Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for ~18 million deaths annually (WHO data). The term CVD gathers a group of different disorders involving the heart, its constituent structures, and the blood vessels. Among CVDs, coronary heart disease and stroke are responsible for four out of five CVD-related deaths, and one third of these deaths occur prematurely in people aged 70 or younger. The progressive or sudden obstruction of the coronary arteries is responsible for the onset of myocardial infarction which initiates a detrimental cascade of events finally leading to heart failure. More in detail, heart failure results from the continuous remodeling of the scar tissue replacing the beating heart muscle in the infarcted region, and represents a chronic condition in which the heart muscle progressively loses its ability to pump enough blood to fulfill the needs of all the body's compartments. Heart failure thus represents the main cause of morbidity and mortality of myocardial infarcted patients in the long term. Within this context, cardiac tissue engineering/regenerative medicine (TERM) strategies could arise as cutting-edge therapies in the management of myocardial infarcted patients, opening the way to the possibility to replace the damaged heart tissue and recover its functionality. Such an approach could effectively represent a valid alternative to the gold standard heart transplantation, encompassing all issues related to donor shortage and the need for life-long administration of immunosuppressive therapies. Among other common cardiovascular diseases, we also recall valve heart diseases and cardiomyopathies. It must be highlighted the strong associations existing between different cardiovascular diseases, such as coronary heart disease and valvular heart disease, cardiomyopathy and heart failure. This observation indicates that a multiple regenerative medicine approach, which considers different diseases, could be an effective strategy in the management of CVDs. The Research Topic “Advanced Therapies for Cardiac Regeneration” aims at presenting a series of articles summarizing the latest research updates on cardiac TERM approaches which combine cells, biomaterials, hydrogels, tissue engineered scaffolds/patches and physico-chemical stimuli to achieve the ultimate goal of regenerating the injured heart tissue. The issue is comprised of 19 peer-reviewed manuscripts  derived from the many fields involved in the topic, namely (bio)materials science and engineering, biology, biotechnology, and biomedical engineering.Montero et al. presents an in-depth overview of the specific characteristics of the myocardium that determine the needs and requirements cardiac TERM has to meet, with particular emphasis on heart tissue components, architecture and biophysical properties. The authors also briefly revise the key components required to design new cardiac TERM approaches, namely cells, materials, maturation stimuli, and scaffold fabrication techniques. Given the central role of biomaterials in the establishment of cardiac TERM therapies, Bar and Cohen focus their review on the current application of biomaterials in the field of cardiac regeneration, mainly discussing their use as forming materials for nano-carriers and matrices for cardiac regeneration induced by biomolecule release, injectable hydrogels for cell delivery, and cardiac patches. Further and more detailed insight on the use of specific biomaterials in cardiac TERM are provided by Cattelan et al. and Gonzalez De Torre et al., with particular emphasis on their application as hydrogel constituents. Being three-dimensional highly hydrated networks showing mechanical properties similar to soft tissues, hydrogels hold great promise in cardiac TERM. Cattelan et al. elucidate the promising properties of alginate in cardiac regeneration strategies and the outcomes of clinical trials in which this material has been tested to treat myocardial infarcted patients. An additional demonstration of the suitability of this material for cardiac TERM is provided by Bloise et al. who develop alginate hydrogels for the controlled release of immunomodulatory and reparative cytokines (anti-inflammatory interleukins 4/6/13 and colony-stimulating factor) to direct immune cell fate and control the wound healing process in the ischemic heart. The therapeutic ability of the proposed treatment has been proved in rat models by macrophage polarization toward healing and the improved global cardiac functionality. Differently, Gonzalez De Torre et al. discuss on the potential of elastin-based biomaterials as constituents of hydrogel scaffolds, injectable systems, or complex devices  to treat CVD-affected patients. In this regard, Fernàndez-Colino et al. investigate the use of elastin-like recombinamers as forming materials of small caliber compliant vascular grafts. The authors describe material processing into macroporous three-dimensional structures favoring cell homing, extracellular matrix (ECM) deposition and endothelium development, while exhibiting non-thrombogenicity and elastic properties mimicking the native elastin. Graft textile components are finely designed to confer proper suture retention, long-term structural stability, burst strength and compliance. The proper selection of the biomaterials used as constituents of cardiac scaffolds/patches/devices or hydrogels thus represents the first step toward the engineering of successful regenerative strategies for the management of CVD-affected patients. Indeed, biomaterials strongly affect the possibility to provide the resulting devices with proper biological signals and physical stimuli to achieve the final goal of recovering the initial organ/tissue functionality. In this regard, Belviso et al. report on the potential of decellularized human skin as scaffold to restore the native ECM which plays a pivotal role in guiding heart compliance, cardiomyocyte maturation, and function. The authors argue that a dermal matrix could effectively represent a viable alternative biological scaffold in cardiac TERM, with the key advantages of being autologous, easily accessible, and cost-effective. On the other hand, decellularized organ-derived ECM (dECM) can also be exploited to design hydrogels, thus combining the ECM characteristic pro-regenerative properties with the possibility to shape the material according to specific requirements. Liguori et al. investigate the differences among dECM hydrogels prepared starting from cardiovascular tissues of different origin, namely left ventricle, mitral valve, and aorta. The authors demonstrate that the source of dECM plays a pivotal role in determining cell differentiation and vascular network formation, because of the different molecular and biomechanical cues provided to cells. For instance, ventricular dECM hydrogels exhibit more robust vascular network formation, while aorta-derived dECM drives adipose-derived stromal cell differentiation toward a myogenic phenotype in the absence of TGF-β1 supplement to the culture medium. The readers can also find a thorough survey on the use of decellularized cardiac ECM as biomaterial for cardiac TERM in the review by Maghin et al. Besides the physical stimuli provided by the designed cell-culture platforms through their architecture and their mechanical properties, the possibility to mechanically stimulate cellularized constructs by mimicking the native milieu also promotes the development and maturation of cardiac tissue analogs, as described by Massai et al. The authors develop an automated bioreactor platform for tuneable cyclic stretch of cellularized samples coupled with a specific set-up for the in situ monitoring of the mechanical response of in vitro engineered cardiac tissues. Another key form of stimulus playing a central role in cardiac regeneration is represented by biochemical cues, as thoroughly surveyed by Cassani et al. Particularly, the authors mainly gather their attention on the crucial need of specific carriers  to deliver such biochemical cues, which would ensure an efficient delivery of the therapeutic cargo, inhibit its metabolic inactivation, and avoid potential side effects. According to a different approach, proper biochemical stimuli driving cardiac repair and regeneration can be also applied by cell-secreted soluble factors. The literature review by Maghin et al. reports on the therapeutic profiling of stem cell secretome, with regard to the cardio-active ability of cell-released extracellular vesicles, including exosomes carrying cardioprotective and regenerative RNA molecules. An in-depth analysis of the potential and the mechanisms of action of miRNAs as mediators of cardioprotection is provided by Nazari-Shafti et al. The authors highlight the crucial need to identify all miRNAs involved in cardioprotection and remodeling, as well as their composition and exact mechanism of action in view of their application in clinical practice. A class of miRNAs has also been reported to drive the direct reprogramming of fibroblasts into cardiomyocyte-like cells. In the perspective of a clinical translation of this approach, Paoletti et al. test the capability of miRcombo  to guide the direct reprogramming of adult human cardiac fibroblasts into induced cardiomyocytes, as previously observed with murine fibroblasts. Other repair and regenerative strategies currently explored in literature target different aspects of the cascade initiated by myocardial insults. For instance, Seclì et al. concentrate on the different role exerted by chaperone proteins in the healthy and injured heart. In detail, in the healthy heart chaperone proteins contribute to the regulation of myocardial physiology, while under stress conditions or during cell damage events, they are secreted by myocardial cells and induce inflammation and cardiomyocyte apoptosis. Blocking chaperone activity has been already demonstrated to produce beneficial effects on heart function in preclinical models. Hence, combining the release of specific antibodies blocking extracellular chaperones with TERM approaches may represent a future innovation to treat infarcted patients. Differently, Bigotti et al. discuss the possible exploitation of agrin as mediator of cardiac regeneration. Recently published works have demonstrated that agrin can promote heart regeneration in murine models through cardiomyocyte de-differentiation and proliferation. Moving from these data, the authors survey the currently available literature on this topic, and critically discuss on the potential clinical translation of agrin-based therapies.To better contextualize the Research Topic, the review by Mazzola and Di Pasquale provide an overview on the differentiation potential of iPSCs toward cardiomyocytes and their application in cell-based therapies, highlighting the typical drawbacks of such approaches in terms of poor cell retention and risks of occurrence of adverse effects . A possible route to overcome these issues consists in combining the use of iPSCs with bioengineering technologies which can retain cells in the therapeutic target while favoring their phenotypical maturation and integration in the host tissue. iPSCs can also be successfully used as cell source in the establishment of cardiac tissue models. In addition, their autologous origin can be exploited to recapitulate in vitro human cardiac pathophysiology. In this regard, Jelinkova et al. report the design of an in vitro human model of the cardiac tissue of Duchenne muscular dystrophy (DMD)-affected patients. DMD is a severe genetic disorder associated with progressive dilated cardiomyopathy which culminates with heart failure onset, finally leading to patient death. The authors demonstrate that the designed cardiac models effectively recapitulate DMD-induced functional defects and cardiac wasting, thus offering a promising tool for a better understanding of DMD-associated cardiomyopathy and its treatment.Last but not least, a new chapter in the TERM field has been opened over the last two decades with the discovery of induced pluripotent stem cells (iPSCs). These cells offer the advantage of being of autologous origin, while retaining the proper characteristics of embryonic stem cells, thus completely overcoming both ethical issues and rejection risks. Taghizadeh et al. exhaustively review the different types of biomaterials applied in the treatment of valvular heart diseases which represent another CVD with high incidence in developed countries, and in particular among the elderly. In the U.S. about 28,000 deaths are due to heart valve disease every year, with approx. the 60% of deaths due to disorders involving the aortic valve (data from the Centers for Disease Control and Prevention). Strict requirements in terms of material physico-chemical properties should also be fulfilled when vascular stents are engineered. Learning from a recent FDA report dealing with the adverse cardiac events and thrombosis observed at the 2-year follow-up on the first FDA-approved bioresorbable vascular stents, Pacharra et al. characterize a series of polyethylene glycol-functionalized poly-L-lactide-co-ε-caprolactone copolymers as a new generation of stent-forming materials, with improved cyto- and hemo-compatibility. As an outcome of the research work, the authors identify promising candidates for cardiovascular implant fabrication, with the additional advantage of being suitable for an easy translation of the synthesis procedure (a one-step synthesis strategy) toward industrial practice.Finally, to complete our survey on the topic, The reader can easily appreciate from the brief summaries of the contributions comprised in this issue that the addressed Research Topic is broad in nature and represents a still open question in the TERM field. The definition of regenerative approaches to fight the detrimental consequences of CVDs is still in its infancy. However, the cross-contamination of new ideas coming from complementary but not overlapping fields, as highlighted in the papers published within this issue, will likely lead in the future to the effective and successful repair and regeneration of injured myocardial tissue.MB wrote the editorial, which was revised, proof-read, and approved by all authors.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.


Ischemic stroke is a worldwide major cause of mortality and disability and has high costs in terms of health-related quality of life and expectancy as well as of social healthcare resources. In recent years, starting from the bidirectional relationship between autonomic nervous system (ANS) dysfunction and acute ischemic stroke (AIS), researchers have identified prognostic factors for risk stratification, prognosis of mid-term outcomes and response to recanalization therapy. In particular, the evaluation of the ANS function through the analysis of heart rate variability (HRV) appears to be a promising non-invasive and reliable tool for the management of patients with AIS. Furthermore, preclinical molecular studies on the pathophysiological mechanisms underlying the onset and progression of stroke damage have shown an extensive overlap with the activity of the vagus nerve. Evidence from the application of vagus nerve stimulation (VNS) on animal models of AIS and on patients with chronic ischemic stroke has highlighted the surprising therapeutic possibilities of neuromodulation. Preclinical molecular studies highlighted that the neuroprotective action of VNS results from anti-inflammatory, antioxidant and antiapoptotic mechanisms mediated by α7 nicotinic acetylcholine receptor. Given the proven safety of non-invasive VNS in the subacute phase, the ease of its use and its possible beneficial effect in hemorrhagic stroke as well, human studies with transcutaneous VNS should be less challenging than protocols that involve invasive VNS and could be the proof of concept that neuromodulation represents the very first therapeutic approach in the ultra-early management of stroke. Cerebrovascular disease ranks as one of the leading causes of disability and mortality worldwide ,2. AcuteAutonomic nervous system (ANS) dysfunction and ischemic stroke have an intricate and deep interconnection. The disruption of autonomic regulatory pathways may determine clinical complications during both the acute and chronic phases of stroke, leading to a worse prognosis . On the The nature of the cause–effect relation between ANS dysfunction and stroke is currently not entirely established. Nevertheless, even a partial unravelment of such an ambivalent role played by ANS, linking both risk factors and clinical features of stroke, may be essential, constituting a premise for the better therapeutic management of primary and secondary prevention of AIS in clinical practice. In this context, we here review the emerging evidence linking the ANS to the pathological process of stroke, the effects of ANS dysregulation after the ischemic event and the potential role of heart variability (HRV) analysis, a non-invasive method to assess ANS function, in the diagnostic workup of stroke patients. In addition, we will discuss the promising role of invasive and non-invasive vagal stimulation as a therapeutic approach in acute and chronic phases of stroke.The 2009 standardized case–control INTERSTROKE study in 22 countries worldwide found that hypertension, current smoking, diabetes, abdominal obesity, poor diet and physical inactivity accounted for more than 80% of the global risk of both ischemic and hemorrhagic stroke; other risk factors included excessive alcohol consumption, dyslipidemia, cardiac causes  and psychosocial stress/depression . The majHeart rate variability (HRV) is a non-invasive method to explore cardiac ANS function; thus, it is not surprising that the majority of the scientific literature employs this tool to predict and monitor cardiovascular disease and associated risk factors. HRV is based on the identification of oscillatory rhythmic components: the low-frequency oscillation (LF—ranging from 0.04 to 0.15 Hz) and the high-frequency oscillation (HF—ranging from 0.15 to 0.4 Hz and synchronous with respiration). The power of the HF band is determined by parasympathetic modulation and the power of the LF band reflects sympathetic modulation and cardiac autonomic outflows by baroreflexes. Changes in sympatho-vagal balance can be detected by the LF/HF ratio ,12. CondDifferent reports suggest that reduced HRV, assessed during both daytime and night-time recordings, is strongly associated with incident stroke in apparently healthy subjects with no history of cardiovascular disease or stroke  and in tHypertension is the most prevalent risk factor of ischemic stroke . An assoAF is a very common cardiac arrhythmia, affecting approximately 1–3% of the general population and whose prevalence rises with age . Its assLarge vessel atherosclerosis, involving both extracranial and intracranial arteries, is reported to be the cause of approximately 15% of ischemic strokes . The patRecent reports suggest that up to one third of stroke patients suffer from diabetes mellitus (DM) . The ANSReduced HRV has also been shown to be inversely associated with carotid stenosis severity , suggestG20210A mutations, deficiencies of protein C, protein S and anti-thrombin) and acquired hypercoagulable disorders (notably anti-phospholipid syndrome and neoplastic thrombophilia) may be a commonly unrecognized cause of ischemic stroke, particularly in young patients [Finally, it would be helpful to mention that ischemic stroke may be the initial manifestation of hematologic diseases and also that both inherited   and its Physiological studies on humans, examining the effect of either stimulation or inactivation of the two hemispheres, suggest a crucial role of the right hemisphere in establishing sympathetic tone, and of the left hemisphere in determining parasympathetic tone ,82. A hiOther possible predictors of ANS dysfunction are represented by the severity of stroke, as expressed by the National Institutes of Health Stroke Scale (NIHSS) ,98, and It is not clear whether the autonomic derangement visible in the acute phase of AIS can be completely reversible. Korpelainen et al. suggested that, within 6 months after AIS, the circadian rhythm of HRV may be restored , but theThe most recent research has shown that the analysis of HRV is a useful tool not only in risk stratification for stroke onset but also as a marker of post-stroke long-term outcomes. Abnormal HRV in the acute phase of stroke has been shown to predict unfavorable outcomes in the first 3 months after the ischemic event ,106. TheAs we have previously described, ANS dysfunction may increase the risk of AIS; long-term autonomic derangement due to AIS could itself be a cofactor of the risk of recurrence of cerebrovascular events in stroke survivors. In the paper by Nayani et al., patients with ANS dysfunction had greater stroke severity at discharge, and more frequent cerebrovascular recurrences at 3-month follow-up, compared with patients without ANS dysfunction . To concThe molecular mechanisms and structural changes that involve the brain ischemic tissue have been extensively described over the years. Due to the rapid progression of the ischemic injury cascade, patients with AIS require immediate medical attention and urgent care. Thus, pharmacological (intravenous thrombolysis (IVT)) and mechanical (thrombectomy) treatments are recommended for selected patients affected by AIS and should be started as soon as possible. Recombinant tissue plasminogen activator (rt-PA) must be administrated within 4.5 h from the onset time of stroke symptoms, whilst surgical interventions should be performed within the first 6 h. However, less than one third of patients are eligible for these treatments ,120,121.ANS is extensively involved in the initiation, progression and outcome of stroke and, starting from the study of this intimate connection, it was possible to draw the basis for new therapeutic approaches. In particular, several pathophysiological aspects of stroke  are influenced by the activity of the vagus nerve . In addiThe putative role of VNS goes beyond the function of re-establishing sympatho-vagal balance in cardiovascular autonomic control, lost following AIS, as seen in the previous chapter ,132. It Neuroinflammation plays a major role in the pathophysiology of stroke and represents one of the major targets for the treatment of stroke in current clinical and experimental research. The strong pro-inflammatory stimuli deriving from ischemic tissue induce the activation of microglia, astrocytes and endothelial cells, which start to produce pro-inflammatory cytokines. Leukocytes are recruited from the vessels and infiltrate the tissue by extravasation. Thus, cytotoxic substances are produced in large quantities  and oxidative stress is aggravated ,138. TheThe recruitment of the same anti-inflammatory cholinergic pathway seems to have antioxidant effects, as well. As a matter of fact, VNS significantly counteracted the oxidative process through the regulation of glutathione and superoxide dismutase levels in a rat model of focal cerebral ischemia and reperfusion . α7nAchRRecent studies suggest that in addition to limiting the damage caused by inflammation and oxidative stress, VNS exerts direct antiapoptotic effects. The protein levels of both α7nAChR and p-Akt were significantly higher and the expression of cleaved caspase-3 was significantly less in the ischemic tissue of the VNS-treated group than in the untreated group . FurtherIn addition to the recruitment of mechanisms for neuroprotection in the acute phase of stroke, VNS has been shown to restore neuronal function, enhancing neuroplasticity and stimulating neurogenesis. Mounting evidence from studies has demonstrated that VNS, especially the non-invasive type, boosts axonal plasticity and reorganization of synaptic connectivity ,156. In The efficacy of VNS has been demonstrated both in attenuating tissue damage, when performed in the acute phase of ischemic stroke, and in restoring lost functions, when applied during the chronic rehabilitation phase. VNS performed 30 min after reperfusion determined an approximately 50% reduction in infarct volumes compared to the untreated control group in all preclinical models of cerebral ischemia and reperfusion ,178,179.Chronic impairment of the arm and hand is one of the most common consequences of stroke. Therefore, an additional effort in stroke research aims to develop new approaches targeting functional recovery that can be applicable and effective even long after the event. VNS has proved to be effective not only in the acute phase but also as a plasticity-enhancing therapy coupled with rehabilitation. In animal models, VNS performed during rehabilitative tasks restored pre-lesion successful rates, whereas training without VNS failed ,184. TheIn humans, two randomized clinical trials confirmed the applicability of VNS paired with rehabilitation to improve limb function in chronic stroke patients with moderate to severe upper limb impairment ,166. StaThe results of preclinical and clinical studies concerning non-invasive stimulation are particularly encouraging. However, while clinical trials on the effect of VNS in the chronic phase of stroke are numerous, to date, no studies on the application of VNS in humans during the acute phase of stroke are available. Since non-invasive VNS does not require surgical implantation and, given the proven safety of the treatment in the subacute phase, the recruitment and applicability of human studies with transcutaneous VNS should be less challenging than protocols that involve invasive VNS. Further research with similar populations, experimental design and timelines is needed to establish whether the efficacy of the two types of treatment is comparable. If so, non-invasive VNS could be introduced as an add-on treatment to the standard therapy in the acute phase of AIS, thus increasing the success rate of recanalization therapies and reducing reperfusion injury. Furthermore, considering the ease of its use and its possible beneficial effect in hemorrhagic stroke as well, non-invasive VNS could represent the very first therapeutic approach in the ultra-early management of stroke.In the last few decades, researchers and clinicians have dedicated tremendous effort to reducing morbidity and mortality in ischemic stroke patients. However, the mortality rate remains considerably high and the chronic consequences of stroke impact severely the health-related quality of life of AIS survivors. There is a bidirectional loop between stroke and ANS, as the major risk factors for AIS are consequences of cardiovascular autonomic dysfunction and central nervous system lesions can lead to alterations in cardiovascular autonomic control. Moreover, ANS dysfunction following acute cerebrovascular events is strongly linked to poor prognosis in stroke patients. Therefore, cardiovascular autonomic control assessment by means of HRV analysis could have a key role in risk stratification for ischemic stroke and in prognosis of mid-term outcomes and clinical response to recanalization therapy. More recently, neuromodulation interventions, which aim to enhance the parasympathetic modulation of ANS, have emerged as a novel strategy for the management of stroke patients, both in the acute and chronic phases, due to their pleiotropic effects. In the latter area, little but promising data are available and an intriguing possibility for progress is offered by this new non-invasive stimulation methodologies.


On behalf of the scientists and surgeons of one of the biggest orthopedic institutes in Italy, located in Milan, we feel the urge to share our position about the recent warnings on the use of anti-inflammatory drugs (NSAIDs) in this coronavirus “era.” Similarly to the antihypertensive drugs angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and beta blockers, and the oral antidiabetic drugs thiazolidinediones, some author have suggested that NSAIDs, particularly Ibuprofen, may induce increased sensitivity to more severe clinical features in coronavirus (Covid-19) infection.NSAIDs are widely used worldwide. In musculoskeletal medicine, they keep under control osteoarthritis, tendinopathies, bursitis, muscle soreness, radicular pain, and others. In Italy, about 12 millions of adults have osteoarthritis or rheumatoid arthritis, a number estimated to grow substantially in the next years [The warning about the possible role of NSAIDs in worsening the severity of Covid-19 infection was very powerful because it was spread by a representative of the national medical authority of one of the European countries strongly interested by this pandemic. The warning also suggested to the population already under anti-inflammatory drugs to ask their general practitioners for advice, unleashing a wave of panic among the population, especially through social media. Other official communications by the national health systems and emergency management agency (EMA)  attempteThe effects of this warning has been very deleterious on our patients, especially on those who need to use these drugs for long periods for their medical conditions: NSAIDs allow these patients to lead a good quality of life, although we acknowledge that the prolonged use of these drugs may cause side effects, including kidney failure, liver failure, gastric ulcers, asthma attacks, heart and circulatory problems, and a partial immune depression .Being based in Milan, we, more than others, are directly experiencing the devastating effects of this pandemic. We have been ready, and we still are now, to take any useful measures to reduce its impact on our population, without underestimating any possible new scientific information. Nevertheless, albeit in the midst of a general intellectual and emotional confusion, we try to maintain our scientific judgment, and would like to underline the danger of unproven and unfounded information, especially when spread out through mass and social media, which directly reach the general population without any filter.p = 0.049). Nevertheless, given the lack of consistent results about the direct effect of NSAIDs on the immunological response and the lack of controlled randomized studies in human, the authors could not conclude a direct effect of NSAIDs in the development of the complications of CAP [The current body of scientific literature reports just a few articles analyzing the relationship between exposure to NSAIDs and possible complications in patients with pneumonia. A recent article investigated whether exposure to NSAIDs prior to hospital admission among patients suffering from community-acquired pneumonia (CAP) was associated with the development of pleural complications or a lung abscess. NSAIDs intake was independently associated with the development of pleuroparenchymal complications , pointing in any case out that, in this period, other NSAIDs and paracetamol could also mask the pyrexial signs of COVID-19, resulting in a delayed diagnosis of the infection.The risks deriving from the use of NSAIDS and the Covid-19 infection should be analyzed from two different perspectives: (i) risks related to exposure to NSAIDs before the patient has been infected and (ii) risks related to the use of a drug during the COVID-19 infection. These two scenarios are dramatically different, and pose different scientific questions, which should be addressed by adequate studies. In other words, would the administration of this drug before being infected causes the same effect of taking it during the infection period? We also have to distinguish between the possible increase in susceptibility to the virus, especially in terms of severity, and the increase in aberrant host response.Patients continue to produce the virus for weeks: the treatment of infected patients with immunosuppressant such as corticosteroids might increase the amount of virus replication . TherefoAs recently stated , there iFollowing the invitation by EMA for timely epidemiological studies to provide adequate evidences on the effects of NSAIDS on prognosis of Covid-19 infection, our institute is about to start an epidemiologic study using our pathology registries . In part


The gut is an efficient barrier which protects against the passage of pathogenic microorganisms and potential harmful macromolecules into the body, in addition to its primary function of nutrient digestion and absorption. Contrary to the restricted macromolecular passage in adulthood, enhanced transfer takes place across the intestines during early life, due to the high endocytic capacity of the immature intestinal epithelial cells during the fetal and/or neonatal periods. The timing and extent of this enhanced endocytic capacity is dependent on animal species, with a prominent non-selective intestinal macromolecular transfer in newborn ungulates, e.g., pigs, during the first few days of life, and a selective transfer of mainly immunoglobulin G (IgG), mediated by the FcRn receptor, in suckling rodents, e.g., rats and mice. In primates, maternal IgG is transferred during fetal life via the placenta, and intestinal macromolecular transfer is largely restricted in human neonates. The period of intestinal macromolecular transmission provides passive immune protection through the transfer of IgG antibodies from an immune competent mother; and may even have extra-immune beneficial effects on organ maturation in the offspring. Moreover, intestinal transfer during the fetal/neonatal periods results in increased exposure to microbial and food antigens which are then presented to the underlying immune system, which is both naïve and immature. This likely stimulates the maturation of the immune system and shifts the response toward tolerance induction instead of activation or inflammation, as usually seen in adulthood. Ingestion of mother's milk and the dietary transition to complex food at weaning, as well as the transient changes in the gut microbiota during the neonatal period, are also involved in the resulting immune response. Any disturbances in timing and/or balance of these parallel processes, i.e., intestinal epithelial maturation, luminal microbial colonization and mucosal immune maturation due to, e.g., preterm birth, infection, antibiotic use or nutrient changes during the neonatal period, might affect the establishment of the immune system in the infant. This review will focus on how differing developmental processes in the intestinal epithelium affect the macromolecular passage in different species and the possible impact of such passage on the establishment of immunity during the critical perinatal period in young mammals. The gut, in addition to its primary function of nutrient digestion and absorption, constitutes an effective barrier to protect against the invasion of pathogenic microorganisms and passage of potential harmful macromolecules into the body. However, as opposed to the restricted macromolecular passage in the adult, enhanced transfer of macromolecules across the immature intestinal epithelium takes place during the fetal and neonatal periods . The higThis property of the intestinal epithelium is largely lost with the progress of development and maturation, during the fetal and neonatal periods, until macromolecular transepithelial transfer ceases at the time of the so-called “gut closure.” In the mature intestine, after gut closure, some macromolecular transfer still takes place, but the extent of transfer is limited and it is restricted in terms of intestinal area, with transfer occurring mainly in the follicle-associated epithelium, especially over the membranous cells (M-cells), overlaying the immune-cell rich regions in the small intestine known as the Peyer's patches . Recent This review will focus on the developmental processes taking place in the intestinal epithelium, highlighting the high macromolecular passage during the critical perinatal period and the impact of such passage on the development of immunity in the young, with possible effects later in life. In addition, the review will describe the differences in timing and amount of such macromolecular transfer between species, in particular those used as animal models for humans, and the impact this could have when comparing different species.The extent and time period of macromolecular transfer over the immature intestine, before gut closure, is dependent on animal species . In euthIn the lagomorph rabbit and in precocious rodent species, i.e., the guinea pig, macromolecular transfer takes place from uterine secretion via the everted yolk sac during the fetal period , 29. Evein utero during the late fetal period, via the endocytic cells of the everted yolk sac endoderm. However, this is quantitatively less important compared to the postnatal intestinal transfer , highly endocytic fetal-type enterocytes express FcRn receptors that bind and mediate the transepithelial transfer of IgG, as well as minor quantities of other milk proteins , 47–49. In contrast, in the distal small intestine (ileum), the fetal-type epithelium internalizes luminal material via the apical endocytic complex and forms large digestive vacuoles that make up most of the cytoplasmic content , 52, allAt about 2 weeks of age, when pups open their eyes and start to become interested in nibbling solid food in addition to suckling milk, adult-type epithelial enterocytes with drastically reduced endocytic capacity and decreased FcRn expression appear in the crypts and move up the villi, in both the proximal and distal parts of the small intestine. By 3 weeks of age, at weaning , this maPrecocious intestinal maturation may be induced by premature weaning  or by luper se stimulates endocytosis  placenta. Due to the “simple” human placenta being formed by one epithelial layer between fetal and maternal circulations, it allows for maternal-fetal macromolecular transfer. This transfer is selective and takes place by receptor-mediated transcytosis, binding of IgG to FcRn, across the syncytiotrophoblast cells . The train utero over the intestine, since vacuolated enterocytes have been observed in the mid-distal intestine from about 13–14 weeks of fetal life  and consequently, their relative immaturity after birth. Thus, it is established that the lymphoid architecture forms prenatally in humans while it occurs mainly during the postnatal period in rodents, and for that reason newborn rodents resemble and can be used as a model for prematurity in humans. However, overall, in both cases, they develop with similar schemes .In rodents, and particularly in rats, development of inductor lymphoid aggregates, as Peyer's patches, and effector immune cells found scattered in the epithelial layer  or in the lamina propria , have been well characterized . Almost It has been proposed that T-cells are mediators of intestinal epithelial cell differentiation and they contribute to the maintenance of the epithelial barrier function . It is aIn ungulate pigs, the complex maternal-fetal placental separation prevents the transfer of maternal immune components, such as Ig or cytokines, to the fetus, thus making intestinal passage during suckling essential for development of the immune system. The structure of innate immunity in pigs is similar to that of other mammals, with natural killer cells (NK) being the key cell-type in early life, even though their functionality is only achieved after birth when immunoregulatory bioactive molecules appear during breastfeeding . AlthougIn human fetuses, B- and T-cells are found in the intestine as early as between 12 and 14 weeks gestation and their abundance and maturation increases until birth . It has In early life, the immaturity of the immune system is paralleled with the immaturity of the intestinal barrier function, resulting in a higher passage of antigens across the intestine. Thus, within this particular time period it is essential that a balance is reached between avoiding infection while at the same time activating the immune system and inducing antigen tolerance.In all mammalian neonates, breast milk is the principal nutrient source, but also the main driving factor in the maturation, activation and expansion of immune cells, particularly at mucosal sites. Even though 87% of breast milk content is water, the remaining 13% contains nutrients, as well as important bioactive compounds that have beneficial, non-nutritional functions . These cMaternal immune cells are also present in breast milk and have been shown to be able to survive and pass the intestinal epithelial barrier and translocate into the newborn's tissues –101. TraBreast milk contains an array of cytokines and growth factors involved in the newborn's immune system development, i.e., IL-6 stimulates differentiation of IgA-producing cells; IL-10 promotes tolerance to dietary and microbiota derived antigens, downregulates inflammation and promotes healing of damaged intestinal cells . In addi5-107 bacteria from the milk  cells . NKT celIn newborns, microbial antigens from the intestinal environment cannot be counteracted by a typical antibacterial and antiviral T helper type 1 (Th1) cell response, they induce a T helper type 2 (Th2) response instead, activating T cells and neutrophils via interleukin 8 (IL-8) . In contOverall, the time window for appropriate host-microbe cross-talk and therefore immune imprinting by microbiota, mainly takes place from birth to weaning, a period that involves a large expansion and diversification of the intestinal microbiota  and whicThe microbial and immunomodulatory components, at both the maternal and infant levels, are dependent on several factors which are considered as positive or beneficial when they promote the growth of bacterial genera, such as lactobacilli and bifidobacteria. Factors which positively shape the neonatal microbiome include vaginal delivery, maternal health status, long duration of lactation and the absence of antibiotic treatment during both pre- and post-natal periods. In contrast, factors that cause alterations in the infant's microbiota are C-section delivery, maternal disease status, especially infections that require antibiotics use; or absence of lactation that results in formula feeding , 121. ThThe microbial colonization of the intestine after birth will generate a diversity of new antigens that will play an important role in stimulating epithelial function and establishing the offspring's immune system . WeaningAt birth, when the offspring leaves the safety of the mother's womb, the intestinal barrier function is of utmost importance due to its dual function in preventing the passage of potentially harmful molecules and selectively allowing bioactive molecules and antigens to pass through. The barrier function is accomplished by several components including the luminal content, formed by endogenous secretions; the microbiota and the mucus layer; the intestinal epithelium itself, and the underlying immune system. The intestinal epithelium, a single layer of polarized cells, forms a key portion of the selective barrier, due to the endocytic capacity of the immature intestinal epithelium to select and shuttle those molecules which are allowed to pass through and reach the sub-mucosa and general circulation. Among the important macromolecules that require selective passage are the maternal antibodies, especially IgG, as well as the bioactive components in the amniotic fluid and breast milk and the microbial and dietary antigens .The transfer of antibodies from immune competent mothers, either during fetal life or after birth, independent of route, provides newborns with effective immune protection, especially taking into account that they will most likely be exposed to the same environmental pathogens. These passively acquired maternal antibodies enter the blood circulation and provide the same protective function as actively produced antibodies. The benefit of acquiring the mother's cumulative antibody repertoire has been shown in both human and animal studies , 124. FoIn addition to immune protection, passively acquired maternal antibodies reduce the metabolic costs of generating an active immune response in the offspring. Thus, passive immunity minimizes growth-suppressive effects that may be caused by activation of the immune system in species with rapid growth after birth . HoweverRecent results also indicate the existence of extra-immunological effects of maternal antibody transfer in addition to the classical protective immune effects, including direct effects on growth and functional development of the brain, the gut and possibly other organ systems in the neonate –132. TheIn addition to the passive immunization by maternal transfer of antibodies (IgG), milk cells and bioactive molecules; enhanced macromolecular passage over the intestines during the fetal and neonatal periods also results, although with some variation in quantity and timing between various species, in increased microbial and dietary antigen exposure and presentation to the underlying immature immune system. The outcome of antigenic exposure and the resulting immune response during this critical time window will be dependent on the maturity of the immune system and can either result in effector cell activation or tolerance induction.During the fetal and neonatal periods, intestinal luminal digestion is generally low due to the immature stomach and pancreatic function and the presence of milk-borne protease inhibitors , 55, 133The antigen format, free vs. immune-complex bound, is a factor of importance for the immunological outcome. Thus, transepithelial passage of antigens, bound in complex to maternal IgG and transported via FcRn, has been observed in both rodents and humans, and the subsequent interaction of these immune-complexes with lamina propria antigen presenting cells might be facilitated by their binding to Fc-receptors on these cells , 136–138Free dietary antigens may also pass materno-fetal barriers, since proteins experimentally fed to pregnant rats have been detected in antigenic form in both amniotic fluid and fetal blood . This maExperiments in neonatal rats have shown that anti-nutritional factors, such as lectins, block intestinal absorption , and oraChanges in paracellular permeability are caused by the disassembling of intestinal tight junctions, regulated by proteins such as claudins, zonulin, and occludin, causing a loosened pore diameter which in turn allows “larger” molecules to pass through –154. TigIn general, it would seem that the pathway of antigen transfer across the intestinal epithelial barrier could affect the immune system response. Transcellular passage of antigen complexes act as an educational pathway for the newborn's developing immune system, whereas paracellular passage would cause a primary immune response against those free antigens. Hence, during this critical period of early life, there is a continuous balance between tolerance induction and effector immune responses .The communication between the luminal content, including microorganisms and dietary macromolecules, and the immune system has been studied mostly with regards to their interaction via the intestinal epithelial cells and their receptors, while less emphasis has been placed on the direct interaction of antigenic molecules after passage across the intestinal epithelium. Moreover, the route for transepithelial passage of antigens, either by the transcellular endocytic pathway  or by paEarly studies on tissue transplantation suggest that the fetal/neonatal immune system is immature and predisposed to induction of tolerance . TransfeDietary macromolecules that induce Treg cells at weaning appear to be required to suppress the immune response to dietary antigens that are important in controlling inflammatory or allergic responses . MoreoveNatural weaning has been classified as “physiological inflammation,” with the recruitment of immune cells to the gut, mediated by chemokines and cytokines released by the enterocytes . T-cell As discussed above the route of transepithelial passage of antigens is important in determining the immune response/outcome, where transcellular passage with enterocyte processing and FcRn biding may be preferable compared to paracellular passage (leakage), which instead might result in inflammation and/or sensitization. The high endocytic activity of fetal-type enterocytes and increased macromolecular intestinal permeability might contribute to the increased susceptibility of preterm and term neonates to enteric infection and allergies. The immaturity of the intestine, together with the high epithelial endocytic activity, is possibly involved in the high risk of developing necrotizing enterocolitis (NEC) in preterm infants , 174.Selecting an appropriate animal species to serve as an ideal model to study the gut and immune system of human infants is not an easy task. The neonatal suckling rat , 82 and This review contributes to a better understanding of how macromolecules of dietary and microbial origin in the intestinal lumen are delivered to the underlying immune system and how this transfer changes during development in perinatal life. Any disturbances in the timing and/or balance of these parallel processes, i.e., intestinal maturation, gut immune maturation and luminal microbial colonization, e.g., due to preterm birth, perinatal infection, antibiotic use or nutrient changes during the neonatal period, might affect the establishment of the immune system in the infant. Understanding the development of intestinal macromolecular permeability, in different animal models, will increase our understanding of intestinal barrier function and provide strategies for the prevention of infection and inappropriate antigen transport affecting the immune response. This might also contribute to a better understanding of the etiology of inflammatory diseases of the gastrointestinal tract and in potential preventive and therapeutic strategies.BW planned the manuscript. BW, EA, and F-JP-C researched and wrote the manuscript and made the figures and tables. BW, EA, KP, SP, and F-JP-C edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present review aims to introduce current knowledge and prospective applications of nanotechnology for improving some of major applied assisted reproductive techniques (ART) in farm animals. Throughout the review, the raised question will be how nanotechnology, as a new emerging biotechnology, can be used to address the concept of smart reproductive management. The hypothesis will be discussed in the light of the few published studies in this field. This is in terms of leading the way for reproductive biologists to develop and innovate ART using nanotechnology scientific bases to maximize reproductive performance of farm animals, which finally serves the increasing consumer demand for animal products.Many biotechnological assisted reproductive techniques (ART) are currently used to control the reproductive processes of farm animals. Nowadays, smart ART that considers technique efficiency, animal welfare, cost efficiency and environmental health are developed. Recently, the nanotechnology revolution has pervaded all scientific fields including the reproduction of farm animals, facilitating certain improvements in this field. Nanotechnology could be used to improve and overcome many technical obstacles that face different ART. For example, semen purification and semen preservation processes have been developed using different nanomaterials and techniques, to obtain semen doses with high sperm quality. Additionally, nanodrugs delivery could be applied to fabricate several sex hormones (steroids or gonadotrophins) used in the manipulation of the reproductive cycle. Nanofabricated hormones have new specific biological properties, increasing their bioavailability. Applying nanodrugs delivery techniques allow a reduction in hormone dose and improves hormone kinetics in animal body, because of protection from natural biological barriers . Additionally, biodegradable nanomaterials could be used to fabricate hormone-loaded devices that are made from non-degradable materials, such as silicon and polyvinyl chloride-based matrixes, which negatively impact environmental health. This review discusses the role of nanotechnology in developing some ART outcomes applied in the livestock sector, meeting the concept of smart production.  The current review aims to afford and connect the current knowledge in two emerging fields, which may become essential for animal production in the next few years: smart reproductive management and nanotechnology.The picture for animal production in these early years of the 21st century is far from the scenario previously settled up during the 20th century. Animal production in the 20th century, agriculture on the whole, was influenced by the need of providing food and clothes to a growing population after a period of wars in the developed countries and after substantial changes in demographics and lifestyle in the developing areas. Strategies for providing food and clothes were implemented at any cost and included intensification of the use of natural resources to the limit and the extensive use of chemical substances , which act as xenobiotics.http://www.fao.org/fileadmin/user_upload/esag/docs/AT2050_revision_summary.pdf). However, currently, we have learnt that natural resources are limited and that any action on the environment has long-lasting consequences. Hence, agriculture needs to adapt to a changing environment and to avoid further changes in such environment, by improving its efficiency and sustainability and reducing its impact [The needs of world population will continue growing during the present century. The Food and Agriculture Organization (FAO) estimates a population of about 8500 million in 2030 and around 9150 millions of people in 2050, which means that food production should be increased by 70% , is a fast-growing field, which is essential for the manipulation of reproduction in the livestock sector. The main aim is to develop and implement relevant tools for improving fertility of farm animals with easily applied, affordable and effective techniques. Currently, such philosophy has to take into account the concept of smart production, which incorporates not only concepts as technique-efficiency and cost-efficiency, but also animal welfare, human health and environmental safety . TherefoNanotechnology is an innovative discipline, combining the sciences of physics, chemistry, biology, mathematics as well as engineering and computer sciences. Nanotechnology has been used for the development of diagnostic and therapeutic agents in human medicine, but its application in animal medicine and production is still scarce. The concept of this technology is to transform chemical molecules into small-scale size particles (range 1–200 nm). Consequently, new physical and chemical properties are achieved, including greater cellular uptake, reactivity, surface area and charge, and binding properties which might have opened a new platform for biosciences innovation ,4. Nano-3O4 NPs), characterized by their bio-compatibility and bio-function [Specifically, for the male, at post-collection semen handling for AI, specific nanomaterials are being designed to obtain semen doses with high sperm quality, by optimizing both semen purification (removing of defective sperm cells) and semen preservation (cooling or freezing) processes. For example, nano-magnetic pods of iron oxide  and therefore to evoke oxidative stress in spermatozoa, impairing integrity of spermatozoon membranes, mitochondrial membranes and nuclear DNA content, and thus leading to a significant reduction in its aptitude to fertilize the oocyte .During cooling/freezing processes, spermatozoa are kept in synthetic extenders which are created to provide nutrients and also to protect against cold/freezing shock and microbial attack. Thus, there have been intensive attempts to ameliorate negative effects of cooling/freezing preservation techniques on spermatozoa structure and function, by manipulating the chemical composition of semen extenders. In this respect, nanotechnology has been employed to modify semen extender properties, improving the uptake of nutrients and/or supplemented active components. One interesting study in this field was carried out by Murawski et al. , who dec2 NPs, an oxygen storing molecule) during semen storage at 4 °C for 96 h improves sperm motility characteristics after 48 h, and even up to 96 h of incubation, and protects the integrity of plasma membranes and DNA of spermatozoa [Enrichment of the semen extender with antioxidant agents, such as antioxidant minerals, has been reported to improve semen quality properties of cooling or post-thawing sperm cells, particularly if they are in nanoforms. It is currently known that the supplementation of semen extenders with cerium oxide nanoparticles  and the removal of defective cells, contaminations and debris within an ejaculate. These techniques may be especially helpful in recovering spermatozoa from sub-optimal ejaculates from animals of high genetic values but, moreover, removal of dead spermatozoa is highly beneficial, because of the negative effects on motility and membrane integrity of live spermatozoa . The mai3O4 NPs) that are characterized by their small size (10–100 nm), great superparamagnetism and surface area and bio-compatibility and bio-functionalization properties have been employed as an easy separation method. These NPs can be functionalized with specific probes and directed to defective spermatozoa using an external magnetic field, allowing the separation of defective spermatozoa [3O4 NPs probes used for semen purification are shown in A novel approach for semen purification that relies on the detection of specific biomarkers raised by defective spermatozoa is developed . Using frmatozoa . The idePisum sativum agglutinin lectin (PSA), annexin V and anti-ubiquitin antibodies have been successfully used in bulls for removing defective spermatozoa [3O4 NPs to purify frozen-thawed bull spermatozoa: a particle coated with antibody against ubiquitin, which is present on the surface of defective spermatozoa, and a particle coated with the PNA, which binds to glycans exposed by acrosomal damage. The results of AI in 798 cows in a field trial showed that the conception rate obtained using 10 × 106 PNA-nano-purified sperm doses (64.5%) was similar to the rate obtained using 20 × 106 non-purified full sperm doses (63.3%) and significantly higher than using a 10 × 106 non-purified sperm dose (53.7%). Similarly, Fe3O4 NPs coated with PNA/PSA lectins were used for the nanopurification of boar semen and resulted in semen doses with high sperm motility and viability [Studies in the purification of semen of farm animals have supported the efficiency of this technique; Fe3O4 NPs functionalized with probes of peanut agglutinin lectin (PNA), rmatozoa . In contrmatozoa  have devIn summary, previous studies have shown that sperm purification using nano-magnetic based techniques may overcome the inconveniences of other semen purification techniques such as extensive semen manipulation, labor and additional costs and run time (> 1 h to prepare a purified semen sample), without needing expensive equipment . These pIn this section, the advantages of nano-drug delivery technologies for smart management of the reproductive cycle in females will be discussed, with particular focus on the mechanisms involved. In conventional drug delivery systems, the bioavailability of the drug relies on several factors, such as the permeability throughout the epithelial and endothelial cells, the solubility, the passage through blood barriers, the clearance rate by liver and kidney and the resistance against systemic and circulating degradation enzymes. Thus, smart nanostructured delivery systems, particularly those depending on the polymerization of bioactive molecules using biodegradable and biocompatible polymers, are engineered with different physicochemical properties acting via different mechanisms, to efficiently deliver drugs from the site of administration to the sites of action, overcoming the unfavorable drug properties and the biological barriers . One of the mechanisms by which NP-based drug delivery systems can protect drugs from enzymatic degradation and rapid clearance depends on the ability of the nanoformula to absorb and/or encapsulate the drugs. The other mechanisms by which NP-based drug delivery systems can improve drug pharmacokinetic and pharmacodynamic properties are related to the physicochemical properties of the nanoformula such as shape, particle size, surface charge  and particles polydispersity (PdI). Particle size can play significant roles in the determination of cellular uptake and degradation and elimination processes of the drug. Small sized-Nps can escape from reticuloendothelial and mononuclear phagocytic systems, leading to an increase in total blood circulation time and bioavailability. Additionally, surface charge characteristics can affect the degree of NPs uptake according to the charge of tissue cells. Many cellular membranes are negatively charged; thus, cationic NPs facilitate membrane attraction and adhesion, which create favorable properties for cellular uptake, via endocytosis or other mechanisms .Based on the previously mentioned scientific facts, many drugs used for managing reproductive processes in females were developed to have new properties meeting the smart reproductive management aims.Procedures for implementing ART in farm animals are based on the administration of exogenous reproductive hormones  for controlling the reproductive cycle, improving reproductive efficiency and treating some reproductive disorders ,32,33. HA summary of studies connecting the nanotechnology and management of female reproduction is shown in In addition to allowing the use of a decreased dose, NHDS may also be used to change the route of administration  and therFinally, conventional hormonal treatments are also upsetting environmental issues, due to the release of hormonal residues and carrier materials into the environment. The most evident example is the use of progesterone-impregnated intravaginal devices  which were developed for cattle, and afterwards for small ruminants, since the 1970s and which are currently available worldwide ,41. The In view of these circumstances, Helbling and coworkers  developeHowever, the few studies performed in this respect have shown promising results regarding the replacement of non-biodegradable progesterone carriers with biodegradable ones using advances of NHDS; the application of such technology on farm scale needs more efforts to be justified . This isPregnancy and delivery are pivotal facts for the reproductive management and productivity of farm animals. One of the main complications of pregnancy in most farm animals, as in other many species including humans, is the occurrence of intrauterine growth restriction (IUGR) ,46,47. IThe main therapies for IUGR, in the case of farm animals, are based on maternal nutritional supplementation with amino acids favoring placental and fetal neoangiogenesis and tissue development, with vitamins favoring protein synthesis and antioxidant capacity and with other antioxidant agents like polyphenols . In humaIn this field, the infusion of the therapies throughout the umbilical cord, the amniotic sac or the placenta may be clearly optimized by the used nanoparticles. Nanoparticles can be directly used as a tool for the targeted delivery of therapeutics to the placenta. In this sense, surface-functionalized nanoparticles focused on receptors exclusively expressed at the placenta are giving promising results for their future use in the targeted therapy of pregnancy complications. In this sense, we have to highlight the works using plCSA binding peptide (plCSA-BP) nanoparticles , which tductus arteriosus and causing hydramnios, enterocolitis and hemorrhagiae). The objective of nanotechnology, in this case, would be to use molecules of a larger size that do not cross the placenta and act focally on the uterus [On the other hand, pregnancy complications may not directly affect the fetus but indirectly, through negative effects on the the maternal side, the uterus and maternal placenta. In this case, the objective would be to treat the mother without affecting the fetus. A classic example is preterm labor, which needs to be treated with tocolytic drugs like indomethacin which freely crosses the placenta and seriously affects the fetus  of NPs, in both biological systems and environments . This ma


In 1910, Raymond Jacques Adrien Sabouraud, a French dermatologist, was the first to report the morphologic characteristics of dermatophytes. During the decades that followed, taxonomy of dermatophytes has gone through revolutionary changes, mostly due to the advent of molecular diagnosis. Although studies performed in the 21st century have resulted in further classification changes and consolidation of new species, debates regarding the taxonomy of dermatophyte agents persist.


Orthopedic biofilm infections are difficult to treat and require a multidisciplinary approach to diagnostics and management. Recent advances in the field include methods to disrupt biofilm, sequencing tools, and antibiotic susceptibility tests for bacteria residing in biofilm. The observation of interclonal differences in biofilm properties of the causative microorganisms, together with considerations of comorbidities and polypharmacy in a growing aging population, calls for a personalized approach to treat these infections. In this article, we highlight aspects of precision medicine that may open new perspectives in the diagnosis and management of orthopedic biofilm infections. Biofilms generally form on a nonliving surface. In bone and joint infections, microorganisms adhere either to dead bone (sequesters) or to implants. Hence, orthopedic biofilm infections include chronic osteomyelitis and implant-associated infections. They represent a serious threat for the patient and a substantial burden on the global health care industry. More than a million knee and hip arthroplasties are performed every year in the United States . ProjectBiofilm development plays a pivotal role in implant-associated infections and allows microorganisms to survive in an environment protected by antimicrobial agents and the host immune system . SuccessAn optimal and individualized approach considers several factors, including stability of the implant, causative pathogen and type of infection, patient's concomitant comorbidities, and surgical procedure limitations. The concept of “precision medicine” is the new frontier of the modern health care industry and it combines multiple fields of expertise  . It recoIn orthopedic biofilm infections, precision medicine applies to biofilm properties of the causative microorganisms. Identifying factors associated with so-called low or high biofilm production can influence treatment strategies. The ability of bacteria to adhere to nonliving surfaces and the antibiotic susceptibility patterns of biofilm—together with host and surgical factors—may aid decision making regarding implant removal. In this article, we examine diagnostic advances in the identification of causative microorganisms and antimicrobial susceptibility testing of biofilm bacteria, putting into perspective how these methods can help individualize the management of bone and joint infections.Culture of bone or peri-implant tissue samples is the gold standard for identifying the organism causing the infection. Low-frequency ultrasound  is a useful tool for the clinical microbiologist in the diagnosis of biofilm-associated infections. Culture of sonication fluid increases the sensitivity and microbiological yield by disrupting the bacterial biofilm and is less affected by prior antimicrobial treatment than are prosthetic tissue samples , 11.Molecular diagnostic tests directly applicable to clinical samples have rapidly developed in recent years, improving the sensitivity of PJI diagnosis. They can be applied to both tissue and sonication samples. The utility of NGS/mNGS in providing a clinically useful diagnosis was first demonstrated in infections of the central nervous system , 44. It Multiple factors promote the implementation of NGS-based tests in routine clinical service. These include decreasing costs of NGS technology, cost savings from the replacement of other diagnostic tests, amount of information provided in a single test , and availability of portable rapid sequencing technology offering real-time data analysis, i.e., Nanopore Technologies , 49.Because of the high sensitivity of NGS, findings must be interpreted in the clinical context, as detection of DNA is not sufficient to conclude that an identified microorganism is the cause of the infection. On the other hand, sensitivity is critically affected by the background level generated by human DNA present in the sample. Implementation of NGS-based tests in clinical settings requires standardized protocols and validation of each step, from DNA extraction and library preparation to bioinformatics analysis and validation and interpretation of sequencing results. The US Food and Drug Administration (FDA) has published general guidelines for the validation of infectious diseases with NGS-based diagnostic tests; mNGS tests have been meanwhile successfully validated by several groups in Clinical Laboratory Improvement Amendments-certified clinical laboratories –55. The Biofilm formation is influenced by strain-specific properties. It is an interaction between environmental factors, surface structure, bacterial growth phase, and genetic determinants. Several methods have been published for the visualization or quantification of biofilm, including crystal violet staining and absorbance measurement, growth on polymethylmethacrylate beads followed by washing and sonication, scanning electron microscopy, and others. The physical and chemical surface properties in these assays may not necessarily reflect those in human infections, but they are responsible for the amount and structure of the biofilm . Bacteriin vitro to infection manifestations in humans has not yet been established. For individual patients, it is important to know whether their infection is caused by a low or a high biofilm producer. Of note, these terms are schematic expressions without precise definition. Additional questions include whether or not there is an antimicrobial agent that is active against the causative microorganism, as well as which antibiotic concentrations are needed, and for how long, to cure the infection  system in Staphylococcus aureus controls the expression of MSCRAMMs  and regulates the quorum-sensing system along with the P2 and P3 promoters , and CovR/S mutants show increased adherence to host cells and biofilm formation –68. PatrEnterococcus faecalis in  of antibiotics are routinely determined by using planktonic bacteria and do not match the concentrations that are effective in preventing, inhibiting, reducing, or eradicating biofilm bacteria . Differein vivo and in vitro when interpreting MBEC results. For Gram-positive organisms, MBECs of beta-lactams and glycopeptides are typically several 100 to 1000 times higher than the corresponding MIC of planktonic bacteria  . The metThe challenge reoccurs when transferring MBEC results to antibiotic dosing in patients and treatment concept. It may seem logical that high MBEC results are associated with high biofilm production, failure to achieve the required antibiotic concentration at the site of infection, and hence, clinical failure. Conversely, low MBEC results may be associated with low biofilm production, success in achieving the required antibiotic concentration at the site of infection, and considerable chance of clinical cure. Unfortunately, there is no such simple linearity because many technical, material, and environmental factors influence the microbiological result, as outlined earlier. Nonetheless, a similar concept was recently applied on a case basis in a context other than orthopedic infections, namely, in a patient with a cardiac device-related infection and conduit valve prosthesis endocarditis caused by nutritionally variant streptococci . BiofilmThe concept of considering clone-level (rather than only species-level) bacterial properties in the medical decision-making process represents a promising perspective. The more invasive the surgical procedure for removing a device, the higher the complication rate. If we are able to identify microorganisms with poor biofilm production by using reliable methods prior to the planned surgical intervention, patients could potentially benefit from conservative treatment. Conversely, unsuccessful attempts at implant retention could be avoided in the presence of microorganisms with strong biofilm production. Lack of clonal analysis beyond species identification for predicting outcome in established treatment concepts reflects a knowledge gap, and findings supporting this approach will affect further research beyond the field of septic surgery. Implementation of such a concept will have cost-saving effects, considering the duration of hospitalization expenditures associated with avoidable surgical infections and the complications associated with these interventions.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.RB drafted and co-wrote the manuscript and performed the literature review. PS revised the manuscript, supervised the work, and co-wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this review, we discuss the growing literature demonstrating robust and pervasive associations between the microbiome and metabolome. We focus on the gut microbiome, which harbors the taxonomically most diverse and the largest collection of microorganisms in the human body. Methods for integrative analysis of these “omics” are under active investigation and we discuss the advances and challenges in the combined use of metabolomics and microbiome data. Findings from large consortia, including the Human Microbiome Project and Metagenomics of the Human Intestinal Tract (MetaHIT) and others demonstrate the impact of microbiome-metabolome interactions on human health. Mechanisms whereby the microbes residing in the human body interact with metabolites to impact disease risk are beginning to be elucidated, and discoveries in this area will likely be harnessed to develop preventive and treatment strategies for complex diseases. Trillions of microbes reside in the human body. The collective genomic contents (or microbiome) of the human population comprises upwards of 45 million non-redundant genes ,2. When In this review, we will give an overview of methods for integrative analysis of microbiome and metabolome data, discuss the growing body of evidence to support a strong relationship between the microbiome and metabolome, and how microbiome-metabolome interactions impact human disease.Advances in nucleic acid sequencing and informatics data analysis techniques now allow for comprehensive analysis of the microbiome ,10. MajoOne technique to interrogate the function of the microbiota is to examine the genomic contents of the detected microbial taxa, which can either be directly measured by shotgun sequencing or inferred from the databases of known bacterial genomes. Identified genes can then be assigned functional and pathway annotations . While tHowever, integration of human microbiome and metabolome data is challenging for several reasons . In termWe will briefly discuss methods used for integrative analysis of microbiome and metabolome data. Further details have been recently reviewed elsewhere ,21. MultOf special interest are methods for ascertaining which metabolites are microbial-derived, and among those, which specific taxa are responsible for metabolite production. For example, a recently reported network method for determining the associations between metabolites and microbes in multi-omics data, when applied to metabolomic and metagenomic data from multiple studies, was able to assign metabolites to specific phylogenic clades that are presumably responsible for their production . SimilarThe microbiome has a major impact on the metabolome, and as we discuss later in this review, this has relevance for human health. Mouse studies comparing germ free and colonized mice demonstrate the profound influence of the intestinal microbiome on the metabolome even at distant body sites including the kidney, liver ,33, and Mouse studies have shown that the intestinal metabolome is particularly tightly linked to the intestinal microbiome. Of 179 fecal metabolites, 70% significantly differed in abundance between germ-free mice and ex-germ-free mice who were inoculated with feces of specific pathogen-free mice . Fecal aMost recently, investigators comprehensively queried the impact of the microbiome on the metabolome throughout the body: the profiling of 750 samples from 96 sites in 29 organs from 4 germ-free and 4 colonized mice revealed that the microbiota affected the chemistry of every organ . The gutThese findings have been recapitulated in human studies, providing additional evidence that fecal metabolome composition is strongly influenced by the microbiome. In an analysis of colon samples from 47 human subjects, there was significant concordance between the cecal, and to a lesser extent, sigmoid metabolome and microbiome in terms of the variance and distribution of metabolites and microbes . CorrelaThe fecal microbiome has the strongest association with the fecal metabolome, and less robust but still significant associations with metabolomes distant from the gut, including urinary and plasma ,42. In aBilophila wadsworthia, a species associated with inflammation. In a separate randomized trial of Mediterranean diet in 82 overweight and obese adults with low habitual intake of fruits and vegetables, the Mediterranean diet led to a decrease in fecal bile acids among other metabolite shifts [Bilophila wadsworthia, which also decreased with the Mediterranean diet intervention. These findings suggest that individualized features of the microbiome may impact the magnitude of benefit conferred by interventions such as adherence to a Mediterranean diet.The close relationship between the metabolome and microbiome has been leveraged in analyses of diseases that are thought to be caused, at least in part, by microbiome perturbations. For example, shifts in fecal microbiome and metabolome composition were observed in association with different phases of colorectal cancer . In parte shifts . InteresProfiling of the microbiome can also lead to personalized optimization of health-relevant metabolites. For example, glucose increases in response to meals were tracked longitudinally in 800 people and varied even when individuals consumed identical meals . Fecal PFuture studies will benefit from more comprehensively querying the microbiome to including not only bacteria, but viruses, fungi, and archaea. For example, a study that challenged healthy but sedentary adults with a short-term exercise regime with or without daily whey protein consumption included profiling of fecal bacteria, viruses, and archaea. It found that whey protein consumption led to significant changes to fecal viral composition . InteresThe first phase of the Human Microbiome Project (HMP), which began in 2007, included 300 subjects who provided samples from a variety of body sites for microbial gene sequencing . MicrobiThe more recent integrative Human Microbiome Project (iHMP), launched in 2012, has turned the focus towards (1) integrative omics including metabolomics, and (2) specific disease states that had been previously associated with the microbiome: prediabetes, inflammatory bowel disease, and preterm birth . ResultiIn iHMP analyses of inflammatory bowel disease (IBD), fecal metabolites, and metabolic pathways were associated with IBD, including bile acids, short-chain fatty acids, sphingolipids, acylcarnitines, triacylglycerols, and tetrapyrroles ,57. A muPrevotella copri, abundance of which correlated with abundance of branched chain amino acids [Metagenomics of the Human Intestinal Tract  was a European consortium that focused on the gut microbiome and its relation to obesity and IBD. Data from MetaHIT were used to identify metabolites, metabolite pathways, microbial genomic pathways, and bacterial microbes that associated with insulin resistance and metabolic syndrome. Specific species were identified that contributed to disease-relevant metabolic pathways, including no acids . Taken tThere are now well-characterized pathways in which members of the human gut microbiota influence metabolite abundances with relevance to human disease, even at distant body sites. For example, gut microbial metabolism of dietary choline results in increased plasma trimethylamine-N-oxide (TMAO), which has been reproducibly associated with development of cardiovascular disease . MembersWhile it is clear that the gut microbiota influences a broad variety of human diseases through production or modification of metabolites, it remains an open question as to the relative importance of metabolite-mediated mechanisms compared to others, like the engagement of host receptors such as Toll-like receptors by pathogen-associated molecular patterns. Additionally, host-derived metabolites, cytokines, or other signaling molecules may impact microbiota composition as another mechanism of association between the microbiome and the metabolome.Microbiome-metabolite interactions are pervasive in the human body. Recent evidence has revealed that the microbiome has a profound influence on the metabolome both near and at distant body sites, and the metabolome in turn can affect the microbiome. It is now clear that crosstalk between the microbiome and metabolome has an impact on many human diseases through a variety of mechanisms. This knowledge can be used to develop rational microbe- and metabolite-targeted treatments and preventive strategies, including probiotics, prebiotics and other dietary modifications, supplementing or inhibiting microbial-derived metabolites, and fecal microbiome transplant . As unde