Background: Oxidative stress is crucial in experimental autoimmune myocarditis (EAM)-induced inflammatory myocardial injury. Ursolic acid (UA) is an antioxidant-enriched traditional Chinese medicine formula. The present study aimed to investigate whether UA could alleviate inflammatory cardiac injury and determine the underlying mechanisms.Methods: Six-week-old male BALB/c mice were randomly assigned to one of the three groups: Sham, EAM group, or UA intervention group (UA group) by gavage for 2 weeks. An EAM model was developed by subcutaneous injection of α-myosin heavy chain derived polypeptide (α-MyHC peptide) into lymph nodes on days 0 and 7. Echocardiography was used to assess cardiac function on day 21. The inflammation level in the myocardial tissue of each group was compared using hematoxylin and eosin staining (HE) of heart sections and Interleukin-6 (IL-6) immunohistochemical staining. Masson staining revealed the degree of cardiac fibrosis. Furthermore, Dihydroethidium staining, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used to determine the mechanism of cardioprotective effects of UA on EAM-induced cardiac injury, and the level of IL-6, Nrf2, and HO-1.Results: In EAM mice, UA intervention significantly reduced the degree of inflammatory infiltration and myocardial fibrosis while improving cardiac function. Mechanistically, UA reduced myocardial injury by inhibiting oxidative stress . Interestingly, UA intervention upregulated the expression of antioxidant factors such as Nrf2 and HO-1. In vitro experiments, specific Nrf2 inhibitors reversed the antioxidant and antiapoptotic effects of ursolic acid, which further suggested that the amelioration of EAM by UA was in a Nrf2/HO-1 pathway-dependent manner.Conclusion: These findings indicate that UA is a cardioprotective traditional Chinese medicine formula that reduces EAM-induced cardiac injury by up-regulating Nrf2/HO-1 expression and suppressing oxidative stress, making it a promising therapeutic strategy for the treatment of EAM. Myocarditis is an inflammation of the myocardium caused by viruses, bacteria, or parasites, as well as non-infectious causes such as autoimmunity, hypersensitivity, and cardiotoxicity . It can Traditional Chinese medicine has long been uniquely effective in treating inflammatory diseases. Ursolic acid (UA) is a pentacyclic triterpenoid compound found in many natural plants, such as apple peels, herbal medicines, and other edible plants . UA has Several works have demonstrated the modulation of Nrf2 by UA as one of the main mechanisms behind their beneficial effects in pathological models like cigarette smoke-induced emphysema  and renaIn the present study, an EAM murine model was developed to investigate the regulatory role of UA in anti-inflammatory and cardiac antioxidant pathways. The present study investigated whether UA can reduce EAM-induced oxidative stress injury and whether this effect is mediated through Nrf2 and HO-1 signaling pathways. The present study provides new insights for developing therapeutic targets to improve the prognosis and treatment of myocarditis patients.Male BALB/c mice aged 6–7 weeks (weighing 18–20 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., LTD. (China) and housed at 25°C ± 2°C with free access to water and food on a 12 h light/dark cycle. The Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (China) approved all experimental procedures that complied with the National Institutes of Health Guidelines on the Use of Laboratory Animals .All mice were anesthetized using 1.5% isoflurane before the procedure to minimize animal stress. Mice were immunized with a cardiac-specific peptide (α-MyHC peptide: Ac-RSLKLMATLFSTYASADR-OH) purchased from GL Biochem Ltd (China). The peptide was dissolved in saline and emulsified in a 1:1 ratio with Complete Freund’s Adjuvant  (n = 30). On day 0, 200 μg peptide in 0.2 mL of the emulsion was injected subcutaneously into one side of the axillary and inguinal lymph node region. On day 7, the same dose was injected subcutaneously into the opposite side. Sham group (n = 12) was injected with a physiological saline solution instead of myosin. Mice immunized with cardiac peptide were randomly divided into UA treatment (n = 16) and PBS (n = 14) groups. From day 7–21, mice were given intragastric gavage of UA , 100 mg/kg) (UA intervention group) or PBS containing 7.5% DMSO  (sham group and peptide–immunized mice without UA). Therefore, three experimental groups were formed: 1) a sham group, 2) an EAM group, and 3) an EAM + UA group. On day 21, all mice underwent echocardiography. The blood was then obtained through cardiac puncture. Body weight (BW) and tibial length (TL) were measured. Finally, hearts were removed from the chest and subjected to subsequent experiments.On day 21, the cardiac function was assessed using an echocardiographic imaging system  with a transducer MS400 (30 MHz). The mice were anesthetized with 1.5% isoflurane, and two-dimensional echocardiographic views of the mid-ventricular short axis at the papillary muscle tips below the mitral valve were obtained. The Vevo 1,100 software calculated the left ventricular ejection fraction (EF) and fractional shortening (FS).Tissue (10 mg) was homogenized in PBS on ice for ELISA. The samples were centrifuged for 10 min at 5,000 × g to remove any insoluble material. Plasma samples were collected by cardiac puncture. Interleukin-6 (IL-6), cardiac troponin I (cTnI), and brain natriuretic peptide (BNP) levels were measured using ELISA kits  according to the manufacturer’s instructions.6) samples were added to 200 µL MDA Lysis Buffer. The samples were incubated at 100°C for 15 min before being cooled in an ice bath to room temperature. The samples were centrifuged at 1,000 g for 10 min at room temperature to remove any insoluble material. Then, 200 µL of supernatant was pipetted into a 96-well plate, and the absorbance was measured at 532 nm (A532).MDA was measured using the MDA assay kit  as directed by the manufacturer. Plasma (100 µL) or homogenized cells . To induce inflammation, cells were rendered quiescent by serum starvation for 12 h prior to stimulation with rIL-6 (25 ng/mL) for 1 h (H9c2 cells were obtained from the cell bank of the Chinese Academy of Sciences (China). The cells were cultured in dulbecco’s modified eagle medium (DMEM) (high glucose) medium  supplemented with 10% fetal bovine serum (FBS) and 1% of penicillin-streptomycin  in a humidified atmosphere of 5% CO for 1 h . In orde for 1 h . The cel450). Each sample was replicated thrice. The cells that stained positive for CCK-8 solution was thought to be viable. The results are presented as a percentage compared with sham group.Cell viability was determined using the Cell Counting Kit-8 (CCK-8)  assay following the manufacturer’s instructions. The cells were seeded at a density of 5,000 cells/well in the 96-well plates. After 24 h, cells were treated with different concentrations of UA. The CCK-8 solution was added to each well, and the cells were incubated at 37°C for another 2 h. The absorbance was measured at 450 nm  of mice at the papillary muscle level were harvested, fixed in fresh 4% paraformaldehyde, embedded in paraffin, and serially sectioned (5–6 μm). Dewaxed and hydrated heart sections were stained with hematoxylin and eosin staining (HE) for general morphology or with Masson trichrome stain kit for fibrosis detection. The blue stained area (collagen deposition) was quantified and normalized to the total section area using ImageJ  software to determine cardiac fibrosis.2O2 for 10 min to remove endogenous enzymes. After 1 h of blocking with goat serum at room temperature, heart slides were incubated overnight at 4°C with the following primary antibodies: α-Smooth Muscle Actin (α-SMA) (1:100) , IL-6 (1:50) . Samples were incubated with secondary antibodies combined with Alexa Fluor 488 and 555  for 1 h at 1:500, followed by 4′,6-diamidino-2-phenylindole (DAPI)  for 15 min at room temperature. Finally, the sections were washed with PBS 1x and covered with coverslips.The paraffin sections were baked for 1 h in a 65°C oven. The sections were dewaxed and antigen repaired before incubating in 3% HMyocardial apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining by TUNEL Apoptosis Assay Kit with fluorescein  following the manufacturer’s instructions. Green fluorescein staining was used to identify apoptotic nuclei, while DAPI stained total nuclei. The apoptotic index was expressed as the percentage of apoptotic nuclei to total nuclei.Cardiac tissues or cells were homogenized in ripa lysis buffer (include protease and phosphatase Inhibitor) with a sonicator on ice, and the total protein concentrations were measured using the BCA Protein Assay Kit . As previously reported, cytosolic and nucleic proteins were separated for analysis of Nrf2 release. Depending on the molecular weight of target proteins, equal amounts of protein lysates (20–40 μg/lane) were subjected to 10%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted to PVDF membranes . After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated at 4°C overnight with respective primary antibodies against Nrf2  , HO-1 , LaminB1 , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , β-actin  and washed. The protein bands were visualized using enhanced chemiluminescence  after incubation with the corresponding secondary HRP-conjugated antibodies. Densitometry values were determined using ImageJ software .® Premix Ex TaqTM Perfect Real Time Kit . Relative expression was computed using the CT method. TRIzol Reagent  was used to isolate total RNA from flash-frozen myocardial tissues or H9c2 cells. cDNA was synthesized using a reverse transcription reagent kit  and then amplified in the Bio-rad CXF CONNECT Detector system using the SYBRThe crystal structure of Nrf2 protein (PDB ID: 7O7B) has been obtained from the RCSB protein data bank. For protein preparation, the natural ligand was extracted and the resulting crystal structure was then freed of the water molecules and added the polar hydrogen atoms. The 3D structure of ursolic acid has been obtained from pubchme database. This structure was optimized and polar hydrogen atoms are added. Subsequently, the docking procedure was conducted by using Autodock software and its conformation was optimized by using a genetic algorithm based on the principle of minimizing docking energy.p-value <0.05.The data were analyzed with GraphPad Prism software . All data are normally distributed. The multiple-group comparisons were performed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test for homogeneity of variance and Games-Howell’s multiple comparisons test for heterogeneity of varianc. All values were presented as mean ± SD. The differences were considered statistically significant with a The ratios of heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) were elevated and heart was enlarged in EAM mice compared to sham group, which was alleviated by UA treatment . H&E-staIn EAM model, heart sections stained with Masson’s trichrome to detect collagen fiber deposition revealed some peripheral fibrotic areas in EAM mice that were reduced by UA administration . ImmunofOn day 21, echocardiography was performed to assess cardiac function in each group ; Table 2Cardiac dysfunction has been associated with increased cardiomyocytes death, which eventually debilitates the contractile capacity of the heart. Findings indicated that the proportion of TUNEL-positive nuclei increased significantly in EAM group . In addiHearts from EAM animals were also exposed to elevated levels of oxidative stress that overwhelms the capacity of antioxidant systems to cope with this severe inflammatory scenario. Therefore, cytosolic reactive oxygen species (ROS) sensor DHE was used to characterize intracellular ROS, and MDA content was measured to assess lipid peroxidation end products. The findings revealed that EAM mice had higher ROS levels and lipid peroxidation levels than sham group, while UA-treated mice had lower levels .Nrf2 is an important transcriptional factor and the Nrf2/HO-1 axis is an important antioxidative stress pathway in the body. The present study identified that EAM reduced Nrf2/HO-1 protein expression of mice while UA could restore it . Furtherin vitro experiments. To investigate the role of Nrf2 in the UA-mediated beneficial effects, we performed a set of experiments in which we exposed IL-6-stimulated cells to UA  for 4 h and quantified Nrf2 total protein levels in H9c2 cell  was employed for the receptor and ligand docking analysis. An interacting complex of Nrf2 and ursolic acid was obtained after docking the molecular proteins with different conformations and poses. The 3D molecular simulation illustrate the binding modes of ursoilc acid with Nrf2 via the hydrogen bond and other interactions . The binIn the present study, we found that UA could reduce EAM-induced myocardial injury through the oxidative stress pathway. UA ameliorated EAM-induced myocardial injury by scavenging ROS and activating the Nrf2/HO-1 pathway.Despite the host-activated immune response, many studies have confirmed the presence of multiple anti-myocardial antibodies in patients with myocarditis and DCM, the most common of which are anti-cardiac myosin antibodies . In the In an abnormal immune response in the myocardium, antigen presence stimulates the differentiation and proliferation of many T cells in the peripheral immune organs, which migrates from the blood circulation to the heart. Lymphocytes infiltrate the myocardium and release inflammatory cytokines such as IL-6, IL-17, and IL-23, causing cardiomyocyte hypertrophy or death, resulting in myocardial hypertrophy or fibrosis and cardiac systolic or diastolic dysfunction . IL-6 plIn recent years, Nrf2 has been shown to have a protective role in major cardiovascular diseases (CVDs) in murine models. It can improve cardiac function and reduce efferocytosis and the release of pro-inflammatory cytokines, thereby improving the pathological phenotype . Nrf2 isCurrently, the prevailing view is that oxidative stress exacerbates the dysfunction and structural remodeling of myocarditis. There are also many studies for the effects of antioxidants. Meanwhile, more and more molecular targets and new drugs have been discovered. However, due to the difficulty of manufacturing the drugs and safety, clinical translation cannot be achieved. In contrast, ursolic acid has following advantages, which make it promising as an antioxidant to assist in EAM treatment: 1) UA is a natural product ingredient that can be extracted from natural plants  in plentiful quantities and at a relatively cheap price; 2) UA probably provides a better safety profile in clinical use, even people with a high risk of myocarditis may benefit from taking UA as a health supplement. Furthermore, the findings of the present study on the regulatory role of UA in EAM broaden our understanding of the multiple functions of UA in the heart. These findings could serve as a model for drug development in autoimmune myocarditis. The results, along with previous studies on the role of UA in myocardial infarction and atherosclerosis, suggest that UA may be useful for various clinical indications in CVDs.However, there are several limitations to present study. To further validate the role of Nrf2/HO-1 signaling pathway in H9c2, we selected Nrf2 inhibitor, ML385, rather than Nrf2 siRNA, which is relatively less persuasion. Furthermore, the antioxidant mechanisms of ursolic acid are complex and still need further study.In conclusion, we have demonstrated that UA administration could alleviate EAM-induced myocardial injury, as evidenced by improvements in cardiac functions, cardiac inflammation, and structural remodeling, partially by attenuating oxidative stress in a Nrf2/HO-1 pathway-dependent manner . The pre


Background Cerebral changes occur in individuals with human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy (HAM) and seem to predominate in subcortical areas. Little is known about the cognitive decline in the elderly living with HTLV-1.Objective To evaluate the cognitive aging of individuals infected with HTLV-1 aged ≥ 50 years.Methods This is a cross-sectional study of former blood donors infected with HTLV-1 who have been followed in the cohort of the Interdisciplinary Research Group on HTLV-1 since 1997. The groups of study consisted of 79 HTLV-1 infected individuals aged ≥ 50 years, with 41 of them presenting symptomatic HAM and 38 being asymptomatic carriers, and 59 seronegative individuals (controls) aged ≥ 60 years. All were submitted to the P300 electrophysiological test and neuropsychological tests.Results Individuals with HAM presented delayed P300 latency in relation to the other groups, and this latency delay increased progressively with aging. The performance of this group in the neuropsychological tests was also the worst. The HTLV-1- asymptomatic group performance was similar to that of the control group.Conclusions Individuals with HAM presented cognitive decline that progressed with aging and, although HTLV-1-asymptomatic carriers appear to present cognitive aging similar to that of healthy elderly people, concern about a subclinical cognitive impairment is warranted in this population. The human T-cell leukemia virus type 1 (HTLV-1) is endemic in some regions of the world, and Japan, Africa, the Caribbean islands, and South America are the areas of greatest circulation of this virus. In these areas, the prevalence gradually increases with age, especially among women.Human T-cell leukemia virus type 1 can cause HTLV-1-associated myelopathy (HAM), a neurological disease in which diffuse loss of myelin and axons occurs in the medulla, especially in the thoraco-lumbar region.The literature is scarce regarding the cognitive performance of HTLV-1-infected people over 50 years of age, and, then, we aimed at evaluating the performance of people living with HTLV-1 over 50 years of age in terms of attention, memory, general cognitive ability, executive functions, motor and psychomotor speed, and manual dexterity. Our hypothesis is that HTLV-1-infected people, especially those who develop HAM, present cognitive impairment parallel to the spinal cord damage.This research was conducted in accordance with the principles expressed in the Declaration of Helsinki and was approved by the research ethics committee from Universidade Federal de Minas Gerais (COEP UFMG), logged under protocol CAAE 92928518.3.0000.5149 and number 2898825. All participants provided voluntary written consent and declared that they were aware of the study procedures and their choice to participate.All participants in this study take part in the cohort of the Interdisciplinary Research Group on HTLV-1 (GIPH). They are seronegative blood donors and former blood donors infected with HTLV-1 from the state of Minas Gerais, Brazil, who have been followed up since 1997. In this study, 138 individuals from this cohort were included and distributed into the groups with and without HTLV-1 infection.The study groups consisted of 59 HTLV-1-seronegative blood donors (controls), as assessed by enzyme-linked immune sorbent assay (ELISA), and 79 HTLV-1-infected ex-blood donors, diagnosed by ELISA and confirmed by Western blot analysis  or by real-time polymerase chain reaction (RT-PCR). The HTLV-1 group was divided into 41 HAM and 38 HTLV-1-asymptomatic carriers, according to the neurological assessment.A cross-sectional, comparative study was carried out, nested to an open cohort, in which HTLV-1 seropositive adults ≥ 50 years of age were compared to HTLV-1 seronegative adults ≥ 60 years of age in relation to cognitive abilities that were assessed using the P300 electrophysiological test and neuropsychological tests. The decision to evaluate individuals infected with HTLV-1 who were 10 years younger than the seronegative controls was based on the comparison of the aging of these patients with the pattern of premature aging that occurs in HIV infection, in which individuals above 50 years of age are classified as elderly people.The individuals recruited for this research were initially submitted to a clinical and neurological evaluation and, later, to an audiological evaluation that consisted of tonal and vocal audiometry, imitanciometry with 226 Hz probe , and brainstem auditory evoked potential (BAEP) at 90 dB hearing level (dB HL) . Finally, the participants included in this study were submitted to cognitive assessment.The exclusion criteria were coinfection with HIV and/or HTLV-2, patients with diagnoses of dementia, alcoholism, or drug addiction, mental illness, sequelae due to opportunistic infections in the central nervous system (CNS), hearing loss greater than 40 dB HL at frequencies from 500 Hz to 4,000 Hz, any alteration in the middle ear or deformities in the external auditory canal, alteration of the auditory pathways to the brainstem, and patients with depressive symptoms scored above 5 on the geriatric depression scale of 15 items.The event-related potential (ERP) is an electrophysiological method that allows the capture of human neuroelectric activities when the individual is subjected to a specific event, such as an auditory stimulus. The P300 complex of the ERP consists of a large positive waveform whose peak occurs around 300 milliseconds (ms) after the stimulus presentation. The P300 is generated by a complex neural network, in which connections occur involving the thalamus, temporal-parietal cortex, prefrontal cortex, hippocampus and limbic region, being closely related to short-term memory and attention.oddballparadigm was used, in which 2 stimuli were presented randomly, with one of them occurring frequently and the other in a rare way (target). The individuals were instructed to identify and mentally count the number of rare stimuli (target). The parameters defined for the stimulus characterized as frequent were a tone burst sound in the frequency of 1,000 Hz, 50 cycles of duration, 20% rise and decay time, 60% plateau, trapezoidal envelope, and alternating polarity. For the tone burst stimulus characterized as rare, the parameters were a frequency of 2,000 Hz, 100 cycles of duration, 20% rise and decay time, 60% plateau, trapezoidal envelop and alternating polarity. For each exam, 300 stimuli were presented, with a rate of 0.8 stimuli per second, with 80% of frequent stimuli and 20% rare. The amplifiers were configured with the 200 µV full scale, the 01 Hz high-pass filter, the 20 Hz low-pass filter and the 60 Hz notch filter. The time window was 1,000 ms and the electroencephalogram was enlarged 50,000 times. Before starting the exam, the stimuli were presented so that the participants became familiar with the test. The procedure was repeated once more to guarantee the replication of the tracing. P300 analysis was performed separately by two independent examiners who were experienced in electrophysiological examinations. The analysis was masked in relation to the group.The auditory P300 test was performed with the individual in a seated and comfortable position, with their eyes open, in an electric and acoustically treated room with dim light, using the MASBE/ACTPlus equipment  10-20 standard, with the negative electrodes placed on the left (A1) and right (A2) earlobes, the ground electrode on the forehead (Fp1), and the active electrode on the forehead (Fz).The P300 component is the largest positive peak between 250 and 500 ms following the presentation of the stimulus, and it occurs after the N100, P160, and N200 components. The study of the P300 can regard its amplitude or latency. Amplitude can be interpreted as the number of cognitive resources that an individual allocates in a cognitive task and, therefore, refers to mental effort.After being submitted to the auditory P300 test, the participants underwent neuropsychological tests to assess specific cognitive domains. The tests applied were Raven colored progressive matrices (RAVEN), which assesses general cognitive abilityThe measurement of interrater agreement of the P300 result was applied using the intraclass correlation coefficient (ICC), in which a value ≥ 0.70 indicates good reliability.p = 0.224) and gender (p = 0.895). As expected, the average age of the control group was higher than the one of the HTLV-1-infected groups (p˂ 0.001). These data are summarized inThe groups were similar regarding educational level  clonazepam, 4 (3%) baclofen, 3 (2%), gabapentin, and less than 1% reported oxcarbazepine, carbamazepine, pregabaline, or phenobarbital. No association was found between cognitive performance and the use of these medications.p < 0.001). So, the P300 measures were shown to be adequate. The P300 latency was delayed in the HAM group when compared with the HTLV-1-asymptomatic and control groups  and in the younger age group among those with HAM (50–59), the difference remained . In fac0–79 and The P300 latency maintained a pattern related to the aging that varied according to the HTLV-1 group. Differently from the results presented by the individuals with HAM, the P300 latency remained stable with the aging in HTLV-1 asymptomatic carriers, similar to what was observed in the control group . A progWhen a comparison was made between the HAM and HTLV-1 asymptomatic groups, an unexpected P300 latency similarity appeared in the comparison between the HAM youngest age group and the eldest HTLV-1-asymptomatic age group . This iRegarding the predominance of the female gender in the GIPH cohort, which was represented by the predominance of women in the present work, this reflects the epidemiology of HTLV-1 in Brazil and in the world.The limitation of the present study was the selection of neuropsychological tests that focused on subcortical dementia and not on a global cognitive evaluation. The reason was that the studied population included fragile HAM elderly people who would not tolerate a long-time test. So, the time taken to run the entire battery of cognitive tests was a limiting factor. The inclusion of P300 was important because this test is easy to apply, it is not tiring and, finally, changes in the electrophysiological tests precede changes observed in the neuropsychological tests.In conclusion, HAM was associated with a cognitive decline, possibly of subcortical onset, that seems to worsen with aging. Those infected with HTLV-1, classified as asymptomatic carriers, did not appear to exhibit a cognitive decline that was different from healthy seronegative elderly. However, HTLV-1-asymptomatic individuals with any cognitive complaint, especially regarding memory, should be submitted to a battery of neurocognitive tests in order to investigate cases of possible subclinical manifestation of the disease.


Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine that has emerged as a critical player in the development and progression of allergy and asthma. It is primarily produced by epithelial cells and functions as a potent immune system activator. TSLP acts through interaction with its receptor complex, composed of the TSLP receptor (TSLPR) and interleukin-7 receptor alpha chain (IL-7Rα), activating downstream complex signalling pathways. The TSLP major isoform, known as long-form TSLP (lfTSLP), is upregulated in the airway epithelium of patients with allergic diseases. More research is warranted to explore the precise mechanisms by which short-form TSLP (sfTSLP) regulates immune responses. Understanding the dynamic interplay between TSLP and the dysfunctional epithelium provides insights into the mechanisms underlying allergy and asthma pathogenesis. Targeting TSLP represents an important therapeutic strategy, as it may upstream disrupt the inflammatory cascade and alleviate symptoms associated with allergic inflammation. The cytokine TSLP was initially identified in conditioned medium from a murine thymic stromal cell line with a medullary phenotype as a growth factor for B and T cells and co-stimulator for thymocyte proliferation, consequently suggesting its role as lymphopoietin. The human TSLP homolog was found using in silico methods, detecting the homology to mouse TSLP by a computational screen of the human genomic databases ,2. TSLP This review is intended to be an educational resource for researchers, clinicians and students interested in cytokine and epithelial cell biology, asthma and allergic diseases, by summarizing and discussing the current state of knowledge on the multifaceted roles of TSLP and its isoforms, and by focusing more on molecular approaches for a better understanding of the topic in its complexity and significance.TSLP is a four α-helical type I cytokine which plays critical roles in diverse physiological and pathological conditions ,2.Various stimuli, including mechanical injury, ligands for Toll-like receptors TLR2, TLR3) and NOD2, pro-inflammatory cytokines and allergen proteases can induce the expression of the alarmin cytokine TSLP in epithelial cells ,3,4. and NOD2In the lungs, TSLP production by epithelial cells with a rapid cellular release is also triggered by viral infections, including those with respiratory syncytial viruses, rhinoviruses, influenza viruses and metapneumoviruses, which incite other danger signals and exacerbate inflammation ,5,6.κB signal pathway, leading to elevated cytokines including TSLP [Moreover, when challenged by allergen molecules, barrier cells such as bronchial and intestinal epithelial cells can overproduce reactive oxygen species (ROS) by up-regulating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and activating the nuclear transcription factor NF-ing TSLP ,8,9.TSLP, mainly produced in response to pathogenic stimuli by lung and intestinal epithelial cells and skin keratinocytes, induces complex immune responses at barrier surface level by activation of dendritic cells (DCs), mast cells, eosinophils and lymphocytes into a type 2 polarizing phenotype ,11. TherTSLP activates intracellular signalling by forming a complex with its specific receptor, TSLPR (encoded by CRLF2) and IL-7Rα. The cognate receptor TSLPR allosterically triggers TSLP to potentiate the recruitment of the shared IL-7 receptor α-chain (IL-7Rα) by leveraging the cytokine molecule’s conformational heterogeneity, flexibility and electrostatics ,14,15,16TSLP has a four-helix structure stabilized by three disulfide bridges, in which the four α-helices, designated αA to αD, are linked via a BC loop and two longer AB and CD loop regions. TSLP, positively charged, binds to TSLPR, negatively charged .TSLP engages its C-terminal short tail, C-terminal half of the helix αD and continuous 10 residues in the long AB loop region , part ① Following the capture and rearrangement of TSLP by TSLPR at the cellular surface, IL-7Rα is recruited to initiate intracellular pro-inflammatory JAK-STAT pathways , part ②.κB (nuclear factor κB), MAPK (mitogen-activated protein kinases), PI3K (phosphoinositide 3 kinase), SRC (sarcoma tyrosine kinase) pathways. TSLP activates JAK1 (via IL-7Rα) and JAK2 (via TSLPR). JAK1 and JAK2 then activate signal transducer and activator of transcription 5 (STAT5) and, to a lesser extent, STAT3 and other transcription factors [TSLP intracellular signalling cascades are initiated by JAK1/JAK2, molecules involved in phosphorylation of STAT3/5, but also include NF- factors ,14,15,16Finally, the transcription of target genes is promoted , part ③,Altogether, the binding of TSLP to its heterodimeric receptor complex TSLPR and IL-7Rα initiates intracellular activation of several downstream protein tyrosine kinases with phosphorylation of transcription factors which initiate the transcription of some target genes including those encoding pro-inflammatory cytokines involved in allergic and eosinophilic inflammation ,16.TSLP engages the C-terminal part of the helix αD (residues 142–152), the C-terminal tail extending from αD (residues 153–158), with an important triplet of arginine residues, and a continuous sequence of 10 residues in the AB loop region (residues 60–69) to interact with a complementary epitope shaped at the cytokine-binding homology region of the receptor TSLPR . Key intTezepelumab, a fully human monoclonal IgG2λ antibody currently indicated for the treatment of severe asthma, specifically binds human TSLP at the level of its ligation site for TSLPR, thereby impeding the TSLP-TSLPR interaction . The comIn humans, there are two TSLP variants: long-form TSLP (lfTSLP or TSLP variant 1) and short-form TSLP (sfTSLP or TSLP variant 2), their genes consisting of four and two exons, respectively, and are derived from the activity of two promoter regions . The disThese two TSLP isoforms have been identified initially in human bronchial epithelial cells, the lfTSLP, upregulated by a TLR3 ligand (poly I:C), and sfTSL, constitutively expressed in normal tissues . The forAmong the two TSLP isoforms, the lfTSLP has been more extensively studied, and it is recognized as a critical regulator of immune responses, particularly in inflammation and allergic reactions. The human TSLP gene is located on chromosome 5q22.1 near the atopic cytokine cluster. lfTSLP is closely related to IL-7, with which it shares a biological profile that is similar, but not identical. It binds to the previously described heterodimeric receptor complex consisting of IL-7Rα and TSLPR. TSLP and IL-7 are considered paralogues, likely having derived from a gene duplication event .The two TSLP isoforms are not the product of alternative splicing of the same transcript, but are instead controlled by two different promoter regions . The lfTThe functional activity of lfTSLP is regulated by post-translational alterations. Proprotein convertase enzymes can truncate it between residues 130 and 131 to generate a heterodimeric short-lived metabolite, TSLP (29–130 + 131–159), which powerfully activates myeloid DCs and group 2 innate lymphoid cells (ILCs) compared to mature lfTSLP, thus driving type 2 inflammation. Carboxypeptidase N (CPN) then digests six AA from the C-terminus of the lfTSLP to produce a more stable dimerized form, TSLP (29–124 + 131–159). These truncations are primate specific and homeostatic. ,22.lfTSLP matches to the commercially available recombinant TSLP produced by prokaryotic cells. sfTSLP overlaps the lfTSLP in its terminal region, and the majority of the available anti-TSLP antibodies for different scientific immunoassays used to detect TSLP do not distinguish between the two isoforms. Therefore, a quantitative polymerase chain reaction using specific primers is the only way to determine the two TSLP variants at the molecular level. Several studies have examined the expression and functions of the two TSLP variants in different human diseases, but additional research is needed .The lfTSLP isoform is primarily produced by epithelial cells, especially in tissues like the lung, gastrointestinal tract and the skin. DCs, basophils, mast cells, keratinocytes and fibroblasts also produce lfTSLP following stimulation ,20.The functional receptor for lfTSLP is expressed on many cells, including DCs, T and B cells, natural killer T cells (NKT), eosinophils, basophils and epithelial cells. Activation of the TSLPR induces multiple transcription factors including those of the JAK/STAT intracellular pathways, and thus lfTSLP impacts many immune responses and is associated with several immune pathologies, such as allergic diseases and intestinal inflammation. lfTSLP-stimulated DCs are involved in the inflammatory cells production of T2 cytokines such as IL-5, IL-4 and IL-13. This inflammatory type 2 phenotype is induced by upregulating OX-40 ligand expression on lfTSLP-treated DCs. Therefore, in respiratory allergy, exposure to lfTSLP of DCs potently augments allergen-specific TH2 responses. In atopic dermatitis (AD), lfTSLP is highly expressed in acute and chronic lesions but it is not detectable in non-lesional skin ,20,23,24lfTSLP is induced at sites of inflammation having important proinflammatory actions. Thus, it can be vastly upregulated in inflammatory diseases, including asthma and AD. The expression of lfTSLP is triggered by TLR ligands, including poly I:C, FSL-1, and flagellin, and proinflammatory cytokines, such as IL-4, IL-13 and tumour necrosis factor-α (TNF-α) ,27,28,29Two single nucleotide polymorphisms (SNPs) in the TSLP promoter, rs2289276 and rs2289278, confer enhanced binding by the AP1 transcription factor and augmented lfTSLP production, explaining why these SNPs are associated with higher incidence of pediatric atopic disease and adult asthma .lfTSLP damages airway barrier function in a murine model of asthma, suggesting its important pathogenesis contribution in the disease. Particularly, lfTSLP contributes to house dust mite (HDM)-induced bronchial epithelial dysfunction and the chromobox CBX4 regulates lfTSLP-mediated airway inflammation through SUMOylation  in HDM-induced asthma . In an HThe pathology-dependent pattern of expression of the lfTSLP compared with sfTSLP is presented in Escherichia coli, upregulate lfTSLP, whereas the opposite occurs after challenge with a commensal strain [Interestingly, lfTSLP exerts antimicrobial activities and this is related to the last 34 AA of the cytokine. This could mean that the dysbiosis observed in many barrier surface pathologies could impact the homeostatic balanced expression of TSLP. Some highly immunogenic bacterial strains, such as adherent-invasive l strain .Additional research into lfTSLP’s precise mechanisms of action and its interactions with other cytokines and cells will offer a better and more detailed understanding of immune regulation and inflammation processes along with improved approaches for therapeutic interventions. The emergence of lfTSLP as a central orchestrator of type 2 responses capable of initiation of inflammation and allergy has preferentially positioned a new therapeutic targeting of the TSLP-mediated signalling in asthma and atopic diseases .Compared to lfTSLP, sfTSLP has been less studied in humans, and its detailed physiological roles are still unknown. However, emerging evidence suggests that sfTSLP might play a unique position as a significant inflammation regulator. The sfTSLP mRNA seems human specific, as there are no published data of a similar variant in other species . TranscrThe sfTSLP is not upregulated by inflammation and plays a homeostatic role being constitutively expressed in a steady state in different tissues. Since it has homeostatic and anti-inflammatory effects, this was considered when designing targeted pharmacological strategies. Ideally, therapeutical monoclonal antibodies produced to neutralize TSLP should not interact nor hamper the known sfTSLP homeostatic functions. ,20,34.sfTSLP has been reported to be expressed in several types of cells, including T cells and mast cells. sfTSLP mRNA is also constitutively expressed in human epithelial cells, lung macrophages and lung fibroblasts. In addition, it is expressed in the skin and oral mucosa keratinocytes and in salivary glands, at the mRNA and protein level. Distinctive roles were suggested, the sfTSLP appearing to have the opposite effect to lfTSLP in immune modulation. Therefore, sfTSLP is considered to act as an inflammation suppressor with anti-inflammatory functions. This short TSLP isoform may counterbalance the effects of the long one, mitigating excessive immune responses and inflammation ,17,21,35Administration of sfTSLP decreases airway hyperreactivity and ameliorated inflammation in an HDM-induced murine model of asthma ,31. sfTSA specific receptor for sfTSLP has yet to be discovered. The striking difference between the two TSLP isoforms is that sfTSLP does not bind to the TSLPR and it cannot block the binding of lfTSLP to this receptor. The short isoform induces phosphorylation of p38 mitogen-activated protein kinase (MAPK) p38-α and extracellular signal-regulated kinase ERK 1/2 but has no significant effect on STAT5 .sfTSLP exhibits potent antimicrobial activity, exceeding that of many known antimicrobial peptides (AMPs). As sfTSLP can be downregulated by inflammation, this might contribute to an aggravation of local infections. In healthy barrier surfaces such as skin and intestine, sfTSLP is the main isoform detected; therefore, it is expected to play an important local role against infection and in inflammation regulation. Vitamin D upregulates only sfTSLP in epithelial cells, a putative vitamin D receptor binding site being identified in intron 3 of the TSLP gene ,35.E. coli strain downregulates the sfTSLP, whereas the opposite happens after challenge with a commensal strain [The expression of sfTSLP in the skin, oral mucosa, salivary glands and the intestine is part of the defence barrier that helps in controlling both commensal and pathogenic microbes . For insl strain .Furthermore, it is important to underline that the sfTSLP is not only necessary for homeostasis, but it is also involved in carcinogenesis ,29,36. IThe dysfunctional airway epithelium and its derived cytokine TSLP represent a very dynamic upstream level within the multifaceted interplay of cellular and molecular complex pathways leading to airway inflammation, either type 2 one, including allergic and non-allergic asthma phenotypes, mostly outlined by eosinophilic inflammation, or T2-low one, featured by neutrophilic or paucigranulocytic patterns .Exposure of the respiratory epithelium to allergens, viruses, bacteria, pollutants or mechanical injuries as environmental stimuli can promote loss of integrity as well as anchorage of the airway epithelium, causing the release of the alarmin cytokines represented by IL-25, IL-33 and TSLP . AlarminThe cellular sources of TSLP are in fact those presented as sources for lfTSLP, and include mainly epithelial cells, along with DCs, basophils, mast cells and fibroblasts. TSLP exhibits diverse effects on multiple cell types ,46,47, iTSLP promotes allergic responses by acting on DCs and inducing their expression of OX40 ligand (OX40L), CD80 and CD86, promoting the differentiation of naive CD4+ T cells into pro-inflammatory TH2 cells, which are capable of producing IL-5, IL-4, IL-13 and TNF-α ,24. MoreTSLP also promotes the release of T2 cytokines and chemokines by eosinophils, mast cells and macrophages ,52,53. AkB pathways, but not STAT5 and STAT3, which are usually activated in other effector cells upon TSLP stimulation. Moreover, TSLP also induces the release of IL-6 and the chemokines CXCL1, CXCL8 and CCL2 from eosinophils, as well as promoting the migration of neutrophils and non-hematopoietic cells [Eosinophils are significantly affected by TSLP, since this alarmin delays eosinophil apoptosis, up regulates the expression of adhesion molecule ICAM-1 and CD18, enhances eosinophil adhesion onto fibronectin and induces the release of inflammatory cytokine IL-6 and several chemokines such as CXCL8, CXCL1 and CCL2. TSLP regulates these effects through the activation of p38 MAPK, ERK and NF-ic cells ,58.The underlying mechanisms of epithelial cell interplay and disruption may be related to the differential capacity of inducing alarmins by allergen molecules with particular enzymatic and/or ligand-binding activity. HDM, Alternaria and cockroach allergen-derived proteases can induce TSLP, although experimental studies did not address specifically lfTSLP . ProteasEpithelial injuries mediated by such proteases together with their sensing by protease-activated receptors (PARs) elicit strong inflammatory responses inducing the release of TSLP and other pro-T2 cytokines  and danger-associated molecular patterns . Importantly, Der p 1 degrades occludin and claudin proteins in tight junctions (TJ), while Asp f 13 also degrades E-cadherin in adherens junctions (AJ). Moreover, PARs, widely expressed in the airway epithelial cells, have been shown to be activated by protease allergens. Der f 3 activates PAR1, Der p 3, Der f 3 and Der p 9 activate PAR2 and Der p 3 can activate PAR4. Pen c 13 can also activate PAR1. In addition, Der p 1 induces thrombin-dependent activation of PAR1 and PAR4 by its capacity to mature prothrombin into thrombin, while a new fungal allergen epithelial-sensing mechanism converges on the caspase 8-ripoptosome activation and IL-33 maturation as contributors to type 2 innate immune responses. The nonselective ion channel TRPV1  is also important in the airway epithelial secretion of IL-33 in response to airborne protease allergens. Last but not least, proteolytic cleavage of fibrinogen can trigger TLR4 signalling, and cleavage of various cell surface receptors further optimize the TH2 polarization. Der p 1 and Per a 10 selectively shed cell surface receptors such as the low-affinity IgE Fc receptor CD23 (FcεRII) on B cells and the IL-2 receptor CD25 on both CD4 and CD8 T cells, while Der p 1 also sheds CD40 directly from DCs surface and cleaves DC-SIGN (CD209), a C-type lectin receptor primarily expressed on target cells ,60. C-tyAsthma risk is influenced by the combined effects of TSLP genotype and TSLP expression in the nasal epithelium rather than circulating TSLP. The joint effect of TSLP rs2289277 and rs11466750 genotypes and TSLP (sfTSLP and lfTSLP) expression in nasal epithelial cells is associated with increased childhood asthma . Such loAs previously mentioned, the central role of TSLP in airway inflammation is to activate CD11c+ myeloid DCs to express OX40L, which then primes naïve CD4+ T cells to differentiate into a pro-inflammatory T2 phenotype expressing IL-5, IL-4, IL-13 and TNF-α. When activated, CD4+ T cells are maintained and expanded through TSLP activated DCs expressing the PGD2 receptor CRTH2, which contribute to the proliferation of TH2 memory cells ,64,65,66In severe asthma, TSLP is a key epithelial cytokine positioned at the top of the inflammatory cascade. For some patients with severe asthma, the epithelium is a starting point for an overactive immune response to various insults including allergens, pollutants/irritants, viruses, bacteria and other external stimuli. The heterogenous response to asthma treatment is likely directly related to differences in patterns of airway inflammation. The overexpression of TSLP can result in pathologic inflammation pathways driving various inflammatory phenotypes such as allergic, eosinophilic, neutrophilic and paucigranulocytic, which cause symptoms and asthma exacerbations ,64,67,68In allergic asthma, by activation of DCs, TSLP promotes the differentiation of TH2 lymphocytes secreting T2 cytokines targeting B cells, eosinophils, mast cells and airway smooth muscle cells. In non-allergic eosinophilic asthma, TSLP stimulates ILC2 to release IL-5 and IL-13, while in neutrophilic asthma, TSLP induces DCs to induce neutrophil-activating TH17 lymphocytes. Moreover, in paucigranulocytic asthma, TSLP mediates multifaceted cross talks between inflammatory cells, such as mast cells and airway structural cells, such as epithelial cells, smooth muscle cells and fibroblasts . ImportaHuman clinical trials of TSLP inhibition with the biologic tezepelumab completed to date have produced favourable results in patients with different asthma phenotypes, who experienced substantial improvements in lung function, symptom control and quality of life as well as reductions in exacerbation rates; this supported its recent approval for clinical practice in adults and adolescents with severe asthma .Examination of nasal polyps from individuals with nonsteroidal anti-inflammatory drug (NSAID) aspirin-exacerbated respiratory disease (AERD)—NERD and those with chronic rhinosinusitis (CRS) without NERD/AERD showed that TSLP mRNA expression was significantly increased in NERD/AERD ,75,76,77In allergic rhinitis, TSLP plays a central role in the initiation and persistence of allergic responses. The histamine H4 receptor regulates TH2-cytokine profile through TSLP in nasal allergy and the ubiquitin-specific peptidase 25 (USP25) downregulation involved in inflammation and the immune responses enhances the TSLP signalling in the nasal epithelium via decreased expression of TNF receptor-associated factor 3 (TRAF3), thereby exacerbating inflammation in allergic rhinitis . RecentlIn addition, TSLP may be an important factor involved to the development and progression of ocular allergy. TSLP is significantly expressed in the epithelial cells and some inflammatory cells of giant papillae from vernal keratoconjunctivitis (VKC) a rare, severe phenotype of chronic ocular allergy. Using immunohistochemical staining, OX40L and CD11c immunoreactive cells largely infiltrates such giant papillae. TSLP activates DCs to prime CD4+ T cells to differentiate into TH2 type and triggers T2-dominant allergic inflammation via TSLP/OX40L/OX40 signalling as part of pathogenesis of VKC .Because it is known that TSLP induces mast cell development, prevents apoptosis in skin mast cells and it is markedly upregulated in the wheals of patients with chronic spontaneous urticaria, the anti-TSLP monoclonal antibody tezepelumab is undergoing clinical trial assessments for this disease .In AD, the overexpression of TSLP in keratinocytes is involved in type 2 inflammatory responses. DNA demethylation of a specific region of the TSLP promoter increases the expression of TSLP in skin lesions of patients with AD and reduces the expression of filaggrin, a structural protein for which the loss-of-function mutations are associated with epidermal barrier defects and more severe AD. ,85. GeneTSLP is not found in the lesions of nickel-induced allergy contact dermatitis .Eosinophilic esophagitis (EoE) is associated with polymorphisms in the gene encoding TSLP. A mouse model of EoE-like disease was developed independently of IgE, but dependent on TSLP and basophils. Therapeutic monoclonal antibody-mediated TSLP neutralization or basophil depletion ameliorated the established EoE-like disease in animals. In human subjects with EoE, elevated TSLP expression and exaggerated basophil responses in oesophageal biopsies were noticed, and a gain-of-function TSLP polymorphism was associated with increased basophil responses in such patients. Therefore, it was suggested that the TSLP-basophil axis contributes to the pathogenesis of EoE and has the potential to be therapeutically targeted .In food allergy, alarmin-targeted monoclonal antibodies may be potentially useful in selected patients, but there is not yet enough certainty to support them. Etokimab, an antagonist of IL-33, has been recently studied for IgE-mediated peanut allergy, while no clinical trials with tezepelumab in food allergy have been reported. Large, randomised trials are needed to assess the safety, efficacy, posology and the most suitable candidates for such treatments ,93. The Bacteroidales (Prevotella), along with Bacillales and Lactobacillales, and oral Prevotella spp. abundance is correlated with decreased local secretion of TSLP [Finally, growing evidence indicates the significant role of commensal microbiota in susceptibility to food allergies . Exposur of TSLP .kB signalling pathway [Important lessons may be learned from animal studies using RNA sequencing and mouse models with deleted TSLP or TSLPR. Both murine and human TSLP exert their biological activities by binding to a high-affinity TSLPR complex, the heterodimer of TSLPR chain and IL7Rα ,14, and  pathway .-/-) have no apparent immune developmental deficiency [-/-) it was revealed that TSLP activates different immune cells such as ILC2s, but also DCs and CD4 + T cells, in the framework of innate and adaptive type 2 inflammation [Because TSLP is mainly an epithelium-derived alarmin, with a critical upstream role in the initiation of different immune responses , in tranficiency  and TSLPficiency . In TSLPammation . In addiammation .Levels of both TSLP mRNA and protein are increased within the airway epithelium and submucosa in asthma compared with healthy controls . In a suSeveral pharmacological strategies used for this purpose targeted TSLP or TSLPR as monoclonal antibodies or antagonists, and even fusion protein vaccines, for the treatment of inflammatory diseases . BispeciTezepelumab is the first-in-class fully human IgG2 monoclonal antibody against TSLP. It has been developed for the treatment of severe asthma, chronic obstructive pulmonary disease, chronic rhinosinusitis with nasal polyps, chronic spontaneous urticaria and eosinophilic oesophagitis . This anTSLP is a pleiotropic cytokine that plays a pivotal role in the development and advancement of allergy and asthma. It is primarily produced by epithelial cells and serves as a potent immune system activator. TSLP exerts its effects by selectively binding to a high-affinity heteromeric complex composed of TSLPR and IL-7Rα, initiating downstream complex intracellular signalling pathways. Its isoforms exhibit distinct roles and functions, with lfTSLP as a potent inducer of immune responses and sfTSLP as a potential inflammation suppressor. Their biological functions and interactions could be a decisive factor in numerous conditions, such as allergy and asthma, offering valuable approaches for therapeutic development. A deeper understanding of the actions of TSLP isoforms and interplay with epithelial cells and immune cells is critical for developing safe and effective TSLP-targeted therapies in inflammatory disorders and beyond.


Atopic dermatitis (AD) is a kind of chronic skin disease with inflammatory infiltration, characterized by skin barrier dysfunction, immune response dysregulation, and skin dysbiosis. Thymic stromal lymphopoietin (TSLP) acts as a regulator of immune response, positively associated with AD deterioration. Mainly secreted by keratinocytes, TSLP interacts with multiple immune cells , following induction of Th2-oriented immune response during the pathogenesis of AD. This article primarily focuses on the TSLP biological function, the relationship between TSLP and different cell populations, and the AD treatments targeting TSLP. Atopic dermatitis (AD) is one of the most frequent chronic and relapsing inflammatory skin diseases, which is featured by intense itching along with various clinical symptoms and signs . Over thThe family history of AD or asthma and seasonal allergies is closely related with increased risk of AD in childhood , 7. AparEmerging evidence has revealed TSLP induces Th2-type immune responses via activation of DCs and mast cells; therefore, TSLP is considered as a key molecular in AD physiopathology . In thisα-chain (IL-7Rα) so as to get involved with biological activities. According to sequence analysis, TSLPR is closely related to the common receptor-γ chain (γc) [α induces high affinity for TSLP and triggers subsequent signaling transduction. Amino acid sequence revealed that the identity of TSLP between murine and human is 43% and TSLPR is 39%, suggesting TSLP and TLPLR are homologous between human and mouse [TSLP was first found in the supernatants of a murine thymic stromal cell line in 1994  and was ain (γc) . Interesnd mouse , 23.The biological role of TSLP-TSLPR axis is rather conserved in human and mice. On the one hand, TSLP is similar to IL-7 that not only activates the same transcription factor, signal transducer, and activator of transcription 5 (STAT5) but also induces common genes expression. On the other hand, the mechanisms in STAT5 activation are distinctly different between TSLP and IL-7 . Isaksenβ and TNF-α) and TLR ligands , gene expression of TLSP is increased in both human and mouse airway epithelial cells [κB and interferon regulatory factor 3 (IRF3) signaling through TLR3 in human airway epithelial cells [β and TNF-α in human airway smooth muscle cells [α, TLR3, and TLR4 ligands also increased TSLP in synovial fibroblasts isolated from rheumatoid arthritis and osteoarthritis patients [in vivo and in vitro experiments demonstrated that notch-deficient skin keratinocytes lead to persistent defect in skin differentiation/barrier formation which subsequently release high levels of TSLP into circulation system [γ (IFN-γ), transforming growth factor-β (TGF-β), and IL-17 treatment [TSLP is abundantly expressed in the skin, intestine, thymus, lung, and tonsils. TSLP is found in epithelial cells, stromal cells, dendritic cells, and mast cells , 27 but al cells . The expal cells . Apart fle cells . TNF-α, patients , 35. Allpatients . What isn system . Of notereatment .TSLP has become as an important factor positively correlated with AD. Serum TSLP levels in adults and children with AD are significantly higher compared to those in healthy people , 39. In In vitro experiments demonstrated that the TSLP expression is significantly elevated in TNF-α-inflamed HaCaT [in vitro findings, skin-specific overexpression of TSLP spontaneously leads to an AD-like phenotype, accompanied with an increase of inflammatory cell infiltration in eczematous lesions, elevated frequency of CD4+ Th2-expressing cutaneous homing receptors, and higher levels of IgE in serum in comparison with WT littermates [Keratinocytes are the most abundant cells located in epidermis, serving a significant role in promoting AD . TSLP ised HaCaT  and ployed HaCaT . Recent ed HaCaT  and histed HaCaT  while need HaCaT  and aryled HaCaT . Furthered HaCaT . Consisttermates . Their ftermates , 52. Li εRI via specific IgE, subsequently driving Th2 polarization along with dominant secretion of IL-4, IL-5, and IL-13 during AD acute phase [+ DCs and the production of CC chemokines  by CD11c+ subpopulation of DCs to attract Th2 cells [γ (PPARγ) apparently serves as an important downregulating factor against TSLP-initiated immune responses through inhibiting DC functions and TSLP production in NC/Tnd mice [γ from T cells [It is well known that DCs play a pivotal role in the immunologic cascade, prompting the AD initiation and development. Allergens commonly stimulate DCs through the binding of Fcte phase . It is rte phase . Even thh2 cells . Elentneh2 cells . It is wTnd mice . Further T cells , 58, 59. T cells , 61, thuIn vitro experiments have exhibited that mouse TSLP serves as a potent cytokine to support T cell growth. The association between TSLP and T cell lymphopoiesis was further supported by the evidence that γc/TSLPR double-deficient mice developed significantly severe hindrance concerning T cell development compared with γc single-deficient mice [−/− mice [−/− mice, implying T cell development not strictly dependent on the TSLP-TSLPR pathway under normal conditions [ent mice . In line−/− mice . Howevernditions .+ T cells in mice. Omori and Ziegler found that TSLP preferentially contributes to Th2 cell differentiation from naïve CD4+ T cells due to the induction of IL-4 expression in CD4+ T cell without the presence of the addition of IL-4 and antigen presenting cells. Thus, the blocking of IL-4 suppresses TSLP-induced Th2 polarization [+ T cells and promotes the upregulation of Th2 cytokine. It is evidenced by the observation that human TSLP not only contributes to the activation and differentiation of CD8+ T cells into cytotoxic T cells but also acts as a survival factor for CD8+ T cells [+ T cells, thus boosting CD8+ T cell survival and inducing the expression of antiapoptotic protein, Bcl-2, while hardly exerting function on CD8+ T cell homeostatic proliferation [Previous studies have uncovered the biological function of TSLP to induce Th2 differentiation due to the direct interaction on CD4rization . Additio T cells . What isferation .β−/− background [In vitro experiments found that the proinflammatory Th2 cytokines, including IL-5, IL-6, and IL-13 produced and secreted by human mast cells, are attributed to the synergistical stimulation of TSLP, IL-1β, and TNF-α [εRI during allergic rhinitis development [Beside from the discussion of TSLP with keratinocytes, DCs, and T cells, TSLP is also closely associated with other immune cell populations. A growing body of evidence has demonstrated that skin inflammatory infiltration cells, including mast cells and eosinophils, are remarkedly increased accompanied with skin epidermal thickening in the K5-TSLP transgenic mice with TCRckground . In vitrnd TNF-α . What isnd TNF-α . Recent nd TNF-α . In turnelopment .−/− mice. Single TSLP treatment can increase the migration of eosinophil, and the combination of TSLP with other cytokines can further enhance eosinophil migration via the phosphorylation of L-plastin [The elevation of TSLP expression mediates eosinophils, DCs, and mast cells in MC903-induced RAG-plastin .TSLP has been found to promote basophil hematopoiesis, when cultured with bone marrow progenitor cells for 5 days. TSLP is capable of maturing a functionally distinct basophil subgroup without strictly requiring IgE. In addition, TSLP-induced basophils would release IL-4, thus triggering the Th2 response and accelerating AD progression , 72.ILC2s, as one of the ILC subsets, are distributed in healthy human skin and found abundantly enriched in lesional skin of people suffering with AD . In AD mλ human monoclonal antibody with the aim at circulating TSLP. In terms of mechanism, tezepelumab binds to the TSLP receptor, which therefore interferes the interaction of TSLP with its receptor and ultimately blocks the downstream inflammatory pathways. Tezepelumab has conducted the phase 1 clinical trial (NCT00757042) and phase 2a (NCT02525094) randomized, double-blinded, and placebo-controlled studies in AD patients.As described, accumulating evidence indicated that TSLP is a key molecule in the pathogenesis of AD. Released by epidermal keratinocytes due to a series of stimulation, TSLP promotes Th2 responses as well as the pathway for pruritus via interacting with a subpopulation of sensory neurons. Thus, TSLP is a promising therapeutic target for ameliorating or preventing AD progression. Tezepelumab (AMG-157/MEDI9929) is an IgG2p = 0.091). Moreover, multiple improvements were observed among patients at clinical endpoint, and biomarker-defined subgroups displayed greater efficacy of tezepelumab, indicating the potential benefits of TSLP blocking in treating AD patients. Nevertheless, no statistically significant improvements were achieved, including EASI50 response, 1EASI75 response, and pruritus numeric rating scale. Apart from efficacy verification, researchers also evaluated the safety of tezepelumab that the occurrence of treatment-emergent adverse events (TEAEs) was similar between two treatment groups. This clinical trial explored a potential agent to ameliorate AD, but with limited samples. In the light of the critical role of TSLP in AD pathogenesis in the previous preclinical studies, more clinical studies with tezepelumab application are required to confirm these findings in the phase 2a trial. Besides, a phase 2b trial of tezepelumab enlisted AD patients to further testify the safety and effectiveness of tezepelumab as monotherapy and adjunct therapy in combination with TCS (NCT03809663) [NCT01732510) but was hauled on the halfway of the trial without statistic recorded [NCT02054130) and has two ongoing phase 3 trials in patients with uncontrolled, severe asthma [NCT03138811) among patients suffering with mild asthma [In the phase 2a trial, 113 patients with moderate to severe AD were randomized 1 : 1 at first, following either subcutaneous tezepelumab (280 mg) combined with class 3 topical corticosteroids (TCS) treatment or a placebo combined with TCS treatment every 2 weeks . At week3809663) . It is we asthma . What isThe association between TSLP and AD has been confirmed by rising evidence. The SNPs of TSLP gene are positively correlated with the high occurrence of AD among people. The current available evidence proves that the overexpression of TSLP in keratinocytes deteriorates AD-like lesions in mice, while ablation of AD impedes the development of AD. As TSLP is involved with numerous cell populations during AD , TSLP no


Head and neck cancers (HNC) encompass a broad spectrum of neoplastic disorders characterized by significant morbidity and mortality. While contemporary therapeutic interventions offer promise, challenges persist due to tumor recurrence and metastasis. Central to HNC pathogenesis is the aberration in numerous signaling cascades. Prominently, the Wnt signaling pathway has been critically implicated in the etiology of HNC, as supported by a plethora of research. Equally important, variations in the expression of non-coding RNAs (ncRNAs) have been identified to modulate key cancer phenotypes such as cellular proliferation, epithelial-mesenchymal transition, metastatic potential, recurrence, and treatment resistance. This review aims to provide an exhaustive insight into the multifaceted influence of ncRNAs on HNC, with specific emphasis on their interactions with the Wnt/β-catenin (WBC) signaling axis. We further delineate the effect of ncRNAs in either exacerbating or attenuating HNC progression via interference with WBC signaling. An overview of the mechanisms underlying the interplay between ncRNAs and WBC signaling is also presented. In addition, we described the potential of various ncRNAs in enhancing the efficacy of chemotherapeutic and radiotherapeutic modalities. In summary, this assessment posits the potential of ncRNAs as therapeutic agents targeting the WBC signaling pathway in HNC management. Head and neck cancers (HNC), encompassing malignancies arising from the oral cavity, oropharynx, nasopharynx, hypopharynx, and larynx, represent a significant global health burden, as underscored by their pronounced prevalence . The higOne of the core signaling processes responsible for regulating cell proliferation, cell polarity, and cell fate determination in embryonic development and tissue homeostasis is signaling via the Wnt family of secreted glycolipoproteins ,19,20,21In recent years, the emergence of non-coding RNA (ncRNA) has profoundly expanded our understanding of cellular and molecular biology. Intriguingly, based on Ensemble1 (v76) data, a mere 34% of the human transcriptome encodes proteins, while the residual 66% encompasses non-coding genes. This segment includes entities such as ncRNAs, antisense RNAs, and pseudogenes, among others ,29,30. CThe components of the WBC signaling pathway were initially elucidated through genetic investigations in Drosophila . In 1982ZD), forming a larger cell surface complex together with lipoprotein receptor-related protein (LRP) 5/6 co-receptors. This complex activates the dishevelled (Dvl) proteins, which recruits the Axin complex to the receptors, leading to the displacement of GSK3β from the Axin complex. Consequently, β-catenin escapes degradation, accumulates in the cytoplasm, and translocates to the nucleus [The WBC signaling pathway operates through a complex network of interactions and signaling events. At the center of this pathway is β-catenin, a protein that plays a dual role as a core component of the pathway and a transcriptional co-factor . In the  nucleus . In the  nucleus . Given the essential roles played by WBC signaling in development and overall well-being, it is no wonder that mutations to the components of this pathway have been linked to several congenital disabilities, cancer development, and other serious diseases . The strThe development of HNC consists of sequential alterations to the cellular and molecular pathways in the squamous epithelium, leading to gradual proliferation from pre-malignant lesions to tumors . The WBCThe 21st century witnessed the discovery of transcripts that do not code for any proteins due to the successful achievements of the Human Genome Project and the subsequent initiative termed ENCODE (The Encyclopedia of DNA Elements project) in 2005  . The majlncRNAs are a highly heterogenous group of transcripts that are more than 200 nucleotides in length and do not code for any protein ,54. TheyAnother subclass of ncRNAs is represented by circRNAs, which are ubiquitously distributed in the blood, various bodily fluids, and tissues . circRNAA growing body of literature has demonstrated the importance of ncRNAs in human malignancies ,59. TheyResearch indicates that anomalies in Wnt signaling can induce unbridled cellular proliferation, thereby fostering tumor initiation and advancement . SpecifiA comprehensive body of research exemplifies the involvement of lncRNAs and miRNAs in mediating cell proliferation in HNSCC cells through the modulation of the Wnt signaling pathway . For exaFurthermore, another study demonstrated that SLCO4A1-AS1 functions as a competing endogenous RNA (ceRNA) binding to miR-7855-p, leading to SETD7 upregulation. This lncRNA also upregulated the WBC pathway proteins, which augmented cell proliferation while inhibiting apoptosis . FurtherSeveral studies have reported the role of lncRNAs in modulating the WBC signaling pathway and its components by functioning as “miRNA sponges”. For instance, Jin et al. (2020) reported that overexpression of the lncRNA TIRY in cancer-associated fibroblasts (CAFs) activated the WBC signaling pathway, promoting the proliferation of OSCC cells. This upregulation was attributed to a sponging mechanism that reduced miR-14 expression . Concurr7 (FZD7), Wnt-7b, phospholipase D1 (PLD1), and CXCR4 [An emerging number of studies have investigated the role of ncRNAs in the suppression of proliferation and survival, and the induction of apoptosis of HNSCC cells by modulating the WBC signaling pathway. For instance, a recent study demonstrated that an siRNA-mediated knockdown of circRNA hsa_circ_0136839 markedly enhanced the cell cycle progression and cell proliferation of NPC cells by upregulating Wnt signaling proteins such as β-catenin and cyclin D1. However, this was significantly reduced by overexpressing this circRNA. Therefore, this study reinforced the notion that the aberrant expression of circRNA results in the development of HNSCC . Additiond CXCR4 ,87,88,89nd CXCR4 . Furthernd CXCR4 . Anothernd CXCR4 . Likewisnd CXCR4 .Curcuma longa) inhibited the proliferation of OSCC cells by upregulating miR-9 and repressing WBC signaling [Glycyrrhizae radix), suppressed NPC cell growth and proliferation as well as induced apoptosis by suppressing miR-32 and downregulating canonical Wnt signaling [Interestingly, a range of natural compounds has demonstrated anticancer properties against diverse cancer types, including HNC ,101,102.ignaling . In addiignaling . TherefoEMT is a biological process characterized by the transformation of epithelial cells into a more mobile mesenchymal phenotype, allowing them to invade and migrate to new tissues . ImportaNumerous investigations have elucidated the pivotal role of ncRNAs in modulating Wnt signaling, which in turn influences the invasive phenotype, indicating their central function in directing the invasion and migration of HNSCC cells . For insFurthermore, another study demonstrated that the lncRNA TIRY activated the WBC signaling cascade within CAFs. This activation promoted EMT, invasion, and metastasis in OSCC cells by diminishing miR-14 levels through a sponging mechanism. At a mechanistic level, TIRY was observed to amplify the expression of molecular markers including Snail, Zeb1, α-SMA, Wnt-3a, and β-catenin . Ji et aA myriad of investigations has elucidated the pivotal role of ncRNAs in hampering EMT, invasion, migration, and metastasis in HNSCC. For instance, the tumor suppressor miRNA, miR-503, was shown to curtail HNSCC cell invasion by downregulating WBC pathway constituents, including Wnt-3a and MMPs 3, 7, and 9. This miRNA also decreased the expression of invasion-related genes, including fibroblast growth factor 7 (FGF7) and connective tissue growth factor (CTGF) . AdditioChemotherapy and radiotherapy constitute the cornerstones of therapeutic intervention for individuals afflicted with advanced or metastatic stages of HNSCC, as substantiated by pertinent research findings ,121. CisAdditionally, a couple of studies have also demonstrated the modulation of radioresistance in HNSCC cells by lncRNAs via WBC signaling. For instance, the lncRNA LINC00473 enhanced the radioresistance of LC cells by activating WBC signaling. This study also demonstrated that the silencing of LINC00473 and X-ray treatment induced apoptosis and suppressed colony formation and the proliferation of LC cells . In addiHNC encompasses a spectrum of malignant conditions affecting the oral, nasal, nasopharyngeal, hypopharyngeal, and laryngeal regions. Deviations in the expression of proteins involved in various cellular activities and pathways constitute a significant factor in the advancement of HNSCC. The WBC signaling pathway exemplifies the complex cellular communication systems vital to both developmental and pathological contexts. While the WBC pathway is fundamental for cellular proliferation, morphogenesis, and homeostatic balance, its aberrations can lead to severe consequences. Genetic alterations within this pathway are associated with diverse cancers, including HNC. Over recent years, ncRNAs have gained prominence as potential therapeutic targets in numerous cancers, including HNC. Emerging research underscores the instrumental role of ncRNAs in HNC, modulating various signaling pathways, including WBC. Consequently, ncRNAs impact key oncogenic processes in HNC, such as cellular proliferation, differentiation, invasion, EMT, migration, and metastasis. Clinical trials are increasingly evaluating the utility of several ncRNAs as diagnostic and therapeutic indicators in oncology. For instance, one noteworthy study highlighted the potential of MALAT1 targeting miR-124 as a diagnostic biomarker for OSCC, as denoted in trial NCT05708209. The investigation encompassed 20 OSCC patients and 20 healthy controls, assessing MALAT1 and miR-124 levels in unstimulated saliva samples. Furthermore, ncRNAs critically influence the chemotherapeutic and radiotherapeutic resistance of HNSCC cells. Given the importance of ncRNAs in HNC, a thorough understanding of their specific roles is vital for the innovation of refined therapeutic strategies. This review describes the central role of ncRNAs in modulating cancer cell behavior, particularly through the WBC signaling mechanism. Nonetheless, further studies are essential to thoroughly discern the contributions of ncRNAs in HNC progression and to design groundbreaking therapeutic interventions for this malignancy.


In the realm of molecular biology, few terms evoke as much curiosity, fascination, and determination as RAS. For more than four decades, researchers have relentlessly pursued a deeper understanding of RAS signaling, unravelling its intricate web of interactions in health and disease. Today, we stand at a remarkable juncture, where ground-breaking advancements have allowed us to finally drug this once elusive oncogene. With this Special Issue on RAS signaling in health and disease, we celebrate the culmination of efforts that have brought us to this historic milestone.One of the key aspects of RAS signaling lies in its multimeric interactions on the cellular membrane. The review article “Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation”  sheds liThe emergence of RAS dimers as pivotal players in the RAS-ERK pathway has opened up new avenues for exploration. “RAS Dimers: The Novice Couple at the RAS-ERK Pathway Ball”  exploresPI3K has emerged as a critical component in the RAS signaling network, influencing diverse cellular processes. “The Importance of Being PI3K in the RAS Signaling Network” [After years of intense research and ingenuity, we have finally witnessed ground-breaking advances in the targeting of RAS in cancer. “Drugging the Undruggable: Advances on RAS Targeting in Cancer”  showcaseThe convergence of the RAS and RHO signaling pathways presents a captivating nexus with profound implications for cellular transformation, motility, and contraction. “The Crossroads between RAS and RHO Signaling Pathways in Cellular Transformation, Motility, and Contraction”  delves iRas GTPases have emerged as critical contributors to kidney fibrosis, a condition of significant clinical importance. “Dissecting the Involvement of Ras GTPases in Kidney Fibrosis”  disentanAs we reflect on the journey of RAS research, “40 Years of RAS—A Historic Overview”  offers aWhile oncogenic RAS has been the focus of intense scrutiny, the role of wild-type RAS in the transformation process cannot be overlooked. “The Role of Wild-Type RAS in Oncogenic RAS Transformation”  sheds liThe RAF signaling pathways hold immense potential as therapeutic targets for blocking oncogenic RAS signaling. “Hidden Targets in RAF Signaling Pathways to Block Oncogenic RAS Signaling”  uncoversWith these extraordinary review articles on RAS signaling in health and disease, we celebrate the tireless efforts of scientists, researchers, and clinicians who have dedicated their careers to unlocking the mysteries of RAS. These are special times indeed, as we are witnessing long-awaited breakthroughs in drugging the “undruggable” oncogene. Let us seize this moment of triumph to not only reflect on the arduous journey but also to envision the future possibilities that lie ahead, as we continue to decode the complexities of RAS signaling and strive to transform lives through scientific discoveries.Together, let us embark on this fascinating exploration of RAS signaling, uniting our knowledge and embracing the special times we are living in.


Functional neurological disorders (FND) lead to increased care requirements and costs, negatively impacting healthcare budgets. Healthcare expenditure in FND has escalated beyond other neurologic disorders during the past decade.To assess inpatient costs in adults admitted to the neurology ward at Universitas Academic Hospital (UAH) in central South Africa.n = 29) and a systematic sample of other neurological disorders were included in the comparison group (n = 29). Data were obtained from the Meditech billing system and clinical records.A retrospective observational study with a comparative component was conducted on patients admitted during 2018 and 2019. All FND cases , translating to approximately half the total costs of patients admitted for other neurological disorders.The daily median cost was similar for FND and other neurology-related admissions. The lower overall inpatient costs for FND patients were only related to significantly shorter durations of stay, which may reflect new diagnostic approaches resulting from changes in the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-5) diagnostic criteria. The prevalence of FND was similar to those reported in previous studies conducted at neurology clinics.The study contributes towards better understanding the prevalence and cost of FND in local neurology inpatient care settings. In the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5), an identifiable stressor is no longer a prerequisite for the diagnosis. This change should improve diagnostic accuracy in many cases.1 Functional neurological disorders are often correctly diagnosed after considerable delays and are a common source of disability in medicine.2The symptoms of functional neurological symptom disorder (FND) are inconsistent with the range of clinical manifestations of other neurological, medical or mental disorders.7 Functional neurological disorders are of unknown aetiology and often require investigation-intensive hospital admissions.6 A key concern has been the fear of misdiagnosis, possibly contributing to unnecessary and costly evaluations and inappropriate treatments.2 Furthermore, FND is characterised by high rates of psychiatric comorbidity and other medical conditions.6 Long-term studies suggest a chronic recurring course with low remission rates and persisting impairment in most patients (31% – 71%).10Evidence suggests that primary, emergency and inpatient healthcare use by patients with FNDs is high and increases more than for other neurological disorders, with significant cost implications.7 Given the over-stretched healthcare resources and planned implementation of the national health insurance (NHI) in South Africa, knowledge of the cost of FNDs to the healthcare system is essential. Knowledge of the costs incurred for FND patients is pertinent to clinicians working in a resource-constrained environment. There is currently a lack of published data on the local costs of FNDs, and therefore, their contribution to healthcare spending is unknown.Locally, it is unknown if incurred spending for FNDs mimics international study results that indicate higher and increasing costs compared to other neurological diseases.1 Before DSM-5, the lack of a clear definition hampered accurate epidemiological findings.2 The incidence rate for mixed FND is estimated at 4–12 per 100 000 population per year.17 Stone et al.18 identified functional and psychological symptoms in 16% of patients referred to neurology clinics over 15 months. Furthermore, up to 30% of patients in their study had symptoms that could not be explained or fully explained based on a known disease.18 Low socioeconomic status and female gender have been associated with FND, with women comprising 60% – 75% of FND cases.8Because of a historical lack of diagnostic clarity, the epidemiology of FND is complex. Although transient conversion symptoms are common, the precise prevalence of FND is unknown.Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM IV)19 and was incorrectly assumed to primarily be a psychiatric disorder and was associated with underlying emotional stressors.20 Recent abnormal findings in functional brain imaging studies, and the absence of identifiable stressors in many cases, confirmed the clear biological underpinnings of FND.21 These findings suggest increased brain activity in regions responsible for motor execution and inhibition, including the supplementary motor area and the right temporoparietal junction (TPJ). The right TPJ is responsible for self-agency, hypothesised to be defective in FND. These abnormalities are postulated to result in an exaggerated response to perceived social threats that may result from an over-sensitivity to emotional stimuli.20Functional neurological disorder was previously known as conversion disorder in the The objectives of the study were: (1) to establish the prevalence of FND among inpatients admitted to the neurology ward at Universitas Academic Hospital (UAH) in Bloemfontein, South Africa, for the period 01 January 2018–31 December 2019; (2) to describe the demographic profile of inpatients diagnosed with FND at the neurology ward, during the study period and (3) to compare the cost of neurology inpatients with FND and those with other neurological disorders during this period.A retrospective observational study with a comparative component was conducted.For the first objective, all patients aged 14 years and above admitted to the neurology ward of UAH from 01 January 2018 until 31 December 2019 were included as the denominator. All patients who fulfilled the following criteria were included in the numerator for the first objective and the FND group for the second and third objectives: (1) a diagnosis of FND made during the study period; (2) admission to the neurology ward and (3) ≥ 14 years of age. Only patients 14 years and older are admitted to this ward.All patients diagnosed with FND were included in the study. For the third objective, a comparative group of similar age was randomly selected from 501 patients admitted to the same ward during the study period. A systematic sample of every 12th patient who did not have FND was selected for the comparative group until the numbers in the two groups matched. Patients’ clinical records that did not contain all the required demographic and clinical variables were excluded from the study.22 were searched on the Meditech database.Secondary data were collected from patients’ notes on Meditech, an electronic database containing clinical information for all patients admitted to UAH. The total number of admissions to the neurology ward from 01 January 2018 to 31 December 2019 was recorded. In addition, clinical files of patients admitted to the neurology ward during the period under investigation were reviewed to identify all patients diagnosed with FND. The ICD 10 codes F44.4 (conversion disorder with motor symptom or deficit) and F44.9 The patients’ accounts containing all costs incurred during the admission period were retrieved from the revenue section of UAH. The first author entered all demographic, clinical and financial data during July 2020 into a Microsoft Excel data collection spreadsheet designed for this study. The following variables were transcribed on the data form: age, gender, clinical presentation, length of stay, medical comorbidities, psychiatric comorbidities, blood investigations, cerebrospinal fluid investigations, radiological investigations, neurophysiological investigations, medications administered, allied health services received and H classification.23 The UPFS is applied by the revenue section of UAH and contains all costs incurred by the hospital. These costs include the ward and professional fees, laboratory fees, professional imaging fees, pharmacy fees, medication fees and allied health service fees.Regarding cost, the Uniform Patient Fees Schedule (UPFS) proposed by the National Department of Health is used for billing all patients. Their H classification determines the amount billed to the patient according to their annual income. The categories for H classification range from H0 to H4 .  per dollar. Therefore, patients categorised as H1 earned approximately $0.00–$4781.00 per annum, category H2 patients earned $4781.00–$17 076.00 and category H3 patients made >$17 076.00 per annum; information obtained from Currency Converter live rates.Data analysis was conducted by the Department of Biostatistics at the University of the Free State using SAS Version 9.4 . Medians with interquartile ranges (IQR) were reported for the patient cost of FND. Medians and quartiles were used to summarise other numerical variables with skew distributions. Frequencies and percentages summarised categorical variables. Ninety-five percent confidence intervals (95% CI) were calculated for primary outcomes. Groups were compared using chi-squared and Fisher’s exact tests for categorical variables and Mann–Whitney tests for numerical variables.Approval to conduct the research was obtained from the Health Sciences Research Ethics Committee (HSREC) of the University of the Free State (ethics clearance number UFS-HSD2020/0342/2909). Permission was also obtained from the Free State Province Department of Health. All data were obtained from patients’ hospital account files; therefore, no informed consent was required.Data were stored safely and securely on a password-protected computer. The Excel spreadsheet was also password-protected to ensure the safety of the data. Data will be retained for five years after completion of the study according to HSREC regulations and facilitate information for subsequent follow-up studies. After five years, data records will be destroyed by the principal investigator.n = 29)  of 530 inpatient admissions to the neurology ward during the period under investigation. The median age reported for FND was 28 years (IQR 19–36), while the median age of the other neurological disorder (non-FND) patients was 34 years (IQR 25–45). The 95% CI for the median difference in age was −4 to 0 years, p = 0.046. However, no significant differences in age categories or gender were observed between the groups (p = 0.008).Functional neurological disorder accounted for 5.5% , while epilepsy  was the most common among non-FND. Major depression was diagnosed in 17.2% (n = 5) and 10.3% (n = 3) of FND and non-FND patients, respectively , psychogenic non-epileptic seizures , hemiplegia , movement disorder , visual disturbance  and quadriplegia . The most common symptoms in the non-NFD group were ataxia  and visual disturbances .Medical comorbidities were more prevalent in the comparison group, but the difference failed to reach statistical significance . The mosectively . SymptomOver 86% of participants in each group had laboratory investigations, and over half were referred for imaging studies. No significant differences related to laboratory, radiological or neurophysiological investigations were observed between the groups . Involvep = 0.008) (p = 0.604).The median total expenditure during the admission period was R12781.03 (approximately $873.00) for FND; and R25069.71 (about $1712.00) for the comparative group (= 0.008) . Ward adMost patients were classified as H1 (low-income category) in both groups, namely 72.4% of FND patients and 86.2% of non-FND patients .9 Universitas Academic Hospital is a tertiary hospital for the Free State Province, with many patients referred from rural areas where investigation-intensive hospital admissions are not feasible. Therefore, patients often need to be admitted for investigations.The prevalence of FND is complex and has been hampered, until recently, by the lack of a clear definition. The study’s FND prevalence of 5.5% is consistent with the 5% – 6% reported in the literature for referrals to neurology clinics.24 Although daily expenditure was similar for both groups, FND patients had a significantly shorter length of stay, contributing to cost savings. The shorter length of stay may reflect the clarification of diagnostic criteria in the DSM-5 and clinicians’ ability to identify neurologic signs specific to FND accurately.Delayed diagnosis, investigation-intensive hospital admissions and re-admissions are cost drivers in FND and increase the risk of iatrogenic harm.25 have reported a wide age range of onset of FND, while other studies27 reported a younger onset of FND symptoms. The study’s median age of FND patients was younger than the comparison group. Although unclear at present, this may result from improved diagnostic clarity and earlier recognition by referring primary healthcare clinicians. Paraplegia, the most common clinical feature in our FND patients, represents a particularly robust clinical presentation of FND, which may have contributed to earlier recognition than in other populations. However, paraplegia as the most common presenting symptom is consistent with previous reports.28 The relatively young age, lower expenditure and shorter length of stay of FNDs in our study suggest appropriate early recognition and limitation of iatrogenic harm in the local setting. The significantly lower pharmacy cost of FND patients further supports this notion.Previous studies26 Understanding of the higher prevalence in women remains obscure. Some authors have postulated that FND may be related to subconscious defence mechanisms employed more frequently by women in male dominated societies.25 Notably, the gender distribution was similar in the comparison group, and conclusions could not be drawn regarding the predominance of women in the FND group.As in some previous studies, more women were represented in the FND group.18This study is the first to examine the prevalence of FND in Neurology inpatients in the Free State. The prevalence of FND is currently unknown, and the study contributes towards a better understanding of the prevalence in tertiary health care settings. The period for which data were collected is comparable to previous epidemiological studies and could be regarded as sufficient.29 Patients attending the Universitas Hospital mostly cannot afford private care; therefore, this finding is not surprising. Unfortunately, obtaining data on the prevalence of FND in private healthcare facilities was beyond the scope of this study.Most patients in our study were from low-income categories , previously associated with FND and the risk of poor health outcomes.Information on outpatient costs before and after hospitalisation was not collected, and the results of this study should be interpreted with caution. Outpatient services required to manage FND in a multi-professional context could be considerable. Although patients are often admitted for confirmation of the diagnosis, the treatment of FND patients mainly occurs in outpatient settings. Prospective studies should investigate both in- and outpatient costs incurred in managing FND patients.2Including patients in private healthcare facilities would have enhanced demographic variability but make cost comparisons difficult. The results indicate the prevalence of FND among lower-income categories only. Although data on neurophysiologic investigations, allied health and consultation-liaison psychiatric services were often incomplete, it was for both the FND and comparative group and is unlikely to have influenced the cost findings. Because of the retrospective nature of the data collection, the investigator relied on previously documented clinicians’ diagnoses in the neurology wards. Therefore, the investigator could not use standardised diagnostic criteria  to confirm the diagnosis of FND. Literature suggests that FND, when diagnosed by neurologists, are accurate and remarkably consistent, and incorrect diagnoses of FND are unlikely to have influenced study results.Diagnostic and Statistical Manual of Mental Disorders, 5th edition changes have improved diagnostic clarity and understanding of the prevalence of FND. Functional neurological disorders are common and can be accurately diagnosed by neurologists on the clinical presentation without requiring extended hospital admissions or extensive and costly investigations. Educating students and clinicians about clinical syndromes outside the simplistic mind–body dichotomy should go a long way towards improved empathy, diagnosis, management and limiting iatrogenic harm.32


Sarcoptes scabiei var hominis, a microscopic ectoparasite mite with no non-human reservoir. Scabies is transmitted through skin-to-skin contact, with higher numbers of mites conferring greater infectiousness. Like human T-lymphotropic virus type 1 (HTLV-1), scabies is an ancient infection, clustering in socio-economically marginalised populations in Africa, Oceania and South America, with outbreaks often found in institutional settings and other overcrowded areas [Scabies is an infestation of ed areas . The gloed areas . Here weClassical, or typical, scabies infection causes pruritus and characteristic lesions. The global prevalence of classical scabies peaks between the ages of five and 25 years , attribuMycobacterium tuberculosis and Strongyloides stercoralis [HTLV-1, the first human retrovirus discovered, has a global distribution that reflects both ancient and modern human migration [rcoralis ,5. EstimP = 0.001)  and post-streptococcal glomerulonephritis (PSGN), with these implicated in the development of rheumatic heart disease (RHD) and chronic kidney disease (CKD) [Streptococcal skin contamination and haematuria, an indicator of kidney disease.Impetigo and pyoderma, particularly due to se (CKD) . In somese (CKD) , while fP = 0.001) [P < 0.001) [The global distribution of ARF, RHD and PSGN mirror those of scabies and HTLV-1, with the highest rates seen in some Australian First Nations populations ,19,21. T= 0.001) , perhaps< 0.001) , The exaMycobacterium tuberculosis and Strongyloides stercoralis [Cutaneous manifestations of HTLV-1 are common, and include cutaneous lymphomas, inflammatory and infective conditions, such as infective dermatitis of HTLV-1 . There ircoralis .Individuals with scabies, particularly those with crusted scabies, can transmit to others in close proximity, regardless of the recipients’ immune status. With its atypical presentation, delayed diagnosis and treatment of crusted scabies is common, facilitating ongoing transmission within communities, particularly in the context of poverty and overcrowding. Indeed, the conditions in which scabies infestations thrive: those of poverty, marginalisation and overcrowding, are common in the communities most affected by HTLV-1. More work is required to assess the extent to which scabies and HTLV-1 overlap due to shared social determinants of health compared to underlying biological pathways.In areas endemic for scabies, such as Fiji and the Solomon Islands, community-wide treatment programmes are often used, as relying only on the treatment of symptomatic individuals may not identify all cases, allowing for ongoing reinfection. Individuals living with HTLV-1 are at heightened risk of both atypical and recurrent scabies infections. Rates of HTLV-1 increase with age, due to the combination of vertical and horizontal transmission . HoweverHigh rates of renal dysfunction and rheumatic fever are known to occur not only in scabies-endemic areas but other areas of HTLV-1 endemicity. Skin barrier breakdown is common in HTLV-1, through related skin conditions like infective dermatitis, structural changes like acquired ichthyosis/xerosis, or infestations like scabies. These may lead to the development of bacterial superinfection with species associated with the immune sequelae of acute rheumatic fever and post-streptococcal glomerulonephritis, and the subsequent development of chronic cardiac and renal disease. Antimicrobials for scabies and bacterial skin infections may reduce risks of systemic sequelae but the likelihood of reinfection is high with concurrent HTLV-1.Streptococcal skin infections, and between these skin infections and immune-mediated renal and cardiac sequelae. This Viewpoint draws on this evidence and hypothesises additional mechanisms by which HTLV-1 infection could be a driver in the maintenance of scabies and its disease sequelae within communities. Further studies are needed to assess the impact of HTLV-1 not only on scabies, but also on its associated renal and cardiac sequelae, at both individual and population levels.There are known associations between HTLV-1 and scabies, between scabies and Group A Although HTLV-1 is not yet curable, its identification allows strategies to be implemented to prevent onward transmission, improving health outcomes for future generations in some of the world’s most marginalised populations.