the physiology and different cell wall types of the various species. However, we find it more likely that differences in the promoters used in the various studies, including their spatial and temporal expression profiles can explain many of the observed differences. Studies that resolve the results obtained with a larger set of promoters in a single species are required and would help to better predict outcomes in future studies. Future studies also need to determine the impact of environmental factors on the outcomes. If indeed the transgene expression profiles are very important, then we may also expect that the effect on plant development, biomass composition and recalcitrance could be strongly influenced by growth conditions. For the practical deployment of approaches such as those reviewed here, it is very important to determine if observations in the laboratory and greenhouse translate to the field condition, and to understand the mechanisms responsible where differences are observed.AB and HS developed the idea of the work and wrote the manuscript. AB illustrated AB and HS are inventors on a patent application related to the work described here on xylan reduction.


A wide range of comorbid diseases is associated with Alzheimer's disease (AD), the most common neurodegenerative disease worldwide. Evidence from clinical and molecular studies suggest that chronic diseases, including diabetes, cardiovascular disease, depression, and inflammatory bowel disease, may be associated with an increased risk of AD in different populations. Disruption in several shared biological pathways has been proposed as the underlying mechanism for the association between AD and these comorbidities. Notably, inflammation is a common dysregulated pathway shared by most of the comorbidities associated with AD. Some drugs commonly prescribed to patients with diabetes and cardiovascular disease have shown promising results in AD patients. Systems-based biology studies have identified common genetic factors and dysregulated pathways that may explain the relationship of comorbid disorders in AD. Nonetheless, the precise mechanisms for the occurrence of disease comorbidities in AD are not entirely understood. Here, we discuss the impact of the most common comorbidities in the clinical management of AD patients. Alzheimer's disease (AD) is the most common neurodegenerative disease affecting around 50 million people worldwide , and overt AD  is the most prevalent metabolic disease affecting 422 million people worldwide. Hyperglycemia and insulin resistance are characteristic features of the disease. Numerous lines of evidence support the association between T2D and AD. T2D is a well-established risk factor for AD, and AD is sometimes referred to as diabetes type 3  levels, diabetes status, and accelerated cognitive decline over 10 years of follow-up  have been investigated as potential AD therapies. For example, long-term and high dose metformin use was associated with a lower risk of incident AD in T2D patients  from a single center for 18 months. The small sample size and the early termination of the trial may explain the negative outcomes. A randomized placebo-controlled trial with 38 AD patients showed that liraglutide, another GLP-1 agonist, increased the blood-brain glucose transport capacity in the AD treated group compared to placebo  receptor agonists, have shown neuroprotective effects in animal models of AD  is another condition within the cardiovascular disease spectrum that has been implicated in AD. CHD is the most common heart disease and one of the leading causes of death worldwide. There is evidence that CHD increases the risk of cognitive impairment and dementia but there are some discrepancies among the studies. For example, a longitudinal cohort study revealed that the incidence of dementia was higher in subjects with CHD, particularly in those with peripheral arterial disease, compared to normal subjects  is associated with an increased risk of dementia. A population-based study including 32,298 patients with irritable bowel syndrome showed an increased risk of dementia in patients older than 50 years , alpha 2 macroglobulin (A2M), paraoxonase 2 (PON2), and microtubule-associated protein 4 (MAP4), were closely related to genes associated with cardiovascular disease, including catechol-O-methyltransferase (COMT), cystathionine beta synthase (CBS), and WNK lysine deficient protein kinase 1 (WNK1) suggesting both diseases are linked through shared molecular networks . In the context of gastrointestinal diseases, an integrative meta-analysis of 3 microarrays from patients with Crohn's disease identified ELAV-like RNA binding protein 1 (ELAVL1) and APP as the most significantly, upregulated and downregulated, respectively, in the blood of patients with Crohn's disease (Li et al., APP are known to cause familial AD (Weggen and Beher, ELAVL1 has been reported in AD, and its alteration may be related to APP processing (Amadio et al., In regards to cardiovascular and gut-related diseases, some bioinformatic studies have explored the connection with dementia. For example, one study showed that genes associated with AD such as apolipoprotein E (Several comorbidities associated with AD may be involved in the disease pathogenesis and progression and thus, may have important clinical implications in the management of patients. For example, disease comorbidities like T2D and depression are associated with poor prognosis in AD patients (Li J. Q. et al., Despite the substantial evidence provided by epidemiological studies regarding the linkage between AD and some of its comorbidities, the precise mechanism explaining their coexistence with AD is still poorly understood. Epidemiological studies are useful in providing the basis for understanding disease risk factors and comorbidities but causal relationships are more difficult to disentangle. Because of the shared genetics, environmental factors, and strong competing risk of death, the mechanisms underlying the association between AD, dementia, and other diseases remain a challenging task for scientists and clinicians. Integrative bioinformatic approaches combining epidemiological, genetic, transcriptomic, proteomic, and metabolomic data may be key to better understand comorbidities in AD (Santiago and Potashkin, Inflammation appears to be a central mechanism linking AD with other chronic diseases, however, it remains unclear whether inflammation plays a causative role or it is a consequence of neurodegeneration (Pugazhenthi et al., While some genetic factors are strongly connected with the development of late-onset AD, genetics alone does not explain the vast majority of the AD cases. The fact that many of the comorbidities associated with AD are related to dysregulated metabolic pathways implies that lifestyle factors play a role in the disease pathogenesis. In this regard, lifestyle modifications including exercise and diet may interact with genetic susceptibility genes and improve cognitive abilities in AD patients (Liang et al., CRI, CLU, and PICALM but not in APOEε carriers (Martinez-Lapiscina et al., In the context of diet, adherence to a Mediterranean diet has been associated with a decreased risk for AD (van den Brink et al., JS and JP wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.JS is employed by the company NeuroHub Analytics, LLC. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.