Temporal artery biopsy (TAB) is a widely used method for establishing a diagnosis of Giant Cell Arteritis (GCA). The optimal TAB length for accurate histological GCA diagnosis has been suggested as 15 mm post-fixation (15–20 mm pre-fixation). The aim of this study was to determine the relationship between a histological GCA diagnosis and optimal TAB length in the South Australian (SA) population.Pre-fixation TAB length (mm) was reported in 825/859 of all samples submitted to SA Pathology between 2014 and 2020 from people aged 50 and over. When more than one biopsy was taken, the longest length was recorded. Analyses of both TAB length and TAB positive proportions were performed by multivariable linear and logistic regression analysis, including covariates sex, age, and calendar year.p = 0.001), increased with age (p = 0.006), and a small positive linear trend with calendar year was observed (p = 0.015). The TAB positive proportion was related to older age  and to TAB length , but not sex or calendar year. Comparison of models with TAB length cut-points at 5, 10, 15, 20 mm in terms of diagnostic yield, receiver operating characteristics and Akaike Information Criteria confirmed ≥ 15 mm as an appropriate, recommended TAB length. However, only 383 (46%) of the biopsies in our study met this criteria. The diagnostic yield at this cut-point was estimated as 25% which equates to an expected additional 30 histologically diagnosed GCA patients.The median age of participants was 72  years, 549 (66%) were female. The TAB positive proportion was 172/825 (21%) with a median biopsy length of 14 mm . Biopsy length (mm) was shorter in females (This study confirms that TAB biopsy length is a determinant of a histological diagnosis of temporal arteritis, and confirms that a TAB length ≥ 15 mm is optimal. Approximately half the biopsies in this study were shorter than this optimal length, which has likely led to under-diagnosis of biopsy-proven GCA in SA. Further work is needed to ensure appropriate TAB biopsy length. Giant Cell Arteritis (GCA) is an autoimmune condition causing inflammation of medium and large blood vessels, known as vasculitis. GCA is the most common vasculitis affecting the elderly. When presented with a case suggestive of the diagnosis of GCA, there is a need to initiate further investigations to exclude or confirm the diagnosis, historically based on the criteria set out by the American College of Rheumatology (ACR) , and morWith a specificity of 100%, a temporal artery biopsy (TAB) with histopathology demonstrating necrotizing arteritis characterized by a predominance of mononuclear cell infiltrates or a granulomatous process with multinucleated giant cells, has been a mainstay for a diagnosis of GCA . The aveThe aim of this study was to determine the relationship between optimal TAB length and a histopathological diagnosis of GCA in the South Australian (SA) population.n = 70, 78%), intimal hyperplasia , and adventitial inflammation  were frequent findings. Over 80% of positive TAB reported inflammatory infiltrates involving all layers of the temporal artery. Those reporting inflammatory infiltrates without the specific term “transmural infiltration” had other strong characteristics of GCA such as the presence of Giant Cells. A specimen with significant eosinophilic infiltrate was excluded.We retrospectively analyzed the results of all TAB reports from January 2014 to December 2020 for biopsies from people aged 50 years and over submitted to the SA public health sector pathology laboratory (SA Pathology). A total of 859 biopsy reports were reviewed, with 825 (96%) reporting pre-fixation TAB length (mm). When more than one biopsy was taken, the longest length was recorded. Age at biopsy and sex information was also collected on all biopsies. The details of the biopsy report including presence of inflammatory cell infiltrate, presence of Giant cells, disruption of internal elastic media, intimal hyperplasia and involvement of vasa vasorum were taken into account. The final opinion of the pathologist determined the assignment of the biopsy into either positive or negative categories. Although the TAB reports were unstructured, a review of specific details reported in 90 positive TAB indicated that giant cells . Multivariable regression analyses were used to determine covariates for TAB length (linear regression), TAB length ≥ 15 mm (logistic regression), and TAB positivity (logistic regression). All analyses included additional covariates sex, age, and calendar year. Both linear and quadratic terms were evaluated for the linear covariates age and TAB length, but a quadratic term was only retained for age in the logistic regression model for a TAB positive outcome based on the Akaike Information Criterion (AIC). Results were interpreted as marginal population-averaged predictions of the outcome for each covariate, which for the logistic regression model for TAB outcome, was the predicted proportion of positive TAB results. Additional models for the TAB outcome were estimated with the TAB length covariate dichotomized at 5, 10, 15, and 20 mm, and these models were compared using the AIC, the area under the receiver operating curve (AUC-ROC), and diagnostic yield .n = 143), the first year of the COVID pandemic, was not decreased compared to previous years (103 TAB in 2019 and 140 in 2018).The median age at biopsy of the 825 patients included in the study was 76 years, 549 (66%) were female, with 172 (21%) TAB positive . The agep = 0.015). To put this in context, the difference in TAB length for 2017 onward compared to pre 2017 was 1.4 mm , after adjustment for age and sex.The overall median biopsy length was 14 mm . Analysis of biopsy length (mm) using a multivariable linear regression model  demonstrp = 0.002, which equates in an increase in the marginal probability/proportion of a positive TAB result of 0.6%/mm , and the recommended TAB length must balance the risk of biopsy with the probability of a positive result. Importantly, we identified that a TAB length of at least 15 mm was optimum for the predictive performance of our model, a result which is supported by two prior studies , 19. A hImproved awareness of the importance of TAB length may not only decrease diagnostic and treatment delay, but may also obviate the need for a contralateral biopsy when there is a negative result, except when there is high index of clinical suspicion , 25. TheThe strengths of this study are that it is a large study with all TAB processing and testing performed at a single laboratory which handles approximately 75% of TAB in South Australia. Therefore, it is a representative sample of suspected GCA cases in South Australia, without additional variability in sample processing and reporting. Some limitations include that there was no follow-up on the final clinical diagnosis, and therefore the effect of TAB length on the sensitivity/specificity of TAB for GCA diagnosis could not be evaluated. Further, there was no information on corticosteroid treatment at the time of biopsy, which may have also modified the relationship between TAB length and a positive TAB.In conclusion, TAB remains a mainstay for a diagnosis of GCA, and TAB length is an important determinant of a positive result. We recommend a minimum pre-fixation length of 15 mm to obtain the maximum diagnostic yield for this procedure, yet approximately half of the biopsies in our study, and likely most published studies, did not meet this criterion. While there are some indications that awareness of the importance of TAB length is increasing amongst vascular or ophthalmologic surgeons, further emphasis is required. Standardization of TAB harvesting, processing techniques and reporting (including length) may also contribute to optimal diagnosis of biopsy-proven GCA.catherine.hill@sa.gov.au.Data is not available for privacy reasons. Requests to access the datasets should be directed to CH, The studies involving human participants were reviewed and approved by the Central Adelaide Local Health Network Human Research Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CR, CH, SLy, JN, and SLe: contribution to study conception and design. CR, JN, KD, SLe, TD, and CH: contribution to data acquisition. CR, JN, SLy, JT, RB, SLe, and CH: contribution to data analysis and interpretation. CR, JN, KD, SLy, JT, RB, TD, SLe, and CH: drafting the manuscript and critical revision and final approval of manuscript. All authors contributed to the article and approved the submitted version.


Immunotherapy, represented by immune checkpoint inhibitors (ICIs), has made a revolutionary difference in the treatment of malignant tumors, and considerably extended patients’ overall survival (OS). In the world medical profession, however, there still reaches no clear consensus on the optimal duration of ICIs therapy. As reported, immunotherapy response patterns, immune-related adverse events (irAEs) and tumor stages are all related to the diversity of ICIs duration in previous researches. Besides, there lacks clear clinical guidance on the intermittent or continuous use of ICIs. This review aims to discuss the optimal duration of ICIs, hoping to help guide clinical work based on the literature. In recent years, immune checkpoint inhibitors (ICIs), represented by programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) monoclonal antibodies, have revolutionized the treatment of malignant tumors. Consequently, patients are accessible to more treatment options and acquire longer overall survival (OS). Despite the significant efficacy, ICIs simultaneously trigger off a growing number of issues, such as the management of immune-related adverse events (irAEs), the mechanism and the management strategies of immunotherapy resistance, valid predictive biomarkers of ICIs treatment, the optimization of ICIs-based combination therapies and using ICIs in special populations, all of which not only puzzle both oncologists and patients but remain further exploration. Moreover, there exists no clear consensus on the optimal duration of ICIs therapy  of a patient exceeded three times longer than the median PFS of the same group . DurableAccording to published clinical consensus, patients with advanced malignant melanoma who have achieved both complete response (CR) and ICIs treatment for at least six months can consider ceasing ICIs. If the efficacy is assessed as partial response (PR) or stable disease (SD) after two years of ICIs treatment, cessation may be taken into account , 9, 10. In addition, if there remains a durable response after ICIs cessation (including programmed cessation and cessation for reasons such as economic conditions), restarting ICIs treatment may be considered in the situation of relapse or progressive disease (PD). Clinical studies researching the efficacy of re-challenging ICIs after early discontinuation exist as well. In conclusion, the optimal duration of ICIs is still debatable for patients with a durable response and needs to be further explored with prospective studies.The tumors can present a transient increase in volume or number of lesions (temporary progression) after ICIs treatment, followed by PR or SD, which is defined as pseudoprogression . PseudopPseudoprogression, as an unusual but beneficial response pattern of ICIs treatment, should be emphasized and carefully recognized in clinical trials. To assist oncologists screen out patients more likely to experience pseudoprogression rather than real progression, auxiliary examinations including radiological evidence, biomarker predictors and biopsies are very useful. Only with correct diagnosis can we avoid incorrect discontinuation of effective ICIs treatment . PatientSome patients can be discovered with accelerated disease progression after the initiation of ICIs therapy, thus the concept of hyperprogression was proposed . There iIn the perspective of hyperprogression, both the patient’s survival and access to other alternative treatments are limited. A case report revealed that a lung cancer patient’s rib metastasis progressed rapidly after receiving ICIs-based combination therapy, and the diagnosis of hyperprogression was then set with early imaging and pathological examinations. Significant shrinkage of the metastatic lesion occurred after one month timely salvage treatment . For patGiven the perspective of clinical practice, it is necessary to figure out whether a rapid progression is hyperprogression or not. Once progression occurs, patients should be reassessed immediately and prepared to transfer to the salvage therapeutic strategy. When hyperprogression is confirmed, ICIs treatment should be stopped as soon as possible, followed by a radiologic examination to assess the patient’s condition and decide the treatment alternatives. Chemotherapy could allow a rapid tumoral response before the timepoint of the anti-tumor immune response, or even counterbalance the deleterious effect of ICIs treatment . As a reThe dissociated response is characterized by some portion of tumor lesions progressed while the other portion shrank after ICIs treatment. This kind of response pattern is similar to mixed responses seen with chemotherapy and targeted therapy . The disThe dissociated response is discovered a relatively common and unique response pattern during ICIs treatment. It is regarded as a preferable treatment response and a signal of better clinical prognosis which brings longer OS than typical progression . When diWhile achieving good efficacy, ICIs treatment may lead to some irAEs. The longer patients are on ICIs treatment, the more likely they are to develop irAEs. In most cases, irAEs emerge within 1-6 months after the initiation of ICIs treatment. Favara et al.  put forwIrAEs often result in the discontinuation of ICIs treatment and the administration of immunosuppressant therapies. The best strategy to manage irAEs is to identify them early and stop ICIs as soon as possible, which helps to avoid or minimize the risk of rare fatal outcomes . The 202Management of irAEs and ICIs treatment are not completely contradictory. When G1 irAEs appear, ICIs treatment can usually be continued while treating the toxic side effects. When G2 irAEs appear, ICIs treatment generally needs to be suspended while managing toxic side effects. In addition to certain cases, when the G2 irAEs reduce to ≤ G1, the resumption of ICIs is worth considering. After G3-G4 irAEs are properly treated, especially for G3-G4 cardiotoxicity, pulmonary toxicity, and neurotoxicity, it is generally recommended that ICIs should never be restarted. According to retrospective research, 68 (14%) of NSCLC patients treated with anti-PD-L1 therapy discontinued due to irAEs and 38 (56%) of these patients restarted ICIs after treating irAEs . Since tCurrent clinical trials show that the duration of ICIs varies depending on the tumor stage. A brief summary is as follows   of patients who underwent surgery was twice that of neoadjuvant chemotherapy, and the safety was good. In 2021, a Phase III clinical trial CheckMate 816 reported that nivolumab combined with chemotherapy neoadjuvant therapy showed a significant improvement in pathological CR rates . The FDAPostoperative adjuvant therapy for patients with early and middle stage tumors. The duration is usually one year. The therapy is applied in various tumors such as esophageal cancer, breast cancer, malignant melanoma, uroepithelial cancer, renal cancer, etc. Taking NSCLC as an example, based on the IMpower 010 study, on March 16, 2022, the National Medical Products Administration (NMPA) approved atezolizumab as adjuvant therapy for patients with stage II-IIIA NSCLC with ≥ 1% tumor cell PD-L1 expression, surgically removed and platinum-based chemotherapy . RecentlIn the medical community, there exists a pair of opposite perspectives on the optimal duration of ICIs. On the one hand, ICIs treatment induces a durable response in the body, allowing the previously activated immune system to regress tumor growth. In addition, short-term ICIs treatment can also avoid the toxic side effects attributed to long-term use. Therefore, some experts advocated discontinuing ICIs after a period of treatment. On the other hand, insufficient ICIs treatment duration may result in disease progression or relapse following remission. Therefore, other experts advocated continuing ICIs treatment to improve patients’ long-term PFS and OS. Numerous clinical trials and other studies have set the duration of ICIs, thus we can determine the optimal duration of ICIs more properly based on the results of these trials. The results of these clinical trials are shown in At present, more studies are standing for this view. Jansen et al.  found thTo verify the above, there are at least two prospective investigations currently in progress. The DANTE trial was designed to determine whether time-limited therapy could improve clinical outcomes by reducing toxicities while maintaining treatment benefits. The results supported time-limited therapy for patients with metastatic melanoma continuously remaining progression-free after two years of ICIs . The DutTherefore, there is a premature suggestion for melanoma-early discontinuation of ICIs can be considered in CR patients ready to receive additional treatment for 6 months after achieving CR . HoweverDespite the perspective of limited ICIs treatment, other studies have suggested that stopping ICIs after two years of treatment may result in disease progression. In the KEYNOTE-189 study, half of the 56 patients who completed 35 cycles (approximately two years) of pembrolizumab progressed after stopping ICIs treatment . In the Arbitrary discontinuation may result in disease relapse in the absence of reliable response markers and predictors of long-term benefit. In a prospective trial, 17 patients dropped out after six months of anti-PD-1 treatment, and 14 (82%) of those experienced relapse . In the vs. 100%, 2-year group vs. > 2-year group) and the lower 3-year OS rate in the < 2-year group (49%), suggesting that the clinical benefit is likely to be seen in patients who had been on continuous treatment for more than two years  study to determine the optimal duration of dual immunotherapy of ipilimumab and nivolumab in patients with advanced NSCLC, patients who do not progress after six months of dual immunotherapy will be randomly assigned to a group to continue ICIs treatment until disease progression, or to the other group to stop ICIs treatment . A figurThe problems associated with long-term continuous ICIs treatment include the potential risk of late toxicity, the financial burden of the high cost and the poor life quality of patients due to irAEs, etc. There emerge growing interests in two aspects: predicting the long-term prognosis of discontinuing ICIs, and re-challenging anti-PD-1 therapy when the disease progresses.Restarting ICIs when disease progresses in patients who initially benefited from ICIs treatment is considered safe and effective and can achieve disease control during ICIs treatment. In the phase III KEYNOTE-006 study, 12 of 27 patients who progressed after completing two years of pembrolizumab treatment were re-treated with pembrolizumab, and the best overall response was 3 CR, 3 PR, 3 SD, 1 PD, and 2 with evaluation pending . In the According to existing studies, there is no conclusive evidence regarding the optimal duration of ICIs. A growing number of studies have explored the timing of discontinuing and restarting ICIs in malignant melanoma and NSCLC based on efficacy and irAEs, but there is not yet sufficient evidence to answer this question. For melanoma, the recommended optimal duration of ICIs is an additional 6 months of ICIs treatment after the patient achieving CR. The existing consensus suggests that the optimal duration of ICIs should be considered based on the response pattern, irAEs, and tumor stages. Meanwhile, combining some necessary examinations such as PET-CT, liquid biopsy  or tissue biopsy can help determine when to discontinue ICIs. As more and more prospective studies are completed and published, the optimal duration of ICIs will be found.JY, YS and JT collected and analyzed the literatures. JY and YS drafted the manuscript. JT revised the manuscript. BZ came up with the origin idea and supervised the work. All authors contributed to the article and approved the final version.This research was funded by National Natural Science Foundation of China (Grant No. 82272928), CSCO-BMS Cancer Immunotherapy Research Foundation (Grant No. Y-BMS2019-003) and Wuhan Municipal Science and Technology Bureau Knowledge Innovation Special Project (Grant No. 2022020801010475).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Understanding how urban environments influence people’s health, especially as individuals age, can help identify ways to improve health in the rapidly urbanizing and rapidly aging populations.To investigate the association between age and self-reported health (SRH) in adults living in Latin-American cities and whether gender and city-level socioeconomic characteristics modify this association.Cross-sectional analyses of 71,541 adults aged 25–97 years, from 114 cities in 6 countries , as part of the Salud Urbana en America Latina (SALURBAL) Project. We used individual-level age, gender, education, and self-reported health (SRH) data from harmonized health surveys. As proxies for socioeconomic environment we used a city-level socioeconomic index (SEI) calculated from census data, and gross domestic product (GDP) per-capita. Multilevel Poisson models with a robust variance were used to estimate relative risks (RR), with individuals nested in cities and binary SRH (poor SHR vs. good SRH) as the outcome. We examined effect modification by gender and city-level socioeconomic indicators.p-value of interaction < 0.001). In gender stratified models, the association between older age and poor SRH was more pronounced in men than in women, and in those aged 25–65 than among those 65+ . Living in cities with higher SEI or higher GDP per-capita was associated with a lower risk of poor SRH. GDP per-capita modified the association between age (25–65) and SRH in men and women, with SEI the interaction was less clear.Overall, 31.4% of the sample reported poor SRH. After adjusting for individual-level education, men had a lower risk of poor SRH  compared to women, and gender modified the association between age and poor SRH (Across cities in Latin America, aging impact on health is significant among middle-aged adults, and among men. In both genders, cities with lower SEI or lower GDP per-capita were associated with poor SRH. More research is needed to better understand gender inequalities and how city socioeconomic environments, represented by different indicators, modify exposures and vulnerabilities associated with aging.The online version contains supplementary material available at 10.1186/s12889-022-13752-2. By 2021, 56,6% of the global population lived in urban areas. Urbanization is especially high in Latin America, where the urban population is currently over 80%, together with North America among the highest across regions of the world . BetweenSelf-rated or self-reported health (SRH) has long been considered a reliable indicator of biological and mental health , 8, and SRH varies widely between –14 and wMost studies on SRH differences have analyzed individuals within countries –24, withLoss of physical function, chronic pain, and the onset of chronic diseases result in worsening average SRH as populations age , 28. ManFurthermore, several cross-sectional studies have shown that women tend to have poorer SRH than men –36. SeveTo our knowledge, very limited evidence is available regarding the association between age and SRH in Latin America cities, and the extent to which these relationships vary by gender or are modified by social and economic features of cities. An existing review of 11 studies in Latin America suggests that features of the built and social environment (where two studies used neighborhood socioeconomic variables) are associated with SRH or health-related quality of life . HoweverTo fill these gaps, we examined the association between age and SRH across all cities of 100,000 residents or more in six Latin-American countries . We further evaluated if gender and city-level socioeconomic conditions are associated and/or modify the association between age and SRH. We used two main contextual social environment exposures: 1) a SALURBAL-derived Social Environment Index (SEI), which measures city-level material conditions of households  and educational level which is a non-material household asset; and GDP per-capita, as a measure for cities SES, capturing dynamics of employment, growth, commerce, and business.. The average number of observations per city was 628, ranging from 16 to 3487.This is a cross-sectional, multi-level, multi-country study using data from the SALURBAL  study. The SALURBAL study has integrated and harmonized health outcomes and physical and social environment data from 371 cities with more than 100,000 inhabitants in 11 Latin American countries . The finHealth outcomes and socio demographic information at the individual level were obtained from existing national surveys in each country and harmonized across countries using standardized definitions. For the current analyses, we used data for adults (25–97 years) from the following countries surveys (year of survey): Argentina (2013), Brazil (2013), Chile (2010), Colombia (2007), El Salvador (2004), and Guatemala (2002)  with ID #1612005035 and by appropriate site-specific IRBs.In general, would you say your health is…”. Argentina, Chile, Guatemala, and El Salvador used the exact same 5-point rating scale response including: “Poor”, “Fair”, “Good”, “Very Good” and “Excellent”. In Brazil and Colombia, the response rating scale included: “Very Poor”, “Poor”, “Fair”, “Good” and “Very Good”. During the data harmonization process, SRH was dichotomized into “Poor”  and “Good” . This categorization facilitates comparison with other SRH epidemiological studies [SRH was measured on a 5-point Likert scale in each country. Although the question asked was fairly similar across countries, the response options differed. Adults were asked to answer the question “ studies , 44–48.Although SRH may be considered a more subjective measure of health compared to some objective measures of morbidity, we selected SRH as a proxy of global health status. SRH has shown to have high prognostic and predictive value of subsequent mortality and therefore represents a good proxy of overall health , 11, 49.Age (in years) was the primary individual-level exposure of interest and was obtained from the health surveys. Gender was our primary individual-level effect modifier, and it was inferred from self-reported sex from the survey responses . We defined that if gender was an effect modifier, we would perform gender stratified analyses when investigating secondary effect modifiers.The two co-primary city-level socioeconomic exposures included: Social Environment Index (SEI), and GDP per-capita.The Social Environment Index, SEI, is a SALURBAL summary measure of city social development, which was already available at the time of the study . SEI wasGDP per-capita was obtained from a gridded global dataset , where sConceptually, these 2 city-level exposures can impact SRH directly or may modify the aging effect on SRH , thus we examined both the main effect of these exposures as well as their effect modification of the association between age and SRH.Individual education was included as proxy for individual socioeconomic level as a covariate. This was categorized as: “Less than Primary Education”, “Primary Education Completed”, “High-School Completed”, and “University Completed or a Higher level”. City size (number of people in the city) as estimated for the survey year based on country projections was also used in descriptive analyses.We used descriptive statistics to characterize the study population by outcome (Poor SRH and Good SRH), age categories (18–65 and 65 +), SEI and GDP per-capita tertiles. We used continuous age (in years) divided by ten, so that coefficients reflect differences in SRH associated with 10 years of age. To accommodate the non-linear association between poor SRH and age, we used a linear spline with a knot at 65 years. We selected the knot at 65 years of age (coinciding with the retirement age in most countries), based on descriptive analyses of the relation between age and SRH. Furthermore, since the association differed by gender, all analyses were gender stratified.We conducted two-level Poisson regression models, with individuals nested within cities, and with robust variance estimation to account for misspecification of the variance by fitting count model for the binary outcome . We usedp-values was < 0.05. The analyses were performed using STATA 16.1 .Associations and interactions were considered significant if the The final analytical sample comprised 71,541 individuals, resident of 114 cities in 6 countries in Latin America. The mean age (SD) of the sample was 46.3 years (15.1) and 58% were women. Overall, 31% reported poor SRH. There were substantial variations in the proportion of poor SRH across the six countries. Those reporting poor SRH were older, more likely to be women and had lower educational attainment, as compared to those with good SRH. People reporting poor SRH were also different in terms of their city-level characteristics: they tended to live in cities with lower GDP per-capita, lower SEI, and smaller populations  such that men showed stronger increases associated with age than women. Therefore, all subsequent analyses were stratified by gender.In an initial model pooling both genders and adjusting for education (not shown in table), age was non-linearly associated with poor SRH . Men had lower risk of poor SRH (RR = 0.76 CI 0.73–0.78), than women. Furthermore, gender modified the association of age with SRH  and men , respectively. There was a positive association between age and poor SRH in women and men, and in both genders the associations were stronger among people aged 25–65 , than among those over 65 years . After adjusting for individual education and city-level socio-economic characteristics (models 2–5), age remained associated with poor SRH among those aged 25–65 years although the associations were slightly attenuated. In this age group, for each ten additional years the prevalence of reporting poor SRH increased 21% in women  and 30% in men . In contrast, age was not associated with poor SRH in women 65 years and above and was weakly associated with poor SRH in men 65 years or above . Model 3 for women and men shows that living in cities with less GDP per-capita (lowest tertile vs. the highest tertile) was associated with increased risk of poor SRH, even after adjusting for individual age and education level . Model 4 shows the adjusted association of tertiles of SEI and poor SRH, and demonstrates significant positive associations between cities with lower SEI (tertiles 1 and 2) and poor SRH compared to those with high SEI (tertile 3), in both men and women. To examine the independent association between SEI and GDP and SRH, we used a model that incorporates both factors (Model 5) and found that although there was some attenuation in the associations, these city-level variables remain significantly associated with poor SRH, for both men and women.Tables Results examining the role of SEI and GPD per-capita as effect modifiers are shown in Figs. p-value for interaction between age and GDP per-capita =0.001 for women and < 0.001 for men). For men and women aged 65 and older no interaction between age and GDP was observed. Additional files Figure Based on cross-sectional health surveys from six Latin-American countries, we found that higher age was associated with poor SRH in men and women. In both genders the association between aging and poor SRH was stronger in middle-aged adults (25–65 years), than in older adults (65+) and remained significant even after accounting for individual level education and city-level socio economic characteristics. We further found that associations of age with poor SRH were stronger in men than in women. In both genders, both lower levels of city SEI and GDP per-capita were associated with poor SRH, even after adjusting for each other, with somewhat stronger associations for SEI as compared to GDP per capita. In addition, we found that GPD per-capita modified the associations between age and poor SRH, such that in middle-aged adults the age-SRH associations were stronger in cities with higher GPD per-capita. This interaction was less clear for city-SEI.In our sample, after adjusting for education, the prevalence of poor SRH was 31.6% higher in women than in men. These findings are in line with previous studies showing that women are more likely to report poorer SRH levels than men –32. The Our results show that the impact of aging on SRH begins in early to mid-adulthood, we found stronger associations of higher age with poor SRH among young and middle-aged adults (25–65) than in older adults (65–97). Most previous studies analyzed the age effects on SRH focus on older population , 29–31, We also found that the association with age seemed to be stronger in men than in women. Reasons for these findings should be further studied in the region. A study from 2000, reported gender differences in SRH assessments in the elderly , noting After adjusting for individual education, we found an independent association between both city-level SEI and GDP per-capita with SRH, in both men and women. These results suggest that these two social economic constructs have different mechanisms by which they impact SRH. SEI can exert a more direct influence of SRH as the index comprises crucial household assets , needed to accomplish basic levels of health and wellbeing, while GDP per-capita representing wealth, constitutes cities potential to achieve better health in a paradigm of economic welfare, perhaps, in a more indirect way. As stated in the introduction SEI approaches follow better (though not perfectly) the capabilities approach while GDP per-capita constitutes the classical welfarist approach to health and healthcare , 42. OneOther studies examining how urban contexts impact SRH have focused on neighborhood level factors , 63. WitFinally, we found that GDP per-capita (more than SEI) modified the association of age and SRH in men and women (25–65 years). Among people aged 25–65 we found stronger associations of age with SRH in cities with higher rather than lower GDP. The potential mechanisms for these results may include larger inequalities among cities with higher GDP per-capita and construct bias regarding self-rated health . Which sOur research has some limitations. Firstly, SRH may be considered a subjective measure of health compared to some objective measures of morbidity. Furthermore, to control for SES at the individual level, we relied only on educational level as a proxy for individual SES. Secondly, the cross-sectional nature of the study limits our ability to make causal inferences. Thirdly, although we had few missing data  and the census year (source of the SEI). The gap between survey data and census was largest for Chile (8-year difference), and smallest for Colombia, Argentina, Brazil and El Salvador (2–3 year difference). Although there may have been differences in the prevalence of poor SRH over time, we do not anticipate differences in the associations that were examined in this study. Another related limitation is the fact that Guatemala data is the oldest (2002), but similarly, we do not expect changes in the associations over time. Finally, the role of built environment characteristics of the cities  on SRH, was beyond the current scope of this manuscript. Future studies in SALURBAL are very well position to address this research question.Despite these shortcomings, this study has important strengths. This is one of the largest studies to date, with a harmonized dataset, focusing on a wide and heterogeneous range of Latin American cities (114 cities from 6 countries), most of the countries are classified in the category of middle-income by the World Bank. Our sample includes adults with a wide range of ages and thus extends our knowledge of SRH across adulthood. Finally, the study multi-level structure, nesting people within cities, allows accounting for unmeasured city contextual factors which may have a role in shaping SRH as people age.In Latin American cities aging was associated with higher risk of poor SRH, but this association was stronger in adults from 25 to 65 than in those over 65. Although women are more likely to report poor SRH, we found that the association between aging and poor SRH was more pronounced among men. City SEI and GDP per-capita were associated with SRH with stronger and most consistent effects for SEI. GDP per-capita modified the association between age and poor SRH such that high GDP was associated with stronger age association.The main policy and research implications are the need to better understand the gender differences in the experience of perceived health with aging in Latin American cities. Women rate their health worse than men, but men feel a stronger deterioration in SRH as they age. Future research could explore the extent to which gender-based cultural, social, or political issues shape these trajectories in Latin American cities. In addition, more research  is needed to better understand how city socioeconomic environments represented by different indicators  exert potentially independent influences on the age effect on SRH, suggesting different mechanisms behind these associations. Studies should also investigate what Latin American people consider when rating their health. On the other hand, realizing that the age effect on SRH is stronger in younger adults rather than people over 65 years is a key finding that should guide the design of better policies that support healthy adulthood in urban settings.Additionally, actions to improve (perceived) health status should begin at early ages in adulthood considering the different gender trajectories as people age and the role of cities in delaying and attenuating the impact of age in SRH in Latin America countries.Additional file 1. Derivation of the analytical sample. SALURBAL StudyAdditional file 2. Data sources per country and analytical sample Additional file 3. Socioeconomic characteristics of the cities in the study, by countryAdditional file 4. Characteristics of the study population by SEI tertiles. SALURBAL Study Additional file 5. Characteristics of the study population by GDP per-capita (in constant 2011 USD) tertiles. SALURBAL Study Additional file 6. Association between individual age and poor self-reported health by tertiles of Socioeconomic Index, age, and gender.Additional file 7. Association between individual age and poor self-reported health by tertiles of GDP, age, and gender


Importantly, varlitinib significantly diminished LPS-mediated microglial nlrp3 inflammasome activation in BV2 microglial cells. In primary astrocytes, varlitinib downregulated LPS-evoked astroglial il-1β mRNA levels, AKT signaling, and nlrp3 inflammasome activation. In LPS-treated wild-type mice, varlitinib significantly reduced LPS-stimulated glial activation and IL-1β/NLRP3 inflammasome formation. Moreover, varlitinib significantly reduced micro- and astroglial activation and tau hyperphosphorylation in 3-month-old tau-overexpressing PS19 mice by downregulating tau kinase DYRK1A levels. However, in 6-month-old tau-overexpressing PS19 mice, varlitinib only significantly diminished astroglial activation and tau phosphorylation at Thr212/Ser214. Taken together, our findings suggest that varlitinib has therapeutic potential for LPS- and tau-induced neuroinflammatory responses and the early stages of tau pathology.The FDA-approved EGFR/HER2 inhibitor varlitinib inhibits tumor growth and is used in cancer treatment. However, the neuroinflammatory response associated with EGFR/HER2 and its underlying mechanism have not been elucidated. This study evaluates the impact of varlitinib on LPS- and tau-mediated neuroinflammatory responses for the first time. In BV2 microglial cells, varlitinib reduced LPS-stimulated  Neuroinflammation is an inflammatory response within the central nervous system (CNS) and is mediated by the release of cytokines, chemokines, and reactive oxygen species by CNS glia . CNS gliLPS evokes neuroinflammation in mammals by stimulating toll-like receptor 4 (TLR4) signal transduction . Recent AD is a neurodegenerative disease that is correlated with neuroinflammation . Both amin vitro and in vivo  levels. However, in 6-month-old tau-overexpressing PS19 mice, the effects of varlitinib on tau-mediated gliosis, tau hyperphosphorylation, and DYRK1A levels were smaller than those in 3-month-old tau-overexpressing PS19 mice. Overall, the present study reveals that varlitinib has therapeutic effects on neuroinflammation and tau-related disease by inhibiting neuroglial NLRP3 inflammasome assembly and tau kinase DYRK1A, respectively.The small molecule varlitinib is an EGFR/HER2  inhibitor that is FDA-approved for the treatment of breast cancer and is known to cross the blood–brain barrier . The bre in vivo , 15. NumAll experiments were approved by the institutional biosafety committee (IBC) and performed in accordance with approved animal protocols of the Korea Brain Research Institute .in vitro or 20 mg/kg  injection in 5% DMSO, 10% PEG, 20% Tween 80) in vivo. The TLR4 inhibitor TAK-242  was used at a dose of 500 nM (1% DMSO) in vitro. LPS from Escherichia coli  was used at a concentration of 200 ng/ml (in PBS) in vitro or 250 μg/kg or 10 mg/kg  in vivo.Varlitinib  was used at a dose of 5 μM (1% DMSO) 4 cells/well) were incubated for 1 h in FBS-free medium and then treated for 24 h with varlitinib  or vehicle . In addition, the effects of longer treatment durations on cell viability were assessed by treating cells with 1% DMSO or 5 μM varlitinib for 24 h, 48 h, or 72 h. After treatment, the cells were incubated with MTT solution for 3 h, and the formazan product was dissolved in DMSO at room temperature for 20 min. Finally, the absorbance of formazan at 570 nm was measured in a microplate reader  using a reference wavelength of 660 nm.The MTT assay was conducted to assess the effect of varlitinib on BV2 microglial cell viability. Cells in 96-well plates , and 5% fetal bovine serum .The microglial cell line BV2 was a generous gift from Dr. Kyung-Ho Suk. The culture conditions were as follows: 37°C, 5% COThe effects of varlitinib on LPS-induced proinflammatory responses were assessed in mouse primary astrocytes isolated as previously described –8, 16–20RT-PCR was performed to assess the effects of varlitinib on LPS-evoked inflammatory cytokine levels in BV2 microglial cells and primary astrocytes. Total RNA extracted from the cells using QIAzol Lysis Reagent  was used to synthesize cDNA by reverse transcription. RT-PCR was then performed using Prime Taq Premix (GeNet Bio) with the cDNA template as previously described , 16. Thenlrp3 in BV2 microglial cells, primary astrocytes, and LPS-treated wild-type mice were measured by real-time PCR  siRNA . First, siRNA at a final concentration of 30 nM in Opti-MEM  was incubated with 1 µL of Lipofectamine® RNAiMAX (Thermo Scientific) for 40 min. Second, BV2 microglial cells in a 24-well cell culture plate (2 x 105 cells/well) were transfected with the siRNA mixture for 24 h. Finally, the transfected cells were treated with 200 nM LPS or PBS for 30 min and 5 μM varlitinib or 1% DMSO for 23.5 h, and real-time PCR was performed using previously described primers  of each cell was automatically measured by CD11b or GFAP fluorescence intensity using the thresholding tools of Image J software. The selected ROIs were overlaid on matching red fluorescence images , and the fluorescence intensity inside the overlaid ROIs was measured. The fluorescence intensity of p-EGFR, p-AKTs473, p-FAK, and p-NF-kB is presented as the measured red fluorescence intensity divided by the selected ROI area. The analysis of 5–10 individual images of each sample was performed in a blinded manner.The effects of varlitinib on neuroinflammatory responses induced by LPS were assessed in BV2 microglial cells and mouse primary astrocytes fixed in 4% paraformaldehyde (PFA) for 10 min. The fixed cells were washed with 1x PBS three times and incubated overnight at 4°C with anti-p-EGFR, anti-p-AKT, anti-p-FAK, anti-p-NF-kB, or anti-GFAP antibodies in GDB buffer . The cells were subsequently washed three times with 1x PBS and incubated for 1 h at room temperature with Alexa Fluor 488- or Alexa Fluor 555-conjugated antibodies . Images of cells mounted in DAPI-containing solution  were obtained by a DMi8 fluorescence microscope  in a single plane. To measure p-EGFR, p-AKT2, 10 mM KCl, 0.5 mM DTT, 300 mM sucrose, 0.5 mM PMSF and 0.1% NP-40). The lysate was centrifuged at 10,000 rpm and 4°C for 1 min, and the supernatant (cytosolic fraction) was removed. Nuclear fractionation buffer  was added to the pellet, which was incubated on ice for 15 min and then centrifuged at 10,000 rpm and 4°C for 15 min. The nuclear fraction was used to detect nuclear NF-kB levels by western blot.Nuclear NF-kB levels were assessed in BV2 microglial cells. First, the cells were treated with 200 ng/ml LPS or PBS for 30 min and 5 μM varlitinib or 1% DMSO (vehicle) for 5.5 h. The treated cells were then lysed for 5 min in cytosol fractionation buffer , anti-AKT , anti-p-FAK , anti-FAK , anti-NF-kB , or anti-PCNA . Finally, the membrane was incubated for 1 h with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG , and ECL Western Blotting Detection Reagent was added for detection . Fusion Capt Advance software (Vilber Lourmat) was employed to acquire and analyze images.For western blotting, BV2 microglial cells, primary astrocytes, or mouse brain tissues were lysed in ProPrep lysis buffer  and centrifuged at 12,000 rpm for 15 min. The protein concentration in the supernatant was quantified by reference to a standard solution of BSA. Next, 10 μg of protein was loaded onto an 8% SDS gel, separated by electrophoresis, and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature and then incubated overnight at 4°C with anti-p-AKTin vivo, wild-type mice exposed to LPS were pre- or post-treated with varlitinib. For varlitinib post-treatment, wild-type mice were i.p. injected with LPS (250 μg/kg) or PBS followed by varlitinib  or vehicle  daily for 7 consecutive days. For varlitinib pretreatment, mice were i.p. injected with varlitinib (20 mg/kg) or vehicle  daily for 7 consecutive, and on the 7th day, 10 mg/kg LPS or PBS was injected followed 30 min later by the final administration of varlitinib or vehicle. Eight hours after the final injection on day 7, mouse brains were perfused and fixed for immunofluorescence staining.Three-month-old wild-type C57/BL6 male mice  were maintained under a 12 h photoperiod in a pathogen-free facility with free access to food and water. To determine the effects of varlitinib on neuroinflammation The effects of varlitinib on age-dependent tau pathology and tau-mediated neuroinflammatory responses were examined in 3-month-old or 6-month-old tau-overexpressing PS19 mice PS19Vle/J; Stock No. 008169, Jackson Laboratory, Bar Harbor, ME, USA). Genomic DNA from a tail snip was used for genotyping. To minimize the effects of hormones, only male mice were used. Regardless of age, the mice received the treatment regimen daily for 14 consecutive days, which consisted of i.p. injection of varlitinib (20 mg/kg) or vehicle . After the final injection on day 14, mouse brains were perfused and fixed for immunofluorescence staining. All animal experiments were performed in accordance with approved animal protocols and guidelines established by the Korea Brain Research Institute Animal Care and Use Committee .After final injection, wild-type and tau-overexpressing PS19 mice were anesthetized using 2,2,2-tribromoethanol (150 mg/kg)  in 2-methyl-2-butanol  for 5 min, perfused with PBS for 5 min, and fixed with 4% paraformaldehyde (PFA) for 7 min using a Cole-Parmer PTFE-Tubing pump . After PFA fixation, brains were immediately post-fixed in 4% PFA for 2 days, which was then replaced with 30% sucrose in PBS solution for 2 days. Finally, the brains were embedded in OCT compound  and sliced at a thickness of 30 μm at -23°C using a cryostat .® Antifade Mounting Medium with DAPI (Vector Laboratories). To reduce individual variance and batch-to-batch effects, brain sections from each group  were randomly selected for immunofluorescence staining. For each group, immunofluorescence staining was conducted using equivalent reagents, the same processing and fixation times, the same antibodies, and identical conditions were used to capture images.To validate tissue-specific fluorescence intensity, brain sections were washed 3 times with PBST (1% Triton-100 in PBS) for 5 min, blocked with 10% normal goat serum (NGS) in PBST for 2-4 h, and incubated with primary antibodies in PBST at 4°C for 24-48 h Table 1.To assess the effects of varlitinib on glial activation, we measured Iba-l and GFAP fluorescence intensity. In addition, to investigate the effects of varlitinib on glial migration and morphology, we quantified the number of GFAP-positive cells and the percent GFAP-positive area, respectively , 21.To quantify the levels of Iba-I, GFAP, IL-1β, NLRP3, AT100, AT180, DYRK1A, p-GSK3β, or pCDK5 in the cortex and hippocampus, the area of each region was measured by drawing a region of interest (ROI) in a DAPI fluorescence image using Image J software. The selected ROIs were overlaid on matching red or green fluorescence images, and the fluorescence intensity inside the overlaid ROIs was measured. The levels of these markers are presented as the measured fluorescence intensity divided by the selected ROI area as previously described . The parComparisons of two groups were performed with unpaired two-tailed t-tests with Welch’s correction. For multiple comparisons, parametric one-way ANOVA or non-parametric one-way ANOVA   followed by Tukey’s test (parametric) or Dunn’s test (non-parametric) was used, with significance set at p < 0.05. Means ± SEMs are presented . Detailed information is listed in The EGFR/HER2 inhibitor varlitinib is used for the treatment of triple-negative breast cancer (TNBC), and its impact on LPS-induced neuroinflammation and the underlying mechanism of action have received little attention. Thus, we examined the effects of varlitinib on LPS-mediated proinflammatory responses. Before conducting further experiments, we assessed the impact of varlitinib on the viability of BV2 microglial cells by performing MTT assays of cells treated with vehicle or varlitinib  for 24 h. No decrease in cell viability was observed after incubation for 24 h with varlitinib concentrations of up to 50 μM  or PBS for 30 min followed by varlitinib (5 μM) or vehicle (1% DMSO) for 5.5 h. Immunocytochemistry demonstrated that LPS-induced p-EGFR fluorescence intensity in BV2 microglial cells were significantly downregulated by varlitinib  or PBS for 30 min followed by varlitinib (5 μM) or vehicle (1% DMSO) for 5.5 h. Subsequent RT-PCR showed that post-treatment with varlitinib (as a curative treatment) significantly decreased LPS-induced il-1β and inos mRNA levels but not cox-2 and il-6 mRNA levels  were assessed in BV2 microglial cells treated with varlitinib (5 μM) or vehicle (1% DMSO) for 30 min followed by LPS (200 ng/ml) or PBS for 5.5 h. RT-PCR showed that pretreatment with varlitinib only significantly reduced LPS-mediated in vitro, we next investigated whether varlitinib suppresses LPS-induced proinflammatory cytokine levels via TLR4 signaling. BV2 microglial cells were treated sequentially with TAK-242  or vehicle (1% DMSO) for 30 min, with LPS (200 ng/ml) or PBS for 30 min and finally with varlitinib (5 μM) or vehicle (1% DMSO) for 5 h. RT-PCR showed that similar to the results in il-1β and inos mRNA levels were significantly reduced in cells treated with varlitinib compared with LPS-treated cells  or PBS for 30 min and varlitinib (5 μM) or vehicle (1% DMSO) for 5.5 h. Immunocytochemistry showed that varlitinib significantly reduced LPS-stimulated microglial p-AKT and p-FAK fluorescence intensity  or PBS for 30 min as before, but the treatment time with varlitinib (5 μM) or vehicle (1% DMSO) was extended to 23.5 h. Real-time PCR indicated that varlitinib significantly diminished LPS-stimulated microglial nlrp3, pro-il-1β, and il-1β mRNA levels in BV2 microglial cells  or scramble siRNA for 24 h were treated with LPS (200 ng/ml) or PBS for 30 min and varlitinib (5 μM) or vehicle for 23.5 h. Subsequent real-time PCR revealed that transfection with nlrp3 siRNA significantly decreased nlrp3 mRNA levels compared with transfection with scramble siRNA, indicating that nlrp3 siRNA transfection was successful  or PBS for 30 min and varlitinib (5 μM) or vehicle (1% DMSO) for 5.5 h. Real-time PCR demonstrated that varlitinib significantly decreased the LPS-evoked increase in We then examined the effect of varlitinib on downstream AKT signaling in primary astrocytes, as varlitinib decreased LPS-induced microglial AKT signaling. Primary astrocytes were treated with LPS (200 ng/ml) or PBS for 45 min and varlitinib (5 μM) or vehicle (1% DMSO) for 45 min. Western blotting showed that varlitinib significantly downregulated LPS-mediated astroglial AKT phosphorylation  or PBS for 30 min and varlitinib (5 μM) or vehicle (1% DMSO) for 5.5 or 23.5 h. Real-time PCR analysis showed that treatment with varlitinib for 5.5 h did not affect LPS-induced in vivo, we used LPS, which is known to induce neurodegenerative disease as well as neuroinflammation  or PBS followed 30 min later by varlitinib  or vehicle . Eight hours after the final LPS or PBS injection on day 7, the mice were perfused and fixed, and brain sections were subjected to immunofluorescence staining with anti-Iba-1 or anti-GFAP antibodies. Under chronic neuroinflammation conditions, varlitinib post-treatment significantly inhibited microglial Iba-1 fluorescence intensity, the number of Iba-1-positive cells, and the percent Iba-1-positive area in the cortex and hippocampus  or PBS followed by varlitinib  or vehicle  daily for 7 days as shown in il-1β and nlrp3 mRNA levels. Three-month-old C57BL/6 mice were injected with varlitinib  or vehicle for 7 days. On the 7th day, varlitinib  or vehicle was injected, and 30 min later, LPS  or PBS was administered. Eight hours after the last injection, the mice were sacrificed, and real-time PCR was conducted. Under these acute neuroinflammation conditions, pretreatment with varlitinib significantly decreased LPS-induced il-1β mRNA levels in the hippocampus only  or anti-AT180 (p-TauThr231) antibodies showed that varlitinib significantly inhibited tau phosphorylation at Thr212/Ser214 in the cortex and hippocampus  or vehicle  daily for 2 weeks. Immunofluorescence staining with anti-AT100  or vehicle  daily for 2 weeks. Immunofluorescence staining with anti-AT100 or anti-AT180 antibodies showed that varlitinib significantly inhibited tau phosphorylation at Thr212/Ser214 in the cortex and hippocampus  were injected with varlitinib  or vehicle  daily for 2 weeks. Immunofluorescence staining with anti-Iba-1 and anti-GFAP antibodies showed that varlitinib significantly suppressed Iba-1 fluorescence intensity, the number of Iba-1-positive cells, and the percent Iba-1-positive area in the cortex and hippocampus  and EGFR inhibitor regorafenib in BV2 microglia is partially dependent on TLR4 signaling  and other neuroinflammation-associated molecular targets in primary astrocytes. Future studies will address this issue.Because there is a clear association between EGFR and NLRP3 in cancer cells , 59, we trocytes Figure 4trocytes . These oin vivo, but acute and chronic LPS treatment differentially affect glial activation and morphology. Under acute neuroinflammation conditions in vivo, LPS activates glial cells, which migrate to the lesion and secrete growth factors  and pro- and anti-inflammatory cytokines  activate chronic microglial and astrocyte activation and vice versa , 64. In K670N;M671L/PS1M146L mutant mice  and performed in accordance with approved animal protocols of the Korea Brain Research Institute .Study conception and design: MM and H-SH. Acquisition of data: JK, S-JK, H-RJ, and J-HP. Preparation of all figures: JK and S-JK. Preparation of tables: JK Writing of manuscript: JK, S-JK, and H-SH. Revision and replication experiments: JK. Writing of revised manuscript: JK and H-SH. All authors contributed to the article and approved the submitted version.This work was supported by the KBRI basic research program through KBRI funded by the Ministry of Science, ICT & Future Planning , the National Research Foundation of Korea , and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea . This work was supported by a National Research Council of Science & Technology (NST) grant funded by the Korean government .in vitro work and in vivo studies.Confocal microscopy  data were acquired at the Advanced Neural Imaging Center at the Korea Brain Research Institute (KBRI). We thank previous and current members of the neurodegenerative diseases lab for editing and valuable comments on our manuscript and fortechnical assistance with The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Massive amounts of data from nucleic acid sequencing have changed our perspective about diversity and dynamics of marine viral communities. Here, we summarize recent metatranscriptomic and metaviromic studies targeting predominantly RNA viral communities. The analysis of RNA viromes reaffirms the abundance of lytic (+) ssRNA viruses of the order Picornavirales, but also reveals other (+) ssRNA viruses, including RNA bacteriophages, as important constituents of extracellular RNA viral communities. Sequencing of dsRNA suggests unknown diversity of dsRNA viruses. Environmental metatranscriptomes capture the dynamics of ssDNA, dsDNA, ssRNA, and dsRNA viruses simultaneously, unravelling the full complexity of viral dynamics in the marine environment. RNA viruses are prevalent in large size fractions of environmental metatranscriptomes, actively infect marine unicellular eukaryotes larger than 3 µm, and can outnumber bacteriophages during phytoplankton blooms. DNA and RNA viruses change abundance on hourly timescales, implying viral control on a daily temporal basis. Metatranscriptomes of cultured protists host a diverse community of ssRNA and dsRNA viruses, often with multipartite genomes and possibly persistent intracellular lifestyles. We posit that RNA viral communities might be more diverse and complex than formerly anticipated and that the influence they exert on community composition and global carbon flows in aquatic ecosystems may be underestimated. Viruses are vital parts of the marine ecosystem. They are major contributors to processes such as remineralization of carbon and nutrients and continuously regulate the diversity and abundance of complex microbial communities in the ocean, consisting of bacteria, archaea, and unicellular eukaryotes, protists ,2,3,4. PRiboviria. Within this realm, kingdom Pararnavirinae consists of RNA viruses with the reverse transcriptase (RT) and kingdom Orthornavirinae contains RNA viruses with the RNA-dependent RNA polymerase (RdRp). Kingdom Orthornavirinae splits in into five phyla on the basis of the phylogenetic analysis of RNA-dependent RNA polymerase (Lenarviricota includes ssRNA with positive (+) genome polarity, such as RNA bacteriophages from family Leviviridae and their eukaryote-infecting descendants, Narnaviridae and Mitoviridae, simple RNA viruses without a capsid that are transmitted vertically. Phylum Pisuviricota is sometimes called Picornavirus supergroup, and it includes five orders of (+) ssRNA viruses and one order of dsRNA viruses with bipartite genomes, Durnavirales. Phylum Kitrinoviricota includes only (+) ssRNA viruses infecting eukaryotes, mostly terrestrial plants, and animals. Phylum Duplornaviricota, the fourth branch, consists of dsRNA viruses infecting both eukaryotes and prokaryotes. Finally, the fifth branch forms phylum Negarnaviricota and includes all known RNA viruses with negative (−) genome polarity .,16.RibovRNA viruses in the aquatic environments may infect a wide range of hosts with important consequences. RNA viral pathogens of marine mammals, seabirds, fish, crustaceans, and bivalves can decimate the populations of wild and farmed marine animals with large economic and ecological impacts . RNA virRoutine methods for assessing viral abundance in aquatic environments, such as epifluorescence microscopy and flow cytometry, currently lack the sensitivity to detect the small genomes of RNA viruses ,22. ConsTraditionally, RNA metaviromics has been the key method to study diversity and dynamics of aquatic RNA virus communities. A comprehensive review summarized gene-marker studies targeting the RNA-dependent RNA polymerase gene and the first RNA metaviromics studies revealing consistent dominance of picorna-like RNA viruses in the marine environments . KnowledWe explore in detail 15 recent studies conducted in aquatic environments and 4 studies focusing on individual protists and macroalge, in culture and in natural environments, and discuss how they have broadened our understanding of RNA viral diversity. The focus of this review is on RNA viruses, but if other viral groups were concurrently detected in a referenced study, they are discussed as well to get a more comprehensive overview of the viruses present in a specific environment. Finally, we address the remaining challenges and provide suggestions for future explorations of the ecological relevance of RNA viruses in aquatic ecosystems.Two commonly used approaches for studying viral diversity in aquatic environments are metaviromics  and data mining of environmental metagenomes and metatranscriptomes for virus discovery . These aLipothrixviridae or bacterial Inoviridae that can be up to 3.5 µm long [Metaviromics  is the method of sequencing extracellular total virus-like particles of either DNA or RNA viruses . Enrichm µm long ,30,36. T µm long ,38,39. C µm long  and are  µm long .Cells of single-celled eukaryotes and prokaryotes from the environment are typically collected onto filters of different pore size, with 0.22 µm filter being the smallest filter size used. Typically, extracellular DNA and RNA viruses will pass through these filters, though aggregates or particle-associated extracellular viruses can still be retained. Viral sequences can be retrieved from both metagenomes and metatranscriptomes derived from the cellular fraction ,49,50,51Narnaviridae, Hypoviridae, and Endornaviridae [Metatranscriptomics, which is the sequencing of RNA transcripts from environmental microbial communities, is well suited to study the active viral infection in diverse viral groups with structurally different genomes . As a paaviridae ,38,39. Taviridae ,53. The aviridae . While taviridae . Limitataviridae ,56 and tAdditional complexity in metatranscriptomics arises from viral genes being transcribed sequentially and in mono- or polycistronic transcription units, which can lead to uneven genome coverage . The higStandard methods for reverse transcription used in RNA sequencing are inefficient in transcribing dsRNA molecules as the presence of complimentary strand will prevent the binding of random primers. Therefore, compared to ssRNA viruses, the dsRNA viral lineages may be underrepresented in both metatranscriptomic and viral RNA metagenomic datasets . An apprRecently, a novel dsRNA sequencing approach, fragmented and primer-ligated dsRNA sequencing (FLDS), has been developed and applied to marine environmental samples, demonstrating the method’s potential to capture RNA viral diversity that previously went undetected . After cIn synthesis, there is currently no method that will comprehensively capture all viral diversity. The comparative study of RNA viromes and metatranscriptomes has highlighted tremendous differences between the fractions as well as between different methodological approaches, which will be further discussed below . This obPicornavirales in temperate, tropical, and polar oceans, often clustering with known RNA viruses that infect diatoms. Very few sequences of dsRNA viruses were typically recovered, and RNA phages or negative-sense RNA viruses were absent. Only five RNA metaviromic studies in the water column have been performed to date, predominantly in coastal productive environments, with only one study having an extensive geographical distribution [The first environmental viral metagenomic surveys consistently showed that most sequences in the RNA viromes were unknown. Among known sequences, typically more than 90% of RNA viral sequences identified as (+) ssRNA viruses from order ribution ,58,59,60ribution .Picornavirales were most abundant, mainly consisting of families Marnaviridae and Dicistroviridae. Two complete viral genomes (JP-A and JP-B) were phylogenetically similar to the diatom-infecting Rhizolenia setigera RNA virus RsRNAV and could have a protist (diatom) host. In the Strait of Georgia library, the majority of viral sequences fell into Tombusviridae, a family of viruses from the phylum Kitrinoviricota [Picornavirales, which encompassed 95% of the assignable reads, with a minority of reads being identified as dsRNA viruses. Similarly, the full genomes recovered from the dataset were phylogenetically related to diatom-infecting viruses, or viruses from families Marnaviridae and Dicistroviridae, consistent with the previous RNA metaviromic study in temperate waters [In RNA metaviromics study of coastal, temperate waters of British Columbia, 98% of the identifiable reads represented (+) ssRNA viruses . In the viricota . Tropicae waters . Picornavirales. Out of five full genomes assembled from the dataset, three  clustered together with diatom-infecting RNA viral isolates or marine RdRp sequences, and two (PAL 438 and PAL_E4) contained highly divergent RdRps. There was a smaller proportion of viral sequences identified as Marnaviridae or Dicistroviridae compared to the tropical RNA viral metagenomes of Kaneohe Bay. Instead, the relative abundance of diatom-infecting RNA viruses from the genus Bacilarnavirus surpassed 90% in three samples or reached around 50%, with unclassified Picornavirales taking up the rest. The relative abundance of RNA viruses in the total virioplankton was estimated to range 2–79% in samples on the basis of nucleic acid content of gradient-purified fractions [The trend continued with the overwhelming majority of identifiable RNA viral sequences (97.8%) from polar RNA viral metagenomes collected during a spring diatom bloom in West Antarctica Peninsula  being clractions .The largest spatially extensive study of RNA viral metagenomes in the water column to date explored the biogeography of the six marine picorna-like RNA viruses: three complete genomes of picorna-like viruses recovered in this metagenomic study itself , two comPicornavirales constituting only around 26% of RdRp sequences in the sample. Within the phylum Pisuviricota, the largest number of RdRp sequences (n = 854) grouped with the aquatic picorna-like viruses  encompassing Marnaviridae and other protist-infecting viruses. Protopotyviruses, ancestors of complex plant-infecting Potyviruses, formed a new clade that was not previously detected in marine environments. Within the phylum Kitrinoviricota, many highly divergent RdRp sequences formed a new, phylogenetically well supported clade with previously “orphan” RdRPs of RNA viruses found in marine invertebrates and wastewater, with multiple new orders and families such as Weiviruses, Yanviviruses, Zhaoviruses, and Shangaiviruses, significantly expanding the presence of this phylum in aquatic environments, previously limited to detection of Tombusviridae in one metagenomic library. Within the phylum Lenarviricota, a high number of sequences had similarity to the Leviviridae, (+) ssRNA bacteriophages, and the simple eukaryote-infecting Ourmiaviridae, documenting for the first time the presence of (+) ssRNA bacteriophages as members of marine RNA viral communities. Capsidless endogenous RNA viruses, such as Mitoviridae and Narnaviridae, were absent from the extracellular RNA virome as they would have been removed together with cells during filtration. The largest RdRp cluster was affiliated with the Marnaviridae, viruses of diatoms and stramenopiles and, together with the use of the alternative (ciliate) genetic code, might suggest that a considerable fraction of RNA viruses infect protists [More recently, a study recovered 4593 near-full-length RdRp sequences clustered within 2192 clusters at 75% amino acid identity (AAI), in one water sample from a single location in China . Consistprotists .Orthornavirinae and Pararnavirinae, RdRp genes, and RT genes, respectively. Most of the identifiable viral RNA sequences were classified as RT-containing Retroviridae and Metaviridae. Out of RNA viruses with the RdRp gene, the two most abundant families were surprisingly dsRNA viruses from families Totiviridae and Cystroviridae, in contrast with water column RNA viromes that are typically dominated with ssRNA viruses. Single-stranded RNA viruses from family Leviviridae were the most abundant ssRNA viruses detected, supporting the idea that RNA bacteriophages are important members of marine RNA viral communities in the water column as well as in sediments. However, most of the viral sequences could not be assigned to known viral groups, suggesting that the deep sea might represent a rich reservoir of unexplored viral diversity. Alternatively, this might just reflect the current limitations of the available software programs in assigning and classifying viral sequences from environmental samples [The high numerical abundance of viral particles and the tremendous impacts of viral infection on bacterial and archaeal communities in marine sediments have been well documented ,66. As w samples . Picornavirales have very high burst sizes [It is still unclear what fraction of the large differences in the estimates of RNA viral diversity between different studies and different habitats is real rather than a result of methodological biases. RNA viruses from order st sizes , and it st sizes . Consistst sizes . Similarst sizes . It is est sizes  uncoverest sizes ,58,59,60st sizes . More rest sizes  also hadst sizes ,74. WhetMetatranscriptomics can capture the active viral infection of diverse viral groups and has been increasingly used in studies of RNA virus community diversity and ecological dynamics in the last few years. Most studies were performed in coastal, productive environments, either on temporal or on spatial resolution  and emplAureococcus anophagefferens take place in Quantuck Bay. Active infections of eukaryotic viruses were tracked during the initiation, peak, and demise of the bloom in Quantuck Bay and in the steady-state, non-bloom conditions in Narraganset Bay using marker gene approach [Mimiviridae and Phycodnaviridae were continuously present in both locations, with two distinct dynamics. Some giant viruses were steadily expressed across sampling dates, and others exhibited sharp peaks in abundance, or a “bloom and bust” type of expression. In the non-giant virus community, between 62% and 74% of contigs in both bays were unclassified (+) ssRNA viruses in the order Picornavirales, followed by classified families Picornaviridae, Secoviridae, and Dicistroviridae from the same order. Few dsRNA viruses resembling Totiviridae, Partitiviridae, and Hypoviridae were detected. The viral community (excluding giant viruses) in Narraganset Bay was steadily dominated (>90%) by Picornavirales. In Quantuck Bay, both ssDNA and unclassified Picornavirales were present in the Aureococcus anophagefferens bloom samples in equal relative abundances, around 50%. Unclassified Picornavirales viruses took over (>90%) when bloom disintegrated, possibly infecting other protists in the bloom succession. Co-occurrence analysis between viruses and host marker genes found that most of the hosts might be phytoplankton or heterotrophic protist, but also fungi [Picornaviridae, Virgaviridae, and Reoviridae with putative protist or invertebrate hosts. The low number of contigs retrieved might be due to low sequencing depth of 1–5 million reads per library [One of the first studies that used environmental marine metatranscriptomes followed active infections of diverse viral groups  in two highly productive sites in the US, Narragansett Bay and Quantuck Bay. Seasonal diatom blooms occur in Narragansett Bay, and harmful brown tide blooms caused by unicellular eukaryotic algae approach . Contigsso fungi . Another library .Mimiviridae , Phycodnaviridae, and Iridoviridae. Major RNA viral groups in the dataset were Picornavirales as well as Narnaviridae, Tombusviridae, Virgaviridae, and dsRNA Partitiviridae. Giant viruses and RNA viruses that were positively correlated with carbon export efficiency were abundant in the oceanic regions with high carbon export efficiency, such as the Indian Ocean and the Mediterranean Sea. Hosts of these giant viruses are predicted to be picoplankton  and haptophytes. Hosts of RNA viruses were either copepods or diatoms from the order Chaetocerales, both groups being important contributors to the biological carbon pump [Multiple metatranscriptome size fractions and a metagenome picoplankton size fraction from the Tara Ocean expedition dataset were screened for eukaryotic viruses using marker gene approach: PolB for giant viruses, RdRp for RNA viruses, and RepB for ssDNA viruses . Interesbon pump .Microcystis-dominated harmful algal bloom in a temperate Lake Tai in China was characterized using the same viral marker genes approach [Microcystis, and RNA viruses constituted the remaining 42% of viral transcripts. Though very diverse, giant viruses take up only 8% of the transcripts. Surprisingly, less than 1% of detected viral transcripts was identified as ssDNA bacteriophages or dsDNA bacteriophages infecting heterotrophic bacteria, though Microcystis bloom is typically accompanied with an abundant heterotrophic bacterial community. Interestingly, this is one of the first metatranscriptome studies reporting substantial diversity and abundance of dsRNA viruses. Of the total RNA viral contigs, 28% were identified as dsRNA, mainly Partitiviridae and Picobirnaviridae, and 72% were ssRNA viruses of the Picornavirales order, largely Marnaviridae and Discistroviridae [More recently, the active viral community within a approach . As expeoviridae . This nooviridae .Myoviridae, 9% to giant viruses infecting eukaryotic phytoplankton from the family Phycodnaviridae, and 8% to the filamentous ssDNA bacteriophages from family Inoviridae. In high-chlorophyll cellular metagenome samples collected during the bloom, the proportion of giant viruses rose to 93% of the DNA reads and previously dominant Myoviridae dropped to 3%. In high-chlorophyll metatranscriptomes collected during the bloom, the proportion of (+) ssRNA viruses from family Picornaviridae increased to 38%, mostly composed of PAL E4 and PAL 156 viruses, both lytic viruses, and the latter closely related to diatom-infecting RNA viruses from the genus Bacilarnavirus [Chaetoceros sp. RNA virus 02 exhibited only one peak, and heterotrophic labyrinthulid protist-infecting Aurantiochytrium single-stranded RNA virus 01 peaked five times during the sampling period. This suggests that RNA viruses are important players in regulating the microbial host communities on hourly scales in steady-state systems not perturbed with phytoplankton blooms [Both metagenomes and metatranscriptomes of the microbial community collected during a diatom bloom succession at Chile Bay in the West Antarctic Peninsula were mined for viruses . Viral rrnavirus . A similrnavirus . Taken trnavirus  providedrnavirus  were mapn blooms .Mononegavirales and a high proportion of Retroviridae-like sequences. At two high-salinity sites, DNA viruses infecting the protist Ostreococcus outnumbered the bacteriophages, amounting to 20%–40% of total viral transcripts, which indicated an ongoing infection. The sample collected in the lake Tornetrask contained virus transcripts mostly belonging to ssDNA bacteriophages from family Microviridae, and two samples had a large proportion of ssDNA viruses infecting pigs that could have originated from nearby animal farms. Collectively, these results provide a valuable snapshot into virus diversity at multiple locations and demonstrate that low-abundance viruses can rise to high abundance if conditions allow [A spatial metatranscriptome study in the Baltic Sea at 11 locations spanning a salinity gradient in the brackish Baltic Sea and one freshwater lake identified a wide distribution of ssDNA and RNA populations and high transcriptional activity of fish viruses in different microbial size fractions . Both ssns allow . Phycodnaviridae) and bacteriophages dominated the viral portion of the Baltic Sea metatranscriptome  [Studies using the size-fractionated approach show differential enrichment of viral groups in different size fractions ,50,51. INarnaviridae, and dsRNA viruses from families Picobirnaviridae and Reoviridae. The ssRNA extracellular virome of the coastal site was dominated by (+) ssRNA viruses from the order Picornavirales, as reported by other studies [Picobirnaviridae and Reoviridae, dsRNA viruses with a capsid and an extracellular phase. Many contigs resemble viruses with protist hosts such as diatom colony-associated dsRNA virus-1 (DCADSRV-1) [Micromonas pussila reovirus [The earlier metatranscriptome studies described above recovered mostly ssRNA viruses and very few dsRNA viruses, in agreement with the diversity found in extracellular RNA viromes. A combined metatranscriptomics and RNA metaviromics study compared RNA viruses in the cellular and purified viral particle fractions in four pelagic sampling sites and one coastal sampling site off the coast of Japan . For bot studies ,58,59,60ADSRV-1)  and Micrreovirus . There areovirus . The secThere have been several new viral discovery studies in metatranscriptomes of (mostly) axenic protist cultures that have discovered an array of small dsRNA and ssRNA viruses, often with multipartite genomes that coexist with the host and do not cause lysis of the host cell. These persistent infections can be chronic or latent (silent). The advantage of viral discovery in cultured metatranscriptomes over broad environmental metatranscriptomics surveys is that the host can be assigned with certainty and low-titre viruses, which are frequently missed by environmental metaviromics and metatranscriptomics, can be picked up. Chlorophyta) and chlorarachniophytes [Ostrebium sp., comprising dsRNA viruses falling into families Partitiviridae and (+) ssRNA Mitoviridae—non-encapsidated small RNA viruses with tiny genomes. Fewer viruses were detected within chloroarachniophytes, possibly due to the clade being divergent and poorly characterized itself, and the highly divergent RdRps might have gone undetected. Additionally, segmented (+) ssRNA viruses from family Virgoviridae were detected along with the first detection of a (−) ssRNA virus resembling family Bunyaviridae in phytoplankton culture, though the confirmation that the chloroarachniophyte algae are the true host requires additional work [The first report of RNA viruses in the cultured metatranscriptomes came from six species of phytoplankton belonging to two distant phylogenetic groups, green algae  ssRNA non-encapsidated viruses from families Narnaviridae and Mitoviridae, known for persistent lifestyles [Totiviridae that were previously considered to be fungal pathogens. A member of Mononegavirales, a negative-sense RNA virus, was detected in the Pseudo-Nitzchia culture, but again, host assignment requires additional experimental work [Recently, the metatranscriptomes of cultured phytoplankton obtained from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) were screened for the presence of viral RNA-dependent RNA polymerase hallmark gene . Thirty festyles . The doutal work .Totiviridae, Picobirnaviridae, Cystoviridae, Partitiviridae, and Endornaviridae) and four families of ssRNA viruses . Typically, the viral sequences were very divergent, exhibiting less than 50% AAI to known viral RdRp sequences [Two studies screened diatom holobionts and marine macroalge for RNA viruses with the FLDS dsRNA sequencing approach ,78. The equences ,78.Amalgaviridae, Chrysovirdae, Totiviridae, Partitiviridae, Endornaviridae, Mitoviridae, and Narnaviridae are all associated with persistent, asymptomatic infections in plants and fungi [These metatranscriptome studies focused on single protist or macroalgae holobionts have demonstrated that (+) ssRNA, dsRNA, and potentially (−) ssRNA viruses are distributed across many diverse protist groups and went previously undetected in “bulk” environmental approaches. These viruses have small genomes , may be segmented, and some lack capsid proteins. Viral families nd fungi  and likeSeveral studies discussed in this review demonstrate the plethora of information available when different sequencing approaches are combined ,50,56, bDespite the immense new RNA viral diversity uncovered with multi-omic approaches, it is evident that RNA viruses in aquatic environments are very sparsely sampled . In addiMost metatranscriptome studies focus on productive areas during algal blooms . EffortsAnother area that deserves attention is standardization and validation of the pre-sequencing and sequencing sample preparation protocols. Enrichment methods, extraction kits, and library preparation can significantly affect the number of viral reads, and consequently the assembly, and skew the relative abundances of particular viral groups and diversity of the viruses present in the sample ,85. To ePicornavirales are a stable component of the aquatic RNA viral communities, but are perhaps not as dominant as previously thought. (2) All three phyla of positive-sense ssRNA viruses are represented in comparable proportions in the aquatic ecosystem as well as the dsRNA viruses. Highly divergent RNA viruses are being discovered frequently, and are infecting protist hosts that were previously thought only to host DNA viruses. (3) Negative-sense ssRNA viruses, which are traditionally associated with multicellular animals and plants, were found for the first time in the metatranscriptomes of some single-celled eukaryotic phytoplankton cultures. The articles discussed above demonstrate significant progress in characterizing aquatic RNA viral communities, but also highlight how undersampled RNA viral communities are. However, as more data accumulate, we expect to paint a more comprehensive picture of RNA viral diversity, abundance, and host range across the aquatic environments.Multi-omic sequencing approaches have advanced our understanding of RNA viral diversity and expanded our understanding of their ecological roles in aquatic ecosystems. Three common messages have emerged from the synthesis of all these studies: (1) lytic ssRNA viruses from the order