Prions are infectious proteins that self-propagate by changing from their normal folded conformation to a misfolded conformation. The misfolded conformation, which is typically rich in β-sheet, serves as a template to convert the prion protein into its misfolded conformation. In yeast, the misfolded prion proteins are assembled into amyloid fibers or seeds, which are constantly severed and transmitted to daughter cells. To cure prions in yeast, it is necessary to eliminate all the prion seeds. Multiple mechanisms of curing have been found including inhibiting severing of the prion seeds, gradual dissolution of the prion seeds, asymmetric segregation of the prion seeds between mother and daughter cells during cell division, and degradation of the prion seeds. These mechanisms, achieved by using different protein quality control machinery, are not mutually exclusive; depending on conditions, multiple mechanisms may work simultaneously to achieve curing. This review discusses the various methods that have been used to differentiate between these mechanisms of curing. Yeast has long served as a model system for studying neurodegenerative diseases, including prion disease, due to cellular pathways being highly conserved from yeast to mammals plus yeast having the advantages of a short generation time and ease of genetic manipulation. Prion proteins exist in two conformations, a normal folded conformation and a misfolded prion conformation, the latter of which is typically characterized by the occurrence of β-rich amyloid fibrils. It is the misfolded conformation that is the infectious agent in prion disease . In mammSaccharomyces cerevisiae are [PSI+], [URE3], and [PIN+], which are the misfolded protein conformations of Sup35, Ure2, and Rnq1, respectively , the two conformations can be distinguished in yeast with a nonsense mutation in the adenine biosynthetic genes. When Sup35 is in its amyloid conformation, there is enough read-through of stop codons to prevent accumulation of intermediates in the adenine pathway, resulting in colonies having a white color. However, when Sup35 is in the normal folded conformation, the nonsense mutations cause intermediates in the adenine synthesis pathway to accumulate, which causes the colony to have a red color [URE3] and the [PIN+] prions  prions ,14. In tractions . Alternaractions . Finallyractions .HSP104 since Hsp104 is a non-essential gene  yeast strain was cured by overexpression of a dominant negative Hsp104 mutant, there is a reduction in the number of foci as a function of generation time  has also been used to monitor the curing of the prions by Hsp104 inactivation in real-time. When a [ion time B consistPSI+] prion, while inactivation of Hsp104 with guanidine cured [PSI+] in ~10 generations [URE3] and some variants of [PIN+] by depleting Ssa1, which has been achieved by overexpressing Ydj1, a member of the Hsp40 family [URE3] curing when Ydj1 is mutated at its J-domain to prevent its binding to Ssa1 [PSI+]  .PSI+], an observation made more than two decades ago by Chernoff et al. [PSI+] curing by Hsp104 overexpression, imaging was used to examine the fluorescent seeds in different [PSI+] strains. As shown in PSI+] based on the plating assay. Since the foci are evident in some cells and not in other cells, as well as being visible in all cells at the start of the experiment, rapid movement of foci during the curing process does not appear to explain the lack of foci in some of the cells. The prion seeds become visible again after the cells are stressed by starvation, which perhaps causes an increase in the size of the prion seeds by altering the chaperone balance in the cell. Therefore, the seeds are present all along but were too small to resolve unless their size was increased by stressing the cells  and Hsp104 (D184S). In agreement with previous results [PSI+], and also not to trim the prion seeds [PSI+] or trim the prion seeds [PSI+] by Hsp104 overexpression was also obtained when we overexpressed Hsp104 homologs from either Candida albicans or Schizosaccharomyces pombe. These homologs neither trimmed the prion seeds nor cured the strong [PSI+] variants, but they did trim the prion seeds and cure the weak [PSI+] variants [PSI+] by Hsp104 overexpression in the absence of trimming.Further evidence that the curing of  seeds, which would ultimately cause their dissolution. This proposal is supported from in vitro studies that showed that increasing Hsp104 concentration caused fragmentation of amyloid fibers, composed of the N-terminal prion domain, the domain that forms the amyloid core, and the M-domain of Sup35 [PSI+] by Hsp104 overexpression. However, since guanidine did not significantly affect the rate of curing of [PSI+] by Hsp104 overexpression  and weak [PSI+] variants [URE3] and weak [PSI+] variants showed that DnaJB6 overexpression caused a loss of resolvable foci or seeds in cells that were still not cured, suggesting that in both cases, the seeds were becoming smaller before they completely disappeared [PSI+] by reducing the size of the prion seeds  variants by protein solubilization involving an increase in Hsp104 expression [PSI+] variants by DMSO addition was significantly faster than the rate of curing by Hsp104 inactivation. This curing was accompanied by an increase in Hsp104 expression along with an increase in free Sup35. In addition, even when cell division was arrested by the addition of alpha factor, which inhibits cell division, there was significant curing by DMSO. This suggests that curing may be due to disassembly of the prion seeds since curing by disassembly should depend on time of incubation with the disassembly agent rather than cell division. On the other hand, when [PSI+] is cured either by Hsp104 inactivation or asymmetric segregation, cell division is necessary to reduce seed number. As they noted, curing by DMSO could be due to disassembly of the prion filament or to accumulation of newly synthesized Sup35 monomer that cannot bind to pre-existing filaments or to newly obtained cured cells in the population.Finally, the Manogaran laboratory recently reported that the chemical reagent dimethyl sulfoxide (DMSO) cures the weak  prion by asymmetric segregation when the yeast were imaged with either the N-terminal prion domain of Ure2 or full-length Ure2 labeled with GFP [URE3] by overexpression of Hsp42, Cur1, or Ydj1, suggesting that, here too, asymmetric segregation has a role in curing [URE3] [URE3], overexpression of Cur1 aggregates the prion seeds in weak [PSI+] yeast, which contributes to curing by asymmetric segregation [PSI+] yeast, which, in turn, leads to curing by asymmetric segregation [PSI+] variants. Another example of asymmetric segregation was observed upon overexpression of a C-terminal Hsp104 truncation mutant from S. pombe, Hsp104ΔC32. This fragment cured both the weak and strong [PSI+] variants by asymmetric segregation , and the proteins Upf1 and Upf3, components in a complex involved in nonsense-mediated mRNA decay [PSI+] colonies were formed than in the wild-type strain. As expected, when these proteins were expressed in their respective deletion strain, there was a marked reduction in [PSI+] colonies, suggesting that these proteins cure some [PSI+] variants. Similarly, genetics identified a function for the proteasome as well as the cochaperones, Sti1 and Cpr7, in the curing of [PSI+] by Hsp104 overexpression  . Interes] prions . The WicNA decay ,62. The pression ,63.In vitro studies using purified proteins have provided insights into the mechanism by which chaperones cure prions. Generally, this reductionist approach has used the prion domain or other misfolded substrates such as denatured luciferase, rather than the full-length prion protein. At least when expressed in the cell, the prion domain shows much greater tendency to aggregate than the full-length protein ,65. In pPSI+] by inhibiting severing of the prion in the former case and causing dissolution or perhaps asymmetric segregation of the prion in the latter case. Furthermore, we also found that overexpression of a truncated Hsp104 fragment from S. pombe cured [PSI+] by asymmetric segregation.This paper has focused primarily on the curing of the canonical prion proteins that misfold into a β-sheet amyloid conformation. For the most part, by using multiple complementary techniques, a good understanding has been obtained of the curing of these prions under varying conditions. Interestingly, the same protein can sometimes cure a yeast prion by more than one method, that is by inactivation, dissolution, or asymmetric segregation. For example, inactivation or overexpression of Hsp104 both cure [PSI+] is affected by the cochaperones, Sti1 and Cpr7, but the role of these cochaperones in this curing is still unclear [PSI+] by Hsp104 overexpression even though there is no evidence of ubiquitination of Sup35, which would be expected if the proteasome is involved. It is not even clear whether overexpression of Hsp104 cures weak and strong [PSI+] variants by the same mechanism, since the curing of these variants differ in their dependence on members of the Hsp40 family [PSI+] curing by Hsp104 overexpression is due to the use of different [PSI+] variants in the relevant experiments. Surprisingly, however, even though the Tuite laboratory proposed that the curing of [PSI+] is due to asymmetric segregation and not dissolution, they only observed a significant difference in seed number between mother and daughter cells when the yeast were ~50%, but not ~30% cured  is not understood. Imaging of the fluorescently labeled Ure2 prion domain, as well as full-length Ure2, showed that overexpression of Btn2, Cur1, or Hsp42 cures [URE3] and causes aggregation of the [URE3] seeds  seeds ,42,60. T3] seeds ,72,73. OURE3] by sequestering Ssa1 [URE3] seeds [URE3] by Ydj1 overexpression depends on inhibition of severing, since severing by Hsp104 requires Ssa1, and how much it depends on asymmetric segregation because Ssa1 causes aggregation of the [URE3] seeds  seeds . Therefo3] seeds .An emerging field with many unanswered questions is the mechanism of propagation of non-canonical prions that have two conformations, a regular conformation and a prion transmissible conformation that is not an amyloid. Instead of the glutamine/asparagine (Q/N)-rich amyloid domain present in the canonical prions, these new prion proteins contain an intrinsically disordered domain (IDD). As occurs with the canonical prions, the altered conformation of these IDD-prions is self-templating, which, in turn, enables this altered conformation to be passed from mother to daughter cells. This new class of prion proteins was discovered by the Jarosz laboratory using a screen in which they expressed proteins at high concentrations under stress conditions in yeast . From thSMAUG+], a prion recently identified by the Jarosz laboratory [SMAUG+] is the misfolded conformation of the yeast protein, Vts1, a member of the Smaug family of post-transcriptional regulators that bind RNA. Furthermore, like other proteins with IDD-domains that form condensates in a concentration-dependent manner, condensates formed from purified Vts1 are liquified when the Vts1 concentration is decreased. This suggests that the local concentration of Vts1 in the cell may regulate the curing of [SMAUG+]. The discovery of the new non-amyloid prions has revolutionized the prion field and undoubtedly will lead to new mechanisms of curing.Proteins with IDD-domains tend to undergo a phase transition from a liquid to a gel under crowding conditions in vitro. Based on this property, one would expect that some of the new prions may undergo such a phase transition in vivo. In fact, [boratory , forms c


Autoimmune rheumatic diseases are systemic diseases frequently affecting the heart and vessels. The main cardiovascular complications are pericarditis, myocarditis, valvular disease, obstructive coronary artery disease and coronary microcirculatory dysfunction, cardiac failure and pulmonary hypertension. Echocardiography, including transthoracic two and three-dimensional echocardiography, Doppler imaging, myocardial deformation and transesophageal echo, is an established and widely available imaging technique for the identification of cardiovascular manifestations that are crucial for prognosis in rheumatic diseases. Echocardiography is also important for monitoring the impact of drug treatment on cardiac function, coronary microcirculatory function, valvular function and pulmonary artery pressures. In this article we summarize established and evolving knowledge on the role of echocardiography for diagnosis and prognosis of cardiovascular abnormalities in rheumatic diseases. Autoimmune rheumatic diseases (ARD) are immune-mediated diseases targeting the connective tissues. Cardiovascular complications are frequent and almost all cardiac structures may be affected. The main cardiovascular manifestations in ARD are (a) pericardial, myocardial and vascular inflammation, (b) coronary artery disease (CAD) and dysfunction of the coronary microcirculation, (c) structural and functional abnormalities of the heart valves and (d) elevated pulmonary artery pressures ,2.Anti-inflammatory treatment reduces inflammation and disease-associated morbidity and mortality. However, the incidence of cardiovascular (CV) events is higher in ARD patients in comparison with the general population resulting in worse prognosis ; thus, tEchocardiography is an established and widely available imaging technique for the detection of cardiovascular involvement and for monitoring the effects of treatment on cardiac and vascular structure and function in ARD patients. In this article, we aim to review the role of echocardiography for diagnosis and prognosis in ARD associated with increased incidence of cardiovascular complications and higher cardiovascular risk  includinCardiovascular complications are common including (a) pericardial involvement (in 30–50% of RA patients) , (b) myoSystemic Lupus Erythematosus is mostly complicated by (a) valvular heart disease in 13–100%) [3–100% 1, (b) perIn Systemic Sclerosis (SSc) the main cardiovascular complications are (a) pulmonary artery hypertension (PAH) ,20 and (Psoriasis and psoriatic arthritis are characterized by (a) accelerated atherosclerosis and increased CV risk because of linked causative pathways with CAD including inflammation and oxidative stress  (in psorThe main cardiovascular manifestations in AS include (a) aortitis with aortic and mitral valve involvement ,27 and  and Doppler imaging ,29. Swin2 and tricuspid regurgitation maximum velocity >2.8 m/s are findings consistent with elevated filling pressures [As some studies have indicated that the systolic function of the LV may be moderately reduced in RA patients suggesting progression to heart failure , assessmressures . Myocardressures ,34.Although extensively validated and used in daily practice, left ventricular ejection fraction (LVEF) is not the ideal marker for the evaluation of myocardial function because a reduction in LVEF is indicative of a relatively advanced myocardial dysfunction. Speckle tracking deformation analysis is considered a more sensitive technique for the identification of subclinical alterations in myocardial contractility, providing a more comprehensive study of myocardial function .It has been demonstrated that subclinical myocardial dysfunction may be present in very early RA patients without CV risk factors. Impaired values of global longitudinal and circumferential strain were observed in RA patients with preserved ejection fraction and high disease activity compared with healthy controls ,37.Inflammatory cytokines including tumor necrosis-α (TNFα), interleukin-1 (IL-1) and interleukin-6 (IL-6) have a pivotal role in RA and may induce myocardial and vascular dysfunction and promote LV remodeling and fibrosis ,39,40.Speckle tracking deformation analysis can be applied in ARD patients with unexplained dyspnea and a preserved ejection fraction. Impaired global longitudinal strain (GLS) values in combination with echocardiography findings of elevated filling pressures in the absence of valvular disease or pulmonary abnormalities are consistent with heart failure with preserved ejection fraction ,35. SpecTreatment with an interleukin-1 (IL-1) inhibitor (anakinra) resulted in improved LV global longitudinal strain .LV twisting-untwisting in RA patients, in parallel with a reduction in oxidative stress markers and interleukin-6, particularly in patients with coexisting CAD ,41,42,43The impairment of circumferential systolic and diastolic strain has been associated with disease activity and the degree of inflammation and diffuse fibrosis as expressed by expansion of extracellular volume by cardiac magnetic resonance (CMR) T1 mapping ,46.Finally, LV hypertrophy is associated with an increased prevalence of cardiovascular events in the general population and in RA patients. TNFα seems to be a significant determinant of LV remodeling . It has Patients with SLE myocarditis may have global or segmental wall motion abnormalities by 2D echo and low systolic Tissue Doppler imaging velocities of the mitral annulus ,49. ImpaTransthoracic 2D echo is a valuable tool for the assessment of heart function in SSc, whereas 3D echo may offer additional information on ventricular volumes and EF especially in case of PAH affecting the right ventricle (RV). Structural and functional abnormalities of the LV and RV have been reported in up to 23% and 21% of SSc patients, respectively . Primaryp > 0.01)) and circumferential strain values ). In patients with SSc, impaired GLS and circumferential strain values each correlated with worse functional capacity and rhythm disturbances [Patients with SSc and preserved EF as compared with controls had impaired GLS  .Impaired myocardial strain correlated with disease activity and the amount of focal and diffuse myocardial fibrosis by CMR T1 mapping  and has It has been demonstrated that GLS is impaired in a similar degree in psoriasis and CAD patients compared with normal controls (−16.5% in psoriasis vs. −16.2% in CAD vs. −21.9% in controls). This is consistent with the presence of subclinical myocardial dysfunction in psoriasis due to a similarly elevated inflammatory and oxidative stress burden as indicated by elevated IL-6 and malondialdehyde values . TreatmeTwo and three dimensional transesophageal echocardiography is an accurate technique for the diagnosis and assessment of the severity of valvular heart disease ,7,63. RhValvular heart disease may be identified in 30–50% with transthoracic and 60–70% with transesophageal echocardiography in SLE patients . ValvulaLibman–Sacks vegetations are detected in 10% by transthoracic and 30–40% by transesophageal echocardiography in SLE patients. Libman–Sacks vegetations are typically located on the tips of the left heart valves as inflammatory formations of >2 mm in diameter, or thrombotic or firm masses that may resolve or relapse . Thus, pAortitis is a frequent and serious complication of AS, occurring late in the course of the disease. The inflammatory process begins from the aortic root wall and may expand to the aortic cusps causing thickening and fibrosis . Mitral RA is characterized by a higher incidence of CAD and cardiovascular events compared with the general population due to systemic inflammation and the high prevalence of traditional CV risk factors ,68. PatiThe detection of carotid atherosclerosis has been shown to reclassify patients with RA into a higher risk group . In ARD Coronary flow reserve can be calculated non-invasively by Doppler echocardiography of the left anterior descending artery as the ratio of peak diastolic velocity after adenosine infusion to peak diastolic velocity at rest . CoronarDecreasing CFR values correlated with elevated values of symmetric dimethylarginine, a marker of endothelial dysfunction that acts as an endogenous inhibitor of nitric oxide synthase . Treatment with an interleukin-1 (IL-1) inhibitor (anakinra) resulted in improved CFR in RA patients, particularly when CAD coexisted, indicating a beneficial effect of the anti-inflammatory treatment on coronary microcirculatory function in RA ,41,42,43The calculation of CV risk based on the traditional risk scores is inaccurate in SLE . SystemiCoronary flow reserve was similarly impaired in psoriasis and CAD patients after adjustment for atherosclerotic risk factors in parallel with elevated markers of inflammation and oxidative stress . MoreoveSeveral studies have indicated an increased CV risk and accelerated atherosclerosis in AS ,87. A meInflammatory cytokines, oxidative stress, traditional CV risk factors and the toxic effects of non-steroid inflammatory drugs have been considered to cause aggravation of the atherosclerotic process in AS ,90,91. ETransthoracic echocardiography is a first line modality for PAH screening and for the detection of RV dysfunction because RV remodeling has a major role in the prognosis of PAH patients . ContinuMoreover, poor contractile reserve and impaired GLS during exercise stress echo in SSc patients has been associated with reduced functional capacity and higher values of pulmonary artery systolic pressures .Due to the complex triangular-crescent shape of the RV, a structural and functional assessment of the RV by 2D echo is based on geometrical assumptions . 3D echoPatients with ARD can have excess CV risk and increased incidence of CV complications. Echocardiography is the first line imaging technique for the detection of cardiovascular involvement and for monitoring the effects of treatment in ARD. Additionally, 3D echo may have an added value in volumetric evaluation especially of the right ventricle, whereas speckle tracking may accurately identify subclinical cardiac dysfunction. It remains to be elucidated in future studies whether the improvement of echo markers of myocardial deformation and microcirculatory function by reduction of the inflammatory burden will reduce adverse cardiac events and improve the prognosis of ARD patients.


Streptomyces bacteria are known for their prolific production of secondary metabolites, many of which have been widely used in human medicine, agriculture and animal health. To guide the effective prioritization of specific biosynthetic gene clusters (BGCs) for drug development and targeting the most prolific producer strains, knowledge about phylogenetic relationships of Streptomyces species, genome-wide diversity and distribution patterns of BGCs is critical. We used genomic and phylogenetic methods to elucidate the diversity of major classes of BGCs in 1,110 publicly available Streptomyces genomes. Genome mining of Streptomyces reveals high diversity of BGCs and variable distribution patterns in the Streptomyces phylogeny, even among very closely related strains. The most common BGCs are non-ribosomal peptide synthetases, type 1 polyketide synthases, terpenes, and lantipeptides. We also found that numerous Streptomyces species harbor BGCs known to encode antitumor compounds. We observed that strains that are considered the same species can vary tremendously in the BGCs they carry, suggesting that strain-level genome sequencing can uncover high levels of BGC diversity and potentially useful derivatives of any one compound. These findings suggest that a strain-level strategy for exploring secondary metabolites for clinical use provides an alternative or complementary approach to discovering novel pharmaceutical compounds from microbes. Streptomyces (phylum Actinobacteria) are best known as major bacterial producers of antibiotics and other useful compounds commonly used in human medicine, animal health and agriculture2. Beginning in the 1940s when microbiologist Selman Waksman discovered streptomycin (the first effective drug against tuberculosis) produced by the soil-dwelling Streptomyces griseus3, Streptomyces species have received enormous attention in microbial sampling efforts. In the 21st century, majority of all antibiotics have been developed from secondary metabolites produced by Streptomyces4. However, the last few decades saw a steep decline in the development and introduction of new medically relevant drugs to the market. This decline is partly due to the repeated re-discovery of the same molecules in the same ecological niches (often soil) and the high costs associated with drug development5. The increasing public health burden caused by multidrug resistance and the continuing need to find new treatments against non-communicable (chronic) diseases means that the search for bioactive compounds with novel mechanisms of action or with new cellular targets is greater than ever.Members of the bacterial genus 9. These genes encode for the enzymes involved in peptide assembly, regulation, resistance, and synthesis of a secondary metabolite, and are physically clustered into groups called biosynthetic gene clusters (BGCs)10. Recent studies involving mining of large-scale genomic datasets have highlighted the tremendous potential of discovering novel and potentially relevant compounds from microbes13, which can allay some of the challenges in antibiotic development today15. While it has been estimated that many more novel natural products remain to be uncovered and functionally characterized, particularly those from poorly studied ecological niches, it is uncertain what the true number is or whether ongoing drug discovery efforts are reaching BGC saturation16. Moreover, a genome-wide study of representative Actinobacteria showed that Streptomyces genomes possess 25–70 BGCs, much more than any other actinobacterial genera, but only a small fraction of these bioactive products are produced when strains are cultivated in the laboratory6. This suggests that the full inventory of the chemical weapons possessed by a single bacterial strain remains poorly studied. Today, with the cost-effective and rapid increase in the number of bacterial genomes sequenced, one challenge encountered in current drug discovery efforts is how to effectively prioritize those strains with the greatest ability to produce new compounds and understand the extent of biosynthetic potential that exists in nature. Hence, knowledge about evolutionary relationships, BGC diversity, and distribution patterns of BGCs is crucial.Genome mining approaches, which often involve identifying the genes involved in secondary metabolite production, have revealed an unprecedented biosynthetic potential in many microbial speciesStreptomyces, the largest BGC study of a single genus to date. Genome mining reveals the presence of hybrid BGCs, variable distribution of antitumor BGCs and inter-strain differences in BGC content that together expand the repertoire of secondary metabolites that are potentially encoded by individual Streptomyces strains. Genome mining of closely related taxa can therefore greatly facilitate the discovery of novel pharmaceuticals and untapped sources of chemotherapeutic agents. These findings also highlight the importance of strain-level drug discovery approaches, exploring multiple genomes of closely related strains, rather than focusing on one strain representative of the entire species.In this study, we aim to determine the diversity of BGCs and their phylogenetic distribution among 1,110 genomes of Streptomyces genomes using antiSMASH17 , with Streptomyces rhizosphaericus NRRL B-24304 (n = 83 BGCs), Streptomyces sp. NRRL B-1347 (n = 82 BGCs), Streptomyces sp. PRh5 (n = 82 BGCs), Streptomyces milbemycinicus NRRL 5739 (n = 81 BGCs) and Streptomyces sp. NBS 14/10 (n = 79 BGCs) having the highest number of BGCs , Streptomyces thermoautotrophicus H1 (n = 9 BGCs) and S. thermoautotrophicus UBT1 (n = 11 BGCs). We observed a weak but significant positive correlation between genome size and the number of BGCs per genome   Fig. .Figure 1Streptomyces rimosus subsp. rimosus), while the average number of NRPS per genome is 17.66. Other BGCs that were commonly found in Streptomyces include butyrolactone (present in 503 genomes), Type 2 PKS (499 genomes), bacteriocin (419 genomes), and Type 3 PKS (366 genomes). Some BGCs were rarely found and were present in only a handful of genomes. These include bottromycin (17 genomes), furan (15 genomes), phosphoglycolipid (12 genomes), homoserine lactone (8 genomes), cyanobactin (7 genomes), acyl aminoacids (2 genomes), and resorcinol (1 genome).The most common BGCs were non-ribosomal peptide synthetases , type 1 polyketide synthases , terpenes (697 genomes), other ketide synthases  and lantipeptides (540 genomes) Fig. . These f21. The origins and specific roles of these hybrid BGCs are not fully known, but they provide additional structural and chemical modifications in major classes of BGCs and may produce medically beneficial derivatives of a compound23. If we subdivide the 34 BGCs into their hybrid types, we obtain a total of 541 unique BGCs. The most common hybrid BGCs were Type 1 PKS-NRPS (present in 753 genomes), Type 1 PKS-other types of KS (237 genomes), terpene-NRPS (189 genomes), bacteriocin-lantipeptide (143 genomes), and Type 3 PKS-NRPS (131 genomes) . However, it must be kept in mind that the draft nature of many of these genomes is likely to affect BGC prediction by antiSMASH.Some BGCs contain genes that code for more than one type of scaffold-synthesizing enzymes and are called hybrid BGCses) Fig. . Of the Streptomyces species are known to harbor BGCs that encode for secondary metabolites with antitumor activity, which we refer to as chemotherapeutic gene clusters (CGCs) to differentiate them from other BGCs. Using individual genes of each CGC obtained from DoBISCUIT . These minimum threshold values were selected to maintain a conservative approach to detecting CGCs and account for possible genome sequencing errors. We also searched the NCBI database for the genome sequences of strains listed in DoBISCUIT. Of all the strains from the DoBISCUIT database that encode the 38 CGCs, only two strains (Streptomyces globisporus C-1027 for the compound C-1027 [synonym: lidamycin] and Streptomyces neyagawaensis ATCC 27449 for concamycin) have genome sequences available in NCBI and were included in our dataset. To ensure that our approach of CGC detection is accurate, we searched for the BGCs that encode for these compounds in the genomes of the two Streptomyces species. Results indicate that we were able to retrieve the CGCs for C-1027 and concamycin in these genomes, thereby ensuring the reliability of our output.Some Streptomyces phylogeny  and Jurkat cells through its ability to induce apoptosis25. Oligomycin is another macrolide whose antitumor capabilities originate from the inhibition of the F0 site of ATP synthase, blocking proton conduction and ultimately inducing apoptosis26.We found that CGCs are widely but differentially distributed in the eny Fig. . The mos28. One of the most commonly used anthracycline for AML chemotherapy is daunorubicin30. It was first isolated from Streptomyces sp. C5 and acts mainly through intercalation with DNA and inhibition of macromolecular biosynthesis31. We found 40 Streptomyces genomes that carry the BGC encoding for daunorubicin , and nogalamycin from Streptomyces nogalater ATCC 27451 (=NBRC 13445) were also found in 37, 32, and 2 genomes, respectively.Induction chemotherapy based on anthracyclines remains a part of standard of care therapy for acute myeloid leukemia (AML) patientsStreptomyces genomes harbor one or few of the genes of steffimycin and tautomycin CGCs. We also found that sequence similarity and patterns of presence/absence vary across the individual genes in each of the 38 CGCs  and tautomycin (tetronic acid derivative) in any of the genomes in our dataset. However, we hypothesized that these genomes are likely to carry some of the genes of these three CGCs but may not have the minimum 90% of the genes of a CGC, which we used as a cut-off to define the presence of a CGC  and 16 are unique to an individual strain33.In this study, we aimed to explore the phylogenetic distribution and diversity of BGCs in 1,110 Streptomyces genomes, is indicative of recent gene loss and horizontal gene transfer (HGT) events36, which is prevalent in Streptomyces38. Furthermore, the existence of hybrid BGCs, composed of different scaffold-synthesizing enzymes from the major BGC classes and joined in a variety of combinations23, further reinforce the role of these two processes in generating the remarkable BGC diversity in Streptomyces. That BGCs have undergone frequent loss and HGT is not surprising. Genes that encode phenotypes that are subjected to weak selection are likely to be lost, but can be regained via HGT when they confer immediate positive benefit in certain conditions39. Hence, inter-strain BGC variation should be taken into consideration when developing approaches to discover microbe-derived pharmacological compounds and that reliance on a single gene  is likely to result in sampling strategies that overlook possible novel compounds with drug potential. Moreover, our conservative threshold values  are likely to overlook the presence of BGCs in majority of the Streptomyces genomes and our results may only be the tip of the BGC iceberg found in this genus. Future work should therefore emphasize deep sequencing methods  to precisely clarify the presence and gene content characteristics of specific BGCs in a genome.The highly variable phylogenetic distribution of BGCs, even among closely related Salinispora, the different ways by which BGCs are re-assorted between strains to produce hybrid BGCs suggest the major role of HGT in the production of structurally and/or functionally unique secondary metabolites18. This likely holds true for Streptomyces as well and may likely be medically relevant. A previous study reported the production of multiple antibiotics by symbiotic Streptomyces harbored by beewolf digger wasps that the insect uses to protect their larvae and cocoons40. Comparable to the combination antimicrobial prophylaxis commonly used to treat human patients, this strategy carried out by the bacterial population, and not simply by individual strains, makes use of the synergistic action of eight different compounds, thereby providing an important long-term and more efficacious defense against multiple diseases40. Hence, personalized medical strategies in the future can be developed towards using specific combinations of Streptomyces bacteria to produce drugs that will target multiple medical conditions. However, this concept remains a hypothesis and the extent in which bacteria make use of a population-level BGC variation clearly requires a more in-depth investigation and sequencing a greater number of closely related, co-existing strains.One possible explanation to account for the existence of the differential distribution of BGCs between strains is that it may prevent the complete loss of a BGC from the population. Even a rare BGC can potentially be beneficial to the entire population and subsequently becomes more common via frequent HGT between strains. As has been observed in the genus Streptomyces genomes carry some of the individual genes in a CGC while only a few harbor the nearly complete set of genes, may be explained by a cooperative strategy conceptualized in the Black Queen hypothesis42. Here, accessory genes, including BGCs, can be viewed as a shared resource in which members of a microbial population or community make use of the compounds or functions derived from close relatives or other taxa42. Hence, mutual sharing of common goods will lead to mutual dependencies and cooperation among members of a group42. On the other hand, the differential distribution of individual genes of the 38 CGCs in Streptomyces suggests that there exists a common set of genes and pathways in the production of each CGC43. The differences in the distribution of other components of the CGCs may suggest that each species or strain may produce different derivatives of each of the 38 chemotherapeutic natural products43, which may prove useful in discovering more effective drugs for a variety of diseases. However, we acknowledge that this is a hypothesis and future work should therefore focus on elucidating the dynamics of ecological interactions between strains in a population.The observation that majority of the rpoB) phylogenetic tree. While rpoB has been used in initial identification and classification of Streptomyces in previous work46, future Streptomyces studies will certainly benefit from using genome-based data  to clarify evolutionary relationships within and between species. We also point out that while this work does not aim to detect novel antibiotic compounds and other secondary metabolites, it provides important insights into the tremendous biosynthetic potential of the genus Streptomyces even below the species level. Future studies on inter-strain genomic variation as well as the ecological and evolutionary processes that shape it will have broad and positive impact on current efforts to explore the biosynthetic potential that exists in nature.Our study presents several caveats. A major caveat of this study is that current methods of BGC identification, including methods used in this study, are largely dependent on the composition of the BGC database being used for comparison. This means that BGCs that encode for metabolites with previously unrecognized functions or cellular targets may be missed. Moreover, the draft nature of many of the genomes can have a major bearing on the ability of antiSMASH to accurately predict BGCs, particularly so in correctly identifying hybrid BGCs. Hence, improved sequencing quality is likely to alter some of our results. Second, our analyses included only the major classes identified by antiSMASH, which represents the broad diversity of BGC secondary metabolite products. Future work should therefore emphasize the extent of BGC diversity at a more fine-scale resolution . Another weakness of the study is the use of a single-gene  were downloaded from the National Center for Biotechnology Information (NCBI). Accession numbers and genomic information  are shown in Supplementary Table 49. To ensure the utilization of up-to-date and discover potentially novel annotations for BGC prediction as well as maintain consistency in gene annotations, we re-annotated the Streptomyces genomes using Prokka, a pipeline comprising several programs that include locating open reading frames (ORFs) and RNA regions on contigs, translating ORFs to protein sequences, searching for protein homologs and producing standard output files for downstream applications50. A total of 47 genomes were excluded from downstream analyses due to poor quality of assemblies, highly divergent rpoB sequences, and failed antiSMASH analyses17.A total of 1,157 genomes of rpoB sequences from the genome assemblies and aligned them using MAFFT51. Out of the 1,110 genomes, three were missing the rpoB gene and were not therefore included in the phylogenetic tree reconstruction. The rpoB sequences were used to build a maximum likelihood phylogeny using the program RAxML v.8.2.1152 with a general time reversible (GTR) nucleotide substitution model53, four gamma categories for rate heterogeneity, and 100 bootstrap replicates  for two reasons. First, Streptomyces are known to harbor multiple copies of the rRNA operon, with as many as six copies and some of which are divergent55. Second, sequence variation in rpoB permits species differentiation and has been widely used for initial taxonomic identification of multiple Streptomyces species in previous studies46. We also ran RAxML on each of the two subclusters in Fig. 56.We extracted the 17. Sequences of BGCs known to encode natural products with antitumor properties were obtained from DoBISCUIT (Database of BIoSynthesis cluster CUrated and InTegrated)57 . We refer to these BGCs as chemotherapeutic gene clusters (CGCs) to differentiate them from BGCs that encode non-antitumor compounds. A total of 47 CGCs were listed in DoBISCUIT. However, we restricted our analyses to CGCs that were first isolated from Streptomyces bacteria. The compounds geldanamycin and salinomycin were listed twice in DoBISCUIT and each was reported to have been derived from two different Streptomyces strains. For clarity, we only used one copy of geldanamycin and salinomycin BGCs. At the end, we used a total of 38 CGCs in our analysis. We searched for the presence of these 38 CGCs representing 17 major classes of antitumor drugs (Supplementary Table Streptomyces genomes using BLASTX and a minimum e-value of 10−10. Individual genes in a CGC were used as query sequences (Supplementary Table BGCs encoding secondary metabolites were predicted and annotated using the standalone version of antiSMASH 4.1, which identifies BGCs using a signature profile Hidden Markov Model based on multiple sequence alignments of experimentally characterized signature proteins or protein domainsSupplementary Figure S1.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.


B cell hyperactivity and breach of tolerance constitute hallmarks of systemic lupus erythematosus (SLE). The heterogeneity of disease manifestations and relatively rare prevalence of SLE have posed difficulties in trial design and contributed to a slow pace for drug development. The anti-BAFF monoclonal antibody belimumab is still the sole targeted therapy licensed for SLE, lending credence to the widely accepted notion that B cells play central roles in lupus pathogenesis. However, more therapeutic agents directed toward B cells or B cell-related pathways are used off-label or have been trialed in SLE. The anti-CD20 monoclonal antibody rituximab has been used to treat refractory SLE during the last two decades, and the anti-type I IFN receptor anifrolumab is currently awaiting approval after one phase III clinical trial which met its primary endpoint and one phase III trial which met key secondary endpoints. While the latter does not directly affect the maturation and antibody production activity of B cells, it is expected to affect the contribution of B cells in proinflammatory cytokine excretion. The proteasome inhibitor bortezomib, primarily directed toward the plasma cells, has been used in few severe cases as an escape regimen. Collectively, current clinical experience and primary results of ongoing clinical trials prophesy that B cell therapies of selective targets will have an established place in the future personalized therapeutic management of lupus patients. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can affect multiple organ systems . The treFirst, the pronounced heterogeneity of clinical phenotypes poses challenges in developing outcome measures which unanimously and reliably capture response to treatment regarding activity in the afflicted organs, and which also reflect the global SLE disease activity. As a result, the lack of reliable measures for treatment evaluation makes it challenging to design clinical trials to assess drug efficacy. Recruitment of participants has been slow and inadequate in organ-specific trials, whereas the applicability of currently available outcome measures has been questioned in clinically heterogeneous study populations. Borrowed from e.g., rheumatoid arthritis (RA), the treat-to-target concept has also gained attention in SLE , and comUsing such tools, the first successful trials , 11 resuThe complex SLE disease is characterized by loss of self-tolerance, which leads to immune responses toward endogenous nuclear and cytoplasmic material. In response to these autoantigens, clones of plasma cells produce autoantibodies, which are considered a hallmark of the disease. Autoantibodies may induce inflammation through the formation of immune complexes and through activation of Fc-γ receptors. Arguing for a pathogenic role, autoantibodies such as anti-Smith (Sm) and anti-double stranded DNA (anti-dsDNA) are associated with the clinical presentation of the disease , and theApart from the production of autoantibodies, B cells play additional roles in the pathogenesis of SLE. In lupus prone mice, B cells that do not secrete autoantibodies are still important to disease progression . This inB cells initially develop in the fetal liver and adult bone marrow and can be characterized by the use of surface markers such as CD19, CD20 and CD22, expressed at different stages of maturation. The development and survival of B cells depend upon stimulation by the B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF), also known as B lymphocyte stimulator (BLyS). BAFF is a member of the TNF ligand superfamily of proteins, and is mainly produced by myeloid and stromal cells . StimulaTo inhibit B cell responses in SLE, two main pathways are currently used, i.e., (i) BAFF inhibition, and (ii) B cell depletion targeting the cell surface receptor CD20. The BAFF inhibitor belimumab was the first biological medication approved in 2011 by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for use in SLE. Belimumab is a recombinant human IgG1-λ monoclonal antibody that inhibits the soluble form of BAFF, preventing its interaction with BAFF receptors, thus inhibiting B cell survival and maturation. In contrast, rituximab is a chimeric anti-CD20 IgG1 monoclonal antibody that targets the CD20 molecule on the surface of B cells. This leads to B cell depletion through apoptosis, antibody dependent cell mediated cytotoxicity (ADCC), or antibody-dependent phagocytosis (ADP). Pharmaceuticals directly and indirectly targeting B cells that are used or have been trialed in SLE are illustrated in The chimeric anti-CD20 monoclonal antibody rituximab was approved by the FDA in 2006 for use in RA, and has been used off-label in the treatment of refractory SLE . The iniBased on experience from rheumatoid arthritis (RA), the most commonly used regimen for rituximab in clinical practice consists of two intravenous infusions of 1,000 mg each, given 14–21 days apart. In the EXPLORER study, 257 patients with moderate to severe non-renal SLE were randomized to receive rituximab or placebo. Rituximab in EXPLORER was administered at a dose of 1,000 mg at week 0, 2, 24, and 26 on a background of azathioprine, methotrexate, or mycophenolic acid therapy. At week 52, there was no difference between the active treatment and placebo groups in the primary endpoints , which cDespite the negative clinical trials, the European League Against Rheumatism (EULAR) recommendations for the management of SLE prompt consideration of rituximab for organ-threatening SLE that has been refractory or shown intolerance to standard of care immunosuppressants . MoreoveIndeed, targeting CD20 with rituximab has been endorsed in several centers where it is used as an off-label therapeutic option in SLE, mostly for refractory renal disease, either alone or as an add-on treatment to cyclophosphamide or mycophenolic acid –43, but Besides rituximab, some additional biological therapies targeting B cells have been trialed in SLE. The anti-CD20 humanized monoclonal antibody ocrelizumab was evaluated in a phase III trial which included 381 cases with severe lupus nephritis. However, the trial was terminated early due to an imbalance in serious infections in the treatment arm, and ocrelizumab has not been studied further . EpratuzObinutuzumab is another humanized anti-CD20 monoclonal antibody with superior B cell cytotoxic effects over rituximab implicated for patients with RA and SLE. This drug has been studied in a phase II clinical trial of lupus nephritis , designed to evaluate the safety and efficacy of the type II anti-CD20 monoclonal antibody obinutuzumab in patients with proliferative kidney disease. The first results were reported in the form of a conference abstract, where greater frequencies of complete and partial renal response were observed among patients who received obinutuzumab vs. placebo, both as an add-on to mycophenolate mofetil and glucorticoids . FinallyDue to its important role in B cell homeostasis, BAFF has been of central interest as a target molecule in B cell pharmacotherapy in SLE. Belimumab, formerly known as Lympho-Stat B, was the first drug to be licensed for SLE in more than 60 years, and is still the sole biological agent approved for use in adult SLE since 2011 and pediatric and adolescent SLE since 2019. The efficacy of belimumab in reducing lupus activity was first shown in two phase III randomized, placebo-controlled clinical trials , 11, andEarly trials of belimumab in SLE were inconclusive. A phase II trial that comprised 449 patients failed to meet its primary endpoints , 71, whiThe first successful randomized controlled trial of belimumab in SLE was the BLISS-52 trial. BLISS-52 comprised 865 patients with a moderate to severe SLE and positivity for immunological markers. Modest but consistent improvements through week 52 were displayed in patients who received belimumab across various clinical outcomes, and the trial met its primary endpoint, i.e., a significantly greater proportion of patients who received belimumab 10 mg/kg at week 0, 2, 4 and thereafter every fourth week met the SRI-4 criteria for response compared with placebo . Since tpost-hoc analysis demonstrated that these patients benefited from belimumab with regard to several organ-specific aspects, including rates of renal flares   and the Autoreactive B cells are indubitably key cells in the pathogenesis of SLE, but the theoretical merit has hitherto seldom culminated in the anticipated outcomes in drug development. The lack of success in clinical trials has not been for lack of trying. Apart from pharmaceuticals which predominantly exert effects on B cells, numerous other therapeutic modalities have been trialed for SLE, several of them expected to indirectly impact on B cells and B cell functions. For example, in lupus prone mice, targeting other B cell stimulating cytokines, such as IL-6, decreased disease progression, but this strategy did not succeed in subsequent clinical trials . TargetiActivation of the type I IFN pathway is prominent in the pathogenesis of SLE, and type I IFNs stimulate BAFF production. In patients with SLE, the type I IFN pathway is overexpressed, and the IFN-α protein in particular has shown associations with both disease activity  and riskThe first reports to support the efficacy of direct IFN-α inhibition in SLE originated from a phase IIb clinical trial of sifalimumab . The resFollowing promising results in a phase II clinical trial , the typAn interesting trend is targeting B cell intracellular signaling, such as through inhibition of Bruton's Tyrosine Kinase (BTK), which is a strategy approved for the treatment of B cell malignancies. Inhibition of BTK has shown efficacy in lupus prone mice, which resulted in reduced kidney damage and increased survival . AnotherTo summarize, B cell hyperactivity and breach of tolerance constitute hallmarks of SLE, and it is widely accepted that B cells play central roles in the pathogenesis. However, the contribution of B cells to disease initiation and perpetuation is less well understood. B cells in SLE constitute the main autoantibody producers and probably facilitate the priming of autoreactive T cells and function as antigen-presenting cells, as well as constitute a source of the cytokines involved in immune dysregulation . As a reEven though drug development in the field of SLE has been slow, B cell-targeting therapies have been increasingly used during the last two decades and contributed to improved management and improved prognosis. The amount and primary results of ongoing clinical trials prophesy that B cell therapies of selective targets will have an established place in the future personalized therapeutic management of lupus patients.All authors contributed to the manuscript draft, critically reviewed all parts of the manuscript, accepted its final version prior to submission, and account for its content.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.


Atopic dermatitis (AD) is the most common chronic inflammatory skin disorder of childhood. Underlying factors that contribute to AD are impaired epithelial barrier, alterations in the lipid composition of the skin, immunological imbalance including increased Th2/Th1 ratio, proinflammatory cytokines, decreased T regulatory cells, genetic mutations, and epigenetic alterations. Atopic dermatitis is a multifactorial disease with a particularly complicated pathophysiology. Discoveries to date may be considered the tip of the iceberg, and the increasing number of studies in this field indicate that there are many points to be elucidated in AD pathophysiology. In this review, we aimed to illustrate the current understanding of the underlying pathogenic mechanisms in AD, to evaluate available treatment options with a focus on recently discovered therapeutic agents, and to determine the personal, familial, and economic burdens of the disease, which are frequently neglected issues in AD. Currently available therapies only provide transient solutions and cannot fully cure the disease. However, advances in the understanding of the pathogenic mechanisms of the disease have led to the production of new treatment options, while ongoing drug trials also have had promising results.  Atopic dermatitis (AD), also known as atopic eczema, is the most common chronic inflammatory skin disorder of childhood and is characterized by pruritus, dryness of skin, and scratching [1]. The disease presents with eczematous, itchy lesions that show distinct distribution in different pediatric age groups, and episodes of clinical exacerbation called flares or flare-ups [1]. AD affects 5%–20% of children [1], and the prevalence differs among geographical regions [2]. The disease usually occurs in early childhood; about 85% of cases are observed during the first 5 years of life [3], and the disease alleviates substantially by the age of 7 [4]. In a small percentage of patients, the disease may begin in adulthood [5]. In addition, AD may be the first manifestation of “atopic march”, which is characterized by development of asthma and allergic rhinitis at a later age [6]. AD is a multifactorial heterogeneous disorder that results from the interaction of genetic and epigenetic factors, environmental agents, immunological defects, and epithelial barrier dysfunction [7]. As understanding underlying disease mechanisms is critical for the development of effective treatment strategies, studies focusing on the pathogenesis of AD have increased in recent years [8]. This review focuses on the pathogenesis of the disease and recently discovered novel therapies, in addition to classical treatment methods. Although AD lesions were well described in antique texts [9], the disease was first described and named by Wise and Sulzberger in 1933 [10]. There are many studies on the frequency and pathogenesis of this common disease; however, the International Study of Asthma and Allergies in Childhood (ISAAC) can be considered the most comprehensive and largest study on AD. According to ISAAC, prevalence of AD varies considerably in different regions [11], and the frequency of childhood AD was reported to range from 0.2% to 24.6% worldwide [1]. Phase 3 of the ISAAC study [12] showed that AD occurs at a higher, relatively stable ratio in developed countries and urban areas, whereas the prevalence is steadily rising in developing countries. Likewise, Civelek et al. reported that the frequency of AD is lower in developing rather than developed Mediterranean countries. In the same study, risk factors and progression of AD also showed differences in developing Mediterranean countries [13]. Compared to ISAAC phase 1, the ISAAC phase 3 study showed stable or decreased AD frequency in developed countries [2], while low-income countries were surprisingly shown to have an increase in frequency. This increase has been attributed to changes in lifestyle [14], as the short time period between phases 1 and 3 would have been insufficient for the emergence of substantial genetic changes [15]. Therefore, it is believed that a modern lifestyle may have caused changes in daily practices, eating habits, and the perception of hygiene among people living in developing countries. The use of household cleaning products containing strong chemicals and increased frequency of soap, shampoo, and shower gel usage are among the factors believed to have contributed to the increased prevalence of AD in these countries [2]. AD is diagnosed by clinical examination mainly based on morphological features and distribution of lesions; however, no laboratory or pathological findings specific to the disease have been discovered to date. Therefore, strict application of standard diagnosis criteria is important and even necessary to prevent misdiagnoses of atopic dermatitis in cases of other kinds of dermatitis. Until 1960, there were no criteria for the diagnosis of AD. In 1961 Georg Rajka established the first criteria, followed by Jon Hanifin, who published 13 features for the diagnosis of AD in 1977 [16]. After these initial efforts, Hanifin and Rajka proposed modified diagnostic criteria for AD in 1980 [17]. This modified set of criteria has been shown to have high sensitivity, up to 93% to 96% according to several studies , and this set of criteria has been used as a basis for later versions of AD diagnostic criteria [20]. According to the original Hanifin and Rajka criteria, a patient was diagnosed with AD when at least 3 of 4 major and at least 3 of 23 minor features were met [16]. However, the minor criteria were not practical to use in clinical practice [21]. The Hanifin and Rajka criteria were used by Kang and Tian , Schultz Larsen , Japanese Dermatological Association (JDA) (in 1994), Danish Allergy Research Center (DARC) (in 2005), Korean Dermatological Association (KDA) (in 2006), and American Academia of Dermatology in 2003 [22]. Unfortunately, the Hanifin and Rajka criteria were deemed unsatisfactory for epidemiological surveys [20]. Therefore, the United Kingdom Working Party diagnostic criteria  [23] and the ISAAC criteria (in 1995) [24] were used for epidemiological studies. Although the UK criteria were appropriate for clinical practice, they were not applicable for small children, especially infants. The ISAAC criteria have been utilized as the gold standard diagnostic criteria in global surveys for all childhood age groups since the 1990s . SCORing Atopic Dermatitis” [26]. This scoring system comprises both objective  and subjective symptoms (C. Pruritus and loss of sleep) [26], and total score is calculated with the formula A / 5 + 7B / 2 + C [26]. The final score is extremely variable and is highly dependent on the physician’s examination. Consequently, an objective SCORAD which omits the subjective C criteria was put forth in 1997 [27]. Other than these scoring systems, a simplified score named Three-Item Severity (TIS) which evaluates erythema, edema, and excoriation [28], SASSAD  that encompasses 6 signs , each on a scale of 0 (absent), 1 (mild), 2 (moderate), or 3 (severe), at each of 6 sites  [29]; finally, the EASI (Eczema Area and Severity Index) has been developed [30]. Nevertheless, TIS is less sensitive than SCORAD [31]; SASSAD does not have the parameters of pruritus and loss of sleep and has disadvantages in calculation of an objective score; EASI scoring is a time-consuming and rather complex system [32]. Nowadays, objective SCORAD has been widely used and is considered the most accurate scoring system for AD [32]. Assessment of disease severity is particularly important in clinical practice when determining appropriate treatment for the patient, identifying the time of treatment cessation, and for epidemiological surveys. For this purpose, a comprehensive scoring system was published in 1993 based on a consensus by the European Task Force Group on Atopic Dermatitis (ETFAD), with participation of over 20 experts [26]. This scoring system was named SCORAD, an acronym for “AD may be confused with many other skin diseases, and differential diagnosis should be made with these disorders. Several diseases, including diseases causing immunodeficiency, may present with eczematous skin rash and could mimic AD. Seborrheic dermatitis, especially in infancy, dermatitis herpetiformis, irritant contact dermatitis, nummular dermatitis, and psoriasis are common inflammatory skin disorders that are often mistaken for AD . Other skin conditions which may be ascribed to AD include allergic contact dermatitis, scabies, molluscum contagiosum, tinea corporis and capitis, mycosis fungoides, Langerhans cell histiocytosis, and pityriasis lichenoides chronica [35]. Additionally, a long list of immunodeficiency disorders, such as hyperimmunoglobulin E syndrome, Wiskott–Aldrich syndrome, Netherton syndrome, immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX), severe combined immunodeficiency (SCID), and Omenn syndrome may be diagnosed in children who present with symptoms suggestive of AD . AD is a multifactorial and heterogeneous skin disorder resulting from genetic predisposition, environmental factors, skin barrier defects, and immunological abnormalities [8] . Elucidating the underlying pathogenesis and understanding the mechanism that causes itching are essential for determining the most effective treatment.There are many molecules in the structure of the epidermis that are critical in preventing water loss and providing protection against environmental factors [37]. Mutations in the genes encoding for proteins responsible for normal barrier function are among the main reasons for AD development [37]. Defined mutations are located in filaggrin (FLG), desmoglein-1 (DSG1), corneodesmosin (CDSN), serine protease inhibitor Kazal-type 5 (SPINK5), Matt [38], and lymphoepithelial Kazal-type–related inhibitor (LEKTI) [37]. The SPINK5 gene encodes LEKTI which is involved in the pathogenesis of AD-like disorders, such as Netherton syndrome, a syndrome characterized by severe dermatitis, allergic diseases, and high serum IgE levels [39]. Skin barrier integrity is maintained by fillagrin via keratinocyte formation. Degraded end products of fillagrin maintain water balance, low acid pH, and barrier function of the skin [40]. Decreased or defective fillagrin molecules lead to high pH and enhance the function of serine protease kallikrein (KLK) [41]. KLK binds to its receptor and thus induces the production of thymic stromal lymphopoietin (TSLP), which promotes inflammation [42]. Inflammation causes further degradation of fillagrin end products and decreases fillagrin production [43]. As a result of decreased fillagrin and its end products, allergen penetration is enhanced [44], as well as bacterial colonization [45]. Changes in the lipid composition of the stratum corneum [46] and looseness of tight junctions are other factors that substantially contribute to AD pathology [47]. Tight junction defects and the claudin-1, claudin-23, and ZO-1 molecules have been associated with skin problems in AD . Additionally, polymorphisms in the CLDN-1 gene which encodes claudin-1 have also been linked to AD pathology [47].Sensitization with food allergens can occur not only via the gastrointestinal tract but also through defective and inflamed skin even before the ingestion of the allergen foods [49]. By this route, 35% of the children with AD sensitize to foods by the age of 2 years [50]. The association between inhalant allergens and AD has been demonstrated in about 65% of patients at the age of 6 years old [51].Staphylococcus aureus occurs at up to 90% of lesion sites [53], while it is found at lower percentages in healthy skin. IL-4, an important cytokine of the Th2 pathway, is crucial for adhesion of Staphylococcus aureus onto skin [54]. Staphylococcal α-toxin induced keratinocyte death was shown to be increased by IL-4 and IL-13 through activation of signaling transducer and activator of transcription 6 (STAT6) [55]. It has also been shown that toxins of the bacteria increase inflammation in the skin, thereby exacerbating symptoms [56]. Fillagrin has a neutralizing effect on Staphylococcus aureus α-toxin in healthy skin [57], and fragmented filaggrin products inhibit the growth of S. aureus [58]. Additionally, enterotoxin B of the bacteria induces the production of IL-31, one of the major cytokines responsible in pruritus, amplifying the severity of symptoms [59].Many bacteria and fungi colonize on the skin; however, in AD, the bacterial composition of skin completely changes due to an increased adhesion of various bacteria to the skin of those with AD [52]. Staphylococcus aureus in the epidermal layer [60]. Although sphingosine prevents the colonization of the bacteria in normal healthy skin, its levels are diminished in lesion sites [61]. Furthermore, the high pH, water loss, and dryness that are characteristic of the disease are known to ease the adhesion of Staphylococcus aureus to skin [61]. Lastly, the increase in fibronectin deposition in the stratum corneum of patients with AD serves as another mechanism that enables adhesion of Immunological background is rather complicated in AD; however, cytokine profile has been shown to have an important effect on the life cycle of keratinocytes [62]. Keratinocytes express pattern recognition receptors including Toll-like receptors (TLRs), NOD-like receptors (NLRs), C-type lectin receptors (CTLRs), and RIG-I-like receptors (RLRs) [63]. These receptors recognize pathogenic antigens, subsequently causing an increase of proinflammatory cytokines [63]. Cells receive signals from their environment and transduce these inputs through intracellular signal molecules. When a relevant signal arrives to the cell surface, the cell responds by producing intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which increase the phosphorylation of protein kinase A (PKA). This pathway leads to a decline in the levels of proinflammatory molecules such as IL-2, IL-4, IL-6, IL-10, and TNF-α, thereby suppressing innate immunity [64]. Phosphodiesterase type 4 (PDE4) inhibitors prevent cAMP degradation [65] and can therefore be used to reduce the levels of inflammatory cytokines [66]. On the other hand, keratinocytes contribute to innate immunity by producing antimicrobial peptides (AMPs)  which damage microbial cellular membranes [67]. Abnormalities in the production of these molecules may contribute to worsening of AD.In patients with AD, cytokine profile shifts from T helper 1 (Th1) to T helper 2 cells (Th2). In the acute phase of AD, there is an excess of Th2 cytokines including IL-4, IL-5, IL-9, IL-13, IL-31 [68], as well as IL-22, which is also produced by Th22 lymphocytes [69], a type of lymphocyte which is crucial in epidermal immunity. These cytokines upregulate inflammatory status and cause overproduction of immunoglobulin E; IL-22 is known to inhibit keratinocyte differentiation [70]. IL-22 is also responsible for skin barrier integrity, and its level has been shown to be correlated with AD severity [71]. TSLP, an IL-7–like cytokine, is expressed by epithelial cells and binds to antigen-presenting cells (APCs) including dendritic cells, macrophages, monocytes, and T and B lymphocytes in the epidermal layer of the skin [72]. Moreover, TSLP increases IL-4 levels, which then further increases TSLP production [73]. Keratinocytes in lesion sites express TSLP, IL-25, and IL-33, which increase Th2 cytokines by activating dendritic cells (DCs) in the skin [74]. These stimulated DCs then migrate to regional lymph nodes and are instrumental in the conversion of naïve T cells to Th2 lymphocytes [75]. Furthermore, TSLP, IL-25, and IL-33 promote the production of innate lymphocyte group 2 cells (ILC2), increase their response, and induce the production of cytokines, including IL-5 and IL-13 [74]. On the other hand, ILC2s elicit IgE isotype class switching in cells [76]. IL-4 and IL-13 are also responsible in the chronic phase through their contribution to tissue remodeling [77]. IL-5 is primarily produced by Th2 and mast cells and participates in eosinophil growth, chemotaxis, and survival [78]. Recent studies have shown that IL-31 has a substantial effect on the development of pruritus [79]. IL-31 is mainly produced by Th2 lymphocytes and to a lesser extent by dendritic cells upon activation [80]. Intriguingly, eosinophils in patients with AD express IL-31 more intensely compared to healthy individuals [81]. IL-31 binds to its receptor IL-31 receptor RA (IL31RA) on sensory nerves and induces an itching sensation, as well as facilitating the elongation and branching of sensory nerves [79]. Th17 lymphocyte is another cell type that contributes to the acute phase [62]. This cell produces IL-17 and IL-22, which increase proinflammatory molecules, S100 proteins, and other types of AMPs in keratinocytes [82]. S100 proteins act as AMP and damage-associated molecular pattern molecules, which initiate the inflammatory cascade [83]. The level of S100 protein increases in acute and chronic AD; thus, it is thought to be an important parameter in preserving enhanced inflammatory status [83]. Furthermore, increases in IL-17 lead to eosinophil- and neutrophil-mediated inflammation [84], while reduced IL-17 levels are correlated with reduced AMP, which has been reported to be associated with increased susceptibility to skin infections [85]. In the chronic phase, interferon gamma (IFNɣ), a Th1 cytokine, is believed to be more prevalent, along with Th2 and Th22 cell responses [86].Cytokines and other chemical molecules that cause itching (pruritogens) are released from eczematous skin areas where they activate the relevant sensory nerves and cause pruritus; thus, the patient feels the need to scratch these areas [87]. Hyperesthesia in AD is due to aberrantly elongated sensory nerves in the upper layer of the skin which are increasingly exposed to environmental factors such as dryness, irritation, and chemicals [88]. Histamine, substance P, TSLP, and IL-31 act on these elongated nerve fibers, contributing to the itching sensation . Heat is an important aggravating factor for itching in AD [91]. Artemin  is a substance which has been shown to increase in AD lesions and is reported to cause itching, especially in warm temperatures [92].Pruritus has detrimental effects on sleep and may reduce the length and quality of sleep, which leads to a decrease in melatonin levels. Decreased melatonin levels have been suggested to promote further itching [93]. Additionally, decreased cortisol level and increased inflammatory status due to elevated IL-2 levels are other factors that contribute to itching at night [93]. Sweating is another factor that exacerbates itching in AD, due to the normal effects of excess sweat on the skin and also the abnormal composition of sweat in patients with AD [94]. Conversely, keratinocyte debris may obstruct sweat glands and prevent normal sweating; this also leads to raised body temperature and could increase pruritus [95].Fillagrin mutation is the most important predisposing genetic change in AD. However, it is found in only 10%–50% in patients with AD, and in 9% of the healthy population [69]. Therefore, AD cannot be explained only by Fillagrin mutation. Genome‑wide association studies (GWAS) have been initiated to elucidate the pathogenesis of AD. Many novel genes have been identified in patients with AD, especially after the first GWAS published by Esparza-Gordillo et al. in 2009 [96]. Genes responsible for the pathogenesis of AD are given in Table 1. TRAF6, RAG1, RAG2, SOCS1, NGFR, 4q27 IL2/IL21, 11p13 PRR5L, 16p13.13 CLEC16A/DEXI, and 17q21.32 ZNF652 are the novel genes that have been reported in GWAS studies on AD [97]. Additionally, several genetic polymorphisms have been found to be related to AD [98]. These include polymorphism of vitamin D pathway molecules [99] and polymorphisms of cytokines IL-4, IL-5, IL-9, IL-13, and IL-31, as well as their receptors [100]. Aside from genetic mutations, epigenetic mechanisms have been implicated in AD pathogenesis [101]. Epigenetic mechanisms regulate expression of genes without altering the DNA sequence [102]. These mechanisms are mainly DNA methylation, microRNA (miRNA), and histone tail modification [102]. It has been shown that epigenetics affect gene expression through environmental factors such as pollutants, tobacco smoke, aging, and diet [103]. DNA methylation differences between normal epidermis and AD epidermis have been shown in a study focusing on epigenetic factors . miRNAs have fundamental roles in cell proliferation, differentiation, apoptosis, signal transduction, and organ development [106]. miRNAs are upregulated in the skin of patients with AD [107]. Children who were exposed to smoking during pregnancy have high miRNA-223 and low T regulatory (Treg) lymphocytes [108]. These children were found to have a higher tendency to develop AD during the first 3 years compared to children without exposure to smoking during pregnancy [108].Treatment of AD is aimed at suppressing inflammation, eliminating identified triggers, reducing pruritus, and combating the development of xerosis, a major feature of AD  . For this purpose, conventional therapy is the first-line treatment approach. However, new therapeutic strategies and agents are continuously being developed with the help of advances in the understanding of AD pathophysiology. Basically, topical moisturizers are the mainstay of AD treatment, as they prevent dehydration, soften the skin, and increase water retention. Above all, they serve to minimize the need for pharmacological drugs for AD . Moisturizers are the first line of therapy in mild AD and constitute an important and primary step in moderate and severe forms of AD [112]. Moisturizing the skin with different types of emollients, occlusive agents, and humectants decrease inflammation, pruritus, and xerosis, and also reduce the requirement for topical steroids and antibiotics [110]. There are many types of products in the form of ointments, lotions, creams, emollients, oils, and gels. Even though there are some differences with regards to their contents, the most important aspect of these moisturizers are to ensure that they are perfume- and fragrance-free and do not contain harsh chemicals [110]. In addition to moisturizers, daily bathing with warm water is an extremely important step for hydration of skin, as well as removing allergens, irritants, crusts, and bacteria [113]. Applying moisturizing agents immediately after bathing has been a fundamental approach for treatment in patients with AD [113]. Otherwise, bathing without moisturizing has a negative impact on AD. Staphylococcus aureus on the skin, preventing secondary skin infections [116]. In this therapy, the child is soaked in water containing a certain amount of bleach (1/4 cup of bleach into 35–40 L of water) for 5 to 10 min . However, bleach baths were shown to be ineffective in the treatment of AD in some studies [119]. Other than bleach or sodium hypochlorite, there are antiseptic agents including triclosan, potassium permanganate, and chlorhexidine gluconate used for both the management of infected skin and the prophylactic treatment of AD. Bathing with antiseptic agents has been shown to be useful in diminishing the bacterial load on the skin of patients with AD. Furthermore, no significant effect on reducing bacterial load on inflamed skin in AD has been identified in a few studies [120]. Therefore, combination therapy of antibiotics along with antiseptic agents has been shown to be particularly effective in the treatment of clinically infected skin in AD [115]. Wet-wrap therapy in combination with topical agents is an effective method used in the treatment of AD flare-ups [114]. Generally, topical agents are applied on the skin followed by a layer of tubular wet bandage and an outer layer of dry bandage [110]. This technique intensifies the effect of moisturizers by providing a smooth skin texture and preventing water loss [110]. A bleach bath was thought to be effective in the treatment of AD, because some physicians reported that eczematous lesions benefited from the chlorinated water of swimming pools [110]. Additionally, Huang et al. showed the efficacy of bleach bath combined with intranasal mupirocin treatment twice a week compared to placebo [115]. Bleach baths have been considered to reduce the colonization of Staphylococcus aureus, Streptococcus pyogenes, Herpes simplex, Varicella zoster viruses, papillomavirus, and molluscum contagiosum . S. aureus mostly colonizes the skin and the nasal mucosa, and is frequently associated with worsening of the eczematous lesions [122]. Mupirocin and fusidic acid are effective antibiotics for the prevention of S. aureus colonization when used twice a day for a week . Furthermore, application of mupirocin twice a day in both nasal orifices for 5 days in a month for 3 to 18 months has been shown effective in eradicating S. aureus [124]. Systemic antibiotics are given in the presence of clear evidence of bacterial infection, not to prevent colonization . The first choice of antibiotic treatment is beta-lactam antibiotics for 7–14 days [127].AD exacerbations and acute flares are mostly associated with bacterial and viral infections, including Herpes simplex and Varicella zoster viruses even after immunization . The clinical picture caused by Herpes simplex virus is eczema herpeticum, which requires administration of systemic acyclovir for treatment [128]. Prompt initiation of acyclovir treatment significantly reduces the mortality of eczema herpeticum [128]. About 3% of AD cases are prone to severe viral infections, especially Antiinflammatory therapy with topical calcineurin inhibitors (TCI), including tacrolimus and pimecrolimus, as well as topical corticosteroids (TCS), is reported to be the most effective approach in the treatment of acute flares [118]. Excessive inflammatory response in the skin is minimized by antiinflammatory treatment agents during flares [118]. Both TCS and TCI suppress T lymphocytes and inhibit proinflammatory cytokine release from immune cells . Therefore, these agents are useful in controlling inflammation, pruritus, and skin eruptions [118]. TCSs are widely used; they are the first-line treatment for acute flares in AD . They are indicated for eczematous lesions unresponsive to daily skin care and proper usage of emollients, creams, and ointments [116]. TCSs exhibit their effect by inhibition of T lymphocytes [110], thus reducing inflammation on the skin; they are also known to abate pruritus. In addition, topical corticosteroids are not only used for acute attacks but also for prevention of relapses [110]. TCSs are divided into 7 categories according to their potency [110]. Corticosteroids with higher potency should be applied for acute flares, whereas low potency corticosteroids are highly recommended for chronic long term usage . The optimal suggested period of application is twice daily for 2 weeks, and only on affected surfaces of the skin for protection from local adverse effects of corticosteroids [116]. Many local side effects have been reported, including hypertrichosis, striae, telangiectasia, and skin atrophy for long term skin application, as well as glaucoma and cataract after use on the periorbital region [116]. Therefore, TCIs are recommended for thin skin surfaces such as eyelids and periorbital areas [132]. Studies have demonstrated that skin atrophy is extremely rare in children [133]. Systemic adverse effects, including the suppression of the hypothalamic–pituitary–adrenal (HPA) axis, have been reported after use of high potency topical corticosteroids in children [134]. Use of low to moderate potency topical corticosteroids for 3 to 4 weeks has been demonstrated to rarely affect the HPA axis [134]. Additionally, the possibility of growth retardation in children has been researched in many studies; however, results have mostly revealed only insignificant delays in growth [135]. Streptomyces genus that inhibits calcineurin-dependent T-cell activation and proinflammatory cytokines. They exert their effects by inhibiting mast cells, dendritic cells, and T lymphocytes [137]. In studies, TCIs have been shown to be more effective than mild TCSs and as effective as moderate TCSs . Therefore, TCIs are suggested for severe, TCS-unresponsive AD lesions, and also for sensitive areas such as eyelids and the face, and when concerned about steroid-related adverse effects . However, erythema, burning, and itching sensations have been frequently reported with TCIs [141]. Additionally, many carcinogenic effects were reported during trials on animals [136]. Systemic absorption of TCI has been shown in patients with AD through the disrupted skin barrier and other skin areas [136]; therefore, side effects may be seen more frequently in pediatric age groups [136]. Pimecrolimus 1% and tacrolimus 0.03% are approved for children who are between 2 and 15 years old; 0.1% ointment of tacrolimus is not allowed for pediatric patients . Both pimecrolimus and tacrolimus are contraindicated in acute viral skin infections, suspicion of skin malignancy, or immunodeficiency [136]. Although the exact relationship between malignancy and calcineurin inhibitors cannot be demonstrated, skin tumors and lymphoma have been reported [142]. Therefore, long-term use of these agents in all age groups should be avoided. In clinical trials, tacrolimus and pimecrolimus have been shown to be effective on moderate to severe and mild to moderate AD, respectively [143]. TCI should be used twice a day over 2 to 3 weeks, then should be reduced to once a day for 2 weeks [144]. At the beginning of the treatment, burning and itching are significant problems for patients and may reduce treatment compliance. Concomitant steroid therapy may overcome this issue [144]. Although safe for use and very effective, there is a risk for rebound after the cessation of therapy with TCIs; in the event of a rebound or flare-up, TCIs may be restarted twice a day again [144]. Tacrolimus and pimecrolimus were approved for AD treatment in 2000 and 2001, respectively [136]; they are safe for use in adults and children over 2 years old [136]. TCIs are a product of the Systemic therapy for AD is considered when the skin lesions do not ameliorate after usage of topical treatments [145]SCS therapy has many side effects, and therefore it is applied for only a week, especially in adults [146]. It has little efficacy on improvement of lesions, and its adverse effects usually outweigh its benefits [147]. Cushing syndrome, hyperglycemia, osteoporosis, and peptic ulcer are primary side effects of systemic steroid therapy [148]. Due to these adverse effects, they are not recommended for routine treatment and recommended for use only for a short time until symptoms are alleviated in severe cases . Cyclosporine exhibits its effect by inhibiting calcineurin receptors which induce the proliferation of IL-2 cytokine. This cytokine is vital for T helpers, T regulatory lymphocytes, Natural Killer cells, and monocytes. By blocking the production of IL-2, the activity and proliferation of T lymphocytes are hampered [151]. Patients with severe, topical therapy-resistant lesions have been shown to benefit from oral cyclosporine treatment [152]. Recommended dosage is 2.5 mg/kg/day in 2 divided doses, not exceeding a maximum dose of 5.0 mg/kg/day [153]. Serum levels of cyclosporine should be monitored routinely and managed according to clinical symptoms and serum levels of the drug. If there is clinical advancement, duration of the therapy may be extended to 12 months [151]. In case of the clinical improvement, cyclosporine therapy is reduced and terminated in 2–3 months [151]. Most frequently observed side effects are headache, hypertension, renal dysfunction, hypertrichosis, hepatotoxicity, and gingival hyperplasia in adults [152]. However, these are extremely rare in children [154]. Cyclosporine has been well tolerated by children and is particularly effective in this age group [155]. Azathioprine acts as an immunosuppressant on proliferating cells. It is a purine analogue inhibiting DNA/RNA synthesis and prevents proliferation of both T and B lymphocytes [156]. Azathioprine has been used with substantial benefits for AD treatment in adults and children [156]. Azathioprine is generally used for a short period in patients who are refractory to cyclosporine [155]. Its effect arises 2–4 weeks after initiation of the therapy, and it is thus considered to be a secondary preference for treatment of AD [149]. The most substantial adverse effects of azathioprine are hepatotoxicity, increased risk of malignancy, and myelosuppression [157]. The recommended dose is 2–4 mg/kg/day, and whole blood count should be monitored for cytopenia regularly [158]. Methotrexate is a folic acid antagonist which inhibits cell division and DNA/RNA synthesis and suppresses the immune system, which are similar to the effects of azathioprine [149]. The efficacy of the drug has been shown in children and it is well tolerated in the pediatric age group [159]. It has been suggested to be effective in improving skin lesions with few side effects and is considered a safe option for children [159]. However, it may cause bone marrow suppression, increase transaminase levels, and result in cytopenia. Furthermore, although extremely rare, pulmonary fibrosis has been shown to be a complication of the drug [149]. Beginning of action may be prolonged up to 3 months, as is sometimes the case with azathioprine [149]. There is little data on the application of methotrexate in children. In reports, a low dose  has been demonstrated to be effective in alleviating lesions . MMF is an immunosuppressive drug derived from mycophenolic acid. It regulates purine synthesis and inhibits T and B lymphocytes [161]. MMF was used in adult patients for the first time in 1999 [162], and was applied to children over 2 years old with refractory AD in 2007 [163]. Pediatric results were promising and no significant undesirable effects were reported. The most frequent adverse effects seen in children were nausea, vomiting, and diarrhea [163]. In addition to these side effects, hepatotoxicity, myelosuppression, and susceptibility to infections may be seen during treatment [163]. Daily recommended dose is 600 mg/m2/day in 2 divided doses in children [164]. Extracorporeal photopheresis and intravenous immunoglobulin therapies have been applied to patients with AD . There are limited data available about these methods, and results of studies which utilized these methods showed that they had little efficacy on AD. Itching is the most intolerable symptom of AD and it has devastating effects on social life and sleep. First-generation antihistamines may be beneficial due to their sedation properties [167], but second-generation antihistamines have less sedative effect [167]. Therefore, only first-generation drugs may be used for severe cases of pruritus and overcoming sleep disturbance.Atopy is defined in up to 70% in patients with AD and exposure to aeroallergens have been shown to cause acute flares . Removal of allergens from patients with AD with underlying etiology of allergy is an important step in treatment, and avoidance of aeroallergens has been suggested in the literature to decrease flare-up frequency and the severity of AD [130]. There are many studies on immunotherapy and AD; however, outcomes of the reports are conflicting, and contrary to studies on allergic rhinitis, efficacy is questionable in AD [169]. Atopic march, a severe problem in children with AD, should be prevented; however, the preventive role of allergen immunotherapy has not been demonstrated yet [170]. Nevertheless, patients with severe AD and accompanying allergic rhinitis and/or asthma may be considered for allergen immunotherapy [171]. IFNɣ inhibits T helper 2 lymphocytes and reduces IgE production [172]. In clinical trials, patients with moderate to severe AD have been shown to benefit from IFNɣ therapy . The dose used in the pediatric age group was 50 µg/m2 for 22 months; clinical improvement was observed . However, a specific recommended dose for IFNɣ for AD has not been determined and the Food and Drug Administration (FDA) has not approved the use of IFNɣ for AD [172]. Omalizumab is a recombinant humanized mAb (rhmAb) that inhibits binding of free, unbound IgE to the high-affinity IgE receptor (FcεRI) on mast cells and basophils [175]. Its first use for severe asthma was assessed in the Global Initiative for Asthma Guidelines study in 2003 [176]. Furthermore, omalizumab was initially used on children who were between 6 and 12 years old in 2009 for severe asthma [177]. Later, Barrios et al. reported the efficacy of omalizumab in their pediatric group of patients with AD in 2013 [178]. Doses were not standardized and varied from 150 mg/dose to 375 mg/dose every 2 to 4 weeks in various studies . The drug was well tolerated in children without any particular side effects, but clinical trials on the use of omalizumab for AD are very few, and they report debatable efficacy . Rituximab is an anti-CD20 monoclonal antibody. It was shown to be beneficial on severe AD lesions in 2008 [183]. However, that was followed by a report on the clinical experience of 2 patients with severe AD who had only minimal transient clinical benefits from rituximab therapy [184]. There is no standardized recommended dose for the usage in patients with AD and further studies with higher numbers of patients are required to evaluate its efficacy in AD. Dupilumab is a human monoclonal antibody against a subunit of the IL-4/13 receptor [185]. In a 2014 clinical trial, significant improvements in patients were observed at the end of the 3-month treatment period [186]. The FDA approved dupilumab for use in adults with moderate-to-severe AD in March 2017 [172]. Clinical trials on children are ongoing [172]; preliminary results indicate that results with dupilumab could be effective in the treatment of AD. The dose of the drug was 2 mg/kg in patients aged 6–11 years and 4 mg/kg in patients aged 12–17 years, or 200–300 mg at intervals of 2–4 weeks in clinical trials [187]. IL-5 is critical for eosinophil growth and differentiation [188]. Eosinophils are essential contributors to AD pathophysiology and have been shown to be elevated in the serum of patients with AD [188]. Therefore, it was suggested that mepolizumab, a human IL-5 mAb, may have an important effect on the treatment of AD [172]. However, initial studies in 2005 did not yield positive results and no clinical improvement was seen . . Omalizumab, rituximab, interferon gamma, and mepolizumab have already been tried, while new agents that function by affecting the various stages of pathogenesis are under ongoing investigation. The primary agents among these new potential treatments are nemolizumab, ustekinumab, tralokinumab, lebrikizumab, antithymic stromal lymphopoietin receptor (TSLPR), and phosphodiesterase (PDE) inhibitors.Biological agents are new therapeutic drugs targeting the molecules responsible in the pathogenesis of diseaseNemolimumab (CIM331), a humanized anti-IL-31 receptor A mAb, binds to IL-31 receptor A, therefore inhibiting IL-31 signaling [190]. This promising agent is thought to be especially effective for pruritus, and therefore may alleviate problems such as sleep disturbance and psychological concerns in both patients and parents. In clinical trials, pruritus has been reported to be reduced 2-fold compared to placebo . Ustekinumab is a humanized mAb that binds to the p40 subunit of IL-12/IL-23 cytokines. These molecules have a pivotal role in the development of Th1 and Th17 cells [192]. Trials have demonstrated that ustekinumab reduces the infiltration of T lymphocytes, dendritic cells, and mast cells in the skin [172]. However, no meaningful clinical efficacy has been reported . Tralokinumab is a recombinant IgG4 neutralizing mAb that binds to IL-13 and interferes with the interaction between IL-13 and its receptor [195]. In trials, the drug has not shown any particular difference from placebo [196]. However, various studies are still ongoing [172]. . Lebrikizumab is another IL-13 binding mAb, but its efficacy and safety profile have been thought to be different from other drugs that bind IL-13. Initial results on the drug’s efficacy have shown superior results compared to placebo with repeated doses [197] and no significant adverse effect has been reported [197]TSLP is an IL-7–like cytokine that is primarily expressed in epithelial cells, keratinocytes, and ocular cells, and has a role in allergic inflammatory disorders [198–200]. Secretion of the cytokine can be stimulated by allergens, viruses, trauma, and smoke [201]. Myeloid dendritic cells (mDCs) are activated by TSLP, and begin a cascade of events that lead to the differentiation of naive T cells into Th2 lymphocytes [202]. Blocking of TSLPR was first explored in asthma treatment [203]; other trials comprised of patients with AD have also begun [196]. TSLP’s role in the exacerbation of pruritus via sensory neurons has been demonstrated [90]. Studies on AD have revealed significant improvement in the severity scores of the patients [196].In the 1980s, enhanced PDE activity was determined in blood cells as well as cord blood cells of patients with AD [204]. Since then, the effects of PDE inhibitors in patients with AD have been demonstrated [205]. Topical forms of PDE inhibitors are crisaborole, OPA-15406, RVT-501, and roflumilast, while systemic  forms of the drug are apremilast and roflumilast [204]. In December 2016, FDA approved crisaborole for the treatment of mild-to-moderate atopic dermatitis in patients aged ≥2 years [204]. AD mostly begins in childhood and may persist throughout adulthood [206]. Therefore, it may have both economic and psychosocial impacts on patients and their families [207]. Evaluation of quality of life (QoL), social, academic, and occupational impacts, and financial costs are important in capturing the whole picture when assessing the burden of AD. Patients with AD were shown to have lower overall QoL [208], especially in the form of mental health and social function [208]. A study by Solomon et al. showed that children with AD had severe sleep difficulties and were more dependent on their caregivers than patients with other chronic disorders [209]. Chamlin et al. also showed that these children were avoided by other people, causing social isolation [210]. Many studies have shown that sleep deprivation, isolation from social activities, and feelings of embarrassment due to itching and appearance of the eczematous lesions were the major problems faced by children with AD . In a study performed on children and adults with AD, it was reported that 87.1% of patients had difficulties falling asleep, and 73.5% reported that severe itching was the cause for waking up [213].AD also affects parents. The parents of children with AD also reported sleep disturbance [210] and stress [214], and were found to spend a significant amount of time on daily skin care [210] and hygiene [215]. Furthermore, the time spent by parents with the siblings of children with AD was also reported to be decreased [216], suggesting that AD has detrimental effects on the entire family. Lifestyle changes including modified dietary habits and usage of cleaning products and moisturizers for the skin in patients with AD were also shown to cause a substantial burden on the family budget [217]. Additionally, regular physician visits may cause loss of worktime for parents, while spending excessive time and effort on the care of a child with AD may also cause social isolation for the parents [217]. Assessing the exact financial burden of AD on the family budget and the health system is very difficult due to the indirect costs as well as direct expenditures. Additionally, the total costs of AD are also dependent on disease severity and may differ significantly from country to country [218]. Indirect costs include loss of labor and decrease in productivity at work [207]. Direct costs consist of pharmacy expenditures, follow-up visits, applications to emergency departments, and spending on special diet programs [207]. A striking result reported by Storan et al. indicated that 86% of the children admitted to their inpatient clinic were diagnosed with AD [219]. This study demonstrates the financial strain caused by AD on the health system. According to studies on economic costs of chronic diseases, AD was in fourth place among skin diseases [220]. In 2004, the annual cost of AD was 4.228 billion dollars in the United States [220], and had increased to approximately 5.297 billion dollars in 2015 [207]. Although indirect costs are not exactly estimated, the direct costs of AD have increased significantly over the years. The complexity of AD pathogenesis, the lack of a definitive treatment, and the fact that available therapies only provide transient well-being are among the factors that fuel the research for a cure for AD. Mainly due to this enthusiasm, experts have explored many novel molecules, mutations, and genes responsible for the disease. New treatment modalities are reported to be promising for the treatment of AD. However, it is also important to consider that current treatments (and the majority of proposed treatments) do not aim to prevent the development of atopic march. For this reason, researchers on this field concentrate not only on antiinflammatory drugs, but also on immunological and biological agents. With our knowledge today, it is evident that AD has a significant immunological background, and if the immunological dysregulation underlying the disease can be elucidated and targeted in treatment, success in AD treatment and prevention will be achieved. Pruritus is one of the most important problems of AD and suggested treatments must address this problem. Pruritus causes exhaustion, despair, and sleep deprivation in patients. Unfortunately, current drugs are insufficient for the control of pruritus. Therefore, development of antipruritic drugs with higher effectiveness should be regarded as high priority. The current therapeutic approach to AD, the advantages of treatments, and unmet needs are summarized in Table 2.The aims of new therapies for AD are not only to procure a definite treatment but also to enhance the adherence of patients and parents to the therapy [221]. Other factors to consider as treatment goals in AD are the prevention of secondary infections, providing support to patients with psychological problems, and increasing the awareness of the population and physicians to a disease with severe consequences on the patient, family, and country.