Compared to control antigen-activated cells, UA enhanced the production of tumor necrosis factor-α at the mRNA and protein levels. However, secretion of this cytokine was inhibited. Further analysis showed that UA enhanced tyrosine phosphorylation of the SYK kinase and several other proteins involved in the early stages of FcεRI signaling, even in the absence of antigen activation, but inhibited or reduced their further phosphorylation at later stages. In addition, we show that UA induced changes in the properties of detergent-resistant plasma membrane microdomains and reduced antibody-mediated clustering of the FcεRI and glycosylphosphatidylinositol-anchored protein Thy-1. Finally, UA inhibited mobility of the FcεRI and cholesterol. These combined data suggest that UA exerts its effects, at least in part, via lipid-centric plasma membrane perturbations, hence affecting the functions of the FcεRI signalosome.Pentacyclic triterpenoids, including ursolic acid (UA), are bioactive compounds with multiple biological activities involving anti-inflammatory effects. However, the mode of their action on mast cells, key players in the early stages of allergic inflammation, and underlying molecular mechanisms remain enigmatic. To better understand the effect of UA on mast cell signaling, here we examined the consequences of short-term treatment of mouse bone marrow-derived mast cells with UA. Using IgE-sensitized and antigen- or thapsigargin-activated cells, we found that 15 min exposure to UA inhibited high affinity IgE receptor (FcεRI)–mediated degranulation, calcium response, and extracellular calcium uptake. We also found that UA inhibited migration of mouse bone marrow-derived mast cells toward antigen but not toward prostaglandin E FcεRI is a tetramer composed of the α subunit, which binds IgE, two γ subunits, which generate the downstream signal through the process initiated by tyrosine phosphorylation, and one β subunit, which amplifies the activation signals. FcεRIs, various enzymes, ion channels, and transmembrane adapter proteins involved in the FcεRI signaling form a functional complex embedded within the phospholipid bilayer of the plasma membrane, called the FcεRI signalosome. The FcεRI signalosome is a highly dynamic complex that interacts with and is regulated by various plasma membrane and cytoplasmic components and plays a crucial role in propagating the signal from FcεRI into the cytoplasm and the nucleus. This leads to degranulation of preformed secretory granules and ediators , 3, 4, 5A), a pentacyclic triterpene that is mainly found but not limited to peels of fruits and some herbs  and rat basophilic leukemia (RBL) cells and examined the sensitivity of FcεRI-initiated signaling events in the presence or absence of UA. We focused on tyrosine phosphorylation of the FcεRI-β subunit and other kinase and phosphatase substrates, calcium response, degranulation, and production and secretion of tumor necrosis factor (TNF-α). We also examined UA-induced changes in chemotaxis toward various chemoattractants and changes in the physical properties of the plasma membrane. Our data indicate that UA interferes with the earliest signaling events in FcεRI triggering. This effect seems to be, at least in part, the result of UA-induced changes in the properties of the plasma membrane. In this way, our data support the lipid-centric hypothesis of UA action at the earliest stages of mast cell signaling.B and C) showed that a small fraction of BMMCs exhibited significantly increased propidium iodide (PI) fluorescence (Q1) 15 min after exposure to 50 μM UA  or 75 μM UA (7.4% ± 1.1%). A small fraction of BMMCs also showed increased cell surface expression of phosphatidylserine, as reflected by an increased number of annexin V-FITC positive cells (Q3) 15 min after exposure to 50 μM UA (2.6% ± 0.4%) or 75 μM UA (3.1% ± 0.3%). Interestingly, only a small number of cells simultaneously exhibited both markers (Q2) after the treatment with 50 μM UA (2.5% ± 1.0%) or 75 μM UA (3.2% ± 0.8%). These and other data (see later) suggested that short-term pretreatment of the cells with UA at the concentrations used had minimal toxic effects. The observed increased staining with PI and annexin V-FITC could reflect changes in the plasma membrane caused by an interaction of UA with the plasma membrane lipids and not because of a toxic effect of the drug  and interleukin (IL)-3. More than 95% of the cells were mast cells, as deduced from the expression of both FcεRI and KIT receptors (not shown). Exposure of the BMMCs for 15 min to UA at a concentration up to 75 μM had no toxic effect, as determined by the trypan blue dye exclusion test (not shown). Detailed analysis by flow cytometry , B and Cthe drug , 33.A). Almost complete inhibition of degranulation was observed with 75 μM UA. It should be noted that at all concentrations tested, UA did not affect the spontaneous release of β-glucuronidase from nonactivated cells. This observation supports the notion that short-term exposure of BMMC to UA at concentrations up to 75 μM is not toxic to BMMCs. In further experiments, we used UA at a concentration of 50 μM, which inhibited degranulation by approximately 50%.Pretreatment of IgE-sensitized BMMCs for 15 min with UA at 25 to 75 μM significantly inhibited antigen-induced degranulation in a dose-dependent way A. Almost2+ from intracellular stores by inhibiting the endoplasmic reticulum Ca2+ ATPase and, in this way, bypasses all FcεRI-mediated early activation steps. When BMMCs were exposed to 1 μM thapsigargin, strong degranulation was observed (B). Pretreatment with 50 μM UA for 15 min significantly inhibited this degranulation (B).Mast cells could also be activated by thapsigargin, which induces the release of Caobserved B. Pretrenulation B.2+]i with a Fura-2 probe. We found that 50 μM UA significantly inhibited the antigen-induced calcium response in cells kept in the presence of 1.8 mM calcium (C). Cells activated by antigen in the absence of extracellular calcium exhibited only a small increase in [Ca2+]i, which was significantly inhibited by UA. After the addition of 1 mM calcium, a rapid rise in [Ca2+]i was observed in control cells, as described in previous studies (D). The reduced calcium response in UA-treated cells was, at least in part, caused by reduced uptake of extracellular Ca2+, as determined by 45Ca-binding assay. In this assay, antigen-induced activation led to an increase in 45Ca uptake, which was significantly inhibited by 50 μM UA (E). When compared to antigen-activated cells, thapsigargin-activated cells exhibited higher 45Ca uptake, which was inhibited by 50 μM UA to the levels comparable to those in UA-treated and antigen-activated cells (E).To determine whether the calcium response is also affected by 15 min exposure to UA, we monitored concentrations of free cytoplasmic calcium ).Antigen-mediated activation of BMMCs leads to enhanced secretion of cytokine gene products , 2. To dFig. 3B). To determine whether the reduced amount of secreted TNF-α in UA-treated cells reflects the decreased amount of total cellular TNF-α, we examined the presence of TNF-α by flow cytometry in saponin-permeabilized cells. We found a significantly increased percentage of TNF-α–positive cells in the UA-treated and antigen-activated cells than in activated control cells (C and D). In antigen-activated and vehicle-treated cells, the geometric mean of fluorescence intensity (geo MFI) was 1143 ± 70 (mean ± SEM). In cells treated with UA and activated with antigen, the geo MFI was increased 3.7 times to 4250 ± 506; the difference was significant (p < 0.002). When the assay was performed with cells not permeabilized with saponin to measure surface TNF-α, we found only 1.4 higher geo MFI of TNF-α at the cell surfaces of activated and UA-treated cells when compared to activated control cells, and the difference was not significant (E). The data suggested that the reduced secretion of TNF-α is not caused by an accumulation of pro-TNF-α at the plasma membrane due to reduced activity of ADAM metallopeptidase, an enzyme responsible for pro-TNF-α cleavage and mature TNF release . Treatment of the cells with UA alone (without antigen activation) also induced increased tyrosine phosphorylation of a similar set of proteins. FcεRI-mediated activation of UA-treated cells did not cause a further increase in global tyrosine phosphorylation.The first biochemically well-defined step in FcεRI signaling is tyrosine phosphorylation of FcεRI β and γ subunits by the LYN kinase, followed by the formation of the FcεRI signalosome and enhanced phosphorylation of numerous substrates , 39. NexFig. 4B and C). In antigen-activated cells, tyrosine phosphorylation of FcεRI-β was significantly higher in UA-pretreated cells than in control cells (B and C).Next, we examined phosphorylation of individual proteins using SDS-PAGE-fractionated whole cell lysates followed by immunoblotting with phosphoprotein-specific antibodies. Alternatively, we immunoprecipitated proteins of interest and analyzed them by immunoblotting with the mAb PY-20-HRP conjugate. To determine whether UA interferes with tyrosine phosphorylation of the FcεRI β subunit, we immunoprecipitated FcεRI from UA- or vehicle-treated cells that were activated or not with antigen. UA had no significant effect on tyrosine phosphorylation of the FcεRI β in nonactivated cells , B and Col cells , B and C519 and Tyr520 of mouse SYK  are located in the activation loop of the kinase, and their phosphorylation is crucial for SYK functioning . SYK kinase phosphorylates the linker for activation of T cells (LAT)1 and LAT2 adapter proteins in antigen-activated cells (F–I).An essential protein-tyrosine kinase in the FcεRI signaling is SYK, which is phosphorylated on several residues in antigen-activated cells. Tyrctioning . We founanalyzed , D and Eed cells ; therefoed cells F–I.783 by SYK kinase enhances the enzymatic activity of PLCγ .It is known that phosphorylation of PLCγ at Tyr of PLCγ . In agrentervals , A and BTyr204, which correlates with ERK activation, we examined ERK phosphorylation in control and UA-treated cells. We found significantly increased phosphorylation of ERKTyr204 in UA-treated nonactivated cells when compared to control cells. After activation of the cells by FcεRI triggering, ERKTyr204 phosphorylation was significantly lower in UA-treated cells than UA-untreated cells in all time intervals analyzed (C and D).A critical signal transduction molecule involved in the activation of many transcription factors is ERK. To determine whether UA is involved in the phosphorylation of ERKanalyzed , C and DTyr700 in both nonactivated cells and cells activated with antigen for 1 min. Cells activated for 5 or 15 min and treated or not with UA showed similar phosphorylation of CBLTyr700 (E and F). mTOR is a serine/threonine-protein kinase, which is phosphorylated on Ser2448via the PI3 kinase/Akt signaling pathway and regulates mast cell degranulation, cytokine production, and migration .We also analyzed phosphorylation of E3 ubiquitin-protein ligase, CBL, and mammalian target of rapamycin (mTOR) proteins. The tyrosine-phosphorylated CBL protein is involved in the negative regulation of FcεRI-mediated signaling by promoting ubiquitination of the activated receptor subunits and associated protein tyrosine kinases , 44. PreBLTyr700 , E and Figration , 46, 47. vehicle , G and H397 stabilizes the activation loop of the catalytic domain and increases LYN activity. In contrast, phosphorylation of Tyr508 at the C terminus of LYN by C-terminal Src kinase (CSK) stabilizes the inactive conformation of the catalytic domain. We found similar levels of LYN with phosphorylated Tyr397 and Tyr508 in UA-treated and control cells and no significant changes in phosphorylation of these regulatory tyrosines in antigen-activated cells (A–C). The data suggest that the observed changes in phosphorylation of the FcεRI β subunit (B and C) is not due to the enhanced activity of the LYN kinase. Rather, UA-induced changes in the spatial arrangement of plasma membrane molecules and their mobility could be involved.Finally, we examined the phosphorylation of the LYN kinase. The catalytic activity of this enzyme is regulated in mice by phosphorylation/dephosphorylation of two conserved tyrosines at positions 397 and 508 , 49, 50.ed cells , A–C. Th subunit , B and CA and E). Exposure of the cells to UA resulted in an increased fraction of tyrosine-phosphorylated LYN, LAT1, and LAT2 in DRMs (B and E). Activation with antigen in control cells reduced the fraction of phosphorylated LYN and PAG in DRMs (C and E), as expected (D and E). These data indicate that UA interferes with the distribution of tyrosine-phosphorylated proteins in DRMs.Previous studies with mast cells showed that acute cholesterol lowering or changes in the plasma membrane lipid composition resulted in changes in the properties of detergent-resistant membranes (DRMs) , 52, 53. in DRMs , B and E in DRMs , C and Eexpected , 56. In expected , D and EA and B; Ctrl-F). In the presence of filipin, the cells showed fluorescence that was similar to the control cells (Ctrl+F), cells pretreated with vehicle (0.1% DMSO+F), or containing 50 μM UA and 0.1 % DMSO. As a control, we also included cells pretreated with methyl-β-cyclodextrin (MβCD), which removes cholesterol from the cells and disrupts detergent-insoluble glycolipid domains . Reduced mobility by UA was also observed when antibody-mediated aggregation of the Thy-1.1 glycoprotein was analyzed (C and D).In our previous study, we found that pretreatment of RBL-2H3 cells with MβCD, which removes plasma membrane cholesterol, resulted in a transient increase in tyrosine phosphorylation of several proteins, increased antigen-induced and spontaneous degranulation, and increased plasma membrane mobility of the antibody-aggregated glycosylphosphatidyl-inositol–anchored protein Thy-1.1 . In furtanalyzed , C and DA and B). From the FRAP data obtained, we calculated that control cells pretreated with vehicle (DMSO) showed a lower number of immobile FcεRI-IgE-FITC complexes  when compared to cell pretreated with UA (64.00 ± 5.16 %). The intergroup difference was significant (p < 0.001).To quantify the effect of UA on the lateral diffusion of the FcεRI and cholesterol, the fluorescence recovery after the photobleaching (FRAP) method was used. When RBL-2H3 cells were exposed for 15 min to 50 μM UA, relative fluorescence of the FcεRI-IgE-FITC complexes in the photobleached area was significantly reduced when compared to cells pretreated with DMSO vehicle; , A and BC and D). In control cells, the immobile fraction was lower (43.00 ± 6.47 %) than in UA-pretreated cells (73.20 ± 2.93 %).Mobility of cholesterol–filipin complexes was also reduced in UA-pretreated cells , C and DData presented in this study show that short-term exposure of BMMCs to UA interferes with antigen-mediated effector functions. To understand the molecular mechanisms of the effect of UA on mast cell activation events, we examined various signaling pathways and targets. Several lines of evidence suggest that UA interferes with the function of the FcεRI-signalosome and support the lipid-centric theory of UA action at least in the earliest stages of mast cell activation.et al. have shown that PI does not bind only to dsDNA in the cell nucleus but also to dsRNA . Similar inhibition of degranulation and calcium response was observed in UA-treated cells activated by thapsigargin. Since thapsigargin bypasses all early FcεRI-mediated signaling steps, it is likely that a key factor leading to UA-mediated inhibition of degranulation in antigen-activated cells is due to reduced Ca2+ uptake. The negative effect of UA on calcium channel activity could be related to UA-mediated changes in lipid bilayer properties (2+ (SOC) channels. Insertion of UA into the plasma membrane could inhibit SOC channels through direct interaction of UA with Orai1 protein in a similar way as was described for inhibition of Orai1 by cholesterol , ERK1 , phosho-ERKY204 , CBL , phosho-CBLY700 , actin , as well as HRP-conjugated goat antimouse IgG  and goat anti-rabbit IgG  were obtained from Santa Cruz Biotechnology Inc antibodies specific for phospho-SYK Y525/Y526 , m-TOR , phospho-mTOR S2448 , phospho-SFKY416  phospho-LynY507  were obtained from Cell Signaling. HRP-conjugated anti-phospho-tyrosine-specific mAb  was purchased from BD Biosciences. Mouse-specific TNF-α-conjugated to R-phycoerythrin  was from BD Pharmingen. TNP-bovine serum albumin (BSA) conjugate (15–25 mol TNP/mol BSA) was produced as described  was purchased from the Institute of Isotopes Co, Ltd Fura-2 acetoxymethyl ester (Fura-2-AM) and Alexa Fluor (AF) 488–conjugated donkey antimouse IgG  were from Life Technologies. RT-PCR reagents were obtained from Top-Bio. Annexin V-FITC (EXB0024), annexin-binding buffer , and PI (EXB0018) were purchased from EXBIO. UA (U6753), saponin (47036), filipin (F9765), DMSO (D8418), 4-methylumbelliferyl-β-D-glucuronide hydrate (M9130), MβCD (332615), and all other reagents were from Sigma–Aldrich.The following mAbs were used: mouse IgE mAb specific for 2,4,6-trinitrophenol (TNP), clone IGEL b4.1 , anti-FcMice were bred and maintained in a specific pathogen-free facility of the Institute of Molecular Genetics, used in compliance with the Institute guidelines, and approved by the Institute Commission for Animal Welfare and Protection . BMMC precursors were isolated from the femurs and tibias of 6- to 8-week-old mice of the C57BL/6 genotype. They were cultured for 8 to 12 weeks in a complete growth medium consisting of RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 71 μM 2-mercaptoethanol, minimal essential medium, nonessential amino acids, 0.7 mM sodium pyruvate, 2.5 mM L-glutamine, 12 mM D-glucose, recombinant mouse SCF (15 ng/ml), recombinant mouse IL-3 (15 ng/ml), and 10% (vol/vol) fetal calf serum. In experiments presented in 2, 1 mM MgCl2, 5.6 mM glucose) supplemented with 0.1% BSA. The assay was performed on a 96-well plate. Cells (0.25 × 106) in 100 μl BSS-0.1% BSA were exposed to various concentrations of UA for 15 min at 37 °C, centrifuged at 4 °C and washed with ice-cold 1× annexin-binding buffer. The cell pellets were resuspended in 50 μl of ice-cold 1× annexin V–binding buffer supplemented with 5 μl annexin V-FITC or 1× annexin V buffer alone for unstained control cells. Cells were incubated for 30 min on ice in the dark, and 5 min before flow cytometry analysis, 50 μl of the binding buffer with or without PI was added. Samples were measured in LSRII FACS DIVA (BD Bioscience). Annexin V-FITC binding was analyzed by flow cytometry using the FITC signal detector (FL1) and PI staining by the phycoerythrin emission detector (FL2).The annexin V–binding assay was used for quantification of phosphatidylserine surface expression in BMMCs treated with various concentrations of UA. Briefly, the cells were washed with BSS  were transferred into wells of a 96-well cell culture plate. The cells were pretreated for 15 min with various concentrations of UA or vehicle (0.1% DMSO) in BSS-0.1% BSA added in 100 μl aliquots and challenged with 20 μl of antigen (TNP-BSA conjugate) at a concentration of 250 ng/ml. When the UA-treated cells were activated by thapsigargin, the sensitization step was omitted. The degree of degranulation was determined as the amount of β-glucuronidase released into the supernatants as described . After 12 to 14 h of incubation, cells were washed and resuspended in BSS-0.1% BSA at a concentration of 1.5 × 10escribed  using 4-2+]i were analyzed using cytoplasmic reporter Fura-2-AM as described (2+ levels were determined by an Infinite M200 microtiter plate reader in a NUNC white 96-well plate (Thermo Scientific) with excitation wavelengths at 340 and 380 nm and constant emission at 510 nm. To determine the effect of UA on antigen-induced changes in [Ca2+]i in the absence of extracellular calcium, the same method was used, except that BSS without CaCl2 was used, and after antigen triggering, CaCl2 was added as indicated in Figure 2D.Changes in the [Caescribed . Brieflyescribed . After 36) were washed in BSS-0.1% BSA, resuspended in 100 μl BSS-0.1% BSA with 1 mM Ca2+, and mixed with 100 μl UA. After 15 min incubation at 37 °C, the cells were activated for 10 min at 37 °C by adding 20 μl BSS-0.1% BSA supplemented with a mix of 45Ca2+ and antigen  or thapsigargin . The reaction was terminated by placing the tubes on ice, and cells with bound 45Ca2+ were separated from free 45Ca2+ by centrifugation at 1200g for 15 min at 4 °C through 12% BSA as described  were added into the transwell insert. Chemoattractants (antigen (250 ng/ml), PGE2 (100 nM), or SCF (50 ng/ml)) were added to the lower compartments in 0.6 ml aliquots of chemotaxis medium. After 6 h incubation , cells migrating into lower compartments were counted in 50 μl aliquots using the Accuri C6 flow cytometer (BD Bioscience).Chemotaxis responses were assayed in a 24-well transwell chamber (Corning) with 8 μm pore size polycarbonate filters in the upper wells as previously described . TNP-spe3VO4, 2 mM EDTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mM PMSF) supplemented with 0.2% Triton X-100 (for FcεRI immunoprecipitation). Lysis buffer of the same composition but with 25 mM Tris–HCl, pH 7.5, supplemented with 1% n-dodecyl-β-D -maltoside and 1% Nonidet P-40 was used for LAT1 and LAT2 immunoprecipitations. After incubation on ice for 30 min, the lysates were centrifuged , and the proteins in the postnuclear supernatants were immunoprecipitated with the corresponding antibodies prebound to UltraLink-immobilized protein A . The immunoprecipitated proteins were size fractionated by SDS-PAGE and analyzed by immunoblotting with PY-20-HRP conjugate or with protein-specific antibodies, followed by HRP-conjugated antimouse or anti-rabbit IgG antibodies. Several tyrosine-phosphorylated proteins were quantified by direct immunoblotting of SDS-PAGE–fractionated whole cell lysates with phosphoprotein-specific antibodies. For loading controls, protein-specific antibodies were used. The primary antibodies were detected with HRP-conjugated secondary antibodies. The HRP signal was detected by Luminescent Image Analyzer LAS-3000 (Fujifilm) and quantified with AIDA software (Raytest GmbH). The levels of phosphorylated proteins were normalized to the loading controls run in parallel gels  for different time intervals. Toward the end of the activation period, the cells were centrifuged and the pellet was resuspended in ice-cold immunoprecipitation buffer  were pretreated or not with UA for 15 min and then activated or not with antigen (250 ng/ml) for 5 min. After centrifugation, the cells were lysed on ice in 0.8 ml lysis buffer  supplemented with 1% Brij-96. The gradient was formed by adding 0.5 ml of 80% sucrose stock solution to the bottom of a polyallomer tube  followed by 1.5 ml of 40% sucrose containing the cell lysate, 2 ml 30%, and 1 ml 5% sucrose. After 4 h centrifugation at 4 °C , 10 fractions were collected from the top of the gradient, and individual fractions were analyzed by SDS-PAGE followed by immunoblotting with PY-20-HRP and protein-specific antibody.Sucrose density gradient separations were performed as previously described . BrieflyAfter 30 min incubation at 37 °C with shaking, the cells were washed and fixed for 10 min at 22 °C (in the dark) with 4% paraformaldehyde in staining buffer. The fixed cells were washed twice in staining buffer, exposed or not to 50 μl filipin (50 μg/ml PBS), and incubated for 30 min in the dark at 22 °C. After incubation, filipin was washed out twice, the cells were resuspended in staining buffer, and cholesterol levels were quantified using a BD FACSymphony flow cytometer with UV light laser (355 nm) and UV detector (450/50 nm).2 for 2 min at 85 °C, followed by 1 min incubation steps at temperatures 5 °C lower than in the preceding step with the final step at 25 °C. Next, 10 μl of the hybridization reaction was mixed with 1.25 U of SplintR ligase (New England BioLabs), 0.14 μl of 100 mM ATP, and 4 μl of 2× concentrated TP buffer (Top-Bio) containing 15 mM Tris–HCl, pH 8.8, 40 mM (NH4)2SO4, 0.4 M trehalose, 2 M 1,2-propanediol, 0.02% Tween 20, and 5 mM MgCl2  TCT GAG TGT GAG GGT CGG AGA CAC GCA GGG CTT AA -3′. PCR primers for the cytokine forward primer: 5′- GCT CGA CCT CTC TAT GGG C-3′; reverse primer: 5′ –TTA AGC CCT GCG TGT CTC C-3′. Oligonucleotides for GAPDH mRNA detection are as follows: GAPDH probe A: 5′- CCC GTA ATC TTC ATA ATC CGA G TGG TGC AGG ATG CAT -3′; GAPDH probe B: 5′- (phos) TGC TGA CAA TCT TGA CTG AAT GGC ATC GAG TAC -3′. PCR primers for GAPDH: forward primer: 5′- CCC GTA ATC TTC ATA ATC CGA G -3′; reverse primer: 5′- GTA CTC GAT GCC ATT CAG -3′. The underlined probe sequences are the sites for PCR primer annealing.IgE-sensitized or nonsensitized cells of the BMMC line were pretreated with 50 μM UA or vehicle (0.1% DMSO) for 15 min and then activated or not with antigen  for 60 min. For cytokine mRNA detection, we used the RNA-mediated oligonucleotide annealing, selection, and ligation (RASL)-quantitative PCR (qPCR) protocol, adapted from the miRNA detection method described previously . BrieflymM MgCl2 . The sammM MgCl2  diluted 6) were sensitized with IgE, then exposed to 50 μM UA or vehicle for 15 min, followed by activation or not with 250 ng/ml of TNP-BSA for 4 h. Then, the cells were centrifuged, and the supernatants were used for TNF-α quantification using the bead-based immunoassay  according to manufacturer’s instructions. BD Symphony flow cytometer equipped with 488 and 637 nm lasers was used for data collection. The data were processed with LEGENDplex data analysis software.BMMCs  for 15 min at 21 °C. After washing in PBS, the cells were permeabilized with 0.1% saponin and further processed as described . After 2.5 h, the cells were centrifuged at 805escribed . To quanhttps://imagej.nih.gov/ij/). Data are presented as a fraction of fluorescence localized centrally.For the FcεRI mobility assay, 15-well multitest microscopy slides  were coated overnight with fibronectin (10 μg/ml in PBS), blocked with 4% BSA in PBS (15 min at 37 °C), and washed twice with PBS. IgE-sensitized BMMCs were left to attach to fibronectin-coated slides for 30 min in BSS-0.1% BSA at 37 °C. Then, the cells were exposed to 50 μM UA or vehicle (0.1% DMSO) in BSS-0.1% BSA. After 15 min incubation at 37 °C, the cells were exposed to AF 488-labeled anti-IgE for 10 min at 37 °C in the presence of 50 μM UA or vehicle and then washed and fixed with 4% paraformaldehyde. Alternatively, the cells were incubated with UA or vehicle as aforementioned, washed, fixed, and then labeled for 10 min with AF 488-labeled anti-IgE. For Thy-1.1 mobility assay, RBL-2H3 cells were grown on sterilized 8-well multitest slides  for 18 h, washed with BSS-0.1% BSA, and treated with anti-Thy-1.1 mAb. After 30 min, the antibody was washed out and the cells were exposed for 15 min at 37 °C to 50 μM UA or vehicle. Then, the cells were exposed to AF 488-labeled anti-IgG for 10 min, washed, and fixed with 4% paraformaldehyde. As controls, anti-Thy-1.1-labeled cells were exposed to 50 μM UA or vehicle, fixed, and labeled with AF-labeled secondary antibody. The cells were washed and the samples were mounted using 90% glycerol supplemented with 5% n-propyl gallate. The mobility of FcεRI and Thy-1.1 was determined in the analytical volume comprising the apical part of the attached cells defined by pinhole size 1.5 AU. Images of the cells were acquired with a confocal laser scanning microscope Leica TCS SP5 equipped with X40/1.40 NA. oil-immersion objective. In each experiment, all images were acquired at identical microscope settings. The distribution of FcεRI and Thy-1.1 in individual cells was evaluated, and the fraction of fluorescence detected in the cell periphery (1 μm width) and centrally was calculated using ImageJ (2 concentration (5%) and temperature (37 °C). The cells were grown in glass-bottomed wells of the 96-well plate , and the photobleaching was achieved by a 100 ms laser pulse at 488 nm with 100% of the laser power level. Fluorescence intensity was monitored in a circular area of 1 μm in diameter. Confocal images were obtained from a single optical plane corresponding to the point of contact between the cell and the glass. The obtained time lapses were analyzed using LasX software (https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/). Subsequently, the background was subtracted from the intensities of the FRAP area, and curves were corrected for bleaching. The resulting FRAP curves (15–18 measurements for each experiment) were fitted individually biexponentially to obtain an immobile fraction  and an environmental chamber controlling COfraction , 108. AlB and D were calculated with one-way ANOVA with Tukey’s multiple comparison test. In experiments where only two groups were compared, the statistical significance of differences was evaluated with Student’s unpaired, two-tailed t test. All values are expressed as means ± SEMs. Significance levels: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.Statistical significance of intergroup differences was determined by one-way ANOVA with Tukey’s post-test using Prism version 5.04 graphics and statistic software package (GraphPad). Data in All data are contained within this article and in the supporting information.This article contains The authors declare that they have no conflict of interest with the contents of this article.


Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated myelopathy-tropical spastic paraparesis (HAM/TSP) with a poor prognosis. This study aimed to evaluate the cost-effectiveness and health impact of HTLV-1 antenatal screening.A state-transition model was developed for HTLV-1 antenatal screening and no screening over a lifetime horizon from a healthcare payer perspective. A hypothetical cohort of 30-year-old individuals was targeted. The main outcomes were costs, quality-adjusted life-years (QALYs), life expectancy life-years (LYs), incremental cost-effectiveness ratios (ICERs), HTLV-1 carriers, ATL cases, HAM/TSP cases, ATL-associated deaths, and HAM/TSP-associated deaths. The willingness-to-pay (WTP) threshold was set at US$50,000 per QALY gained. In the base-case analysis, HTLV-1 antenatal screening  was cost-effective compared with no screening . Cost-effectiveness was sensitive to the maternal HTLV-1 seropositivity rate, HTLV-1 transmission rate with long-term breastfeeding from HTLV-1 seropositive mothers to children, and the cost of the HTLV-1 antibody test. HTLV-1 antenatal screening was cost-effective when the maternal HTLV-1 seropositivity rate was greater than 0.0022 and the cost of the HTLV-1 antibody test was lower than US$94.8. Probabilistic sensitivity analysis using a second-order Monte-Carlo simulation showed that HTLV-1 antenatal screening was 81.1% cost-effective at a WTP threshold of US$50,000 per QALY gained. For 10,517,942 individuals born between 2011 and 2021, HTLV-1 antenatal screening costs US$785 million, increases19,586 QALYs and 631 LYs, and prevents 125,421 HTLV-1 carriers, 4,405 ATL cases, 3,035 ATL-associated deaths, 67 HAM/TSP cases, and 60 HAM/TSP-associated deaths, compared with no screening over a lifetime.HTLV-1 antenatal screening is cost-effective and has the potential to reduce ATL and HAM/TSP morbidity and mortality in Japan. The findings strongly support the recommendation for HTLV-1 antenatal screening as a national infection control policy in HTLV-1 high-prevalence countries. Human T-cell leukemia virus type 1 (HTLV-1) is a carcinogenic retrovirus that causes adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated myelopathy-tropical spastic paraparesis (HAM/TSP), an unremitting and progressive neurological disorder that presents with spastic paraparesis, neurogenic bladder, sphincter dysfunction, and mild sensory disturbance in the lower extremities. HTLV-1 is endemic in the Southwestern part of Japan, sub-Saharan Africa, South America, the Caribbean area, foci in the Middle East, and Australia-Melanesia. Based on published seroprevalence rates, which are missing or sparse for up to 6/7th of the global population, an estimated 5 to 10 million people worldwide are infected with HTLV-1. Treatment of ATL and HAM/TSP is very difficult and no vaccine is available. HTLV-1 transmission patterns include mother-to-child transmission through breastfeeding, horizontal transmission through sexual intercourse, and direct contact transmission through blood. HTLV-1 antenatal screening is effective to prevent mother-to-child transmission of HTLV-1. The probability of mother-to-child transmission of HTLV-1 through long-term and short-term breastfeeding can be reduced from 20.3% and 7.4% to 2.5% by withholding breastfeeding. However, Japan is the only country in the world that has implemented HTLV-1 antenatal screening. This study demonstrates that HTLV-1 antenatal screening is cost-effective and has the potential to reduce the number of cases and deaths from ATL and HAM/TSP in HTLV-1 high prevalence countries. Human T-cell leukemia virus type 1 (HTLV-1) is a carcinogenic retrovirus that causes adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated myelopathy-tropical spastic paraparesis (HAM/TSP), an unremitting and progressive neurological disorder that presents with spastic paraparesis, neurogenic bladder, sphincter dysfunction, and mild sensory disturbance in the lower extremities. These diseases develop after a long incubation period and have a poor prognosis . An estiJapan is the highest-endemic country in the world, where at least 1 million people are estimated to be HTLV-1 carriers ,8,9. TheCost-effectiveness regarding HTLV-1 antenatal screening warrants evaluation as a national infection control policy for HTLV-1.This study aimed to assess the cost-effectiveness and health impact of HTLV-1 antenatal screening compared with no screening.A state-transition model was developed for two intervention strategies: HTLV-1 antenatal screening and no screening. Decision branches were directly connected to one Markov node per intervention strategy and the first event was modeled in a Markov cycle tree. The cycle length was set to one year. A half-cycle correction was applied. Incremental cost-effectiveness ratios (ICERs) were calculated and compared to a willingness-to-pay (WTP) threshold of US$50,000 per quality-adjusted life-year (QALY) gained ,22.A hypothetical cohort of 30-year-old individuals born to mothers who had or had not received HTLV-1 antenatal screening was targeted from a healthcare payer perspective with a lifetime horizon.The main outcome measures were costs, QALYs, life expectancy life-years (LYs), ICERs, HTLV-1 carriers, ATL cases, HAM/TSP cases, ATL-associated deaths, and HAM/TSP-associated deaths.The model was constructed using TreeAge Pro 2022 . As this was a modeling study with all inputs and parameters derived from the published literature and Japanese statistics, ethics approval was not required.For individuals born to mothers with positive HTLV-1 antibody tests at HTLV-1 antenatal screening, withholding breastfeeding reduces the probability of mother-to-child transmission of HTLV-1 from 20.3% for long-term breastfeeding and 7.4% for short-term breastfeeding to 2.5% ,24. TherIn individuals born to mothers with positive HTLV-1 antibody tests, the probability of being infected with HTLV-1 through breastfeeding is 20.3% for long-term breastfeeding, 7.4% for short-term breastfeeding, and 2.5% for bottle feeding, making them HTLV-1 carriers. Patients with ATL and HAM/TSP receive treatment according to the Japanese practical guidelines for each disease.Clinical probabilities were collected using MEDLINE from 2000 to December 2022 . To obtaCosts were calculated based on the Japanese medical fee schedule  and adjuHealth status was included to represent nine possible clinical states: (i) HTLV-1 uninfected state, (ii) HTLV-1 carrier, (iii) acute-type ATL, (iv) lymphoma-type ATL, (v) unfavorable chronic-type ATL, (vi) favorable chronic-type ATL, (vii) smoldering-type ATL, (viii) HAM/TSP, and (ix) death . Health To determine which strategy would be more cost-effective if one variable was tested over the widest possible range, holding all other variables constant, one-way sensitivity analyses were conducted on variables such as the maternal HTLV-1 seropositivity rate, the incidence of ATL and HAM/TSP among HTLV-1 carriers, the mortality of ATL and HAM/TSP, the cost of HTLV-1 antibody test, treatment cost of ATL and HAM/TSP, and health utilities . A two-wThe Markov cohort analyses determined the cumulative lifetime probability of ATL cases and deaths, HAM/TSP cases and deaths, and HTLV-1 carriers in HTLV-1 antenatal screening and no screening. The cumulative lifetime number of ATL cases and deaths, HAM/TSP cases and deaths, and HTLV-1 carriers were obtained by multiplying the cumulative lifetime probabilities of the two strategies by the total number of births from 2011 to 2021  .HTLV-1 antenatal screening  was cost-effective compared to no screening  .ICER tornado diagram showed that HTLV-1 antenatal screening is cost-effective compared with no screening at a WTP threshold of US$50,000 per QALY gained when the maternal HTLV-1 seropositivity rate is greater than 0.0022, HTLV-1 transmission rate with long-term breastfeeding from HTLV-1 seropositive mothers to children is greater than 0.154, HTLV-1 transmission rate with short-term breastfeeding from HTLV-1 seropositive mothers to children is greater than 0.043, HTLV-1 transmission rate with bottle feeding from HTLV-1 seropositive mothers to children is lower than 0.044, the health utility value of HTLV-1 carriers is lower than 0.77, and the cost of HTLV-1 antibody test is lower than US$94.8 . The twoFor 10,517,942 individuals born between 2011 and 2021, HTLV-1 antenatal screening costs US$785 million, increases 19,586 QALYs and 631 LYs, and prevents 125,421 HTLV-1 carriers, 4,405 ATL cases, 67 HAM/TSP cases, 3,035 ATL-associated deaths, and 60 HAM/TSP-associated deaths, compared with no screening over a lifetime .This study demonstrated that HTLV-1 antenatal screening is cost-effective compared with no screening in Japan. The main reason for the superior cost-effectiveness of HTLV-1 antenatal screening is that withholding breastfeeding for HTLV-1 seropositive mothers significantly reduces the mother-to-child transmission rate of HTLV-1 infection, resulting in fewer HTLV-1 carriers and subsequently fewer ATL and HAM/TSP cases and deaths.The study showed that the lifetime per person cost of HTLV-1 antenatal screening was US$74.67 higher than that of no screening. However, further cost savings may be expected in the future by promoting low-cost HTLV-1 antenatal screening through high-volume screening worldwide.To the best of my knowledge, this is the first modelling study in HTLV-1 high-prevalence countries to evaluate the cost-effectiveness and health impact of HTLV-1 antenatal screening. Malik et al suggested, based on a modelling study, that HTLV-1 antenatal screening meets the cost-efficacy standards in the UK, one of the HTLV-1 low-prevalence countries . This stThe main HTLV-1 highly endemic regions are localized in the world: the Southwestern part of Japan, sub-Saharan Africa and South America, the Caribbean area, and foci in the Middle East and Australia-Melanesia . Djuicy Recently, horizontal transmission of HTLV-1 through sexual intercourse has also become an important route of HTLV-1 infection . A largeUnfortunately, no vaccine is currently available, likely due to the low interest of pharmaceutical companies associated with the restricted markets in industrialized countries . HoweverThere are negative impacts of not breastfeeding. For mothers, it increases the long-term risk of cancer , endometriosis, diabetes, osteoporosis, hypertension, cardiovascular diseases, metabolic syndrome, rheumatoid arthritis, Alzheimer disease, and multiple sclerosis. For infants, it increases the risk of otitis media, upper and lower respiratory tract infection, asthma, RSV bronchiolitis, atopic dermatitis, gastroenteritis, inflammatory bowel disease, diabetes, leukemia, and sudden infant death syndrome ,50.This study has several limitations. First, the cost of health supervision and counseling for pregnant women with confirmed HTLV-1 infection at HTLV-1 antenatal screening was not taken into account. Second, the costs of hospitalization, rehabilitation, and complications from treatment of ATL and HAM/TSP were not included. Third, HTLV-1 horizontal transmission rate was not taken into account in the model. The HTLV-1 horizontal transmission rate from males to females is greater than the HTLV-1 horizontal transmission rate from females to males . Fourth,This study demonstrated that HTLV-1 antenatal screening is cost-effective and has the potential to reduce ATL and HAM/TSP morbidity and mortality in Japan. The findings strongly support the recommendation for HTLV-1 antenatal screening as a national infection control policy in HTLV-1 high-prevalence countries. 10 Oct 2022Dear Dr. Kowada,Thank you very much for submitting your manuscript "Cost-effectiveness and health impact of HTLV-1 antenatal screening for prevention of mother-to-child transmission" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Eugenia Corrales-AguilarSection EditorPLOS Neglected Tropical DiseasesEugenia Corrales-AguilarSection EditorPLOS Neglected Tropical Diseases***********************Reviewer's Responses to QuestionsKey Review Criteria Required for Acceptance?As you describe the new analyses required for acceptance, please consider the following:Methods-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?Reviewer #1: Methods need clarification as per comments on the Summary and General Comments session.Reviewer #2: (No Response)--------------------Results-Does the analysis presented match the analysis plan?-Are the results clearly and completely presented?-Are the figures  of sufficient quality for clarity?Reviewer #1: Please see comments on Summary and General Comments session.Reviewer #2: (No Response)--------------------Conclusions-Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?Reviewer #1: Please see comments on Summary and General Comments session.Reviewer #2: (No Response)--------------------Editorial and Data Presentation Modifications?Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”. Reviewer #1: Minor Comments.1. The estimate of 20 million people infected with HTLV-1 worldwide is from de The & Bomford(3), not Gessain & Cassar(4)– who estimated 5 – 10 million based on published seroprevalence but acknowledged that data pertinent to 6/7th of the global population were missing or sparse.2. Regions of high prevalence include Iran and Central Australia – this is mentioned in the discussion but omitted from the introduction.3. In the author summary an explanation of the type of disease would be helpful for HAM/TSP (as is given for ATL).4. Line 123 countries which screen – please provide a reference.5. Line 365 – How do you know that the proposed large scale education campaign will be effective in preventing sexual transmission? Perhaps compare to HIV campaign and reference.6. The term “we” is used in the manuscript but there is only one author. In which case either use I or write in the third person.Reviewer #2: (No Response)--------------------Summary and General CommentsUse this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.Reviewer #1: The addition of a cost effectiveness study from Japan on antenatal screening for prevention of mother-to-child HTLV-1 transmission is welcome. Indeed, it is interesting that no such study has previously been published either leading up to the decision to extend HTLV antenatal screening nationally circa 2011 or since. The only previous study, from the UK– a low prevalence country – concluding that even in the UK screening could be cost effective(1). The paper by A. Kowada concludes that antenatal screening in Japan meets the cost criteria for screening based on provider willingness to pay values of US$ 50,000 or 100,000 per QALY. These are higher than in the UKMajor Comments.1. The life-time risk of HAM/TSP cited in the introduction is 0.18 – 1.8%  have on the cost effectiveness? Noting that this cannot be attributed to deaths from ATL or HAM5. In the model structure the assumption is that all mothers breast-feed. Is this the case in Japan and if so for how long?6. What is the negative impact (QALY cost) of not breast-feeding?7. The effectiveness of the intervention is given as reducing transmission from 20% to 3% but the rate of transmission is determined by the duration of breast feeding. This should be taken into account in the model.8. The cost of the antibody screening is given as US$ 76.4 per person inclusive of confirmatory test. How was this calculated? It is taken from a table of medical fees but high-volume screening is likely to be cheaper in terms of the reagents and in personnel as it is added to other screening assays. Has this been considered?9. The impact of sexual transmission was considered. What was estimate of life-time risk that a person infected through in early life would transmit HTLV-1 through sexual intercourse? How was it calculated? Was this different for males and females?10. In the section on cumulative lifetime health effects the number of cases of ATL and HAM prevented seem small for ATL with an expected ATL rate of 2.8% only and very small for HAM with a lifetime risk of just 0.15%. A detailed explanation of these rates is required for the reader to understand the findings. 11. In the analysis how are twins accounted for?12. In Figure 2C the pivot point for effectiveness seems to be at about $25,000 per QALY). Why consider $100,000 per QALY?13. Table 3 is confusing with cumulative life-time cost of HTLV-1 antenatal screening given as US$ 866 million and various sums presented as reduction regardless of whether these are losses or gains.14. Furthermore the reduction in cases in Table 3 requires explanation. Why is the reduction in disease not at the same as the reduction in cases of infection?15. In the discussion reference is made to the implementation of a national antenatal screening programme in 2011 and that recently maternal seroprevalence has been decreasing. Clearly an antenatal screening programme from 2011 cannot have impacted maternal seroprevalence already especially as the average age of mothers in Japan is 30 years. Comment should be made on the extent of screening in Japan in the preceding years noting the introduction of HTLV antenatal screening in Nagasaki was early as the late 1980s?16. In the comment on depression and anxiety whilst acknowledging that all diagnostic tests and screening programmes are inevitably associated with a degree of anxiety the current statement implies that this would be particularly a feature of HTLV screening and does not take into account the depression and anxiety that occurs when parents discover that they were not offered testing and interventions for a preventable infection causing cancer and neurological disease (as HTLV-1) and see these occurring in the children (even as adults). It is important to discuss both elements since the psychological aspects are not included – not just the potential negative aspects.1. Malik B, Taylor GP. Can we reduce the incidence of adult T-cell leukaemia/lymphoma? Cost-effectiveness of human T-lymphotropic virus type 1 (HTLV-1) antenatal screening in the United Kingdom. Br J Haematol. 2018;184:1040-3.2. Schierhout GM, S; Gessain, A; Einsiedel, L; Martinello, M, Kaldor, J. The association between HTLV-1 infection and adverse health outcomes:a systematic review and meta-analysis of epidemiologic studies. Lancet Infect Dis. 2019.3. de The G, Bomford R. An HTLV-I vaccine: why, how, for whom? AIDS Res Hum Retrovirol. 1993;9(5):381-6.4. Gessain A, Cassar O. Epidemiological Aspects and World Distribution of HTLV-1 Infection. Front Microbiol. 2012;3:388.Reviewer #2: 1. As this paper is written, it appears to have an overwhelmingly major flaw: It seems to assume that prevention of an HTLV-1 infection increases the utility of the person in question in by far most cases from 0.712 to 1. The author appears to assume that a person who is not an HTLV-1 carrier, is in perfect health and has a utility of 1. That assumption is incorrect because there are many other factors and conditions than HTLV-1 that can cause less than perfect health. Correction would greatly reduce the number of QALYs gained by screening, possibly by a factor greater than 10, so that cost-effectiveness of screening would change from well within what is generally considered cost-effective, to well outside of that. Correction would therefore reverse the main conclusion of the paper and require a major revision of the text and tables.Some comments of less importance than the above:2. The author mentions several times “more cost-effective” while the paper only compares screening with not-screening, therefore there is only one cost-effectiveness ratio at play, so that there is no comparison (“more”) of cost-effectiveness ratios possible.3. The text seems often unclear about whether a number mentioned concerns incidence or prevalence. E.g. line 78 seems to describe prevalence, while line 111 seems to quote the same number but describes it as incidence.4. At line 155: are those individuals all pregnant women? If so, please say so.5. At line 171: as I read the paper, compliance rate does not affect the estimated cost-effectiveness ratio6. At line 191 under the heading ‘no screening’: is the probability of sexual transmission not considered in the screening arm?7. It is generally left unclear how sexual transmission affects the cost-effectiveness of screening. Does it have any influence on the presented estimates?8. Does direct transmission play any role in this model?9. The one-way sensitivity analysis does not seem to just involve changing one parameter at the time , but to involve only one parameter altogether.10. At line 207: Reference 20 does not describe test specificity as 100%. Furthermore, the false positivity rates mentioned there, would cause that around half of test positives are false positives.11. At line 336: This paper does not demonstrate that screening reduces morbidity and mortality but rather, makes literature based quantitative assumptions concerning effects on morbidity and mortality in order to estimate cost-effectiveness.12. At line 379: The paper leaves unclear how a gender difference in horizontal transmission affects the cost-effectiveness of screening.13. At lines 386-389: As mentioned under 1., the paper seems to assume that HTLV-1 carriers have a much lower utility than non-carriers therefore it seems incorrect to state that other diseases than ATL and HAM/TSP were not taken into account, on the contrary, it seems that their effects are gravely overestimated.--------------------what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at  25 Oct 2022AttachmentResponse to Reviewers comments.docxSubmitted filename: Click here for additional data file. 6 Dec 2022Dear Dr. Kowada,Thank you very much for submitting your manuscript "Cost-effectiveness and health impact of HTLV-1 antenatal screening for prevention of mother-to-child transmission" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations. Thank you for addressing point by point the reviewers' comments. This has been most helpful. Further clarification of how onward sexual transmission was incorporated into the model is required as well as how the contribution of sexually acquired transmission to the total number of cases of HTLV-1 in Japan has been factored into the incidence data. See the points below and the further comments from Reviewer 1.Line 131 – are 4000 infections in adults and adolescents diagnosed each year or is this the estimated number of new infections per year?Line 217 and Table 1– the horizontal infection rate is 0.000046 on the 2016 paper of HTLV-1 incidence in Japanese blood donors. This is the infection rate but not the transmission rate. A carrier infected in early life will have potentially more opportunity to transmit than a carrier who does not become infected until they become sexually active. Please also confirm that the transmission rate when incorporated into the model describes the life-time risk of transmission following early life infection and not the risk per 100,000 person years. This is not clear at the moment. I note too that the work of Satake et al has recently been updated in J Clin Virol 2022. than in men . The number of seroconversions per 100,000 person-years was 1.54 for men and 4.21 for women – satake et al J Clin Virol . 2022 Dec;157:105324. doi: 0.1016/j.jcv.2022.105324.Although the seroconversions per 100,000 person years is lower than in the 2016 report the sero-conversion rate amongst adolescents and Young adults had increased.Line 307 – “The sexual transmission rate didn’t affect the cost-effectiveness of screening”. Do you mean that the number of transmissions attributed to early life infection was so low that if did not increase the cost-effectiveness of screening. How many sexual transmissions would have been prevented by the avoidance of mother-to-child transmission. This should be added to the section on cumulative lifetime health effects.In this section the estimate on ATL-associated deaths is 64% of the total of ATL cases whereas the data input was that the 4 year survival of 16%. Why is the mortality rate in the model so low. On the other hand 71 HAM associated deaths were prevented out of 177 cases – a 40% mortality. The suggestion in the text is that these numbers may be wrong. What is their contribution to the cost-effectiveness?In the response to Reviewer one you state Yes, the association of ATL risk with acquisition of HTLV-1 in early life was considered in the analysis. In the model, HTLV-1 is transmitted mainly through mother-to-child transmission (MTCT), even if horizontal transmission through sexual intercourse is being considered. However, the data used for ATL risk was the published incidence rate – which would include all persons infected with HTLV-1 regardless of the age of infection. Whilst ATL has been reported following infection in adult life a strong association with infection in early life remains. The relative contribution of infection in early v adult life is therefore likely to impact significantly in the model. What proportion of all HTLV-1 infection in adults in Japan did you consider to be related to mother-to-child transmission? The increasing rates of infection with increasing age, especially among females, indicate a considerable infection after early life. In your response to point 16 Reviewer 1 you have stated that anxiety related to screening would be a particular feature of HTLV-1 screening. It is not clear that this was your intention. If not, please amend. If so, why for example would HTLV-1 screening cause more anxiety than other antenatal screening tests – eg for Down’s syndrome or HIV?Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments. Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Graham P Taylor, MB, DScAcademic EditorPLOS Neglected Tropical DiseasesEugenia Corrales-AguilarSection EditorPLOS Neglected Tropical Diseases***********************Thank you for addressing point by point the reviewers' comments. This has been most helpful. Further clarification of how onward sexual transmission was incorporated into the model is required as well as how the contribution of sexually acquired transmission to the total number of cases of HTLV-1 in Japan has been factored into the incidence data. See the points below and the further comments from Reviewer 1.Line 131 – are 4000 infections in adults and adolescents diagnosed each year or is this the estimated number of new infections per year?Line 217 and Table 1– the horizontal infection rate is 0.000046 on the 2016 paper of HTLV-1 incidence in Japanese blood donors. This is the infection rate but not the transmission rate. A carrier infected in early life will have potentially more opportunity to transmit than a carrier who does not become infected until they become sexually active. Please also confirm that the transmission rate when incorporated into the model describes the life-time risk of transmission following early life infection and not the risk per 100,000 person years. This is not clear at the moment. I note too that the work of Satake et al has recently been updated in J Clin Virol 2022. than in men . The number of seroconversions per 100,000 person-years was 1.54 for men and 4.21 for women – satake et al J Clin Virol . 2022 Dec;157:105324. doi: 0.1016/j.jcv.2022.105324.Although the seroconversions per 100,000 person years is lower than in the 2016 report the sero-conversion rate amongst adolescents and Young adults had increased.Line 307 – “The sexual transmission rate didn’t affect the cost-effectiveness of screening”. Do you mean that the number of transmissions attributed to early life infection was so low that if did not increase the cost-effectiveness of screening. How many sexual transmissions would have been prevented by the avoidance of mother-to-child transmission. This should be added to the section on cumulative lifetime health effects.In this section the estimate on ATL-associated deaths is 64% of the total of ATL cases whereas the data input was that the 4 year survival of 16%. Why is the mortality rate in the model so low. On the other hand 71 HAM associated deaths were prevented out of 177 cases – a 40% mortality. The suggestion in the text is that these numbers may be wrong. What is their contribution to the cost-effectiveness?In the response to Reviewer one you state Yes, the association of ATL risk with acquisition of HTLV-1 in early life was considered in the analysis. In the model, HTLV-1 is transmitted mainly through mother-to-child transmission (MTCT), even if horizontal transmission through sexual intercourse is being considered. However, the data used for ATL risk was the published incidence rate – which would include all persons infected with HTLV-1 regardless of the age of infection. Whilst ATL has been reported following infection in adult life a strong association with infection in early life remains. The relative contribution of infection in early v adult life is therefore likely to impact significantly in the model. What proportion of all HTLV-1 infection in adults in Japan did you consider to be related to mother-to-child transmission? The increasing rates of infection with increasing age, especially among females, indicate a considerable infection after early life. In your response to point 16 Reviewer 1 you have stated that anxiety related to screening would be a particular feature of HTLV-1 screening. It is not clear that this was your intention. If not, please amend. If so, why for example would HTLV-1 screening cause more anxiety than other antenatal screening tests – eg for Down’s syndrome or HIV?Reviewer's Responses to QuestionsKey Review Criteria Required for Acceptance?As you describe the new analyses required for acceptance, please consider the following:Methods-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?Reviewer #1: Methods are adequate.Authors need to clarify if they have included secondary vertical transmissions, or horizontal transmission only --------------------Results-Does the analysis presented match the analysis plan?-Are the results clearly and completely presented?-Are the figures  of sufficient quality for clarity?Reviewer #1: Figure 1 A needs editing as it is not clear. Consider changing scale.--------------------Conclusions-Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?Reviewer #1: Authors should keep both thresholds  in the discussion.--------------------Editorial and Data Presentation Modifications?Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”. Reviewer #1: The study is well designed, clear and important. I would strongly advise to keep both thresholds in the discussion 50,000 and 100,000. This information is important as it shows that HTLV antenatal screening is not only cot-effective, but highly cost-effective, as authors have used a conservative threshold.--------------------Summary and General CommentsUse this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.Reviewer #1: The paper is well written, timely and important. Only minor comments as above. The inclusion of short discussion adding both threshold is important in a broader context of cost-effectiveness of such intervention in other settings.--------------------what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at ReferencesPlease review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice. 13 Dec 2022AttachmentVer2 Response to Reviewers comments.docxSubmitted filename: Click here for additional data file. 9 Jan 2023Dear Dr. Kowada,Thank you very much for submitting your manuscript "Cost-effectiveness and health impact of HTLV-1 antenatal screening for prevention of mother-to-child transmission" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you further modify the manuscript according to the review recommendations. The focus of concern is mainly (but not only) on the impact of horizontal transmission of HTLV-1 in the two scenarios and how the reduced transmission from the screened pregnant lady after diagnosis of HTLV-1 infection has been calculated based on the expected behaviour change as well as the horizontal transmission that does not occur during their lifetime from the infants in whom HTLV-1 was prevented. If either or both of these have not been included in the analysis, and there is insufficient data to include in the revision, then this should be made clear as a limitation and an underestimate of the potential benefit of the antenatal screening.Please also address the other, minor points.Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments. Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Graham P Taylor, MB, DScAcademic EditorPLOS Neglected Tropical DiseasesEugenia Corrales-AguilarSection EditorPLOS Neglected Tropical Diseases***********************Reviewer's Responses to QuestionsKey Review Criteria Required for Acceptance?As you describe the new analyses required for acceptance, please consider the following:Methods-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?Reviewer #1: Line 166-167 author say that a hypothetical cohort of 30-year individuals was targeted. Is the author referring to pregnant women? The model should focus on pregnant women, since they are the target population of the intervention. This needs to be clearLine 191 “Patients with favorable and smoldering types of ATL continue to be followed up without treatment”. How long are they followed up for? What rate of transformation to the more aggressive forms of ATL were imputed in the model.ine 194-195 Author states "The probability of HTLV-1 transmission through sexual intercourse is considered", does it refer to the sexual transmission in the 30-year-old cohort  or the sexual transmission that may occur during the lifetime of the babies born from those mothers? If it refers to the 30-year-old cohort, it is not expected that sexual transmission would affect or be affected by antenatal screening program and should not be considered. However, prevention of sexual transmission to a partner can also be prevented by safer sex (condom use), once the women is aware of their serostatus due to antenatal screening. If it refers to secondary sexual transmission throughout the babies` life author should not use the incidence rate in blood donors. The horizontal transmission rate used is that of the incidence found among persons of reproductive age however all models of infection have shown the prevalence of infection to increase with age and that the rate of increase is higher in older age. Therefore the model should not use the same horizontal transmission rate throughout the person’s life as this under-estimates the number of infections prevented.The transmission rate of HTLV through sex is expected to be much higher than incidence in blood donors at reproductive age, but would vary according to different variables, such as gender of the infected individual, number of partners, frequency of unprotected sex, co-infection with other STI, HTLV proviral load, etc. The rate of sexual transmission was estimated as 60% from male to female and 0.4% from female to male in a 10y period, but this is an estimation only and has a lot of uncertainties. In the Miyazaki cohort of sero-discordant couples the transmission rate over 5 years was 7% with a male-to-female rate 4-fold higher than females-to-males. Some references regarding sexual transmission ratehttps://pubmed.ncbi.nlm.nih.gov/15809908/https://pubmed.ncbi.nlm.nih.gov/8418183/https://pubmed.ncbi.nlm.nih.gov/2877031/In terms of those who are infected in early life – there is clearly a longer period of risk of horizontal transmission that those who are infected in later life, but we do not know whether this translates into a greater overall risk of horizontal transmission as behavioural factors may differ between the two risk groups.If horizontal transmision is being considered the model should include the transmissions relating to the unscreened pregnant lady and transmissions relating to the infants who become infected as a result of the non-screening strategy. As there is only one row for horizontal transmission it seems that only the mother is considered but this is not completely clear.--------------------Results-Does the analysis presented match the analysis plan?-Are the results clearly and completely presented?-Are the figures  of sufficient quality for clarity?Reviewer #1: Line 304-305 "Cost effectiveness is not sensitive to the horizontal sexual transmission rate". See comment above.Table 3. the cumulative lifetime HTLV-1 carriers with horizontal transmission is higher if antenatal screening is implemented, with a gain of 91 cases. This does not seem to be correct. Higher number of infected people is expected without antenatal screening, and therefore, more people would be able to transmit HTLV through sex. There is no reason that could explain higher number of sexual transmission if antenatal screening is implemented. Table 3. ATL cases are primarily associated with vertical transmission. Therefore, the reduction of number of ATL cases should be proportional to the reduction of number of infections that would be prevented. In table 3 number of carriers with vertical transmission reduces from 158,244 to 32,822, while number of ATL reduces from 14,804 to 10,392. Even considering horizontal transmission, the number of ATL seem to be too high. Please explain the model for generating the number of cases of ATL. It would appear from the numbers that the model considered most cases of ATL to be consequence of horizontal trasmission.Table 3 – the LY gain is 456 despite the 4412 fewer cases of ATL – this amounts about 1 month of life expectancy gain for each case of ATL prevented – again this seems difficult to explain.--------------------Conclusions-Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?Reviewer #1: (No Response)--------------------Editorial and Data Presentation Modifications?Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”. Reviewer #1: (No Response)--------------------Summary and General CommentsUse this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.Reviewer #1: The study addresses an important gap in the literature. It is not clear, however, how horizontal transmission was considered in the model. This is a major limitation and should be addressed.--------------------what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at ReferencesPlease review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice. 17 Jan 2023AttachmentVer3 Response to Reviewers comments.docxSubmitted filename: Click here for additional data file. 31 Jan 2023Dear Dr. Kowada,We are pleased to inform you that your manuscript 'Cost-effectiveness of HTLV-1 antenatal screening for prevention of mother-to-child transmission' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. 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All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.Best regards,Graham P Taylor, MB, DScAcademic EditorPLOS Neglected Tropical DiseasesEugenia Corrales-AguilarSection EditorPLOS Neglected Tropical Diseases*********************************************************** 13 Feb 2023Dear Dr. Kowada,We are delighted to inform you that your manuscript, "Cost-effectiveness of human T-cell leukemia virus type 1 (HTLV-1) antenatal screening for prevention of mother-to-child transmission," has been formally accepted for publication in PLOS Neglected Tropical Diseases.We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. 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The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases. Best regards,Shaden Kamhawico-Editor-in-ChiefPLOS Neglected Tropical DiseasesPaul Brindleyco-Editor-in-ChiefPLOS Neglected Tropical Diseases


It has been well known that a variety of pro-inflammatory cytokines and related signaling pathways are involved in the procession of DKD. Additionally, some anti-hyperglycemic agents and mineralocorticoid receptor antagonists (MRAs) that are effective in alleviating the progression of DKD have anti-inflammatory properties, which might have beneficial effects on delaying the progression of DKD. However, there is currently a lack of systematic overviews. In this review, we focus on the novel pro-inflammatory signaling pathways in the development of DKD, including the nuclear factor kappa B (NF-κB) signaling pathway, toll-like receptors (TLRs) and myeloid differentiation primary response 88 (TLRs/MyD88) signaling pathway, adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathways, inflammasome activation, mitochondrial DNA (mtDNA) release as well as hypoxia-inducible factor-1(HIF-1) signaling pathway. We also discuss the related anti-inflammation mechanisms of metformin, finerenone, sodium-dependent glucose transporters 2 (SGLT2) inhibitors, Dipeptidyl peptidase-4 (DPP-4) inhibitors, Glucagon-like peptide-1 (GLP-1) receptor agonist and traditional Chinese medicines (TCM).Diabetic kidney disease (DKD) is a common complication of diabetes mellitus and a major cause of end-stage kidney disease (ESKD). The pathogenesis of DKD is very complex and not completely understood. Recently, accumulated evidence from  Diabetic kidney disease (DKD), which is also known as diabetic nephropathy before, is the most common microvascular complication of diabetes mellitus . The numPrevious studies demonstrate that many factors are implicated in the progression of DKD, such as metabolic abnormalities and hemodynamic changes caused by hyperglycemia and insulin resistance, etc . It has Inflammation is a biologically conserved immune defense mechanism . In humaIn this review, we discuss the role of some novel and key pro-inflammatory signaling pathways in the progression of DKD, such as the nuclear factor kappa B (NF-κB) signaling pathway, toll-like receptors (TLRs) and myeloid differentiation primary response 88 (TLRs/MyD88) signaling pathway, adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathways, inflammasome activation, mitochondrial DNA (mtDNA) release and hypoxia-inducible factor-1(HIF) signaling pathway. We also introduce the role of anti-hyperglycemic drugs, non-steroidal selective MRA and traditional Chinese medicine in anti-inflammation during the treatment of DKD.Hyperglycemia and insulin resistance are the most common pathological characteristics in patients with DKD and can lead to systemic low-grade inflammation . HyperglThe transcription factor NF-κB is a key mediator of inflammation and NF-κB remains inactive while binding to the proteins of the IkappaB (IκB) family (mainly IκBα) . There aIt has been observed that activated NF-κB signaling pathway is associated with inflammation and fibrosis in DKD . ForestoToll-like receptors (TLRs) is an evolutionarily ancient family of pattern recognition receptors and an important component of innate immunity . ActivatIt has been demonstrated that the high glucose (HG) could activate TLRs-MyD88 signaling pathway in the kidneys of DKD, which is the initiator of renal interstitial fibrosis . Liu et Inflammasome was first proposed by Tschopp et al., in 2002, which could modulate inflammation by the activation of caspases . There aAccumulated data suggests that the inflammasome contributes to the progression of DKD . UpregulOther novel inflammasomes such as NLRP2, NLRP6, NLRP10, NLRP12, and AIM2 are also found to be involved in the regulation of inflammation and their expressions are upregulated in various kidney diseases . It has AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is involved in regulating cellular metabolism by activating energy-producing pathways  Wang et, In addiInterestingly, AMPK signaling pathway was related to the renal inflammation . PreviouWhen mitochondria are dysfunctional, the mitochondrial DNA (mtDNA) translocates to cytoplasm and initiates the inflammation through various signaling pathways  Zhang e. First, As we know that proximal tubular cells of the kidney are rich in mitochondria and mitochondrial dysfunction and mtDNA release have been recognized in various kidney disease . A largeHypoxia-inducible factor (HIF) is conserved in biological evolution and is an important mediator of inflammation . The strRecent studies have shown that HIF signaling pathway is closely related to the development of renal interstitial fibrosis in DKD . The actIn addition to be involved in the pathogenesis of DKD, inflammation has recently been shown to play a role in diabetic cardiomyopathy . Under hThe treatments of DKD mainly include management of hyperglycemia, hypertension and hyperlipidemia. Actually, these drugs especially the nonsteroidal selective mineralocorticoid receptor antagonist (MRA) finerenone and some Chinese traditional medicine could alleviate the development of DKD by regulating the inflammation.Metformin has been used for treatment of diabetes mellitus since 1950s, and is now the most widely used anti-hyperglycemic drug . There’sNonsteroidal selective MRA is a novel drug with cardio-renal protection. Compared with traditional MRAs such as spironolactone and eplerenone, nonsteroidal selective MRA has significantly improved efficacy and safety. Finerenone  is currently approved by FDA for treatment of DKD. A series of clinical studies confirmed the reno-protective effect of finerenone in DKD. The phase III FIDELIO-DKD trial has shown that finerenone significantly delayed renal failure and eGFR decline. In addition to finerenone, several nonsteroidal MRAs are currently in development , such asIt has been well known that overactivation of MR is a key event for chronic inflammation by increasing the recruitment of neutrophil, macrophage and Th1 & Th17 cells and upregulating the expression of pro-inflammatory factors and fibrotic-related factors including TGF-α, endothelin 1, PAI-1, CTGF . There i2+ concentration and activating AMPK signaling pathway (Sodium-glucose cotransporter 2 inhibitor (SGLT2i) is a novel anti-hyperglycemic drug by inhibiting reabsorption of glucose in the kidney . Several pathway . Recent  pathway . Interes pathway . Birnbau pathway . In concGlucagon-like peptide 1 (GLP-1) is an incretin hormone that stimulates insulin secretion in response to food intake . GLP-1 bAstragalus, Cordyceps sinensis, Tripterygium wilfordii, Fructus arctii, Panax notoginseng, Berberine, etc. are also powerful weapons against DKD  such as inst DKD . A systeinst DKD . Furtherinst DKD . Some stinst DKD . The Triinst DKD . Guo et inst DKD . In addiinst DKD . All of In addition to TCM, nutraceuticals derived from Chinese herbal medicines could prevent DKD, for example, boswellic acid, curcumin and quercetin . A systeHowever, TCM has limitations such as low bioavailability, which greatly limits its broad application in the treatment of DKD . The stuMesenchymal Stem Cells (MSCs) and extracellular vesicles (EVs) are current research hotspots and pre-clinical agents for the treatment of DKD . PreviouThe EVs include exosomes, microvesicles and apoptotic bodies. It has been found that the EVs play an important role in the progression of DKD . Under tInflammation plays an important role in the progression of DKD . In thisIn addition, MSCs and EVs have shown the curative effect and anti-inflammatory effect in the treatment of DKD animal models . However


Injury to the ocular lens perturbs cell-cell and cell-capsule/basement membrane interactions leading to a myriad of interconnected signaling events. These events include cell-adhesion and growth factor-mediated signaling pathways that can ultimately result in the induction and progression of epithelial-mesenchymal transition (EMT) of lens epithelial cells and fibrosis. Since the lens is avascular, consisting of a single layer of epithelial cells on its anterior surface and encased in a matrix rich capsule, it is one of the most simple and desired systems to investigate injury-induced signaling pathways that contribute to EMT and fibrosis. In this review, we will discuss the role of key cell-adhesion and mechanotransduction related signaling pathways that regulate EMT and fibrosis in the lens. The vertebrate ocular lens is a highly specialized transparent tissue of ectodermal origin that separates the anterior of the eye from the posterior. The lens is encased in its own basement membrane and is mainly composed of epithelial cells, which occupy the anterior part of the lens, and the fiber cell mass that makes up the remaining lens volume. Any perturbation in the structural organization or function of these cells results in opacification of the otherwise transparent ocular lens, causing a cataract. Cataract continues to be the second largest cause of visual impairment leading to blindness across the world, affecting nearly 94 million people with an overall financial burden of US $6.9 billion . Currentvia Smad-dependent pathways (TGFβ is a cytokine that has been shown to be a key modulator of the fibrotic cataracts PCO and anterior subcapsular cataract (ASC). Indeed, the aqueous humor from patients having undergone cataract surgery exhibits increased levels of active TGFβ , and TGFpathways . Howeverpathways .Although active TGFβ has been shown to be a major inducer of lens EMT and fibrosis, lens injury studies suggest that earlier, upstream events are required for activation of TGFβ and its sustained expression . The seca and β subunit, including 18 a and eight β subunits identified in mammals that can partner to form 24 different integrin receptors that bind specific ligand or set of ligands . Studies that have directly targeted αV integrin through conditional knockout in the murine lens have demonstrated its role in EMT and lens fibrosis . For exaRecent studies have also demonstrated that changes in the lens capsule, the matrix of the lens, can also contribute to EMT. For example, studies have shown that advanced glycation end products (AGEs) present in the lens capsule can potentiate TGFβ2-mediated EMT in human LECs and this occurs through the upregulation of both the canonical and noncanonical pathways . Interesin vitro and in vivo model systems  -actin assembles to form filamentous (F) –actin. During this process, actin binding proteins (ABPs) including myocardin-related transcription factors (MRTFs) dissociate from G-actin complex and translocate to the nucleus. Within this compartment, MRTF forms a complex with serum response factor (SRF) to regulate the expression key EMT genes . Therefof MRTF-A . A follof MRTF-A . These af MRTF-A . These oactility .et al performed a series of immunohistochemistry (IHC) analysis on lenses from mice upon needle injury. The lens epithelium of these mice showed increased expression of TGFβ1, fibronectin and a-SMA. As expected, the epithelium of injured lenses also showed increased activation of MLC9, and thus Rho signaling, suggesting an important role of Rho kinase signaling in the induction of EMT in lens upon injury . Taken together, these studies show that Rho/ROCK signaling is central to EMT induction in the lens either upon injury or induced by growth factor, and therefore modulating the expression and/or activation of RhoA/ROCK signaling might serve as a possible prognosis for EMT-mediated fibrosis in the lens and PCO.One of the important outcomes of Rho kinase signaling is actin cytoskeletal remodeling, which mainly occurs through polymerization of G-actin into F–actin fibers. F-actin, along with actomyosin, forms stress fibers that upon its interaction with focal adhesion points and cell junctions play an important role in cell motility, shape, and morphogenesis . Epithel-catenin . The forring EMT . Stabili in LECs  , mainly integrins, to the F-actin in the actin cytoskeleton and fibronectin expressed in the ECM by myofibroblasts . The foclocation . Recent via Rho/ROCK pathway. Inhibition of Rho/ROCK pathway also prevented cell elongation along with a decrease in Piezo1 density at the cell extension points (One of the major causes of EMT-mediated age-related fibrotic cataract is modulation of interaction of LECs with its basement membrane, the lens capsule, resulting from the changes in the matrix architecture of the lens capsule due to aging . Such chn points . These sInjury-induced repair results from collective migration of epithelial cells to the wounded area, a process that is controlled by partially transformed mesenchymal-like leader cells . These mUsing a mock cataract surgery model in chick, Walker et al observed increased expression of vimentin intermediate filaments in the lamellipodia of the mesenchymal-like leader or repair cell population located in the lens equatorial region . These cRepair cells following mock cataract surgery show increased presence of soluble vimentin, specifically in the wound activated mesenchymal cells that localize to the leading edge of the wound. The soluble vimentin is released extracellularly in response to injury where it is known to mediate the differentiation of leader cells to myofibroblasts . The preAnother mechanism through which extracellular vimentin might be contributing to the increased cell migration and invasion is FAK signaling. In the mock cataract injury model, vimentin colocalized with prominent paxillin-rich adhesion plaques at the tips of the lamellipodia of migratory leader LECs . The colIn recent years, there has been increased focus on understanding how injury contributes to EMT-mediated fibrosis in the lens. These studies point towards cell adhesion molecules as key players, responsible for the induction and progression of EMT in the lens, either independently or through activation of TGFβ signaling. For example, adhesion molecules such as the integrins have been shown to play an upstream role in lens induced-EMT and do so, at least in part, through activation of the conventional TGFβ signaling pathways following injury. Interaction of lens matrix AGEs with RAGE is also known to play a critical role during TGFβ-induced EMT in the lens. Furthermore, injury to the lens modulates mechanotransduction-mediated signaling including activation of YAP and Piezo1, both of which require Rho/ROCK-induced FAK signaling. FAK-mediated Rho/ROCK signaling may play an important role in enhanced cell migration, mediated by increased expression of extracellular vimentin during wound healing in the lens. As outlined in this review, many of the cell-adhesion mediated signaling pathways coordinate with one another to induce EMT and fibrosis in the lens. However, further studies are needed to understand how these complex signaling pathways crosstalk with one another during both early events of lens injury as well as in the later progression of EMT and fibrosis.