Chemotherapy and/or head and neck radiotherapy are frequently associated with oral mucositis. Oral pain, odynophagia and dysphagia, opioid use, weight loss, dehydration, systemic infection, hospitalization and introduction of a feeding tube should be mentioned as the main determinated effect of oral mucositis. Oral mucositis leads to a decreased quality of life and an increase in treatment costs. Moreover, oral mucositis is a life-threatening disease. In addition to its own direct life-threatening consequences, it can also lead to a reduced survival due to the discontinuation or dose reduction of anti-neoplasm therapy. There are numerous strategies for the prevention or treatment of oral mucositis; however, their effectiveness is limited and does not correspond to expectations. This review is focused on the ghrelin and obestatin as potentially useful candidates for the prevention and treatment of chemo- or/and radiotherapy-induced oral mucositis. Oral mucosa is constantly exposed to mechanical, chemical and thermal trauma, as well as to infectious agents. Even in physiological conditions, during the chewing process, minor traumas occur and inflammatory infiltration is frequently observed in the histological slides of the gum . EpithelMoreover, oral mucosa may be a source of anti-inflammatory cytokines and growth factors, reducing the development of oral mucositis and accelerating the regeneration. On the other hand, oral epithelial cells can also secrete pro-inflammatory cytokines in the presence of pro-inflammatory factors, such as Tumor Necrosis Factor-α (TNF-α) or Pathogen-Associated Molecular Patterns (PAMPs) ,4,5,6,7.Saliva is a fluid present in the oral cavity and produced by salivary glands. Saliva contributes to the digestion of food and plays an essential role in maintaining oral hygiene. The presence of mucous prevents the mechanical trauma of oral mucosa and aids in bolus formation. The protective function of saliva is related to the presence of ingredients with digestive and anti-infective effects . NumerouThe important role in the protective and healing effect of saliva in injures is played by biologically active peptides called growth factors. Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF) and Fibroblast Growth Factors (FGFs) seem to play an especially essential role in this respect.EGF was originally isolated from the submandibular glands of the mice ; later iThe administration of exogenous EGF inhibits the development of experimental acute pancreatitis in rats  and acceNGF, a member of the neurotrophin family, is typically synthesized as a high molecular weight complex (130 kDa) consisting of β-NGF and two subunits α and γ. Subunits α and γ belong to the kallikrein family of serine proteases. β-NGF is a large precursor of NGF (pro-NGF) and is processed by α and NGF subunits to generate functional NGF . NGF plaIn addition to its effects on the nervous system, NGF has been shown to accelerate the healing of skin wounds and corneal ulcers ,47,48. NThere are two classes of NGF receptors, the high-affinity receptor: Tropomyosin receptor kinase A (TrkA) and the Low–affinity Nerve Growth Factor Receptor (LNGFR) also known as the p75neutrophin receptor. The activation of TrkA is essential for inducing cell survival and differentiation caused by NGF. Activating LNGFR may lead a cell to die by apoptosis; however, this effect may be counteracted by the activation of TrkA with antiapoptotic effects. Previous studies have shown that NGF receptors are expressed in the soft tissues of the oral cavity ,51. The Fibroblast Growth Factor-2 (FGF-2) known as also basic Fibroblast Growth Factor (bFGF) is a member of the Fibroblast Growth Factors Family (FGFs) . FibroblThe irradiation of the head region leads to the severe damage of salivary glands and a decrease in the saliva output in clinical  and expeContrarily, there are studies showing the elevated levels of FGF-2 in saliva and serum in recently diagnosed and untreated patients with oral squamous cell carcinoma ,61. ThesHistatins are the next group of factors present in saliva and demonstrate a potential protective and therapeutic effect in the oral cavity . HistatiOral mucositis is defined as an inflammation of the oral mucosa, characterized by the presence of erythematous areas and severe inflammation subsequently merged with ulcerations . Oral muChemo- and radiotherapy should only target neoplastic cells; however, these therapies also exhibit an antimitotic effect on the rapidly dividing progenitor cells in different parts of the body. In the past, it was believed that oral mucositis is solely the result of the nonspecific damaging effect of chemo- or/and radiotherapy on rapidly dividing cells in the basal layer of the oral mucosa ,6. CurreChemotherapy and radiotherapy cause DNA injury and reactive oxygen species generation, leading to basal cell damage in the endothelium. Affected cells release endogenous damage-associated molecular pattern molecules (DAMPs) which initiate the signaling phase with immune response including the activation of several hours ,81. The Oral mucositis leads to a significant lowering of the life quality and an increase in the cost of treatment. The presence of oral lesions is responsible for the development of local and systemic detrimental effects such as severe oral pain, difficulties with the swallowing of solid and liquid food (dysphagia), a pain or burning sensation on swallowing (odynophagia), trouble with (dysarthria), opioid use, dehydration and tube feeding. Moreover, mucosal lesions may be a gateway for opportunistic infections, systemic inflammation and, finally, death. These complications are associated with morbidity and mortality. Patient death may be a result of the above-listed direct life-threatening consequences of oral mucositis or the limitation of cancer control due to cancer treatment interruption or a reduction in the chemo- or/and radiotherapy doses .The grading scale for oral mucositis plays a crucial role in the objective comparison of the toxicity of anticancer therapies and the efficiency of therapeutic methods used in the prevention and treatment. There are several independent systems for assessing the severity of oral mucositis, but the scoring scale proposed by the World Health Organization is most often used. The World Health Organization Oral Toxicity Scale measures the anatomical, symptomatic and functional signs of oral mucositis  and gradDuring the initial visit before chemo- or radiotherapy, the patient should be assessed for the presence of risk factors for the development of oral mucositis . Some riAmong the recommended evidence-based methods for the prevention of radiotherapy and chemotherapy-induced mucositis, the following ones should be mentioned: good oral hygiene, palifermin, antioxidant agents (amifostine and GC4419) and low-level laser therapy/photobiomodulation .Good oral hygiene is mandatory for minimizing the incidence and severity of mucositis in cancer patients. Shieh et al. have shown that an appropriate oral care protocol applied 1 week before radiotherapy delays the onset of mucositis and reduces the grade of this inflammation . Good orPalifermin  is a trunked recombinant human Keratinocyte Growth Factor (KGF) . KGF belAmifostine, as a free radical scavenger, exhibits antioxidant and cytoprotective activities . Its impThe excess free radicals play an essential role in the development of oral mucositis evoked by radio- or/and chemotherapy. GC4419 is a synthetic manganese-based small molecule that mimics the antioxidant enzyme Superoxide Dismutase (SOD). GC4419, like SOD, converts a superoxide anion to a hydrogen peroxide or oxygen molecule and no other reactive oxygen species . The cli2 and no more than 6 J/cm2 on the tissue surface treated,; administered two to three times a week up to daily [There is a large number of evidences showing that photobiomodulation, formerly known as low-level laser therapy, is useful in the prevention and/or treatment of oral mucositis evoked by radiotherapy for head and neck cancer or chemotherapy . The preto daily . The mosto daily .Among the methods that were or are used to protect the oral mucosa against the development of inflammation during radio- or/and chemotherapy should also be mentioned: the oral administration of PTA , therapy with Granulocyte Colony-Stimulating Factor (G-CSF) or Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) and cryotherapy.The effect of treatment with PTA on the development of oral mucositis remains unclear in patients receiving radiotherapy or chemotherapy. Clinical placebo-controlled double-blind studies, performed during last 20 years, have shown that treatment with an oral paste containing PTA  or the aThere is no definite and convincing evidence that the use of the Granulocyte Colony-Stimulating Factor (G-CSF) or the Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) before or during radiotherapy of head and neck cancer or chemotherapy exhibits therapeutic benefits in oral mucositis. Clinical trials on the systemic or topical application of G-CSF or GM-CSF on oral mucositis in these patients are confusing. Some especially early studies have suggested that the administration of GM-CSF ,105,106 In contrast, clinical double-blind placebo controlled randomized studies exhibit a minimal or no effect of GM-CSF ,111,112 Oral cryotherapy involves the cooling of the oral cavity using ice cubes, ice chips, cold water or ice cream . It has The treatment of oral mucositis is mainly symptomatic and should reduce the side effects of chemo- or/and radiotherapy and oral mucositis. Patients suffer, among others, from a loss of taste, xerostomia, nausea and vomiting, odynophagia or dysphagia. If patients are able to eat, a diet restriction should include the elimination of acidic, salty and spicy food, as well as alcohol . In the Pain control plays an essential role in the maintenance of life quality and oral feeding. Some authors recommend the use of anesthetics and analgesics for a reduction of the pain in oral mucositis. Most therapeutic regimens are empirical with no scientific basis. The administration of local anesthetics such as lidocaine , bupivacaine or dyclonine leads to a transitory pain relief in oral mucositis. Various lidocaine preparations, in the form of gels, sprays or viscous solutions, are currently used for the local anesthesia of the oral cavity ,122. TheRadiotherapy for head and neck cancer leads to oral mucositis, the damage of the salivary glands and xerostomia. Also, chemotherapy is associated, in most cases, with the development of oral mucosistis and xerostomia . For thi®, Orabase®, Episil® and MuGard® are the most commonly used protectants for the oral mucosa membranes. They reduce pain in the mouth, but unlike typical mouthwashes, the effect lasts longer after a single administration. Gelclair® is produced by Helsinn Healthcare SA  and contains as active ingredients, among others, maltodextrin, propylene glycol, polyvinylpyrrolidone (povidone), sodium hyaluronate, hydroxyethylcellulose and glycyrrhetinic acid [®, produced by ConvaTec, contains gelatin, pectin and carboxymethylcellulose sodium in plasticized hydrocarbon gel (polythene and liquid paraffin). Episil®  is composed of glycerol dioleate, phosphatidylcholine (soy lecithin), ethanol, propylene glycol, polysorbate 80 and peppermint oil. MuGard® contains purified water, glycerin, benzyl alcohol, sodium saccharin, Carbomer Homopolymer A, potassium hydroxide, citric acid, polysorbate 60 and phosphoric acid.Some relief of pain in oral mucositis may be reached by bioadherent oral gels or ointments. They adhere to the surface of the oral mucosa, creating a barrier between the light of the mouth and oral lesions. This effect protects the mucosa membrane against oral irritating and the harmful effect of oral content. Gelclairnic acid . Orabase® does not significantly improve oral food intake or oral pain control in comparison to the effects of treatment with a rinsing solution containing chlorhexidine, benzydamine and salvia (control group) in patients with oral mucositis after allogeneic stem cell transplantation. On the other hand, the duration of pain relief after a single dose of Gelclairx® was statistically longer than after rinsing the mouth with the control solution. Moreover, Gelclair® significantly reduced the colonization of the oral cavity by Enterococcus faecalis and Candida spp. [® does not significantly reduce the severity of oral mucositis and oral pain in comparison to standard mouthwash in patients undergoing hematopoietic stem-cell transplantation [The role of Gelclair in oral mucositis was tested in some clinical studies. Studies performed by Barber et al. have shown that Gelcleir does not produce better therapeutic effects than standard therapy with surcralfate plus Mucaine in relieving oral pain in radiotherapy-induced oral mucositis. There was also no reduction in pain on speaking and no improvement in the ability to eat and drink . Partly ida spp. . Also, santation .® in cancer patients with oral mucositis has been reported by Cheng et al. [® [® effectively mitigates oral mucositis symptoms such as oral soreness and World Heath Organisation (WHO) oral muscositis score on the last day of radiation therapy in patients treated with chemoradiation therapy for head and neck cancer [The local analgesic effect of Episilg et al.  who haveg et al. . A similt al. [® ; this muk cancer .Some clinical studies with a low number of participants suggest that the systemic and especially topical administration of vitamin E reduces oral mucositis induced by cancer chemo/radiotherapy ,140. SomCurrently, natural organic agents are under investigation to determine their protective and therapeutic activity in the management or oral mucositis in cancer patients . Some ofMelatonin is the next new strategy for prevention and treatment of oral mucositis evoked by chemo- or/and radiotherapy. Melatonin is synthesized in the pineal gland from the amino acid tryptophan. The synthesis of melatonin occurs also, among others, in the oral mucosa and salivary glands . MelatonThe effectiveness of current therapeutic methods is limited. They are not able to prevent the development of oral mucositis in cancer patients treated with radio- and chemotherapy. On the other hand, some of them can delay the development of this inflammation, reduce its severity and duration and improve the quality of patient’s life. However, it is not in line with patients’ expectations. Efforts should be made to find new methods for the prevention and treatment of oral mucositis in patients at risk of developing this inflammation. Experimental studies suggest that two hormones, ghrelin and obestatin, may be useful in solving this problem.In 1999, ghrelin was discovered in human and rat stomach by Kojima et al. . Ghrelinq/11 α-subunit that results in the activation of phospholipase C and the production of Inositol Triphosphate (IP3) and that releases Ca2+ from the endoplasmic reticulum [Currently, GHS-R1a is called the ghrelin receptor to follow the convention of naming receptors after the endogenous agonist . The ghreticulum ,161. Theeticulum . The ghreticulum . Moreoveeticulum .In the circulation, ghrelin exists in two different forms, as acylated ghrelin (acyl ghrelin) and non-acylated ghrelin (des-acyl ghrelin) . Acyl ghStudies in humans and on animals have shown that ghrelin strongly and dose dependently stimulates the release of growth hormone from the anterior pituitary . It is mNumerous animal and human studies have evaluated the effect of ghrelin on food intake, body weight, energy expenditure and glucose homeostasis . GhrelinThe food intake-promoting (orexigenic) effect of ghrelin involves central and peripheral mechanisms. The hypothalamic arcuate nucleus mediates the anorectic effects of leptin and orexogenix effect ghrelin, and the neurons of the arcuate nucleus exhibit a high density of ghrelin receptors . The lepGhrelin and cholecystokinin (CCK) are gastrointestinal hormones regulating feeding. The first step in the integration of hunger and satiety signals is related to the peripheral level of ghrelin and CCK and takes place at the level of the vagus nerve ,194. DatPrevious studies have also shown that the regulation of ghrelin secretion by energy balance, as well as the influence of ghrelin on food intake and weight gain is age-dependent and imperfect at an early stage of life. For example, the study performed on prepubertal children has shown that ghrelin secretion in childhood is refractory to the inhibitory effect of feeding ,196. SimAmong the physiological functions of ghrelin should also be mentioned the stimulation of gastric motility and gastric emptying ,200. TheThe administration of ghrelin inhibits pancreatic exocrine pancreatic secretion . HoweverNumerous animal studies have demonstrated that exogenous ghrelin exhibits a protective effect in several organs such as the heart , kidney Accelerating the healing in gastric and duodenal ulcers is related to an improvement of the gastri and duodenal mucosal blood flow, an increase in mucosal cell proliferation and antioxidant defense, as well as a reduction in mucosal oxidative stress and inflammatory response. Treatment with ghrelin increased the serum level of GH and IGF-1 ,219.Ghrelin exhibits a protective and therapeutic effect also in the colon. Ghrelin reduces the severity of colitis evoked by trinitrobenzene sulfonic acid ,224. TheNext, experimental studies have shown that ghrelin administered intraperitoneally after the induction of colitis evoked by acetic acid leads to the acceleration of healing of colitis . This efIn contrast to the above observation, in vitro studies performed by Zhao et al. suggest that ghrelin may participate, in some conditions, in the induction or aggravation of colonic inflammation . The expAnimal studies have also shown that the administration of ghrelin protects the liver, pancreas and remote organs against direct damage and oxidative injury evoked by either the bile duct or common pancreaticobiliary duct ligation . The proGhrelin exhibits a therapeutic effect in the course of animal models of acute pancreatitis. The administration of ghrelin after the development of acute pancreatitis accelerates the recovery in acute pancreatitis induced by cerulein  and pancClinical observations have shown that acute pancreatitis affects the serum level of ghrelin. However, the character of changes remains unclear. First of all, there are large differences in the levels of ghrelin and the direction of changes in the level of ghrelin between reports. Secondly, in most cases, there is no control group without acute pancreatitis. Moreover, there are differences in the time of blood sample collection. For example, Liu et al.  collecteThese observations are partially contradictory to those obtained by Daniel et al. . Daniel Ulger et al.  carried A higher level of ghrelin on the day of discharge than admission in patients with acute pancreatitis was found by Lee et al. . Moreovep < 0.05). Not to mention, the serum ghrelin level becomes progressively higher during the first five days of hospitalization. The authors stated that the receiver-operating characteristic (ROC) curves demonstrating serum ghrelin level at the first day of hospitalization had some predictive value for acute pancreatitis severity. In addition, the results presented by Wang et al. show that the serum ghrelin level in patients with acute pancreatitis exhibits an upward trend, but this effect was statistically insignificant, probably due to short time of observation [Wang et al.  have repervation .Only one observation regarding the level of ghrelin in acute pancreatitis has been confirmed by most authors. Almost all of them stated that the level of ghrelin during pancreatitis grows systematically and reaches the highest value at discharge or on the last day of observation according to the study design. This observation suggests that endogenous ghrelin is involved in the recovery process in the course of acute pancreatitis.There are numerous important finding showing the involvement of endogenous ghrelin in physiology and pathology of the oral cavity. Ghrelin is produced and released by parotid and submandibular salivary glands but not sublingual salivary glands ,248. GhrGhrelin receptors (GHS-R1a and GHS-R1b) have been detected in oral epithelial cells , periodoGhrelin is present in saliva in similar or even higher concentrations than in plasma or serum ,254,255,Fusobacterium nucleatum, a pathogen involved in peridontitis development, leads to an initial upregulation and the subsequent downregulation of ghrelin receptor in periodontal cells [Clinical data indicate that the ghrelin level in the gingival crevicular fluid is lower in patients with chronic periodontitis than in healthy individuals . The stual cells . Furtheral cells .Experimental studies performed on rats have shown that the intraperitoneal administration of ghrelin significantly accelerated the healing of oral ulcers and that this effect occurs in both non-sialadenectomized and sialadenectomized rats. The beneficial effect of ghrelin is associated with a reduction of mucosal IL-1β concentration and an improvement of mucosal blood flow, cell vitality and proliferation. These finding have been confirmed and extended by studies performed on animals with intact or removed pituitary glands . In pituThe observations presented above strongly suggest that treatment with ghrelin could be useful in the prevention and/or therapy of oral mucositis. The administration of ghrelin in oral mucositis should be recognized as a supplementation in the ghrelin deficiency and the restoration of physiological condition. This concept is supported by the following facts:The concentration of ghrelin in saliva is similar or even higher than in plasma ,254,255;In addition to the potential protective and therapeutic effects of ghrelin in oral mucositis, there is one more benefit arising from its use. Oral mucositis, chemotherapy, radiotherapy and cancer, the primary cause of chemo- and radiotherapy, reduce food intake, leading to weight loss. The administration of ghrelin increases food intake and appears to be a promising therapeutic option for cancer cachexia .On the other hand, it is necessary to determine what form of ghrelin should be used in oral mucositis, as well as, the question about of the route of it administration: As a peptide or synthetic non-peptide analog? Parenterally or orally? Only new studies could answer to these questions.4 and Gln15, and His19 and Ala22, respectively. These α-helical structures are critical for in vivo activity [Obestatin was discovered by Zhang et al. in 2005 in the rat stomach which is a main source of endogenous obestatin . The namactivity ,267. Obeactivity . Besidesactivity . Moreoveactivity  have addactivity , who havactivity . Morettiactivity  have expactivity . Volanteactivity  have repactivity . Alnema activity  have exaactivity .Zhang et al. ,274 and Previous studies have reported that obestatin inhibits the motility of gastrointestinal tract ,285, theIn the central nervous system, obestatin inhibits anxiety and improves memory  and incrObestatin, like ghrelin, shows antioxidant and anti-inflammatory effects in many organs in the human body. Therefore, the effects of obestatin in the prevention and/or treatment of various diseases have been extensively investigated. The protective or therapeutic effect of obestatin has been observed in the heart ,292,293,Mechanisms of protective and therapeutic effects of obestatin involve, among others, the improvement of blood flow in exposed to damaging factor organs, the amelioration of cell vitality and cell proliferation, a reduction of the expression and presence of pro-inflammatory IL1-β and TNF-α, a decrease in leukocytosis and tissue activity of myeloperoxidase ,299, theOral mucosistis is a common and severe side effect of anticancer therapy. The presence of oral lesions is responsible for the development of local and systemic detrimental effects such as severe oral pain, difficulties with the swallowing of solid and liquid foods (dysphagia), trouble with speaking (dysarthria), opioid use, dehydration and the introduction of tube feeding. Moreover, mucosal lesions may be a gateway for opportunistic infection and systemic inflammation. Oral mucositis and its complications are associated with morbidity and mortality, the lowering of the quality of life and the increase in the cost of treatment. There are numerous strategies for the prevention or treatment of oral mucositis; however, their effectiveness is limited and does not correspond to expectations. Ghrelin and obestatin exhibit anti-inflammatory, antioxidative and antiapoptotic effects. Numerous experimental studies have shown that ghrelin and obestatin exhibit a protective and healing-promoting effect in different models of organ damage, including mucosa in the gut. These findings suggest that ghrelin and/or obestatin may be useful in the prevention and treatment of oral mucositis. Hence, further animal and clinical studies should be performed to clarify their usefulness in this disease.


The risk of oxidative stress is high in preterm newborns. Room air exposure of an organism primed to develop in a hypoxic environment, lacking antioxidant defenses, and subjected to hyperoxia, hypoxia, and ischemia challenges the newborn with oxidative stress production. Free radicals can be generated by a multitude of other mechanisms, such as glutamate excitotoxicity, excess free iron, inflammation, and immune reactions. Free radical-induced damage caused by oxidative stress appears to be the major candidate for the pathogenesis of most of the complications of prematurity, brain being especially at risk, with short to long-term consequences. We review the role of free radical oxidative damage to the newborn brain and propose a mechanism of oxidative injury, taking into consideration the particular maturation-dependent vulnerability of the oligodendrocyte precursors. Prompted by our observation of an increase in plasma Adenosine concentrations significantly associated with brain white matter lesions in some premature infants, we discuss a possible bioenergetics hypothesis, correlated to the oxidative challenge of the premature infant. We aim at explaining both the oxidative stress generation and the mechanism promoting the myelination disturbances. Being white matter abnormalities among the most common lesions of prematurity, the use of Adenosine as a biomarker of brain damage appears promising in order to design neuroprotective strategies. Oxidative stress is the consequence of an imbalance in the ratio among pro-oxidants and anti-oxidants in the cell . Free raWhen the production of ROS exceeds the antioxidant defenses, or antioxidant levels are low, as is the case in the preterm newborn, oxidative stress usually occurs to the detriment of all of the cellular macromolecules . In adulThe oxidant/antioxidant status balance is a process that begins before birth , and pre2) of an organism primed to develop in a hypoxic  environment as the womb is. For this reason, current indications on neonatal resuscitation highlight the importance of starting respiratory support using the lowest oxygen concentration to reduce the postnatal oxidative stress , in preterm infants compared with term infants . Along wCerebral WM injury is a full-spectrum of lesions named periventricular leukomalacia (PVL), that occurs in two overlapping forms: cystic PVL, in which the periventricular focal necrosis is macroscopic and evolves to multiple cysts; and non-cystic PVL, in which the focal necrosis are microscopic and evolve principally to glial scars . EvidencIt has been reported that brain injury mostly affects WM, being oligodendroglial death the most important cause of PVL and PWML , 48, 49.In addition, oxidative stress reduces the expression of differentiation-promoting genes, such as Olig1, Olig2, and Sox10 in pre-OL, and increases the expression of differentiation-inhibiting genes (ID2 and ID4), resulting in the interruption of OL maturation .Although there is evidence of an imbalance between antioxidant and oxidants, the ultimate cause of oxidative stress and molecular bases for the maturation-dependent vulnerability of the pre-OL to injury in a window of time ranging from 30 to 34 weeks of gestational age is yet unknown. We have recently proposed a bioenergetics hypothesis, correlating the oxidative stress generation to the significant increase of plasma Adenosine (Ado) concentration observed in some VLBW infants . Ado proFree radical production consequent to hypoxia and ischemia/reperfusion, together with the low caloric intake after birth in the premature babies may cause a slowing down of the oxidative phosphorylation, diminishing high-energy compounds , 55.Many authors accept the hypothesis that free radicals persists to the damage of the premature brain. Consequently, to prevent long-term sequelae of oxidative stress, it is necessary to early diagnosis the presence of an oxidative stress damage by a validate panel of biomarkers, which could also represent the first step in delineating potential therapeutic interventions. To date, different biomarkers have been proposed to measure oxidative stress in the newborn. Plasma prostanoids were validated as biomarkers of oxidative stress injury to neurons . VisfatiRecently, novel treatment strategies have been proposed to counteract damages induced by oxidative stress in preterm infants (see Table In conclusion, despite gaps still present in our knowledge of the mechanism of oxidative stress production in the pathogenesis of brain damage in the premature newborn, this organ remains at major risk especially for the prolonged vulnerability of white matter at certain gestational ages during which preterm newborns undergo intensive care treatment. There is a need for new and accurate neonatal biomarkers of brain injury that can foresee those babies at higher risk of developing brain injury thus needing neonatal neuroprotection, by new therapeutic interventions centered on reversal of the processes that promote dysmaturation, one of the more important being oxidative stress.Both LR and IP devised main conceptual ideas and outlines. IP took the lead in writing the manuscript. All authors provided critical feedback and discussed 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.


Multiple Sclerosis (MS) is a demyelinating and neurodegenerative disease. Though a specific antigen has not been identified, it is widely accepted that MS is an autoimmune disorder characterized by myelin-directed immune attack. Pharmacological treatments for MS are based on immunomodulatory or immunosuppressant drugs, designed to attenuate or dampen the immune reaction, to improve neurological functions. Recently, rehabilitation has gained increasing attention in the scientific community dealing with MS. Engagement of people with MS in exercise programs has been associated with a number of functional improvements in mobility, balance, and motor coordination. Moreover, several studies indicate the effectiveness of exercise against fatigue and mood disorders that are frequently associated with the disease. However, whether exercise acts like an immunomodulatory therapy is still an unresolved question. A good tool to address this issue is provided by the study of the immunomodulatory effects of exercise in an animal model of MS, including the experimental autoimmune encephalomyelitis (EAE), the Theiler's virus induced-demyelinating disease (TMEV-IDD) and toxic-demyelinating models, cuprizone (CPZ), and lysolecithin (LPC). So far, despite the availability of different animal models, most of the pre-clinical data have been gained in EAE and to a lesser extent in CPZ and LPC. These studies have highlighted beneficial effects of exercise, suggesting the modulation of both the innate and the adaptive immune response in the peripheral blood as well as in the brain. In the present paper, starting from the biological differences among MS animal models in terms of immune system involvement, we revise the literature regarding the effects of exercise in EAE, CPZ, and LPC, and critically highlight the advantages of either model, including the so-far unexplored TMEV-IDD, to address the immune effects of exercise in MS. Rehabilitation is a supportive therapy increasingly required to manage symptoms and improve quality of life (QoL) in many neurological disorders, including Multiple Sclerosis (MS), the most common neurodegenerative disease affecting young adults. MS patients encounter a number of symptoms, from sensory-motor impairments to psychiatric comorbidities, which heavily impair daily life activities and make compulsory the search for effective therapies . DiseaseExercise has been convincingly associated with functional recovery of several disease outcomes, such as walking , balanceIn this mini-review, we will briefly summarize the key elements involved in immune response in human and experimental MS to provide a general overview of the immune component involved and possibly targeted by experimental paradigms of exercise in rodents. Next, we will revise animal studies exploring the immunomodulatory action of exercise, critically highlighting possible future directions.Cells of both the adaptive and the innate immune response have been claimed to play a crucial role in the initiation and the progression of the disease . AlthouAn animal model is predicted to recapitulate the main pathological and clinical features of a given human disease. Different animal models of MS have been developed to account for the difficulty to reproduce the whole spectrum of pathological events occurring in MS and the different phenotypes in a unique experimental system .The experimental autoimmune encephalomyelitis (EAE) is the most used to investigate immunological events in the brain  and consSeveral viral models have been developed to account for the environmental susceptibility factors linked to MS etiopathogenesis . The mosThe toxic-demyelinating models, namely lysolecithin (LPC) and cuprizone (CPZ), are used to study demyelination and remyelination processes and reproduce autoimmune-independent myelin damage. Lysolecithin (LPC) is a detergent supposed to be toxic for myelin. Injected directly into a specific brain area, LPC induces a focal demyelination, which triggers a fast recruitment of macrophages and microglia followed by T and B cells to repair myelin damage . CPZ is Different paradigms of behavioral interventions can be applied to rodents. Environmental enrichment (EE), consisting in rearing mice or rats in extra-size cages containing toys, running wheels, tunnels, and other devices, is considered a positive modulator of both motor and sensory systems known to modulate both synaptic activity and immunity , 24. In Other approaches are intended to simulate the effects of physical training in humans and fall into two categories, forced (FE) and voluntary (VE) exercise . In FE, The main differences between FE and VE rely on the amount of stress that FE can induce to animals  and the Most of the studies addressing the effects of exercise in experimental MS, have been conducted in EAE and to a lesser extent in CPZ and LPC, while none has been performed in TMEV-IDD. Such studies have focused on immunomodulation and/or regulation of neurotrophic factors in the brain as underlying putative mechanisms  Table 1Table 1. The first reports showed that 10-day treadmill training initiated soon after EAE induction was effective in reducing clinical disability in RR-EAE rats , 35. ThiMore recently, two elegant studies have corroborated the idea that exercise-mediated beneficial effects on EAE course occur through a peripheral immunomodulation , 39. AdoFE has been tested in CPZ model , 41. TwoMore consistent data have been obtained with swimming. A preventive swimming training started 4 weeks before EAE induction and maintained until 10 days post-immunization (dpi) attenuated EAE severity, without changing the number of infiltrating mononuclear cells and promoting a reduced and an increased pro-inflammatory environment in brain and in spinal cord of exercising mice, respectively . SwimminMore recently, preventive high-intensity swimming has been shown to reduce EAE disability and the number of spinal cord infiltrating cells . ExercisTherapeutic treadmill exercise has also been tested. In CR-EAE, exercise which started at 20 dpi for 4 weeks improved hippocampal demyelination and neurogenesis, memory function, and BDNF levels . In rat Finally, a recent study deserves attention. Bernardes et al.  faced, fFew studies have tested the effects of EE in MS models. The first paper showed that EE initiated the day of EAE induction improved motor dysfunction and induced mobilization of precursor cells in demyelinating areas. The latter effect was also confirmed in LPC injured-mice. However, the anti-inflammatory action of EE was not assessed . The benad libitum free access to wheel. Notably, 1 h per day of free access to running wheel was found to delay disease onset, without affecting disease severity, and to reduce microgliosis and T cell infiltration in spinal cord (VE has been invariably associated with recovery of motor function in EAE mice, but its immunomodulatory action has been faintly investigated, thus providing contrasting results. Indeed, VE started the day of EAE induction  did not nal cord , 54.VE has been tested in demyelinating toxic models. VE initiated soon after LPC injection in spinal cord induced an early shift from a pro-inflammatory to an anti-inflammatory phenotype of microglia/macrophages, which, by removing myelin debris, allowed the proliferation of OPCs . The comRecently, we have investigated the effects of VE in CPZ . ExercisUnderstanding the DMT potential of exercise in human and experimental MS is a challenging topic, which is gaining increasing interest. Nevertheless, the lack of standardization of both exercise protocols and evaluation scales in human studies does not allow a comprehensive analysis of the effects of rehabilitation in MS subjects . RehabilOverall, pre-clinical studies performed in different MS models suggest that exercise may involve multiple, parallel, and non-exclusive immunoregulatory mechanisms . On one Exercise appears to exert a pre-conditioning effect to the development and/or progression of the disease, raising two clinically relevant and interconnected questions. The first refers to the immunomodulating role of a therapeutic regimen of exercise and the possibility that exercise activates different mechanisms respect to the preventive regimen. The second regards the association of exercise to currently approved DMTs and the possible synergistic effects on pathological hallmarks of the disease. Both issues are barely addressed.Finally, we envisage the importance of understanding in depth the immunomodulatory action of EE and VE, since they are reported to exert a control on immune and nervous systems in healthy condition , 24, 50 AG, DC, and GM conceived and designed the manuscript. All the other authors made significant contributions, reviewed, and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In Alzheimer's disease (AD), tau pathology manifested by the accumulation of intraneuronal tangles and soluble toxic oligomers emerges as a promising therapeutic target. Multiple anti-tau antibodies inhibiting the formation and propagation of cytotoxic tau or promoting its clearance and degradation have been tested in clinical trials, albeit with the inconclusive outcome. Antibodies against tau protein have been documented both in the brain circulatory system and at the periphery, but their origin and role under normal conditions and in AD remain unclear. While it is tempting to assign them a protective role in regulating tau level and removal of toxic variants, the supportive evidence remains sporadic, requiring systematic analysis and critical evaluation. Herein, we review recent data showing the occurrence of tau-reactive antibodies in the brain and peripheral circulation and discuss their origin and significance in tau clearance. Based on the emerging evidence, we cautiously propose that impairments of tau clearance at the periphery by humoral immunity might aggravate the tau pathology in the central nervous system, with implication for the neurodegenerative process of AD. Cognitive decline and memory loss, with related clinical symptoms affecting everyday activities, signify the advanced stage of the pathology, preceded by preclinical and mild cognitive impairment (MCI) stages. MCI is a prodromal process, characterized by the onset of the earliest cognitive symptoms including memory dysfunctions and other cognitive impairments, which in most of the cases gradually evolve into a clinical AD . In-dept disease , 3. Tau  disease . It play disease , contrib disease –9. Underβ peptides and tau protein are of special research and translational interest [β peptide-reactive antibodies [β treatments turned disappointing, suggesting also other players [β-related impairments [Due to the critical involvement of the immune system in the pathobiology of AD, antibody-based therapies are under close scrutiny as a promising disease-modifying strategy. To this end, circulating immunoglobulins reactive with Ainterest . While mtibodies . Neverth players , 13. Tauairments . Accordiairments , 16.Throughout this review, we discuss emerging data on tau-reactive antibodies in circulatory systems and consider their relevance to tau homeostasis and dysregulations in AD. We highlight advances and stress the lack of consensus in the field, along with urgent need of in-depth research, to elucidate the role of humoral immunity in tau homeostasis. Finally, we discuss the possible significance of tau-reactive antibodies for countering the progression of the neurodegenerative process in AD.Although tau is an intracellular protein with the main localization in neuronal axons , it alsoIn the CNS, tau protein occurs in six isoforms due to alternative splicing  and as aUntil recently, there was no possibility of measuring tau protein in peripheral fluids, due to lack of sensitive methods. Research for blood-based biomarkers of neurodegenerative diseases, however, facilitated the development of a Simoa-HD1-based novel single-molecule ELISA capable of detecting minuscule amounts of tau . With thBecause of physiological tight protection of the brain by blood-brain barriers (BBB), an increase in the amount of neural proteins in peripheral blood circulation suggests impairments of the barriers . Like ADThe increased levels of tau protein in the peripheral circulation of AD patients could potentially reflect two pathological processes occurring in the brain: (1) BBB disruptions and (2) extensive axonal damage with neurodegeneration. Despite the rise of the level of tau protein in the blood, as noted above, levels of tau-reactive antibodies in serum at MCI and clinical AD remain unchanged –57. One The presence of tau-reactive autoantibodies of both IgG and IgM has been proven in sera and cerebrospinal fluid of AD patients as well as in healthy controls , 60–64, Relatively similar levels of tau-reactive antibodies in AD and controls detected in most studies call into question their general physiological significance and potentially beneficial role under pathological circumstances. The ubiquitous occurrence of tau-reactive antibodies in sera and cerebrospinal fluid of healthy individuals and in IVIG products suggests that they are unlikely to be harmful , 65. ButOverall, while overwhelming data indicate the presence of tau antibodies in circulatory liquids in the brain and at the periphery, debate persists over their origin and relevance to normal physiology and pathobiology of AD. Whether these immunoglobulins are naturally occurring antibodies, mostly with the poly/cross-reactive profile, or whether they are generated as more specific antibodies after the exposure of tau protein as an antigen with newly exposed epitopes to the peripheral immune system remains to be determined. As discussed below, an in-depth analysis of these important issues has led recently to several interesting discoveries, bringing more clarity in complex affairs of the immune system and the brain under healthy and diseased conditions.The generalized assumption that low-affinity polyreactive natural anti-tau antibodies have beneficial functions while high-affinity nonreactive autoantibodies are harmful, while instructive, requires rigorous experimental validation. With growing recognition of major immunogenic components in AD and other chronic neurodegenerative diseases and interest in immunotherapy, there is a pressing need in the research of naturally occurring endogenous antibodies directed to brain antigens , 70. WheThe prevalence of tau-reactive natural human antibodies shown recently in peripheral circulation presents a major advance . Their oBeside predominantly beneficial natural antibodies, autoantibodies causing a range of chronic autoimmune disorders can be also produced, typically due to dysregulations of the immune system , 78. TheTo summarize, antibodies reactive with tau protein are more likely to be represented by a mixture of both, the natural antibodies without antigen-dependent maturation and more specific antibodies formed after the exposure to tau as an immunogen. The crucial question that remains to be addressed is whether the antibodies against pathological tau forms may help their clearance or whether they bind with them and thus contribute towards the inflammation and harmful immunological response unfolding in AD.Using circulating tau-reactive antibodies from the peripheral system, it was shown that these antibodies can recognize abnormally modified tau forms and vary in a great deal in their characteristics , 67. TheAberrations of tau protein conformation play a major role in the pathobiology of AD; therefore, antibody-based immunotherapy directed against structurally modified and aggregated tau variants presents an attractive therapeutic strategy . The patin vitro binding affinity for antibodies might also contribute to the general variability of the research outcome [To the best of our knowledge, to date, only five reports analyzed IVIG products containing tau-reactive antibodies –68, summ outcome . Finally outcome .Throughout this study, we reviewed recent evidence concerning the origin and functionality of tau-reactive antibodies in the circulatory systems of the brain and periphery and discussed their relevance to pathophysiology and therapy of AD. Although much progress has been made in this direction, numerous basic and translational questions remain unanswered, requiring new thinking and in-depth research. There is also a major need in new assays with higher sensitivity, for the detection and quantification of a wide variety of tau proteins, as well as antibodies reactive to tau, which will allow better substantiation of the research and therapeutic interventions, including the use of IVIG immunotherapy. Important lessons should be learned from disappointing outcomes in clinical trials, which testify the incompleteness of our understanding of the biology of autoantibodies and pathophysiology of AD. Arguably, the most exciting outcome of recent studies is the recognition that throughout evolution, a powerful natural mechanism has been set to maintain the homeostasis of tau protein and facilitate its clearance in peripheral circulation when in excess, which can be exploited and guided for therapeutic benefit. With incentives getting higher for effective AD-modifying therapies, reviewed herein are advances in tau-reactive antibodies and their role in tau clearance is likely to become an important part of gradually emerging complex solution to this debilitating disease in the foreseeable future.


What problems were addressed?Abstraionalism, Undergraduate medical student, Content analysis, In-depth interview Informal learning through interaction between a teacher and a learner could not be replaced or compensated by formal education . In factWhat was tried?A core committee of 12 clinical staff and ethicists was developed. All of these professors had previously attended the faculty development workshops in the field of professionalism. Each member chose at least one topic out of the list of defined topics: the concept of professionalism, excellence, justice, responsibility, altruism, respect, autonomy, honor and integrity, conflict of interest, and ethical reasoning, and prepared for presenting it in peer-review sessions which was held monthly. After each peer-review session, feedback was provided on the content, teaching method, and teaching materials used by the presenter by all participants. Concurrently, faculty development workshops were held in Educational Developmental Center (EDC) of the Tehran University of Medical Sciences (4 times a year) by these empowered trainers. After each workshop, it was announced that any interested professors were welcomed to join the group; so the group was actively expanding. So far, 14 peer-review sessions attended by 42 participants with a combination of faculty members from the Ethics and Clinical Departments have been held to peer-review and provide feedback on the sessions. Finally, 16 new trainers in 25 person/topics have been prepared for teaching at the workshops for the empowerment of clinical professors in the context of professionalism. This team has held ten workshops at the EDC and 8 workshops in 6 subsidiary hospitals of the university, and has empowered a total of more than 300 faculty members in the professionalism and its related topics.What lessons were learned?With regard to the skills and capabilities of the medical practice, such as professional commitment and communication skills, impact of lessons provided by clinical trainers -familiar with real challenges-, is higher. So, potent trainers can be trained using the peer-review sessions with active participation of a team consisted of both clinical disciplines and ethicists. This method combines the advantage of being familiar with real challenges and enough self-confidence of the clinical trainers.


Brain natriuretic peptide (BNP) is an important biomarker for patients with heart failure, hypertension and cardiac hypertrophy. Although it is known that BNP levels are relatively higher in patients with chronic kidney disease and no heart disease, the mechanism remains unknown. Here, we review the functions and the roles of BNP in the heart-kidney interaction. In addition, we discuss the relevant molecular mechanisms that suggest BNP is protective against chronic kidney diseases and heart failure, especially in terms of the counterparts of the renin-angiotensin-aldosterone system (RAAS). The renal medulla has been reported to express depressor substances. The extract of the papillary tips from kidneys may induce the expression and secretion of BNP from cardiomyocytes. A better understanding of these processes will help accelerate pharmacological treatments for heart-kidney disease. The Brain natriuretic peptide (BNP), which is a component of the natriuretic peptide (NP) system and also known as B-type NP, is mainly secreted from the cardiomyocytes in response to cardiac stretch and ischemia, and plays an important role in cardiorenal protection ,2,3,4. TCongestive heart failure (CHF) is a complex syndrome characterized by sodium and water retention through the activation of different neurohormonal systems, such as the RAAS and the sympathetic nervous system (SNS), and also importantly, the NP system. Several experimental and clinical studies have implicated BNP in the pathophysiology of the unbalanced cardiorenal axis in CHF. In CHF patients, BNP plasma levels are in excess of 100 pg/mL . Due to Growing evidence suggests that in CHF, coronary artery disease and/or left ventricular hypertrophy etc., are increasingly associated with CKD patients. More recently, the cardiorenal syndrome (CRS), which is a complex pathophysiological condition that involves an association between acute heart failure (AHF) or CHF and renal impairment, has received much more attention. However, this condition is more than just a simultaneous cardiac and renal disease . A recenBNP acts as a compensating agent in the early stages of disease progression by inducing natriuresis and dieresis, and reducing RAAS and SNS. Similar to that seen with severe disease states like HF or CRS and despite high levels, endogenous BNP becomes resistant and is no longer able to compensate for volume overload in such cases. Thus, the supportive role of BNP in counteracting the unwanted effects of activated RAAS and SNS in these diseases provides the rationale for using this peptide as a potential therapeutic agent ,24. As iHuman BNP mRNA is translated to preproBNP of 134 amino acids, from which proBNP of 108 amino acids is processed and cleaved by the serine proteases corin and/or furin, yielding a biological inactive amino-terminal fragment, 76-amino acid proBNP (NT-proBNP) and an active carboxy-terminal fragment, 32-amino acid BNP . It has It has been well established that BNP plays important roles in the kidneys, and provides multiple beneficial effects involving renal function . AbnormaThe functions of NPs are mediated by their interactions with specific surface receptors on the target cells. In the kidney, BNP increases GFR by relaxing the mesangial cells and inhibits the tubular fractional reabsorption of sodium ,35 Figu. BNP alsBNP infusion may play an important role in preventing the development of CKD. McKie et al. recently examined BNP’s role in the pathophysiology of cardiorenal dysfunction and found that in asymptomatic systolic HF patients, chronic subcutaneous BNP therapy for 12 weeks improved renal function along with favourable hemodynamic effects in response to volume expansion . A recenThe vascular effects of NPs are site-specific . ImportaIn CKD patients, impaired renal function restricts the use of NPs, as plasma BNP levels are elevated to ~200 pg/mL in CKD patients without HF. Whether these elevated BNP levels in CKD promote the activation of the NP system and have an effect on the target organ still remains unclear. Downregulation of NPR-A and upregulation of NPR-C expression in the renal medulla and renal cortex, respectively, may be responsible for the resistance of NPs including BNP in CKD, thereby resulting in its limited use in treating CKD patients with HF ,65.BNP clearance is associated with two major pathways. The first involves binding to the NPR-C receptor, while the second one is involved with the degradation by neprilysin (NEP), a zinc metallopeptidase. NPR-C, which is devoid of any guanylyl cyclase activity and coupled to the adenylyl cyclase inhibition , plays aSympathetic stimulation activates the RAAS, including the augmentation of angiotensin II (Ang II) and aldosterone production, as a result of the elevation of vasopressin and norepinephrine plasma levels, as well as the secretion of renin. SNS and RAAS augment the cardiac output by increasing heart rate, contractibility, preload and afterload at the expense of increased oxygen consumption . InhibitBNP has been shown to directly inhibit renin production from the kidney before affecting RBF or GFR , similarExperimental studies using rat cardiomyocytes reported that both endogenous and exogenous BNP reduced aldosterone synthase (CYP11B2) mRNA expression, which may lead to the inhibition of the RAAS and attenuation of cardiac hypertrophy and fibrosis . Further2 has demonstrated that over 12 months, the combination of sacubitril and valsartan was well tolerated and had similar effects on kidney function and albuminuria compared to ARB irbesartan, with a BP and cardiac biomarker lowering effect, suggested that this combination could have the potential for reducing cardiovascular risk in CKD [Although BNP is progressively activated in HF, its response may often be insufficient to counteract the sodium retention and vascular constriction due to activation of RAAS and SNS. Therefore, more thorough approaches were undertaken both experimentally and clinically in an attempt to minimize the dysregulation of these neurohormonal systems. Ongoing strategies in promoting NP include synthesis or using agonists to increase its bioactivity and inhibition of NEP to reduce its catabolism ,91. Nesik in CKD .LCZ696 lowered BP more effectively than valsartan in hypertensive patients . The PARIn spite of having numerous benefits of ARNi so far, however, some concerns raised that limits its clinical use, such as the elevation of bradykinin levels with ARB, the development of angioedema , and theKidneys play an important role in regulating blood pressure. In addition to volume control via urination with balanced water and electrolytes excretion, the kidneys possess several substances that are used to control blood pressure  Figure . The mosEndothelin-1 (ET-1), a 21 amino acid vasoconstrictor, is also produced by the endothelial cells, tubular cells and inner medullary collecting duct cells . In physProstaglandin (PG) is a lipid mediator involved in a variety of physiological and pathological processes in the kidneys . PGE2 isTissue kallikrein is mainly expressed in the submandibular gland, pancreas and kidneys. Kallikreins are primarily produced in the distal convoluted tubule ,119, theIt has been reported by several independent groups that the renal medulla possesses a depressor substance that acts as a counterpart to RAAS ,127. ThiNPPB gene from −1000 to +136 and demonstrated that this promoter was specifically activated in the papillary tips of the kidneys and was not accompanied by the BNP mRNA expression [We recently developed a BNP reporter mouse carrying tdTomato under the mouse promoter of the 1136-bp fragment of the mouse pression . No evidpression ,133,134.pression .The pBNP-tdTomato-positive cells were interstitial cells and were not proliferative. The papillary medulla has been reported to possess the ability to decrease blood pressure due to its vasodilatory activity ,127. To BNP plays an important role as a major player in the heart-kidney connection via its inhibitory effect on the RAAS, especially in the heart and the kidneys. Kidneys possess several substances involved in regulating the blood pressure in addition to volume control via urination. Furthermore, the papillary tips may play important roles in regulating the BNP expression from cardiomyocytes. Additional investigations will need to be undertaken in order to determine the relationship between the renal depressor system and BNP regulation, especially in terms of cardiovascular diseases, such as heart failure, hypertension and CKD.


The use of the rabies vaccine for post-exposure prophylaxis started as early as 1885, revealing a safe and efficient tool to prevent human rabies cases. Preventive vaccination is the basis for the control of canine-mediated rabies, which has already been eliminated from extensive parts of the world, including Europe. Plans to eliminate canine-mediated human rabies by 2030 have been agreed upon by international organisations. However, rabies vaccines are not efficacious against some divergent lyssaviruses. The presence in European indigenous bats of recently described lyssaviruses, which are not neutralised by antibody responses to existing vaccines, as well as the declaration of an imported case of an African lyssavirus, which also escapes vaccine-derived protection, leaves the European health authorities unable to provide efficacious protective vaccines to some potential situations of human exposure. All these circumstances highlight the need for a universal pan-lyssavirus rabies vaccine, able to prevent human rabies in all circumstances. Carnivora, is caused worldwide by members of the lyssavirus genus, principally rabies virus (RABV), the same virus used as the immunogen in existing human and animal vaccines. Other viruses within this genus are present in bat populations across much of the globe. Interestingly, North and South America are the only continents where RABV is the only lyssavirus present, causing rabies in bats, dogs and other mammals. Elsewhere, across the old world, thirteen different lyssaviruses are known to infect bats and can cause the disease rabies. European bat lyssaviruses type-1 (EBLV-1) and -2 (EBLV-2), Bokeloh bat lyssavirus (BBLV), Aravan lyssavirus (ARAV), Khujand lyssavirus (KHUV), Irkut lyssavirus (IRKV), West Caucasian bat Virus (WCBV), Lleida bat Lyssavirus (LLEBV) and Gannoruwa bat lyssavirus (GBLV) are present in bat populations in Eurasia, Australian bat lyssavirus (ABLV) in Australian bat populations and Duvenhage lyssavirus (DUVV), Lagos Bat lyssavirus (LBV) and Shimoni bat lyssavirus (SHIBV) in African bats [Rabies is one of the most feared infectious diseases worldwide due to its extreme lethality in animals and humans . For thican bats   and Mokola Virus (MOKV), have been described but have never been detected in bats. Genetic evidence of a putative lyssavirus species detected in Finland, Kotalahti bat lyssavirus, awaits isolation and classification. Lyssaviruses cause neurological disease in mice when infected intracranially under laboratory conditions , but onlVespertilionidae. Cases of EBLV-2 infection have been detected in Myotis dasycneme and Myotis daubentonii, cases of BBLV infection detected in Myotis nattereri [Myotis brandtii. EBLV-1 is associated with more than 95% of the infected bats diagnosed in Europe and, with the exception of sporadic cases, with the serotine bat (Eptesicus serotinus), or its sibling species (Eptesicus isabellinus) in the southern Iberian Peninsula. Both Eptesicus species are synanthropic, which probably drives the higher incidence of EBLV-1 cases detected, since rabies passive surveillance is based on bats submitted by the public and consequently is biased to dead or diseased individuals of the species present near human habitats. In fact, most of the bats submitted for rabies diagnosis are common bats (Pipistrellus pipistrellus/pygmaeus), which have been very rarely shown to be infected with lyssaviruses and are not considered rabies reservoirs [Myotis blythii) and KHUV (Myotis mystacinus). Rabies vaccine-induced antibodies neutralise all these viruses and consequently PEP is believed to be efficacious for the prevention of human infection by these viruses [All phylogroup I lyssaviruses presently known in Europe are associated with specific bat species belonging to the family attereri  and the servoirs . Some of viruses . Miniopterus schreibersii) captured in the northern region of the Caucasus. More recently, another lyssavirus, LLEBV, was reported in the same bat species in the city of Lleida, at the north-east of the Iberian Peninsula in Spain [In 2003, a new lyssavirus, WCBV, was detected in a cave bat  imported from Africa into Belgium and then transferred to the city of Bourdeaux in France was shown to be infected with LBV  and also on the island of Cyprus (Eastern Mediterranean). Interestingly, the barrier effect of the Strait of Gibraltar on bat populations is different depending on the species considered and connections between African and European populations have been demonstrated in some cases, including some that are known to be reservoirs of lyssaviruses, as in the case of E. isabellinus [In 1999, a fruit bat (with LBV . LBV is bellinus . This miRousettus aegyptiacus are bred locally in Europe and Miniopterus schreibersii is not usually linked to urban or suburban environments and rarely interacts with humans. In fact, both bat species are underrepresented in the series of passive surveillance in Europe. However, although low, the risk of human exposure to their associated lyssaviruses exists. This risk must be considered in the global plan to eliminate human rabies, especially should the elimination of dog-mediated human rabies by the 2030 target be successful. New lyssaviruses are discovered with increased frequency. Moreover, there are several studies reporting lyssavirus antibodies on a wide spectrum of bat species in Europe [Bat rabies has been considered a threat that can be safely managed in Europe with the combination of good surveillance and immediate PEP. However, the presence in European indigenous bats of recently described lyssaviruses, which are not neutralised by antibody responses to existing vaccines, as well as the declaration of an imported case of an African lyssavirus which also escapes vaccine-derived protection, leaves the European health authorities unable to provide efficacious protective vaccines to some potential situations of human exposure to phylogroups II and III bat lyssaviruses. It is true that captive n Europe , suggestn Europe . Technoln Europe . The req


ESE  is widely used for the treatment of digestive diseases. The dental floss traction (DFT) method has been successfully used to facilitate ESE to resect mucosal lesions such as early gastric cancer. DFT has not been used in ESE to remove submucosal masses. This study aimed to examine the efficacy of DFT-assisted ESE (DFT- ESE) for the removal of submucous masses. From March 2017 to May 2017, a total of 12 patients with gastric submucosal masses at the First Affiliated Hospital of Nanchang University, Jiangxi, China, were enrolled. The tumor characteristics, en bloc resection rates, complications, and outcomes on follow-up were evaluated for all patients. The 12 submucosal tumors were completely removed by DFT- ESE. Nine were gastrointestinal stromal tumors. Two were Schwannoma, located in the greater curvature of the gastric corpus. One was gastric ectopic pancreas. All the resected tumors were removed completely with intact tumor capsules. There was no more bleeding or perforation after the endoscopic closure of the perforation or the wound after the DFT-ESE, and no recurrences were identified at the time of follow-up.ChiCTR-OOC-15005833). The DFT method efficiently and safely facilitated the ESE procedure during the resection of gastric submucosal tumors. This study was registered with Chinese Clinical Trial Registry under Registration number  Endoscopic submucosal excavation (ESE) has been widely used for resection of the early gastric cancer, gastric submucosal masses, and colonic laterally spreading tumor (LST) . The proFrom March 2017 to May 2017, twelve patients with gastric submucosal masses located in the gastric fundus or at the greater curvature of anterior gastric corpus wall were enrolled in the study, since lesions located in those locations are difficult to remove by ESE without traction.The gastric masses were examined by endoscopic ultrasound and computed tomography before ESE; all masses were confirmed to be localized in the submucosal or muscular layer without distant metastasis. The mass characteristics, en bloc resection rate, and complications were reviewed. Informed consent was obtained from each patient.The DFT-ESE procedure is depicted in the images presented in Figures First, the mass was labeled and injected in multipoint with lifting solution  by injection needle through an endoscope  channel. The mass body was usually identified after the mucosa was dissected along the label margin. Second, dental floss was knotted to the titanium clip , which was then delivered to the lesion through the biopsy channel of the endoscope. The titanium clip was clamped at the side of the mass, and the lifting position of the mass was kept in front of the endoscopic view while pulling. Lastly, we used varying levels of strength to pull the dental floss according to the exposure extent of the mass. In this way, the hook knife  could easily enter into the gap between the mass and normal tissue and therefore, the mass could be easily resected en bloc. During ESE, hemostasis was achieved with HybridKnifes  or Coagrasper . The method used for closing the wound or perforation depended on its size. Small wounds or perforations were directly closed with titanium clips, whereas for large ones, we used endoscopic nylon loop and a titanium clips pouch suture technique to close them, which we have reported in our previous study , 8.After the operation, all patients who underwent full-thickness resection or had perforation during the operation were fasted for 24 hours and received antibiotics for 24-48 hours and proton pump inhibitor (PPI) therapy for 4-6 weeks. For the patients without perforation, they fasted for 24 hours after the procedure and were given PPI for 4-6 weeks without antibiotics.From March 2017 to May 2017, our group had completed 12 cases with gastric submucosal mass by DFT-ESE.The details are shown in Gastric submucosal masses include gastric stromal tumor, leiomyoma, heterotopic pancreas, neuroendocrine tumor, and lipoma. Some of the lesions, such as gastric stromal tumors, have malignancy potential. At present, endoscopic submucosal dissection (ESD), ESE, and endoscopic full-thickness resection (EFR) are used to remove those tumors –12, but The key step that ensures DFT-ESE successful is that the transparent cap attached at the tip of the endoscope can get close to the gap between the lesion and the normal tissue after traction. For lesions located in the gastric fundus or in the greater curvature of anterior gastric corpus wall, it is difficult to reach to lesion with the endoscope during conventional ESE procedure. For lesions arising from the serous layer, the transparent cap does not maintain a good view by separating the lesion from the normal tissue because the scope and cap cannot reach the gap between the lesion and the normal tissue, resulting in difficulty and even failure to remove the lesions.DFT can assist in exposing the gap between the lesion and the normal tissue by lifting the lesion, thereby making resection possible, even if the endoscope cannot reach the lesion or the transparent cap cannot enter into the gap between the lesion and the normal tissue, even if, during the process of gastric full-thickness resection of a lesion, DFT can maintain a good view and prevent the lesion from falling into the abdominal cavity. Since DFT makes ESE easier, it improves the success rate of ESE and reduces the complications such as bleeding and perforation which happen during the operation.We recommend using DFT-ESE for resection of gastric submucosal masses located in some anatomic areas, such as the gastric fundus; the greater curvature of anterior gastric corpus to increases the success rate of resection.


Diet is a key lifestyle factor that can have long-term effects on ocular health. This Special Issue of Nutrients entitled ‘Nutrition and Eye Health’ contains 12 articles, including reviews and primary research studies, that report on a diverse range of topics relating to the role of nutrition in maintaining eye health, and the potential use of nutritional interventions for preventing or treating ocular disease. Collectively, these papers span a spectrum of ocular conditions, including corneal angiogenesis ,4, diabeGlobally, approximately 250 million people suffer from varying degrees of vision loss . LeadingAMD is characterised by degenerative changes within the macula, the central area of the retina that is responsible for high-resolution vision, in people aged 55 years or older. AMD is a leading cause of severe vision impairment in European-derived populations. In the UK, the disease is responsible for over 50% of certifiable vision loss . AlthougIn terms of primary research studies, the highest quality evidence to evaluate the efficacy and safety of therapeutic interventions derives from randomised controlled trials (RCTs). In RCTs, participants are randomly allocated to receive either the intervention or a comparator , which minimises the potential for bias in the intervention assignment . There iOther RCTs have investigated whether high-dose antioxidant vitamin and mineral supplements can slow the progression of AMD . Most ofCataract is defined as any visible opacity within the otherwise clear crystalline lens of the eye. Cataract can be further classified as cortical, nuclear, or posterior sub-capsular, depending on the anatomical location of the opacity. Globally, over 60 million people are visually impaired due to cataract, however cataract-associated blindness shows significant geographical variation, accounting for less than 22% of blindness in high-income countries compared to more than 44% in South East Asia . Age is In conclusion, age-related eye diseases, including cataract and AMD, are of global public health concern. Acquired vision loss associated with these conditions can be devastating to the individual through its detrimental impact on quality of life, and also impart substantial societal burden. Although the pathogenesis of these conditions is not fully understood, there is increasing evidence that their impact can, to some extent, be mitigated by targeting modifiable risk factors. Since diet and nutrition have been linked with the most common diseases affecting the elderly, dietary modification and nutritional supplementation for the prevention and treatment of these diseases has attracted a considerable amount of scientific attention. As evidenced by the quality and diversity of the contributions in this Special Issue, the role of nutrition in eye health remains a highly topical area, with scope for future research to enhance our understanding of the role of nutritional strategies for optimising eye health.


The use of transgenic animals as bioreactors for the synthesis of therecombinant proteins secreted into milk is a current trend in the developmentof biotechnologies. Advances in genetic engineering, in particular theemergence of targeted genome editing technologies, have provided newopportunities and significantly improved efficiency in the generation ofanimals that produce recombinant proteins in milk, including economicallyimportant animals. Here, we present a retrospective review of technologies forgenerating transgenic animals, with emphasis on the creation of animals thatproduce recombinant proteins in milk. The current state and prospects for thedevelopment of this area of biotechnology are discussed in relation to theemergence of novel genome editing technologies. Experimental and practicaltechniques are briefly discussed. For many years, genetically modified laboratory animals have been an effectivetool for studying the functional properties of genes, proteins, and othermolecules, and their importance as human disease models in biomedical researchcan hardly be overestimated. Such animals can be used to study the pathogenesisand molecular features of diseases, for the identification and validation ofnew therapeutic targets, and for effective search and development of new drugs,including preclinical studies. At the same time, genetically modified animalsare becoming increasingly attractive objects in fields such as livestockfarming, where genome changes can be used to correct economically importantanimal traits. Finally, transgenic animals can serve as bioreactors for thesynthesis of the recombinant proteins secreted into milk, which enables theproduction of recombinant proteins in substantially larger amounts and at muchlower costs than the production of proteins in eukaryotic cell cultures. AccordiThe classical method for the generation of transgenic mammals, which wasdeveloped in the early 1980s and has been in wide use up to the present time,entails the microinjection of a transgene-containing DNA fragment into thepronucleus of a fertilized oocyte, followed by transplantation of the oocyte tocompetent (pseudo-pregnant) animals. In the scheme, the transgene-containingDNA fragment is randomly integrated into the recipient genome during naturalprocesses of genomic DNA breakage and repair.TransgeTherefore, during classical transgenesis, the transgene is randomly integratedinto the recipient’s genome; in this case, the number of integratedtransgene copies, including incomplete transgene fragments, is uncontrolled. Ifproduction of a recombinant protein is required, the transgenic constructshould include a full expression module that provides autonomous transcriptionof the transgene in target tissues of the organism and proper mRNA processing,because of random transgene integration. When using alternative methods oftransgenesis and technologies of targeted genome editing (see below), thisrequirement is not mandatory.The key determinant that provides tissue specificity in transgene expression isthe promoter. A number of promoters of genes encoding milk proteins have beensuccessfully used for the production of recombinant proteins in the mammarygland. Promoters that enable production of the target protein at a sufficientlyhigh level in milk (up to tens of grams per liter of milk) include promoters ofgoat and cow β-casein, cow α-s1-casein, rabbit whey acidic protein(WAP), human α-lactalbumin, and sheep β-lactoglobulin genes. However,the level of protein production depends not only on the promoter, but also on anumber of other factors. In this case, promoters of one animal species canprovide effective transcription of the transgene in the mammary gland cells ofanother animal species due to conservatism of the transcription factorsregulating the production of milk proteins in mammary cells-19.As the experience of generating transgenic animals has demonstrated, effectiveproduction of a recombinant protein often requires, apart from atissue-specific promoter that ensures a high level of transgene transcription,inclusion of introns into the transgene. Inclusion of introns into thetransgene in some experimental systems enabled a 400-fold increase in thetransgene transcription level, compared to that from intron-free cDNA, whilethe effect of intron inclusion is minimal in other systems, 21.Difcis-elements that are designed to protect the transcription ofthe transgene from the influence of its environment. One of the most commonlyused cis-elements is the chicken β-globin locus HS4 insulator[Even the optimal expression cassette design does not guarantee effectivetransgene expression, which is due to the random site of transgene integrationinto the recipient genome. The surrounding chromatin, depending on thetransgene integration site, can have a negative effect on transgenetranscription. In addition, widespread tandem integration of several transgenecopies can lead to suppression of their transcription due to transcriptionalinterference by neighboring copies .Therefonsulator, 31.Incnsulator.Therefore, the classical transgenesis used since the early 1980s has a numberof significant drawbacks that are primarily due to the high variability oftransgene expression caused by the randomness of its genomic integration site.Thus, to select an animal line with satisfactory parameters of recombinantprotein production, a sufficiently large number of primary transgenic animalsshould be available. This may be a significant technical problem whengenerating transgenic livestock that produce recombinant proteins in milk,which is due to the need for a large number of embryos to generate a line oftransgenic animals with satisfactory parameters of target recombinant proteinproduction.In addition to these drawbacks of classical transgenesis, the randomness oftransgene integration into the recipient genome and uncontrolled variability inthe number of transgene copies create certain difficulties specific totransgenic animals intended for practical use in the real economy. Namely,registration of modified organisms requires mandatory identification of thetransformation event (the exact integration site of the transgenic constructinto the genome) unique to the line of transgenic animals. In the case ofclassical transgenesis, identification of the transformation event for eachtransgenic animal line is a separate experimental problem whose solution iscomplicated if multiple transgene copies are integrated into the genome.The randomness of transgene integration into a recipient genome anduncontrolled variability in the number of transgene copies are significantdrawbacks of the “classical” approach to the creation of transgenicanimals. These drawbacks have stimulated the development of alternativetechnologies enabling transgene integration into a specific genomic site. Untilrecently, transgene integration into a specific genomic site using homologousrecombination either in embryonic stem cells with a subsequent injection ofgenetically modified cells into blastocysts or in somatic cells, followed bysomatic cell nuclear transfer (SCNT) into the oocyte, was the alternative toclassical transgenesis. In both cases, genetic manipulations are performed withcells in culture, which enables a characterization of the accuracy of transgeneintegration before the generation of transgenic animals. In addition, aqualitative improvement of the classical transgenesis technology was atransgene integration into a pre-determined site of the genome, usinghomologous recombination through flanking of a transgenic expression cassettewith genomic regions . It should be noted that the genetic elementsenabling the production of a recombinant protein in milk are identical for thedescribed approaches and classical transgenesis. The drawbacks of theseapproaches include the need for selective markers for picking cell clones witha genome-integrated transgene and a laborious clonal selection process thatrequires the analysis of a large number of cell clones  even when negative selection is used, which is due to the low efficiencyof homologous recombination. In this case, even upon subsequent removal of aselective marker from the expression cassette, e.g., by means of site-specificrecombination when the marker is flanked with appropriate recombination sites,exogenous DNA sequences, along with the target transgene sequences, inevitablyremain in the genome, which may be undesirable.in vitro. In this case, to generate a genetically modifiedanimal, stem cells carrying a genetic modification are injected intoblastocysts, with their subsequent implantation and creation of transgenicmosaic animals [Embryonic stem cells can be used as recipient cells for genetic manipulations animals . The des animals -36.Howe animals ) into mi animals , 38, 39.In addition to the methods based on homologous recombination of the transgeneand on the use of cultured cells in combination with positive and negativeselection for picking cell clones with homologous recombination, anotheralternative for targeted insertion of a transgene into a recipient genome isthe use of site-specific recombinases. In general, the concept of usage ofsite-specific recombinases is based on the generation of a line of transgenicanimals carrying recognition site(s) for recombinase in the genome. Suchrecognition sites can be integrated into a specific genomic site by means ofhomologous recombination or into a random genomic site by means of classicaltransgenesis. In the latter case, lines of transgenic animals with differentvariants of transgene localization in the genome are subjected to selection ofa line with the transgene integrated into the genomic site that provides therequired properties of transgene expression. This animal line is then used as auniversal recipient for the insertion of different transgenes into a specificgenomic site via site-specific recombination. For this purpose, a geneticconstruct containing a transgene flanked by recombination sites ismicroinjected into the fertilized egg of a transgenic animal containing thesame recombination sites in the genome, together with a vector for theexpression of recombinase or its mRNA, 41.ThiSaccharomyces cerevisiae, and phage φC31 integrase[In practice, three recombination systems have been commonly used forsite-specific transgenesis: phage P1 Cre recombinase, Flp recombinase ofntegrase, 43.In ntegrase, 44.Intntegrase. The prontegrase-48can bntegrase, 50.ROSA26, Cd6, Hipp11, and some others[An alternative approach to ensuring effective and stable transgene expression,in particular in mammary gland cells, using appropriate tissue-specificpromoters is the use of so-called “safe harbors” as transgeneintegration sites. These harbors are genomic loci that are, on the one hand,insignificant for the development and functioning of the organism, whichenables harmless transgene insertion into this locus and, on the other hand,provide a high level of transgene expression in the presence of appropriateregulatory elements in transgene. Examples of these genomic loci are the locie others,51, 52.In addition to the listed advantages, the use of site-specific recombinases andintegrases for targeted transgene insertion into an animal genome has aregulatory significance in the case of transgenic animals intended forpractical use, due to the significant simplification of the characterization ofthe transformation event (the site of transgene integration into the recipient genome).The emergence of targeted genome editing technologies using site-specificnucleases has resulted in significant advances in the field of animaltransgenesis that enable much more efficient transgene integration into aspecific site of the recipient genome compared to the sole use of sequences forhomologous recombination flanking the transgene . Below, Targeted transgene integration using site-specific nucleases is based on asignificant increase in the site-specific efficiency of transgene integrationinto the recipient genome during the repair of double- or single-strand DNAbreaks . TargeteThere are several classes of artificial nucleases that are used for targetedgenome editing and the production of transgenic animals: zinc finger nuclease(ZFN) , transcrZFN was the first nuclease designed for use in genetic engineering for targetedgenome editing. The nuclease contains DNA-binding domains of the zinc fingerprotein (ZFP) that provides highly specific binding to the target DNA sequence,as well as the catalytic domain of FokI restriction endonuclease thatintroduces a double-strand break into the binding site. Each zinc fingerrecognizes a certain triplet of nucleotides. Three to six zinc fingers are usedto construct the DNA-binding domain of ZFN. Their combination can be used togenerate ZFN for almost any DNA sequence . The strXanthomonas contain a DNA-binding domainconsisting of a series of monomers. Each monomer binds to one nucleotide in thetarget nucleotide sequence. Monomers are tandem repeats of 33–35 aminoacid residues, except for the last “half-repeat” that consists of20 amino acid residues. The amino acid residues located at positions 12 and 13of the monomer are highly variable and responsible for recognizing a specificnucleotide: Asn-Ile, Asn-Gly, Asn-Asn, and His-Asp bind to the nucleotides A,T, G, and C, respectively. Like ZFN, artificial TALEN nuclease is a chimera ofthe TALE DNA-binding domain consisting of 20-30 monomers and the FokI nucleasecatalytic domain [Later, a simpler, compared to ZFN, code of transcription activator-likeeffector (TALE) was deciphered ,65. TALc domain , which iIn the CRISPR/Cas9 system, the target is recognized as a consequence of thecomplementary interaction between CRISPR RNA (crRNA) and the target DNA site.In this case, a complex of trans-activating crRNA (tracrRNA), crRNA, and Cas9nuclease is assembled and then a double-strand break is introduced into aRNA/DNA duplex by Cas9 nuclease .TherefoIt should be noted that the artificial nucleases ZFN and TALEN do not possessabsolute specificity. To a larger extent, this problem relates to theCRISPR/Cas9 system. The problems of DNA cleavage by artificial nucleases innon-targeted genomic sites can be solved in various ways that allow forincreasing the specificity of the changes introduced into the genome andreducing the probability of unprogrammed genetic changes. However, the problemof nonspecific modifications in the recipient genome is not so critical upongeneration of transgenic animals as compared to targeted genome editingtechnologies in the field of clinical applications, since accidental changesthat occurr in non-targeted genome sites can be excluded during breeding.In eukaryotic cells, double-strand breaks introduced by site-specific nucleasescan be repaired through several mechanisms; in particular byhomologous-directed repair (HDR), where a repair template is the sister orhomologous chromatid as well as the donor DNA with 200–800 bp homologyarms , which eThe key mechanism for the repair of breaks introduced by site-directednucleases into a specific site of the genome is homologous recombination thatenables integration of the transgene located between homology arms into aspecific genomic site. This approach has been successfully implemented inanimal transgenesis using double-strand templates containing a transgeneflanked by 1- to 3-kb homology arms, with a repair efficiency of 0.5 to 20%-80. BecaAt the same time, alternative technologies have been developed which ensuretargeted integration of extended (up to 10–15 kb) DNA fragments into apre-determined genomic site without using the HDR mechanism. For example, oneof such technologies exploits NHEJ-based repair of breaks by ligating thecomplementary overlapping single-strand DNA ends of the genomic target site andthe repair template comprising a transgene, which are generated by a ZFNnuclease pair upon cleavage of the targeted sequences of the genome and repairtemplate , 86. InsTherefore, the rapid development of targeted genome editing technologies hasallowed researchers to avoid a number of the drawbacks inherent to classicaltransgenesis: e.g., random transgene integration into the genome anduncontrolled variability in the number of transgene copies. The CRISPR/Cas9technology enables the generation of transgenic animals with transgeneintegration into a specified genomic site, which, together with the use ofhomologous recombination, determines the controlled number of transgene copies.In particular, one of the most promising approaches to the generation ofanimals producing recombinant proteins in milk is the CRISPR/Cas9-targetedintegration of the transgene into the genes that encode milk proteins in such away that transgene expression is controlled by the endogenous regulatorysequences of the recipient gene. Application of these technologies willsimplify and standardize the technologies for generating transgenic animals forthe production of recombinant proteins. This will make the transgenesis processmore efficient and reduce costs in the generation of economically importanttransgenic animals. Genome editing technologies will allow researchers togenerate transgenic animals with one transgene copy integrated into a specificgenomic site, which will enable a reliable comparison of the influence ofcertain genetic elements present in a construct for transgenesis on recombinantprotein production in milk, which cannot be done using classical transgenesisdue to the integration of an uncontrolled number of transgene copies intodifferent genomic loci in different lines of transgenic animals.The development of targeted genome editing technologies has opened newprospects for the generation of transgenic animals at a whole new level. Thestandardization of the generation of transgenic animals with specified andstable target traits is becoming possible thanks to the use of knowledge on themolecular genetic mechanisms of regulation of gene expression and genomefunctioning, as well as the available technologies of genetic engineering. Thatis fully applicable to the production of recombinant proteins in milk for themanufacturing of pharmaceuticals, biologically active additives, etc.Taking into account the set of technologies generated to date, which are stillunder active development, the optimal direction of studies in the field ofrecombinant protein production in the milk of economically important animalspoints to the creation of animal lines  whose genome is modified bythe insertion of sequences for asymmetric directed recombination of theexpression cassette into a milk protein-encoding gene . The promoter, and other regulatory sequences, of this gene will providea high level of transgene expression. This insertion can be achieved with highefficiency through a microinjection into the oocytes of a genetic constructcarrying an expression cassette, together with the corresponding recombinantintegrase or its mRNA. These animal lines can be created with previouslyinaccessible efficiency by means of genome editing technologies us ing theCRISPR/Cas9 system or its analogues, thanks to the simplicity of implementationand design of this system. At the same time, the technologies of syntheticbiology allow one to use mini-genes with artificial introns as a transgene, butnot full-length gene copies, to facilitate efficient expression of thetransgene and production of the target protein, thus simplifying the design andcreation of genetic constructs for transgenesis.Prospects for the development of such a direction inthe production of recombinant proteins, primarily for medical needs, aresupported by the two marketed drugs that are based on recombinant proteinsobtained from the milk of transgenic animals. It should be noted that there isthe possibility of producing significant quantities of recombinant proteins atcosts substantially lower than those required for production in cellularsystems. The use of modern technologies significantly simplifies compliancewith regulatory requirements for describing the transformation event. At thesame time, the requirements to the biological safety of recombinant proteinproduction in milk will require a revision of the standards for the welfare offarm animals and veterinary control to exclude the presence of zoonotic andanthropozoonotic infectious agents, as well as controlled parameters of themanufactured drugs.