Adenosine is an extracellular signaling molecule that is particularly relevant in times of cellular stress, inflammation and metabolic disturbances when the levels of the purine increase. Adenosine acts on two G-protein-coupled stimulatory and on two G-protein-coupled inhibitory receptors, which have varying expression profiles in different tissues and conditions, and have different affinities for the endogenous ligand. Studies point to significant roles of adenosine and its receptors in metabolic disease and bone health, implicating the receptors as potential therapeutic targets. This review will highlight our current understanding of the dichotomous effects of adenosine and its receptors on adipogenesis versus osteogenesis within the bone marrow to maintain bone health, as well as its relationship to obesity. Therapeutic implications will also be reviewed. Adenosine is a nucleoside that is released by all cells, including adipocytes in vitro and the subcutaneous adipose tissue in vivo ,2. AdenoAdenosine is found extracellularly under pathologic situations including hypoxia, ischemia or cell damage. Cells release adenosine into the extracellular space through equilibrative nucleoside transporters (ENTs). Adenosine can also be formed by the conversion of extracellular ATP, released from damaged cells, by ectonucleoside triphosphate diphosphorylase (CD39) and ecto-5′-nucleotidase CD73) enzymes. Adenosine can also be metabolized to inosine by adenosine deaminase or phosphorylated to AMP by adenosine kinase. As one can imagine, the coordination of adenosine release is well regulated at baseline and, during times of cellular stress, can become a signal of disequilibrium . Similar3 enzymesThe expression of adenosine receptors is increased in various pathologic conditions . TheBone-marrow-derived mesenchymal stem cell (MSCs), precursors to adipocytes and osteoblasts, express the A2bAR and the A2aAR . In testThe A1AR is the most highly expressed adenosine receptor in white adipose tissue, followed by the A3AR, A2aAR and A2bAR ,32,33. AAdipogenesis results from the differentiation of adipocyte precursors into mature adipocytes capable of storing lipid in lipid droplets and secreting adipokines as well as cytokines either within the bone marrow or within adipose tissue depots. Adipocyte precursors are found in the bone marrow and within the stromal vascular fraction of peripheral adipose tissue depots. In the bone marrow, adipocytes differentiate from the MSCs, which are common progenitors that are also capable of differentiating into osteoblasts and chondrocytes. The process of adipogenesis requires a cascade of transcription factors. The master regulator of adipogenesis is the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is necessary for adipogenesis; mice lacking PPARγ and humans with dominant negative mutations in PPARγ have severe lipodystrophy and are insulin-resistant ,42. MoreOne of the first studies investigating the role of adenosine receptors in adipogenesis found that the treatment of rat primary preadipocytes with the adenosine analogue, NECA, promoted differentiation . In boneWithin the bone marrow, there is thought to be a dichotomous relationship between adipogenesis and osteogenesis, whereby the MSC is directed to one lineage or the other. The control of this fate decision is still being investigated. Some research points to adenosine receptors playing a role in dictating the formation of adipocytes or osteoblasts. Clinically, these decisions may manifest during aging, when the bone marrow contains more fat than younger marrow, or in osteoporosis, where there is loss of bone and an accumulation of fat. Transcription factors important for bone development include runt-related gene family members, such as Runx2. Osteoblast differentiation is known to be stimulated by fibroblast growth factor, insulin-like growth factor, bone morphogenic proteins and the Wnt/β-catenin pathway.There has been interest in the role of adenosine and adenosine receptors in bone metabolism. Using pharmacologic inhibitors and A2aAR knock out (KO) mice, the A2aAR was shown to promote proliferation of bone-marrow-derived MSCs . During The ectonucleotidase CD73, which regulates levels of extracellular adenosine through the metabolism AMP to adenosine, plays an important role in bone metabolism. CD73 KO mice have impaired osteoblast differentiation and decreased bone formation with the development of osteopenia . In vitrWhile the above studies focus on the cellular mechanism of osteogenesis, further direct investigation is needed on the effect of adenosine and its receptors on bone density in humans. Due to the beneficial effect of activation of adenosine receptors on bone density in various mice models ,62,63, ZThe function of the A1AR in inhibiting lipolysis has been extensively studied. It is thought that endogenously released adenosine maintains a constant inhibitory signal to adipocyte lipolysis. When adenosine deaminase, which removes endogenous adenosine by degrading it to inosine, is added to the culture of adipocytes from fasted rats, lipolysis reaches maximal levels ,65. It hThe A2bAR has also been studied in the context of lipid and glucose homeostasis. Koupenova et al. showed that lack of the A2bAR in ApoE-deficient mice fed a HFD resulted in elevated liver and plasma cholesterol and triglycerides, hepatic steatosis and atherosclerotic plaques . FurtherThe A2aAR has also been implicated in adipose tissue dynamics, obesity and inflammation. Mice fed a HFD for 12 weeks had increased expression of the A2aAR in macrophages within the adipose tissue as compared to mice on a low-fat diet . These m11C]TMSX, which binds specifically to the A2aAR, lean men were found to have reduced brown adipose tissue radioligand binding (a marker of A2aAR receptor density) following cold exposure [An exciting study explored the effect of the A2aAR on brown adipose tissue. The authors found that the A2aAR is the most highly expressed adenosine receptor in brown adipose tissue . A2aAR Kexposure . Howeverexposure , suggestGnad et al. evaluated the effect of A2bAR stimulation or suppression in major energy dissipating tissues . Their sAdenosine receptors play a role in regulating adipogenesis in peripheral adipose tissue and the bone marrow, and influence whole-body metabolism in terms of lipid storage and glucose homeostasis. Given the potential therapeutic benefits of adenosine receptors, this is an exciting time in adenosine biology. Given the complex biology and diverse, and, at times, contradictory effects of the adenosine receptors, continued development and study of drugs that target specific adenosine receptors will be key to the ultimate goal of creating therapeutic agents that lack potential adverse influences (thoroughly reviewed in ).As shown in Other exciting potential targets include the A2bAR, given its reported role in improving glucose and insulin homeostasis, and the A2aAR, given its reported ability to promote thermogenesis and browning of the white adipose tissue. However, given the potential for alterations in bone metabolism and other off-target effects, caution should be applied when evaluating adenosine receptor modulators. Much effort has been devoted to the synthesis of therapeutic drugs that target adenosine receptors, but several clinical trials have failed, and few such drugs are approved. Adenosine itself and regadenoson, an A2aAR agonist, are both approved for coronary stress imaging and for the treatment of paroxysmal supraventricular tachycardia in the case of adenosine. The first adenosine receptor antagonist to be FDA-approved is the A2aAR antagonist istradefylline (Nourianz), which is used with levodopa/carbidopa in Parkinson’s disease . While i


Lynx lynx carpathicus) was reintroduced to Switzerland in the 1970's. Health monitoring of the reintroduced population started in the late 1980's. Since then, six lynx have been found affected by a myocardial disease. The earliest case was an animal that died after a field anesthesia. Two lynx were found dead, two were euthanized/culled because of disease signs, and one was hit by car. Two had a heart murmur at clinical examination. At necropsy, the first animal showed only lung edema but the other five had cardiomegaly associated with myocardial fibrosis. Three had multisystemic effusions. Histological examination of all six lynx showed mild to severe, multifocal, myocardial interstitial and perivascular fibrosis along with multifocal myocyte degeneration and loss, and replacement fibrosis. Moderate to severe multifocal arteriosclerosis with associated luminal stenosis of the small and medium-sized intramural coronary arteries and the presence of Anitschkow cells was also observed. The heart lesions may have led to sudden death in the first case and to a chronic right-sided heart failure in the remaining. None of the lynx showed lesions or signs suggestive of an acute or subacute infection. Given the common geographic origin of these animals and the severe loss of heterozygocity in this population, a genetic origin of the disease is hypothesized.The Eurasian lynx (subspecies  Cardiovascular disease is considered the leading cause of mortality in people worldwide . SpontanIn both domestic animals and humans, the term cardiomyopathy has been long used to refer to idiopathic myocardial disorders, but cardiomyopathies were subsequently defined as clinicopathological entities resulting either from genetic anomalies or from nutritional deficiencies, with cardiomegaly and myocardial fibrosis as most typical features . MeanwhiThe most common type of cardiomyopathy and frequent cause of morbidity and mortality in domestic cats is HCM. This disease concerns mainly older cats and is often associated with a loud systolic heart murmur. Although it frequently remains subclinical, a majority of affected cats develop congestive heart failure, others arterial thromboembolism, and a minority die unexpectedly in absence of clinical signs , 18–20. Lynx lynx) is a wild felid endemic to Europe and North-central Asia, which has undergone a massive population reduction in Europe in the 19th century , which currently only occurs in small fragmented populations in Western, Central and Eastern Europe, is one of the nine currently proposed Eurasian lynx subspecies (L. lynx lynx) in Scandinavia, Finland and European Russia, the Balkan lynx (L. lynx martinoi) in the Balkan region and the Caucasian lynx (L. lynx dinniki) in Turkey. Although the separation into these subspecies remains controversial  for post-mortem examination in the framework of the lynx health monitoring program in Switzerland . AccordiThe lynx population in the northwestern Swiss Alps originated from probably <10 of the 26 individuals released in Switzerland in the 1970's , 37. TheSix lynx older than 1 year and of both sexes submitted for necropsy to the FIWI between 1988 and 2014 were included in this study. The four most recent cases were examined by the authors and documented with photographs. The two earliest cases were retrieved from the archives. Selection criterion was the record of information suggestive of cardiomyopathy in the case history, macroscopic pathologic observations and/or histological descriptions. Lynx were aged as accurately as possible by cementum annuli counts, tooth wear evaluation and body measurements, and placed into age classes as previously described , 41.TMLittmann® 3200) and later confirmed by a board-certified cardiologist. Physical examination, hematology and blood chemistry, radiographs of the thorax and echocardiography in clinical settings, as well as pathological examination after traumatic death (necropsy and histology) were also consistent with a healthy heart.Since there are no published reference values for the morphology of the Carpathian lynx heart, and those generated for Northern lynx do not include the ratios necessary to overcome body size and weight differences , 42, a hMacaca mulatta)  and ventricular wall thickness (mm). The right ventricular free wall, interventricular septum and left ventricular free wall were measured on a transverse cross section (perpendicular to the long axis of the ventricles) from the epicardium to the endocardium, excluding papillary muscles, as a single measurement, using a millimeter rule. This procedure was the same as reported in domestic cats  except tmulatta) .Representative samples of the lung, heart, kidneys ; liver, spleen, genital organs ; stomach, intestine , adrenals (case 3–6); lymph nodes ; skeletal muscle ; pancreas (case 2 and 4); thyroid (case 4 and 5); urinary bladder ; and brain (case 5) were collected and fixed in 10% neutral-buffered formalin. Tissues were then embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin (H&E) for histologic examination following the accredited protocols of the Institute of Animal Pathology, Vetsuisse Faculty, University of Bern. Archived paraffin blocks and/or histological sections were available for case 2–6. Additional heart sections were stained using the Masson's trichrome (MT) and Van Gieson (VG) protocols for a qualitative and semi-quantitative evaluation of collagen proliferation and vascular changes.Histological assessment of the available heart sections was performed with board-certified pathologists, focusing on the left free ventricle wall and septum, which were shown to be more severely affected than the right free ventricle wall in a preliminary retrospective histological study . The twoGeneral information on the six cases and the control are given in Case 1 was found dead after a field anesthesia with xylazine and ketamine  for fittCase 2 was found dead. The original diagnosis of feline infectious peritonitis was not confirmed by immunohistochemistry  and the Case 3, first captured in September 1999, was recaptured twice (2 and 4 years after the first capture). At the second capture in February 2001 it still appeared healthy and was transported to a quarantine station to be subsequently translocated to northeastern Switzerland , 46. It Case 4 was caught in February 1998 and had shown a normal post-capture spatial and hunting behavior until its radio-collar stopped working early March 2001. In February 2012 (14 years after capture) it was found lying close to a road without escape reaction and therefore assumed to be ill and culled.®, Chassot AG, Switzerland) and arrived still alive at the University of Bern, where it was euthanized. Heart auscultation prior to euthanasia confirmed the presence of a loud systolic heart murmur nearly completely covering the heart sounds.Case 5 was in good body condition (24 kg) when captured mid-March 2013, and all recorded health and anesthesia parameters were normal except for a heart murmur. It was re-captured and euthanized 7 weeks later because of abnormal behavior (apathy and ataxia) and severe emaciation. It developed respiratory insufficiency during transport but could be stabilized with an intramuscular injection of 40 mg Doxapram  had cardiomegaly together with multifocal fine light tan striations coalescing to prominent tan foci in the myocardium . Cardiomegaly was associated with marked dilation of either the right (case 2) or left ventricle (case 4), or both (case 3 and 5), while bilateral ventricular hypertrophy was present in case 6 .Case 3 additionally had a subaortic stenosis with a distinct fibrotic ridge running across the left ventricle wall ~1.5 cm from the root of the aorta. A diffuse ectasia of the aortic arch was observed associated with the dilation of the left atrium and ventricle. The endocardium was thickened, particularly above the stenotic ring  and in tCase 2–5 showed a decreased body condition, ranging from moderately emaciated  up to cachectic , and they all had an empty stomach indicating the absence of recent food intake. All four presented with changes consistent with a right-sided congestive heart failure, including large amounts of serosanguinous fluid with fibrin deposits in the abdominal cavity  and multifocal atelectasis of varying severity in four lynx each . Furthermore, heart-failure cells were seen in two (case 2 and 3). Additional findings included a subpleural hemangioma (case 2), multifocal emphysema (case 1 and 5), mild to moderate hemorrhages (cases 2 and 6), multifocal mild calcifications (case 4 and 6), and a mild multifocal histiocytic to lymphocytic interstitial pneumonia (case 4).Microscopic liver changes were observed in four lynx (case 2–5). Three animals showed Ito-cells hyperplasia (case 2–4), with additional moderate to severe hemosiderosis in case 2 and 3, which were both found dead in a status of cachexia. Furthermore, the liver of case 3 was characterized by a severe centrolobular to panlobular fibrosis, together with multifocal moderate arteriosclerosis and mild cholestasis. In case 4, there was moderate centrolobular degeneration and mild centrolobular fibrosis together with mild neutrophilic and lymphocytic infiltrates, mild hemosiderosis, cholestasis and sinusoidal dilation.Additional findings were acute necrotizing pancreatitis with fibrinous and plasmacellular peripancreatitis (case 2), peritonitis (case 2 and 3), interstitial edema in the hind leg muscles (case 3), mild to moderate lymphoid follicle hyperplasia in the mesenterial lymph nodes (case 2), spleen lymphoid depletion (case 3 and 4), mild membranous glomerulopathy , and mild multifocal lymphoplasmacytic meningitis (case 6).A comparative summary of the main pathological findings, data on heart morphology and scoring of histological heart lesions are presented in This study reveals the existence of previously undescribed cardiac disorders in Eurasian lynx from a reintroduced population in Switzerland. The pathological features consistent throughout the cases were the presence of myocardial fibrosis with associated Anitschkow cells, and coronary arteriosclerosis with intimal proliferation, which were pronounced in all lynx except case 2. Despite inter-individual differences, shared pathological features suggest that the six lynx were affected by the same disease entity.While case 1 unexpectedly died during anesthesia and presented with a severe acute lung edema, case 2–4 had macroscopic pathological changes characteristics of a congestive right-sided heart failure. The most advanced disease picture, including liver fibrosis and congestion, was observed in case 3, the animal with a subaortic stenosis, which had presented with a heart murmur at clinical examination and died spontaneously. Case 4, which was even older but had been culled due to disease signs, seemed to be in an intermediary position between case 2 and 3 in term of disease severity and chronicity. In line with the clinicopathological features, relative heart weight and both heart wall ratios of case 3 and 4 (no available morphologic heart data for case 2) strongly differed from those of the control animal. Case 5 (euthanized) and 6 (hit by car) had major macroscopic and histological cardiac lesions although they died prematurely, but they presented with fewer (case 5) and no (case 6) major systemic changes. Accordingly, case 5 may have been in an earlier disease stage than case 2–4, and disease may have been subclinical in case 6. In these two last cases, relative heart weight and right/left ventricular wall ratios were similar to those of the control but they had bilateral ventricular alterations , possibly explaining that the wall ratios did not diverge much from the control ratio. In case 6, all three walls were over the average values of adult males without evidence of cardiac disease, in agreement with a bilateral ventricular hypertrophy, although the similar relative heart weight was not consistent with an increased heart mass. It is conceivable that this animal with hypertrophic heart features may have been at an earlier disease stage than cases 2–5, which showed heart dilation. In animal models, concentric myocardial hypertrophy and fibrosis precede dilation, which marks progression toward decompensated heart failure and systolic dysfunction .Scores of the histological main lesions were highest in case 5, followed by case 1 , 6, 3, and 4, and they were lowest in case 2, not paralleling the clinicopathological findings. It is known that there is not necessarily full correspondence between the severity of gross and microscopic abnormalities within the heart and the presence of clinical signs attributable to them. While major structural abnormalities may be found incidentally after death, there may be relatively mild but critically located lesions resulting in marked functional disturbance . Sudden Capture procedure and anesthesia likely were stressing factors in case 1. Anesthesia is a procedure associated with an increased risk in cardiac patients, whether they present with clinical signs of heart failure or not. Anesthesia was performed using an alpha-2 adrenergic drug, a category of sedatives that is considered as contra-indicated in patients with cardiac disease . Alpha2-The three lynx with chronic heart disease had cardiomegaly matching the criteria of an eccentric hypertrophy, considering that the cardiac volume and mass were increased, although the ventricular free walls were of normal to decreased thickness and the papillary muscles were attenuated , 49. HowCase 1, 3, 4, and 6 had valvular disease additionally to the lesions in the myocardium and coronary arteries. Subvalvular aortic stenosis and mitral endocardiosis are common in dogs but not in cats. They are typically associated with a left-sided cardiac dysfunction that may end in a congestive heart failure characterized by interstitial and alveolar pulmonary edema. Myocardial fibrosis may develop with both valvular pathologies and may be associated with arteriosclerosis. However, animals with aortic stenosis are mainly prone to sudden death, and endocardiosis is a frequent incidental finding at necropsy , 49. WhiAll lynx had varying degrees of fibrosis in the myocardium. Myocardial fibrosis is associated with increased ventricular stiffness, which compromises ventricular systolic (and diastolic) function, abnormal cardiac remodeling and development of ventricular dilation, and activation of compensatory mechanisms that increase sodium and water retention to increase venous return to the heart , 57, 58.The origin of the observed myocardial lesions is unknown. In animals and man, fibrosis is a common feature of most myocardial diseases. The adult mammalian heart has negligible regenerative capacity, and once myocytes are lost, there is progressive scavenging of the necrotic remnants and replacement by fibrosis, mainly by activation of resident interstitial myofibroblasts, a process that aims at preserving the structural integrity of the heart. Pathophysiological mechanisms leading to fibrosis development are various, some being acute as in myocardial infarction, others being progressive. Accordingly, not only cardiomyocyte death but also injurious stimuli such as pressure and/or volume overload, metabolic dysfunction, myocardial inflammation, as well as aging, may cause interstitial and perivascular fibrosis in the absence of infarction . HoweverChlamydia sp. infection, and dogs experiencing sudden death  and found a significant difference in prevalence of combined myocardial fibrosis and arteriosclerosis , which affected mainly adult males . In a seWe found Anitschkow cells in all six lynx, differently from animals without evidence of cardiac disease, including the control animal. Anitschkow cells are characteristic mononuclear cells with “caterpillar” - like nuclei, which are usually observed in the myocardium and in coronary vessel walls and may represent modified myocytes. They are typically found in humans affected by rheumatic heart disease and other cardiac pathologies, and in animals of various species with myocarditis, myocardial necrosis, degenerative and inflammatory endocardial diseases, or arteriosclerosis , 60, 68.Given the absence of obvious signs of infection  in all studied lynx, a genetic origin of this cardiomyopathy should be considered. Genetic factors are increasingly recognized as a cause of cardiac disorders, including cardiomyopathies with and without associated intramural coronary artery wall abnormalities , 4, 69. In conclusion, the investigated lynx were affected by a myocardial disease with coronary arteriosclerosis, potentially leading either to unexpected death or to a right-sided congestive heart failure, and associated with a heart murmur in the two cases that could be examined clinically. Heart function was likely further compromised by valvular disorders in four of six cases. As the heart alterations did not match any of the three main cardiomyopathy categories , they must be regarded as a cardiomyopathy of non-specified phenotype with potential genetic background. The cases presented here span over 20 years, illustrating the value of long-term health monitoring of reintroduced wildlife populations and of performing necropsies on wild animals that die during capture events. This study also highlights the importance of veterinary investigations and standardized necropsy protocols in the framework of conservation programs.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.Ethical review and approval was not required for the animal study because investigations were performed on dead animals submitted to necropsy. Data previously collected on live animals were obtained in the framework of ecological studies run independently of the present study and conducted with the necessary authorizations of the Swiss cantonal ethics committees according to the Swiss law on animal experiments.M-PR-D designed, performed the study, and wrote the manuscript. NR and FO provided pathology expertise and contributed to pathology data collection. FO described the histological pictures. M-PR-D, MP, and RM carried out clinical examinations. M-PR-D, SZ-G, and RM performed necropsies. UB initiated veterinary examinations of lynx in Switzerland, coordinated the ecological projects, and contributed to field work. AR organized the captures and radio-tracked the lynx. AK provided cardiology expertise and performed an in-depth cardiological examination of the control animal. All authors critically read and approved the 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.


The unfolded protein response (UPR) is an evolutionarily conserved adaptive signaling pathway triggered by a stress of the endoplasmic reticulum (ER) lumen compartment, which is initiated by the accumulation of unfolded proteins. This response, mediated by three sensors-Inositol Requiring Enzyme 1 (IRE1), Activating Transcription Factor 6 (ATF6), and Protein Kinase RNA-Like Endoplasmic Reticulum Kinase (PERK)—allows restoring protein homeostasis and maintaining cell survival. UPR represents a major cytoprotective signaling network for cancer cells, which frequently experience disturbed proteostasis owing to their rapid proliferation in an usually unfavorable microenvironment. Increased basal UPR also participates in the resistance of tumor cells against chemotherapy. UPR activation also occurs during hematopoiesis, and growing evidence supports the critical cytoprotective role played by ER stress in the emergence and proliferation of leukemic cells. In case of severe or prolonged stress, pro-survival UPR may however evolve into a cell death program called terminal UPR. Interestingly, a large number of studies have revealed that the induction of proapoptotic UPR can also strongly contribute to the sensitization of leukemic cells to chemotherapy. Here, we review the current knowledge on the consequences of the deregulation of UPR signaling in leukemias and their implications for the treatment of these diseases. About one-third of human genes encode secreted or transmembrane proteins as well as proteins resident of the endoplasmic reticulum, the Golgi apparatus, and lysosomes. Most of these proteins are targeted to the ER. The endoplasmic reticulum is a complex network of membrane-enclosed tubules and vesicles, extending from the nuclear membrane throughout the cytoplasm. ER is the largest organelle of most eukaryotic cells, as its membrane may account for at least 50% of all cell membranes and even more for specialized secretory cell. Its total area is 10–30 times that of the plasma membrane. ER constitutes the first compartment of the secretory pathway in which secreted and transmembrane proteins are folded and post-translationally modified .,75.74,75The IRE1α activation level, stability, conformation, and oligomerization status appear to be also regulated by the interaction with many different protein partners such as for example HSP47 which facilitates the dissociation of BiP from its luminal domain thus helping in activation of IRE1α signaling under low stress conditions ,84,85.IRE1α associates also with additional partners through its cytosolic domain to induce different signaling pathways. TRAF2 (TNF receptor-associated factor), an adaptor protein, associates with IRE1α’s kinase domain. The IRE1α/TRAF2 complex was found to interact with ASK1  to activate the c-Jun N-terminal kinase (JNK) and induce apoptosis ,86. ThusRecent work has demonstrated that the activity of PERK and ATF6α can also be regulated by specific interacting proteins (reviewed in ). These 12 blood cells daily in a healthy adult. The hematopoietic system functions as a pyramid-like hierarchy organized from a hematopoietic stem cell (HSC) at the top, able to self-renew or differentiate to produce all the cells of the hematopoietic system  is a group of phenotypically and genetically heterogeneous diseases, which is among the most common adult leukemia (it accounts for about 80% of leukemias in adults), with an average age of first diagnosis over 60 years . This isAcute lymphoblastic leukemia (ALL), also called acute lymphocytic leukemia, is a rare genetically heterogeneous clonal malignant disorder of the bone marrow characterized by immature lymphoid precursors proliferation leading to the crowd out of normal hematopoietic cells . ALL, whThe genetics of T-ALL is extremely heterogeneous, with chromosomal abnormalities in nearly all patients. Mutations in the NOTCH1 gene leading to constitutive activation of NOTCH signaling is the main oncogenic pathway found in the majority of patients. These alterations are generally associated with loss of p16 (INK4A) and p14 (ARF) suppressor genes at the CDKN2A locus. In addition, in 50% of patients with T-ALL, chromosomal translocations affect genes encoding oncogenic transcription factors like TAL1, TAL2, MYC, MYB, LYL1, TLX1 (HOX11), TLX3 (HOX11L2), or HOXA genes, placing these genes under the control of powerful T cell specific activators .As for the AML example, it is not possible to exhaustively list all the genetic alterations and different combinations encountered, so we refer the reader to references dealing more specifically with this pathology ,116,117.Chronic myeloid leukemia (CML) is a slow-growing myeloproliferative neoplasm characterized in more than 95% of cases by the t  chromosomal translocation leading to the formation of the Philadelphia chromosome (Ph*), resulting in the BCR-ABL1 gene fusion. The subsequent BCR/ABL1 chimeric protein is a constitutively active tyrosine kinases oncoprotein which activates transduction pathways involved in cell growth and differentiation such as RAS, MYC, STAT, AKT RAF, or JUN, and is therefore capable of transforming hematopoietic stem cell into neoplastic one ,119.Before targeted therapies became available, the main treatment options for CML included allogeneic stem cell transplantation and chemotherapy. However, the prognosis for CML improved considerably since the use of tyrosine kinase inhibitors (TKIs), most notably imatinib in the early 2000s, which inhibit the BCR-ABL1 fusion protein by blocking its kinase domain . SeveralChronic lymphocytic leukemia (CLL) is characterized by a clonal proliferation and accumulation of mature but defective lymphocytes in the blood, bone marrow, lymph nodes, and spleen. CLL is the most common form of leukemia in Western countries. It is highly heterogeneous in its evolution, with some patients needing chemotherapy early after diagnosis and others never requiring specific treatment and having a survival rate similar to the general population. More than 95% of people with CLL develop the B cell type . CLL is A variety of targeted drugs including BCR signaling pathway inhibitors, anti-CD20 antibodies and BCL-2 inhibitors have been used in therapeutics and have significantly improved the management of this disease ,133. HowHematopoietic stem cells (HSCs) are sitting at the apex of the hematopoietic hierarchy. They are the most immature cells and are capable of replenishing all hematopoietic cell types ,135. As Increased ERAD has also been recently reported to actively participate in the maintenance of proteins homeostasis in HSCs and appeared to be essential for stem cell pool maintenance . In addiRecent lines of evidence link activation of the three UPR branches to most hallmarks of cancer and especially those aimed to protect the cells against the numerous aggressions they undergo during their growth inside tumors . This isIn AML patients, increased expression of XBP1, BiP, and Calreticulin has been detected in 17.4% of cases . SchardtInterestingly, UPR activation in some hematological malignancies is not always the consequence of stress integration but can also be induced through aberrant pathway activation. For example, in chronic lymphoid leukemia (CLL), UPR activation is observed in response to surface immunoglobulin M stimulation and activation of the kinases BTK and SYK . In pre-In addition, mutations in epigenetic splicing factors, which are considered as first hit mutations, have pleiotropic effects that might be linked to ER stress activation . The comFurthermore, transcription of ER stress-related proteins by oncogenic pathways also participates in the UPR activation in leukemia. For instance, the MAPK pathway promotes the transcription of Xbp1 through STAT5 activation. In pre-B acute lymphoblastic leukemia (ALL), BCR-ABL1 or NRASG12D signals through MAPK-STAT5-XBP1 . IndirecCompared to HSC, in which both cytoprotective (IRE1α-XBP1) and cell death-promoting (PERK) UPR pathways can be activated in the basal state, the activation of an adaptive UPR  appears to be preferred in leukemic cells. However, the increase in basal UPR could sensitize these cells to additional stress induced, for example, by chemotherapy treatment (see next chapter). By analogy with what is observed in HSCs under normal growth conditions, the UPR response may represent a real checkpoint influencing cell fate of leukemic cells experiencing chemotherapy: either the stress can be resolved via an adaptive phase and cancer progresses or the damage accumulates and becomes unrecoverable. In this latter case, excessive or prolonged stress triggers proapoptotic signaling through a terminal process ,151,160.Despite the numerous pieces of evidence of reticulum stress activation in multiple cancers, the question of whether UPR reduces or promotes tumor growth in patients is still the subject of intense debate . Two theAs mentioned above, various studies have shown that leukemic cells often possess basal UPR activity with a cytoprotective function, which favors tumor progression and additionally may increase chemoresistance of the cells to various drugs. For example, the ER stress sensor BiP was found to be highly expressed in B-ALL and its pharmacological inhibition by epigallocatechin gallate (a polyphenolic compound purified from green tea) sensitized cells to the anti-leukemic drug vincristine . In the Altogether, the data presented above have largely validated the inhibition of adaptive UPR as an effective means of fighting leukemia and a significant number of pharmacological inhibitors of central UPR effectors are currently under preclinical studies or clinical trials ,150.However, these promising results should not lead us to neglect the other strategy aimed at inducing a cytotoxic response in the cell through terminal UPR induction, the “second edge of the sword”. Indeed, leukemic cells, which usually experience unfavorable growth conditions and maintain increased levels of ER stress and basal UPR, may show an increased susceptibility to enter terminal UPR in response to different treatments. Indeed, artificially increasing the unfolded protein load can lead to a cytotoxic cellular response in some leukemic models. Thus, in ALL treatment with the drug pevonedistat, which inhibits the NEDD8 conjugation pathway and impairs degradation of misfolded proteins by the proteasome, induces a reorientation of UPR towards apoptosis . InteresFew studies describing the induction of cell death in leukemic cells by a strategy deliberately aimed at redirecting the cell response towards terminal UPR has yet been described. However, the analysis of data published over the last two decades and describing the use of antileukemic drugs shows, strikingly, that for many of them see  their moTherefore, it seems important to further investigate terminal UPR induction, on its own as well as in combination with other pharmacological treatments, for the improvement of therapeutic strategies in leukemia.We have reviewed the currently available data in the literature dealing with the various roles played by UPR in leukemia. We also presented some of the UPR-mediated molecular processes that can induce cytoprotection of leukemic cells or direct them towards cell death through apoptosis induction. In the light of all of the currently reported data from studies carried out to dissect the role of UPR in the progression of leukemia, it is clear that the cytoprotective/cytotoxic balance regulation is a complex, highly dynamic machinery, still poorly understood and that a wide and integrative approach is needed to discover the genuine mechanisms underlying this crucial process. The specific networks that regulate ER stress-induced cytoprotection or apoptosis may be dependent on the nature, the intensity and the length of the stimuli. It is probable, even if contradictory results have sometimes been published, that it also depends on the cell type being stressed.A better understanding of the UPR mechanisms acting in response to chemotherapy appears also essential to provide new therapeutic pathways aimed to eradicate neoplastic cells either by inhibiting the adaptive UPR, or by activating UPR-mediated cell death pathways .


The use of mandibular overdentures (MO) for the rehabilitation of totally edentulous individuals with limited bone availability is widespread and has proven clinical success. Narrow diameter implants (NDI) are available on the market as MO retainers to solve problems related to limited bone availability and bone thickness, providing a low-cost, minimally invasive treatment option. This technique evolved over the years, and changes frequently involved the number of implants used as MO retainers, as the adoption of a smaller number of implants can generate biomechanical disadvantages, contributing to the increased stress in peri-implant tissues, which may accelerate marginal bone loss (MBL), in addition to reducing masticatory capacity and satisfaction with rehabilitation. Some studies pointed out that the use of 3 or more implants as MO retainers improves the biomechanics. Thus, the objective of this study was to report 3 different clinical cases where 3 or more NDI were adopted to retain mandibular overdentures in association with diverse loading protocols: (i) 3 implants adopting conventional loading, (ii) 4 implants using progressive loading, and (iii) 4 implants with hybrid loading. The case with 4 implants and progressive loading showed a slight worsening of masticatory function at 1 year, in addition to a more pronounced MBL compared to other cases, but with improvements in satisfaction and oral health-related quality of life. Thus, NDI can be used as MO retainers with predictability and clinical success, using different numbers of implants and loading protocols. Complete denture wearers often report complaints related to lack of stability and retention, especially of the mandibular prostheses . These cAccording to the McGill consensus, mandibular overdentures (MO) retained by 2 implants should be the minimum treatment offered to this profile of patients, due to the benefits that this type of rehabilitation promotes, such as increased bite force, decreased discomfort during function, improvements in masticatory function, and neuromuscular control, thus ensuring greater quality of life and patient satisfaction . Severalrd or 4th implant would guarantee greater retention and stability, in addition to reducing anteroposterior movement [Narrow diameter implants (NDI) have become an alternative rehabilitation for patients with limited bone availability, especially for elderly patients, guaranteeing the rehabilitation of edentulous patients with residual ridge atrophy, who often could not undergo an extensive surgical procedure with prolonged healing time . The Facmovement –14.Thus, the objective of this study was to explore the effect of using different numbers of NDI (Facility-Equator system) to retain MO combined with various loading protocols in a series of clinical cases describing the predictability of this system by monitoring clinical, functional, and patient-centered outcomes after 1 year of use. The following combinations of implant numbers and type of occlusal loading were adopted in these 3 clinical cases: (i) 3 NDI with conventional loading, (ii) 4 NDI with progressive loading, and (iii) 4 NDI with hybrid loading.First, all individuals that agreed to participate in this study were asked to sign an informed consent form. This case series reportfollowed the CARE Guidelines . In all MO retained by NDI (Facility-Equator) was indicated for all patients; 3 or 4 implants were installed between the mental foramen, depending on the patients' preference, desired degree of retention, financial situation, and bone availability. The implant insertion torque determined the type of loading used in the 3 cases: (i) immediate loading was used when the implant insertion torque was ≥32 N, (ii) conventional loading when the insertion torque was <32 N, and (iii) hybrid loading was applied in cases where 4 implants were used and where at least 2 implants achieved the minimum insertion torque required for immediate loading, and the remaining cases were loaded 3 months after surgery. In all cases, implant installation followed the bone drilling protocol recommended by the manufacturer , adoptinThe patient presented an ovoid mandibular ridge with a rounded asymmetric residual ridge and a medium muscle insertion. In this case, 3 implants were installed because the patient is younger and requested a higher degree of retention. The patient was unsatisfied with his prosthetic experience after extraction of the lower teeth, as he was unable to adapt to the immediate mandibular total prosthesis in the short period of edentulism (3 months). Conventional loading was applied since no implant achieved the minimum insertion torque of 32 N during installation. Patient 2 preoperatively presented a Kennedy Class I modification I type mandibular arch. After extraction, the patient had an ovoid mandibular ridge with an asymmetric, rounded residual ridge with a high muscle insertion close to the ridge. In this case, we opted to install 4 implants due to the patient's young age and request for high retention. However, because the patient's lower teeth were extracted at the time of surgery, adequate bone beds for all 4 implants were initially unavailable, so we opted to install implants 2 and 4 first, followed by installation of implants 1 and 3 after 2 months. The type of loading was conventional and progressive. Neither the first 2 implants (implants 2 and 4) nor the last 2 (implants 1 and 3) reached a minimum torque of 32 N, and all implants were thus loaded conventionally after 3 months of osseointegration. Patient 3 preoperatively presented a Kennedy Class I, modification II type mandibular arch. After extraction, the patient had an ovoid mandibular ridge, with an asymmetric, knife edge-type residual ridge with a high muscle insertion close to the ridge. In this case, we opted to install 4 implants immediately after dental extractions in the lower arch. The bone availability and bone bed conditions at the time of surgery permitted to install all 4 implants simultaneously. However, unlike in case 2, patient 3 received hybrid loading: two implants reached torque > 32 N (implants 2 and 4) and received immediate occlusal loading, while the other two (implants 1 and 3) did not reach the minimum torque and received conventional loading. At the time of surgery, 4 Equatorattachments were installed but only two were immediately loaded. Assessments of the masticatory function and oral health-related quality of life (OHRQoL) were carried out before installation and after 1 year of MO use. All individuals had high expectations for the treatment and reported complaints related to masticatory function and showed dissatisfaction with their own oral health and quality of life. Peri-implant health and marginal bone loss (MBL) around the implants was monitored to verify the presence of differences in these outcomes in cases with different numbers of installed implants.Masticatory function was assessed via the swallowing threshold test wherein individuals chewed a standardized portion of test food  until they felt the desire to swallow . AfterwaAll patients showed signs of healthy peri-implant conditions during healing and postloading. The patient in case 2 showed the greatest peri-implant bone loss . The ST_Treatment with dental implants has become an integral part of modern dentistry, even in challenging situations, such as completely edentulous elderly patients with systemic impairments, long periods of edentulism, mandibular bone atrophy, and/or poor-quality bone. The specialized literature shows that MO use for rehabilitation of totally edentulous individuals with low bone availability is well established and has satisfactory success and survival rates alongside a major positive impact on patients' quality of life , 19, 20.Studies of this research group that investigated the Facility-Equator NDI system have observed that patients show a fast and significant increase in masticatory function 30 days after installation of 2 implant-retained MO; after three months, there is an OHRQoL improvement in the functional, oral comfort, and psychosocial domains , 11. We α and IL-1β were more stable in the CC group [The biological performance of the Facility-Equator system in the early and late osseointegration period was also evaluated according to conventional loading (CL) and immediate loading (IL) . During CC group . The driCC group .There is currently a tendency to increase the number of implants used to retain MO, as some studies have shown that MO retained by 2 implants between the mental foramen have biomechanical disadvantages compared to MO retained by 3 or 4 implants, as 2 implants permit anteroposterior movements of the prosthesis, increasing the tension around the implants , accelerIt is clear that MO retained by 3 or 4 NDI loaded with conventional, immediate, progressive, and hybrid loading protocols can promote improvements in masticatory function, OHRQoL, and MBL of totally edentulous patients with limited bone availability. However, since a longitudinal clinical study  demonstrThe strengths of these approaches include the predictability of the treatment using narrow diameter implants that is attributed to (i) detailed case planning that took into account the clinical conditions along with the patient's preference and expectations and (ii) consistent clinical, functional, and OHRQoL improvements, regardless of the number of implants. We emphasize that MO retained by a smaller number of implants can fulfill their role adequately, with positive bone tissue, chewing, and quality of life responses, as in our case with 3 NDI. Therefore, this option can be strongly indicated for individuals with low bone availability and a reduced budget. The limitations for the proposed approaches are (i) the financial aspect: both the initial costs and the maintenance costs scale with the number of implants  and (ii) with progressive aging and cognitive and motor decline, the increased retention of MO promoted by the greater number of implants can result in difficulties removing them; conversion to retention by 2 implants is recommended in these cases.Finally, narrow diameter implants can be successfully used as MO retainers in diverse clinical scenarios where mandibular ridge atrophy is present. The available treatment options in these cases include the use of 3 or 4 implants and adopting conventional, conventional progressive, or hybrid loading, illustrating the versatility of the available clinical options. We recommend clinicians to analyze each case individually, taking into account the clinical conditions and the patient's preferences, together with the literature recommendations. Furthermore, we suggest that MO retained by 3 NDI can be indicated in the clinical routine as an optimal option when the patients wish increased retention and stability at a lower budget.


SMC1A. Thus, the micronucleus formation assay described here is a versatile and efficient alternative to more laborious cytological approaches, and greatly increases throughput, which will be particularly beneficial for large-scale chemical or genetic screens.Micronuclei are small, extranuclear bodies that are distinct from the primary cell nucleus. Micronucleus formation is an aberrant event that suggests a history of genotoxic stress or chromosome mis-segregation events. Accordingly, assays evaluating micronucleus formation serve as useful tools within the fields of toxicology and oncology. Here, we describe a novel micronucleus formation assay that utilizes a high-throughput imaging platform and automated image analysis software for accurate detection and rapid quantification of micronuclei at the single cell level. We show that our image analysis parameters are capable of identifying dose-dependent increases in micronucleus formation within three distinct cell lines following treatment with two established genotoxic agents, etoposide or bleomycin. We further show that this assay detects micronuclei induced through silencing of the established chromosome instability gene,  Micronuclei are small, membrane bound, DNA-containing bodies located outside the primary cell nucleus and whose presence is synonymous with genome instability. Micronuclei are typically defined as having a diameter ≤ 1/3 the size/area of the primary nucleus ,3. In thAlthough micronuclei arise through a variety of mechanisms, they generally manifest from chromosome mis-segregation events stemming from the stress response (reviewed in ). In somThe underlying molecular events driving micronucleus formation are often associated with chromosome instability , an In this study, we describe the development, optimization, and application of a single cell quantitative imaging microscopy (scQuantIM) approach used to enumerate micronuclei in various experimental and cellular contexts. We first present the critical concepts behind the automated approach, then highlight the key experimental parameters requiring optimization that will enhance the accuracy and reproducibility of the approach. Using two genotoxic compounds and an established CIN gene, we demonstrate the broad-spectrum utility of the scQuantIM approach to rapidly enumerate micronuclei in multiple cellular contexts. We end by highlighting how this approach can be easily adopted to evaluate query compounds (drugs) or candidate genes, in either direct tests or as part of screening libraries. Accordingly, this scQuantIM approach, coupled with the quantitative data it provides, will enable novel studies aimed at determining the severity and impact novel compounds have on genome stability, and will identify novel genes and pathways with implications in CIN and cancer pathogenesis. Although not shown, this automated approach can be easily adjusted and applied in various clinical contexts  to assess the induction and prevalence of micronucleus formation, which will ultimately enable novel health outcome analyses aimed at assessing the clinical utility of micronuclei as novel biomarkers of disease. In summary, the ability of the scQuantIM approach to rapidly identify and enumerate micronuclei in various cellular contexts is critical to enable the innovative studies performed and glean unprecedented insight in a diverse array of experimental and clinical settings.2.HCT116 colorectal cancer cells were purchased from American Type Culture Collection  and grown in McCoy’s 5A medium (HyClone) supplemented with 10% fetal bovine serum. FT194 and FT246 fallopian tube secretory epithelial cells were generously provided by Dr. R. Drapkin  and were grown in DMEM/F12 medium (Gibco) supplemented with 2% Ultroser G . Cell lines were authenticated based on growth, morphology and spectral karyotyping. All cells were grown at 37 °C in a humidified incubator containing 5% CO2O, respectively). Due to differences in doubling times, cells were permitted to grow for an additional four (HCT116 and FT194) or six (FT246) days, so that equivalent population doublings occurred. Cells were fixed  and counterstained  and subjected to scQuantIM as described in the Results.Dose-dependent changes in micronucleus formation were evaluated by treating cells with the established genotoxic agents, etoposide  and bleomycin . Briefly, 1000 HCT116, FT194, or FT246 cells were seeded into 96-well optical bottom plates, permitted to attach for 24 h and subsequently treated in triplicate with a two-fold serial dilution of etoposide (39 nM to 10 μM) or bleomycin (70 nM to 18 μM) and vehicle control  was applied to eliminate partial nuclei located along the image periphery. Figure panels were assembled in Photoshop CS6 .To quantify the micronuclei, nine central, non-overlapping images were acquired from each well using a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek) equipped with a 20× objective  and a 16-bit gray scale charged couple device camera. Gen5  image analysis software (Image Prime and Spot Counting software features) was used to automatically detect and enumerate the total numbers of micronuclei and primary nuclei within each image series from each experimental condition. To optimize micronucleus enumeration, a maximal mean fluorescence intensity filter was applied to each image to eliminate brightly labeled apoptotic or mitotic cells, while a size inclusion filter was employed to limit the analyses to micronuclei with a diameter of ≤1/3 the size of an average (control) nucleus. In this regard, the average nuclear area for each cell line (control conditions) were empirically determined from which the maximum 1/3 area (size) threshold was calculated. Finally, an SMC1A silenced and siControl conditions were statistically different, with a p-value < 0.05 considered significant. All descriptive statistics , dose response curves, dot plots, and MW tests were generated in Prism v7 .To identify changes in micronucleus formation in any given condition  relative to the controls, the mean number of micronuclei/nucleus was calculated by determining the total number of micronuclei/the total number of nuclei ± standard deviation (SD). Dose response curves were generated wherein a dose-dependent mean ± SD was derived from all three technical replicates (at a given dose) that is presented relative to the vehicle control. For gene silencing, the mean frequency of micronuclei for each technical replicate of each experimental or control condition was determined. The fold change in micronucleus formation is presented relative to the siControl and is determined by dividing each technical replicate value by the mean micronucleus frequency of the siControl. Mann–Whitney (MW) tests were performed to determine whether the mean of the ranks between the The appearance of micronuclei in cells has traditionally been employed as a beacon that signals an underlying genome instability event or the impact of a genotoxic agent or stress ,3,4. As Determining the frequency of micronucleus formation is critical to establish and rank the severity of a specific experimental condition , and is essential to determine the clinical utility of micronuclei as potential biomarkers of disease progression, treatment response, and patient outcomes. To enable these types of studies mandates the development of an unbiased, reproducible, and automated scQuantIM approach capable of accurately quantifying micronuclei (or the frequency of micronuclei) within a diverse array of experimental and clinical contexts. The central concept of scQuantIM is that the qualitative differences existing between various conditions can be quantified in such a manner that statistical comparisons can be used to identify significant impacts on genome stability. To enable these quantitative comparisons requires all images to be collected in an identical fashion, that is, the image exposure times must be optimized and maintained constant throughout the entire image acquisition process for a given experiment.To accurately quantify micronuclei, three key image features  need to be accurately identified through a process referred to as image segmentation . To segmx,y image boundary exclusion filter to prevent the inclusion of partial nuclei located along the image periphery; and (4) a Hoechst signal intensity threshold to prevent brightly stained apoptotic or mitotic bodies from being erroneously included in the analyses. The use of additional user-defined inclusion/exclusion criteria may also be critical to ensure accurate image quantification. Size filters including minimum and maximum areas for primary (nuclear) and secondary (cell body) masks, along with spot (micronucleus) detection, can greatly enhance image segmentation and the accuracy of data extraction. For example, the use of a defined ring width for the secondary (cell body) mask will limit the detection of spots (micronuclei) to a defined region surrounding each primary (nuclear) mask. Alternatively, cell boundaries can be readily defined through the use of membrane dyes or the use of antibodies recognizing the cell surface markers; however, these approaches can be time-consuming, costly, and may require optimization prior to experimental execution.To enhance feature recognition and data extraction, several image filters/thresholds should be optimized prior to their consistent application within a given data series. These filters include: (1) A primary (nuclear) mask size filter to ensure only intact nuclei are included in the analyses; (2) a spot (micronucleus) detection size filter set to ≤ 1/3 the size of the average nucleus to ensure only micronuclei are scored; (3) an If the image filters/thresholds are not properly optimized prior to data extraction, then a variety of image segmentation and data extraction errors may occur (detailed below). In this regard, many image features may be cell type/line dependent, and thus, will require independent optimization. For example, HCT116 cells generally have smaller nuclei and cell bodies that benefit from smaller mask sizes (particularly for the cell body mask), while FT194 and FT246 typically have larger nuclei and cell bodies that benefit from larger mask sizes. Finally, as CIN is frequently associated with large changes in chromosome complements , the ultimate thresholding parameters employed, especially for the primary (nuclear) mask, must be empirically optimized for each cell line or condition. To assist in this initial optimization step, The ability to accurately define and detect image features of specific sizes and signal intensities is of paramount importance, as inaccurate image segmentation can lead to both type I  and type II  errors. On one hand, false negative errors A may arix and y dimensions) from the image periphery (20× image) is highly effective at eliminating these types of errors. Finally, since apoptotic bodies and mitotic chromosomes often fluoresce brighter than interphase nuclei and micronuclei [Many of the common errors listed above are overcome by adjusting the thresholds and filters employed for a given condition. For example, it is critical to optimize mask (primary and secondary) and spot (micronucleus) detection to enhance feature extraction. Conceptually, inappropriately low thresholds may inadvertently cluster multiple distinct objects (nuclei or micronuclei) into a single object, or fail to recognize small or weakly stained micronuclei. Conversely, overly high thresholds may segment single objects into multiple objects/spots, or erroneously include irrelevant background signals as micronuclei. Beyond mask and spot detection thresholds, image periphery filters should also be applied to prevent partial nuclei from being recognized as micronuclei. In general, restricting the analyses to primary (nuclear) masks located at least 30 μm  these include: (1) A ‘reduce primary mask’ option designed to improve the spatial resolution between the primary nucleus and a proximal micronucleus; (2) a background subtraction option to eliminate non-specific or autofluorescent features that may adversely impact nucleus or micronucleus detection; and (3) a ‘rolling ball’ option to enhance micronucleus detection by better distinguishing non-specific background signals. Finally, As many common cell lines exhibit genome (karyotypic) instability and/or harbor high levels of micronuclei, it is critical to assess the utility of each cell line prior to evaluating the impact that drug administration or gene silencing may have on micronucleus formation. Accordingly, it is necessary to determine the frequency of micronuclei (on a per cell basis) for each line to be employed in a given study. For demonstration purposes, we carefully selected three different and karyotypically stable cell lines in which to conduct this work: HCT116, FT194, and FT246. Briefly, HCT116 is a malignant colorectal cancer cell line and has been used extensively in CIN-based studies ,29,36, w0 due to high confluency, which can adversely impact image segmentation. Cells are permitted to attach to the vessel, treated (drugs or siRNAs) and grown for up to 6-days (assay dependent), at which point cells are fixed  and counterstained (Hoechst). Cells are subjected to scQuantIM, where the image exposure time (Hoechst) is first optimized so that the maximum exposure time that produces ~80% of saturated values within typical interphase nuclei is identified. This specific exposure limit allows for dimly stained micronuclei to be more easily quantified, but also accounts for biological variation whereby some nuclei may stain brighter than others.To execute the scQuantIM approach, cells  are seeded into individual wells of a 96-well plate in triplicate such that they are ~80% confluent on the day of imaging. This ensures that cells are actively growing and are not arrested in GOnce determined, the optimal exposure time is set and maintained at constant throughout the acquisition process. In general, nine non-overlapping images (20×) are collected from the central region of each well to eliminate edge effects like cell stacking along the well periphery. At this magnification, nine images typically provide data on ≥2000 HCT116 primary nuclei from, or ≥1000 FT194/FT246 primary nuclei.Next, image segmentation is optimized and performed as detailed above and key image feature data are extracted including the total numbers of primary nuclei and micronuclei. To eliminate technical errors and ensure accurate image segmentation (including primary/secondary mask and spot detection), it is imperative to establish the accuracy of the image segmentation through manual  confirmation of a subset of randomly selected images from both the control and experimental conditions. If the image features are not accurately detected/scored, then subsequent rounds of optimization are required prior to data extraction. To account for potential differences in cell confluency resulting from distinct experimental conditions, the frequency of micronuclei in a given well is calculated and expressed as a percentage of the total number of nuclei analyzed within that well. Once collected, all data are imported into a statistical software program  where statistical tests  are conducted. In general, untreated HCT116, FT194, and FT246 have low frequencies of micronuclei ranging from 1.0% to 3.8% and 2.8%, respectively. These low frequencies (<10%) are consistent with genome (karyotypic) stability and thus identifies each as a suitable cell line model in which to conduct subsequent work.To demonstrate the scQuantIM approach, we purposefully selected two well-characterized genotoxic agents, etoposide and bleomycin , whiSMC1A, an established CIN gene [SMC1A encodes an essential member of the sister chromatid cohesion complex, which has established roles in DNA double-strand break repair [SMC1A silencing. Briefly, cells were seeded, allowed to attach and grow for 24 h prior to transfection with siRNAs targeting SMC1A or a non-targeting control (siControl). Cells were permitted to grow for an additional four  or six (FT246) days, so that equivalent population doublings occurred. Cells were subsequently fixed, counterstained, imaged, and subjected to the scQuantIM analyses, as detailed above  of micronucleus formation, which may be useful in distinguishing or ranking the level of genotoxic stress ascribed to query compounds or genes. Although the assay was performed using a Cytation 3 Cell Imaging Multi-Mode Reader and Gen5 software (BioTek), the general principles and approaches are readily extractable to virtually any fluorescent microscope  equipped with image acquisition and analytical software.Currently, the cytokinesis block micronucleus (CBMN) assay is perhaps the most common approach used for toxicological assessment. In this approach, cytochalasin B (disrupts actin filament formation and cytokinesis) is added to cells to induce the formation of binucleated cells that are subsequently scrutinized for the presence of micronuclei . While tThe scQuantIM approach is a versatile and rapid tool that will expedite biomedical research. Here, we established the utility of the scQuantIM approach through direct tests  involving both genotoxic agents and a CIN gene. A fundamental limitation of this approach is that it requires the use of adherent cell lines, or that suspension cells be adhered to substrates (detailed below). In addition, the scQuantIM approach is unable to detect micronucleus clusters that generally arise due to mitotic slippage (reviewed in ). Althoul-lysine or fibronectin. Alternatively, the frequency of micronuclei could be determined within solid cancers following the isolation of cancer cells from primary or metastatic sites, or through the interrogation of tissue microarrays housing tumor samples isolated from hundreds of patients. However, this would require additional optimization to account for the 3D nature of nuclei within tissue samples. In any case, the information gleaned from this work will enable novel studies aimed at assessing the clinical utility of micronuclei, or the frequency of micronuclei, as novel disease biomarkers. In this regard, health outcome analyses are easily envisioned that would determine the association between the frequency of micronuclei and disease progression, treatment response, drug resistance, or overall survival. Accordingly, the scQuantIM approach described herein has tremendous implications in cancer, as it will uniquely enable a myriad of future studies in both experimental and clinical settings.A fundamental benefit of the scQuantIM approach is that it can be easily optimized and applied in a variety of contexts, particularly in clinical settings. For example, this approach could be adapted and applied in hematological malignancies where cancer cells are easily isolated. Similarly, circulating tumor cells could be isolated from blood and readily assessed, and they represent a minimally invasive resource that could enable ongoing monitoring of key clinical features including disease progression or treatment response. In both of these circumstances, the suspension cells would need to be adhered to glass microscope slides or within wells prior to imaging, and is readily accomplished using standard approaches including the use of cytospins (centrifugation of cells onto slides), or slides specially coated with poly-In summary, we present a scQuantIM approach that accurately detects and determines the frequency of micronuclei within a variety of cellular texts. Importantly, this automated approach represents a significant advancement over traditional manual approaches that are frequently impacted by user bias and fatigue. Through the use of two genotoxic agents and a validated CIN gene, we demonstrated the utility of this approach in quantifying increases in micronucleus formation following treatments. We further highlight that with minimal optimization, this approach is easily scaled to assess compound or gene libraries, and will enable novel studies to identify, rank, and validate those experimental conditions inducing the most severe phenotypes. We also highlight the potential utility of this approach in various clinical settings, highlighting the potential utility of micronuclei as biomarkers of disease. As such, this scQuantIM approach has tremendous potential in a myriad of experimental and clinical contexts and will undoubtedly shed unprecedented insight into the molecular determinants driving disease development, progression, and outcomes.


Cancer is a leading cause of mortality, with its incidence only expected to rise with an increasingly aging population. Dietary interventions, primarily caloric restriction (CR), lower cellular energy metabolism and have long been utilized to slow the aging process and protect against age-related diseases, including cancer. However, due to the stringency of CR, dietary alternatives that offer the same beneficial outcomes in cancer prevention and longevity have become increasingly attractive. Periodic cycles (4 days twice a month) of low caloric intake followed by a standard ad libitum (AL) diet was previously shown to promote health-span in mice and humans and protect against primary tumorigenesis and enhanced the effects of chemotherapy. The aim of our study was to compare the tumorigenic potential of 4T1 cells, a murine model of stage IV breast cancer, in young and aged female BALB/c mice fed either periodic cycles of low caloric diets versus chronic 20% CR. Compared to AL controls, we found a significant delay in primary tumor growth in mice regardless of diet composition by the 4:10 cycles of very low caloric intake. However, unlike in CR, CR-alternative diets were not protective against lung metastases in the absence of chemotherapy. Our study sheds light into the underlying differences of calorie-based interventions in the absence of chemotherapy.


Uncovering the intricacies of the gut microbiome and how it interacts with the host immune system has opened up pathways in the search for the treatment of disease conditions. Alcohol-associated liver disease is a major cause of death worldwide. Research has shed light on the breakdown of the protective gut barriers, translocation of gut microbes to the liver and inflammatory immune response to microbes all contributing to alcohol-associated liver disease. This knowledge has opened up avenues for alternative therapies to alleviate alcohol-associated liver disease based on the interaction of the commensal gut microbiome as a key player in the regulation of the immune response. This review describes the relevance of the intestinal immune system, the gut microbiota, and specialized and non-specialized intestinal cells in the regulation of intestinal homeostasis. It also reflects how these components are altered during alcohol-associated liver disease and discusses new approaches for potential future therapies in alcohol-associated liver disease. The significance and intricacy of the intestinal immune system cannot be underscored. Specialized cells within the intestine working with lamina propria immune cells are vital for protection against invading pathogens. The gut microbiota, comprising many different bacterial species is a major influencer on the intestinal immune system and is a necessary cog for proper intestinal immune function. Research studies continue to uncover new knowledge about the functionality of specialized cells within the intestine and how they interact with the gut microbiome and the immune system. This has allowed for further understanding to search for potential targets against conditions that are exacerbated due to dysfunctional intestinal homeostasis comprising a disrupted intestinal immune system and an altered intestinal microbiota such as the different stages of alcohol-associated liver disease (ALD). These different stages include steatosis, steatohepatitis, fibrosis, alcoholic hepatitis (AH), alcohol-associated cirrhosis (AC), and hepatocellular carcinoma. Steatosis in ALD is marked by the presence of lipids accumulating in the hepatocytes within the liver due to activation of lipogenic pathways, inhibition of fatty acid oxidation, alteration of very-low-density lipoprotein (VLDL) secretion from the liver, and alteration of triglycerides metabolism which accumulates in the form of lipid droplets The physical barriers of the intestine offer several lines of defense from potentially harmful pathogens as well as luminal contents and commensal bacteria from infiltrating the body under normal healthy conditions. Each barrier offers a distinctive structure that collectively functions to maintain a balance for both a healthy host as well as a healthy gut microbiome known as intestinal homeostasis. The intestinal epithelium, the innermost layer of the mucosa, is one of three major physical barriers that include specialized cells such as enterocytes, goblet cells, intestinal microfold (M) cells, enteroendocrine cells, Tuft cells, stem cells, and Paneth cells which are located in the crypts.-/- mice exacerbates ethanol-induced liver injury, steatosis, inflammation, and induces expansion of intestinal Enterococcus faecalis  that produce a pore-forming toxin called cytolysin 25. These results suggest that modulation of intestinal α1-2-fucosylation could be used as a therapeutic approach to prevent ALDs.Enterocytes are present in large numbers in the small intestine but are also found in the large intestine, providing surveillance for protection Intestinal stem cells (ISCs) are an important structural and functional component of the intestinal immune system due to the ability to replenish themselves along with replenishing intestinal epithelial cells which can also differentiate to other specialized intestinal cell types Paneth cells offer an imposing barrier to microbial products trying to pass the intestinal epithelial layer. Located predominately in the crypts of the small intestine adjacent to ISC in the small intestine, some Paneth cells can also be found in the proximal portion of the large intestine; however, during disease conditions, Paneth cells have been found in the distal portion of the large intestine Goblet cells also distribute AMPs such as resistin-like molecule ß (RELM-ß) and trefoil factor (TFF). RELM-ß acts to promote inflammation and immune response to invading pathogens through its ability to increase interferon-gamma (IFN-γ) expression in CD4 T cells M cells are associated with Peyer's patches of the small intestine, isolated lymphoid follicles, and colonic patches Enteroendocrine cells (EECs) are a very small part of the epithelium, but their function is far-reaching within areas such as the small intestine, colon, and gut microbiota. In response to nutrients, EECs secrete peptide hormones like somatostatin, motilin, cholecystokinin, neurotensin, vasoactive intestinal peptide, enteroglucagon, gastric inhibitory peptide (GIP), glucagon-like peptide-1, peptide YY, oxyntomodulin and glucagon-like peptide-2, histamine, and secretin to control gastrointestinal function and can also release cytokines in response to microbial metabolites and pathogenic molecules Tuft cells, similar to EECs constitute a very small number of cells within the epithelium and are also found throughout the colon and small intestine; however, their relative number is higher is the distal portion of the small intestine Figure  The impact of alcohol abuse on the colon is illustrated in Figure  Major cells in the intestinal epithelium along with functions and associations with ALD are summarized in Table The overall structure of the small intestine with changes on a cellular and molecular level in the context of ALD is illustrated in The lamina propria is another major physical barrier underneath of the intestinal epithelial cells consisting of noncellular connective tissue elements (collagen and elastin), myofibroblasts, and diverse immune cells such as CD4+ T lymphocytes, NKT lymphocytes, IgA plasma cells, B lymphocytes, innate lymphoid cells (ILCs), macrophages, mast cells, and DCs high, major histocompatibility class (MHC)IIhigh+ CD11b+ DCs causing secretion of IL-6 which, in turn, induce T helper cells (Th) 17 differentiation 3CR1- DCs produce retinoic acid and TGF-ß to stop any unnecessary inflammation LP-DCs within the lamina propria are classified based on their expression. DCs are defined as CD11cThe CD103+ CD11b- subset of LP-DCs is similar to CD8+ DCs in lymphoid organs 3CR1+ DCs promote inflammation through IL-6 and tumor necrosis factor-alpha (TNFα) + CD11b- and CD103- CD11b+ cDCs can drive Th1 differentiation rather than the CD103+ CD11b+ DCs 3CR1+ LP DCs can extend their dendrites to receive and monitor antigens from the lumen 3CR1 also allows for proper transportation of these antigens to other DCs, as mice without CX3CR1 have reduced capability of pathogen uptake and transportation from DCs to areas such as the mesenteric lymph nodes The CD103- CD11b+ DCs subset is closely related to macrophages, is always found in the lamina propria, and activate nearby immune cells by releasing inflammatory cytokines Uncovering the structure and function of LP-DCs has offered insight into one of the crucial components of the intestinal immune systems adaptive response. Antigen-specific T cell responses and accessory cell function of myeloid DCs is greatly strained in human blood by alcohol drinking ILCs are also present in the lamina propria and include ILC1s, ILC2s, and ILC3s Intestinal macrophages throughout the lamina propria have similarities in the membrane gene expression profile to macrophages in other tissues as well as monocytes, however, several key differences differentiate intestinal macrophages from macrophages of other tissues CD4+ T cells are another important component of immune cells within the lamina propria. Th1 cells, a subtype of CD4+ T cells are noted for controlling immune responses responsible for killing intracellular parasites and perpetuating autoimmune responses through inflammatory cell activation via IFN-γ and for T and B cell growth through IL-2 interaction with T and B cells Another type of CD4+ T cell, Th17, plays a key role in inflammation. Th17 cells, along with Paneth cells in the intestine produce IL-17 which was previously discussed as a pro-inflammatory cytokine IgA was previously discussed due to its importance in controlling bacteria after being transported to the lumen with the assistance of pIgR. IgA production in the intestine is markedly higher than other areas in the body, with two subclasses IgA1 and IgA2 existing in humans Table Mucosa-associated invariant T (MAIT) cells also play an important role in immune response as once activated in response to bacterial infection, MAIT cells secrete cytokines such as IFN-γ, TNFα, and IL-17 to destroy bacteria-infected cells While these physical barriers and their components offer critical defense from pathogen invasion of the gut, they must also work in harmony with the microbiome for optimal health. The influence of the gut microbiome in training the immune system is a hallmark of a healthy intestinal immune system.The presence of a diverse set of microbes in the gut creates a mutualistic relationship with the host allowing proper gut and immune function. Intestinal microbiota gain a habitable environment and nutrients in exchange for their functions within the gut. These functions include nutrient metabolism, xenobiotic and drug metabolism, protection against pathogens, maintenance of the integrity of the mucosal barrier, and regulation at the molecular, genetic, and cellular levels of the intestinal environment which impacts the immune system The cruciality of the intestinal microbiome has long been accepted for health and proper immune response. This has been illustrated in many studies done on germ-free mice lacking microbiota. Germ-free mice have significant deficiencies in the immune system. Specifically, the lack of gut microbiome is associated with fewer intraepithelial lymphocytes, less IgA secreting plasma in the lamina propria, smaller Peyer's patches, fewer Tregs, and markedly reduced mRNA expression of AMPs from the Paneth cells such as Angiogenin-4 A common method of treatment in preclinical models of ethanol-induced liver disease is the use of antibiotics. The main purpose of antibiotics is to combat bacterial overgrowth and avoid translocating bacteria. The differences in antibiotics and the history of their success have led to the many different viewpoints about their current use and effectiveness in treating ALD. Antibiotics have direct impacts on the immune system within the gut due to changes in the GAPs in the colon. Colonic GAPs are typically closed to prevent bacterial translocation; however, following the administration of antibiotics, GAPs can be opened leading to a higher level of inflammation by induction of CX3CR1+ cells after recognizing higher levels of microbes and antigens A major function of gut microbes is converting dietary carbohydrates into organic acids. Lactate and SCFAs such as acetate, propionate, and butyrate can all be end-products created by the gut microbes The gut microbiota influences the expression of neutrophil chemokines through the promotion of naïve CD8+ to CD4+ T cells. In the mucosa of the small intestine, specific microbiota induces the differentiation of T-helper cells and secretion of IL-22 along with IL-17 which specifically induces the expression of neutrophil chemokines Gut microbiota can also influence DCs through Fut2 mediated α1,2-fucosylation. Certain bacteria can produce energy from fucose metabolism. This fucosylation has been found in the intestinal epithelial cells to increase during acute inflammation which causes the DCs to produce IL-23 T. muris infection invading pathogens to fight the infection Clostridia class modulates the levels of RA in the gut by reducing the expression of retinal dehydrogenase 7 (Rdh7) in intestinal epithelial cells and consequently disrupting the RA signaling immune cells, reducing the levels of IL-22 and altering antimicrobial responses which in turn protects the commensal microbiota Gut microbes can also provide influential benefits on gene-level expression in the intestine. An example of this is the regulation of the immune response via regulation of the retinoic acid receptor alpha (RARα) Clostridia production of SCFAs has been related to the induction of TGF-β and induction of Treg cells Bacteroides fragilis is required for the differentiation of functional Tregs producing IL-10 expression and prevent colitis Bifidobacteria has also been involved in Th17 differentiation along with regulating the balance of Th1 and Th2 responses and stimulation of CD8+ cells Segmented filamentous bacteria of the microbiota lead to the induction of Th17 cells through the stimulation of reactive oxygen species (ROS) enhancing IL-1β secretion, IL-6, and TGF-β The microbiota also can perform quorum sensing in the gut which regulates gene expression due to microbe cell-population density fluctuations. Microbiota creates and releases chemical signals called autoinducers that operate on a positive feedback loop with cell density During ALD, disruptions occur within the microbiome that accelerates and exploit damage previously mentioned in the intestinal barriers. The most common impacts involve bacterial overgrowth within the intestine along with compositional changes Candida albicans  causing a decrease in intestinal fungal diversity C. albicans were associated with progression of ethanol-induced liver disease in mice C. albicans and the resulting pro-inflammatory and immune response contributes to increased intestinal permeability further exacerbating liver damage and was associated with increased mortality in patients with AH, and increased fecal levels of candidalysin which were also associated with higher mortality The collective intestinal fungi that act symbiotically with the host known as the intestinal mycobiome contributes to the immune system and also undergo major impacts in disease conditions Figure 3 with the structure of the liver under healthy conditions and the impacts of ALD changes on the molecular structure. Dendritic cells in the intestine can sample and transport some bacteria to the mesenteric lymph nodes through afferent lymphatics, allowing a more localized immune response CCL21The liver immune system has limited interaction with the microbiome under normal healthy conditions. With the main connection points between the liver and microbiome being only through bile ducts and the portal vein, only select substances can get through the intestinal barrier and move into the liver. This is illustrated in E. faecalis, which is linked to a higher incidence of liver damage and mortality due to the release of the pore-forming toxin cytolysin E. faecalis overgrowth in mice with ethanol-induced liver disease fed a long-term ethanol diet and it is a risk factor for developing ALD in alcohol-dependent patients E. faecalis to the liver was associated with an induction of IL1B secretion by hepatic macrophages and Kupffer cells which further contribute to the disease Disturbances within the host can push the liver immune system to act further in regulating the microbiome to protect itself from a compromised gut barrier. Dysbiosis in patients with AH has been related to overgrowth of The liver can also have an impact on the Th17 and Treg balance in the lamina propria through derivates of the bile acid metabolite lithocholic acid (LCA). 3-oxoLCA can bind to RORγT to prevent Th17 cell proliferation, whereas isoalloLCA produces mitochondrial ROS leading to an uptick in FOXP3 expression which is a master regulator of the differentiation of Tregs Under healthy conditions, the intestinal immune system finds a balance between proinflammatory response to pathogens and preventing the pro-inflammatory responses from causing damage to vital organs. As previously mentioned, this is regulated by the gut microbiota, crucial for the maintenance of intestinal homeostasis. However, under certain conditions, this balance can become skewed, leading to a compromised intestinal immune system. Chronic alcohol abuse causes consequential impacts on the immune system, liver, intestine, and gut microbiota. Excessive alcohol use increases the permeability of the intestine, leads to inflammation, and causes tight junction dysfunction. Intestinal bacterial overgrowth and dysbiosis is also a common effect of alcohol abuse which can lead to high levels of bacterial translocation to the liver, causing irreparable damage Therapeutic approaches targeting ILC3s in mice have become an emerging area of research over recent years. Ethanol-induced liver disease is typically associated with impairment of ILC3s in the gut from properly producing IL-22, thus fueling bacterial translocation The cytokine osteopontin has shown controversial results in animal studies in ethanol-induced liver disease Th17 offers an intriguing option as an alternative therapy approach as their presence within the immune system fights invading pathogens through promoting inflammatory responses Lactobacillus rhamnosus GG supernatant (LGG-s) therapy was also found to increase mRNA expression of tight junction proteins and reducing intestinal permeability in a mouse model of chronic-binge alcohol feeding. This therapy led to a decrease of Escherichia coli in the liver and an overall balance restored of Tregs, Th17s, and IL-17 Lactobacillus rhamnosus GG supplementation will improve their condition and intestinal complications. While some alternative therapies seek to address ALD treatment by significantly reducing bacteria levels overall within the host, prebiotic and probiotic therapies aim to alleviate gut microbe dysbiosis. Prebiotic therapies aim to restore commensal gut microbes through the introduction of dietary supplements. Studies using fructooligosaccharides (FOS) prebiotics in mice after the intragastric feeding model of continuous ethanol infusion showed a lessening of bacterial overgrowth and restoration of AMP levels such as REG3γ Bacteroides and protected from ethanol-induced liver disease Lactobacillus species GG in rats, administration of Akkermansia muciniphila in mice, short-term oral supplementation with Bifidobacterium bifidum in preclinical studies have shown improvement of gut microbe homeostasis and reducing liver damage Lactobacillus plantarum 8PA3 to AH patients, and Lactobacillus subtilis/Streptococcus faecium supplementation in AH patients also have shown improvement of gut microbe homeostasis, less liver damage, and balancing immune response and cytokine production Lactobacillus rhamnosus R0011 and acidophilus R0052 in mice with ethanol-induced liver disease has also been linked to a reduction in TNFα, IL-1β TLR-4 in the liver Fecal microbiota transplantation (FMT) is a growing interest as an alternative therapy to treat conditions such as AH to improve the long-term mortality rate of affected patients. Initial findings from a limited number of studies performing FMT on patients with severe AH found higher survival rates and less toxin buildup in the bloodstream in patients receiving the transplantation versus other patients that received other treatments such as corticosteroids and pentoxifylline C. albicans have been related to the use of certain antibiotics, among them amoxicillin The use of antibiotics to control ethanol-induced bacterial overgrowth and prevent ethanol-induced liver disease in rodent models has been previously demonstrated E. faecalis contributes to liver damage after translocation. The presence of cytolysin in stools correlates with mortality in patients with AH. Mice gavaged with cytolytic E. faecalis exhibited increased levels of cytolysin in the liver and developed a more severe ethanol-induced liver injury. Cytolysin forms pores E. faecalis were found to significantly diminish cytolysin and the impacts of AH such as liver damage and hepatic cell death in mice As previously mentioned, during ALD overgrowth of certain bacteria such a cytolytic The connection of the bile ducts between the liver and intestine offers a potential option to alternative therapy approaches. Restoring the homeostasis of bile acids is a necessary step to preventing further liver injury in patients with ALDs. The potential key target to restoring homeostasis within bile acids is FXR, with a study reporting positive results of reduced liver injury in rats C. albicans clearance and Tregs inductions in mice treated with an antibiotic Clostridia are also related to the induction of TGF-β and Treg cells Nutrition-based therapies are another targeted alternative therapeutic approach to ALD. Given the influence on diet and microbiota derived-metabolites such a SCFAs, MCFAs, and LCFAs on the host, these potential therapies could offer promising results for attenuating ALD. Supplementation of SCFAs, MCFAs, or LCFAs in both mice and rats, have all been linked to reduce liver damage and improve the intestinal gut barrier Other novel therapies have emerged with promising results towards potential ways of alleviating ALD. Purified hyperimmune bovine colostrum (IMM-124E) therapy has been used in phase 3 human clinical trial (NCT02473341) in severe AH patients to reduce bacterial translocation to the liver as it contains IgG anti-LPS antibodies.Therapies using agonists and antagonists have also shown promise and are currently moving through human trial phases. Anakinra, an IL-1 receptor antagonist is currently being tested in phase 2 clinical trial (NCT04072822) in human subjects with AH. If successful, anakinra will be able to reduce TLR4-induced inflammation which depends on signaling from IL-1. Inhibition of IL-1R signally is related with reducing expression of IL-17A by Th17 cells in mice and humans Antioxidant therapy clinical trial results in AH patients using such antioxidants as N-acetylcysteine (NAC) and metadoxine have shown higher survival rates short term, however, the long-term effects and overall efficacy require further study Human clinical trials using regenerative methods such as the previously mentioned IL-22 therapies are also offering positive initial outcomes While many recent studies show promise in advancing treatment towards ALD, further research is needed to combat the disease. Along with targeting ILC3s, and Th17 cells functionality for alternative therapeutic approaches, the LP-associated immune system also offers other potential targets for effective therapy. Continuing research into alternative therapies to these targets offers a promising outlook to both better understanding and treating diseases impacting gut and liver health.