Urticaria is a disorder affecting skin and mucosal tissues characterized by the occurrence of wheals, angioedema or both, the latter defining the urticaria-angioedema syndrome. It is estimated that 12–22% of the general population has suffered at least one subtype of urticaria during life, but only a small percentage (estimated at 7.6–16%) has acute urticaria, because it is usually self-limited and resolves spontaneously without requiring medical attention. This makes likely that its incidence is underestimated. The epidemiological data currently available on chronic urticaria in many cases are deeply discordant and not univocal, but a recent Italian study, based on the consultation of a national registry, reports a prevalence of chronic spontaneous urticaria of 0.02% to 0.4% and an incidence of 0.1–1.5 cases/1000 inhabitants/year.We reviewed the recent international guidelines about urticaria and we described a methodologic approach based on classification, pathophysiology, impact on quality of life, diagnosis and prognosis, differential diagnosis and management of all the types of urticaria.The aim of the present document from the Italian Society of Allergology, Asthma and Clinical Immunology (SIAAIC) and the Italian Society of Allergological, Occupational and Environmental Dermatology (SIDAPA) is to provide updated information to all physicians involved in diagnosis and management of urticaria and angioedema. Urticaria is a disorder affecting skin and mucosal tissues characterized by the occurrence of wheals, angioedema or both, the latter defining the urticaria-angioedema syndrome. The wheal is a skin lesion presenting with a central edema of variable size, surrounded by erythema and associated to itching or, more rarely, feeling of warmth, that are transient, with spontaneous resolution in less than 24 h, and with no relics . Under hThe aim of the present document from the Italian Society of Allergology, Asthma and Clinical Immunology (SIAAIC) and the Italian Society of Allergological, Occupational and Environmental Dermatology (SIDAPA) is to provide updated information to all physicians involved in diagnosis and management of urticaria and angioedema.Classification is based on duration of clinical manifestations and on causative agents.AU is defined by a duration of < 6 weeks while for chronic urticaria (CU) the duration is ≥ 6 weeks. As far as causes are concerned, Table It is estimated that 12–22% of the general population has suffered at least one subtype of urticaria during life , but onlThe diagnosis of urticaria may be complicated by the heterogeneity of its phenotypes. The diagnostic work-up must start with an accurate clinical history defining the trait, the duration in order to distinguish acute from chronic forms, and the possible causative factors , 21. TabIn isolated angioedema, particularly when recurring from years and persisting over 24 h despite corticosteroid treatment, a differential diagnosis with hereditary or acquired angioedema must be performed by the level of C4 and the quantitative and functional measurement of C1-inhibitor .The responsibility of a given causative factor defines the group of chronic inducible urticaria (CIU), which is characterized by the occurrence of wheal or angioedema induced by stimuli including cold, heat, dermographism, pressure, vibration, sunlight and water and represent 20–35% of chronic urticaria. Contact, cholinergic and aquagenic urticaria are not included in this group, because the triggering factor is not physicalThere are some characteristics that are shared in the various forms of inducible urticaria:(1) the clinical manifestation develops only after an adequate stimulus and is regularly reproduced by it; (2) usually the latency time varies from a few minutes (e.g. cold urticaria) to 3–12 h (e.g. pressure urticaria); (3) in general, the single episodes last for about 30–60 min (with the exception of pressure-delayed urticaria) and the symptoms are localized at the stimulus site; (4) after the regression of the lesions without leaving any relic, there is a refractory period that is variable, depending on the nature of the stimulus , probably linked to the exhaustion of chemical mediators , 29; (5)In diagnostic work-up, the aspects of critical importance are clinical history, because the suspicion of an inducible urticaria can arise from the location, distribution and morphology of the lesions, in relation to a specific triggering factor, and the provocation test, that allows us to confirm the diagnosis and to identify the stimulus capable to elicit urticaria, followed by the threshold test that allows the identification of an individual threshold of reactivity to the stimulus . The knoThe epidemiological data currently available on CU in many cases are deeply discordant and not univocal. The main factors involved in the development of this complex framework are the heterogeneity of the studied populations and of the degree of correspondence between the characteristics of each patient sample and those of the general reference population, the heterogeneity of the diagnostic criteria used , 33, andHelicobacter pylori, Mycoplasma pneumoniae) or parasitic infections  are associated with the development of CSU. Some foods  and drugs (e.g. NSAIDs) or even physical or psychic stress, can be identified as causative agents and triggers of urticarial eruptions. However, in a high percentage of cases (55–70%), CSU is defined as idiopathic because the causes remain unknown [CSU is more common in females , which  unknown . A parti unknown . The pos unknown . Patient unknown , 46. Som unknown .Multiple chemical mediators are involved in urticaria, often interacting each other in a complex molecular network. Chemical mediators can be secreted at the systemic or local level following the activation of several cells that are resident in the dermis (mast cells) or recruited from peripheral blood .Mast cells have a key pathogenetic role and can be activated by different mechanisms . The mosStaphylococcus A protein, Peptostreptococcus L protein, HIV gp-120) or endogenous proteins synthesized by the liver during viral infection. These proteins are able to bind non-specifically to surface basal cells IgE interacting with a variable region of heavy chains. The binding of superallergens with mast cell IgE induces the activation of these cells and the secretion of chemical mediators in a way that is similar to that induced by the classic allergens [A new exposure to the trigger factor induces the receptor cross-linking and the activation of the intracellular signaling resulting in mediator’s production. Such mechanism can occur in the absence of re-exposure to the antigen in the presence of anti-FcεRIα or anti-IgE IgG, with a special role for IgG activating the mast cells, that belong mainly to the IgG1 and IgG3 subclasses, able to trigger the complement cascade with production of C5a. This molecule is able to amplify the mast cells response by binding to specific receptors present on their surface. Immunologically activated mast cells quickly release various preformed mediators, including histamine and some proteases , as well as synthesize and release lipid metabolites such as prostaglandins (PG)D2, leukotrienes ((LT)C4 and LTD4) and Platelet Activating Factor (PAF), and, ultimately, cytokines and chemokines, particularly tumor necrosis factor (TNF) α, interleukin (IL)-1, IL-4, IL-6 and IL-13. The activation of mast cells can be done by non-canonical way through molecules known as “superallergens”. Superallergens are proteins of bacterial or viral origin  supports their pathogenic role. These antibodies induce the release of major basic protein (MBP) which activates mast cells and basophils . EosinopConcerning other cells, in lesion biopsies a perivenular infiltrate of lymphocytes is generally found, predominantly CD4+. Cytokines of T helper 2 origin were found in the lesions, such as IL-33 and IL-25. High levels of IL-4 were detected in the serum of patients with CSU. The lymphocyte response in the CSU cannot be attributed to a specific type T helper (Th)1 or Th2 and some authors assume that the lymphocytes infiltrating the lesions actually have a Th0 profile . IncreasIn any case, the pathogenic mechanisms described above have as final results the release of mediators, with a primary role for histamine, which is the main mediator responsible for increased vascular permeability. There are four subtypes of histamine receptors, all of type G protein-coupled receptor (GPCR), but H1 seems to be the most important in the pathogenesis of urticaria. The mechanism of action sees the activation of the G Protein αq pathway and therefore of phospholipase C, which causes an increase in the concentration of Ca2+ cytosolic in the vessels of the dermis. The latter activates the MLCK —dependent Myosin Light Chain Kinase) triggering the contraction of the actin-myosin system with cell retraction. The reactions triggered downstream of the H1 receptor are numerous and, in particular in the endothelial cells, there is an activation of several MAP Kinases, all determining an alteration of the system of intercellular junctions (tight and adherens junctions), with gaps formation and consequent increase in vessel permeability. The amount of histamine present in the lesions correlates with the disease activity. However, histamine is not the only mediator involved in the pathogenesis of CSU. Serum tryptase levels were higher in patients with CSU compared to controls both in phase of quiescence and in acute phase . Some stIn the serum of patients with CSU, higher levels of VCAM1, ICAM1 and CCL5/RANTES have been found: these molecules could have the role of markers of endothelial dysfunction and be implicated in the pathogenesis of CSU. However, their serum levels do not seem to correlate with disease activity nor with the severity of the manifestations .Recently, the Vascular Endothelial Growth Factor (VEGF) and the Calcitonin Gene-Related Peptide (CGRP) have been observed in the lesions of the patients with the highest levels of vasodilatory factors , 59. Of Some metalloproteinases (MMP-9) and extracellular matrix degradation products (Endostatin and Thrombospondin 1) are detectable in sera from patients with CSU at higher concentrations than controls. IL-31 is a cytokine produced by different cell types, which has been proposed to be involved in various chronic skin diseases  capable of activating nitric oxide synthase with release of NO. The role of bradykinin in the pathogenesis of CSU has not yet been sufficiently investigated, although it is known its effect of induction of wheal after subcutaneous injection and its pathogenic role in various inflammatory diseases of allergic interest  .Still largely unknown are several plasma factors, often generically identified as Histamine Releasing Factors (HRF). The HRFs include both high molecular weight molecules (probably immunoglobulins) and low molecular weight molecules (< 30kD) capable of activating mast cells bypassing the classical activation path represented by the FcεRI-IgE axis .The available literature shows that urticaria is more than a troublesome symptom. Patients with urticaria, when compared to healthy subjects or patients suffering from other diseases, have a significantly reduced health related quality of life (HRQoL) score. Such score, as expected, is lower than in healthy subjects, regardless of age, duration of disease, or the presence or absence of angioedema . HoweverBased on the variety of urticaria types, an accurate history collection and a general objective examination constitute a valid starting point . The useHelicobacter pylori, Streptococcus, Staphylococcus, Mycoplasma pneumoniae, Salmonella, Brucella, Borrelia, Chlamydia, Yersinia enterocolitica, herpesviridaae, parvoviridae, caliciviridae, picornaviridae, flaviviridae, hepadnaviridae, Anisakis simplex, etc. [Helicobacter pylori antibodies can be useful, as well as a prick test for Anisakis simplex and the search for IgM to the above-mentioned pathogens [Once the patient’s medical history has been collected, it will be useful to carry out a general objective examination with attention to the dermographism (after antihistamine treatment has been withdraw from some days). Carrying out extensive and costly screening protocols is not recommended given the extremely varied nature of the triggering causes. For example, immunoreactions of the first type  rarely cause chronic urticaria; sometimes the urticaria symptoms could be worsened by a toxic effect of some foods, and then a diet poor in histamine could give relief to the patient . Also, bex, etc. , 72, 73.athogens , 73, 74.athogens .Finally, since the coagulation cascade is activated in both self-reactive and non-reactive patients, it is of interest that high levels of D-dimer can identify patients with more severe pathology; to this end, it would be useful to add to the D-dimer assay also an evaluation of the F1 + 2 fragment of prothrombin, of fibrin and fibrinogen degradation products and of the soluble fibrin monomeric complex –78.As to prognosis, CSU is a transitory condition (average duration 2 to 5 years) and does not endanger the patient’s life, even if it is experienced as a disabling condition . In mostWhile diagnostic tests of standardized provocation are available for the diagnosis of inducible urticaria in accordance with international recommendations, the spontaneous form should be differentiated from vasculitic urticaria, auto-inflammatory syndromes and other forms of urticarial dermatitis based on clinical features, morphological and histopathological. In chronic spontaneous urticaria, the wheals have an asymmetric distribution, they resolve without dyscromic outcome and are associated in about 40% of cases with angioedema.The urticarioid syndromes, on the other hand, are extremely heterogeneous and may be localized only in the skin or be systemic, but in both cases the clinical pictures are characterized by the presence of atypical, persistent wheals, with bilateral and symmetrical distribution, which resolve with residual discolouration, mostly associated to other lesions such as papules, vesicles, or bubbles rarely associated with angioedema .The most important differential diagnosis of spontaneous CU is with vasculitic urticaria, which represents 5–10% of CU and is characterized by skin lesions similar to those of spontaneous urticaria but with histopathological characteristics typical of cutaneous necrotizing vasculitis . VasculiVasculitic urticaria is mostly idiopathic but sometimes drugs such as anti-folate, antidepressants, cimetidine, or physical stimuli like cold, and sun exposure are involved. It may present as a manifestation of a systemic pathology and in particular of connective pathologies such as systemic lupus erythematosus (SLE), paraneoplastic dermatomyositis, systemic sclerosis, or be part of systemic vasculitis such as Wegener’s granulomatosis or Churg-Strauss syndrome . MoreoveThe pathogenetic mechanism underlying the pathology is the formation, by antigens currently unknown, of antigen–antibody complexes that are deposited on the vessel wall, trigger the complementary cascade with subsequent activation of the mast cells, recruitment of neutrophils and vascular damage. Cutaneous biopsy and histological examination with direct immunofluorescence are necessary for diagnosis.If a patient with recurrent wheals reports osteo-articular pain, unexplained fever and general malaise an auto-inflammatory syndrome, that may be hereditary or acquired, should be suspected. Auto-inflammatory syndromes are a group of monogenic disorders in the genes that regulate the innate immune response, resulting in an aberrant activation of mediators and in particular the IL-1 pathway. Most of them are associated with rash, fever in some cases periodic, but may be unpredictable over time without evidence of neoplastic tumor or autoimmune diseases, arthritis, elevation of inflammation indices, abdominal pain, amyloidosis, myalgias and neurological signs. A detailed description of these syndromes goes beyond our objectives and will be limited to the forms in which urticaria lesions such as cryopyrin-associated periodic syndrome (CAPS) and Mediterranean family fever are more frequently present. CAPS are a group of inherited autoinflammatory syndromes that represent the clinical spectrum of different mutations of the CIAS1 gene (cold induced autoinflammatory syndrome) that encodes a protein called cryopirine (NALP3), a component of the inflammatory agent that activates IL-1alpha when the cell receives the danger signal. CAPS include the Muckle-Wells syndrome , the chronic infant with neurologic, cutaneous and articular (CINCA) involvement, the autoinflammatory cooling syndrome with papillary orticarioid lesions after cold exposure accompanied by systemic symptoms and with a negative ice cube trigger test .The Mediterranean family fever affects the populations originating in the Mediterranean more frequently and is associated with a mutant pirine with functional impairment and activation of IL-1beta. The urticaria lesions present in this syndrome are persistent, often bilateral, symmetrical, but there may also be macular, papulose and purpuric lesions accompanied by burning and always with systemic symptoms. .Orticarioid dermatitis was recently defined to describe patients with a peculiar pattern of hyperreactivity. It mostly affects adult patients aged 60 years of age or older, with urticaria and eczematous lesions associated with intense, bilateral and symmetrical pruritus with localization to the trunk and root of the limbs and have a duration of even weeks. Histological examination shows an eczematous reaction with minimal epidermal spongiosis, papillary dermal edema and a superficial perivascular infiltrate of lymphocytes with eosinophils. The etiology is uncertain even though in some cases it has been attributed to a reaction to drugs, antihistamines are not effective, and a short corticosteroid treatment leads to non-definitive remission since the dermatitis can relapse .Drug exanthematic reactions are frequent, occurring after a few days. Although a maculopapulotic rash with erythema initially on the trunk and upper limbs is the most frequent form, the lesions may also be intense with red, persistent orticarioid lesions with bilateral and symmetrical localization and tendency to confluence and residual pigmentation associated with mild itching .Cutaneous mastocytosis, previously called urticaria pigmentosa, is the most common clinical picture of mastocytosis that can mimic urticaria. It concerns pediatric or adult patients with red-brownish macules and papules which become apparent with rubbing (Darier’s sign), with the main site of the trunk and limbs. In pediatric age there may be a urticaria rash with persistent lesions also localized in the face, spontaneous or caused by rubbing, heat or sun exposure. The number of injuries is variable but does not correlate with the risk of systemic involvement. In half of the patients flushing appears and syncopal or anaphylactic episodes can occur. In adults, rarely in children, there may be an asymptomatic involvement of the bone marrow. The diagnosis is clinical and histological with possible recourse to immunohistochemistry for the count of the mast cells. The serum tryptase assay (< 20 ng/ml) is the most important parameter for screening patients with possible systemic involvement and for their follow-up. The prognosis depends on the age of onset and is better for pediatric forms while in adults it is possible an evolution towards a systemic mastocytosis in a percentage ranging from 3 to 30%. Also, in the systemic form there may be episodes of urticaria-angioedema but always associated with other systemic symptoms of the gastro-intestinal, respiratory, cardiological system .In conclusion, urticaria lesions may appear in a variety of skin or systemic diseases.The clinical-evolutionary criterion is fundamental for the differential diagnosis since the lesions of the urticaria syndromes are predominantly bilateral, symmetrical, are persistent and often resolve with dischromic outcomes, are often associated with different skin lesions and systemic symptoms.The clinical-pathological correlation with biopsy and histological examination is the other element that is necessary and in some cases indispensable for a correct classification.The management of urticaria must be faced considering in each patient essentially two aspects, the first related to the identification and elimination of the causes and triggers, the second concerning the effective treatment of symptoms. To treat the patient by avoiding the triggering cause would be the best option, but often in CSU this is not possible because the triggering factor is not recognizable, while it may be possible in the rare cases of IgE-mediated urticaria and partly in the forms induced by physical stimuli. The clinical classification in the various subtypes is important for a correct diagnostic and therapeutic approach. The therapy has the role of reducing the release of mediators and the effect that these exert on the target organs and to induce tolerance.The severity of clinical manifestations and the nature and strength of the stimulus, which causes and perpetuates the symptoms, are different in individual patients and, for example, it is always necessary to evaluate the environmental situation in the inducible forms, such as hot or cold , 89.Identifying the causes is not easy, because often more stimuli can be associated in the same patient. In case of remission after the elimination removal of the suspect agent, only the recurrence of symptoms in the presence of the same agent can lead to the recognition of the cause, thus excluding an accidental factor.Helicobacter pylori, contrasting data were reported about the possibility to resolve urticaria by effective treatment of the infection [Among drugs, antibiotics and NSAIDs are often considered to cause urticaria. When their responsibility is suspected, they must be avoided or replaced to prove their actual responsibility. Drugs may also aggravate pre-existing urticaria . Concernnfection .Food-IgE mediated allergy is rarely associated with chronic urticaria, but if a food allergen is identified as the triggering factor, it must be eliminated from the diet. In some patients there may be a reaction related to non-IgE mediated allergy, but to a hypersensitivity reaction to some foods or ingredients, especially additives . The newIt is recognized that stressful events can trigger or aggravate urticaria, and that urticaria can worsen a pre-existing stress. Psychological support therapies can be considered in addition to the treatment of the disease .st generation anti-histamines, they also have anti-inflammatory effects by inhibiting the release of cytokines from mast cells and basophils [The therapy of AU and/or angioedema, although strongly conditioned by the identification of the cause, is predominantly symptomatic. The aim of this treatment is to reduce the clinical effects of mediators released by mast cells and mainly of histamine, using drugs capable to exert a competitive blockade of histamine H1 receptors. Second generation antihistamines (SGA) are generally considered to be first line drugs in both acute and CU , 95. Comasophils , have leasophils .H1 antihistamines of 1st generation, however, can be used in patients with anxiousness or with insomnia due to nocturnal exacerbations of the skin symptomatology or in the onset phases of urticaria. Taking antihistamines should be regular (not as needed) and should be prolonged for at least 7–10 days after symptom remission.In cases of inadequate therapeutic response to antihistamines, two different strategies are possible, i.e. increasing the dosage of the antihistamine used up to four times the conventional daily dose or the addition of a corticosteroid. The first approach , 98–100 The association with a corticosteroid to ensure greater control of mediators can be considered in the most serious cases when the wheals are very large and widespread and/or associated with angioedema, and in presence of exacerbation of symptoms , 101. InWhen cutaneous and/or mucosal symptoms are associated with one or more systemic symptoms , suggesting an initial anaphylaxis, epinephrine should be prescribed .These drugs are generally administered in a single daily dose, in order to minimize nocturnal sedation. Bilastine, at a daily dose of 20 mg, levocetirizine (active enantiomer of cetirizine) and desloratadine, both administered at a daily dose of 5 mg, appear to be more effective antihistamines and with less side effects (sedation and interaction with other drugs) compared to 1st generation antihistamines. Other SGA tested in CSU are cetirizine, fexofenadine, loratadine, ebastine and rupatadine. Antihistamines do not modify natural history of CVS, but when symptoms control is achieved must be progressively reduced to indentify the minimal effective doseIf the symptoms are severe or associated with angioedema, the old H1 antihistamines with sedative action, such as hydroxyzine (25 to 50 mg/day) and diphenhydramine (25 mg twice a day) may reduce anxiety and insomnia. Adding a second H1 antihistamine or an H2 histamine in patients with little effect of H1 antihistamine treatment. However, treatments combined with H1 and H2 antihistamines or with two different H1 antihistamines do not seem to provide advantages over monotherapy. At present, it is preferred to increase the dose of a single antihistamine as indicated by the 2017 EEACI/GA2LEN/EDF/WAO guidelines [CorticosteroidsTreatment with corticosteroids is generally not recommended in the treatment of CSU. However, there are cases of in which in the pathogenesis of the disease mediators other than histamine are involved, such as PAF, leukotrienes and other cytokines, and the perivasal infiltrate shows lymphocytes in addition to basophils and eosinophils. These cases respond to a short course of corticosteroids and are generally refractory to treatment with antihistamines , 101.Ciclosporin has a direct effect on the release of mediators. The efficacy of cyclosporine in combination with second-generation antihistamines has been demonstrated in open-label and double-blind clinical trials , 103. ItOmalizumab is a humanized monoclonal antibody derived from recombinant DNA that binds selectively to human IgE . It was Usually the side effects caused by Omalizumab are mild or moderate, but like any drug it can have even serious adverse effects . The mosThe therapeutic objective consists in the prevention of exposure to the triggering factors and on the symptomatic treatment in order to obtain complete control of the symptoms and clinical signs and to improve the patient’s quality of life , 21. TheThe prevalence of pediatric urticaria varies between 2.1 and 6.7% and appears to be influenced by the atopic constitution , 112. InThe diagnosis of urticaria in childhood does not present particular difficulties and is based on clinical criteria; its clinical presentation is not different from that of the adult. In AU, the hives can more frequently assume a figurative appearance and hemorrhagic aspects and may result in hyperchromic pigmentary outcomes. The association with angioedema is possible in 15% of cases . PrurituIn case of repeated episodes of AU, a food etiology should be suspected, that must be further researched and confirmed through the compilation of a diary (appearance of symptoms less than an hour after ingestion of a food) and a careful elimination diet. However, food allergies seem to be present in less than 7% of cases of urticaria . The patThe management of urticaria in children is substantially similar to that in adults, as underlined in guidelines and recent reviews , 21, 121The same treatment algorithm used for adults may be applied, although the recommendation is weak and based predominantly on a clinical consensus . The firIf SGA have no effect in controlling pruritus and urticaria, the dose increased up to four times previously reported for adults is not validated in pediatric age , 21, 124The elevated estrogen-progestin levels as well as the physiological anatomical changes that characterize pregnancy have a significant impact not only on the product of conception but also on the health of the mother. As known, some pathologies such as bronchial asthma, can both improve and worsen during these 9 months, therefore requiring an often-multidisciplinary approach in women who are planning or carrying out a pregnancy. Even some skin diseases show a strong link with pregnancy. High hormone concentrations are known to exacerbate episodes of angioedema in women carrying the XII factor mutation. The same hormonal framework may be the trigger of complex systemic diseases such as SLE which very often has the skin as the first involved organ. Actually, urticaria is not a pathology showing during pregnancy a greater incidence of both early diagnosis and recurrence in women affected already in the pre-conception period , 131. ThThe therapy must at the same time protect the health of the fetus from possible iatrogenic damages, especially during the first trimester, but also safeguard the well-being of the mother. It should not be forgotten that urticaria has a very significant impact on the quality of life and psychological well-being of patients , similarThe most important issue, however, is that mothers and often even general practitioners perceive the potential harmful effects from drugs as more severe and more frequent than those caused by the disease itself –136. An It is important to distinguish between acute and chronic forms and to search for possible triggers and etiological agents. The symptoms of urticaria may be the expression of pathologies that could significantly affect pregnancy, such as food allergies as a risk of anaphylactic reactions . In bothFortunately, a large part of the drugs available to treat acute and CU has proved safe even during pregnancy and lactation. The therapeutic algorithm for this particular patient population is the same one applied to the general population affected by both acute and chronic urticaria, as recommended in the European guidelines . In any Concerning antihistamines, SGA must be preferred. In particular, the only ones with proven safety during pregnancy are loratadine and cetirizine, classified by FDA as class B drugs. As with SGA in general, they have a low secretion in breast milk and can therefore also be used during breastfeeding. If it is necessary to use first generation antihistamines, the safest are chlorpheniramine and diphenhydramine, tough their concentration in breast milk is higher, leading to a greater risk of sedation of the newborn. To date there are no studies demonstrating the safety of a possible increase in the dosage of antihistamines both before and second-generation during pregnancy . NeverthCorticosteroid are considered by FDA class C drugs, since prolonged use is associated with the risk of intrauterine infections and prematurity  in additAs far as biologic agents are concerned, Omalizumab was promoted to class B drug according to the FDA . The useFor ciclosporin, no withdrawal is required either during conception or during gestation, as this drug does not appear to reduce fertility  and doesUrticaria is a term referring to a group of diseases which involve the onset of pruritic wheals, angioedema, or both. Urticaria may be divided into acute and chronic forms. AU is defined as the occurrence of spontaneous wheals, angioedema, or both for < 6 weeks. The diagnostic procedures in patients with AU are fundamentally different from those used in patients with CU. The current recommendations are thus based on a precise formulation of the goals and steps in the recommended diagnostics. Because an underlying cause is rarely detected, a causal and/or curative treatment in not available for the majority of patients. Therefore, symptomatic therapy remains the mainstay of treatment. Modern SGA in licensed doses are the treatment of the first choice. In patients who do not respond adequately to standard dosage of SGA, the dosage should be increase up to four times the standard dosage. Patients who fail to respond adequately even to higher dosages of SGA should be given omalizumab (anti-IgE). More effective and safe therapies should be investigated for patients who do not respond to H1-antihistamines or omalizumab.


Here we aimed to determine whether sarco-endoplasmic reticulum (SR/ER)–mitochondria interactions and mitochondrial function were impaired in dystrophic heart at the early stage of the pathology. For this purpose, ventricular cardiomyocytes and mitochondria were isolated from 3-month-old dystrophin-deficient mice (mdx mice). The number of contacts points between the SR/ER Ca2+ release channels (IP3R1) and the porine of the outer membrane of the mitochondria, VDAC1, measured using in situ proximity ligation assay, was greater in mdx cardiomyocytes. Expression levels of IP3R1 as well as the mitochondrial Ca2+ uniporter (MCU) and its regulated subunit, MICU1, were also increased in mdx heart. MICU2 expression was however unchanged. Furthermore, the mitochondrial Ca2+ uptake kinetics and the mitochondrial Ca2+ content were significantly increased. Meanwhile, the Ca2+-dependent pyruvate dehydrogenase phosphorylation was reduced, and its activity significantly increased. In Ca2+-free conditions, pyruvate-driven complex I respiration was decreased whereas in the presence of Ca2+, complex I-mediated respiration was boosted. Further, impaired complex I-mediated respiration was independent of its intrinsic activity or expression, which remains unchanged but is accompanied by an increase in mitochondrial reactive oxygen species production. Finally, mdx mice were treated with the complex I modulator metformin for 1 month. Metformin normalized the SR/ER-mitochondria interaction, decreased MICU1 expression and mitochondrial Ca2+ content, and enhanced complex I-driven respiration. In summary, before any sign of dilated cardiomyopathy, the DMD heart displays an aberrant SR/ER-mitochondria coupling with an increase mitochondrial Ca2+ homeostasis and a complex I dysfunction. Such remodeling could be reversed by metformin providing a novel therapeutic perspective in DMD.Besides skeletal muscle dysfunction, Duchenne muscular dystrophy (DMD) exhibits a progressive cardiomyopathy characterized by an impaired calcium (Ca Dystrop2+) homeostasis is one of the first pathophysiological features associated with dystrophin deficiency. Before any sign of DCM and cardiomyopathy, the calcium channels of the sarcoplasmic reticulum (SR), the type 2 ryanodine receptors (RyR2), are leaky due to posttranslational remodeling  production and oxidative damages are increased in the DMD mdx mouse model and contribute to the pathogenesis of heart failure in DMD  and the porine of the outer membrane of the mitochondria (OMM), VDAC1 (voltage-dependent anion channel 1). These two channels are linked by a chaperone, GRP75, forming a direct Ca2+ channeling between the SR/ER and the mitochondria  complex creating an IP3R1–GRP75–VDAC–MCU Ca2+ transfer axis  comprising MICU1 and MICU2 which give it its Ca2+ sensitivity  and muscle weakness  and the mitochondria, also called mitochondria-associated ER membrane (MAM), is essential for the maintenance of energy metabolism, Camdx mice that show no signs of DCM, the interconnection between SR/ER and mitochondria is modified and whether it may interfere with myocardial mitochondrial function. Our results demonstrate that increased SR/ER–mitochondria interaction is associated with increased mitochondrial Ca2+ content and a disrupted mitochondrial function. One-month metformin treatment reversed these defects providing a novel therapeutic perspective in DMD.The aim of this study was thus to establish whether, in Ministère de l'enseignement supérieur et de la recherche (N° #16473-2018082016141320). Male mdx or WT  mice  were used at 3 months of age. According to the animal-to-human translation factor for drug treatments and as previously shown, a daily dose of metformin of 200 mg/kg/day was given for 1 month in 50 ml of drinking water ] containing 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 12 mM NaHCO3, 10 mM KHCO3, 10 mM Hepes, 30 mM taurine (pH 7.4). Isolated myocytes were then transferred to the same enzyme-free solution containing 1 mM CaCl2 prior experiments.All experiments were conformed to the institutional ethics committee for animal experiments and received the agreement from the national Twenty to thirty micrograms of total or mitochondrial protein was separated on SDS-polyacrylamide gel and electro-blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were saturated with blocking buffer for 1 h at room temperature and incubated overnight at 4°C with monoclonal mouse anti-VDAC , anti-OXPHOS , anti-IP3R1 , anti-NDUFA13 , anti-GAPDH  or with polyclonal rabbit anti-MCU , anti-MICU1 , anti-MICU2 , anti-PDH-E1α , anti-PDHE1α phosphor Ser 293 , anti-PDHE1α phosphor Ser 300 , anti-PDHE1α phosphor Ser 232 , anti-PDK4 (1:1000 Novus Biotech), anti-GRP75 , anti-SIGMA1R , anti-MFN2 , and anti-Hsp60 . Hsp60 and GAPDH were used as loading controls. All immunoblots were developed and quantified using the Odyssey infrared imaging system (LICOR Biosystems) and infrared-labeled secondary antibodies. Band intensities were quantified with ImageJ.Tbp . The primer sequences of ER stress-related genes are Hspa5 forward 5′-CCACCTCCAATATCAACTTG-3′, Hspa5 reverse 5′-ACGATCAGGGCAACCGCATCA-3′; Ddit3 forward 5′-CTGGAAGCCTGGTATGAGGA-3′, Ddit3 reverse 5′-CTCTGACTGGAATCTGGAGA-3′; total Xbp1 forward 5′-GTGCAGGCCCAGTTGTCACC-3′, Xbp1 reverse 5′-TCTGGGTAGACCTCTGGGAG-3′; U-Xbp1 forward 5′-CAGACTATGTGCACCTCTGC-3′, U Xbp1 reverse 5′-TCTGGGTAGACCTCTGGGAG-3′; Atf3 forward 5′-CCAGGTCTCTGCCTCAGAAG-3′, Atf3 reverse 5′-CATCTCCCAGGGGTCTGTTGT-3′; Atf4 forward 5′-TCGATGCTCTGTTTCGAATG-3′, Atf4 reverse 5′-AGAATGTAAAGGGGGCAACC-3′; Atf6 forward 5′-TCGAGGCTGGGTTCATAGAC-3′, Atf6 reverse 5′-CTGTGTACTGGACAGCCATC-3′.Isolated cardiomyocyte RNA was extracted with the TRIzol Reagent (Life Technologies). The level of target mRNAs was measured by reverse transcription  followed by quantitative real-time PCR using a RotorGene (Corbett Research). TATA-binding protein (TBP) mRNA was used as a housekeeping gene The Proximity Ligation Assay (PLA) was performed as previously described  using an o-cresolphthalein complexone assay according to the manufacturer's instructions (TECO Diagnostics). Results are expressed in mM of Ca2+ per μg of protein.Mitochondrial isolation protocols were performed and adapted from Frezza et al. . Briefly2PO4, 3 mM MgCl2·6H2O, 0.5 mM EGTA, 20 mM taurine, 60 mM K-lactobionate, 20 mM HEPES, 110 mM sucrose, and 1 g/L BSA, pH 7.1. The respiratory rates of 10 000 cardiomyocytes were recorded at 37°C using a high-resolution Oxygraph respirometer  after addition of different substrates. Briefly, the aerobic glycolysis pathway was stimulated by addition of pyruvate and malate. Pyruvate, the last substrate generated by glycolysis, and malate activate the Krebs cycle after their conversion in acetyl coenzyme A and oxaloacetic acid, respectively. Under these conditions, the complex I of the mitochondrial respiratory chain was activated by NADH produced. Complex II-driven respiration was obtained by addition of succinate and rotenone (to inhibit the complex I), and complex IV-driven respiration was permitted by addition of antimycin A (to inhibit the complex III), ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) to reduce cytochrome c. In some experiments, the free Ca2+ was set to 400 nM in the respiratory buffer using the Maxchelator program (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/). The respiratory coupling ratio (RCR) was measured in all conditions, and no differences between groups were observed  with the O2k-Fluorescence LED2-Module on isolated mitochondria in the presence of thaspigargin (10 μM). The decay time constant of the calcium green fluorescence was measured after application of 25 μM extramitochondrial Ca2+ and reflects mitochondrial Ca2+ uptake.Respiration was measured on digitonin-permeabilized cardiomyocytes (15 μg per million cells) in the respiratory buffer containing 10 mM KHobserved . MitochoActivity of the pyruvate dehydrogenase (PDH) was obtained by using Pyruvate Dehydrogenase Enzyme Activity Microplate Assay (Abcam ab109902) and 100 μg of mitochondria isolated from heart homogenates according to the manufacturer's instruction. Mitochondrial enzymatic activities of complexes I and IV and citrate synthase were measured on mitochondria isolated from heart homogenates at 37°C using a SAFAS spectrophotometer and standard method  and confocal microscopy as described previously  and its regulatory protein, phospholamban (PLB), and its phosphorylation were also unchanged, although the Ca2+ load of the SR was reduced  inhibits its activity whereas its dephosphorylation by the Ca2+-dependent PDH phosphatase (PDP) increases its activity. We thus measured the PDH phosphorylation in mdx LV mitochondria. The total PDH content and the PDK4 expression were comparable in both groups; however, the phosphorylation levels of the three phosphorylable serines  were significantly reduced  production. We have indeed observed an increase in Mitosox red fluorescence in paced mdx cardiomyocytes but also in the presence of the complex III inhibitor antimycin A, demonstrating an increase in the production of O2.− at the level of complex I compared to WT , one of the downstream targets of AMPK, was significantly increased validating the efficacy treatment with metformin . We next content . The dec232 site . Howevermdx mice . Finallymdx mice , indicat2+ homeostasis and SR Ca2+ leak is unlikely  bind the protein chaperone BiP in the ER lumen stabilizing the ER stress response proteins  on the alpha chain of the E1 subunit is significantly decreased in conjunction with an increase in PDH activity. This increases in PDH activity may account for the previously described elevation in the pyruvate decarboxylation and the shift from fatty acid to carbohydrate oxidation in the heart of both mdx and DMD patients .CA and MP performed and analyzed the experiments. ML and JR performed the experiments. AL designed the experiments, discussed the data, and read the manuscript. JF designed the experiments, collected and discussed the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca2+ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics.The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca On the single-cell level, Ca2+ transients are critical for the contraction of myocytes, the induction of changes in gene expression patterns, and cell proliferation and differentiation, in addition to driving apoptotic cell death ..2+ influ2+ mobilization by promoting the ubiquitination and lysosomal degradation of Orai1. According to the study, the UBL and UBA domains of Ubiquilin 1 seem to be non-relevant for the interaction with Orai1 .,46.2+ sto et al. , capablechannels . Moreovechannels  65,66].,66.2+ st2+. In this regard, initial reports of Srikanth and colleagues linking junctate to CRAC channel activation revealed that mutation of Ca2+ binding domains in junctate leads to cluster formation of STIM1 under store-replete conditions int, but also in persistency. Altogether, CRACR2A likely stabilizes the association of Orai1 and STIM1 upon store depletion as long as intracellular Ca2+ concentrations are low. However, further data are necessary to decipher whether the cytosolic EF-hand protein is actively involved in the accumulation of STIM1 and/or Orai1 at ER-PM junctions or recruited to already clustered proteins in a subsequent step. Although CRACR2A was reported to be more potent in enhancing Orai1-dependent Ca2+ influx, CRAC2B seems to show a functional redundancy with the former . It. It21]. f hSTIM1 . CRAC2A e former ,21. Howe93 cells . For rev2+ signalling, Quintana et al. found a protein in the ER membrane encoded by the TMEM110 gene  family, which is known for its activity as a sheddase ,99. The 2+ influx and in turn, mobility of cancer cells . A3R is in direct concurrence with the engagement of the latter by STIM1, both of which depend on ER-luminal Ca2+ but in a reverse manner and imposing functional consequences on store depletion. In turn, the PI3K/Akt cascade was suggested to promote associations of PC1/STIM1 with IP3R if active, concomitantly restraining interactions between PC2 and the particular Ca2+ release channel to keep the latter quiescent ..2+ depenCDK1| Smyth et al. showed that the cyclin-dependent kinase 1 (CDK1) interacts with STIM1 . During 2+ signalling.Apart from protein–protein interactions executing a modulatory function on SOCE, STIM1 and Orai1 have been shown to comprise an affinity to and thereby have the potential to regulate the activity of other proteins implicated in Ca2+-dependent transcription factors of the nuclear factor of activated T cells (NFAT) family, goes along with downregulations in the PMCA-dependent Ca2+-clearance of the cytosol. PMCA1 and PMCA4 were found to be expressed at higher levels as a consequence of T-lymphocyte stimulation. A series of experimental findings postulates a decrease in PMCA function to be dependent on STIM1, the latter of which is expressed at higher levels upon T cell activation. This seems to be mastered via direct interactions between both, and although co-immunoprecipitation efforts indicated that STIM1 binds to PMCA also under resting conditions, fluorescently labelled forms of both were reported to considerably co-localize just after T-cell activation  and the sodium-calcium exchanger 1 (NCX1) to be expressed at appropriate levels.PMCA| Ritchie et al. revealed that the activation of T-lymphocytes, critically dependent on CRAC channel activation to allow for the activation of Cativation . Furtheresponses . Moreove2+ channels (CaV1.2)| Cav1.2 is a subunit of L-type voltage-dependent calcium channels , yet also to Na/K-ATPase and to nuclear transporter proteins. While the function of POST-mediated STIM1 interactions with importins and exportins remains to be elucidated, the apparent function of POST to ameliorate STIM1 couplings to Ca2+ pumps might allow for cytosolic Ca2+ elevations to be persistent over longer periods of time, since they hinder these in their activity [POST (SLC35G1)| The solute carrier family 35 member G1 (SLC35G1), a transmembrane protein located in the ER membrane and the plasma membrane, also termed as transmembrane protein 20 (TMEM20) or partner of STIM1 (POST), respectively, was identified by Krapivinsky et al. Notwithstanding the latter designation, POST binds to Orai1, yet independently of store depletion . Upon stactivity .2+ entry into close vicinity of the open channel. This includes the scaffold-constituting protein A-kinase anchoring protein 79 (AKAP79). This protein is suggested to associate with calcineurin and cytosolic NFATc proteins, as well as with Orai1 to form a signalling complex [AKAP79| Other proteins capable of binding to STIM1 and/or Orai1 serve as scaffold to bring downstream effectors of Ca complex . Thereby complex ,165.2+ store depletion, upregulating the SOCE-induced adenylate cyclase activity in discrete regions of the plasma membrane. These regions are shielded from other calcium events, such as the IP3-induced Ca2+ release or ionophore-mediated Ca2+ entry [ADCY8| Martin et al. identified colocalization of the cAMP-forming enzyme adenylate cyclase 8 (ADCY8), STIM1, and Orai1 in lipid raft domains. Overexpression of Orai1 and STIM1 in HEK293 cells leads to an increase of ADCY8 activity, indicating that Orai1 and STIM1 play an important role in the cAMP-pathway . Another2+ entry . Sanchez2+ entry .2+ levels is vital on the single-cell level and further in the hierarchy for the health of the entire organism, as is an efficient entry of the ion upon stimulation. Signalling systems such as the CRAC channel complex that are associated with changes in internal Ca2+ conditions are therefore placed into a sophisticated regulatory network with individual proteins exerting a positive or negative effect on store-operated Ca2+ entry by modulating plasma membrane-residence of Orai1, scaffolding ER–PM junctions or altogether by sculpturing the ER membrane, allowing for the dynamic establishment of PM microdomains, affecting translocation and cluster formation of STIM1, as well as by promoting or hindering interactions of STIM1 and Orai1 in different respects. Although such regulatory networks are now beginning to emerge, it needs to be emphasized that these might be, at least in parts, obscured in experimental efforts with unphysiological expression levels of STIM1 and Orai1. Though putatively vital under physiological conditions, such accessory proteins are left in disregard in the majority of generically STIM1/Orai1-centered publications. Moreover, inspection thereof demands careful considerations on the respective expression system, given that a set of regulatory mechanisms is apparently cell-type-specific. Altogether, the herein summarized and further modulators of CRAC channel function that remain to be elucidated and/or identified add further complexity to efforts aiming to delineate a holistic view of the complete sequence of events culminating in CRAC channel gating, inactivation, and deactivation. However, knowledge of the entire process of the CRAC channel cascade, including all regulatory proteins involved therein, is of immense importance for the development of future therapeutic targets in the treatment of various human diseases.Tight control of cellular Ca


There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the “calcium hypothesis” of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration  in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration.Ca Studying human diseases always requires the choice of the best possible model. Of course, humans would be the best subjects to study, but there are practical and ethical limitations that generally prevent them from being used as study subjects. The next obvious choice is other mammals, particularly mice, and they are excellent in many ways, particularly when mimicking or studying behavioral phenotypes. However, they also have important limitations, both in how well they mimic some diseases, and in the slow development of pathologies and the time required to obtain conclusions. This is specially the case for the neurodegenerative diseases that develop at an advanced age, requiring several months of study in mice. On the other hand, disease models in cell cultures offer the advantage of ease of handling and the possibility of full genetic and metabolic manipulation. However, it is much more difficult to obtain information from these studies which is able to be easily translated into the physiological response of an entire organism.Drosophila melanogaster or the nematode C. elegans constitute a reasonable intermediate that has been widely used in recent years, as they combine the ease of study with the presence of a complete organism, albeit with the obvious limitation of the greater difficulty in mimicking human disease.Models of invertebrate organisms such as the fly C. elegans was introduced as a model organism by Sydney Brenner in 1963 ; several mechanisms have been reported for this effect, from the inhibition of Ca2+ extrusion through the plasma membrane Ca2+ ATPase or the Na/Ca2+ exchanger, to the activation of Ca2+ entry through voltage- or receptor-operated Ca2+ channels, and including the possible direct interaction of the Aβ oligomers with the lipid bilayer to form pores  level both by increasing the activity of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) and by reducing the Ca2+ leak from the ER. In fact, it was reported that wild-type presenilins could, in part, mediate that leak by forming passive Ca2+-leak channels in the ER membrane. Mutant presenilins, instead, would have reduced Ca2+-leak activity   ,48,49, w2+] . This phoverload .2+ transfer from the ER to mitochondria, thanks to the presence of IP3R in the ER, voltage-dependent anion channels in the outer mitochondrial membrane, and mitochondrial Ca2+ uniporters in the inner mitochondrial membrane, together with many other proteins that form long-lasting structures that keep ER and mitochondrial membranes at distances of 10–30 nm. It has been shown that presenilins and APP are ER integral membrane proteins that are enriched in the MAMs  is probably the final common step required for necrotic cell death. In some cases, Ca2+ influx to the cytosol is small and ER-Ca2+ release is required for necrosis (mec-4 mutants), and in other cases, Ca2+ influx to the cytosol is enough to trigger necrosis (deg-3 mutants) by itself. In this model, reducing cellular [Ca2+] with EGTA or inhibiting Ca2+ release through RyR with dantrolene reduced neurodegeneration, while SERCA inhibition with high concentrations of thapsigargin increased neurodegeneration by inhibiting Ca2+ reuptake into the ER , leading to an increased ER Ca2+ release that resulted in the neuronal damage.A search for el trp-4 . The phe (crt-1) . Thus, tacr-2 subunit of the C. elegans nicotinic acetylcholine receptor led to loss of motor neurons and paralysis, which was reduced in the absence of both ER-Ca2+ binding chaperone proteins calreticulin (crt-1) and calnexin (cnx-1) (but not only one of them). However, even the protected double cnx-1;crt-1 mutants in the presence of mutant acr-2 finally underwent a delayed but progressive degeneration of motor neurons and paralysis  that activates calpain and aspartyl proteases leading to cell necrosis  and increased cytosolic [Ca2+] activity that was dependent on ER-Ca2+ release. In addition, sel-12 mutants showed mitochondrial morphological and functional defects which appeared to be dependent on the increased ER to mitochondria Ca2+ transfer, as they reverted either by knocking down the IP3R with RNAi, in RyR mutants (unc-68), or in the absence of the mitochondrial Ca2+ uniporter (mcu-1) [membrane . The rol (mcu-1) .2+] using a targeted GCaMP6, and they showed a significant increase in the resting mitochondrial [Ca2+] in the sel-12 mutants, both in body wall muscle cells and in several neurons; this was accompanied by an increased level of ATP and oxidative phosphorylation activity, and increased superoxide production. Again here, decreasing ER Ca2+ release or inhibiting mitochondrial Ca2+ uptake improved mitochondrial function and suppressed neurodegeneration in the sel-12 mutants, suggesting that neurodegeneration in these mutants is caused by elevated ER to mitochondria Ca2+ transfer.  induces the selective loss of a GFP protein fused to nucleoporin in the degenerating neurons   ,48,49,50C. elegans model with Aβ overexpression in glutamatergic neurons, Griffin et al. , and therefore, reduced ER Ca2+ release after stimulation. Thus, reduced ER Ca2+ release could be a common mechanism promoting longevity and neuroprotection. In fact, it has been shown that SERCA (sca-1 in C. elegans) is one of the proteins that undergoes a larger decrease in expression in C. elegans during aging, i.e., nearly 10-fold from day 4 to day 10  in C. elegans neurons, and this effect was blocked by 34 °C preconditioning, by reducing Ca2+ release through either IP3R (itr-1 RNAi) or RyR (dantrolene), or by inhibiting SERCA (sca-1 RNAi). Instead, it was enhanced in pmr-1 null mutants. Thus, Ca2+ pumping into the Golgi by the PMR-1 Ca2+ pump is very important for survival after heat stroke, an effect which was conserved also in mammalian neurons. Importantly, PMR-1-dependent preconditioning was also efficient at protecting against several neurotoxic stimuli, including α-synuclein overexpression ; both effects were prevented in pmr-1 deficient mutant nematodes  in defined cells of worms are now available, and should be increasingly used in the coming years to obtain further and more precise information. Optical imaging of Ca2+ dynamics in C. elegans is possible during the whole life of the animal because it is always transparent. The first dynamic cytosolic [Ca2+] measurements were obtained in 2000 using a genetically-encoded Ca2+ indicator expressed in pharyngeal muscle [2+ sensors have been used to monitor [Ca2+] in pharynx, vulva, and body wall muscles, as well as in several kind of neurons  signal in the pharynx muscle cells of old animals is better maintained than muscle contraction, probably because of sarcopenia. Young animals, however, showed with higher frequency long periods of persistent high [Ca2+] during serotonin stimulation, either by excess stimulation of a very active Ca2+ entry/release machinery, or by excess energy waste through Ca2+ pumping and contraction leading to energy depletion [2+] at all ages, showing that the fast mechanisms of uptake and the release of Ca2+ from mitochondria remain intact during aging  in defined cell populations of live animals, or even in subcellular organelles. The scheme in 2+ signaling mechanisms related to neurodegeneration mentioned in this review.Regarding the research for the role of Ca2+ pathways are involved in the process of neurodegeneration, including Ca2+ uptake into the ER , or into the Golgi apparatus , Ca2+ release from ER, or Golgi through IP3R (itr-1) or RyR (unc-68), Ca2+ uptake into mitochondria through the MCU (mcu-1) and Ca2+ entry through several channels of the plasma membrane . The effects of modulation of these pathways in neurodegeneration are complex and sometimes contradictory in the different models. The activation of plasma membrane Ca2+ channels always induces neurodegeneration. Then, depending of the intensity of the activation of Ca2+ entry, the knock-down of genes for ER Ca2+ storage , ER Ca2+ release channels (itr-1 or unc-68) or mitochondrial Ca2+ uptake (mcu-1) may be protective or not. Similarly, the inhibition of the sca-1 or pmr-1 Ca2+ ATPase pumps may be protective or increase neurodegeneration, depending of the model. Therefore, it is clear that intracellular Ca2+ signaling plays a critical role in the process of neurodegeneration. However, the system is quite complex, and there is only a thin line separating the level of calcium fluxes that can be considered normal and those that may lead to pathology.The evidence available indicates that many Ca2+] occurring during ER to mitochondria Ca2+ transfer in the mitochondria associated ER membranes (MAMs). Many models of neurodegeneration include mitochondrial damage as one of their characteristic features. An increase in ER Ca2+ content/release always produces an increase in mitochondrial [Ca2+] via Ca2+ transfer through the MAM structures. A certain degree of ER to mitochondria Ca2+ transfer may serve to increase mitochondrial metabolism and ATP production, and this may be protective. However, excess Ca2+ transfer may lead to mitochondrial Ca2+ overload and damage.An example of this thin line can be found in the changes in mitochondrial [Ca


S. cerevisiae, nine proteins participate with Spo11 in DSB formation, but their molecular functions have been challenging to define. Here, we describe our current view of the mechanism of meiotic DSB formation based on recent advances in the characterization of the structure and function of DSB proteins and discuss regulatory pathways in the light of recent models.Developmentally programmed formation of DNA double-strand breaks (DSBs) by Spo11 initiates a recombination mechanism that promotes synapsis and the subsequent segregation of homologous chromosomes during meiosis. Although DSBs are induced to high levels in meiosis, their formation and repair are tightly regulated to minimize potentially dangerous consequences for genomic integrity. In  Genomes are continuously damaged by endogenous and exogenous factors and must be accurately repaired to maintain genome integrity and function . HomologMeiotic DSBs are produced by the evolutionarily conserved topoisomerase-derived protein, Spo11, along with a cohort of partner subunits . FollowiEvery aspect of meiotic recombination is tied to the structural organization of the chromosomes . Early iS. cerevisiae. We present recent advances in deciphering the structure and function of proteins required for DSB formation, their interactions and relationships with chromosome organization, and discuss the mechanisms that regulate DSB formation in the light of these new models.Here, we discuss current models for meiotic DSB formation, focusing on the molecular mechanisms in S. cerevisiae hotspots localize within nucleosome-depleted regions at promoters  responsible for nucleotide binding and ATP hydrolysis, a central helix two-turn helix (H2TH) fold and a C-terminal transducer domain with an extended α-helix that connects the B subunit to the 5Y-CAP domain of the A subunit (Topo VIA is composed of a 5Y-CAP domain (related to the DNA-binding domain of the catabolite activator protein) and a Toprim domain  . Both do subunit .Topo VI functions through a two-gate mechanism . In its cerevisiae Spo11 has long been known to closely associate with Ski8, Rec102, and Rec104 based on genetic and cytological evidence. Indeed, yeast-two-hybrid (Y2H) experiments showed strong interactions between Spo11 and Ski8, and between Rec102 and Rec104  stabilize the interactions with DNA, but the catalytic tyrosine (Y135) does not impact DNA binding  within the hotspots . Palindrhotspots , which ahotspots . Hence, hotspots .in vitro  provided further support for end-capping by Spo11-oligonucleotides after resection had initiated  form another sub-group of functionally conserved DSB proteins with enigmatic roles at the molecular level. Although they have long been recognized as meiotic DSB proteins in yeast, the identification of their homologs across the eukaryotic kingdom has been challenging because of sequence divergence. Nevertheless, RMM homologs have now been identified in many species, including mice and humans . While Ror, Mer1 .Rec114, Mei4, and Mer2 localize to chromosomes in leptonema prior to DSB formation and were proposed to act as a complex based on Y2H interactions, coimmunoprecipitation, and partial foci overlap and co-dependencies . NeverthMus musculus REC114 was crystallized and revealed a Pleckstrin Homology (PH)-like fold with an α-helix sandwiched between two anti-parallel β-sheets , could also remain embedded within the condensate .S. cerevisiae , or rad50S). These arise from situations where two Spo11 complexes catalyze break formation in close proximity from one another. Double-cuts account for ∼5–20% of total Spo11 activity in wild-type cells, much higher than expected if the DSBs were independent from one another. Therefore, a mechanism must explain the formation of hyper-localized DSBs.Independent evidence providing strong support for a higher-order assembly model of the DSB machinery came from the analysis of break patterning in revisiae . SequencAn important clue came from their spatial patterning, which shows a periodicity of ∼10.5 bp corresponding to the helical pitch of DNA . TherefoSince DSB formation is potentially dangerous, the activity of Spo11 is controlled to ensure appropriate timing, number, and distribution of breaks . ComplemSPO11, REC102, REC104, REC114, and MEI4) and meiosis-specific splicing of MER2  and Dbf4-dependent kinase Cdc7 (DDK) are both essential for replication origin firing and later for DSB formation . CDK-S aPhosphorylation of Mer2 by DDK is temporally coordinated to DNA replication by tethering of DDK to the replisome component Tof1 . Mer2 phReplication stress downregulates DSB formation through Mec1 via three complementary mechanisms: (1) partial inhibition of Spo11 transcription, (2) inhibition of DDK via Rad53 leading to hypophosphorylation of Mer2, and (3) inhibition of chromatin loading of Rec114 and Mre11 , bottom.S. pombe, blocking DNA replication also abolishes meiotic DSB formation  that abolish SC elongation but not crossover formation show elevated DSBs . This inSC assembly removes Hop1 from the chromosome axis . This isIn mice, reduced SPO11 dosage leads to synaptic defects, and unsynapsed regions display elevated DSB markers . In addiS. cerevisiae, exit from pachytene is controlled by the Ndt80 transcription factor (NDT80 activation leads to the disassembly of the SC and the removal of DSB proteins, which ends the window of opportunity for DSB formation (ndt80 mutants accumulate more DSBs (In n factor . NDT80 aore DSBs . In mutaore DSBs . Therefondt80 mutant leads to a further increase in DSB levels (While the negative feedback loop dependent on homolog engagement is chromosome autonomous, the Ndt80 feedback loop is nucleus-wide. The distinction was demonstrated by epistasis analysis showing that deletion of ZMM proteins in an B levels . TherefoC. elegans and Drosophila oocytes, suppression of crossing over on a single pair of chromosomes lead to nucleus-wide increase in the retention of DSB proteins (In proteins  or crossproteins , respectin vitro, why do their chromatin-association depend on the axis in vivo? Current models provide a molecular framework that will guide future experiments to better understand the mechanism of DSB formation.To conclude, recent studies have brought new insights into the mechanism and regulation of meiotic DSB formation. However, our understanding of the structure, biochemical properties, and regulation of DSB proteins remains limited, and many important questions are yet to be addressed. Why DSB formation requires the collaborative action of so many proteins has been enigmatic for a long time. Our current model provides a tentative and partial response to this question by highlighting the organizational role of Rec114—Mei4 and Mer2 in the assembly of DSB-competent sites along chromosomes. As we have seen, the phase-separation model is consistent with, and explains, many long-standing observations regarding the behavior of DSB proteins. However, it also raises new questions regarding the biophysical properties of the condensates, their assembly and disassembly mechanisms, and how these might be controlled, perhaps through post-translational modifications. What are the minimal components required for DSB formation? In addition to known DSB proteins and essential phosphorylations, is something else needed to trigger Spo11 activity? What is the role of MRX? The rationale that its presence prior to break formation allows coordination with DSB repair is straightforward, but how is it recruited and how does it impact Spo11 activity? What is the relationship between DSB proteins and axis proteins? How are their spatial distributions controlled? Since Rec114—Mei4 and Mer2 bind DNA independently of axis proteins VKY and CCB wrote the manuscript. Both authors approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We constructed a DO model through PBOO and studied the Ca2+ spark activity of detrusor. By way of using confocal microscopy and the patch-clamp technique, Ca2+ sparks and spontaneous transient outward currents (STOCs) in detrusor myocytes were measured respectively. Our results indicated that Ca2+ spark activity and STOCs were significantly reduced in the DO detrusor myocytes compared to unafflicted control cells, and both of these had levels that were remarkably increased by applications of caffeine (10 μM), a RyR agonist, in DO myocytes. In addition, measures of detrusor contractions were also recorded by using freshly isolated detrusor strips. These results indicated that the spontaneous contraction of DO detrusor was significantly enhanced, and that the effect of caffeine (10 μM) upon detrusor contractions was reversed by applications of iberiotoxin (100 nM) which is a BK channel blocker. Western blotting (WB) analyses indicated that the levels of expression of ryanodine receptor type 2 (RyR2) and FK506 binding protein 12.6 (FKBP12.6) in bladder muscle were respectively decreased and increased in the samples from DO rats. Thus, we considered in the rat DO model wherein PBOO, the reduced Ca2+ spark activity in detrusor myocytes partly contributed to overactive detrusor contractions. The impaired Ca2+ spark activity may have resulted from decreased RyR2 expression and increased FKBP12.6 expression. Such novel findings in our research might help to provide means for better treatment outcomes for patients afflicted by bladder dysfunction.We tested whether or not altered Ca Bladder dysfunction has a higher incidence in the elderly , and gre2+ due to the opening of RyRs, which are visualized as Ca2+ sparks, activate large conductance of Ca2+ sensitive K+ channels (BK channels) that generate spontaneous transient outward currents (STOCs) .The STOCs were subsequently successfully recorded in control and DO detrusor myocytes. We then were able to eliminate these STOCs by administration of iberiotoxin (100 nM) which was a BK channel blocker and this suggested that the detected STOCs were generated from BK channels (data not shown). The frequency and amplitude of STOCs were found to have been significantly decreased in DO detrusor myocytes (n/c=6/78) compared to control cells (n/c=6/95) spanning voltages from -40 mV to 0 mV –1C. Thes2+ spark activity may also contribute to decreased STOCs. Already, such a finding has been noted in similar studies on cerebral artery smooth muscle cell .Based on our findings and previous research, it was reasonable for us to test whether or not the effects of RyR regulators on Cativation , 25 and h muscle , 27. Thurequency , F/F0  in DO muscle .We then tested measures of the effects of Ca2+ spark regulators to investigate whether or not decreased measures of Ca2+ spark activity contributed to increased spontaneous contractions in DO muscle. Consistent with the results from our own similarly oriented previous research .In the detrusor strip experiments, we used Caresearch , ryanodi2+ spark related properties. Because overexpression of FKBP12.6 has been reported to decrease Ca2+ spark activity in cardiac myocytes . CaSupplementary Figure 1


Staphylococcus aureus contamination has been described in food-producing animals and farm workers involved in the primary industrial production of rabbits, pigs, cattle and poultry. This study describes the effects of age and colonization of body on S. aureus detection from rabbits raised intensively for meat production, and evaluates these parameters as possible risk factors for lesions by S. aureus. In addition, a genotypic characterization was performed for 96 S. aureus strains out of 595 that were isolated, including three from farm workers. It was observed that the risk of skin lesions increased with the number of colonized body sites and with age. All isolates were low-virulence methicillin-sensitive-Staphylococcus aureus (LV-MSSA). We found five different clonal lineages: spa-t2802, the most frequently detected (53.1%), also in all human samples; spa-t491, the second most detected (38.5%); spa-t094; t605; and spa-t2036. The same spa-type was observed in both animal and worker samples, showing that S. aureus strains could easily circulate in a community. The two most frequent strains were associated with noses, but not with age categories nor with the number of colonized sites. The circulation of LV-MSSA should not be underestimated, as they could determine damage or even acquire and spread resistance genes.S. aureus and to describe the diversity of spa-types within farm, including workers. On 400 rabbits of different age, 2066 samples from the ear, nose, axilla, groin, perineum and lesions were analyzed and 595 S. aureus were isolated. In total, 284 (71.0%) were colonized by S. aureus in at least one anatomical site and 35 animals (8.8%) had lesions. S. aureus prevalence was higher in adults than other age categories. Ear (29.4%) and nose (27.2%) were the most frequently colonized sites. The presence of lesions due to S. aureus was strongly associated with the colonization of at least one body site and the risk of lesions was proportionate to the number of sites colonized by S. aureus. In addition, a genotypic characterization was performed for 96 S. aureus strains randomly selected. All isolates resulted as low-virulence methicillin-sensitive-Staphylococcus aureus (LV-MSSA). Five different clonal lineages were found: spa-t2802, the most frequently detected (53.1%), also in human samples; spa-t491 (38.5%); spa-t094; t605; and spa-t2036. Strains t094, t491 and t2036 have not been isolated in Italy before.A study in an intensive Italian rabbit farm was carried out to assess the effect of age category and anatomical site on detection of  Staphylococcus aureus infections has received growing attention because of their importance in veterinary medicine, and the increased evidence of their zoonotic potential. S. aureus is an extremely flexible organism: it can be a commensal but also a dangerous and easily adaptable pathogen with high persistence and multiplication capacity in a wide variety of environments and conditions, able to cause a wide range of diseases in both humans and animals [S. aureus that associate closely with specific hosts [Oryctolagus cuniculus), staphylococcal infections usually result in skin lesions that often evolve involving subcutaneous tissues, but they can also generate more complex and varied secondary lesions such as pododermatitis [S. aureus. In rabbits, two principal clinical types of S. aureus infections could be distinguished. Low virulence (LV) strains determine infections that remain limited to a small number of animals, and only have little economic importance; high virulence (HV) strains spread throughout the flock, leading to chronic problems and subsequent declines in production performances [In recent decades, the epidemiology of  animals ,2,3,4,5.ic hosts ,7,8. Staic hosts , with a ic hosts  to 70% [ic hosts . In domermatitis ,13, multrmatitis ,15,16. Sormances .Staphylococcus aureus (MRSA) in farmed rabbits [S. aureus population in rabbits [The discovery of emergent methicillin-resistant  rabbits ,19 and c rabbits ,21,22, i rabbits ), has ra rabbits . These sS. aureus colonization in rabbits; (ii) to assess the prevalence of lesions due to S. aureus infection; and (iii) to characterize the main clonal lineages of animal and human strains and relative closeness among them.The aims of this study were as follows: (i) to observe the age and sampling site effect on the outcome of The study was carried out on a commercial medium-sized intensive rabbit breeding farm in Central Italy. It consisted in a large shed where fattening, breeding, and replacement rabbits were all in the same shed. Animals were raised in overlapping cages and the “battery” method on parallel rows separated by corridors. At the time of the research, about 8000 animals, including 1700 breeding rabbits, were present. The reproduction was by artificial insemination, and about 70% and 30% were the internal and external replacement rabbits, respectively. All animals were submitted to a half-yearly MEV vaccination plan and the average annual production was around 40–45,000 slaughter rabbits between 82 and 90 days of age. The average annual mortality of 8–9% in the nest, and 7–8% in fattening, were recorded. Prior to beginning of the study, the rabbits were kept under standard conditions of animal housing, with good management and cleaning of animal cages and watering holes. They had light and dark conditions for each 12 h and temperature of 20–24 °C. The feeding consisted of a single cycle feed with addition of barley, finishing weaning at 35 days of age.In June 2018, a total of 400 rabbits, represented 5% of the total animals reared, were randomly selected: 26 young (40–45 days of age), 312 adults (60–80 days of age), 27 breeding rabbits, and 35 replacement rabbits.Skin swabs from five anatomical sites, nose (N), ear (E), axilla (A), groin (I), and perineum (P), were collected (n = 2000) from each animal. When skin lesions (L) were present, additional swabs (n = 66) were sampled . Lesions were circumscribed, ulcerate skin areas with purulent exudate, in various body sites .®, Roseto degli Abruzzi, Italy) and kept at 4 °C until delivery to the microbiological laboratory, which occurred within 48 h. Animals treated with antibiotics within 30 days were excluded from the study. Nasal and hand skin swabs were also collected from three farm workers, after obtaining their informed consent.Each swab was placed into transport medium  for 6 h at 37 °C and was then inoculated onto nutrient and selective media as Columbia agar  supplemented with 5% ovine blood, Baird-Parker agar , Mannitol Salt Agar  and Chromatic™ Staphylococcus aureus . After 24–48 h of incubation at 37 °C, presumptive S. aureus colonies were studied according to usual bacteriological standards  and biochemical identification was carried out by a commercial kit . Then, S. aureus strains were stored at −80 °C  for molecular analysis.For bacteriological culture, each swab (n = 2066) was enriched with Triptic Soy Broth . Purified DNA was quantified with Thermo Fisher Nanodrop and stored at −80 °C until use. To ascertain S. aureus and MRSA identification, multiple polymerase chain reactions (PCRs) were performed targeting S. aureus specific thermo-nuclease gene (nuc) and methicillin-resistance  genes, as described previously [DNA extraction was performed using InstageneTM Matrix  and low virulence (LV)  strains , a multi strains  was usedspa) gene [The spa-typing method was performed to amplify Staphylococcal Protein A (pa) gene ,28. Sequpa) gene . ClusterS. aureus, partial coding sequences” .The spa-type sequences found were submitted to GenBank as “Immunoglobulin G binding protein A of 2 test to evaluate the significance of differences between qualitative variables, and Fisher’s exact test to deal with empty cells. Odds ratios (ORs) were used as measures of association between outcome and risk factors, with 95% confidence intervals obtained by the Woolf’s method for crude ORs. Mantel-Haenszel OR was used to control for confounding factors, with 95% support intervals obtained by maximum likelihood method (ML). Logistic regression models, chosen by forward selection method, were performed to evaluate the effect on outcome of discrete quantitative and binomial variables as risk factors and confounders, and to predict expected prevalence of lesions by S. aureus. Particularly, the outcome of interest was defined as lesion from which S. aureus was isolated. As possible risk factors for lesions by S. aureus, colonization of body sites, nasal carriage, age category and bacterial lineages (spa-types), were evaluated. Association measures were reported as OR, with 95% support intervals obtained by ML. An association plot was produced using package vcd of software R version 3.6.1 [Statistical analysis was performed by software STATA version 13.0 , using χon 3.6.1 .S. aureus in relation to the age animal category and the anatomical site are shown in S. aureus coming from 284 rabbits , and at least in one anatomical site. Between the different sites, a significant difference in the frequency of isolation was recorded (p < 0.05), while a similar and high isolation was observed in both the auricular (29.4%) and nasal swabs .In total, 2066 skin samples were collected from 400 animals. The sampling distribution and prevalence of S. aureus colonization. A significant difference was recorded for all sites across all age categories (p < 0.05), except between young rabbits (4.6%) and the replacement rabbits .Out of 284 rabbits, 174 (61.3%) had more than one site colonized, distributed as follows: 105 rabbits (37.0%) had two; 49 (17.2%) had three; 15 (5.3%) had four; and in 5 (1.8%) animals all five sampled anatomical sites were positive for S. aureus, and of these, 30 (7.5%) had only one lesion, while five rabbits (1.3%) had two lesions.During sampling, 66 lesions were observed in 58 animals: Only one lesion in 51  rabbits; two lesions in six (10.3%) rabbits; and three in one (1.7%) rabbit. Out of those, 35 (8.8%) animals presented lesions by Staphylococcus spp. were isolated from 54 (81.8%), while S. aureus was identified in 40 of them (74.1%), and it was significantly associated with lesions . In relation to the age category, S. aureus was isolated in 20%, 84.6%, 30%, and 0% of swabs collected from lesions present in young (n = 5), adult (n = 39), breeding (n = 20), and replacement rabbits (n = 2), respectively . The higher number of colonized body sites was associated with an increased risk of lesions due to S. aureus ; nose ; inguinal region . Nose colonization had a higher correlation with colonization than the other sites (Spearman’s rho ranging from 0.129 to 0.216), and therefore swabs from the nose were analyzed more in detail. Although all sites were positively associated to the outcome, nose colonization was significantly associated with increased risk of lesions by S. aureus .Prevalence of colonization differed among anatomical sites, particularly in those that were positive for  regions . Some ofS. aureus were more representative in a particular age category or in animals with more than one colonized body site, a weak association was observed between categories and lesions by S. aureus (Fisher’s exact p = 0.048), and between categories and colonization by S. aureus of at least one body site . In young rabbits, the most contaminated site by S. aureus was the ear (33.3% isolates), with no significant difference towards the prevalence recorded in the other anatomical sites (p > 0.05). In particular, all young and replacement rabbits had none or few sites colonized by S. aureus, and never were there more than three. While in young rabbits only in 20% (n = 5) of the lesions S. aureus was cultured, in replacement rabbits no lesions due to S. aureus were recorded. Among adult fattening rabbits, out of 478 isolates, the nose (29.1%) and the ear (27.6%) were the most contaminated sites . Compared to young rabbits, adult fattening and breeding rabbits had a higher risk of developing lesions by S. aureus, OR = 2.6 (95% CI: 0.3–19.6), and OR = 5.7 (95% CI: 0.6–52.4), respectively. Statistical data evidenced that age category cannot explain all the differences regarding presence of lesions, and other factors, such as the number of colonized sites, seem to play a role. Breeding rabbits had a higher probability to become colonized by S. aureus in at least one body site , showing positive correlations (Spearman’s rho ranging from 0.071 to 0.126), and a positive association with the colonization of all sites, in particular the ear  and nose .Rabbits were unevenly distributed among age categories. When the analysis was carried out to understand if lesions by ed sites . AlthougS. aureus, since it was strongly associated with them , and crude risk of OR = 3.2 (95% CI: 1.5–6.5) was observed. The risk from nasal carriers was due only to adult fattening and breeding rabbits, as young and replacement rabbits added no information. Age-stratified odds ratios showed that the nasal carriage effect varied widely among different age categories, being OR = 2.6 (95% CI: 1.2–5.8) for adult and OR = 4.0 (95% CI: 0.4–41.8) for breeding rabbits.Nasal carriage alone was evaluated as a potential risk factor for developing lesions by S. aureus was the stronger risk factor observed for developing lesions, with a dose–response pattern that reinforces the plausibility of our findings ; 21 strains were isolated from lesions; and three strains came from farm workers. Most of the samples were cultures from nose swabs (n = 64), due to the high correlation found between the colonization of this site with other anatomical regions, and for the spreading role of nasal carriage of S. aureus, because rabbits—similar to many other animals—use this part to reach all other body sites. Paired samples (n = 16) were selected from rabbits with both lesions and nose or ear colonization; some samples (n = 2) were from lesions in rabbits with no colonized sites, while the others were chosen to respect age category and level of colonization distributions.Out of a total of 592 isolates,  a sample of 96 S. aureus strains were negative for the presence of bbp, selm, flank genes of virulence and for mecA and mecC gene detection; therefore, they were classified as LV-MSSA strains.All 96 spa gene was found in all tested isolates (n = 96). We identified 5 different spa-types: t094, t491, t2036, t2802, and t605. A bimodal distribution was identified, with the most frequently spotted spa-type, t2802 (53.1%), also detected in all human samples, and t491 being the next most frequently spotted spa-type (38.5%) (The  (38.5%) .Due to its shortness, t605 could not be clustered in any clonal complex. The other four spa-types were arranged into three spa-CCs using BURP algorithm: spa-CC267, to which belongs t2802; spa-CC084, to which belongs t094 and t491; and spa-CC012, with t2036. The last two clustered together in our study, due to the relative closeness (distance < 4) among the spa-types found.p = 0.005), but their scarce frequencies did not facilitate a generalization of our findings to a larger population. The two most represented spa-types, t491 (n = 37) and t2802 (n = 51), were not associated with animal categories (Fisher’s exact p = 0.192), but t2802 is the only one that has been found in farmers (n = 3).Since spa-type t094 was found only in replacement rabbits, t605 in adult, and t2036 in breeding rabbits, they were significantly associated to age categories (Fisher’s exact p = 0.526). Out of 11 isolates from the auricular region, five were t2802, three were t094, two were t2036, and one was t491; meanwhile, out of 64 isolates from the nose, 39 were t2802, 24 were t491, and one was t094. An overall association with sampling sites was found, with the most represented spa-types being isolated mainly from nose (Fisher’s exact p < 0.000). Spa-types were not associated with the number of colonized sites , even controlling for both age category and number of colonized sites .Spa-type t605 isolates (n = 2) were drawn from lesions, while t094 and t2036 only came from healthy skin swabs. Spa-type t491 and t2802 isolates were drawn from lesions 12 out of 37 and 7 out of 51 times, respectively. t491 was positively associated to lesions by S. aureus, t491 and t605 were isolated once each.Out of the eight rabbits in which both isolates from lesions and nose were analyzed, six had the same spa-type, and two had different spa-types. Particularly, when lesions differed from the nose, t2802 in the nose and t491 or t605 in lesions were observed; when they had the same spa-type in both sites, t2802 was isolated most frequently . In lesions from rabbits which had no other sites colonized by S. aureus in at least one body site was quite high (71.0%), although being within the same range observed by Hermans et al. [S. aureus prevalence of 60.5% in Belgian rabbit farms and 77.6% in Italian farms, respectively. Rabbits were colonized mainly in the nose, which suggests that it was a stable colonization in balance between bacterium and host. The high colonization rate (71.0%) is not only attributable to the intrinsic capacity of certain clones, but also to the crowding of the animals, their frequent manipulation, and the top-down distribution of the pure lines of parents, which favors the dissemination of S. aureus clones within the groups. These phenomena are intrinsic characteristics of the industrial breeding rabbit system rather than the S. aureus clone of interest.In this study, the overall prevalence of rabbits colonized by s et al.  and Agnos et al. , who repmecA and mecC gene presence is still not a widespread problem in this type of breeding in our country, although MRSA was isolated in commercial rabbits in 2014 [All strains, including those isolated from skin lesions, resulted with LV-MSSA, suggesting that the  in 2014 ,31.S. aureus (LVSA) leads the farmer to control it rather than spending resources to prevent it; consequently, little information is available on the role of those bacteria in lowering productivity of the animals. During a clinical experiment, Meulemans et al. [The limited economic impact of low virulence s et al.  demonstrS. aureus can be isolated from the whole body of rabbits: The nose, ear, skin of the interdigital spaces, the skin of the forelegs, the skin of the axillary and inguinal regions, the skin around the nipples, the perineum, the vagina, and the foreskin [S. aureus from rabbits. The ethology indicates that rabbits spend an elevated amount of time washing themselves, eating and performing caecotrophy, meaning that they can reach virtually every part of the body with their nose; thus, the nose should represent the colonizing population of all body sites. As Hermans et al. [S. aureus, and that sampling only the nose could result in a loss of information.As reported previously, foreskin ,34,35,36s et al.  stated, S. aureus, it was not appropriate to give a summary result for all the rabbits, but it was necessary to consider them separately. It is presumable that the greatest number of lesions observed on adult animals derived from the type of cage and the hyper energetic diet, while for breeding rabbits the behavior during mating could induce wounds that could become infected. However, those reasons were not enough to explain the excess of lesions observed, and so the number of infected sites, or the level of body colonization by S. aureus should be considered. Not only was this associated with a higher probability of lesions, but it showed a dose–response effect that also remained, considering the differences related to age categories. On the other hand, nasal carriage, which was higher among adult and breeding rabbits, could partially explain the excess of lesions and suggests a stable bacterium–host interaction. The relatively low prevalence of severe lesions (8.8%) compared to colonization of at least one body site suggests that most of the animals were asymptomatic carriers and that the infection was due to perturbation of this equilibrium. What we observed in this study could reflect the hypothesis that all rabbits, irrespective of age group or production category, the secondary lesions by S. aureus derive from abscessualization of primitive traumatic lesions , and the risk increases when more sites are involved [Adult and breeding rabbits have many differences to young and replacement rabbits, in terms of physiology, weight, diet, time spent in the same environment, type of cage, and behavior. Age category represented a summary measure to consider all of them, and since it was associated in different ways with lesions and colonization by involved .Spa-typing is a fast and powerful tool for epidemiology research, since it allows one to identify the predominant lineages circulating in the flock, and to trace them back to clonal complexes. In this farm, five different spa-types were identified and t491 and t2802 were the most frequently detected. Among rabbit categories, the bimodal distribution of spa-types, namely t2802 and t491, partially reflects the spatial allocation of the subjects within the farm. Spa-type t094 was isolated only from replacement rabbits, and from them all, while t2036 was observed only from breeding rabbits, corroborating the belief that subjects purchased from other farms for breeding purposes could act as carriers of new, and potentially pathogenic, strains.S. aureus strains were identified as t2802, the most frequently represented in animal isolates, thus corroborating the belief that the transmission of S. aureus strains, both HV and LV, between rabbits or from man to rabbits, can take place directly or indirectly, by contact from cage to cage, air, or through the hair and feed [S. aureus strains could easily circulate in a community. Since spa clonal complexes could be related to MLST clonal complexes with good approximation [All human and feed ,26,38. Sand feed , and it ximation , the t28ximation ,41. Spa-ximation ,41. GrouS. aureus.This study cannot claim to be representative of the Italian rabbit population, but it does represent an initial step towards understanding the main risk factors that could increase infections by S. aureus, with its opportunistic behavior, can survive on the skin of animals and humans for a long time, making them agents of the infection.Among rabbit breeding farms, staphylococcosis represents one of the main problems due to its economic impact. S. aureus from rabbits reared intensively for meat production in a standard Italian breeding farm, and shows that those are significantly associated with the risk of skin lesions. Particularly, the S. aureus occurrence is higher in adult rabbits than other categories and ear and nose are the most frequently sites colonized. The presence of lesions due to S. aureus is strongly associated with the colonization of at least one body site, and the risk of lesions due to S. aureus is proportionate to the number of sites colonized by S. aureus.This study describes the effects of age and body colonization on detection of S. aureus, and an association with nose colonization was found with the most represented clonal lineages in the rabbit breeding farm, but no association between spa-types and the number of colonized sites was found. The same spa-types were observed in both animal and worker samples, showing that S. aureus strains could easily circulate in a closed community with the workers-animal interaction.The authors observe that the nasal carriage is significantly associated with an increased risk of lesions due to S. aureus in rabbits, and little is known about their prevalence. Our study focused on the impact of those bacteria on rabbit breeding activity, using a simple yet powerful epidemiologic approach to highlight the main risk factors.Finally, the authors report a circulation only of LV-MSSA strains that should not be underestimated, as they could nonetheless cause economic damage, or even acquire and spread resistant genes. Few studies have been published on low virulence