Factors contributing to the risk for significant morbidity included being male, lesion diameter > 5 mm, volume > 0.3 mL per application, and total injected volume > 0.3 mL. A waiting time of 12 weeks prior to additional EAO application was required in 12 out of 29 lesions for clinical observation of complete regression. It was concluded that sclerotherapy with EAO as monotherapy is easy to apply, safe, and effective within a small number of sessions. Application of <0.3 mL EAO per session, and a waiting time of 12 weeks prior to the second application, would significantly minimize morbidity.Vascular malformations (VMs) are a wide vascular or lymphatic group of lesions common on the head and neck. The objective of this study was to assess the efficacy and morbidity of sclerotherapy for the treatment of VMs in the oral and perioral area. Special attention was given to factors that may contribute to minimizing postoperative morbidity. Data from 25 patients (32 lesions) with oral VMs submitted to sclerotherapy with monoethanolamine oleate (EAO) were included. A structured form was used to collect data. An arbitrary score was determined to evaluate postoperative morbidity. Each of the following signs or symptoms received one point: pain, swelling, hematoma, ulceration, erythema, transient numbness, and transient itching. Pain and swelling were further divided into mild to moderate (1 point) and severe (2 points). Theoretically, the score was in the range of 0–9. Calculated scores ranged 0–4. The patients were further divided into two groups with scores of 0–1 denoting minimal morbidity (MIN) and 2–4 denoting significant morbidity (SIG). The number of lesions in each morbidity-score group were comparable (MIN 17and SIG 15). There were no statistically significant differences between the groups regarding age, number of applications, or average injection volume per mm lesion. Statistically significant differences were noted regarding gender ( Vascular malformations (VMs) are a wide vascular- or lymphatic-system heterogeneous group of lesions common in the head and neck . PathogeRecent studies pointed out the potential role of mediators, e.g., vitamins and asymmetric dimethylarginine (ADMA), in vascular malformations ,14. AnalSclerotherapy is an efficient and conservative method for the treatment of VMs. It is a simple procedure that consists of intralesional injection with a low recurrence rate, good esthetic results, and reasonable morbidity . Among tThe objective of the present study was to assess efficacy and morbidity following sclerotherapy  of VMs in the oral and perioral areas. Special attention was given to factors that may contribute to minimizing postoperative morbidity. The null hypothesis was that postoperative morbidity is dose-dependent. This was a retrospective descriptive study based on data collection from patients with oral VMs submitted to sclerotherapy with EAO at the Department of Oral and Maxilofacial Surgery, Rabin Medical Center, Petah-Tikva, Israel. An electronic and manual database search was performed to locate patients diagnosed with VM in the oral cavity treated by sclerotherapy. The electronic search used the terms “vascular”, “vascular malformations” and “vascular lesions” from 2013 to 2017, resulting in 25 cases that were included in the present study. The hospital ethics committee approved the study in accordance with the Declaration of Helsinki (protocol no. 14-0190). A structured form was used to collect demographic data , diagnostic resources used, location and size (greatest diameter) of the VMs, dosage and number of intralesional applications of EAO, interval between treatment sessions and follow-up sessions, intra- and postoperative complications, response to treatment, morbidity, patients’ satisfaction, recurrence, and total follow-up period. Morbidity was further classified as minor-to-moderate or severe according to intensity and duration of symptoms, patients’ return to the clinic, and the need for further treatment (medications or other).Clinical observation of painless purplish vesicles or bullae with soft consistency on palpation.Diascopy showing changes in coloring, intralesional ischemia, and decrease or alteration in e shape.Lesions were diagnosed as low-flow VM on the basis of a patient’s anamnesis, and the clinical evaluation of the color, consistency, and size of the lesion. Diascopy (performed by applying pressure and observing color changes), the diagnostic injection of a vasoconstrictor agent (observing size and color changes), and aspiratory puncture and auscultation were also performed as needed.High-flow VMs,inadequate available data, andPatient’s medication interfering with wound healing  or specific states preventing the use of EAO .Application of the sclerosing solution followed manufacturer’s instructions An arbitrary score was determined to evaluate postoperative morbidity. Each of the following signs or symptoms received one point: pain, swelling, hematoma, ulceration, erythema, transient numbness, and transient itching. Pain and swelling were further divided into mild-to-moderate (1 point) and severe 2 (points). A morbidity score was calculated for each patient. Theoretically, the score could be in the range of 0–9. Calculated scores ranged 0–4. The patients were further divided into two groups, 0–1 for minimal morbidity (MIN) and 2–4 for significant morbidity (SIG). p > 0.05). Descriptive statistics were produced, and means (M) and standard deviations (SD) were calculated for all continuous measurements. Categorical data were expressed as fractions , and fractions were also expressed as percentages. Statistical analyses of differences between the groups were assessed using Student’s t-test for continuous parameters and the chi-squared test for categorical data. p values lower than 5% were considered statistically significant.Data were collected using a Microsoft Excel 2016 spreadsheet . Statistical analyses were performed using SPSS Statistics for Window and version 22.0 . Kolmogorov–Smirnov tests were conducted to test normal distribution. Measurements showed normal distribution  and 10 males (40%), with a total of 32 VM lesions were included in the study. The most frequently involved area  was the Lesion diameter was in the range of 3–35 mm . The maiThe volume of injected EAO  ranged fThe majority of lesions were treated by a single application of EAO , while 6 (19%) underwent two treatment sessions and one case (3%) needed three treatment sessions. Among the seven cases that underwent more than one treatment, four sessions were held within a 1–2 week interval from the previous session. Follow-up sessions were conducted with a 1–3 week interval and until a total resolution of the lesion was observed. Twenty-nine lesions (~91%) showed total clinical regression , two lesIn most cases , healing was still in process after 1–3 weeks, and the response for treatment could not be determined at that time. In 97% of the cases (28/29 lesions), more than dix weeks were needed for complete resolution, while in 72% (21/29 lesions), complete healing was observed after eight weeks or more. In 41% of the cases (12/29 lesions), the required waiting time to resolution was 12 weeks.Most of the patients  experienced side effects to some degree. Common side effects included pain , swelling , hematoma , and ulceration . Less common side effects included erythema , transient numbness of the lower lip , and transient itching sensation of the lower lip . One patient developed necrotic tissue on the anterior part of the tongue , requirip = 0.05), lesion diameter (p = 0.030), volume of first application (p = 0.007), volume of second application (p = 0.05), and total injected volume (p = 0.03). Patients of male gender with lesion diameter > 5 mm, volume > 0.3 mL per application, and total volume > 0.3 mL showed higher morbidity.The number of lesions in each morbidity-score group were close . There were no statistically significant differences between groups regarding age, number of applications, or average volume per mm lesion . StatistThe subjects included in the present study were adults with a mean age of 59 ± 18 years. Other studies reported a higher prevalence of VMs in the young ,20,21.Sixty-one percent of VMs were in women, as reported in other studies ,21. HoweVMs were most frequently found on the tongue, whereas in most of the literature, the lower lip is the most frequent site ,7,20. OnAll the VMs were venous with slow blood flow. Success rate in such cases is in the range of 70–100% ,26,27,28Ideal EAO concentration and dosing for treatment is still debatable ,8,19,20.Complications in sclerotherapy are dose-dependent ,30,31,32The present study demonstrated that, in most cases (72%), complete healing was observed after 8–12 weeks, while in 41% of the cases, the required waiting time to resolution was 12 weeks. Consequently, no additional applications were required. In the present study, the required interval between treatment sessions and the waiting time necessary for complete healing were longer than previously reported . A totalThere are no standard treatment algorithms available for VMs, and the disorder has significant unmet clinical needs. Improved understanding of the pathogenesis of vascular anomalies may provide insights to the development of new targeted therapies . RecentlTaking into consideration the limitations of the study, such as the reduced number of samples (32 lesions) and the fact that it was a retrospective study, the application of EAO as a monotherapy was an easy, simple, and quick treatment because it was performed as an outpatient procedure with a limited number of sessions. It was well-tolerated by patients and resulted in low morbidity. The data showed that this is an effective treatment in 100% of the cases without the need for surgery .Sclerotherapy with EAO as a monotherapy is easy to apply, safe, and effective within a small number of sessions. Application of <0.3 mL EAO per session with a waiting time of 8–12 weeks before the second application significantly minimizes morbidity.


We present a combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics–statistical approach for the interpretation of nuclear magnetic resonance (NMR) chemical shift patterns in phycocyanobilin (PCB). These were originally associated with colour tuning upon photoproduct formation in red/green-absorbing cyanobacteriochrome AnPixJg2 and red/far-red-absorbing phytochrome Cph1 Organised in biliproteins, they take the role of a chromophore and serve phototrophic organisms as accessory pigments . Exc. ExcZZZsresponse ,39,40,41roducts,  while throducts, ,20,34,42The colour tuning in CBCRs is exceptional, regarding the very limited number of chromophore species they bind. We recently showed that the degree of solvation due to a sealed chromophore binding pocket cannot serve as an explanation model . HoweverIn this work, we present a quantum mechanics/molecular mechanics (QM/MM) study of PCB in a protein environment by means of extensive molecular dynamics (MD) and ab initio proteins : the rederfamily ,64,65,66erfamily ,71,72,73on state ,70,72. Hon state ,44. To dExtensive MD sampling under the QM/MM regime allows for the inclusion of thermal fluctuations of the molecular structure on the femtosecond and picosecond time scale. at 233 K . The simAlthough it seems that the uncertainty in the obtained averages of However, The interconnection of nuclear shieldings and bond lengths is not particularly surprising, but the remarkable “isolation” of Qualitatively, the geometric sensitivity follows the clear rationale that an atom’s chemical shift increases with the length of the originating bonds—and decreases with the next, and increases again with the following bond and so on. In The characteristic sensitivity in In 2015, Song et al. reported a peculiar difference pattern of oproduct . The autions cf.  the chanchanism: ,44 the eThe NMR experimental observations are made amid scarce information on the atomic coordinates of the photoproduct states since no structural data from X-ray diffraction are available. In these terms, the geometric sensitivity may serve to retrieve structural data from the g et al.  We carriComparing the two Ponds cf. . Going oThe presented results of the inversion of the geometric sensitivity of g et al.  We inferConsidering that the shaping of delocalisation of double bonds by means of mesomeric schemes has so far not received due attention, we argue that working out what is actually possible and favourable in terms of bond alternation is a key to fully understand the colour tuning phenomenon in PCB.core conjugation. Assuming that in Pr et al.  There exAnPixJg2 . In any amide resonance pulling electron density from the pyrrole ring and generating a negative partial charge at the carbonyl oxygen atom. However, in pyrrole, resonance does not only include the NH group of the amide bond, but the entire aromatic system. As a consequence another mesomeric form can be found, which is again stabilised through formation of a tertiary carbocation far from the electronegative centres. This has the important consequence that bond CSimultaneously, ring D, being fully detached from the conjugated core, is subjected to We want to note that the methylene bridges are not favourable sites to put a positive partial charge, due to their secondary type, but they still take part in conjugation. Furthermore, it has been argued that a certain single bond character of CWe conclude that a plausible prediction of the geometric bond length conjugation pattern of the PCB in its Pfull conjugation because it resembles the longest possible resonance in PCB. Including ring D in the exchange of the core . We shale charge . Based oIn summary, also for PCB in its PWe have addressed the colour tuning mechanism in the photosensor proteins AnPixJg2 and Cph1Our complementary approach represents an uncommon computational route that is interesting for situations in which clear and reliable structural information  is difficult to obtain due to experimental constraints . The siΔ2: 2VEA  solvatinΔ2: 2VEA . Possiblevidence .FFMD simulations were carried out with the NAMD package  under peSnapshots from FFMD simulations were transferred into a QM/MM setup. AIMD simulations were performed as the Born-Oppenheimer type in the NVT ensemble using CP2K 5.1 . The paron atoms ,84. The on atoms ,86 was eon atoms  using thon atoms , GTH pseon atoms ,90,91 a on atoms ,93. Equion atoms ,94. For NMR calculations were performed in the same setup as AIMD simulations, but the size of the QM/MM part was chosen adaptively such that RMSE of chemical shifts were below 1% cf. . Water mPrincipal component analysis (PCA) and multivariate regression was carried out with R using the packages tidyverse, ade4, and GGally . For PCAs 6; cf. .Multivariate regression and prediction was carried out, inverting the correlation matrix of n matrix ; using t using chemfig 1.56 [Python-based MDAnalysis 1.0.0 ,100 was fig 1.56 .


Purpose: To evaluate safety and tolerability on the ocular surface of an anti-septic formulation containing 0.6% povidone–iodine (0.6% PVI) for a 4 week period.Methods: An observational, prospective study included 20 mild-moderate dry eye disease (DED) patients who enrolled at the Ocular Surface Disease Unit of the University of Messina, receiving 0.6% PVI eye drops for 28 days, 2 drops twice daily (BID). The assessment included the Ocular Surface Disease Index questionnaire; symptoms score (0 = absent to 3 = severe) for burning, ocular dryness, foreign body sensation, watery eyes, tearing, photophobia, and ocular pain; fluorescein tear break-up time (TBUT); and corneal-conjunctival staining, performed at baseline (T0), after 7 (T7) and 28 (T28). Schirmer I-test, corneal endothelial cell count, intraocular pressure, and fundus examination were performed at T0 and T28. The main outcome measures were TBUT and corneal-conjunctival staining as markers of ocular surface homeostasis. For statistical analysis, Student's T-test and Wilcoxon test were used as appropriate.Results: No significant alterations of the safety parameters were found throughout the study. Further, at T28 a significant improvement of burning, ocular dryness, foreign body sensation, and watery eyes (T0 vs. T28 P < 0.03) were observed; corneal-conjunctival staining improved at T28 (T0 vs. T28 P < 0.0001), and TBUT improved already at T7 (T0 vs. T7 P = 0.0008) lasting so till the end of the study. The only adverse event was mild burning at instillation for the first 3 days of treatment in most of the patients.Conclusions: The treatment with 0.6% PVI was safe and well tolerated in a group of patients with a damaged ocular surface. A recent experimental, 8–10Therefore, 0.6% PVI represents a valid prophylactic method of ocular surface antisepsis, as demonstrated by recent studies.PVI applications, but more diluted solutions showed a better tolerability.11Corneal epithelial damage was demonstrated after repeated 5% d-alpha-Tocopherol Polyethylene Glycol (vit E TPGS), seems to contains factors that may favor the ocular surface protection.Considering the growing use of 0.6% PVI, it is important to know not only its effectiveness as an antimicrobial agent but also its tolerability and safety profiles, in particular in subjects with pre-existing alterations of the ocular surface, such as dry eye disease (DED) patients. In fact, its formulation, including substances such as SH, MCT, glycerol, and vitamin E The aim of this study is to evaluate the safety and tolerability of 0.6% PVI on the ocular surface after a long-term treatment period.This observational, prospective, single-center study involved 20 mild-moderate DED patients  recruited at the Regional Referral Centre for the Ocular Surface Diseases of the Department of Biomedical Sciences, University of Messina, Messina, Italy.The study was approved by the Institutional Review Board of the University of Messina, protocol number 86/17, and was conducted in accordance with the tenets of the Declaration of Helsinki.Informed consent was obtained from all participants after the explanation of nature and the possible consequences of the study.I-test <8 mm in 5 min.13 Exclusion criteria were: glaucoma, history of herpes simplex or herpes zoster keratitis, history of corneal ulcer, allergic keratoconjunctivitis, use of contact lenses, and previous ocular treatments within 6 months, including anti-inflammatory and immunosuppressant drugs for the treatment of dry eye. The only treatment allowed was tear substitutes, whose use was stable for at least 3 months before the beginning of the study.Inclusion criteria were: age between 18 and 80 years old, the presence of qualitative or quantitative alterations of the ocular surface and tear film dysfunction demonstrated by tear film break-up time (TBUT) <10 s, ocular surface fluorescein staining (NEI/Industry workshop score) >3, and Schirmer's All patients were treated with 0.6% PVI eye drops  2 times daily (BID) for 28 days, and they underwent assessment at baseline (T0), and after 7 (T7) and 28 days (T28). The composition of the 0.6% PVI solution was reported in 15- Ocular symptoms were evaluated by means of the Ocular Surface Disease Index (OSDI) questionnaire, a test designed to score the severity of dry eye, administered before the ophthalmologic assessment. The questionnaire consisted of 12 questions about the respondent's past-week experience with ocular symptoms, vision-related functioning, and environmental triggers. This was performed at T0, T7, and T28.- A symptom score questionnaire (from 0 = absent to 3 = severe) for burning, ocular dryness, foreign body sensation, watery eyes, tearing, photophobia, and ocular pain was also obtained at T0, T7, and T28.- Best-corrected visual acuity was obtained at T0 and T28.13 The assessment was carried out at T0, T7, and T28.- The Fluo imaging function of Keratograph 5M  was used to study the ocular surface after fluorescein instillation. For TBUT, 1 drop from a fluorescein strip  wetted with saline solution  was instilled onto the lower fornix; the patients were invited to blink 3 times to allow an even distribution of the fluorescein, and then they were asked to stare without blinking as long as they could. The time gap between the last complete blink and the appearance of a dark spot on the corneal surface was recorded as the time of tear film resistance (s) and was calculated, for statistical purposes, as the average of 3 consecutive measurements.12 The epithelial staining was evaluated and photographed, with Keratograph 5M with a score from 0 (absent) to 3 (areas of complete loss of epithelium), for each of the 5 corneal sectors considered, with a total score for corneal staining ranging from 0 to 15 and for conjunctival staining from 0 to 18, since 6 areas were considered . The total score for both corneal and conjunctival stain was added and recorded as 1 single value, indicative of the global epithelial condition, and it was used for statistical purposes. To obtain this value pictures from the cornea and superior, inferior, nasal, and temporal conjunctiva were used. The assessment was performed at T0, T7, and T28. The images obtained were assessed in a masked fashion by 2 experienced observers (P.A. and E.I.P.). An interobserver accordance above 95% was achieved.- Three minutes after the TBUT assessment, the ocular surface fluorescein staining score was obtained according to the scheme proposed by the NEI/Industry workshop.I-test, filter paper strips  were applied at the junction between the outer and the middle third of the lower lid. The moisturized length of the strip was measured after 5 min (mm/5 min). The test was performed at T0 and T28.- For Schirmer The safety profile was further assessed at T0, T7, and T28 by slit-lamp examination to evaluate the anterior chamber, and lens changes were seen as possible signs of an intraocular inflammatory reaction.Further, the corneal endothelial cell count, carried out with a Perseus endothelial microscope ; intraocular pressure measurement, performed with Oculus Corvis ST ; and fundus examination, carried out with Daytona , were performed at T0 and T28.16 Using an alpha error of 0.05 and a beta error of 80%, the population number obtained was 16; therefore, 20 patients were enrolled to allow a possible dropout. However, no dropouts were reported. The main outcome measures were TBUT and corneal-conjunctival staining.Population of the study: To identify the numerosity of the population of the study was considered an improvement of 50% for TBUT, based on the reported effect of the SH solutions.For statistical analysis, the MedCalc version 12.2.1.0 statistical software for Windows  was used. Data were described as mean ± standard deviation.t-test for parametric data was applied to assess the significance of differences between baseline and treatment at T7 and T28 data; Wilcoxon signed-rank test was used for nonparametric data. Values from the worst eye at baseline were taken into account for statistical purposes. A P value <0.05 was considered statistically significant.The Student's The clinical parameters evaluated at T0, T7, and T28 are reported in No changes were found for visual acuity, slit-lamp exam for anterior chamber and lens, endothelial cell count, intraocular pressure, and fundus exam.The only adverse event reported by 16 patients (80%) was a mild burning on the instillation, lasting a few minutes. Such symptom disappeared after about 3 days of treatment.P = 0.0002), ocular dryness (P = 0.0003), foreign body sensation (P = 0.0001), and watery eye (P = 0.03) at the T28 visit with respect to baseline values  and lasting until the end of the treatment (P < 0.0001)  .P < 0.0001 vs. T0)  .I-test did not show significant changes at the end of the study period versus T0 values.Schirmer 17–19The resistance to antibiotics is a worldwide growing emergence mostly derived by their inappropriate use; even their topical administration on the conjunctiva may induce changes of the ocular surface microbiota, not only reducing its diversity but also promoting the proliferation of resistant bacterial genera and an alteration of the microbiota/epithelial cell interaction. This can provoke a further shift toward a state of infection and/or of chronic inflammation, secondary to epithelial activation.20 However, repeated and prolonged exposure to antiseptic agents, such as benzalkonium chloride, chlorhexidine, and polyhexamethylene biguanide (PHMB), could potentially induce toxic side effects to the ocular surface cells.21–25Antiseptics could represent an alternative or adjuvant treatment for the infections of the ocular surface.26 Unfortunately, this outcome is also true for eukaryotic cells, and therefore, the ocular surface epithelium could be damaged by the repeated and/or prolonged instillation of PVI eye drops. Studies demonstrated the toxicity on both epithelial and endothelial corneal cells after treatment with povidone–iodine at concentrations higher than 1% and/or treatment longer than 2 min.Five percent PVI is routinely used in ophthalmic surgery, for the prevention of postoperative infections. PVI solutions release free iodine, which penetrates the microbial membrane, causing death due to the oxidative stress brought on the cells.27–29 In fact, the prolonged exposure to 5% PVI of the damaged corneal epithelium might result in keratocyte death and persistent epithelial defects, due to the disruption of the stromal–epithelial interaction with the consequent retarded re-epithelialization.27Clinical and experimental data showed the toxic effects of topical iodine on the ocular surface epithelium, particularly after repeated exposure.30–32PVI, in several concentrations, proved to have a virucidal activity documented against herpes simplex virus, adenovirus, as well as coronavirus, responsible for the recent epidemic outbreaks such as the severe acute respiratory syndrome coronavirus (SARS-CoV) and the middle-east respiratory syndrome coronavirus (MERS-CoV).11 This effect may also derive from the very low pH of the more concentrated solutions: In fact, the more diluted are the solutions, the higher is the pH, closer to that of tears.6Compared with 5% PVI, more diluted PVI solutions showed a better safety profile in an experimental study carried out on rabbits. After the application of 10 drops of PVI with different dilutions , the corneas were studied at the slit lamp after 30 min. The application of the 2.5% solution resulted in lower damage than the 5% solution, whereas no damage at all was demonstrated for the 1% and 0.5% solutions.33 It was supposed that this effect may be related to the high concentrations of PVI used (5% for 3 min) that can affect the health of the ocular surface epithelium. Other studies proved the efficacy of 0.6% PVI solution in reducing the bacterial load with the presurgical treatment in intravitreal injections, and also in the treatment of infectious keratitis and conjunctivitis.10Saedon et al. reported the detrimental effects of repeated intravitreal injection procedures for macular disorders on symptoms of dry eye.in vitro study proved the antimicrobial efficacy of a more diluted formulation, containing 0.6% PVI, which showed a faster bactericidal activity compared with the traditional 5% PVI solution. The 0.6% PVI revealed in vitro antimicrobial activity against S. epidermidis, S. aureus, P. aeruginosa, and, to a lesser extent, Candida spp.9Further, an 6The greater bactericidal efficacy of 0.6% PVI, compared with more concentrated solutions, has been related to the increased availability of free iodine, which is an active antimicrobial component.However, the possible positive effects of PVI instillation might be thwarted by pre-existing ocular surface alterations, such as those present in DED patients, who could be more sensitive to any toxic effect determined by PVI treatment.To explain the effect of 0.6% PVI treatment on DED, it could be hypothesized that ocular surface microbiota might be involved in it.34–36 The microbiota plays a fundamental role in determining ocular surface immunological homeostasis. In DED patients, the changes in microbiota components may form the basis of the reduced tolerance mechanisms and determine the induction of both innate and adaptive immunity, characteristic of the chronic inflammatory process typical of DED.38 This can contribute to ocular surface damage characterized by corneal-conjunctival squamous metaplasia with goblet cell reduction. In moderate-to-severe DED patients, a greater bacterial presence was described,39–41 with a consequent loss of the ocular surface immunologic tolerance. In fact, goblet cells, through the production of transforming growth factor-β (TGF-β), participate in the immune tolerance process, typical of the healthy ocular surface condition, by selecting tolerogenic dendritic cells, which are able to stimulate the homing of regulatory T cells (T-regs) on the ocular surface.42–48In fact, numerous evidences confirmed its role, stating that the ocular surface microbiota, bacterial biofilm, and bacterial lipases may influence the occurrence of DED and other ocular surface disorders.40The use of antiseptic substances, such as PVI, can abolish germs overgrowth that is responsible for the induction of an inflammatory status, without inducing resistance-related problems. So, in DED patients, the antiseptic effect of PVI can decrease the bacterial population, resulting in a reduction of the inflammatory stimulus on the ocular surface epithelial cells.The formulation of 0.6% PVI tested in this study is characterized by a high dilution with an increased pH, with respect to less diluted formulations; further, the presence of other components may favor good epithelial tolerability.49–57 However, the SH molecule, present in the formulation, has a high molecular weight  but a very low concentration (0.05%). For this reason, it does not have the rheological characteristics of proper artificial tears formulations. Nevertheless, SH is effective in reducing the irritating effects of free iodine on the ocular surface epithelia in a PVI formulation.The tested 0.6% PVI formulation contains an SH molecule; it extensively described the great water retention ability and non-Newtonian viscoelastic behavior of SH that increase the epithelium wettability.58–60Other important components are MCT, which may improve the tear film stability ameliorating the lipid layer.61Glycerol is a compatible solute substance that, together with the mineral salts, contributes toward equilibrating the tear film osmolarity, thus reducing the hyperosmolar damage on the epithelial cells.62–66Another important component is the hydrophilic vit E TPGS, present as a surfactant in the 0.6% PVI formulation; it is a potent antioxidant agent and is responsible for the protection of eukaryotic epithelial cells against the oxidation induced by free iodine through reactive oxygen species derived from the alteration of cell membrane components, such as lipids and proteins, and nucleic acids.The aim of our study was to evaluate the safety and tolerability of 0.6% PVI treatment with an already damaged ocular surface, such as mild-moderate DED patients. Patients enrolled were of both DED types, namely evaporative and aqueous deficient. The results showed that some dry eye-specific symptoms, such as burning, ocular dryness, foreign body sensation, and watery eye, improved at the end of the treatment. However, these improvements did not bring significant changes in OSDI values. It is possible to state that the improvement of the symptoms was linked to the statistically significant amelioration of TBUT and epithelial damage, as demonstrated by corneal-conjunctival stains. No serious adverse events were reported during the study, apart from a mild-moderate burning sensation on the instillation in some patients that disappeared after about 3 days from the beginning of the treatment.67 we considered TBUT as a safety marker, indirectly representing possible changes in vision quality.As to the safety aspects, it was possible to demonstrate the absence of alterations regarding visual acuity, corneal endothelial cells, lens transparency, intraocular pressure, and fundus. Since mild-moderate dry eye patients usually do not show changes in visual acuity but rather a possible reduction of contrast sensitivity, as a consequence of tear film quality alteration,Anterior chamber reaction, demonstrated by the presence of cells and proteins, can be an indicator of anterior segment suffering with iris and ciliary body involvement.Lens transparency is often altered by topic treatments such as those with some NSAIDs.The evaluation of corneal endothelial cells carried out with the specular microscope is important to evaluate possible damage to these cells, described for higher concentrations of PVI.Intraocular pressure and fundus exams are usual safety parameters studied in several trials.We preferred to study the TBUT after fluorescein staining of the ocular surface, because in this way it was possible to obtain the evaluation of the whole corneal surface, differently from the non-invasive BUT (NIK-BUT) function of the Keratograph 5M, which is based on the reflection of Placido rings projected on the corneal surface that do not cover the entire corneal area.Therefore, it is possible to conclude that 0.6% PVI appears to be safe on the ocular surface. Further, its use could improve signs and symptoms in DED patients. It can be hypothesized that 0.6% PVI acting on ocular surface microbiota may rebalance the anomalous bacterial overgrowth typical of DED, thus promoting the prevalence of immune tolerance on inflammatory mechanisms. Moreover, for the aforementioned reasons, the use of 0.6% PVI could be considered in the antiseptic treatment of repeated para-surgical procedures, such as intravitreal therapies.in vivo confocal microscopy, are necessary to better elucidate the possible mechanisms underlying the ocular surface modifications induced by 0.6% PVI treatment.The limitations of the study are that this is an observational, prospective study carried out without a control group. However, our main interest was to evaluate the possible toxic effects brought by the 0.6% PVI formulation on the ocular surface and we chose mild-moderate DED patients as an ideal population to be investigated with regard to this aspect. Nevertheless, further studies specifically addressing the possible use of 0.6% PVI as a complementary treatment for dry eye are warranted. Further studies addressing the ocular surface microbiota changes, the effect on visual function, and the modified corneal-conjunctival structure, evaluated by


Retinoids have numerous applications in inflammatory, dyskeratotic, and oncohematology diseases. Retinoids have now reached the fourth generation, progressively reducing toxicity whilst increasing their efficacy. Trifarotene is a new fourth-generation retinoid with a selective action on RAR-γ. In this review, we reported the trials—both concluded and in progress—including the use of trifarotene in dermatological diseases. Studies were identified by searching electronic databases  from 2012 to today and reference lists of respective articles. Only articles published in English language were included. Randomized trials evaluating trifarotene tolerability, safety, and efficacy in congenital ichthyosis and acne have demonstrated great results and mild side effects, leading to the approval by the FDA of trifarotene for the treatment of lamellar ichthyosis in 2014, and of acne vulgaris in October 2019. No high-quality randomized clinical trials have evaluated the treatment of primary cutaneous lymphomas with trifarotene. Finally, we are hypothesizing future perspectives in the treatment of non-melanoma skin cancers, fungal infections, photoaging, and hand-foot skin reactions with trifarotene. Vitamin A , a cardinal micronutrient in human metabolism, is a lipophilic molecule composed by isoprene units. As an isoprenoid, it is characterized by a hydrocarbon chain containing an ending hydroxyl. The term “retinoid” concerns both natural and synthetic analogues of vitamin A. In synthetic analogues, such as etretinate, acitretin, or tazarotene, a benzene ring substitutes the cyclohexane. According to the International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular Biology, retinoids are characterized by four isoprene units with a head-to-tail structure .It is known that vitamin A and its synthetic analogues have a crucial role in modulating some skin functions; in particular, they regulate epidermal keratinization, differentiation, maturation, and proliferation . Due to Stimulated by Retinoic Acid 6 (STRA6), the cell surface receptor mediates the uptake of vitamin A from plasma . IntraceUpon RA binding, the RAR-RXR heterodimer recognizes specific DNA sequences, named retinoic acid response elements (RAREs) ,8. RecenThe first generation is composed by natural retinoids, obtained modifying polar groups of vitamin A, which do not act selectively: retinol and its metabolites, such as retinal, tretinoin, isotretinoin, and alitretinoin;The second generation is constituted by monoaromatic retinoids, synthetic compounds where a benzene ring replaces the cyclohexene ring: etretinate, and acitretin;The third generation is made up by polyaromatic retinoids, resulting from cyclization of the side chain and characterized by a selective activity towards receptor: adapalene, tazarotene, and bexarotene .Retinoids can be classified into three generations, in respect of molecular structures and properties :The firsIn the skin, RAR-γ is the most represented isoform , sustain1)Skin hydration: trifarotene induces skin peptidyl arginine deiminase 1 and aquaporin-3 channels, and, therefore, influences skin barrier functions;2)Cell adhesion: trifarotene weakens hemidesmosomes, reducing intercellular adhesion. The minor cohesion among keratinocytes explains its comedolytic properties;3)Proteolysis: trifarotene downregulates matrix metalloproteinases (MMPs), which act as proteolytic enzymes on elastin and collagen, thus improving skin texture ,22.Trifarotene influences three different pathways, identified by a large-scale gene expression analysis:Ex-vivo pharmacokinetic models on trifarotene proved the high stability of the compound, with a half-life of >24 h. Despite this, it is quickly metabolized by hepatic microsomal enzymes, with a half-life of minutes, compared to tazarotenic acid, which has a 10-fold higher stability in hepatic microsomes. This is a predictor of a favorable safety profile. The metabolism of trifarotene is catalyzed primarily by cytochrome (CY) P2C9, CYP3A4, CYP2C8, and, to a lesser extent, by CYP2B6 .We performed a comprehensive search in the following databases from 2012 to 2020: Cochrane Central Register of Controlled Trials; MEDLINE; Embase; US National Institutes of Health Ongoing Trials Register; NIHR Clinical Research Network Portfolio Database; and the World Health Organization International Clinical Trials Registry Platform. We studied reference lists and published systematic review articles. We used the following keywords, separately and in a combination: “trifarotene”, “CD5789”, “skin”, “dermatology”. Only English language articles were included in the searches. The search was restricted to studies on humans. Forward citation searching of the reference lists of the original studies and review articles was also conducted.All the studies investigating the use of trifarotene in skin disorders were examined. If a study included trifarotene with other drugs, only trifarotene frame was analyzed. All human studies were included with no restrictions on age, sex, ethnicity, or type of study. Case reports and case series were included if they described the use of trifarotene in diseases not present in reviews or trials.The target intervention excluded the analyses of other pathologies out of the dermatological field, animal studies, and non-English language articles.Eleven trials including trifarotene were identified by this quantitative research. Four resulted completed at the time of this Review, all regarding acne vulgaris, the others have no results published on Trial.gov or Pubmed. Despite the FDA’s 2014 approval, of trifarotene for the treatment of lamellar ichtyosis, no data on large-scale trial are currently available. At present, only one trial has evaluated the safety and tolerability of trifarotene in early-stage primary cutaneous T-cell lymphoma patients, but no results have been published yet.RAR-γ selective retinoid derivatives such as trifarotene, are being investigated as topical agents, which are expected to offer a more favorable clinical profile compared to the dual RARβ/γ drugs currently used in the clinical practice. Trifarotene is a potent and selective agonist of RAR-γ, with significantly less activity on RAR-β and RAR-α , and has no activity on RXRs . AlthougTrifarotene is metabolized in vitro by CYP2C9, CYP3A4, CYP2C8, and, to a lesser extent, by CYP2B6, and excreted in the feces . SystemiHerein we illustrate the rational use of trifarotene in skin diseases.Acne vulgaris is a chronic inflammatory process of the pilo-sebaceous unit. Overproduction and abnormal cohesiveness of desquamated epithelial cells leads to their retention within the hair follicle, with subsequent obstruction of the ostium . Pro-infRetinoids promote keratinocytes differentiation and diminish their proliferation, modulating desquamation . Topicalp < 0.001) in favour of trifarotene, demonstrating its safety, efficacy, and tolerability in acne treatment of both face and trunk [Trifarotene is the latest new retinoid approved for the treatment of acne by the FDA in over 20 years. Topical administration is demonstrated to be safe, well-tolerated, and more effective than vehicle in reducing both non-inflammatory and inflammatory lesions in acne of the face and trunk . We evalnd trunk . Blume-Pnd trunk . Adversend trunk . All subnd trunk .Johnson et al. reported a series of three subjects with moderate face and trunk acne treated with trifarotene 50 µg/g cream for 12 weeks, then evaluated through questionnaires. A reduction in inflammatory lesions was reported. The reduction in the lesions on the face and trunk was 90% and 47% for inflammatory and non-inflammatory lesions, respectively, in the first subject. The second one achieved a 20% reduction in inflammatory lesions and 22% reduction in non-inflammatory lesions both on the face and trunk. In the last subject, a reduction of 66% and 34% was observed in inflammatory and non-inflammatory lesions, respectively .Many trials are still ongoing or have not yet produced results on the use of trifarotene in acne vulgaris .Retinoids can indirectly affect skin microbes, blocking the essential nutrients supply and stabilizing immune system hyperreactivity ,40. In tAutosomal recessive congenital ichthyosis (ARCI) is a heterogeneous family of congenital diseases of keratinization linked to generalized hyperkeratosis, often accompanied by erythroderma. ARCI is infrequent, with 1 in 200,000 births incidence. The most frequent phenotypic subtypes include lamellar ichthyosis, congenital ichthyosiform erythroderma, and harlequin ichthyosis. ARCI is also genetically heterogeneous—with at least nine different genes responsible for the most common forms—but approximately 30% of cases are explained by TGM1 mutations, the gene encoding transglutaminase 1, involved in the development of the cornified envelope . ATP-binThe efficacy of retinoids to treat this disease has been known for some time . VirtaneAs topical trifarotene was demonstrated to be safe and well-tolerated, in 2014, the FDA grated it with Orphan Drug Designation for the treatment of congenital ichthyosis .Cutaneous T-cell lymphomas (CTCLs) are primary skin lymphoproliferative disorders, deriving from mature T cells. CTCLs could be indolent or aggressive processes and this great heterogeneity explains the difficulties in clinical management. Sixty percent of all CTCL cases is represented by mycosis fungoides, while less than 5% consists of Sézary syndrome, a more aggressive form ,54,55,56Retinoids display a crucial role among WHO and EORTC recommended therapies for CTCLs. They have been used in lymphoma of every stage for over three decades . In vitrSidell et al. observed that ATRA could increase IL-2Rα expression in human thymocytes, increasing steady-state mRNA levels . Gorgun Retinoids are largely used for the prevention and treatment of non-melanoma skin cancers (NMSCs). Since the 1920s, when vitamin A deficiency in rats paved the way to investigate its relationship with cancer, retinoids have played a pivotal role in the oncology field . BiochemSince ATRA has been used in acute promyelocytic leukemia treatment, a lower incidence of total episodes of fungemia has been reported in these patients . StartinFrom these studies, trifarotene could pave the way for fourth generation retinoids as agents in respiratory and oesophageal mycosis, due to their high selectivity for these districts, with consequent reduced adverse effects . OesophaCandida spp.), and non-dermatophytes [Onychomycosis is a fungal infection of the fingernails that may involve any component of the nail unit, including the matrix, bed, or plate. Distal and lateral onychomycoses are the most frequent forms of onychomycosis, causing subungual hyperkeratosis that usually limits local penetration of antimycotic drugs. The involved pathogens are dermatophytes, yeasts  to 2.7 ± 0.6. Significant reductions of dark spots (−40%) and severity of wrinkles (−12%) were observed at week 4 compared to baseline. At the end of the study, ultrastructural analyses performed by reflectance confocal microscopy also highlighted significant improvements, with the recovery of the polygonal keratinocytes pattern as the main observed aspect [In the 1980s, Kligman et al. suggested for the first time to use retinoids in the treatment of photo-damaged skin . Howeverd aspect . TrifaroTargeted therapy has improved the oncological management and survival of patients. Sorafenib and sunitinib are two novel, small-molecule multikinase inhibitors that have shown promising results in the inhibition of tumor cell angiogenesis and proliferation, both in vitro and in vivo . DespiteRetinoids are largely applied in cutaneous inflammatory, dyskeratotic, and infectious diseases, besides oncohematology. In this review, we have focused our attention particularly on trifarotene, a new fourth-generation retinoid with a selective action on RAR-γ. Trifarotene has been tested for the treatment of acne and congenital ichthyosis, demonstrating its safety and tolerability. No high-quality randomized clinical trials are currently evaluating the treatment of primary cutaneous lymphomas with trifarotene. Differently from other retinoids, trifarotene acts selectively on RAR-γ reducing RAR-β adverse effects, and offer a more favorable clinical profile compared to the drugs with dual action on both RAR-β and RAR-γ, such as tretinoin and derivatives. As RAR-γ is much more abundant than the other retinoids receptors in the skin, this could be the rationale for increasing studies on trifarotene usage in skin disorders, as already seen with previous generation retinoids.


The therapeutic groups which presented higher detection frequencies and concentrations were anti-inflammatories, antiepileptics, antibiotics and lipid regulators. These results present a broad and specialized background, enabling a complete overview on the occurrence of pharmaceuticals in the aquatic compartments.Potential risks associated with releases of human pharmaceuticals into the environment have become an increasingly important issue in environmental health. This concern has been driven by the widespread detection of pharmaceuticals in all aquatic compartments. Therefore, 22 pharmaceuticals, 6 metabolites and transformation products, belonging to 7 therapeutic groups, were selected to perform a systematic review on their source, fate and occurrence in different aquatic compartments, important issues to tackle the Water Framework Directive (WFD). The results obtained evidence that concentrations of pharmaceuticals are present, in decreasing order, in wastewater influents (WWIs), wastewater effluents (WWEs) and surface waters, with values up to 14 mg L Human pharmaceuticals, presenting different characteristics and, consequently, producing different environmental exposure profiles, represent a group of widely used chemicals that contaminate the aquatic environment. Albeit in trace amounts, they are of concern, since they are designed to perform a biological effect. Moreover, given their continuous introduction into the environment, their impact, both as stressors and as agents of change, is of great importance .−1 . ow superior to 1, the likelihood of predominance of the chemical in the aqueous phase decreases logarithmically, whereas below a log Dow of -1, the likelihood of predominance of the chemical in the aqueous phase increases logarithmically. Therefore, compounds having log Dow values between -1 to +1 could be anticipated to be distributed in both the water and organic phases [With a log Dc phases .ow ranges from -2.23 to 4.6, the log Koc varies between 0 and 3.88 and even solubility goes from 0.1 to 101,200 (mg L−1). These variations are not only observed between different therapeutic groups but also within each group, since, as previously referred, this pharmaceuticals grouping does not correspond to similar chemical structures and there are more than one class per group. This can be seen especially for antibiotics, lipid regulators and anti-inflammatories, where greater fluctuations in these parameters are reported.As seen in In summary, although pharmaceuticals present different physicochemical properties, some are expected to be more lipophilic and others to sorb to soils and sediments, they all have relatively high water solubility, having the potential to contaminate the aquatic environment .After excretion, pharmaceuticals are transported to WWTPs through the sewer system, and no significant removal occurs during transport in sewer pipes to WWTPs . As hotsThe removal of pharmaceuticals in WWTPs is a complex phenomenon with many plausible mechanisms; additionally, these facilities are generally not equipped to deal with complex pharmaceuticals, as they were built and upgraded with the principal aim of removing easily or moderately biodegradable carbon, nitrogen and phosphorus compounds and microbiological organisms ,78. The WWTPs employ a primary, a secondary and an optional tertiary treatment process, being the last one is always associated with a high treatment cost. During primary treatment, physical removal of solids is achieved through a sieve, regularly followed by coagulation-flocculation processes for the removal of particulate matter, as well as colloids and some dissolved substances; however, this process is ineffective for the elimination of pharmaceuticals . In the Besides the type of wastewater treatment, WWTPs’ efficiency in removing pharmaceuticals is influenced by operational and environmental conditions, namely, the hydraulic retention time (HRT) ; solid retention time (SRT) ; environmental temperature  and pH conditions (affecting on the degradation kinetics of the compounds) ,82,84,85As previously mentioned, the physicochemical characteristics of the pharmaceuticals also affect their removal in WWTPs. Since a significant part of the removal process is through sorption or biodegradation in sludge, the ability to interact with solid particles plays a major role. Thus, compounds with low sorption coefficients tend to remain in the aqueous phase, favoring their mobility through the WWTPs and into the receiving waters ,87. Indeow higher than 3, which indicates high sorption potential, tend to be removed through sorption onto sewage sludge, while compounds with log Dow between 1.5 and 3 are removed mainly by biodegradation. The remaining pharmaceuticals with log Dow inferior to 1.5 tend to remain dissolved [ow are more influenced by SRT, while compounds with low log Dow are more influenced by HRT [Generally, during secondary treatment, compounds with log Dissolved ,80,82,88d by HRT . During d by HRT ,90.Currently, besides the conventional treatments, new methodologies have been applied as tertiary treatments with higher removal efficiencies, but some of these new methods have high construction, maintenance and energy costs associated . AdvanceMore recently, the growing trend of improving sustainability and reducing energy demands in WWTPs have encouraged alternative methods, such as algae ponds for secondary effluent polishing, with promising results .Escherichia coli capable of deconjugating the β-glucuronated pharmaceuticals excreted by the human body, resulting in the release of the active pharmaceutical into the wastewater [As previously referred, metabolization in the human body can lead to elimination of pharmaceuticals conjugates. However, these phase II metabolites can be converted back into the parent compound, especially in WWTPs, being infrequently found in surface waters. One of the mechanisms used is the action of a β-glucuronidase enzyme produced by stewater ,89,94,95stewater ,96,97,98Naturally, the type of treatment can affect not only the removal efficiencies but also the metabolites and transformation products generated.This supports the need for the evaluation of metabolites and transformation products and the further development of new treatment techniques to achieve complete mineralization of emerging contaminants ,97. BesiData from 52 publications were collected, and removal efficiencies of the selected pharmaceuticals are summarized in Although, as mentioned, some studies indicate that physicochemical properties set the efficiency of removal of pharmaceuticals in WWTPs, the literature review performed showed that the target compounds present very different removal rates, ranging between negative and high removal rates, and no obvious pattern in behavior was observed, even within the same therapeutic group, implying that factors other than compound-specific properties affect removal efficiency ,85. Negaow (from 2.49 to 3.06), higher than most of the selected pharmaceuticals, predicted large sorption to sludge and higher removal rates, this was not observed in real removal data.Concerning the removal efficiencies of each therapeutic group, anxiolytics present the lowest average, having a small variation due to their similar physicochemical properties, with values ranging from 0% to 25%. Although their log Dow for CIP (-2.23) sorption to sludges, it has been suggested as the primary removal mechanism for fluoroquinolones, whereas, for macrolides, limited sorption to sludge is observed [As for antibiotics, the range observed in the removal efficiencies was from 0% to 100%, similar to anti-inflammatories and hormones. The average removal rates for AZI, CLA and ERY (macrolides) are near 30%, whereas CIP presented higher removal rates (64%). Despite the lower log Dobserved ,132,134.Although the therapeutic group of lipid regulators encloses a statin (SIM) and fibrates (BEZ and GEM) and their removals vary between 0% and 99%, their averages are similar, ranging from 36% to 51%, being also found in sludges .ow (2.28) than anxiolytics and a wide range of removal efficiencies, it is one of the most persistent compounds and is averagely reduced by only 18.1% [For CAR, although presenting a lower log Dly 18.1% ,136. Thily 18.1% ,135.Regarding SSRIs, even though they all belong to the same group, the average removal efficiencies range from 39% to 75%, with ESC, PAR and SER presenting lower values, below 55%, when compared to CIT and FLU that present higher removal rates, 75%.3/H2O2 system shows high efficacy [The most investigated therapeutic group in WWTPs are anti-inflammatories, and despite their high variability, average removal rates are above 77% and up to 96% (PARA), with the exception for DIC (34%) ,135. Excefficacy ,135. On efficacy .Hormones are the therapeutic group with higher log Dow and high average removal efficiency, which ranges from 65% to 82%. This low variation was expected, since the molecules have similar physicochemical properties ,139. It As observed, the WWTPs are unable to completely remove the pharmaceuticals, and through direct discharge of WWEs in surface water or by land application of WWTPs’ sludge or through leaching, these facilities are the major sources of pharmaceuticals in the environment ,140,141.Optimization of wastewater treatment still remains a task of high priority. Biological treatment is commonly unable to remove pharmaceuticals; however, its efficacy can be improved under favorable conditions. Although advanced treatment technologies, such as membrane and advanced oxidation processes, have been promising for pharmaceuticals removal, high operation costs and formation of degradation products still remain an issue .Since WWTPs are not able to completely remove pharmaceuticals, they are disseminated through their WWEs and sludges, mostly, into surface waters. In the aquatic environment, the fate and concentration of pharmaceuticals can be reliant on the receiving water body flow rate, partitioning to sediments, biological entities and consequent degradation, uptake by biota, volatilization, photodegradation or transformation through other abiotic mechanisms, such as hydrolysis ,134,142.When WWEs reach the surface waters, the dilution effect varies significantly due to different flows in different rivers; however, this effect can be relatively low, especially in arid or semi-arid regions due to water scarcity, like some Iberian rivers, where other processes gain relative importance ,144. AltThe fate of different pharmaceuticals has already been studied in surface waters by several authors using estimates of mass loading, dilution and in-stream attenuation, here understood as the reduction of the concentration of pharmaceuticals along the river segment by processes different from dilution ,141,143.ow of a given compound influences its in-stream attenuation; in the case of hydrophobic compounds (with higher log Dow), sorption to suspended particles and sediments is a dominant process leading to in-stream attenuation by reducing the concentration in the aqueous phase along the river segment [ow show not only more differences in attenuation rates between sites but also more temporal differences  within each site [Overall, it is expected that the log D segment . In thisach site . This soach site ,33. In fach site . Moreoveach site . Additioach site .ow of pharmaceuticals influence the variability of rates among rivers, likely due to its effect on sorption to sediments and suspended particles, and therefore, influence the balance between the different attenuation mechanisms  [As already referred, in complex natural waters, electrostatic interactions, chemical bounding and nonspecific forces between ionized molecules and dissolved organic matter can also occur, meaning that we cannot generalize the attenuation of a compound based on its physicochemical properties alone ,143. HowThe attenuation of pharmaceuticals was evaluated in surface water in Spain where the total concentration of pharmaceuticals  decreased about 40% in less than 5 km, although the number of compounds detected only decreased 13% . StudiesBesides the presence of the parent compounds in surface waters, sulphate conjugates of E1 and E2 have already been observed. Although these conjugates no longer possess a significant biological activity, they can act as precursor steroid reservoirs that might be converted into free estrogens ,139. EveIn addition to the parent compounds, some studies also addressed the contribution of WWTPs for pharmaceuticals transformation products in surface waters and confirmed that these facilities were a major source of contamination to the recipients ,98.In summary, on one hand, the emissions from WWEs vary widely because of differences in regional usage of the compounds and efficiency of WWTPs. On the other hand, the processes that drive in-stream attenuation  depend on the different pharmaceutical characteristics, as well as on a series of physicochemical and biological parameters of the river, such as river flow rate, temperature, the vertical hydrological exchange between surface and subsurface compartments, turbidity, dissolved oxygen concentration, biofilm biomass and pH . The magAlong with advances in analytical instruments and techniques, trace levels of various pharmaceuticals and their metabolites have been detected in the aquatic compartment since the latter half of the 1970s .−1 [A literature review on worldwide monitoring programs in recent years, presented in −1 ,147. Usu−1 .Beside the aspects previously referred, several others can influence the concentration of pharmaceuticals in the different aquatic compartments, promoting a great variability in the detected concentrations. In WWTPs, other aspects that can influence the detected concentrations are the flow rate, the time of the year, the temperature, the type of WWTPs, day and the type of sampling, etc. . As for −1) was observed in the anti-inflammatories group, with statistical differences for all of the other therapeutic groups, being the maximum individual concentration observed for IBU (700 µg L−1) [−1, followed by the other groups, with medians under 20 ng L−1.All investigated pharmaceuticals were frequently detected in WWIs, with PARA, CIT, IBU, CAR, BEZ, CLA and α-E2 (E2 isomer) presenting detection frequencies higher than 88%. As for the different therapeutic groups, antiepileptics and anti-inflammatories were the ones with higher detection frequencies, above 86%, followed by lipid regulators (75%) and hormones (74%). Anxiolytics were the group with lower values (31%), much different from the other groups. The highest median concentration (1.7 µg L µg L−1) . Antibio−1. Additionally, the highest detection frequency belonged to LOR, with 38% [Although anxiolytics were the group with the lower detection frequency and median, ALP had concentrations up to 4.7 µg Lwith 38% . These r−1 and with all detection frequencies above 65%. Although some discrepancies in excretion rates, with higher values for CIP, both CIP and CLA have higher consumptions, being this pattern was observed in the occurrence data.Antibiotics were the most homogenous group, with median concentrations ranging from 93 to 324 ng L−1, respectively).Lipid regulators occurrence data was comparable to that of antibiotics, mostly because of similar consumption and excretion rates. Within this group, we can observe that the one with the highest consumption in most countries, SIM, had the lowest detection frequency and median concentration in WWIs. This can be due to a significant difference in excretion data, where BEZ have clearly higher rates than SIM, with excretion values up to 72% and 12.5%, respectively . Therefo−1 [The antiepileptic CAR with excretion rates up to 33%, and whose consumption is only surpassed by anti-inflammatories, had a detection frequency of 89% and concentrations up to 22 µg L−1 ,111.−1. However, this group presented some peculiarities, SER being one of them. This SSRI has the highest consumption in European countries. Nonetheless, due to its very low excretion rate (0.2%), this compound and its metabolite (Nor-SER) present lower median concentrations than CIT and Nor-FLU [Like anxiolytics, SSRIs also had low consumption and excretion rates, which reflected also in low concentrations in the WWIs, with a median concentration of 8 ng L Nor-FLU . On the  Nor-FLU .−1 for DIC, NAP, IBU and PARA, respectively [As referred, anti-inflammatories were the group with higher concentrations in WWIs, not only due to their high consumption but also to significant excretion rates (up to 80%), with median concentrations of 450, 1550, 2680 and 20 601 ng Lectively .−1) when compared to EE2 (0.7 kg y−1) resulted in higher concentrations even for its metabolite E1, being even present in the enantiomer of E2 (α-E2) up to 10 µg L−1 [In the hormones group, although there were lower excretion rates observed for E2, its higher consumption , antibiotics (142 ng L−1) and lipid regulators (126 ng L−1), a similar pattern to that in WWIs but with no statistical significance between them. The remaining three groups had lower medians, with 10, 5.2 and 1.4 ng L−1 for anxiolytics, SSRIs and hormones, respectively. The highest individual mean concentration observed was for DIC 233 ng L−1; however, the maximum concentration regarded CIP, 14 mg L−1. This high value, along with others that are completely offset, were observed in the effluents of pharmaceutical industries and hospitals [In general, regarding the median concentrations, antiepileptics were followed by anti-inflammatories  and individual (438 ng L−1) levels [Anxiolytics were the only therapeutic group with a clear higher median and individual concentrations in WWEs than in WWIs and surpassed the mean concentration of hormones and SSRIs. This is justified by the fact that anxiolytics have the lowest removal efficiencies, and, in some cases, even negative values are found. This increased concentration in WWEs is related to the transformation of metabolites and/or transformation products back into the parent compounds during wastewater treatment ,82. Sinc) levels .−1. The extremely high value found for CIP was observed in the effluent of a pharmaceutical industry [As indicated in industry .−1, respectively [As regard to the antiepileptic CAR, the fact that it does not adsorb to soils and has low removal efficiencies in WWTPs results in a small increased median from WWIs to WWEs, from 193 to 226 ng Lectively .−1).Lipid regulators having removal efficiencies analogous to those observed for antibiotics present an occurrence pattern in WWEs comparable to that of WWIs, again with SIM presenting the lowest median concentration  concentrations were in the same range or higher as the parent compounds [−1, which was also detected in a pharmaceutical industry effluent [The therapeutic group SSRIs had also the same pattern observed in WWIs, with CIT and N-CIT presenting the higher median concentrations of 73 and 107 ng Lompounds . The higeffluent .−1, respectively, meaning that PARA shifted from the highest median concentration in WWIs to the fourth in WWEs, mainly due to the high removal average (96%) presented.Anti-inflammatories had one of the highest removal efficiencies, only comparable to hormones, and although they remain with a high median concentration, the difference to the other therapeutic groups  was significantly reduced. Within this therapeutic group, DIC presented the highest median concentration, followed by IBU, NAP and PARA, with 163, 142 and 10 ng L−1) and the lowest to α-E2 (0.4 ng L−1); the highest individual value was also for α-E2 (4.7 µg L−1), observed in only one study [As for hormones, with average removal efficiencies above 60%, concentrations were also significantly reduced, with the highest median concentration belonging to E1 , transposed into the Portuguese legal system by the Law N 58/2005 of 29 December 2005 (the Water law). Within this framework, the key strategy adopted was the establishment of priority substances or groups of substances due to their persistence, toxicity, bioaccumulation, widespread use and detection in rivers, lakes, transitional and coastal waters. Additionally, a list of environmental quality standards have been issued for these substances, to ensure adequate protection of the aquatic environment and human health ,186,187.According to the Directive 2013/39/EU strategy, all member states shall monitor each substance in the watch list at selected surface waters representative monitoring stations at least once per year. The number of monitoring stations varies within each member state, taking into account the population and area of each country. About 40% of European water bodies still have an unknown chemical status, as not even the monitoring of the EU priority substances have been performed .After reviewing 88 scientific references, as expected, lower median concentrations (ten times lower) were found in surface waters than in WWEs (−1), antiepileptics (28 ng L−1), antibiotics (20 ng L−1) and lipid regulators (16 ng L−1). These four therapeutic groups had statistically significant higher median concentrations than SSRIs and hormones. SSRIs, hormones and anxiolytics, with notably lower values, had the lowest median concentrations of 0.8, 0.4 and 0 ng L−1, respectively. The highest values observed were reported for CIP in India, with a maximum concentration of 650 µg L−1 for CIP [We can observe similar patterns in WWEs, with the same four therapeutic groups presenting higher median concentrations, anti-inflammatories . It also presented two extremely high average concentrations detected for CIP in surface waters near pharmaceutical industries in Pakistan (1.3 µg L−1) and in India (164 µg L−1); however, all the other average concentrations were below 108 ng L−1 [As above mentioned, antibiotics were one of the therapeutic groups with high median concentrations  and presented concentrations up to 12 µg Lalf-life . In factalf-life .−1) and in detection frequencies from 55% to 26%. The highest concentration regarded CIT (76 µg L−1); however, it was found, once again, near a pharmaceutical industry in India [The SSRIs values decreased from WWEs to surface waters in median concentrations (from 5.2 to 0.8 ng L−1), followed by DIC and NAP (34 ng L−1) and IBU (26 ng L−1). The results of PARA, higher than the ones in WWE, move PARA to values in the same range as the other anti-inflammatories. Looking at the detection frequencies, they all fall in the same range, from 52% to 59%. In this group, another extremely high concentration was observed for PARA in Kenya 107 µg L−1 [−1 was the highest concentration found for this metabolite [−1) was twice the purposed value of 100 ng L−1 for the environmental quality standard in 2012-2013. The high values in surface waters possibly raised some issues regarding the establishment of this standard.Anti-inflammatories presented, once again, higher concentrations when comparing with other therapeutic groups . PARA pr7 µg L−1 . Althougtabolite . The ave−1), and the highest average value was detected in China, 180 ng L−1, whereas its detection frequency was slightly decreased (from 57% to 54%) [Within the hormones group, E1 presented the higher median concentration (2.1 ng L to 54%) . Contrar to 54%) ,193.As above mentioned, lower concentrations of pharmaceuticals (ten times lower) were found in surface waters than in WWEs. Surface waters showed an overall trend of higher concentrations in sites influenced by the location of WWTPs ,194.As discussed earlier, the concentrations of pharmaceuticals decrease from the WWIs to WWEs and to surface waters through different mechanisms. However, data collected from 28 references showed that pharmaceuticals can reach groundwaters, seawaters and even mineral waters and drinking waters . RegardiThe concentrations in remaining waters bodies should be lower than the previous ones, since they suffer attenuation mechanisms similar to surface water. Additionally, drinking water has dedicated treatment plants. However, these facilities do not completely remove pharmaceuticals and can also produce transformation products that can be toxic ,173,199.Although susceptible to degradation or transformation, pharmaceuticals’ continuous introduction into the aquatic environment confers some degree of pseudo-persistence, reaching, at extremely low concentrations, all aquatic compartments all over the world, even drinking waters ,91. Howe−1, respectively. Groundwater and seawater were the water bodies with higher detection frequencies and concentrations, and the highest concentration found was of 14 µg L−1 for CIP in groundwater [In undwater . No statA careful literature review was conducted in order to understand the sources, fate and occurrence of pharmaceuticals in the aquatic environment. In this context, a broad and specialized background was obtained, enabling a complete overview of the state-of-the-art in these subjects.The data provided in this review evidenced that WWTPs are the major source of pharmaceuticals contamination. It is also noteworthy that pharmaceuticals belonging to the same therapeutic group can have distinct physicochemical properties, resulting in different behaviours both in WWTPs and in the aquatic environment.The concentrations of pharmaceuticals found in the aquatic bodies were, in decreasing order, WWIs, WWEs, surface water and other water bodies.Overall, these results present a global picture of the pharmaceuticals’ contamination, an important input for setting prioritizing measures and sustainable strategies to minimize their impact in the aquatic environment.


Objective: This study aims to investigate the factors affecting the efficacy of first oral prophylaxis in patients with chronic migraine (CM) and to assess patient compliance with their medication regimens.Method: To identify the therapeutic effect of prevention medication in 740 patients with newly diagnosed CM that did not receive any preventive treatments after 4 weeks in an open-label prospective study with retrospective baseline from January 2016 to January 2018, the factors that may affect the outcomes of preventive treatment were analyzed based on the demographic characteristics, migraine characteristics, family history of headache, and history of medication overuse. Moreover, the patients were followed up to evaluate their compliance with and the side effects of the medication at 4 weeks and at 12 weeks.Results: After 4 weeks of prophylaxis, 94.3% (n = 698) of the patients persisted with taking the medicine. The treatment was effective for 61.7% of CM patients (n = 431) and ineffective for 38.3% (n = 267). The results showed that the effectiveness of the preventive treatment was related to the number of headaches per month, and the effect was better for patients with headaches for 15–20 days/month than for those with headaches for 26–30 days/month . After 12 weeks of treatment, only 34.5% (n = 255) of the patients persisted with taking the medicine. The most common reason for non-compliance in CM patients is appointment difficulty in a headache clinic (31.8%).Conclusion: The effect of CM prophylaxis was related to the frequency of headache. Only 34.5% of the patients continued to take medicine after 12 weeks of treatment, suggesting that patient compliance needs to be enhanced in the prophylaxis of CM. For the Chinese headache society, the best way to increase patient compliance should be treatment at dedicated headache centers and timely visits to headache specialists. Chronic migraine (CM) is a common disabling disease with a global prevalence of ~1–2% . At presData of CM patients who went to the specialized headache clinics of six hospitals  in Chongqing, China, from January 2016 to January 2018 were collected. The CM patients were diagnosed according to the criteria of the International Headache Disease Classification 3rd Edition (ICHD-3 beta) . The patPatients 18 years of age or older with headaches that were diagnosed as CM were included in the study. We recruited prophylaxis-naïve patients with newly diagnosed CM that did not receive any preventive treatments including pharmacological , botulinum toxin injection, or acupuncture therapy before participating in this study.The exclusion criteria were as follows: (i) patients who were younger than 18 years of age, (ii) headache type other than CM, (iii) previous history of migraine prophylaxis before enrollment, (iv) pregnancy or nursing status, (v) hepatic or renal disorder, nephrolithiasis, or other severe systemic diseases, and (vi) incomplete medical records.After receiving detailed information about the adverse reactions of each drug, the patients who met the study criteria were given at least one of the following preventive drugs under the guidance of a headache expert: metoprolol (25–50 mg/day), flunarizine (5–10 mg/day), amitriptyline (25–50 mg/day), and topiramate tablets (75–100 mg/day); all patients' medications were gradually increased from a small dose to the maximum dose within 2 weeks. Furthermore, all patients with medication overuse stopped using painkillers that were previously used before starting the prophylactic treatment.To analyze the effect of preventive treatment on CM patients, the effective treatment aimed to reduce the number of headache days by more than or equal to 50% after 4 weeks of medication, and the clinical characteristics of the patients were analyzed to identify the factors that affected the preventive treatment outcomes. Patients with CM were followed up to evaluate their compliance with the medication regimen after 4 and 12 weeks of treatment. Headache intensity was assessed by a 10-point visual analog scale (VAS) . The side effects experienced by the patients under treatment were recorded, and the reasons for non-compliance were also recorded. The primary endpoints are medication compliance, side effects of the drugs, and the reasons for non-compliance. The secondary endpoints are general information of CM patients and headache-related factors of CM patients.t-tests were used for comparisons between groups. The count data are expressed as a ratio, and the χ2-test was used for comparisons between groups. A multivariate regression analysis model was used to estimate the odds ratios (ORs) of age, sex, CM history, duration of headache attack, headache days per month, and VAS as well as their 95% confidence intervals (CIs). Considering that preventive outcomes were clinically distinct entities that associated with individual risk factors, we chose potential confounding factors that would be relevant for outcomes. Thus, age and sex were adjusted in the multivariate model. A two-sided P < 0.05 was considered as statistically significant.The statistical analysis was conducted using Statistical Package for the Social Sciences (SPSS), version 20.0 . All normally distributed measurement data were expressed as x ± s, and A total of 740 patients met the study criteria, and 698 patients continued to take medicine after 4 weeks, including 156 males and 542 females; 387 patients (55.4%) had a family history of headache, and 468 patients (67.0%) had a history of drug overuse. All the patients received prophylactic medication: 65 patients took metoprolol, 351 patients took flunarizine, 167 patients took amitriptyline, 62 patients took topiramate tablets, and 53 patients took metoprolol and flunarizine at the same time. The efficacy of the medication for the patient was evaluated after 4 weeks of prophylactic treatment; the medication was effective for 431 patients (61.7%) and ineffective for 267 patients (38.3%). Metoprolol was effective for 51 patients (78.5%); flunarizine was effective for 233 patients (66.4%); amitriptyline was effective for 57 patients (34.1%); topiramate was effective for 47 patients (75.8%); metoprolol and flunarizine, used in combination, were effective for 43 patients (81.1%). At the 4-week follow-up, 698 patients continued to take the medicine, and the compliance rate was 94.3%. At the 12-week follow-up, only 255 patients continued to take the medicine, and the medication compliance rate was 34.5%.P = 0.001). The patients in the effective-treatment group had fewer headache days per month than those in the ineffective-treatment group (P = 0.013). The duration of each headache episode was longer in the ineffective-treatment group than in the effective-treatment group (P = 0.038). There were no significant differences in age, sex, body mass index (BMI), education background, age of headache and CM onset, headache history, duration of headache attack, VAS, migraine characteristics, symptoms associated with headache, family history of headache, or medication overuse between patients in the effective-treatment group and patients in the ineffective-treatment group (P > 0.05) (shown in P = 0.006). There were no significant differences in the CM history, duration of headache attack, or VAS (P > 0.05) between the two groups. The therapeutic effect could not be predicted  developed side effects, including 3 patients with metoprolol, 52 patients with flunarizine, 29 patients with amitriptyline, 8 patients with topiramate, and 12 patients with metoprolol and flunarizine combination. The most common side effects are dry mouth, drowsiness, and gastrointestinal reactions. The reasons for non-compliance among 485 patients are side effects, unsatisfied effect, forgot to take the medicine, appointment difficulty in a headache clinic, and pain relief shown in . The mosCM has a higher severe disability rate than episodic migraine, and the efficacy of preventive treatment and the treatment compliance rates are poor , 8. ThisPrevious observational studies have shown that, for patients with CM who receive prophylactic treatment, the medication compliance rate was 65.3–92.4% after 4 weeks of follow-up, 30.9–76.0% after 12 weeks, 21–80% after 6 months, and 31–56% after 12 months , 11. MorThe domestic and international guidelines for CM prevention and treatment recommend that the observation period for preventive treatment should be at least 1–6 months, and the effects of an effective preventive treatment need to last for ~6 months , 14. SomP < 0.01), which indicates that this factor may be related to the effectiveness of the first preventive treatment. However, the current research results on the relationship between the number of headache days per month and the effectiveness of preventive treatment are still inconclusive. Luconi et al.'s  developed side effects. The most common side effects are dry mouth, drowsiness, and gastrointestinal reactions, but the side effects are not the primary cause of non-compliance for CM patients. The most common reason is appointment difficulty in a headache clinic (31.8%) because headache physicians are still relatively rare compared with other physicians in China. How could the patients get formal and standard treatment if they could not get an appointment at a headache clinic? So strengthening the construction of headache clinics and training more headache physicians are the top priorities for the Chinese headache society.There are also some limitations in this study. Firstly, this study was conducted in the six first-class hospitals in Chongqing, China, and there is a higher number of patients with severe CM, which also explains why the patients included in this study had a high number of headaches and a long history of migraine. Secondly, this study has a historical retrospective baseline with high recollection bias, and the results may be influenced by many factors.This study suggests a significantly low compliance rate with CM therapy. The onset of first-time prophylaxis may be related to the number of headache days per month in CM patients, suggesting that prophylaxis for CM should be initiated early. For the Chinese headache society, the best way to increase patient compliance should be treatment at dedicated headache centers and timely visits to headache specialists.All datasets generated for this study are included in the article/supplementary material.Our study was approved by the ethics board at the First Affiliated Hospital of Chongqing Medical University (20151223). All the patients provided written informed consent before enrollment. The trial was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.GT conceived and oversaw the study. YZ, QL, YL, XL, YY, and WL performed data collection. DY performed data analysis. DY and GT wrote manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Extra-articular manifestations (EAMs) are serious outcomes or complications of rheumatoid arthritis (RA) with increasing mortality and morbidity. The purpose was to explore the factors influencing EAMs, tissue-specific/systemic EAMs, and the concurrence of several EAMs.t test, one-way analysis of variance, Mann–Whitney U test, and Kruskal–Wallis H test. Binary and ordinal logistic regression analyses were performed to determine the relationship between influential factors and EAMs, tissue-specific/systemic EAMs, and the concurrence of several EAMs.In total, 519 inpatients with RA were enrolled. The clinical characteristics, laboratory parameters, and medications of RA patients and the details of EAMs were recorded carefully. Differences between groups were examined by a Chi-square test, independent samples The morbidity of EAMs was 44.70%. Male sex, age, and traditional Chinese medicine (TCM) were independent influential factors of EAMs, and a number of statistically significant influence factors were found in the multivariate analysis of tissue-specific/systemic EAMs. Finally, age, CRP levels, number of RA-affected types of joints, and TCM were the factors that independently influenced the concurrence of several EAMs.Influential factors identified in this study could be recommended in clinical work, which is hopeful to decrease the morbidity and mortality of EAMs in RA. Cumulative studies have revealed that severe EAMs could be attributed to environmental factors, chronic systemic inflammation, and treatment ,6,7. EnvBased on the background of EAMs in RA, we hypothesized that some of the clinical characteristics, laboratory parameters, and medications might be connected with EAMs. In this research, we collected general information on RA patients and aimed to explore the factors influencing EAMs and tissue-specific/systemic EAMs. The second purpose of the research was to screen the risk factors or the protective factors for patients suffering from a greater number of EAMs.2Informed consent: Informed consent was obtained from all individuals included in this study.Ethics approval: This retrospective study involving human participants was in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The General Hospital of Western Theater Command approved this study.2.1The retrospective study was performed with 519 inpatients who were diagnosed with RA between January 2014 and August 2019 at the General Hospital of Western Theater Command . The inclusion criteria met the diagnostic criteria that satisfied the American College of Rheumatology 2010 revised criteria [2.22.2.1A series of common clinical characteristics were included, such as patient ID, sex, age, age at disease diagnosis, disease duration, smoking habit, and number of RA-affected types of joints.2.2.2Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), RF, anti-cyclic citrullinated peptide antibody (ACPA), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), complement 3 (C3), complement 4 (C4), and lactate dehydrogenase were recorded carefully.2.2.3Tripterygium wilfordii, Red paeonia, Astragalus membranaceus, Cassia twig, prepared liquorice root, and Ephedra herb [Treatment history of RA patients was recorded, such as hydroxychloroquine (HCQ), biological agents , MTX, sulfasalazine (SAZ), leflunomide (LEF), and steroid hormones. The traditional Chinese medicine (TCM) included dra herb .2.3The EAMs shown in 2.4t test. One-way analysis of variance was used to compare the mean values between multiple groups (three ranked tissue-specific/systemic EAMs). We classified the number of affected tissue-specific/systemic EAMs into three categories: the lowest category indicates 0 tissue-specific/systemic EAMs, the middle one indicates 1 tissue-specific/systemic EAM, and the highest rank indicates 2–4 tissue-specific/systemic EAMs. The skewed distribution continuous variables were presented using the median , and the Mann–Whitney U test and Kruskal–Wallis H test were used to identify the differences in continuous variables between two groups (RA patients with EAMs vs RA patients without EAMs) and multiple groups (three ranked tissue-specific/systemic EAMs). Categorical variables were presented in frequencies/percentages, and distributions of categorical variables between observed clinical outcomes were estimated using cross-tabulations, and Chi-square tests were used to identify differences in categorical variables across the observed clinical outcomes. The associations of influential factors with EAMs and tissue-specific/systemic EAMs were explored using odds ratios (ORs) and 95% confidence intervals (CIs) in binary logistic regression analysis. Additionally, possible influential factors associated with a greater number of EAMs were screened by ordinal logistic regression analysis. Both the binary and ordinal logistic regression analyses have the function of adjusting confounding factors so that the final calculated influential factors are independent. All variables that were found to be significantly associated with tissue-specific/systemic EAMs using univariate analyses were included in binary and ordinal logistic regression analyses. P values ≤ 0.05 were considered statistically significant.All statistical analyses were performed with SPSS version 23.0 . Distributions of continuous variables were examined by the Kolmogorov–Smirnov test. The normal distribution continuous variables were presented using the mean values with standard deviations, and differences in continuous variables between two observed clinical outcomes (RA patients with EAMs vs RA patients without EAMs) were examined by independent-samples 33.1According to the inclusion criteria, 519 RA patients were included (232 RA patients with EAMs and 287 RA patients without EAMs). The morbidity of EAMs among RA patients was 44.70% (95% CI = 40.50–49.10). We compared clinical characteristics, laboratory parameters, and medications between two groups, and there were significant differences between two groups in terms of sex, age, age at disease diagnosis, smoking history, number of RA-affected types of joints, RF, and treatment with SAZ, biological agents, LEF, and TCM (Table S1).3.2In binary logistic regression analysis, through adjusting for the confounding factors , male sex , age , and treatment with TCM  were independently associated with EAMs .3.33.3.1The morbidity of the ocular system was 3.85% (95% CI = 2.30–5.60). There were significant differences between two groups (RA patients with ocular involvement vs RA patients without ocular involvement) in terms of sex, CRP, ACPA, IgG, IgA, IgM, and treatment with HCQ (Tables S2–S4).3.3.2The morbidity of the pleuropulmonary system was 11.75% (95% CI = 8.90–14.60). There were significant differences between two groups (RA patients with pleuropulmonary involvement vs RA patients without pleuropulmonary involvement) in terms of sex, age, smoking history, RF, IgM, and treatment with biological agents, MTX, TCM, and steroid hormones (Tables S2–S4).3.3.3Among the 519 RA patients, the cardiovascular system was the system that was the most affected . There were significant differences between two groups (RA patients with cardiovascular involvement vs RA patients without cardiovascular involvement) in terms of sex, age, age at disease diagnosis, number of RA-affected types of joints, and treatment with TCM and LEF (Tables S2–S4).3.3.4The morbidity of the renal system was 1.54% (95% CI = 0.60–2.70). There were significant differences between two groups  in terms of age, age at disease diagnosis, ESR, and IgA (Tables S2–S4).3.3.5The morbidity of the cutaneous system was 2.50% (95% CI = 1.30–4.00). There were significant differences between two groups (RA patients with cutaneous involvement vs RA patients without cutaneous involvement) in terms of CRP and treatment with MTX (Tables S2–S4).3.3.6The neurological system was the system that was the least affected . The results of neurological involvement could not be calculated because of the limited number of RA inpatients that were affected (Tables S2–S4).3.3.7The morbidity of LNE was 13.49% (95% CI = 10.40–16.40). There were significant differences between two groups (RA patients with LNE vs RA patients without LNE) in terms of sex, smoking history, CRP, ACPA, IgM, and treatment with biological agents, TCM, and SAZ (Tables S2–S4).3.4Through binary logistic regression analysis, several significant influential factors associated with tissue-specific/systemic EAMs were found. In terms of ocular diseases, high levels of ACPA were found to be associated with greater susceptibility to ocular diseases . Greater age , smoking history , high levels of IgM , and history of treatment with MTX , TCM , and steroid hormones  were independently associated with pleuropulmonary disorders. Male sex , greater age , and history of treatment with TCM  were independently associated with cardiovascular involvement. High levels of CRP  and history of treatment with MTX  were independently associated with cutaneous diseases. Smoking history , high levels of CRP , ACPA , and history of treatment with TCM  were independently associated with LNE. However, there were no significant variables that were found to be associated with renal disorders .3.5In an attempt to identify the influential factors associated with a greater number of EAMs, ordinal logistic regression analysis was performed. After adjusting for the confounding factors , age , CRP levels , number of RA-affected types of joints , and treatment with TCM  were found to be related to a greater number of EAMs .4The EAMs of RA are characterized by multiple systemic manifestations and are usually associated with active and severe diseases, which doubles the risk of developing RA and increased mortality. To provide the possible options to prevent the occurrence of EAMs, our research screened the influential factors of EAMs as well as tissue-specific/systemic EAMs. Furthermore, we estimated the risk/protective factors affecting the occurrence of a higher number of EAMs.Based on the inclusion and exclusion criteria, 519 RA patients were chosen. Among the population, EAMs were observed in 232 RA patients with a morbidity of 44.70%, which is slightly lower than that reported by Turesson et al. (50%)  but highOcular manifestations (OMs) are some of the EAMs and usually present as Sjögren’s syndrome. Other inflammatory ophthalmological conditions include keratoconjunctivitis sicca, scleritis, and episcleritis. All of these OMs are both sight- and life-threatening ones because of their association with systemic vasculitis . A risk The overall morbidity rate of pleuropulmonary manifestations (PMs) in RA was 11.75%; pleuritis, interstitial lung disease, and pleural effusion were the most common symptoms in the present study. In general, the frequency of PM occurrence in RA is estimated to range from 4% to 68% . The accCardiovascular manifestations (CMs) were the most common EAMs among RA patients in this study, with a morbidity of 27.55%. Epidemiological studies about the morbidity of CMs in RA patients are rare; however, a series of reports have revealed an increased risk of mortality of up to approximately 50% compared to that of the general population . CMs areCutaneous manifestations (CMs) can be present during the entire disease course, and a mass of CMs is an indication of disease severity and activity, with increased mortality and morbidity . RheumatDraining lymph nodes are responsible for immune surveillance, particularly generation of adaptive immune responses and maintenance of peripheral tolerance . LNE in Severe EAMs often indicate the severity of the condition and are related to markedly elevated mortality in RA patients. To afford more efficient management for severe EAMs and possible predictors of a greater number of EAMs, we ran ordinal regression analysis and found that greater age and higher CRP levels were considered risk factors of the concurrence of several EAMs. Additionally, we discovered the phenomenon that RA patients with fewer types of RA-affected joints were more likely to suffer from severe EAMs, which may indicate that some RA patients present with EAMs as initial symptoms of RA, while the articular manifestations are less serious. The result may, to some extent, depend upon the population selected and the study setting; this is the first study to report the association between the types of RA-affected joints and the concurrence of several EAMs. Further research is needed to confirm the conclusion and explore the pathogenesis. Besides, a history of TCM treatment was found to decrease the risks of a greater number of EAMs. The addition of TCM to RA therapy can benefit RA treatment and prevent EAMs and the occurrence of a greater number of EAMs. We have found that TCM may be helpful in the prevention of all kinds of EAMs, whereas reports concerning associations between TCM and EAMs are rare. More research is needed to determine the relationship between TCM and EAMs.Several limitations are present in this study. These limitations should be acknowledged, and some of them need to be improved in further research. First, our study is a retrospective study, and the casual relationship could not be ascertained because of the coexistence of exposure and outcome. Second, the conclusions would be more convincing with a larger sample. Third, the presence or absence of EAMs we observed would be the result of the comprehensive effect of therapy instead of the sole variable we set, so with regard to the influences of treatment on EAMs, more clinical trials and case studies are needed. The exploration of additional potential predictors and the confirmation of the suggested influential factors require further exploratory research.5In the present study, we found several potential influential factors that can be associated with EAMs, all kinds of EAMs, and even the concurrence of several EAMs, which provide directions for exploratory research to identify potential biomarkers of EAMs. Whereas research studies on EAMs are still lacking, reaching a consensus on the disease definitions of EAMs is urgent. Regular inspection and comprehensive analysis of influential factors are necessary to detect the presence of EAMs and initiate a treatment plan.