A growing number of young people tend to regard their pets as their surrogate children, yet research examining the relationship between pet attachment and fertility intention remains scarce. Moreover, individuals’ fertility intention is affected by economic resources. Therefore, we conducted two studies to examine the interaction effect of pet attachment and subjective socioeconomic status (SES) on childbearing-aged individuals’ fertility intention. In Study 1, we utilized questionnaires to measure Chinese pet owners’ pet attachment, subjective SES, and fertility intention. In Study 2, participants’ pet attachment was experimentally manipulated by reading articles about the benefits of petkeeping. The results of the two studies consistently demonstrated that the effect of pet attachment on fertility intention was moderated by subjective SES. Specifically, pet attachment was negatively associated with fertility intention when individuals had a high level of subjective SES, whereas this effect disappeared when individuals had low subjective SES. These findings suggest an explanation for why individuals with high subjective SES delay or even opt out of childbearing. The limitations and implications of the current study are discussed. Currently, many countries face challenges of low fertility rates. In China, fertility rates have begun to drastically plunge since the 1970s, and the total fertility rate has been well below the replacement level . HoweverAs companion animals, pets can satisfy people’s psychological needs for companionship, friendship, and unconditional love. Hence, an increasing number of people treat pets as their family members . ResearcA large number of studies have recognized the psychological, physical, and social benefits of human–animal interaction, especially for dog or cat owners ,12,13. KOne of the most important reasons why pets have beneficial effects on individuals is that pet owners view pets as their children, and they have a strong pet attachment with them. Pet attachment refers to the intimate and lasting emotional connection between pets and humans ,20. BowlPrevious studies have found that people with higher pet attachment tend to regard their pets as their children ,27. HoweOn the other hand, some studies have suggested that pets can replace children. A qualitative study of 12 childfree companion animal owners between the ages of 18 and 44 suggested that the participants tended to frame their pets as not just family members but as surrogate children and that the human–pet relationship could make them delay or opt out of childbearing . ResearcHowever, the association between pet attachment and fertility intention might be different for childbearing-aged individuals with different levels of subjective SES. Subjective SES represents the perception of one’s own socioeconomic position or rank relative to that of others in the social class hierarchy ,31. SubjThe psychological orientation approach to SES suggests that perceptions of one’s social class rank produce different characteristic patterns of thought, feelings, and behaviors ,37. IndiOverall, we propose two hypotheses: (a) for individuals with high levels of subjective SES, pet attachment is positively associated with fertility intention, while the fertility choices of individuals with low subjective SES are not influenced by pet attachment ) for CN¥5 (CN¥1 = USD 0.15). Seven participants were excluded from the data screening process because they chose the same option on all items. The valid sample in Study 1 consisted of 155 Chinese adults . All the research procedures met the ethical guidelines of the American Psychological Association and were approved by the Academic Ethics Committee of the Faculty of Psychology at Beijing Normal University (20181220).As we were unsure of the appropriate sample size, we recruited as many participants as our resources permitted prior to any data analysis. We recruited childbearing-aged (age range: 20–35 years) participants by distributing survey links through university online forums and social media, such as WeChat. A total of 162 Chinese pet owners completed the survey on a Chinese survey website  on a 4-point scale  . This quFertility intention. We measured the participants’ fertility intention with a four-item scale. This scale was adapted from previous studies  as control variables. The participants reported their education level  on a 4-point scale. The participants’ marriage status was coded into three categories . The participants also reported whether they had children .The descriptive statistics and bivariate correlations are shown in Z scores. In Model 3, after standardizing the variables, we entered the interaction term (subjective SES × pet attachment). The results of the regression analyses showed that gender and children significantly influenced fertility intention. Specifically, women had lower fertility intention. Those who already had children tended to have higher fertility intention. Neither subjective SES nor pet attachment predicted fertility intention . As shown in ∆R2 = 0.036, p = 0.009).Next, we used hierarchical regression to test the hypothesized interaction between subjective SES and pet attachment on fertility intention. To test our hypotheses, we entered the control variables in Model 1 of the regression. In Model 2, we entered the main effects of subjective SES and pet attachment after standardizing them as simpleB = −0.29, SE = 0.10, t = −2.88, p = 0.004. For the participants with low subjective SES, the relationship between pet attachment and fertility intention was non-significant, simpleB = 0.09, SE = 0.11, t = 0.84, p = 0.40. Thus, Hypothesis 1b was supported.We further conducted simple slope analysis and plotted the results for low (mean − 1 SD) and high (mean + 1 SD) levels of subjective SES in Study 1 provided preliminary support for our hypotheses regarding the interaction effect of subjective SES and pet attachment on pet owners’ fertility intention. However, whether the interactive role of subjective SES and pet attachment would also occur in individuals who do not keep pets in their daily lives was still unknown. Thus, Study 2 aimed to replicate and extend the present findings in a childbearing-aged sample who were not required to keep pets to be included in the sample. Moreover, in Study 2, we manipulated the participants’ pet attachment to establish the model of the interaction effect of subjective SES and pet attachment on fertility intention.Study 2 used a two-condition, between-subjects experimental design in which we manipulated the participants’ pet attachment (high pet attachment vs. control) to replicate the interactive role of subjective SES and pet attachment on fertility intention in a sample with both pet owners and non-pet owners.https://www.wjx.cn (accessed on 14 May 2019)). A total of 255 participants completed the survey for CN¥5. Nine participants were excluded because they did not comply with the manipulation as instructed. Twelve participants were excluded because they chose the same option on all items. The valid sample in Study 2 consisted of 234 Chinese adults . All the research procedures met the ethical guidelines of the American Psychological Association and were approved by the Academic Ethics Committee of the Faculty of Psychology at Beijing Normal University (20181220).A priori power analysis using G*Power software  with then = 121) or a control condition (n = 113). Then, the participants’ fertility intention was assessed with the same item as in Study 1. Next, all participants completed the MacArthur Scale of Subjective Social Status and other demographic questions indicating their age, gender, marital status, education level, and whether they had a child, as in Study 1. In addition, we asked the participants to indicate their monthly income  and whether they had pets . We standardized monthly income and educational level and then summed the two scores as a composite measure of objective SES [On the first page of the questionnaire, all participants read that the purpose of the study was to examine people’s attitudes toward pets. After giving their informed consent, the participants were randomly assigned to one of the two pet attachment conditions: a high-attachment condition  . Considet-test revealed that the participants in the high attachment condition were more likely to have a higher level of pet attachment  than those in the control condition ; t (232) = 2.94, p = 0.004, d = 0.38.First, we examined the manipulation effect. An independent-samples Z scores. First, we entered the control variables in Model 1 of the regression. In Model 2, we entered the main effects of subjective SES and pet attachment. In Model 3, we entered the interaction term (subjective SES × pet attachment). The results of the regression analyses showed that gender significantly influenced fertility intention. Women tended to have lower fertility intention. Neither subjective SES nor pet attachment predicted fertility intention . As shown in ∆R2 = 0.014, p = 0.056).Next, we tested the interaction effect of pet attachment  and subjective SES on fertility intention using hierarchical regression. Prior to the analyses, the predictors were all standardized as SD) and high (mean + 1 SD) levels of subjective SES in simpleB = 0.03, SE = 0.17, t = 0.17, p = 0.86. For the participants with high subjective SES, the negative relationship between pet attachment and fertility intention was significant, simpleB = −0.45, SE = 0.17, t = −2.57, p = 0.01. Thus, Hypothesis 1b was supported.We further conducted simple slope analysis and plotted the results for low , may influence their fertility decisions . MoreoveThe present research makes a significant contribution to the literature on how pet attachment influences individuals’ fertility intention. There have been few empirical studies investigating the relationship between pet attachment and fertility intention. More importantly, there have been inconsistent results regarding this relationship. On the one hand, some studies have suggested that strong pet attachment leads people to have a stronger motivation to rear children . On the Several limitations of the present study should also be noted. First, the current research examined the interaction effect of pet attachment and subjective SES on individuals’ fertility intention. A previous study found that actual fertility behavior failed to match reported intention at the individual level . Future Despite these limitations, the study is important in light of the increasing childfree trend in China . Strong


Envisioning a T cell-dependent mechanism, they suggest that stimulation of sebum production by thymic stromal lymphopeietin (TSLP) “pulls” lipids out of the adipose tissue.The World Population Review (worldpopulationreview.com/) ranks obesity rates to one third of the global population. Associated co-morbidities comprise diabetes type 2, ischemic stroke and cardiovascular complications render this epidemic disease the number 1 cause of deaths worldwide (who.int/). Treating obesity encompasses besides classical approaches like dietary restrictions combined with increased physical activity more and more the targeting of molecular pathways involved in the pathogenesis and persistence of obesity. E.g., the discovery of “beige” adipocytes in white adipose tissue (WAT), which share lipid utilization via thermogenesis with “brown” adipocytes, fostered the hype to induce therapeutic “beiging” of WAT by cold stress and pharmacological interventions .1 In the NASH livers, TSLP also reduced hepatocyte damage, which is the consequence of lipid-induced oxidative stress and inflammation. TSLP might directly affect hepatocytes, since it stimulated hepatocyte autophagy after ischemia/reperfusion injury (I/R) by activation of the PI3-kinase pathway. This reduced liver damage in the I/R-induced sterile inflammatory environment,4 a scenario, which is closely linked also to the pathogenesis of the metabolic syndrome.While nutritional control by dietary or pharmacological interventions directly target the adipose tissue with the aim of fat tissue mass reduction, TSLP-stimulation of sebum production indirectly relates to the adipose tissue Fig. . The reg5 Yet, so far it is unclear, which form might be involved in sebocyte regulation in humans. Therefore, fat waste by TSLP-stimulated sebum production remains to be established unequivocally. Albeit the possibility to loose fat through the skin is intriguing, the stringent regulation of sebacious glands by hormones and neuropeptides and their feedback control requires careful evaluation before the clinical launch of TSLP as fat waist option. Hence, be aware of not casting out the Fiend with the devil of inflammatory skin and other organs´ diseases.Mouse and human TSLP are different. While in the mouse only one form exists, humans feature a long and a short isoform of TSLP. In mice, TSLP at physiological levels seems involved in homeostasis regulation, while overexpression supports adipose loss via increased sebum production. In humans, this relationship remains elusive. Current knowledge rather suggests that the long isoform is upregulated in Th2-mediated inflammatory allergic reactions like asthma and atopic dermatitis. The short form is constitutively expressed by, e.g., stimulation by commensal bacteria, and is involved in homeostatic regulation like maintenance of physiological gut barrier functions. The stimulation of epithelial cells by pathogens induces secretion of the long form.


Furthermore, serum and endometrial levels of UA are higher in mice with decidualized uterin horn and in vitro decidualized stromal cells. The existence of monosodium urate (MSU) crystal was also confirmed by immunostaining. Next, the roles of MSU on decidualization were explored by both in vitro and in vivo models. Our data shows MSU crystal but not UA enhances the decidualization response of endometrial stromal cells, via the upregulation of inflammatory genes such Ptgs2 and Il11. inhibiting of Cox-2 activity abolishes MSU crystal induced higher expression of decidualization marker Prl8a2. At last, in women, we observed enriched expression of XDH in decidua compare to non-decidualized endometrium, the serum level of UA is significantly increased in women in very early stage of pregnancy, and drop down after elective abortion. In summary, we observed an increased serum UA level in the early stage of women’s pregnancy, and proved that the increased level of UA results from the expressed XDH in decidualizing endometrium of both human and mouse, leading to the formation of MSU crystal. MSU crystal can enhance the decidualization response via inflammatory pathways. Our study has uncovered the association between UA, MSU, and decidualization during the early stage of pregnancy.High level of uric acid (UA) is the major origin of gout, and is highly associated with various pregnant complications, such as preeclampsia and gestational diabetes. However, UA’s level and role in the very early stage of pregnancy has not been uncovered. This study aims to investigate the relevance of serum UA and decidualization, an essential process for the establishment and maintenance of pregnancy in women and mice during the early stage of pregnancy. In this study, we first proved that expression level of UA synthase xanthine dehydrogenase (XDH) is highly increased along with decidualization of endometrial stromal cells in both  Uric acid (UA) is a product of the metabolic breakdown of purine nucleotides by xanthine dehydrogenase/oxidase (XDH/XOD). Upon achieving concentrations of >0.41 mmol/L (6.8 mg/dL), UA crystalizes and forms needle-like, immunostimulatory monosodium urate (MSU) crystals, a sterile inflammatory mediator, which further cause gout, the most common form of inflammatory arthritis .In humans, risk factors for gout include diet, hyperuricemia, genetics, age, and sex . The preDecidualization is a transformation process during which the fibroblastic endometrium stromal cells differentiate into epithelial-like secretory decidual cells, which is essential for the establishment and maintenance of pregnancy . Failurein situ synthesis of UA led to the crystallization of MSU and further enhanced the decidualization process in both human cells and mouse models, through inflammatory pathways. At last, we observed higher expression of endometrial XDH expression, associated with higher levels of serum UA from women in approximately 6 weeks of gestation compared to that of non-pregnant women, suggesting the in situ synthesis of UA occurred in decidualized women endometrium as well.In the present study, we observed increased UA synthetase Xdh along with decidualization in both natural pregnancy and artificial decidualization model in mice, which resulted in higher levels of UA in endometrial tissue and serum. Furthermore, our data suggested that n = 15) and endometrial biopsies (n = 9) of the non-pregnant group were collected during a regular medical examination. Serums of the pregnant patients (n = 13) were collected during the first visit of antenatal care at 5.93 ± 0.71 weeks of gestation. 6 of them were sampled the 2nd time at about 2 weeks after elective abortion. Decidual tissues (n = 9) were collected from women undergoing an elective abortion in the first trimester of pregnancy. There are no significant differences in gravidity, parity, and BMI between the two groups. Serum levels of UA were measured by Blood Uric Acid Analyzer . Tissues were fixed in 4% PFA and embedded in paraffin for further use. All the patients were written noticed before sampling. Demographic information on the patients who provided samples for this study is included in Collection of human endometrium, decidua, and serum was carried out with the approvals of the Medical Ethics Committee of Nanfang Hospital, Southern Medical University, the Scientific Research Ethics Committee of the Drum Tower Hospital, Nanjing University Medical School, and the Ethical Committee of the Women’s Hospital, Zhejiang University School of Medicine. Serums (All mice (ICR strain) were housed in the SPF Experiment Animal Facility with the approval of the Institutional Animal Care and Use Committee of South China Agricultural University. Mice were maintained in a temperature- and light-controlled environment (12 h light and 12 h dark cycle). Mature female mice (8–10 weeks) were mated with fertile males of the same strain to induce pregnancy; the day of the vaginal plug was marked as D1. To artificially induce decidualization, mice were ovariectomized and treated with E2  for 3 days and P4  plus E2 (6.7 ng) for another 3 days, with 2 days of rest in between to mimic the hormonal environment of normal early pregnancy. 6 h after the last injection, 10 μL sesame oil  or 2 mg/mL MSU  was infused intraluminally into one or two uterine horns of mice to induce decidualization (S). The uninjected horn served as a hormonal control (Non-S). Daily injections of P4 plus E2 were continued for 4 days to maximize the decidual response, the day of injection was marked as AD1. A diagram of the artificial decidualization protocol was shown in In vitro decidualization of mESCs was induced with 10 nmol/L estrogen (E2) and 1 μmol/L progesterone (P4) in DMEM/F12 containing 2% charcoal-treated FBS  in vitro. HESC was obtained from ATCC and cultured in DMEM/F12 supplemented with 10% FBS, and in vitro decidualization of HESC was induced by treatment of the cells with 1 mmol/L medroxyprogesterone , and 500 mmol/L cyclic adenosine monophosphate  in DMEF/F12 supplemented with 2% cFBS. Cells and culture medium were collected, and the UA levels were measured by Mouse UA ELISA Kit .mESCs were isolated as follow: Uteri were washed with Hanks’ balanced salt solution  and then digested by digest solution [1% (w/v) trypsin  and 6 mg/ml dispase  in 3.5 ml HBSS] for 1 h at 4°C, followed by 1 h at room temperature and 10 min at 37°C. After the luminal epithelial cells were removed by HBSS washing, the remaining tissues were digested in 6 ml of HBSS containing 0.15 mg/ml collagenase I  at 37°C for 35 min. The stromal cells were collected and cultured in DMEM/F12  containing 2% heat-inactivated fetal bovine serum . TM Real-Time System . Data were analyzed and normalized to RPL7 for human data and Rpl7 for mouse data. The corresponding primer sequences of each gene used in this study were listed in Total RNAs were extracted from the whole uteri or cultured cells using TRIZOL  method, and cDNAs were reverse-transcribed by PrimeScript reverse transcriptase reagent kit  according to the manufacturer’s introduction. qPCR was performed using an SYBR Premix Ex Taq kit  on the CFX96 Touchin vitro using a digoxigenin RNA labeling kit . In situ hybridization was performed as previously described . Sections were then blocked and incubated overnight at 4°C in an anti-precipitated UA antibody . Next, sections were incubated in respective species-specific fluorochrome-conjugated secondary antibody and then mounted with Vectashield Antifade Mounting Medium with DAPI. For immunohistochemistry, 4% paraformaldehyde-fixed, paraffin-embedded uterine tissues were sectioned into 6 μm. Following deparaffinization and hydration, sections were subjected to antigen retrieval in citrate buffer and hydrogen peroxide treatment. They were then incubated with an anti-XDH primary antibody  overnight. After incubated with secondary antibody, HRP and diaminobenzidine  were used to visualize antigens. Sections were counterstained with hematoxylin. Normal IgG was used as a negative control to validate the specificity of antibodies.Western blot was performed as described previously . BrieflyFrozen uterine sections were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C and washed with 1 × PBS three times for 5 min. The uterine sections were then washed in PBS, followed by incubation at room temperature in an AP substrate solution containing 5-bromo-4-chloro- 3-indolyl phosphate  and nitro blue tetrazolium .t-test or one-way ANOVA, followed by Tukey’s post hoc multiple-range test. p < 0.05 was considered significant. All statistical analyses were performed by GraphPad Prism 8.0 (GraphPad Software). Power analysis was performed by using G × Power. The powers of all the human data in this study were more than 0.85.Data are expressed as means ± SEMs. Mann-Whitney test (unpaired) or Wilcoxon test (paired) was used for human data with two groups. Mouse data were analyzed using the Student’s Xdh, the key synthetase of uric acid, in the mouse uterus from early pregnancy. There was no detectable signal in the uterus during natural pregnancy in mice, from D1 to D4 , while another horn received no stimulation and served as control (Dec Non-S)  . Meanwhic Non-S) .Xdh, we treated ovariectomized mice with either E2 or P4. The results showed that E2 but not P4 induced Xdh expression in only 6 h, indicating a regulation of the estrogen pathway on Xdh  and endometrial tissue UA level (eUA) in both Dec and Non-Dec groups of mice. Compared to Non-Dec mice, which received no stimulation, the sUA levels of decidualized mice were approximately twice higher  were significantly higher in the stimulated uterine horn (Dec S) than the unstimulated horn (Dec Non-S) of the same mouse  . To explric acid . Our resric acid . During ric acid . In contric acid . These rin vitro, together with a well-established decidualization cocktail (E + P). As shown in Prl8a2. Furthermore, MSU crystal induced higher expression of Prl8a2 even without combining with the E + P cocktail  and Interleukin 11   . Previouby Il11) . In this in mESC . Mpges1,ificance . Moreoveastocyst . Further crystal . We also on mESC . Our resin situ synthesis of UA in decidua was also the case in women. We first tested the expression level of XDH in decidual tissue obtained from elective abortion women, which showed dramatic higher XDH expression than non-decidualized endometrium from non-pregnant women, consistent with the observation in mice  treatment  and Il11. Previously, MSU was also reported to significantly stimulated Cox2, IL-11, and other inflammation factors expression in macrophages, osteocyte cell lines, or synovial fluid cells (in situ synthesized MSU may enhance the decidualization via sterile inflammation by up-regulating a network of these inflammatory factors.Evolutionarily, the establishment of pregnancy in mammals, including both placental and non-placental mammals such as opossum, is considered an inflammatory response to the embryo . In humaXDH . Herein,id cells . More imid cells . Moreoveid cells , has beeid cells . On the id cells . These din situ manner in decidualized endometrium, further leading to the formation of MSU crystal in the endometrium. The presence of MSU crystal then enhances the decidualization response via inflammatory pathways. Our study has uncovered the interaction between UA, MSU crystallization, and decidualization during the early stage of pregnancy.In summary, we proved the decidual expressed XDH in both humans and mice, which synthesizes UA in an The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Nanfang Hospital, Southern Medical University the Scientific Research Ethics Committee of the Drum Tower Hospital, Nanjing University Medical School the Ethical Committee of the Women’s Hospital, Zhejiang University School of Medicine. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of South China Agricultural University.Y-YZ, YW, Z-MY, and R-WS designed the study. Y-YZ, YW, S-TC, J-WK, J-MP, X-ZL, and S-YL performed the research. G-JY, A-XL, and Q-TH contributed to the new reagents/analytic tools. Y-YZ, YW, G-JY, A-XL, Q-TH, Z-MY, and R-WS analyzed the data. Y-YZ, YW, Z-MY, and R-WS wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


CSF levels of Tau, NfL, and pNfH were similar between groups, but PrPc and neopterin were elevated in HAM/TSP patients. Most individuals in the control group and all HTLV-1 AC had CSF/serum neopterin ratio < 1.0, and two-thirds of HAM/TSP patients had ratio values > 1.0, which positively correlated with the speed of disease progression and pNfH levels, indicating active neuroinflammation. HAM/TSP patients showed high serum levels of CXCR3-binding chemokines  and elevated CSF levels of CCL2, CCL3, CCL4, CCL17, CXCL5, CXCL10, and CXCL11. Indeed, CXCL10 concentration in CSF of HAM/TSP patients was 5.8-fold and 8.7-fold higher in than in HTLV-1 AC and controls, respectively, and correlated with CSF cell counts. HAM/TSP patients with typical/rapid disease progression had CSF/serum CXCL10 ratio > 1.0 and a higher frequency of CXCR3+Tax+CD4+ T-cells in blood, which indicated a positive gradient for the migration of infected cells and infiltration into the central nervous system. In conclusion, the slow progression of HAM/TSP abrogates the usefulness of biomarkers of neuronal injury for the disease prognosis. Thus, markers of inflammation provide stronger evidence for HAM/TSP progression, particularly the CSF/serum neopterin ratio, which may contribute to overcome differences between laboratory assays.Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease due to axonal damage of the corticospinal secondary to an inflammatory response against infected T-cells. In the present work, we aimed to evaluate biomarkers of neurodegeneration and neuroinflammation in the definition of HAM/TSP prognosis. Neurofilament light (NfL) and phosphorylated heavy (pNfH) chains, total Tau protein, cellular prion protein (PrPc), inflammatory chemokines, and neopterin were quantified in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n=21), HTLV-1 asymptomatic carriers (AC) (n=13), and HTLV-1 seronegative individuals with non-inflammatory non-degenerative neurological disease  (n=9) as a control group. HTLV-1 proviral load in peripheral blood mononuclear cells and the expression of chemokine receptors CCR4, CCR5, and CXCR3 in infected CD4 HTLV-1 infection is endemic in Japan, the Caribbean, Middle East, sub-Saharan Africa, Australia, and South America, particularly in Brazil, and it is estimated that 5 to 10 million people are infected worldwide , 3.HAM/TSP is an inflammatory disorder that affects the spinal cord, causing axonal degeneration and perivascular demyelination that results in a slowly progressive neurological disability and motor impairment without signs of remission , 3. HAM/+ cytotoxic T lymphocytes (CTLs) against infected CD4+ T-cells in the central nervous system (CNS) , there is a predominance of CD8+ CTLs and low virus expression  and tumor necrosis factor α (TNF-α), which are released during anti-HTLV-1 responses by CD8em (CNS) –15. At tpression , 16. It pression .High HTLV-1 proviral load (PVL) in peripheral blood mononuclear cells (PBMCs) has been shown as a risk factor for the development of HAM/TSP –20. IndeBiomarkers of neurodegeneration have been defined for distinct neurological disorders such as multiple sclerosis, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and HIV-associated neurocognitive disorders (HAND) –27. In tin vitro , including the Creutzfeldt-Jakob disease and other diseases such as schizophrenia, bipolar disorder, and major depression –35. PrPcin vitro . To dateThe speed of HAM/TSP progression is typically defined by clinical follow-up and consecutive assessment of the neurological disability using scales to determine the motor impairment . The CSFThis cross-sectional study was conducted at the Instituto Nacional de Infectologia Evandro Chagas, Rio de Janeiro, Brazil, with the approval of the institutional committee of ethics in research (protocol numbers 53518416.9.0000.5262 and 64426517.8.0000.5262). Patients diagnosed with HAM/TSP according to the World Health Organization’s criteria and HTLV-1 asymptomatic carriers (AC) were selected from an open cohort. Initially, patients with HTLV-1 PVL records and on active clinical follow-up were accounted. Then, individuals with HTLV-1 PVL < 1% in peripheral blood leukocytes were excluded. Upon recruiting, 21 HAM/TSP patients and 13 AC agreed to participate in the study after written informed consent Figure 1®-1077  and preserved in liquid nitrogen with 10% dimethyl sulfoxide  in RPMI-1640 medium  with 20% fetal bovine serum .CSF samples were obtained by lumbar puncture into L3/L4 or L4/L5 vertebrae interspace after anesthesia with 1% lidocaine and were maintained in ice bath. CSF cell counts, glucose, and total protein levels were immediately determined by automated routine analysis. CSF samples were centrifuged at 400 × g for 10 min at 4°C, and the supernatant and cellular sediment were stored at -80°C until use. CSF, serum, and blood in EDTA were collected on the same day within a one-hour interval at the maximum. PBMCs were isolated with HistopaqueThe clinical history was obtained from electronic medical records, and HAM/TSP severity was determined with the IPEC-2 disability scale Table 1.The IPEC-2 scale is a variation of IPEC-1 disability scale, which was previously validated , and difCognitive function was evaluated in all patients as part of the routine neurological examination employing the Mini-Mental State Examination ; none ofThe speed of disease progression was defined by the quotient between the disability score and the duration of disease . The first quartile (≤ 0.37 points/year) represented patients with very slow progression, the second and third quartiles (from 0.38 to 1.44 points/year) consisted of patients with typical progression, and the fourth quartile (≥ 1.45 points/year) presented individuals with rapid progression  for the amplification of the HTLV-1 tax and human β-globin genes (tax copies/(β‐globin copies/2). The results are shown as the percentage of infected cells in PBMCs and CSF cells.DNA was extracted from PBMCs and CSF cells with the QIAamp DNA blood mini kit , following the manufacturer’s instructions, and HTLV-1 PVL was determined as previously described . Brieflyin genes  were carCommercially available ELISA kits were used to determine CSF levels of soluble PrPc , NfL , pNfH , and total Tau protein (tTau) . All assays were carried out following the manufacturer’s instructions. Analyte concentrations were determined in Four-Parameter Logistic Curves. Age-adjusted reference values for NfL , and the following pro-inflammatory chemokines were quantified with a multiplex cytometry bead-based immunoassay , according to the manufacturer’s instructions: MCP-1/CCL2; Macrophage inflammatory protein 1α (MIP-1α)/CCL3; MIP-1β/CCL4; Regulated upon activation, normal T cell expressed and presumably secreted (RANTES)/CCL5; Eotaxin/CCL11; Thymus and activation-regulated chemokine (TARC)/CCL17; MIP-3α/CCL20; Growth-regulated oncogene α (GRO-α)/CXCL1; Epithelial-derived neutrophil-activating peptide 78 (ENA-78)/CXCL5; IL-8/CXCL8; MIG/CXCL9; IP-10/CXCL10; and Interferon-inducible T-cell alpha chemoattractant (I-TAC)/CXCL11. Data were acquired in a FACSCanto II flow cytometer . Reference values for neopterin in serum (< 10 nmol/L) and CSF (< 4.2 nmol/L)  were use+ T-cell depletion was performed with Dynabeads CD8  according to the manufacturers’ instructions. CD8-depleted PBMCs were suspended in RPMI-1640 medium with 10% fetal bovine serum and plated at 5×106 cells/well in 24-well microplates. Cells were incubated for 20 h at 37°C with 5% CO2 atmosphere and then stained with Live/Dead Yellow  to determine cell viability, following the manufacturers’ instructions. After, cell surface was stained with anti-CCR4-PE-Cy7, anti-CCR5-PE, and anti-CXCR3-APC-Cy7 monoclonal antibodies (mAbs)  for 30 min at 4°C. Further, intracellular staining was carried out using the FoxP3 intracellular staining kit , and cells were incubated with anti-CD3-BV785, anti-CD4-efluor450 mAbs , and biotin-conjugated anti-HTLV-1 Tax protein mAb . After washing, APC-conjugated streptavidin diluted in 1× permeabilization buffer (1:200) was added to tubes, and cells were incubated for 10 min at 4°C. Cells were washed twice with permeabilization buffer, fixed with 1% paraformaldehyde, and analyzed in a CytoFlex S flow cytometer , with the acquisition of 500,000 events, and data analysis was carried out with CytExpert version 1.2 software . Briefly, live cells (Live/Dead Yellow unstained cells) were gated, and doublets were excluded according to FSC-A and FSC-H distribution. Lymphocytes were gated in FSC-A and SSC-A plots, and the CD3+CD4+ T-cells subset was subsequently defined. HTLV-1 Tax expression was evaluated in the population of CD4+ T-cells, and the frequency of cells expressing CCR4, CCR5, and CXCR3 chemokine receptors was determined in the subsets of Tax+ and Tax− CD4+ T-cells.PBMCs were thawed and washed three times by centrifugation at 300 × g for 10 min at 4°C with RPMI-1640 medium and suspended in phosphate-buffered saline (PBS) with 0.25% bovine serum albumin (BSA) and 2 mM EDTA and kept in ice bath. CD8t-test, with Welch’s correction for distinct variances when applicable, and between three groups using ANOVA test with Bonferroni correction for multiple comparisons. Analysis of nonparametric data of two groups was carried out by Mann-Whitney test, and by Kruskal-Wallis test with Dunn’s post-test for multiple comparisons when comparing three groups. Correlation analysis was performed by Spearman’s rank correlation. The association between qualitative data was evaluated by Chi-square method. Differences with p-value < 0.05 were considered significant. Relative expression of distinct biomarkers between individuals was evaluated by heatmap analysis using the gplots package for R software after log2 transformation of values. Complete agglomeration method and Euclidean algorithm were used to perform clustering and to obtain the distance matrix, respectively.Data were analyzed with GraphPad Prism v.5 and R software v.3.6.1. Descriptive analysis included the frequency of categorical variables and summary measures of quantitative variables: mean, median, standard deviation and interquartile range (IQR). Normal distribution of quantitative data was evaluated with the Kolmogorov-Smirnov test. Comparison of parametric data between two groups was performed by Student’s p < 0.032). All HTLV-1 AC and individuals in the control group presented normal CSF cell counts (≤ 5 cells/mm3), with a median of 1 cell/mm3, while HAM/TSP patients had higher cell counts, with a median of 4 cells/mm3 (p < 0.001), and 38.1% (n=8) presented pleocytosis. In addition, HAM/TSP patients had higher HTLV-1 PVL than HTLV-1 AC in PBMCs  and in CSF cells  . Additionally, at least one of extra motor manifestations was reported in all HAM/TSP patients , while 23.8% (n=5) had moderate, and 14.3% (n=3) had severe neurological involvement Table 2.p = 0.036). Individuals with altered CSF levels of tTau and NfL according to age were observed in both groups. While one HTLV-1 AC and two HAM/TSP patients had altered tTau, elevated NfL concentration was observed in 53.8% (7 of 13) of HTLV-1 AC and 66.7% (14 of 21) of HAM/TSP patients.The CSF of HTLV-1 AC and HAM/TSP patients was examined to determine whether biomarkers of neuronal injury might predict the neurological involvement in HAM/TSP. However, no difference was seen between groups considering the concentration of tTau, NfL, and pNfH (p = 0.059). Elevated neopterin in serum was observed in 85.7% (18 in 21) of HAM/TSP patients. However, 61.5% (8 in 13) of HTLV-1 AC also had elevated levels, and only one individual had an altered concentration in the control group. Neopterin levels were significantly higher in the CSF of HAM/TSP patients  compared to HTLV-1 AC  and HTLV-1-seronegative individuals  , HTLV-1 AC  and HTLV-1-seronegative individuals   was calculated to define the degree of CNS inflammation. All HTLV-1 AC had values < 1.0, indicating that inflammation was more pronounced or restricted to the periphery, with a median of 0.48 (IQR 0.42 - 0.72), and HTLV-1-seronegative individuals presented a median of 0.71 (IQR 0.40 - 1.06) (CSF/serum ratio of 1.24 (IQR 0.82 - 1.59), which was significantly higher , indicating that the speed of disease progression is related to the intensity of CNS inflammation. Although neopterinCSF/serum ratio differentiated most individuals according to the speed of disease progression, it better indicated the disease activity, which is supported by the positive correlation with CSF levels of pNfH .HAM/TSP progression correlated with neopterinChemokines are proteins with cell-type specific chemotactic functions and are involved in cell homing and recruitment to sites of inflammation. It was observed that serum levels of CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CXCL1, CXCL5, and CXCL8 had no significant differences between groups Figure 5The CSF concentration of distinct inflammatory chemokines is shown in The correlation analysis between the concentration of chemokines showed similar patterns in the serum of HTLV-1 AC and HAM/TSP patients, likely associated with immune responses against the infection  and lower (320 pg/mL) cut-offs were proposed 0 pg/mL aFigure 6R = 0.468, p = 0.033), higher pNfH levels , elevated neopterin in the CSF , and increased neopterinCSF/serum ratio . A correlation was also seen between the CXCL10CSF/serum ratio and the disease progression rate , although it did not differentiate between individuals with very slow and typical or rapid progression (data not shown). Moreover, higher CSF levels of neopterin in HAM/TSP patients were associated with elevated CSF concentration of CCL2 , and CXCL8 .The CSF to serum ratio concentration of chemokines was calculated, and values ≥ 1.5 (CSF concentration 50% higher than in serum) were considered suggestive of a positive gradient to attract immune cells to the CNS. A proportion of HTLV-1 AC and HAM/TSP patients presented CCL2, CXCL8, and/or CXCL10 levels in the CSF higher than in serum, which was also observed among HTLV-1-seronegative controls . According to Tax expression, distinct patterns of CCR4, CCR5, and CXCR3 expression in CD4+ T cells were observed , NfL  and pNfH (677.3 pg/mL), and patient 3G displayed high NfL concentration , suggesting the presence of subclinical neuronal injury. Conversely, low relative expression of biomarkers of neuroinflammation was observed.Most HTLV-1 AC (11 of 13) clustered closely. This group was characterized by low relative expression of inflammatory chemokines and neopterin and normal CSF cell counts. On the other hand, no specific pattern was observed for biomarkers of neuronal death  and HTLV-1 PVL. Two HTLV-1 AC that clustered separately , and increased pNfH levels (554.3 pg/mL) confirming ongoing CNS inflammation.The analysis according to the speed of HAM/TSP progression did not reveal any specific pattern for biomarkers of neuroinflammation and axonal degeneration. Only two patients had an intense elevation of tested biomarkers of neuronal death, an HTLV-1 AC (Patient 5C) and a 61-year-old woman (Patient 5B) with early HAM/TSP onset (14 months) and mild disability, which showed high levels of tTau (490.5 pg/mL), NfL , pNfH . Despite normal CSF cell counts, patient 5B had HTLV-1 PVL in CSF cells (9.37%) 15-fold higher than in PBMCs (0.60%). On the other hand, a cluster of patients with typical or rapid disease progression showed pleocytosis and intense relative expression of neopterin and inflammatory chemokines. Among them, patient 4H had 14 years of disease, the highest concentration of tTau (813.4 pg/mL), and increased neopterin levels, although NfL and pNfH levels were unaltered. Patient 5G had moderate neurological disability and was the only one to have a CSF/serum ratio ≥ 1.5 for CCL3 (ratio = 4.16) and had the highest ratio values for CXCL8 (ratio = 18.53) and CXCL10 (ratio = 19.38). CSF analysis also revealed mild elevation of total proteins (63.8 mg/dL), mononuclear pleocytosis . In addition, to overcome differences implicit to neopterin quantification by distinct methods, we propose using neopterinCSF/Serum ratio instead of concentration cut-offs.HAM/TSP is a chronic demyelinating inflammatory disease that typically presents with a slow course. Despite the correlation between HAM/TSP disability severity and illness duration, a small proportion of individuals show a very slow progression with overall mild motor impairment. On the other hand, faster disease progression at early stages may represent an important indicator of deterioration in the clinical picture of HAM/TSP. Thus, evidence-based prognosis may improve the decision for therapeutic intervention and consecutive follow-up. Neopterin and CXCL10 have been proposed as biomarkers for HAM/TSP activity and disease prognosis since increasing CSF levels of these factors correlate with rising scores in the Osame Motor Disability Score –41. AccoCSF/Serum ratio values among HTLV-1 AC were < 1.0, indicating that inflammation was more pronounced or restricted to the periphery.Neopterin concentration commonly correlates with cell-mediated immune processes during inflammation , 48. Neo+ cells to the CNS, which are mostly IFN-γ+CD4+ T-cells infected by HTLV-1  and uninfected CD4+ T-cells. Therefore, HAM/TSP patients have a stronger potential of infected cells to migrate towards the CNS in response to the CXCL10 concentration gradient, confirming the role of this chemokine in the chronic CNS inflammation associated with HAM/TSP.CCL2 binds to CCR2 and directs monocyte/macrophage infiltration into the CNS , while C T-cells , 57. HowIt has been shown that CCL2/CCR2 signaling is upregulated and plays a role in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease, multiple sclerosis, HAND, and in the experimental autoimmune encephalomyelitis, a murine model for inflammatory demyelinating diseases , 65. CCLFree PrPc in the CSF, but not in the serum, represents a specific biomarker for neurocognitive impairment and CNS dysfunction in individuals with HAND . The PrP+ CTLs, and monocytes. In turn, CXCL5 is a ligand of CXCR2, which is expressed by neutrophils  were not helpful to define HAM/TSP prognosis. Still, they evidenced neuronal death associated with exacerbated inflammation, particularly indicated by neopterin levels. Therefore, quantification of neopterin in the CSF and serum to calculate the neopterinThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the committee of ethics in research of the Instituto Nacional de Infectologia Evandro Chagas. The patients/participants provided their written informed consent to participate in this study.OME designed the study. ACCBL, MASDL, MTTS, and AQCA conducted the neurological assessment and clinical follow-up of patients. FSS, NLF, RCT, YCPG, and ILS-F performed assays and data analysis. OME and YCPG carried out statistical analysis. OME, FSS, YCPG, JE-L, ACCBL, MASDL, MTTS, and AQCA drafted and corrected the manuscript.OME was awarded with grants in the INOVA Program - Fundação Oswaldo Cruz (Grant number VPPCB-008-FIO-18-2-39). NLF and YCPG were granted with Master’s scholarships from the Fundação Oswaldo Cruz and the Coordination for the Improvement of Higher Education Personnel (CAPES) of the Brazilian Ministry of Education, respectively. This study was partially supported by the Coordination for the Improvement of Higher Education Personnel  - Finance Code 001.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Leishmania (Leishmania) infantum and treated with UA-NLC showed lower parasitism than the infected controls, animals treated with UA or Amphotericin B (AmB). The therapeutic activity of UA-NLC was associated with the increase in a protective immune response, and it was associated with a high degree of spleen and liver preservation, and the normalization of hepatic and renal functions. These data indicate that the use of lipid nanoparticles as UA carriers can be an interesting strategy for the treatment of leishmaniasis.Ursolic acid, a triterpene produced by plants, displayed leishmanicidal activity in vitro and in vivo; however, the low solubility of this triterpene limits its efficacy. To increase the activity of ursolic acid (UA), this triterpene was entrapped in nanostructured lipid carriers (UA-NLC), physical-chemical parameters were estimated, the toxicity was assayed in healthy golden hamsters, and the efficacy of UA-NLC was studied in experimental visceral leishmanisis. UA-NLC exhibited a spherical shape with a smooth surface with a size of 266 nm. UA-NLC displayed low polydispersity (PDI = 0.18) and good colloidal stability (−29.26 mV). Hamsters treated with UA-NLC did not present morphological changes in visceral organs, and the levels of AST, ALT, urea and creatinine were normal. Animals infected with  Leishmania that is transmitted to mammalian hosts during a phlebotomine vector blood meal. Leishmaniasis has been considered a serious public health problem, given the wide geographical distribution, the number of pathogenic species to humans and other vertebrates, diversity of clinical forms, and the scarcity of drugs available for therapy [Leishmaniasis is a neglected tropical disease, caused by a parasite of the genus  therapy ,2.The effectiveness of leishmaniasis treatment depends on several factors, such as drug choice, host immune response, parasite strain, treatment regimen, as well as patient compliance . The treLeishmania sp., Trypanosoma cruzi, and Toxoplasma gondii [L. (L.) amazonensis, L. (L.) braziliensis, L. (L.) donovani, L. (L.) guyanensis, and L. (L.) infantum [Studies have shown that medicinal plants and their purified molecules, present attractive features to develop new antileishmanial agents, such as selectivity, low toxicity to the experimental hosts, and importantly, high leishmanicidal potential . In thisa gondii ,9,10. Ininfantum ,12,13, sinfantum . AltogetAlthough the activity of this triterpene has been recorded in experimental leishmaniasis, the main drawback of UA is related to the low solubility in common physiological diluents, which may indeed impact its efficacy in the treatment of leishmaniasis. To overcome this disadvantage, UA can be encapsulated into different types of nanocarriers, which would increase the solubility and availability of this molecule in vivo. Additionally, it is well recognized that drug delivery using nanocarriers can overcome physiological barriers, such as the lipidic membrane of cells, and thus a high concentration of drug will be accumulated in the cytosol of cells as well as the phago-lysosomal compartment. This strategy of treatment may improve the efficacy of the therapy and reduce the toxicity, as the drug release happens at specific locations ,16. AmonLeishmania species, this study aimed to develop a nanocarrier loaded with UA to enhance the effectiveness of this triterpene in experimental visceral leishmaniasis caused by L. (L.) infantum.Considering the scarcity of safe and effective approved drugs for the treatment of leishmaniasis and the activity of UA towards ® 80) were purchased from Acofarma® . UA (purity ≥ 98%) and AmB (purity ≥ 99%) were purchased from Cayman chemical company  and Cristalia Laboratory , respectively. For the HPLC assay, acetonitrile was obtained from VWR , ultrapure water  was obtained from EMD Millipore . Cell culture media were bought from Sigma-Aldrich .Solid lipid cetyl palmitate (CP) was provided by Gattefossé SAS , the liquid lipid miglyol 812, and polysorbate 80  at a stirring speed of 10,400 rpm for 5 min. For the NLC formation, the prepared emulsion was quickly transferred to the high-pressure hot homogenizing equipment , being homogenized during five cycles at 600 bar, and then cooled to 25 °C in an ice bath. The samples were stored in glass bottles at 4 °C. Empty NLC was prepared similarly, without adding UA.NLC and UA-NLC nanocarriers were prepared by high-pressure homogenization technique. CP (solid lipid—2%) with the mygliol-812 (liquid lipid—3%) and an aqueous solution of polysorbate 80 were heated to 70 °C, separately. Then, UA (0.1%) was dissolved in the lipid phase and the aqueous phase was added. The emulsion was submitted to a homogenization process in Ultra-Turrax T25, with S25N—18G dispersing element  was used with acetonitrile: water  mixture as the mobile phase, with a flow rate of 1.0 mL·min−1; UA was detected at 210 nm wavelength [In all experiments, UA was quantified using the UltiMate 3000 HPLC apparatus , with a UV-VIS detector, and automatic injector. A reverse-phase C18 column , the supernatant collected, and filtered through a 0.45 µm PTFE syringe filter . The supernatant was diluted (1:6) in eluent solution and applied to the HPLC column and the amount of UA released quantified. The encapsulation efficiency (EE) was calculated according to the following equation:The freshly prepared formulation was diluted in milli-Q water (1:5), filtered through a 5 µm nitrocellulose membrane filter , and diluted 1:10 in ethanol to extract UA from NLC. The mixture was centrifuged at 4620× Z-average), PDI, and ZP were obtained by calculating the mean value of six measurements, performed in three samples.Particle size and distribution (polydispersity index) were analyzed by Dynamic Light Scattering . The zeta potential (ZP) of NLC dispersions was measured by Electrophoretic Light Scattering in a zeta potential analyzer . Samples were diluted (1:200) in milli-Q water, yielding a suitable scattering intensity. The samples were analyzed at room temperature, with a fixed light incidence angle of 90°; the mean hydrodynamic diameter . An aliquot of nanoparticles (10 μL) was placed on nickel gratings with Formvar mesh/carbon film . Samples were contrasted with 1% uranyl acetate solution. Samples were analyzed under a microscope at a voltage of 120 kV. Images were recorded using a CCD digital camera Orious 1100 W Tokyo, Japan.Mesocricetus auratus), 8 weeks old, were obtained from the Anilab . This study was performed in accordance with the recommendations of the guide for Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation. The Ethics Committee of Animal Experiments of the Institutional Committee of Animal Care and Use at the Faculdade de Medicina da Universidade de São Paulo (FMUSP) approved the following protocol 056/16, from 22 July 2016. Hamsters were housed in the Animal Experimental Instituto de Medicina Tropical da Universidade de São Paulo (IMTUSP), according to the standards of the Committee of Animal Welfare.Golden hamsters ; Group 6 was treated with 5.0 mg/kg of AmB  and GrouL. (L.) infantum (MHOM/BR/72/46) was provided by Prof. Dr. Fernando Tobias Silveira, from the cryobank of the Leishmaniasis Laboratory Prof. Dr. Ralph Laison, Department of Parasitology, Instituto Evandro Chagas . Parasites were identified using monoclonal antibodies and isoenzyme electrophoretic profiles. L. (L.) infantum parasites were maintained in Schneider’s Medium , supplemented with 10% heat-inactivated fetal bovine serum and 50,000 IU/mL penicillin, 50 μg/mL streptomycin (S10). Stationary phase promastigotes were used throughout the entire study.7L. (L.) infantum promastigote forms. The noninfected control group was injected with PBS alone. Infected hamsters were divided into 8 groups, with 5 animals each. After 60 days of infection, the treatment was initiated with UA, UA-NLC, AmB or empty NLC. The experimental groups were arranged as follows: Groups 1 and 2 were treated with UA-NLC, containing 1.25 and 5.0 mg/kg of UA, respectively; Groups 3 and 4 were treated with 1.25 and 5.0 mg/kg of UA, respectively; Group 5 was treated with NLC ; Group 6 was treated with 5.0 mg/kg of AmB [Leishmania-specific IgG and IgG2a by Enzyme-Linked Immunosorbent Assay (ELISA) ; AST, ALT, creatinine, and urea were quantified by a colorimetric method on COBAS C111 equipment , as detailed in the Golden hamsters (8 weeks old) were infected intraperitoneally with 2 × 10g of AmB ; Group 7The splenic and hepatic parasitism was quantified by limiting dilution assay . Briefly®VILO™ cDNA Synthesis Kit . Amplification conditions consisted of an initial denaturation phase at 95 °C for 10 min, followed by 40 amplification cycles consisting of 95 °C for 15 s (s); 61 °C for the 90 s, and 72 °C for 30 s. All reactions were performed in a Mastercycler Nexus GSX1 . Before quantification, the efficiency of each reaction was verified using cDNA from the spleen of healthy animals; that was always above 95%. β-actin (endogenous control) was used to normalize the expression level of the genes. qPCR reactions were performed using the GoTaq® 1-Step RT-qPCR System  and 75 nM of primers. The primer sequences were as follows (5′ to 3′): IFN-γ forward: GACAACCAGGCCATCC and reverse: CAAAACAGCACCGACT; interleukin -10 (IL-10) forward: TGGACAACATACTACTCACTG and reverse: GATGTCAAATTCATTCATGGC; enzyme inducible nitric oxide synthase (iNOS) forward: CGACGGCACCATCAGAGG and reverse: AGGATCAGAGGCAGCACATC; β-actin forward: TCCTGTGGCATCCACGAAACTACA and reverse: ACAGCACTGTGTTGGCATAGAGGT [−ΔΔCt over the infected control group [RNA from hamster spleen fragments (~10 mg) was purified using the commercial RNeasy Mini Kit . Isolated RNA was used to synthesize cDNA with the SuperScriptATAGAGGT ,23,24. Qol group . PCR prool group  was alwaL. (L.) infantum was used to analyze the humoral immune response by ELISA. Briefly, promastigote forms in stationary phase of growth were collected by centrifugation at 3000 rpm, 10 min, 4 °C, and the pellet was washed three times with PBS. The pellet was resuspended in 100 μL of PBS and immediately frozen in liquid nitrogen. Following this step, the pellet was thawed at room temperature. This cycle of freeze and thaw was repeated three times, allowing parasite lysis. The lysate was centrifuged at 10,000 rpm, 30min, 4 °C; the supernatant was collected and the concentration of protein was determined using the Bradford method . Ninety-six-well high-binding ELISA plates  were coated with the soluble antigen of the promastigote forms of L. (L.) infantum (1.0 μg of protein/well) in the carbonate-bicarbonate buffer, pH 9.6 , for 18 h at 4 °C. After this period, the wells were washed three times with PBS plus 0.05% Tween 20 (PBST), and nonspecific bindings were blocked with 10% skimmed milk diluted in PBS for 120 min at 37 °C. The wells were washed three times with PBST, and 100μL of animal serum (diluted 1: 1000 in PBST) were added to each well and the plate was incubated for 60 min at 37 °C. After this period, the wells were washed three times with PBST, and the secondary antibodies goat anti-hamster IgG  or IgG2 , both conjugated with horseradish peroxidase—HPR— were added to the wells for 60 min at 37 °C. After this step, the wells were washed five times with PBST, and the substrate 3.3′, 5.5′ tetramethylbenzidine—TMB— was added to the wells for 15 min. The reaction was blocked by the addition of 50 μL/well of sulfuric acid (2N) and the absorbances were read in an ELISA reader at 450 nm wavelength. Serum from animals chronically infected with L. (L.) infantum and healthy animals were used as positive and negative controls of the reactions, respectively.Soluble antigen of t-test. Differences were considered statistically significant at the 5% level (p < 0.05). Values were expressed as mean ± standard deviation from a minimum of three independent experiments.Statistical analyses were performed using the GraphPad Prism 5.0 software  and the nonparametric test Kruskal–Wallis, followed by Dunn’s multiple comparison test, was used. Differences between two groups were analyzed by an unpaired The size of nanoparticles (PS), index of polydispersity (PDI), zeta potential (ZP) and efficacy of encapsulation (EE) are shown in The morphology of the lipid nanoparticles, observed by TEM, revealed that both NLC A and UA-p < 0.05), as observed in Golden hamsters treated with UA-NLC did not show significant changes in the levels of AST A and ALTp < 0.05) compared with the control group .L. (L.) infantum and treated with 1.25 or 5.0 mg/kg of UA-NLC or UA a significant reduction in the number of parasites in comparison with the infected controls was observed (p < 0.05). Furthermore, it was observed that UA-NLC (1.25 mg/kg) showed superior therapeutic activity than UA (1.25 mg/kg) at eliminating splenic and hepatic amastigotes (p < 0.05). AmB also reduced splenic and hepatic parasites in comparison with the infected control (p < 0.05); however, animals treated with 5.0 mg/kg of UA-NLC displayed superior antileishmanial properties than AmB in both organs (p < 0.05).In the spleen A and livHistological sections of the spleen and liver were immunolabelled to observe amastigote forms C,D and lHistological sections from the spleen of infected control groups (infected nontreated or treated with empty NLC), shown in In the animals from the infected control group, NLC, and those treated with 1.25 and 5.0 mg/kg of UA-NLC or UA, the main histopathological finding in the liver was related to periportal inflammation with the presence of parasitized macrophages, and granulomas in the portal space and parenchyma A–H. Addip < 0.05). Additionally, it was found that the groups treated with UA-NLC (1.25 or 5.0 mg/kg) expressed higher levels of IFN-γ than animals treated with UA at the same doses  or UA (5.0 mg/kg) expressed higher levels of IFN-γ A than thme doses A. The exme doses B. A signme doses C. A signme doses C.p < 0.05).The analysis of the humoral immune response showed that only hamsters treated with 5.0 mg/kg UA-NLC produced a significant amount of antileishmanial IgG A and IgGp < 0.05), and AST level was similar with the healthy animals (p < 0.05), and these values were close to the normal levels observed in the healthy group (p < 0.05).Animals treated with UA-NLC or UA did not change the levels of AST and ALT in comparison with the infected control A,B; alth animals A. Animalhy group C. Urea lhy group D, exceptNanotechnology has been considered an important tool to reduce side effects and increase the effectiveness and selectivity of drugs . In thisIn the present study, high-pressure homogenization was used to formulate UA-NLC that exhibited a monodisperse distribution with a suitable mean size (266 nm) and PDI of 0.18, which indicates a homogenous and narrow size distribution of the nanoformulations. Particles with a size between 100 and 500 nm may be used in human and/or animal therapy, since they remain in circulation longer ,29, enabThe properties of UA-NLC on the visceral organs and on the metabolism of the liver and kidney of healthy golden hamsters were investigated. In the histopathological study, it was verified that the treatment of hamsters with UA-NLC did not change the morphology of the spleen, liver, kidney, lung or heart. Hepatic and renal functions of animals were also investigated, since these organs are responsible for the metabolization and excretion of drugs ,34. UA-NIn experimental models of liver damage, it was observed that UA presented hepatoprotective potential when administered by the oral route ,37,38. IIn contrast to UA-NLC, it was observed that AmB caused morphological changes in the medullary area of the kidney along with an increase in creatinine levels in the blood, suggesting that the treated animals developed acute renal failure . A commoL. (L.) donovani, this formulation was 94 times more active than miltefosine, 5 times more than sodium stibogluconate and 2 times more than paromomycin. According to a study conducted by Kar and collaborators [L. (L.) donovani resistant or not to stibogluconate sodium and paromomycin. Additionally, it was observed that oral treatment with cedrol entrapped into NLC displayed higher efficacy in murine visceral leishmaniasis caused by wild or drug-resistant L. (L.) donovani than cedrol or miltefosine. Thus, the data shown herein indicate that NLC is an interesting platform to deliver drugs, and the formulation UA-NLC was more active than UA in visceral leishmaniasis.In infected golden hamsters, it was verified that animals treated with UA-NLC or UA exhibited a significant reduction in splenic and hepatic parasitism, suggesting that both UA and UA-NLC presented high therapeutic potential. The number of parasites reduced as the UA dose increased, and this effect was maximized when UA was encapsulated. It was observed that treatment with UA at 1.25 mg/kg  reduced parasitism by 74.20% in the spleen and 90.04% in the liver, while the same dose of UA loaded into NLC decreased splenic and hepatic parasitism by 98.63 and 99.78%, respectively. Animals treated with 5.0 mg/kg of UA  had a reduction in parasitism by 98.03% in the spleen and 99.79% in the liver, while a reduction of 99.92 and 99.98% in splenic and hepatic parasite loads, respectively, was observed in animals treated with 5.0 mg/kg of UA-NLC. These data suggest that NLC improved the efficacy of UA when compared to UA administered freely, reinforcing that nanoparticles can be good carriers of drugs, as it has been observed in different studies ,49,50,51borators , NLC potIn addition to the antileishmanial activity, UA-NLC also modulated the immune response of golden hamsters with visceral leishmaniasis, increasing the expression of IFN-γ transcripts more efficiently than the groups treated with UA. Effective immunity in leishmaniasis is mediated by IFN-γ, which activates macrophages to a leishmanicidal state . If corrLeishmania IgG, subsequently classified as IgG2 isotype. In experimental murine leishmaniasis, antibodies can be used as markers of resistance, as is the case with IgG2 isotype, which is produced upon the increase in IFN-γ; therefore, it has been associated with resistance and Th1 immune response development [L. (L.) donovani [L. (L.) infantum. In contrast, it was observed that animals treated with AmB displayed a significant decrease in IFN-γ and iNOS gene expression, which may be associated with an inhibition of the inflammatory response, caused by the low number of parasites [In addition to the elevation of the cell immune response, it was observed that infected animals treated with 5.0 mg/kg of UA-NLC increased the levels of anti-elopment ,57. In telopment  or L.  infantum. Importantly, the nanoformulation exhibited superior therapeutic activity than UA given in free form and AmB, which, although it has a significant therapeutic activity, is nephrotoxic, limiting its use in infected animals as well as humans. Thus, the UA loaded into NLCs can be considered an important and promising approach in the treatment of leishmaniasis.Taken together, the results obtained herein demonstrate that UA-NLC was stable and showed a homogeneous morphology and size. Additionally, UA-NLC proved to be safe for use in golden hamsters and it showed therapeutic activity in experimental animals with visceral leishmaniasis caused by


However, impaired oxidant-antioxidant balance and extreme growth conditions in plants are bound to cause increases in the cellular concentrations of radical and non-radical ROS such as superoxide anions (O2•−), hydroxyl radical (OH•), singlet oxygen (1O2), and hydrogen peroxide (H2O2). On the one hand, H2O2 has no unpaired electrons and is moderately reactive. Owing to its relative stability compared to other ROS and its capacity for diffusing through aquaporins in the membranes and over more considerable distances within the cell  of varied types can be yielded in plants at several primary sites  under normal aerobic metabolism 2O2 gets produced in routine in stressed or non-stressed conditions via dismutation of O2•− radicals through superoxide dismutase (SOD) during electron transport in different compartments of the plant cell, and is involved in the regulation of the plant growth, metabolism, and stress tolerance. It has also been noted that at higher concentrations in the cell during oxidative stress, ROS, including H2O2, can oxidize vital biomolecules  and significantly impacts the seed germination process . Among the major abiotic stress factors, several heavy metals provoke increases in the production of ROS through plasma membrane-bound NADPH oxidases. However, the relationship of H2O2 has also been established in heavy metal tolerance in crop plants . H2O2 directly mediates metal-induced oxidative signaling, where the production of H2O2 may involve H2O2 receptors, redox-sensitive transcription factors and inhibition of phosphatases  pathways  can be detrimental for the plant cell leading to premature cell death, a fine-tuning of these signaling molecules (H2O2 and PAs) can result in stress management by coordinating intra-cellular and systemic signaling systems . Polyamine oxidase (PAO)-induced production of H2O2 was found to be involved in the coleorhiza-limited rice seed germination . ROS-specific probe DCFH2-DA enabled confocal laser scanning microscopy revealed a high level of ROS in the stigma at different developmental stages  of scrutinized plants .As a potent signaling molecule HSingh et al.). Notably, NADH oxidase (RBOH), alternative oxidase (AOX), the plastid terminal oxidase (PTOX), and the malate valve with the malate dehydrogenase isoforms are involved in maintenance of the cellular redox homeostasis under salinity stress (Hossain and Dietz). In Arabidopsis cell suspension cultures, anoxia stress/shock led to significant increases in H2O2 ; however, re-oxygenation maintained the components of ROS scavenging machinery like ascorbate-glutathione (AsA-GSH) system, α-tocopherol, and eventual cell survival as result of decreased H2O2 . Eutrema salsugineum  and Arabidopsis thaliana (glycophyte) exhibited a differential pattern of accumulation and scavenging of ROS. In particular, compared to A. thaliana chloroplasts, E. salsugineum chloroplasts showed a constitutive increase and the cell's steady-state regulation of H2O2 level which prepared this plant for ROS-control mainly due to an efficient ROS-scavenging machinery including glucosinolates content and well-coordinated tuning of hormonal signaling . Elevation in the cellular level of H2O2 and its consequences can be controlled by brassinosteroids, a class of plant-specific essential steroid hormones. To this end, in tomato seedlings, brassinosteroid (24-epibrassinolide) ameliorated the impacts of zinc oxide nanoparticles-caused elevated H2O2 by enhancing the activity of enzyme involved in superoxide-dismutation (SOD), H2O2-metabolizing enzymes , increasing GSH-regeneration , finally inducing the transcripts of Cu/Zn SOD, GSH1, CAT1, and GR1 . In a comprehensive in silico study, APX and GSH-peroxidase (GPX) genes/proteins from 18 different plant species were identified and compared in order to unravel their significance in excessive H2O2 management . Notably, APX and GPX were found to be involved in the metabolism of antioxidants and secondary metabolites, redox homeostasis, stress adaptation, and photosynthesis/respiration. The major redox proteins namely plant peroxiredoxins (Prxs) and sulfiredoxins (Srxs) are involved in antioxidant defense and redox signaling in stressed plants. Srxs were are also found to be involved in antioxidant defense and redox signaling in response to environmental stimuli; post-translational modifications of Srxs regulate the ROS-transduction and bioactivity. On the other hand, Prxs are sensitive to glutathionylation. Investigation of the glutathionylation of recombinant chloroplastic 2-Cys Prx and mitochondrial Prx IIF of pea plants revealed glutathionylation-mediated change of the decameric form of 2-Cys Prx into its dimeric glutathionylated form. Additionally, the reduced dimeric form of Prx IIF was glutathionylated without changing its oligomeric state . Thus, glutathionylation was argued to depend on the GSH/GSSG ratio owing to the perceptible difference in the exact effect on the 2-Cys Prx and Prx IIF proteins.During evolution, plants have developed an efficient ROS-scavenging system constituting an array of enzymatic  and non-enzymatic  antioxidants to get rid of excessive ROS in the cell  helps in biotic and abiotic stress tolerance in various crop plants by triggering the ROS scavenging machinery . In a study on mustard (Brassica juncea L.) cultivars, H2O2-induced reversal of the major negative impacts of Ni stress (200 mg Ni kg−1 soil) led to increased photosynthetic nitrogen-use efficiency, sulfur-use efficiency, and GSH content and decreased levels of lipid peroxidation and electrolyte leakage . Notably, H2O2 priming-mediated increased tolerance to cadmium-caused oxidative stress in Brassica napus involved fine-tuning between the glyoxalase system and the components of ROS-scavenging machinery .H2O2, other signaling molecules  and phytohormones  play key roles in stress signaling cascades and crosstalk during plants' stress responses . Notably, the role of SA in adventitious root formation involved H2O2 acting as a downstream messenger . ABA-induced H2O2 accumulation can protect plant parts (such as pumpkin-grafted cucumber leaves) against Ca(NO3)2 via ABA/H2O2 signaling-led induction of ROS-scavenging machinery . S-nitrosoglutathione reductase (GSNOR) determines the level of S-nitrosothiol and thereby regulates NO-signaling in plants .Along with HRecent insights into the double role of hydrogen peroxide in plants,” the contributions discussed the versatile role of H2O2 as a signaling molecule that triggers the upregulation of the components of antioxidant defense machinery and imparts tolerance in crop plants against the variety of environmental cues. The crosstalk of H2O2 with other signaling molecules and phytohormones leads to signal transduction in response to various stresses and regulates plant growth, development, and stress tolerance. Therefore, further understanding on the coordination of H2O2 and other signaling molecules NO, In the current Research Topic “NA and SG prepared the first draft of the manuscript. FC, CO-V, LH, NT, AS, MH, and MF read and revised the manuscript. All authors listed approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Purinergic signaling regulates neuronal and glial cell functions in the healthy CNS. In neurodegenerative diseases, purinergic signaling becomes dysregulated and can affect disease-associated phenotypes of glial cells. In this review, we discuss how cell-specific expression patterns of purinergic signaling components change in neurodegeneration and how dysregulated glial purinergic signaling and crosstalk may contribute to disease pathophysiology, thus bearing promising potential for the development of new therapeutical options for neurodegenerative diseases. Neurodegenerative diseases are characterized by a progressive loss of structure and function of the CNS. An estimated 50 million patients worldwide are currently affected by neurodegenerative diseases and it has been projected that this number will rise to 131 million patients by 2050  (SLC17A9/VNUT) or ultimately by membrane rupture during cellular injury, allowing them to trigger purinergic receptor signaling  . Adenosiignaling , 16.Purinergic receptors are subclassified into two large families: P1 receptors that are activated by nucleosides, and P2 receptors that are activated by nucleotides. P2 receptors are further subdivided into ionotropic P2X receptors and metabotropic P2Y receptors , 18. So-Purinergic signaling controls important physiological processes in the healthy CNS, such as synaptic transmission, cell proliferation and innate immune response , 12. ATPADORA3, P2RY12, P2RY13, and at lower levels P2RX7 and P2RY14 genes) and ectonucleotidases  in the human cortex. There are significant differences in murine microglial gene expression of purinergic components compared to human. For example, Adora1, P2rx4 and Entpd2 show higher expression levels in mouse. Additionally, genes encoding ENTs  are expressed at higher levels in mouse microglia, suggesting that nucleoside transport processes may differ between humans and mice. Similarly, cell-specific expression of some ectonucleotidases, including Entpd1 in microglia are considerably different in human and mouse  and ectonucleotidases . Similar patterns can be found in mouse astrocytes  in oligodendrocytes show a higher expression in mouse  and NT5E (CD73) are expressed in all CNS cell types in the human CNS, albeit in case of CD73 at very low and in case of CD39 at high levels in microglia compared to the other cell types  and Entpd1 (downregulated) that are consistent between several data sets in mice. However, the majority of differentially expressed genes is only detected as differentially expressed in a few studies. Some inconsistencies in expression changes of some genes including upregulation of P2RY6 and P2RY12 in Zhou et al.  and Adora3 (A3) that can be detected in the transcriptomic studies in neurodegeneration  is barely detectable under normal conditions (Ptgs2 (Cyclooxygenase 2) expression  serves, among other functions, as a signal to regulate neuronal activity, since BDNF-mediated disruption of chloride gradient and following disturbance of chloride-dependent GABAergic inhibitory function was reported –125. Exoin vitro  are upregulated in AD mouse models, although not consistently across the different studies, while only Gja1 was found upregulated in human astrocytes in AD , Entpd2, and Enpp4 are downregulated in AD models or human AD. Furthermore, different sets of purinergic genes were found upregulated (P2ry6) or downregulated  in an ALS mouse model.Based on the transcriptomic data we assembled here , it appees in AD . AdditioFunctionally, the P2Y1 receptor signaling pathway is one of the major purinergic signaling pathways that may contribute to altered astrocyte properties in neurodegeneration, especially in AD. P2Y1 mediates calcium waves in astrocytes, which are linked to gliotransmitter release and synchronization of astrocytic syncytium, which is critical for synaptic plasticity –151. UnlENTPD2) at Braak stage III-IV in AD  in astrocytes found in the transcriptomic analyses from AD mouse models and human AD brains we summarized here , connexin-32 (CX32 or Gjb1) or connexin-47 (CX47 or Gjc2) in a manner similar to CX43 in astrocytes cannot be ruled out  together with a distinct set of activated glial purinergic signaling pathways  and loss of homeostatic neuronal and glial purinergic signaling pathways  during neurodegeneration may alter the balance of purinergic signaling homeostasis. This in turn could lead to more cellular damage , loss of homeostatic functions  and promote protein aggregation (reduced phagocytosis of protein aggregates), thus ultimately favoring the progression of disease pathology in neurodegenerative diseases.Recent advances have highlighted the beneficial effect of targeting purinergic signaling in neurodegenerative diseases, notably with the authorization of istradefylline, a selective antagonist of A2A receptors, in the co-treatment of Parkinson's disease in the USA in 2019 , followiP2X7 is one of the most evident targets of glial purinergic signaling in neurodegeneration, being upregulated in both microglia and oligodendrocytes at the protein levels . It was ex vivo hyperactive motility around Aβ plaques in the 5xFAD amyloidosis model (Adenosine receptors also represent suitable targets for glial modulation. Administration of the A2A receptor antagonist preladenant partly decreased is model . In addiis model . Severalis model , 167. Adis model . AltogetApart from purinergic receptors, several studies have highlighted elevated astrocytic CX43 in neurodegenerative diseases, leading to increased ATP release. Together with other nucleotide transporter such as pannexin-1, they could be targets of choice in order to decrease ATP release and aberrant purinergic signaling in neurodegeneration .As outlined here, purinergic signaling in neurodegeneration is not only altered in neurons, but in all CNS cell types, including glial cells. This highlights the potential to target purinergic signaling in a multi-cellular fashion. However, to develop this as a valuable strategy in the future, many functional aspects of purinergic signaling in glial cells need to be further elucidated. In particular, purinergic signaling in astrocytes and oligodendrocytes have remained ill-defined, as well as purinergic signaling pathways in proteinopathies involving FUS or TDP-43 aggregates. Furthermore, novel tools are needed that help to better define the cell-specific and the spatio-temporal aspect of purinergic signaling in neurodegeneration. These challenges will need to be faced in the future to better understand this fascinating system, as within it potentially lies the hopes of tomorrow's treatments.MP, AG, SK, AH, and KC wrote the manuscript. MP, AG, SK, DB, AH, and KC reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Receptor tyrosine kinases (RTKs) are membrane receptors that regulate many fundamental cellular processes. A tight regulation of RTK signaling is fundamental for development and survival, and an altered signaling by RTKs can cause cancer. RTKs are localized at the plasma membrane (PM) and the major regulatory mechanism of signaling of RTKs is their endocytosis and degradation. In fact, RTKs at the cell surface bind ligands with their extracellular domain, become active, and are rapidly internalized where the temporal extent of signaling, attenuation, and downregulation are modulated. However, other mechanisms of signal attenuation and termination are known. Indeed, inhibition of RTKs’ activity may occur through the modulation of the phosphorylation state of RTKs and the interaction with specific proteins, whereas antagonist ligands can inhibit the biological responses mediated by the receptor. Another mechanism concerns the expression of endogenous inactive receptor variants that are deficient in RTK activity and take part to inactive heterodimers or hetero-oligomers. The downregulation of RTK signals is fundamental for several cellular functions and the homeostasis of the cell. Here, we will review the mechanisms of signal attenuation and termination of RTKs, focusing on FGFRs. FGFR1, FGFR2, FGFR3, and FGFR4), and 18 fibroblast growth factors (FGFs) related to the receptors are known nowadays. FGFRs contain in the extracellular domain three immunoglobulin (Ig)-like domains (D1-D3). Moreover, they are characterized by the presence of a highly conserved motif rich in aspartate acids, called the acid box. Interestingly, the extracellular domain of FGFRs has been proved to have an auto-inhibitory function [RTKs are a class of membrane spanning cell-surface receptors that are characterized by an intrinsic protein tyrosine kinase activity. In fact, they catalyze the transfer of the γ phosphate of ATP to hydroxyl groups of tyrosines to target proteins. Therefore, RTKs are important mediators of intracellular signaling and regulate the cell cycle, cell proliferation, cell migration, cell differentiation, apoptosis, fertilization, cell metabolism, and survival . In humaRTKs are located at the plasma membrane (PM) where they are in monomeric form. Their function is based on their ability to bind extracellular ligands and to form dimers or oligomers that show active intracellular kinase domains and determine receptor phosphorylation. In fact, the contact between the KDs of dimers stimulates catalytic activity, the auto-phosphorylation, and the cross-phosphorylation of receptor molecules and substrates . TherefoThe mechanisms that allow modulation of RTK activity and signaling are based on signal attenuation and termination . RTK dowReceptor tyrosine kinase signaling can be terminated by internalization of the receptor after ligand binding at the plasma membrane. This occurs after autophosphorylation of RTKs and phosphorylation of secondary signaling molecules that bind motifs containing phosphotyrosine residues, including phosphotyrosine binding (PTB) and Src-homology 2 (SH2) domains . FirstlyOnce on intracellular vesicles, RTK downregulation is controlled by both ubiquitination and deubiquitination. Ubiquitination in the cytoplasmic domain of RTKs occurs at an early stage and is used as a sorting signal for their sorting and delivery to MVBs . UbiquitMono-ubiquitination and multi-ubiquitination  are the signals for lysosomal degradation of membrane proteins, and several molecules take part in the regulation of RTK degradation, such as the ubiquitin ligase c-CBL or the endocytic regulator proteins Epsin, Eps15, Rabex5, Hrs, STAM, GGA3, and Tsg101 . For insInterestingly, ubiquitinated proteins at EEs are recognized by a complex of two ubiquitin-binding proteins, hepatocyte growth factor-regulated substrate (Hrs) and signal-transducing adaptor molecule (STAM), which belong to the class E Vps (vacuolar protein sorting) proteins and form ESCRT-0. The proteins coded by the VPS genes are fundamental for the normal membrane trafficking to lysosomes. RTKs are then transferred to the endosomal sorting complex required for transport (ESCRT)-I and ESCRT-II and then transported to MVBs with the help of ESCRT-III . It has Deubiquitination enzymes (DUBs) are proteases which specifically hydrolyze the isopeptide bond between Ub and a determined protein or between Ub molecules in case of Ub chains. This class of proteins includes Ub-specific protease Y (UBPY) and associated molecule to the SH3 domain of STAM (AMSH) in mammals and Doa4 in yeast that associates with class E Vps proteins on endosomes. UBPY and AMSH interact with the Hrs-STAM complex and counteract ubiquitination of RTKs. The specific roles of DUBs are to process the newly synthesized Ub precursors in order to generate monomeric Ub, retrieving Ub from proteasome- and lysosome-targeted poly- and mono-ubiquitinated proteins before degradation, disassembling poly-Ub chains into Ub monomers, and counteracting the ubiquitination-dependent regulatory process ,27.FGFRs compose a family of four single-pass transmembrane receptors, FGFR1, FGFR2, FGFR3, and FGFR4. A fifth FGFR-like protein, FGFR5/FGFRL1, lacks the KD necessary for the intracellular signaling; however it can dimerize with other FGFRs ,29,30. AOnce the FGFR is activated, a ternary complex formed of FRS2α, GRB2, and CBL is formed. In fact, the binding of the ligand FGF to FGFR determines the phosphorylation of FRS2, its binding to GRB2, and the recruitment of CBL, which is responsible for ubiquinating the FGFR and FRS2 . InteresAnother mechanism of modulation of FGFR activity that involves FRS2α, GRB2, and CBL regards Sprouty (Spry) proteins. Spry proteins inhibit signaling of the RTK when, after stimulation by growth factors, they reach the PM, become phosphorylated on a conserved tyrosine, and bind to GRB2, therefore inhibiting the recruitment of GRB2-SOS complex either to FRS2 or to Shp2. Intriguingly, the phosphorylated Spry2 inhibited GRB2 from binding FRS2 or Shp2 and blocked ERK signaling. When a dominant negative mutant of Spry was expressed, which was not phosphorylated, a prolonged activation of ERK signaling and relative induced outgrowth of neurites in PC12 cells was detected . InteresIn relation to RTK signaling, variations in strength and duration of the signal affect cell functions. Signal termination is controlled by several molecules, and degradation after internalization of the receptor is not the only way to downregulate the activity of RTKs. The activity of molecules such as kinases and protein tyrosine phosphatases (PTPs) has a regulatory effect on RTKs . MoreoveDownregulation of signaling can occur also through the action of PTPs, which reverse RTK signals by dephosphorylating them. Seven autophosphorylation sites have been detected in FGFR1  and FGFR2 . An eighth tyrosine  is conserved in all FGFRs. Besides this, five conserved tyrosine residues have been discovered in FGFR3  and four in FGFR4  [A fine tuning of RTK signaling is exerted through a spatial and temporal restricted action of the PTP . MoreoveCaenorhabditis elegans, which is named EGL-15. CLR-1 has a complex extracellular region and two intracellular phosphatase domains, and it downregulates the activity of egl-15 by dephosphorylating it [Ptprb is highly expressed in adult mammary stem cells and is responsible for branching morphogenesis in the mammary epithelium. Therefore, knockdown of PTPRB in mammary epithelial cells induces a higher level of FGFR2 activation and ERK1/2 phosphorylation [Interestingly, the PTP CLR-1 (CLeaR) has been associated with a FGFR-related receptor discovered in ating it . Intrigurylation .Interestingly, another phosphatase that affects FGFR signaling is Mkp3. Its transcription is regulated by the activation of FGFRs, then it dephosphorylates MAPK and inhibits MAPK signaling by acting as negative feedback regulator of FGFR .RTK activity can be regulated through the modulation of the action of protein kinases in order to protect the cells from excessive intracellular signaling. Interestingly, in the case of FGFR1, FGF1 ligand binding determined the direct ERK1/2-mediated phosphorylation of a specific serine residue (Ser777) that is present in the C-terminal domain of the FGFR1. The phosphorylation of this site inhibited the phosphorylation of tyrosines in the KD through a negative feedback route. Therefore, FGFR1 activation induces a negative feedback loop via ERK1/2 signaling. Furthermore, inactivation of ERK1/2 abrogated the phosphorylation of FGFR1. Nevertheless, the abolition of the phosphorylation event or the mutation of the serine residue to alanine, which cannot be phosphorylated, determined enhanced FGFR1 activity after FGF1 ligand binding and increased cell proliferation, cell migration and axonal growth. On the contrary, mutation of Ser777 to a phosphomimetic residue reduced the FGFR1 signaling . Interes2-terminal region containing potential binding sites for SH3 domains, a PH domain, an SH2 module, and a COOH tail containing a conserved tyrosine phosphorylation site. They compose a family of structurally and functionally related adapters that share the role of negative regulators of RTKs and cytokine receptors [Termination and fine-tuning of RTK signaling activity occur through different mechanisms and the involvement of other negative regulators such as SOCS, APS, Lnk, SH2-B, and RALT . APS, Lneceptors . For inseceptors .Similarly, Src-like adapter protein (Slap) negatively affects the activity of PDGFR by competing with Src for binding and blocking Src-mediated mitogenic signaling . AnotherAmong the proteins involved in tuning and terminating the receptor signaling, the family of SOCS proteins represents a set of proteins widely involved in the negative regulation of several RTKs . SOCS faAnother inhibitor of FGF signaling is sef (similar expression to fgf genes). Sef is a transmembrane protein that interacts with FGFR1 and decreases the phosphorylation of FGFR1 and FRS2 and reduces FGF-induced cell proliferation. This leads to an inhibition of FGFR1 tyrosine phosphorylation and consequently a reduced phosphorylation state of molecules involved in the signaling of FGFR1, such as Raf-1, MEK 1/2, and ERK . Xenopus oocytes, the mobilization of intracellular calcium was completely blocked due to the non-functional heterodimer [Xenopus FGFR by using FGFR mutants that presented either a point mutation in the ATP-binding site or a truncation of the cytoplasmic domain. Indeed, they discovered a negative action of these receptors on the transduction of the signaling and in the processes of mesoderm formation of Xenopus embryos [Certain tissues express, besides the endogenous full length protein, truncated/shorter variants of the RTK that could affect its activity and the relative signaling. In particular, expression of inactive deletion mutants could determine the formation of hetero-oligomers, which could result in deficient RTK receptors. In fact, the variants would act as dominant negative mutants and inhibit the activity of the heterodimer or hetero-oligomer . Some trerodimer . Other s embryos . Similar embryos . Interes embryos . Intrigu embryos .RTKs are subjected to cleavage and generation of fragments due to the action of caspases and proteases or by following alternative splicing. In particular, RTKs undergo several proteolytic cleavages operated by secretases that generate an extracellular domain, which is released in the extracellular space, and the intracellular domain that is present in the cytoplasm . RTK cleThe mechanism explaining how the fragments of FGFRs may regulate the function of other RTKs has been discovered by Hanneken and colleagues. They studied the inhibition of cellular proliferation and in-vitro angiogenesis induced by the competition of FGFR1 soluble fragments with FGF2 ligand. Furthermore, in this case, the cleavage of FGFR1 was due to a metalloprotease, and its production was tightly regulated. Interestingly, the stimulation with FGF2 increased the shedding of FGFR1 ectodomain, and this effect was dose-dependent, whereas the usage of metalloprotease inhibitors inhibited the release of the FGFR1 ectodomain. Similarly, the effect of the treatment on the RTK cleavage was dose-dependent. Furthermore, they discovered that activators of protein kinase C, such as TPA, enhanced shedding of FGFR1, whereas this was blocked by specific inhibitors of FGFR tyrosine kinases, therefore affecting ERK signaling .Activity of RTKs can be downregulated also through the action of antagonist ligands or molecules able to bind to the extracellular site of the receptor where the ligands normally bind, at the ligand-RTK interface or at the intracellular domain, usually at the KD . An antaAntagonist ligands are biological molecules or chemical drugs that block or dampen a biological response by binding the receptor and inhibiting the normal RTK activation and relative signal transduction. Interestingly, ligand-derived antagonists produced through protein engineering have been developed and used as potential cancer therapeutics. For instance, a developed mutant of angiopoieitin-2 ligand retained the ability to bind the receptor Tie2 but negatively influenced the ligand multimerization and RTK dimerization and activation. The angiopoietin2-derived Tie2 antagonist strongly inhibited Tie2 phosphorylation, affected endothelial capillary tube formation, and influenced cell invasion .In relation to FGFR activity, several small molecule tyrosine kinase inhibitors (TKI), classified as antagonist molecules, target the ATP binding pocket of RTKs and are under preclinical and clinical development or have been accepted for cancer treatment. They can be classified as non-selective FGFR TKIs  and selective FGFR TKIs. Among the multi-target TKI inhibitors, dovitinib, lenvatinib, lucitanib, ponatinib, nintedanib, regorafenib, and pazopanib have been reported ,91,92,93Moreover, antibodies and antibody fragments can be FGFR blockers. The main advantage is that exclusive members or a specific isoform of a receptor can be targeted by recognition of an extracellular epitope of cell surface proteins. For instance, the molecule scFvD2-Fc may inhibit FGFR1 activation and signaling by acting either alone or in combination with the cytotoxic drug monomethyl auristatin E . InteresFurthermore, a peptide named F8, CSLNHTVNC, shared a similar binding site on FGFR1 as the ligand FGF1. In fact, it interacts at the FGF1-FGFR1 interface. Noteworthy, F8 affects FGF1-FGFR1 interaction, inhibits FGFR1 downstream signaling, and FGF1-induced cell proliferation .The modulation of RTK activity is fundamental for all the molecular processes occurring in the cell. A fine balance between activation and inhibition or downregulation of the signaling cascade of RTKs is important in order to avoid the onset of pathologies such as cancer and alterations of cell behavior during cell development, differentiation, and metabolism. While activation of RTKs has been widely studied and several molecules implicated in the signaling pathways of the individual RTKs have been identified, less is known on the attenuation and termination of the signal, especially for FGFRs. In the case of these receptors, the main mechanisms of downregulation of the signal are ubiquitination followed by degradation in the lysosomes and inhibition of RTK due to antagonist molecules ,13,104. Interestingly, deregulation of FGFR signaling affects tissue homeostasis and is responsible for the onset of developmental syndromes . The deg


Burn patients are extremely vulnerable to hospital acquired infections, some of which may originate from environmental surfaces. Enhanced cleaning practices of surfaces varies from hospital to hospital. In a burn unit, patients are frequently exposed to multiple environments including the patient room, hydrotherapy and treatment rooms (treatment spaces), and the operating room. We sought to evaluate the efficacy of current protocols, including standard cleaning (SC) and cleaning with ultraviolet light application (C+UV) in the patient rooms and treatment spaces, and compare to a novel Hybrid Hydrogen Peroxide (HHP) fogging system in the patient rooms.G. stearothermophilus 1x106 spores) were measured.This study was conducted at a regional 12-bed verified adult and pediatric burn center that functions as an intensive care unit and step-down unit, where patients stay during their entire admission. Data from December 2020 to June 2021 was collected from rooms following 17 patient discharges with a minimum of a 2-week hospital stay. Data was collected from 5 preset locations in the patient rooms following either SC or C+UV and after HHP fogging. Baseline data was also collected from 20 preset locations in the treatment spaces following SC and C+UV. Quantitative and qualitative counts , hydrogen peroxide chemical indicators (CIs) and bacterial spore biological indicators . An industry-known location for high bioburden, one sink backsplash swabbed on two separate occasions resulted in >5,700 ACC.No statistically significant difference between SC and C+UV was seen . ACC levels were reduced following HHP fogging demonstrating a 98% improvement over SC and C+UV . ATP demonstrated an 88% reduction after HHP fog without manual cleaning. Biological indicators confirmed a 1x10In patient rooms, HHP fogging resulted in a significant reduction in ACC compared to current protocols of SC and C+UV. Baseline sampling of the treatment spaces resulted in ACC levels similar or greater in range to those seen in the patient room, indicating a similar reduction in bioburden levels could be achieved through implementation of HHP fogging. The efficacy and feasibility of HHP fogging in a patient room setting within a burn unit suggests that a protocol for the use of HHP fogging for treatment spaces will likewise be beneficial.