Biologic sex and gonadal hormones matter in human aging and diseases of aging such as Alzheimer’s – and the importance of studying their influences relates directly to human health. The goal of this article is to review the literature to date on sex and hormones in mouse models of Alzheimer’s disease (AD) with an exclusive focus on interpreting the relevance of findings to the human condition. To this end, we highlight advances in AD and in sex and hormone biology, discuss what these advances mean for merging the two fields, review the current mouse model literature, raise major unresolved questions, and offer a research framework that incorporates human reproductive aging for future studies aimed at translational discoveries in this important area. Unraveling human relevant pathways in sex and hormone-based biology may ultimately pave the way to novel and urgently needed treatments for AD and other neurodegenerative diseases. Biologic sex and gonadal hormones exert profound effects on brain function – and we are only beginning to appreciate the complexities of their actions in Alzheimer’s disease (AD) from studies of humans and mouse models. In 2010, the Institute of Medicine advocated for expansion of neuroscience research to understand sex differences in the susceptibility and progression of key neurodegenerative conditions such as AD.-45.41-45The prevalence of AD, or the total number of cases in a population at a given time, is higher in women compared to men in multiple populations. This isThe incidence of AD, a measure of the risk of developing disease over time, is on the whole similar between men and women. Many epidemiologic studies show increased risk for the development of AD in women compared to men in specific populations, and many do not. A meta-analysis of studies worldwide shows similar risks between men and women that increase dramatically with age, and may increase disproportionately in women after the age of 80 yrs. In paraSexual dimorphism in the progression of AD is a major and meaningful epidemiologic measure that has received very little attention compared to incidence and prevalence. Men are more vulnerable to an aggressive disease course compared to women. This underappreciated sex difference is supported by several studies. First, men progress to death faster than women in both early--43 and lDisease course may be the human epidemiologic factor most relevant to our study of AD in animal models. Since most mouse models of AD involve the transgenic expression of mutated APP with or without mutated tau, outcome measures are focused squarely on the manifestation or disease course rather than the risk or prevalence of disease. Specifically, mouse models enable study of how a manipulation changes the manifestation or severity of AD–related disease measures such as pathology, biochemistry, cognition/behavior, synaptic/network plasticity, or survival.Reproductive aging is a fundamental aspect of the aging process and is accompanied by dramatic decreases in certain gonadal steroid levels in human-60, but d after menopause, 2) by subcutaneous routes which largely bypass the liver, 3) in lower doses, 4) or in other formulations since conjugated equine estrogens without medroxyprogesterone acetate (Premarin) or with it (Prempro) may have differing biological activities from hormones typically used in animal studies (estradiol and progesterone). While future studies may shed light on these complex possibilities, the clinical data to date show that HRT, in its current forms, can be deleterious to cognitive outcomes.The results of randomized, controlled clinical trials, including the Women’s Health Initiative (WHI),66, founBecause AD pathophysiology begins and progresses years before its clinical manifestation,75, it iAndropause is the male correlate of the female menopause, characterized by a gradual but steady decline of certain circulating gonadal steroids. A modest decrease in total testosterone accompanied by an increase in steroid hormone binding globulin (SHBG) results in a major decrease of bioavailable testosterone, averaging 1-2% per year beginning in the third or fourth decade of life,60,76-79Low levels of biologically active testosterone in the aging male may be deleterious to the brain. Despite new findings that SHBG might facilitate steroid delivery to target tissues , botAn understanding of human AD epidemiology and reproductive aging, combined with intelligent research strategies to study effects of sex and hormones, set the stage to simulate the human condition using mouse models of AD. Both mice and humans undergo reproductive aging and subsequent decline in fertility. However two main differences in reproductive aging exists between the species. First, gonadal steroid levels decline in males and females during human aging-60 but nSimulation of a prominent aspect of human reproductive aging in mice can be accomplished by gonadal steroid depletion through gonadectomy or other methods. Since ASince aging is the primary risk factor for AD and development of AD is restricted to the aging brain, the study of sex and hormones in light of reproductive aging, steroid depletion, and hormone replacement represents a translational framework for human-relevant research in mice. With this framework in mind, we propose three research areas, offer optimal research approaches using mice, and consider caveats. Each human-relevant question posed is followed by an optimal research approach using mice.Gonadal hormone depletion. The following questions focus on sex differences  model .Reproductive aging. The following questions focus on effects of reproductive aging.2. a. How does reproductive aging alter vulnerability to AD in females Figure ? Compareb. How does reproductive aging alter vulnerability to AD in males Figure ? CompareHormone replacement. The following questions focus on effects of HRT.3. a. How does hormone replacement in gonadal steroid-depleted females alter vulnerability to AD? Compare vehicle vs hormone treatment in hormone-depleted (gonadectomized) female mice Figure . HRT dosb. How does hormone replacement in gonadal steroid-depleted males alter vulnerability to AD? Compare vehicle vs hormone treatment in hormone-depleted (gonadectomized) male mice Figure .All three areas of research outlined above incorporate the strategy of depleting gonadal hormones though gonadectomy to mimic an aspect of human reproductive aging. This manipulation successfully depletes levels of gonadal hormones, but a few points regarding its limitations and optimal applications deserve consideration. First, gonadectomy decreases hormone levels in a subacute, rather than a slow and progressive manner, as occurs in human reproductive aging. However, whether the rate of hormone decline influences AD-related outcomes is unknown. Second, gonadectomy performed during middle-age (10–15 months in mice) may more closely model reproductive aging, since central nervous system changes that underlie reproductive aging have already occurred; but restricting studies to aging mice is not always practical or economically feasible. Finally, gonadal hormone depletion through natural or surgical means in humans,93-96 orAlternatives to gonadectomy in female mice include genetic and chemical models of reproductive aging. The follitropin receptor knockout (FORKO) mouse is a genetic model that leads to chronic estrogen deficiency early in development,100. An Given current limitations of genetic and toxin models of reproductive aging in studying sex differences in mouse models of AD, we advocate gonadectomy as a relevant and reliable manipulation.If the comparison between gonadectomized males and females reveals a sex difference, further mechanistic dissection can be achieved by determining whether sex chromosomes or organizational effects of hormones (long-lasting effects of hormones that persist in their absence) explain the difference Figure . One strThe necessity of dissecting molecular mechanisms and identifying potential therapies to improve the human condition requires using animal models of disease. A model is only useful, however, if the discoveries generated truly inform us about human disease and potential treatments. Thus, attention to aspects of the AD mouse model that recapitulate clinical manifestations of human AD, such as cognition and its underlying substrates, is essential.While AD is a strictly human disease, the genThe development of new mouse models of AD offers the opportunity to optimize paradigms and tackle unanswered questions. For example, models that express hAPP via targeted insertion and from the gene’s own promoter might produce more physiological results. Yet, such “knock-in” models of AD have shown very mild, if any, tractable AD-like disease progression or dysfunction-108 to dDespite the fact that current mouse models of AD are imperfect representations of the human condition, key features of AD are indeed preserved,102 and The efficacies of therapies, and their relevance to patients and families, depend on whether they can prevent or reverse cognitive decline, the primary clinical manifestation of AD. Thus, its human relevance, combined with the incredible homology between rodent and human memory systems, make coSex-based differences in AD exist and comparisons between males and females that model AD are invaluable. Understanding sexual dimorphisms can inform us about what protects one sex or makes the other more vulnerable. Yet, studies that have compared AD-related measures in male versus female mice have reported quite varying results (cognitive differences reviewed in Table1. If a sex difference exists (or not), it may result from differential activational effects of endogenous gonadal steroids in the male versus the female brain. That is, actions of androgen in the male brain may differ from actions of estrogens or progesterone in the female brain. It is also possible that the sex difference may exist even in the absence of gonadal hormones.2. Androgen levels remain constant in males while estrogen and progesterone levels fluctuate in females across the estrous cycle. Stages of the estrous cycle influence brain functions of females in diametrically opposing ways. Since f3. The ability to detect, repeat, or precisely interpret a sex difference in mouse models of AD can be obscured by points one and two.In light of these complexities, we review the current literature on sex differences in mouse models of AD – and stress that while comparing gonadally intact males and females is a valuable first step, studies that incorporate human-relevant manipulations to model reproductive aging via gonadal steroid depletion in both sexes are urgently needed.For the reasons outlined above, it may not be surprising that reports on sex differences between intact male mice compared to intact female mice in AD-relevant cognitive and behavioral tasks vary considerably, even within the same AD model Table. At baseIt is curious, however, that intact females are impaired slightly more often than intact males when considering all studies together. While these studies have concluded that female mice are more vulnerable to AD-related deficits, we offer two additional interpretations: in mouse models of AD, 1) female gonadal hormones are more deleterious in female brains than male hormones in male brains or 2) male gonadal hormones are more beneficial in male brains than female hormones in female brains. It will be necessary to further dissect these complex possibilities, beginning by simply depleting gonadal steroids and then comparing males to females. Gonadal steroid depletion in male and female mice will enable us to determine what sex differences exists in a hormonal milieu that is more comparable to the human condition Figure .Like cognitive and behavioral data on intact males and females that model AD, there is considerable variation in reports of sex differences in baseline Aβ levels and amyloid plaque deposition: females had higher levels than males,123-132,As we learn more about the pathophysiology underlying AD, we are coming to appreciate that moderate differences between plaque load or neurofibrillary tangle abundance bear less biologic relevance to cognition than once thought. Nonetheless, levels of plaques and tangles, combined with more toxic assemblies of Aβ and tau should be further assessed in gonadal steroid depleted mice.Many studies have examined molecular and biochemical differences between gonadally intact male and female mice that model AD. Among reports are measures of: hAPP processing enzymes and products,134,137,Mice that model AD suffer premature mortality,144-150 While comparing intact male and female mice that model AD is an initial step, we propose that the next step requires modeling human reproductive aging  in mice. We strongly advocate for this research strategy with the following scientific rationales:1. AD is a disease of aging that, in humans, develops in a gonadal steroid-depleted state; thus, depleting gonadal steroids in mice is a human-relevant manipulation.2. Endogenous gonadal steroids profoundly impact cognition and brain function (reviewed in-156); thIn the presence of endogenous gonadal steroids, differences between males and females may result from differential actions of androgens in males, or of estrogen/progesterone in females. Further, hormonal effects in females will vary according to the stage in estrous cycle-113. Thuc  and progesterone effects in female mice since these are the principle, systemic gonadal hormones decreased following menopause; but other steroids, including neurosteroids , maWhile estradiol facilitates synaptic plasticity and several forms of hippocampal-dependent learning and memory in the adult and aging rodent brain, (reviewed in-153), itIn contrast to the watermaze studies, studies in the 3xTg mouse model show impaired working memory in the Y-maze following gonadectomy,163 – anA large body of evidence, with notable exceptions,160,170,Consistent with estradiol-mediated decreases in Aβ levels, estradiol treatment suppresses the Aβ producing β-secretase 1 (BACE), and incStudies have reported survival data in female AD-model mice following gonadal hormone depletion or hormone replacement – and consistently show that estrogens are toxic. Estradiol treatment increased mortality in both intact and hormone-depleted adult female APPswe mice. InteresEstrogens benefit cognition in the normal, aging brain but not in the diseased, AD brain (or in the brain at risk for AD) , ju Figure . In suppWhether androgens, or other forms of hormone replacement in men with AD are beneficial has yet to be determined in rigorous, double-blinded, prospective, and placebo-controlled clinical trials – but clinical observations,173-176 Gonadal steroid depletion, via gonadectomy of male mice that model AD, worsened cognitive impairment. The impIn parallel with protecting against cognitive deficits, androgens also decreased levels of pathogenic proteins and pathology related to AD. While gonadectomy increased Aβ levels and plaque deposition in male AD-model mice,179, elein vitro, cell culture systems, they are not reviewed here.Consistent with androgen-mediated decreases in Aβ levels, elevating brain and serum testosterone via aromatase knockdown, modified important enzymes , and IDE) leading to decreased Aβ production. These dNo studies to date have examined if androgens, or the lack of, alter survival in male mice that model AD.Though the number of studies is limited, they collectively show a deleterious effect of gonadectomy and a protective action of androgens in male mice that model AD. The protective effect existed in more than one AD model, was achieved using genetic or pharmacologic strategies, and modified a directly relevant measure to clinical AD – cognition. Several important questions arise from these studies. How do androgens modulate Aβ-targeting enzymes and can they protect against cognitive deficits in an Aβ-independent manner? Do androgens signal through receptor-dependent or alternate pathways to achieve protection? Can androgen signaling improve AD-related cognitive deficits in both males and females? If so, can we modify androgen signaling to improve cognition without eliciting masculinizing effects? These and other lines of androgen-focused investigation may hold promise for treating the human condition.Given our evolving tools and knowledge base, we put forth the following suggestions for future research in sex- and hormone-based studies in AD mouse models. First, since gonadal steroid depletion is an inextricable aspect of human, but not mouse, aging, it should be vigorously incorporated into our study of AD and other disease models of aging. Gonadal steroid depletion through gonadectomy is a human-relevant manipulation that models the hormone environment in which AD develops and enables clearer approaches to comparing males and females, understanding reproductive aging, and testing effects of hormone replacement. Second, when using mouse models of AD, measures that relate closely to clinical AD, and thus directly reflect human-relevant outcomes, should be included in studies. This means expanding the focus of mouse model research to include cognition, behavior, and measures closely correlated with these, such as synaptic and network function. Third, because clear sex differences exist in human AD epidemiology, specifically in progression to death, it is important to look at survival and related measures in our mouse models. Finally, in light of past, present, and future clinical trials, we should continue lines of research studying how the loss and replacement of hormones affect each sex in models of AD.As we learn more about AD itself, how it manifests differently in men compared to women, and how hormones modify its risk, we must simultaneously recast our questions to reflect high-yield and human-relevant research strategies – with the goal of achieving biomedical discoveries that improve the human condition. This is not an easy task, as it requires merging the complex, emerging field of sex and hormone biology with the study of a complex, devastating disease of aging, AD. We believe this task is not insurmountable, and if taken on mindfully, may reveal novel targets for defeating the disease.aSex is biological classification of living beings as male or female and is used in this review to describe both humans and mice. Gender, a term that is only appropriate when applied to humans, is a cultural expression of sex, shaped by environment and experience.bThis review of sex differences in the epidemiology of AD includes large populations in which the effect of ApoE4, a genetic risk factor that increases AD risk in women, is not specifically examined.cEstradiol is the most biologically active form of estrogen that circulates in high levels in the body prior to the menopause in women. Most studies in animal models use estradiol for hormone treatment. In contrast, clinical studies in humans have used hormone replacement paradigms that include other estrogenic steroids.dDuring the publication process of our review, an observational study showing an association between hormone therapy use started within 5 years of menopause and decreased AD risk was released . Though the findings are highly intriguing in light of the critical window hypothesis, caution must be exercised when interpreting observational studies due to their inherent limitations. Ultimately this study approach can only show associations and not causal links. Whether hormone therapy reduces the risk of AD when given during a critical window will need to be determined through the gold-standard of clinical research – randomized, double-blind, placebo-controlled trials.The authors declares that they have no competing interest.DD, LB and KW reviewed the literature. DD and LB wrote the review. All authors read and approved the final manuscript.Dena B Dubal, MD, PhD is an Assistant Professor of Neurology and Director of the Laboratory for Neuroscience and Aging Research. She holds the David Coulter Endowed Chair in Aging and Neurodegeneration at UCSF. She is a physician-scientist with a clinical and research focus on intersections between aging and neurodegeneration.Lauren Broestl is a Research Associate in the department of Neurology at UCSF.Kurtresha Worden is a Research Associate in the department of Neurology at UCSF.


Despite significant reductions in acute-rejection rates with the introduction of calcineurin inhibitor (CNI)-based immunosuppressive therapy, improvements in long-term graft survival in renal transplantation have been mixed. Improving long-term graft survival continues to present a major challenge in the management of kidney-transplant patients. CNIs are a key component of immunosuppressive therapy, and chronic CNI toxicity has been widely thought to be a major factor in late graft failure. However, recent studies examining the causes of late graft failure in detail have challenged this view, highlighting the importance of antibody-mediated rejection and other factors. In addition, the diagnosis of CNI nephrotoxicity represents a challenge to clinicians, with the potential for over-diagnosis and an inappropriate reduction in immunosuppressive therapy. When graft function is deteriorating, accurately determining the cause of the kidney disease is essential for effective long-term management of the patient. Diagnosis requires a thorough clinical investigation, and in the majority of cases a specific cause can be identified. Despite significant reductions in acute-rejection rates seen with the introduction of tacrolimus, mycophenolate mofetil (MMF) and interleukin-2 receptor antibody induction in the 1990s, improvements in long-term graft survival in renal transplantation have been mixed. In Europe, data from the Collaborative Transplant Study show substantial improvements in graft survival half-life , and eduEven with the gains seen in Europe, long-term graft loss is still a significant problem in renal transplantation. Improving long-term graft survival represents a major challenge in the management of kidney-transplant patients, as a wide range of factors can contribute to transplant loss .Providing immunosuppressive therapy that is effective for the individual patient is an important part of transplant management. Calcineurin inhibitors (CNIs) are a key component of immunosuppressive regimens, yet chronic CNI toxicity has also been widely thought to be a major factor in late graft failure. Recent studies, however, have challenged this view. Findings from the Long-term Deterioration of Kidney Allograft Function (DeKAF) and the Genome Canada studies showed that antibody-mediated injury was a predictor of graft loss, whereas a biopsy diagnosis of ‘CNI nephrotoxicity’ was not –7. AccurA large number of nonimmunological and immunological factors can potentially contribute to deteriorating function and graft loss following renal transplantation ,9. Both Risk-factor analyses of graft failure are after-the-fact evaluations performed in a defined cohort of patients. Although such analyses are valuable in identifying those at risk of graft failure, this is quite different from defining the cause of graft function deterioration in an individual patient. In these cases, the first, and often late, sign of a negative development is an increase in serum creatinine. The first, but sometimes insufficient, step in determining causality is to perform a graft biopsy.de novo or recurrent), chronic hypertension and obstruction can all be identified from biopsies. Chronic CNI nephrotoxicity may be suggested by a number of histological lesions, although these are not specific, so diagnosis relies on elimination of other potential causes [Transplant biopsies may be helpful in determining the potential causes of deteriorating graft function . Early bl causes –15. ChroDetermining the cause of late graft failure presents a considerable challenge, given the interactions between the factors that can affect graft function, and the difficulty of making definitive diagnoses from graft biopsies. Consequently, many cases of late graft failure are classified using the nonspecific term ‘interstitial fibrosis and tubular atrophy (IF/TA), not otherwise specified’, previously known as chronic allograft nephropathy (CAN) . The recThe slow decline in renal function coupled with absence of evidence of acute rejection has led to the view that nonimmunological factors, such as CNI nephrotoxicity, are the most important causes in late graft failure. However, given concerns that the actual causes of late kidney allograft dysfunction and failure may be being overlooked, a number of recent studies set out to examine these causes in detail .et al. showed that most cases of kidney allograft failure could be attributed to a specific cause [n = 153), glomerular diseases were the most common cause (37%), whereas around 30% of cases were described as IF/TA; however, most of these could be attributed to a specific cause  from CNI- (ciclosporin-) treated and CNI-free kidney-transplant patients . AlthougChronic renal failure is a serious problem in nonrenal transplant recipients, raising concerns about the potential effects of CNI therapy on renal function in these patients. An analysis of over 69 000 nonrenal solid organ transplants showed that 16.5% of patients developed chronic renal failure during follow-up (median 36 months), with the 5-year cumulative incidence ranging from 7% to 21%, depending on the type of transplant . MultivaIn a retrospective analysis of renal biopsies in 101 patients following nonrenal transplantation, histological changes characteristic of hypertension, CNI toxicity, primary glomerular disease and thrombotic microangiopathy were observed . Other sOver-diagnosis of CNI nephrotoxicity could lead to an inappropriate reduction in immunosuppressive therapy, putting the patient at risk of increased immunological activity . This haEvidence from clinical trials shows that CNI-based regimens provide similar renal function and equivalent or superior graft survival to alternative regimens. The CAESAR study showed that patients receiving a ciclosporin withdrawal regimen showed similar renal function to low- or standard-dose ciclosporin therapy, but had a higher incidence of biopsy-confirmed acute rejection (BCAR) . In the Evidence shows that under-immunosuppression is associated with increased injury and graft loss. Some studies have shown that reduced immunosuppression therapy is associated with increased IF/TA progression , while aPreventative measures aimed at minimizing the risk of renal dysfunction are an important aspect of patient management following kidney transplantation. Hence, the impact of comorbidities such as diabetes, hypertension and hyperlipidaemia on graft function and overall patient health needs to be considered. For example, hypertension is widespread in kidney-transplant recipients, and treatment with calcium channel blockers has been shown to be well tolerated, reduce graft loss and improve GFR . Other mWhen graft function is deteriorating, accurately determining the cause of the kidney disease is essential for the effective management of the patient. Diagnosis requires a thorough clinical investigation, with nothing taken for granted; as discussed above, a specific cause can be identified in the majority of cases, based on a combination of biopsy diagnosis, antibody testing and clinical information.Investigations should include clinical assessments for dehydration, uncontrolled hypertension and infection, which can all affect renal function, whereas ultrasound should be used to assess outflow obstruction and renal circulation . ProteinBiopsy assessments are a key part of the diagnostic process, with characteristic histological changes allowing the identification of a range of specific causes of graft loss. Studies show that in most cases, a specific cause of late graft failure can be identified ,19. DiagOnce a diagnosis has been made, the treatment approach should be individualized for each patient. For those with evidence of chronic AMR from biopsy or the presence of DSA, for example, immunosuppression should be increased, either by raising the dose of tacrolimus or MMF therapy or adding an mTOR  inhibitor, such as everolimus. Specific measures to reduce DSA levels may also be considered, such as the administration of bortezomib or eculizumab, although these treatment modalities should still be regarded as largely experimental –47. AsseEditorial services of iS Health involved in the preparation of this manuscript were supported by Astellas Pharma Europe Ltd. No honoraria were received by the authors for the preparation of the manuscript.


To evaluate changes in QT duration during low-dose haloperidol use, and determine associations between clinical variables and potentially dangerous QT prolongation.In a retrospective cohort study in a tertiary university teaching hospital in The Netherlands, all 1788 patients receiving haloperidol between 2005 and 2007 were studied; ninety-seven were suitable for final analysis. Rate-corrected QT duration (QTc) was measured before, during and after haloperidol use. Clinical variables before haloperidol use and at the time of each ECG recording were retrieved from hospital charts. Mixed model analysis was used to estimate changes in QT duration. Risk factors for potentially dangerous QT prolongation were estimated by logistic regression analysis.adj 34.9, p = 0.009) and before-haloperidol QTc duration .Patients with normal before-haloperidol QTc duration  had a significant increase in QTc duration of 23 ms during haloperidol use; twenty-three percent of patients rose to abnormal levels . In contrast, a significant decrease occurred in patients with borderline  or abnormal before-haloperidol QTc duration ; twenty-three percent of patients in the borderline group, and only 9% of patients in the abnormal group obtained abnormal levels. Potentially dangerous QTc prolongation was independently associated with surgery before haloperidol use (ORQTc duration during haloperidol use changes differentially, increasing in patients with normal before-haloperidol QTc duration, but decreasing in patients with prolonged before-haloperidol QTc duration. Shorter before-haloperidol QTc duration and surgery before haloperidol use predict potentially dangerous QTc prolongation. Although haloperidol is effective in clinical practice, it has been associated with QTc prolongation on the ECG Haloperidol has for many years been a widely prescribed drug for the treatment of agitation, delirium, acute and chronic psychoses. Haloperidol is a butyrophenon antipsychotic that reduces psychomotor disturbances such as agitation and hallucinations by blocking both dopamine DIn this study, we examined whether there is an association between haloperidol use and QT duration changes in a common population of elderly hospitalized patients with multiple morbidities and co-medications. By analyzing the ECGs of these patients before, during and after haloperidol use, we studied whether QT duration changed during the in-hospital use of haloperidol, taking other clinical factors into account. Also, by comparing patient characteristics and (acute) medical condition before haloperidol use, we aimed to define patient groups at risk for dangerous QT prolongation during haloperidol use. In particular, given that hospitalized elderly patients are often given haloperidol because they are agitated during an acute phase response, after surgery, or during acute hospitalization In this retrospective cohort study, we used a database of all 1788 in-hospital patients for whom haloperidol was prescribed between 2005 and 2007 during admission at the Academic Medical Centre, a university teaching hospital in Amsterdam. We analyzed all patients (N = 237) of whom three ECG recordings were made before, during and after haloperidol prescription, i.e., i) between two weeks and one day before start of haloperidol, ii) between one hour after start of haloperidol and 24 h after the last dose of haloperidol, and iii) >one week after the last dose of haloperidol The study was conducted according to the principles expressed in the Declaration of Helsinki. All data were retrieved from the hospital's databases  and were analysed anonymously. The medical ethics committee of the Academic Medical Center (Amsterdam) waived the need for approval and informed consent.ECG analysis was conducted while the researchers were blinded for haloperidol status. QT durations were measured by hand and corrected for heart rate using Bazett's formula (QTc). All three measurements  were analyzed in the same lead for each patient, based on the best readable recording. In case of atrial fibrillation or irregular heart rates, the mean of all RR intervals in the recording was used for rate correction. Patients who had ECGs with multiple ventricular extrasystoles, pacemaker beats, left or right bundle branch block, or whose QTc duration could not be reliably measured  were excluded from further analysis. Of each patient, we obtained the following information from the hospital records: gender, age, medical history, medical status during hospital admission before haloperidol use , actual administration and given dose of haloperidol, and the use of other QT prolonging drugs  was used for data analysis. Changes in mean QTc duration before, during and after haloperidol use, and differences in the change of QTc duration per subgroup were tested for significance using a mixed model analysis assuming fixed effects, thereby accounting for changes in relevant clinical factors. We evaluated associations with patient variables, haloperidol dose, co-medication, medical history, medical status during hospital admission, electrolyte levels, and ECG parameters. Factors that were univariately associated (p<0.25) with before-haloperidol QTc duration, change in QT duration, or subgroup stratification were entered in the mixed model analysis. The final model estimated changes in QTc duration while accounting for relevant variables (backward selection with p<0.05). To test the possibility that an above normal QRS width influenced observed QTc duration, we performed a second analysis in which we did not use the measured QTc duration as the dependent variable, but the QTc duration minus excess QRS duration .Risk factors for potentially dangerous QTc prolongation were analyzed using logistic regression in SPSS. Potentially dangerous QTc prolongation was defined as an increase in QTc duration by >50 ms or to >500 ms, or the occurrence of Torsade de Pointes From a total cohort of 1788 patients for whom haloperidol was prescribed between 2005 and 2007 , we first extracted all patients of whom an ECG recorded before, during, and after haloperidol prescription was available; this yielded 237 patients . Since the prescription of haloperidol often reads ‘when necessary’, we verified in the patient's medical charts whether haloperidol was actually administered, and whether the ECG was recorded during actual haloperidol use. We excluded 81 patients of whom we could not establish this . We furtWhen we studied mean QTc duration of our overall study group, we found no change in QTc duration upon haloperidol use  and a deDifferences in QTc duration were found significant in the final mixed models analysis when comparing QTc durations before and during the use of haloperidol in the normal and abnormal subgroups, after adjusting for relevant covariates . Changes in QTc duration after haloperidol discontinuation were not significant. Overall, the course of the changes in QTc duration was significantly different for each subgroup (p<0.001). Repeating the analysis with QRS-duration subtracted from the measured QTc duration did not yield different results.In total, 16 patients (16%) developed potentially dangerous QTc prolongation during haloperidol use, 15 (94%) of whom had a normal before-haloperidol QTc duration. In only 2 patients QTc duration increased to >500 ms. No episodes of Torsade de Pointes were found.We analyzed factors associated with dangerous QTc prolongation . Five riIn this real-life study among hospitalized elderly patients with multiple co-morbidities, we found that substantial QTc prolongation upon low-dose haloperidol use occurred predominantly in patients with normal before-haloperidol QTc duration. Conversely, in most patients with borderline or abnormal before-haloperidol QTc duration, an unexpected QTc shortening occurred. Importantly, 94% of patients with potentially dangerous QTc prolongation had a before-haloperidol QTc duration in the normal range. A rise in QTc duration to potentially dangerous levels was not only associated with before-haloperidol QTc duration, but also with surgery (increased risk) and signs of inflammation (decreased risk) before haloperidol use. Although patients with cardiovascular risk factors are reported to have a higher risk of QTc prolongation per se. A more plausible explanation might be that changes in the underlying condition occurred during haloperidol use, and that these changes caused QTc duration to shorten, despite haloperidol use. For instance, although speculative, it is possible that the complex effects of acute illness (both cardiac and non-cardiac), acute phase response, or poor general condition , contributed to QTc prolongation through as yet unknown mechanisms. In support of the view that acute illness may cause QTc prolongation, average QTc durations of all three subgroups were normal after haloperidol use, i.e., when the acute illness had subsided.It is unlikely that QTc shortening in patients with a prolonged QTc interval prior to haloperidol use is due to the effects of haloperidol Unexpectedly, potentially dangerous QTc-prolongation was associated with absence of signs of inflammation, though not significantly. We expected the contrary since inflammation/fever is suggested as a risk factor for QTc prolongation in patients with a decreased hERG function  A limitation of our study is that the patients had different causes and stages of inflammation or acute phase response; thus, different levels and types of circulating cytokines may have been present. Therefore, it remains difficult to link the changes in QT duration during haloperidol use directly to the effects of inflammation. Moreover, the indication for haloperidol prescription  may also be associated with QTc prolongation, independent of haloperidol use. This may be the case in post-operative patients, who, in our population, are at increased risk to develop potentially dangerous QTc prolongation. A stress response has been shown to induce QTc prolongation To our knowledge, this is the first study that attempts to evaluate changes in QTc duration upon haloperidol use, while taking co-morbidities that play an important role in a real-life clinical setting into account. A major limitation of our study, however, is its retrospective design. By limiting our study to patients of whom ECG-recordings were available before, during and after haloperidol use, we excluded patients who were discharged or died during haloperidol use. Moreover, we were biased towards patients in whom multiple ECG recordings were made, thereby probably introducing an overrepresentation of patients with cardiac disease. This might explain the large proportion of male patients in our study population.Our findings would seem to suggest that ECG monitoring of elderly patients who receive in-hospital low-dose haloperidol may not be necessary, as QTc prolongation occurred mostly in patients with normal before-haloperidol QTc duration, while QTc duration generally shortened in those with before-haloperidol QTc prolongation. Overall, potentially dangerous QTc prolongation occurred only in few patients, and Torsade de Pointes was recorded in none. Yet, we see the largely surprising findings of our study  as indication that the real-life clinical effects of haloperidol are not well-established. We therefore conclude that more research (larger prospective studies) is needed to allow for rational clinical decision making on the cardiac safe use of haloperidol.


Triticum spp.) to identify some of the important genetic variants responsible for the phenotypic differences introduced by intense selection during domestication. A Web-based implementation of FATHMM, including a high-throughput batch facility and a downloadable standalone package, is available at http://fathmm.biocompute.org.uk.The rate at which nonsynonymous single nucleotide polymorphisms (nsSNPs) are being identified in the human genome is increasing dramatically owing to advances in whole-genome/whole-exome sequencing technologies. Automated methods capable of accurately and reliably distinguishing between pathogenic and functionally neutral nsSNPs are therefore assuming ever-increasing importance. Here, we describe the Functional Analysis Through Hidden Markov Models (FATHMM) software and server: a species-independent method with optional species-specific weightings for the prediction of the functional effects of protein missense variants. Using a model weighted for human mutations, we obtained performance accuracies that outperformed traditional prediction methods  on two separate benchmarks. Furthermore, in one benchmark, we achieve performance accuracies that outperform current state-of-the-art prediction methods . We demonstrate that FATHMM can be efficiently applied to high-throughput/large-scale human and nonhuman genome sequencing projects with the added benefit of phenotypic outcome associations. To illustrate this, we evaluated nsSNPs in wheat ( Nonsynonymous single nucleotide polymorphisms (nsSNPs) lead to amino acid substitutions (AASs) and have the potential to affect the function of the protein product of a gene via the structure, biochemistry and/or splicing of the protein. Advances in high-throughput sequencing technologies have accelerated the rate at which nsSNPs are now being identified [The 1000 Genomes Project, The majority of computational prediction methods utilize evolutionary sequence conservation and/or structural annotations within homologous  proteins from a database of known sequences and/or structures [Ng and Henikoff, Triticum spp.) to identify some of the key genetic variants responsible for the phenotypic differences introduced by intense selection during domestication and have made this analysis publicly available to the scientific community.Inspired by previous work [Calabrese et al., http://www.hgmd.org) and inherited putative functionally neutral AASs in the UniProt database [Apweiler et al., http://www.uniprot.org/docs/humsavar) were downloaded and used to calculate the pathogenicity weights implemented in our weighted/species-specific method. Next, we obtained two human mutation datasets to assess the performance of FATHMM against the performance of other computational prediction algorithms previously reported in the literature: the VariBench database  used in a comprehensive review [Thusberg et al., BRCA1, MSH2, MLH1, and TP53) used in a recent review [Hicks et al., http://swissvar.expasy.org) and performed an independent benchmark of FATHMM against eight other computational prediction algorithms [Adzhubei et al., A collection of five human mutation datasets from online databases and the literature were downloaded and used in this study . First, JackHMMER component of HMMER3  were used to assess the performance of our models:tp and fp refer to the number of true positives and false positives reported and tn and fn denote the number of true negatives and false negatives reported.where Theoretically, using our prediction formulae, scores approximately equal to zero indicate that there is no significant change in the underlying amino acid probabilities whereas scores less than zero indicate that an unfavorable substitution has been observed, that is, the mutant residue is less likely to be observed than the wild-type residue, and scores greater than zero indicate that a favorable substitution has been observed, that is, the mutant residue is more likely to be observed than the wild-type residue. However, in practice, FATHMM is sensitive to small fluctuations in the amino acid probabilities modeled within the HMMs. For example, the slightest reduction in amino acid probabilities would yield a pathogenic prediction in our unweighted/species-independent algorithm. Therefore, to eliminate the effects of these fluctuations, we plotted the distribution of the predicted magnitude of effect for both disease-associated and functionally neutral AASs within the SwissVar dataset . From thThe performance of FATHMM was compared against the performance of other computational prediction algorithms reported in two previously published reviews [Hicks et al., Next, we used the Hicks dataset to benchmark FATHMM against four other computational prediction algorithms  [Adzhubei et al., Although we recognize the importance of comparing prediction methods in relation to previously established benchmarks, we also conducted our own benchmark  comparing the performance of FATHMM against eight published computational prediction methods [Adzhubei et al., Wd and Wn, if and only when the AAS being evaluated was present in either the HGMD [Stenson et al., The pathogenicity weights incorporated in FATHMM were not directly used to train for, or recognize, pathogenic sequences and/or mutations. We do nevertheless recognize the potential for bias in the performances observed. Therefore, to remove this bias, we performed a “leave-one-out” analysis on all benchmarking datasets. Here, we adjusted our pathogenicity weights, To understand the potential complementarity/redundancy of FATHMM to other methods, we assessed the intersection of disease-associated AASs correctly identified (true positives) by our method and the top-ranking computational prediction algorithms . From thAnticipating a massive increase in the number of available whole-genome and whole-exome datasets, the need for accurate computational prediction methods capable of processing these datasets in a timely fashion is increasingly apparent. As a result, the majority of computational prediction algorithms now offer some form of precomputed facility allowing for near-instant predictions to be returned  are correctly identified via domain-based ontological associations. However, potential issues of using domain-centric ontologies arise when a common domain harbors multiple mutations with distinct and uniquely expressed phenotypes. In these instances, domain-centric ontological associations may have become diluted and should therefore be used with caution. For example, the predicted phenotypic consequences for the R239C mutation in CHRNG  are consistent with the associated syndrome, which is characterized by a decrease in fetal movement and overall muscle weakness. However, phenotypes not associated with (or secondary to) Escobar syndrome, for example abnormalities in temperature regulation, were also predicted. Nevertheless, we foresee that these annotations will be most prominent in protein sequences of unknown function and/or ongoing nonhuman genome sequencing projects, as demonstrated below.As previously alluded to, FATHMM not only predicts the potentially deleterious nature of AASs but is also capable of annotating the molecular and phenotypic consequences of these mutations via domain-centric ontologies. To illustrate this, we evaluated the predicted phenotypic consequences of disease-associated AASs within the SwissVar dataset Supp. . As expeArabidopsis [Ogiso et al., http://www.cerealsdb.uk.net/functional_snps/index.htm, illustrate the potential additional utility of FATHMM in predicting the functional consequences of variants identified in ongoing nonhuman genome sequencing projects (even in species very distantly related to human).As the world's population continues to grow, so does the demand for crops with particular characteristics such as drought resistance, high yield and resistance to pests and pathogens. The cultivation and repeat harvesting of wild “landrace” wheat varieties has led over time to the emergence of domesticated “elite” wheat varieties with desirable phenotypic characteristics. In an attempt to elucidate some of the important genetic variants responsible for these characteristics, we collected single nucleotide variants (SNVs) from four elite UK bread wheat varieties  and have predicted the functional effects of these mutations when compared to four landrace wheat varieties from the Watkins collection held at the John Innes Centre, Norwich, UK . For this analysis, SNVs were mapped onto the draft wheat genome assembly and six-frame translated. For each reading frame, SUPERFAMILY [Gough et al., Here, we have introduced and discussed the FATHMM software and server: a species-independent method with optional species-specific weightings for the prediction of the functional effects of protein missense variants. Inspired by previous sequence-based computational prediction algorithms [Ng and Henikoff, BRCA1, MSH2, MLH1, and TP53) shows Align-GVGD [Tavtigian et al., The performance of FATHMM was compared to the performances of alternative computational prediction methods previously reported in two published reviews [Hicks et al., An important issue to consider when comparing the performance of trained/weighted computational prediction methods is the cross-validation dataset, that is, these prediction methods should ideally be tested using “blind” datasets to minimize the bias in the performances observed. Unfortunately, this level of testing is not possible as it would require retraining/validating all prediction methods with common datasets. However, the majority of disease-associated AASs in the VariBench database were collected from Locus-Specific Databases (LSDB) and are not found in commonly used training datasets, for example, SwissProt/TrEMBL [Apweiler et al., http://fathmm.biocompute.org.uk.A potential disadvantage of our weighted method is the inherited restriction (via the weighting scheme employed) to AASs falling within conserved protein domains. However, protein domain annotations from the SUPERFAMILY and Pfam databases encompass around 80% of the SwissProt/TrEMBL database [Punta et al.,


To address this, investigators have used a variety of tumor models in which MSCs are added exogenously to determine their impact on tumor development. Interestingly, many studies have reported contradicting results, with some investigators finding that MSCs promote tumor growth and others reporting that MSCs inhibit tumor growth. Many mechanisms have been reported to account for these observations, such as chemokine signaling, modulation of apoptosis, vascular support, and immune modulation. In this review, we analyzed the differences in the methodology of the studies reported and found that the timing of MSC introduction into tumors may be a critical element. Understanding the conditions in which MSCs enhance tumor growth and metastasis is crucial, both to safely develop MSCs as a therapeutic tool and to advance our understanding of the role of tumor stroma in carcinogenesis. S Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) are multipotent progenitor cells that exhibit a marked tropism for tumors. MSCs form a tumor's fibrovascular network, differentiating into tumor-associated fibroblasts (TAFs) and vascular pericytes –3. DespiThe tropism of MSC for tumors makes MSCs uniquely destined to function as cellular delivery vehicles for antitumor agents –32. MSCsMSCs have been isolated from many types of adult and fetal tissues using similar methodologies . Bone maThese tissue-derived MSCs share a number of important characteristics with bone marrow-derived MSCs, including cell surface marker expression, plastic adherence, and the capacity to differentiate into cells of mesenchymal lineage  under appropriate conditions . In addiMSCs are thought to endogenously support wound healing and hematopoiesis, but many of the native functions of MSCs remain poorly understood. When engrafted at sites of tissue injury, MSCs differentiate into connective tissue elements, support vasculogenesis, and secrete cytokines and growth factors that facilitate healing. The potential of MSCs to promote tissue repair is being investigated in a diverse array of diseases, including ischemic heart disease, diabetes, and Parkinson's disease .The function of MSCs in tumors is likely to parallel the role of MSCs in wound healing. As in wounds, MSCs differentiate in tumors into fibroblasts and pericytes and, perhaps, endothelial-like or vessel-attached cells , 46. AddMSCs also have complex immunomodulatory effects. MSCs can counteract inflammation, by suppressing host immune responses and preventing fibrosis. The immunosuppressive properties of MSCs are being exploited in an effort to reduce the toxicity of allogeneic bone marrow transplant due to graft versus host disease (GvHD) in which the transplanted cells formulate an immune response against the transplant recipient , 49.Studies reporting that MSCs can promote tumor growth are listed in Bone marrow-derived MSCs have been shown to increase the in vivo growth of colon cancer, lymphoma, and melanoma cells , 6, 50. B16 melanoma cells transplanted into allogeneic mice did not form tumors unless MSCs were coinjected .Adipose tissue is a rich source of multipotent MSCs (referred to as adipose stem cells [ASCs]), which exhibit tumor tropism and are thought to be functionally similar to bone marrow-derived MSCs . MuehlbeHowever, in most of these reports MSCs were added to fixed numbers of tumor cells, resulting in increased total numbers of cells injected including both tumor cells and MSCs. We have demonstrated earlier that MSCs proliferate in the presence of tumor cells in vivo, as evidenced by BrdUrd incorporation in vivo, whereas MSCs implanted without tumors did not proliferate . Hence, Finally, MSCs have been reported to undergo transformation into malignant cells and form tumors in vivo. However, three groups of investigators have now found that the “malignant” MSCs were contaminated with tumor cells used for other projects in their laboratories. Garcia-Castro and coworkers reported initially that MSCs underwent spontaneous transformation but recently found contamination with HT1080 sarcoma cells , 54. RosIn direct contradiction to the studies reporting that MSCs support tumor growth, other studies have shown that MSCs suppress tumor growth . MSCs inHuman fetal skin-derived MSCs inhibited human liver cancer cell lines, with reduced proliferation, colony formation, and oncogene expression both in vitro and in vivo . When thThe same fetal skin-derived MSCs inhibited growth of MCF-7 breast cancer cells in vitro . The resAdipose-derived MSCs were also found to inhibit proliferation of primary leukemia cells . This efMSCs have been shown to suppress pancreatic tumors by altering cell cycle progression. In vitro coculture with MSCs increased rates of G1-phase arrest in pancreatic cancer cells . In vivoThe impact of hyperthermic treatment was tested in an in vitro study in which adipose-derived stem cells were heated . OvarianMSCs have been reported to support the tumor vasculature directly, by differentiating into pericytes and perhaps endothelial cells  and throMSCs also secrete various proangiogenic factors, such as VEGF, fibroblast-derived growth factor, PDGF, and stromal-derived factor-1 (SDF-1). These cytokines promote endothelial and smooth muscle migration and proliferation at the tumor site, facilitating angiogenesis , 65.MSC-secreted factors, such as VEGF, support blood vessel growth. MSCs expressing VEGF increased microvessel density in pancreatic xenografts . HoweverIn what appears to be a direct conflict with this data, MSCs appear to inhibit capillary growth under certain conditions. MSCs migrate in vitro toward endothelial cell-derived capillaries in Matrigel, incorporate into these vessels, and produce reactive oxygen species, resulting in endothelial cell apoptosis . These rFibroblasts, which are the primary cell type comprising tumor stroma, are a critical component of the tumor microenvironment. Tumor fibroblasts (referred to as TAFs) are derived in part from MSCs that may be recruited regionally or from circulating populations , 66. AftThe importance of TAFs in promoting tumorigenesis has been well established in multiple tumor models . TAFs exMSCs are generally thought to have immunosuppressive effects, which may be an important mechanism through which MSCs promote tumor growth or increase incidence of tumor formation in vivo. MSCs can directly impair the function of a variety of immune cells, including B and T lymphocytes, dendritic cells, and natural killer cells –75. ThesMSCs suppress T-cell proliferation through multiple mechanisms. IFN-γ directly enhances the T-cell suppressive effects of MSCs through upregulation of an inhibitory cell surface marker, B7-H1 . A subpoToll-like receptor (TLR) signaling has recently been shown to regulate the immunomodulatory properties of MSCs . TLR resThe immunomodulatory effects of MSCs, if any, are not well understood within tumors. Djouad et al.  reportedKarnoub et al. reported that MSC-secreted CCL5 induced a transient prometastatic effect on breast cancer cells . Tumors MSCs may modulate the epithelial-to-mesenchymal transition (EMT), a developmental process that is subverted by tumor cells resulting in a more invasive phenotype . In breaMSCs may also modify the metastatic niche, supporting the formation of early metastasis through vasculogenesis or growth factor secretion. For example, Corcoran et al.  reportedMSCs secrete a variety of growth factors that are known to influence tumor proliferation, migration, and angiogenesis . In addiAlthough a great deal of research has focused on the role of MSCs as delivery vehicles for antitumor proteins or viruses, targeting endogenous MSCs in tumors also has the potential to be an effective therapy. Studies investigating the effects of MSCs on response to cytotoxic therapy have demonstrated that MSCs can protect tumor cells from chemotherapy. Interactions with MSCs in bone marrow has been shown to promote survival of acute and chronic myelogenous leukemia , 96–98. MSCs secrete high levels of leptin, which has antiapoptotic properties, during adipocyte differentiation . MSC-derMSCs interact with tumor cells in a myriad of ways, which have the potential to support or suppress tumor growth. Additionally, MSCs interact with their tumor-resident neighbors, such as immune cells (macrophages and T cells) and endothelial cells. Other cell types have also been shown to play roles in modulating tumor progression, thereby potentially exacerbating or inhibiting the outcomes of the MSC-tumor interactions.The heterogeneity in MSC is likely a major factor contributing to the inconsistent reports about the effects of MSC in tumors. The MSC used in the studies reviewed here were isolated largely on the basis of plastic adherence. These MSCs exhibited appropriate cell surface marker expression as well as multipotency and yet these cell populations are fundamentally different in critical ways. To address this concern, cell surface markers that permit isolation of a more homogenous population are needed. Several markers, such as NG2 , have beThe studies reviewed here reported both growth promotion and suppression for the same cell types, including adult- and fetal-derived bone marrow, suggesting that the age of the donor does not determine the effect of MSC on tumor progression. Zhu et al.  directlyThe effects of propagating cells ex vivo are also variable and may contribute to these discrepant results. Authors have reported growing MSC in high serum or growth factor supplemented media, which may alter the MSC phenotype. In addition, passage number and confluence may impact MSC function. Studies using endogenously labeled lineages of stromal cells will eliminate these concerns and will help clarify the effects of MSC or MSC-like cell in vivo. Finally, it is not known what proportion of “MSC” cell lines are contaminated with tumor cells, such as reported contamination with osteosarcoma and glioma cells used in the same laboratories . FingerpIn vivo tumor models vary widely and may also account for some of the differences in the findings of these studies. Tumor immunity, hypoxia, angiogenesis, and cytokine secretion are all highly variable in the models described in these studies. However, MCF-7 breast cells and B16 melanoma cells were reported by different investigators to be both promoted and suppressed by MSC delivery, suggesting that the tumor model is not the only factor accounting for the disparate findings.The dose of MSCs delivered has been proposed to be a determinate of MSC effects on tumor growth. In general, studies that reported growth promotion tended to use a higher MSC to tumor cell ratio. However, low percentages of MSCs, such as the 10% used by Yu et al. , promoteThe timing at which MSCs are introduced into the tumor microenvironment may be an important consideration. All three of the studies that introduced MSCs into established tumors reported tumor growth inhibition , 20, 23.Finally, patient-to-patient variability in MSC isolates may also contribute to the conflicting data reported in the literature. It is possible that due to genetic, epigenetic, or environmental effects, MSCs may have different effects on the tumor microenvironment and could conceivably contribute to an individual's cancer risk. To address this, studies of MSCs from patients predisposed to cancer because of somatic mutations, such as BRCA, or environmental exposures, such as cigarette smoking, may be helpful to determine if patient-to-patient variability exists.In summary, no simple paradigm can account for the conflicting findings in the studies of MSCs. Understanding the nuances of the interaction between MSCs and cancer cells is especially critical given the therapeutic potential of MSCs. The possibility of MSCs promoting tumor growth and metastasis raises concerns about the safety of their use as clinical tools. No evidence of tumor formation has been reported in over 1,000 patients treated with MSC for a variety of indications, so far. MSCs that have been engineered to express antitumor cytokines have potent antitumor effects, suggesting that perhaps putative tumor-promoting effects of MSCs can be overcome by manipulating cytokine expression. Additionally, MSCs could also be engineered to express a suicide gene so that MSCs delivered systemically would migrate into tumors, express an antitumor protein, and then be terminated with the delivery of an agent, such as gancyclovir, resulting in selective termination of the exogenously delivered MSCs. Furthermore, therapeutic strategies aimed at disrupting tumor-promoting effects of MSCs opens the door to a novel therapeutic strategy, but this would need to be properly timed to prevent disruption of a growth-suppressive MSC-tumor cell interaction. This review highlights the critical need for studies designed to explicitly test the hypothesis that tissue source, individual donor variability, timing of MSC injection, or expression of critical receptors such as TLR, determine the effect of MSC on tumor progression. Only systematic analysis of the role of these and other factors will resolve this important issue.


Firefighting is a hazardous profession which has claimed on average the lives of 105 US firefighters per year for the past decade. The leading cause of line-of-duty mortality is sudden cardiac death, which accounts for approximately 45% of all firefighter duty-related fatalities. Strenuous physical activity, emotional stress, and environmental pollutants all strain the cardiovascular system, and each can increase the risk of sudden cardiac events in susceptible individuals. Sudden cardiac death is more likely to occur during or shortly after emergency duties such as fire suppression, despite the fact that these duties comprise a relatively small proportion of firefighters' annual duties. Additionally, cardiac events are more likely to occur in firefighters who possess an excess of traditional risk factors for cardiovascular disease along with underlying atherosclerosis and/or structural heart disease. In this review, we propose a theoretical model for the interaction between underlying cardiovascular disease in firefighters and the multifactorial physiological strain of firefighting. It is widely recognized that firefighting is a hazardous profession. Many people outside the fire service mistakenly assume that most on-duty deaths result from burns and/or smoke inhalation. However, in the USA, the leading cause of duty-related deaths in firefighters is sudden cardiac death (SCD). In fact, cardiovascular disease (CVD) accounts for approximately 45% of all firefighter duty-related fatalities . CurrentFor every duty-related SCD event, almost 17 non-fatal cardiovascular events (heart attacks and strokes) occur in other firefighters during the course of their duties . Given tIn this review, we will summarize the factors producing marked cardiovascular strain on firefighters during firefighting activity. We will explore leading hypotheses and provide a theoretical model to explain how the strain of firefighting interacts with underlying CVD to precipitate a CVD event in vulnerable individuals in the US Fire Service.Firefighting leads to a rapid increase in heart rate initiated by sympathetic physiologic arousal from the fire alarm bell. Subsequently, maximal or near-maximal heart rates are achieved during physically strenuous firefighting activities. Perhaps of more functional significance, our work has shown a reduction in stroke volume following firefighting activity. We have reported a 35% reduction in stroke volume (using Doppler echocardiography) following short-term firefighting activity . We haveWe have recently reported that firefighting activity leads to an increase in arterial stiffness . FurtherAs shown in Figure During an emergency fire call, cardiovascular strain begins with initial activation of the sympathetic nervous system following the sound of an alarm bell . SeveralFollowing an initial fire alarm, the sympathetic response continues during transport to the scene and at the fire scene. The fire scene is a chaotic environment characterized by loud noise, multiple auditory signals , and com−1·min−1 ) to perform the essential tasks safely [During structural firefighting, firefighters are regularly required to perform activities combining static and aerobic exertion such as stair and ladder climbing (while carrying heavy equipment), forcible entry, victim search and rescue, building ventilation, and fire attack and suppression. Moreover, these essential tasks are performed while wearing heavy (≥25 kg), multiple-layered and fully encapsulating protective clothing, which add to the metabolic demands of firefighting. For example, von Heimburg and co-workers  reporteds safely ,22.The most obvious physiological changes in response to performing heavy work while wearing encapsulating PPE in a hot environment are elevation of body temperature coupled with muscular and cardiovascular fatigue. We have documented an increase in core temperature of approximately 1.5°C (peaking at 38.6°C) following short-term (18 min) firefighting activity in a structure that contained live fires  and an i−1[Strenuous work in a hot environment while wearing protective clothing also leads to profuse sweating and resultant dehydration. During strenuous firefighting simulations, firefighters have been reported to experience sweat rates up to and greater than 2 l∙ h−1,27. Even−1.Despite the use of self-contained breathing apparatus, firefighters are routinely exposed to fire smoke, which contains toxic gases such as carbon monoxide (CO) and cyanide. When inhaled, CO diffuses through the alveolar–capillary membrane and binds with hemoglobin, forming carboxyhemoglobin. The formation of carboxyhemoglobin reduces the availability of hemoglobin to transport oxygen to the tissues, also binds cardiac and skeletal muscle myoglobin, and alters the intercellular use of oxygen, which can all result in tissue hypoxia . Tissue Fire smoke also contains particulate matter associated with the promotion of arrhythmias, decreased heart rate variability, and increased blood pressure -32. OtheThe factors discussed above all interact with individual characteristics such as health status and fitness level to determine the magnitude of the cardiovascular strain of firefighting and whether responses to the strain are limited to physiologic adaptations or result in the activation of pathophysiologic pathways. Underlying cardiovascular health status is largely determined by fitness and the prevalence of cardiovascular risk factors among firefighters. In recent studies, the prevalence of smoking among general cohorts of firefighters was 10%–18%; however, the proportion of smokers among firefighters who suffered a CHD fatality while on duty is 40%–50% ,34. The −1·min−1, indicating that, in many cases, a mismatch occurs between the fitness requirements of the job and the fitness profile of the firefighter. This is despite the fact that firefighters perceive themselves to have high fitness levels [Given the strenuous nature of firefighting and the widespread concern about sudden cardiac death in the fire service, a high aerobic fitness level is clearly desirable. Yet, it is widely reported ,44,45 ths levels . Lower ls levels ,47,48 ass levels . ConversA typical firefighting shift is characterized by long periods of low-intensity work interspersed with occasional bouts of moderate to high-intensity activity . As manyClinical CVD, subclinical CVD, and the presence of specific CVD risk factors increase the risk of sudden cardiac events. Studies investigating the prevalence of cardiovascular risk factors in firefighters suggest that firefighters often have risk factor prevalence similar to the general population ,19,40,51Numerous studies have shown that a variety of stressful situations can trigger acute CVD events, particularly in susceptible individuals with underlying CHD. Heavy physical exertion -54 or stThe three factors highlighted above  are encountered by firefighters on a regular basis during fire suppression activities. Therefore, it is likely that these multiple stressors may function independently or work in concert to precipitate CVD in firefighters depending on the conditions encountered and the individual firefighter's susceptibility. As shown in Table Sudden cardiac death in firefighters most likely arises from a multifaceted interplay of substrates and triggers. As seen in Figure Although a series of complex pathophysiologic processes are involved in SCD, pump failure usually due to cardiac arrhythmias is ultimately the cause of death. Sudden cardiac death can be caused by many conditions; however, a very large percentage of SCD is caused by CHD  and/or cardiomegaly/LVH. CHD is associated with stenosis, ischemia, plaque rupture, and the formation of an occlusive thrombus. Cardiomegaly/LVH is associated with increased risk of fatal arrhythmias.Coronary heart disease and its consequences account for approximately 80% of all SCD in Western countries -61. HoweAtherosclerosis has long been associated with the gradual and cumulative accumulation of lipid in the arterial wall. Both intravascular ultrasound and wartime autopsy studies provide strong evidence of a high prevalence of subclinical coronary atherosclerosis in young and middle-aged Americans . More reper se are not a pre-requisite for ACS but are an indication of a higher overall plaque burden and, thus, a greater likelihood of having an ACS event. The mechanisms linking a previously quiescent atherothrombotic plaque to an acute coronary syndrome are complex and include factors related to plaque stability and hemostatic balance. Furthermore, there is evidence that external triggers, such as heavy work and emotional stress, may lead to acute changes that precipitate a cardiac event [Many plaque-rupturing events, which cause ACS, occur on thin vulnerable plaques and not necessarily on hemodynamically significant stenoses. Hemodynamically significant stenoses ac event . Studiesac event ,34.Cardiomegaly (increased heart size and mass) and LVH  are structural abnormalities that are associated with SCD. Left ventricular hypertrophy has been shown to be a powerful predictor of cardiovascular morbidity and mortality in population-based studies -68. The Hypertrophic cardiomyopathies have long been associated with SCD in athletes, and recently, Rowland has raised the provocative question about the arrhythmogenic nature of LVH that accompanies athletic training . AlthougThere is mounting evidence that LVH/cardiomegaly is common among US firefighters and plays a major role in CVD events in the fire service. In a seminal 2003 case–control investigation of on-duty CHD fatalities, we found evidence for LVH in 76% of the CHD deaths where the autopsy results were available . In a laThis review has proposed a theoretical model for the interaction between underlying cardiovascular disease in firefighters and the multifactorial strain of firefighting. We have posited that both atherosclerotic heart disease and left ventricular hypertrophy/cardiomegaly are important substrates that markedly increase the risk of sudden cardiac death. Additional research is necessary to understand the risk associated with a given level of atherosclerotic burden, cardiomegaly, or left ventricular hypertrophy. Furthermore, research is necessary to identify when and how firefighters should be screened for these conditions.Firefighting activities involve sympathetic arousal, heavy strenuous work, and adverse environmental conditions that can lead to hyperthermia and dehydration with considerable associated cardiovascular strain on firefighters. The theoretical model proposed in this article is based on diverse lines of evidence, including physiological studies of firefighters during strenuous emergencies, epidemiologic studies linking CVD event risk to specific types of duty, as well as autopsy data confirming the presence of underlying heart disease in almost all victims. The model suggests that in susceptible individuals with underlying structural heart disease (most often CHD and LVH), the cardiovascular strain associated with firefighting may trigger a sudden cardiac event through several biological pathways. Increases in shear stress may cause rupture of vulnerable plaque, resulting in occlusion of coronary arteries, and this may be exacerbated by hyper-coagulability which increases the risk of thrombotic events. Ischemia  may exceed myocardial demand, resulting in electrical, mechanical, and biochemical dysfunction of the cardiac muscle, precipitating fatal arrhythmias. Changes in electrolytes and exposure to environmental conditions (such as gaseous and particulate toxicants in smoke) may also increase susceptibility to arrhythmias -32, partACS: acute coronary syndromes; CHD: coronary heart disease; CO: carbon monoxide; CVD: cardiovascular disease; LVH: left ventricular hypertrophy; METs: metabolic equivalents; NSTEMI: non-ST elevation myocardial infarction; PPE: personal protective equipment; SCBA: self-contained breathing apparatus; SCD: sudden cardiac death; STEMI: ST elevation myocardial infarction.SNK has served as an expert witness in legal cases involving firefighters and was also contracted to revise the Heart Disease Manual of the International Association of Fire Fighters. DLS has served as an expert witness in legal cases involving firefighters. The other author has no competing interests to declare.DLS conceived of the review and collaborated with SNK on the development of the theoretical framework for the manuscript. DLS provided an initial draft of the manuscript. DAB provided significant review and revision to the manuscript draft. SNK reviewed and provided significant contributions to the final manuscript. All authors read and approved the final manuscript.


The special issue “Antimicrobial Polymers” includes research and review papers concerning the recent advances on preparation of antimicrobial polymers and their relevance to industrial settings and biomedical field. Antimicrobial polymers have recently emerged as promising candidates to fight microbial contamination onto surfaces thanks to their interesting properties. In this special issue, the main strategies pursued for developing antimicrobial polymers, including polymer impregnation with antimicrobial agents or synthesis of polymers bearing antimicrobial moieties, were discussed. The future application of these polymers either in industrial or healthcare settings could result in an extremely positive impact not only at the economic level but also for the improvement of quality of life. Microbial contamination is of great concern in many area including medical devices, health care products, textiles, water purification systems, food packaging and storage. Any material, either synthetic or natural, can be, in fact, easily colonized by microorganisms that then proliferate on its surface building up a peculiar tridimensional structure known as a biofilm.Biofilms can cause significant damage to industrial settings with consequent economic losses. For example, in industrial processes for water and wastewater treatment, biofilm formation on filtration membranes can greatly compromise the efficiency of treatment processes. In drinking water distribution systems, microbial colonization of the pipe walls can decrease water quality by increasing bacteria level, reducing the dissolved oxygen and changing water taste and odor.Microbial contamination of food products during storage, processing or distribution is an important hygienic and health problem since it can cause food spoilage and food-borne illnesses.In the biomedical field, biofilm formation of the surfaces of implantable medical devices, such as intravascular catheters, urinary catheters and orthopedic implants, is associated with the development of antibiotic-resistant, often life-threatening, infections.Biofilm eradication is considered a difficult task, since the biofilm-like mode of growth permits the survival of microorganisms under high levels of antibiotic and extreme conditions of temperature, pH, ionic strength and shear stress. Therefore, prevention of biofilm formation represents a great challenge for both industry and research.The present special issue includes research and review papers on recent advances obtained in the development of antimicrobial polymers and their relevance to industrial and healthcare settings. Antimicrobial polymers have recently emerged as promising candidates to fight microbial contamination onto surfaces thanks to their interesting properties. In fact, the versatility of the macromolecular chemistry facilitates the tailoring of polymer physico-chemical properties  to fulfill different applications. Polymers have become the materials of choice in numerous industrial settings and particularly in the biomedical field, where they are used for a wide range of products among which implantable medical devices are the prevailing ones.Two main strategies can be pursued to develop antimicrobial polymers: (i) polymer impregnation with antibiotics or other biocides; (ii) polymer functionalization with groups exerting antimicrobial activity.In the first approach, the polymer acts as a carrier for one or more antimicrobial agent that once released from the polymer exerts its action . In the second approach, bactericidal functionalities, such as quaternary amine containing compounds or phosphonium salts, are introduced in the polymer to obtain intrinsically antimicrobial polymers . Biocidal polymers do not release antimicrobial substances but exert their killing action when microorganisms contact the surface.To obtain antimicrobial agent-releasing polymers, anionic or cationic surfactants, antibiotics, antiseptics or heavy metals can be applied to the polymer by: (a) physical adsorption; (b) impregnation, (c) complexation or (d) conjugation via hydrolytically labile linkages. Particularly, drug impregnation is the most pursued method since it is easy to perform and can permit the loading of significant drug amounts by increasing polymer matrix porosity.b-polydimethylsiloxane block copolymer. Some attractive features of this system are the possibility to tune pore size, to have narrow pore size distribution, and to adjust its chemical and mechanical properties. The obtained SDS-loaded nanoporous polymer films were shown to be more active in preventing bacterial adhesion and reducing biofilm formation when challenged with the gram-negative Escherichia coli than the gram-positive Staphylococcus epidermidis.Li and coworkers  develope2 nanoparticles were impregnated into a biodegradable polycaprolactone (PCL) matrix by Munoz-Bonilla et al. to obtain a bioactive food packaging [2 nanoparticles were well dispersed into the PCL matrix and did not significantly change PCL crystalline structure, phase transitions and thermal stability. Since TiO2 is a light-induced biocidal substance, antibacterial tests were performed by irradiating samples with light at 280 (UV) and 500 (visible) nm for different time periods. Better results were obtained by UV irradiation where a log-reduction ranging from 3 to 5.6, depending on TiO2 content, was observed against E. coli and from 4 to 5 for Staphylococcus aureus.Different amounts of TiOSilver has always attracted much attention for its broad-spectrum antibacterial activity and has become among the most employed antimicrobial agents for impregnation in biomedical applications. Different approaches have been investigated to incorporate silver in polymeric substrates. A conventional approach is by the deposition of metallic silver directly onto the surface of the substrate, for example by vapour coating, sputter coating, or ion beam coating. An approach based on impregnation of materials with silver nanoparticles (Ag-NPs) rather than coating has been more recently developed.Several AgNPs-containing systems have been prepared to promote wound healing , to hindNanogels, cross-linked hydrophilic polymers, have many practical applications including drug delivery systems and as filler materials in the coating industry. Different physical and chemical methods are used for polymer crosslinking. Especially in the case of chemical crosslinking, some drawbacks, such as toxicity and elimination of crosslinker after reaction, can be found.et al. have used radiation technology for preparation of polyacrylic acid-based nanogels containing silver nanoparticles (Ag/PAAc) [+ ions without using reducing agents was easily obtained as shown by FE-SEM-EDX analysis. Particle size analysis showed that the average size of the Ag/PAAc nanogels were approximately 200 nm at 100 kGy. Also, it was verified that while the size of PAAc nanogels decreased with increasing electron beam irradiation doses, the zeta potential value increased demonstrating a poor material aggregation. The obtained Ag/PAAc nanogels exhibited good antibacterial activity against both Gram-negative and Gram-positive bacteria and by in vivo experiments a good healing effect of the Ag/PAAc nanogel pastes was observed.To simultaneously obtain sterilized and crosslinked materials, Choi Ag/PAAc) . The redIt is generally recognized that metallic-based nanoparticles are stabilized by a biocompatible polymer coating. Chitosan (CS), polysaccharide derived from naturally occurring chitin, is widely investigated for many applications thanks to its biocomopatibility, interesting biological properties and its versatility to be prepared into different forms such as nanoparticles, fibers and film.et al. developed a nanocomposite system based on a lactose-substituted chitosan and silver nanoparticles (Chitlac-nAg) [v/v), provided the inhibition of the growth of Streptococccus mutans and Streptococcus oralis planktonic phase, except for Streptococcus mitis that was instead inhibited at one step less. On the contrary, on preformed biofilm, Chitlac-nAg was able to inhibit the bacterial growth at a value of 0.2% on the supernatant phase and on the mature biofilm. For S. mitis the Chitlac-nAg inhibitory concentration on biofilm was 0.1%. The silver nanoparticle polysaccharide system showed a good antimicrobial behavior also when studied against saliva.Di Giulio lac-nAg)  for hindet al. [in vitro antifungal efficiency and physical properties no worse than those of the material without silver were found for composites with concentrations of silver nanoparticles of up to 40 ppm.To reduce the colonization of denture soft lining materials, silver nanoparticles ranging from 0 to 200 ppm were incorporated into composites by Chladek et al. . The effTo prevent infections, silver, iodine, polyhexamethylene biguanide (PHMB) or chlorhexidine are the most commonly used antimicrobial agents in wound dressings. Infected wounds can have serious local and systemic complications. While an important local complication of infected wounds is a non-healing wound, the infection of the surrounding tissues may cause a skin infection or an acute or chronic bone infection. If bacteria get into bloodstream, they may instead cause infection in other areas of the body.et al. [®) loaded with different combinations of cationic and anionic antiseptics, specifically PHMB, Povidone-iodine (PVP-iodine), Octenidine-dihydrochloride (OCT), Betaine and Phenoxyethanol. Altrazeal® is based on dehydrated hydrogel modified into small particles containing a poly-2-hydroxyethyl-/poly-2-hydroxypropyl (pHEMA/pHPMA)-methacrylate backbone and terminal hydroxyl group. In vitro model of porcine wound, contaminated with S. aureus, evidenced that an individually shaped and controlled antibacterial effect may be obtained using the same type of wound dressing but different combinations of cationic and anionic antiseptics. In particular, the highest bacterial reduction was found for PVP-iodine at 24 h followed by OCT at 48 h. Altrazeal® loaded with PHMB alone showed a stable bacterial load up to 120 h while by using the combination PHMB and Betaine, a bacterial growth inhibition during the first 48 h and a slightly increase of bacterial number at 120 h was observed.Forstner et al.  have invThe development of biocidal polymers by introduction of antimicrobial functional groups is proving to be an alternative to the use of low molecular weight antimicrobials. Specially, polymers containing positively charged groups such as phosphonium, biguanide or quaternary ammonium groups have likelihood to succeed in preventing infections and combating multi-drug bacterial resistance.Carmona-Ribeiro and Dias de Melo Carrasco have extensively discussed recent research on macromolecular antimicrobial agents such as cationic polymers, peptoids, peptides and lipids . In thiset al., in their review [Chitosan has been widely used in agriculture, in medicine and in industrial applications as a macromolecular antimicrobial agent. To improve CS antimicrobial properties and its solubility in water over a large range of pH values, different chemical modifications aimed at obtaining CS derivatives have been investigated. Tan r review , have atet al. have investigated the interaction between an antibacterial agent and the lipid bilayer by using a surface sensitive optical waveguide spectroscopy [Generally, the interaction between an antibacterial cationic agent and the bacterial cell wall leads to disruption of lipid bilayer and then to an enhancement of membrane permeability. Horvath troscopy . SpecifiFluorescent labeling is an important technique playing a key role in biological imaging and biological monitoring. Quantum dots (QDs) are a novel class of inorganic fluorophores which are gaining wide recognition as a result of their unique optical and chemical properties.et al. obtained multifunctional hybrid microspheres with magnetic, antibacterial and fluorescent function by using poly(glycidyl methacrylate) (PGMA) as a cladding material [2-PGMA). After further surface functionalization with poly(hexamethylene guanidine hydrochloride) (PHGH), the microspheres exhibited excellent antibacterial activity against both Gram-positive and Gram-negative bacteria. Finally, the authors provided the hybrid microspheres with targeted localization and biological monitoring functions by the quantum dot CdTe introduction.Xiao material . In partRadiology is another monitoring technique widely utilized to evaluate dental restoration and long-term stability of dental implant. Therefore radiopacity property of dental materials is very important. An adequate radiopacity allows dentists distinguishing existing restorations and primary caries, evaluating margins, overhangs and major voids and identifying any recurrent caries.et al. [In order to endow a dental material with antibacterial and radio-opaque functions, He et al.  have incThe main advantage of using antimicrobial agent-releasing systems is the drug release targeted to the infection site. However, most of these systems have a short persistence of antimicrobial action, due to rather quick diffusion controlled release. In addition, the low systemic drug concentration reached after few days of drug release can increase the risk for selecting of antimicrobial resistant strains.Candida species are the most common fungi isolated from device-related infections, and biofilms formed by these fungal organisms are especially associated with drastically enhanced resistance against many antimicrobial agents. This resistance contributes to the persistence of the infection despite antifungal therapy.Fungal infection has become a leading cause of nosocomial infections. The Candida albicans less susceptible to PDI, high doses of photosensitizer and light energy are needed to treat infectious diseases related to this strain.Antimicrobial photodynamic inactivation (PDI) has been developed as an alternative therapeutic treatment against bacterial and fungal local infections. This treatment is particularly utilized in case of drug-resistant strains. Being C. albicans, Chien et al. [C. albicans and three fluconazole-resistant clinical isolates, in planktonic and biofilm forms. The results showed that chitosan, after cell damage by PDI, was able to enhance the effect of PDI mediated by toluidine blue O. In addition, a reduction in the PDI treatment conditions needed to completely eradicate C. albicans was possible with increasing chitosan concentration or incubation time.To potentiate PDI efficacy against n et al.  studied et al. [Staphylococcus epidermidis than both the free drug and the polymer alone. The best results were obtained with the complex based on the lowest molecular weight polymer and containing a low drug content. This improved killing effect was attributed to the reduced size of the complex that allowed its efficient cellular uptake.Another valid approach developed to prevent the emergence of resistant pathogens involves the use of proper antimicrobial combinations having different mechanisms action. Particularly, advantages in the employment of an intrinsically antimicrobial polymer as a drug carrier, rather than of an inert one, have been exploited by Francolini et al.  to reducIn this special issue the promising performance of antimicrobial polymers in fighting microbial contamination onto surfaces and reducing multi-drug bacterial resistance were highlighted. We believe the future application of these polymers either in industrial or healthcare settings could result in an extremely positive impact not only at the economic level but also for the improvement of quality of life.


In recent decades, tumor surveillance by the immune system and its impact on disease outcomes in cancer patients in general and in breast cancer (BC) patients in particular has been documented. Natural killer (NK) cells are central components of the innate immunity and existing data indicate that they play a role in preventing and controlling tumor growth and metastasis. Their biological significance was first recognized by their ability to exert direct cellular cytotoxicity without prior sensitization. This is important in tumors, as transforming events are likely to result in downregulation of self-ligands and expression of stress-induced ligands which can be recognized by NK cells. Their activation also leads to secretion of stimulatory cytokines which participate in cancer elimination by several direct mechanisms as well as by stimulating the adaptive immune system. In this regard, it was recently revealed a dendritic cell (DC)-NK-cell crosstalk which provides another novel pathway linking innate and adaptive immunity. In addition, NK cells are feasible targets of stimulation in immunotherapeutic approaches such as antibody-based strategies and adoptive cell transfer. Nevertheless, NK cells display impaired functionality and capability to infiltrate tumors in BC patients. This review compiles information about NK-cell biology in BC and the attempts which aim to manipulate them in novel therapeutic approaches in this pathology. Natural killer (NK) cells have been first described as innate effector lymphocytes and their biological significance has been first recognized by their ability to exert direct cellular cytotoxicity without prior sensitization  mice lack T, B, and NK cells which makes them a suitable model for tumor engraftment and to investigate the role of NK in tumor growth and metastasis  cells were most efficient in the formation of large tumors within 2–3 weeks in all mice. Moreover, activated NK cells inhibited tumor formation and organ metastasis, suggesting that NK cells are responsible for inhibiting the formation of progressively growing rapid large tumors of BC cells in SCID mice  activities. In contrast, NOD/SCID/γ+, basal subtype, normal breast-like, and the recently introduced Claudin-low subtype, based on their molecular characteristics . However, when quantity was normalized to target cell number, there was a relationship between CD16-158V/F polymorphism and the killing efficiency of CD16+CD56+ cells  are also important therapeutic strategies for cancer. The mAb trastuzumab has been approved for clinical use and has become a mainstay for HER2-positive BC (Slamon et al., Based on its ADCC mechanism, the anti-tumor efficacy of trastuzumab can be enhanced by means of combination with other immunomodulatory approaches such as chemotherapy, radiotherapy, targeted therapy agents, vaccines, or other immunomodulators. Table in vitro that it triggers NK-cell-mediated ADCC (Roberti et al., Experience with trastuzumab arouses the hope that mAbs could be used in other BC subtypes. Particularly important is the need for target therapies in basal BC as these patients are not likely to benefit from anti-estrogen or anti-HER2 therapy, and first-line treatment usually consists of conventional cytotoxic chemotherapy. Although basal BCs are quite sensitive to chemotherapeutic regimens in the preoperative setting, they are nevertheless associated with poor long-term outcomes (Liedtke et al., + T-cell expansion and further differentiation into IFN-γ-producing effector cells (Qi et al., Besides effector functions for tumor elimination and cytokine production, NK cells have mechanisms for stimulating the adaptive immune system, thus, being instrumental for enhancing antigen processing and presentation. Recent studies illustrate the potential of NK cells to increase the immunostimulatory capacity of DCs. It was demonstrated that NK cells can efficiently promote maturation and differentiation of DCs toward a Th1 profile (Mailliard et al., The compiling evidence of NK-cell biology in BC sheds light on the mechanisms involved in NK-cell tumor evasion and highlights the role of NK cells in the control of invasive breast tumors. However, regarding tumor strategies to overcome its elimination by innate immunity, it stresses the necessity of attempts which aim not only to stimulate NK cells directly but to target the tumor counterattack mechanisms in order to obtain clinical success in novel therapeutic approaches to battle this complex pathology.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.