Brachypodium distachyon were exposed to wind and mechanical treatments. We surveyed a wide range of stem‐related traits to determine the effect of the two treatments on plant growth, development, and stem biomass properties. Both treatments induced significant quantitative changes across multiple scales, from the whole plant down to cellular level. The two treatments resulted in shorter stems, reduced biomass, increased tissue rigidity, delayed flowering, and reduced seed yield in both accessions. Among changes in cell wall‐related features, a substantial increase in lignin content and pectin methylesterase activity was most notable. Mechanical stimulation also reduced the enzymatic sugar release from the cell wall, thus increasing biomass recalcitrance. Notably, treatments had a distinct and opposite effect on vascular bundle area in the two accessions, suggesting genetic variation in modulating these responses to mechanical stimulation. Our findings highlight that exposure of grasses to mechanical stimulation is a relevant environmental factor affecting multiple traits important for their utilization in food, feed, and bioenergy applications.Mechanical stimulation, including exposure to wind, is a common environmental variable for plants. However, knowledge about the morphogenetic response of the grasses (Poaceae) to mechanical stimulation and impact on relevant agronomic traits is very limited. Two natural accessions of  Brachypodium distachyon induces a range of changes, several varying quantitatively between accessions, from the cellular and tissue level to whole plant morphology with important implications for fitness, productivity, and quality‐related traits.We show that mechanical stimulation of the grass model  Grasses are widely cultivated for their grains and provide a large part of the diet for most people on Earth , ferulic acid (FA), and Brachypodium distachyon (Brachypodium), a model plant for cereal crops and forage and bioenergy grasses . Vernalization was initiated 14 days after germination and lasted for 7 weeks, with 16‐hr day length at 5°C. After vernalization, plants were grown in the greenhouse with conditions as mentioned above. Plants at the same developmental stage (three tillers) were selected for stress experiments, with 20 biological replicates for each treatment.2.2−1 as measured with an anemometer . In the natural environment, the average wind speed 10–20 cm above the ground is 2–3 m s−1 , and plants were rotated 180° daily.For wind treatment (WT), plants were placed in front of a velocity fan  at a mean distance of 1.5 m where the wind speed reaches 2–3 m sFor MT, plants were flexed twice a day for 3 min (first at 8 a.m. and second at 6 p.m.) at three quarters of the mean plant height by the rapid front to back movement of a stick all‐around each stem. Each treatment consisted of 40 flexures, so at the end of the day, plants were flexed 80 times—40 times in each direction.Control plants were kept in calm conditions, without fan‐induced air movement or MT.Bd21 treatments were initiated 1 day after the vernalization process, whereas the treatments for ABR6 were initiated 3 weeks after the vernalization process. The reason for this was to synchronize the developmental stage between the two accessions at the start of the treatments: stem elongation in ABR6 only started 3 weeks after the transfer from the cold room to the greenhouse environment, whereas Bd21 stem elongation began immediately after the vernalization process.2.3n = 5). Seeds were harvested from basal florets of spikelets from the main tiller, and the lemma and palea were removed before weighing. For seed yield and total seed number, all seeds from the plant were collected (n = 5). Seed‐based measurements were based on Boden et al. (Plant growth and development parameters were taken every 2 days. At the end of the experiment (after 14 days), more detailed measurements were taken, including for tiller number (main stem and tillers), node and leaf number, stem length, internode length, and stem diameter. Flowering time was assessed from the first day of the treatments. Additionally, five plants per treatment for both accessions were left to reach full maturity, and measurements were taken for aboveground biomass yield (based on dry weight after oven drying to constant mass at 70°C) and seed related parameters. Average single seed weight was calculated from the weight of five seeds from each replicate plant  of the first four rows of cells above the bundle sheath of a VB  phloroglucinol in 75% EtOH for 5 min in darkness. The stained sections were flooded with a few drops of 12 N HCl and mounted on glass slides with 30% glycerol. Samples were immediately observed on a Leica LMD6000 microscope and images were captured.2.6For compositional analysis, stem material from five plants per each treatment (both accessions) was harvested and pooled. Material from three independent experiments was used as biological replicates. For enzyme‐linked immunosorbent assays (ELISAs) stem material from three plants per treatment from one independent experiment was pooled. Lignocellulosic biomass was prepared according to the NREL LAP “Preparation of samples for compositional analysis”  and Novozyme 188  was added to 10 mg of AIR at a 4:1 ratio. Per sample, 997 μl of KOAc buffer at 0.025 M (pH = 5.6), 2.4 μl of Celluclast, and 0.6 μl of Novozyme 188, with 0.04% (w/v) sodium azide, were used. Samples were incubated for 48 hr in a shaking incubator set at 50°C and 150 rpm and diluted by adding 9 ml of deionized water (1:10), centrifuged, and the supernatant was collected. Samples were further diluted by taking 100 μl of 1:10 diluted samples and adding 900 μl of distilled water. Before analysis, 400 μl of diluted samples was transferred into 0.45 μm nylon filter vials  and analysed by HPAEC‐PAD on an ICS‐5000 ion chromatography system using the same conditions as described for the “Determination of monosaccharide content,” see . From th2.10p‐CA and FA in AIR was determined by using an alkaline saponification method as described by Buanafina, Langdon, Hauck, Dalton, and Morris  and from the second internode collected from plants immediate after the treatments (n = 10). Tests followed the procedure as described by Anten, von Wettberg, Pawlowski, and Huber (Three‐point bending tests were performed on stem sections (2.5 cm long and a diameter of 0.7–1 mm), cut from the middle of the second and third internodes of fully mature plants  in the two accessions. For Bd21, WT and MT reduced the length of the main stem to less than half that of untreated control plants . Significant shortening (P ≤ .05) was observed for internodes 3–5 (IN3–5) in Bd21 after both stresses, whereas no significant changes were noted for the first  and second  internode. For ABR6, all internodes after both treatments were significantly (P ≤ .05) reduced in length compared with those of control plants (Table P ≤ .05) of the aboveground biomass for the two accessions (Table P = .580) and ABR6 (P = .899), and number of leaves . No consistent differences in stem diameter were found in either accession after mechanical stimulation (data not shown).Both treatments resulted in a significant shortening of the main stem  decrease of about 25% in the area of epidermal cells of ABR6 as compared with controls. The epidermal area of Bd21 was not significantly affected by either treatment  by more than 30% compared with control, whereas MT had no effect. Treatments had no significant effect on the cortex area of ABR6 compared with control, although there was a significant difference between the treatments  decrease in the area of both inner and outer VBs in Bd21, leading to an overall decrease of ~20% in the total VB area (Table P ≤ .05) the area of the interfascicular region by 5% in Bd21, these plants developed significantly (P ≤ .05) more pith area (>30% increase) as compared with control Bd21 plants . Both treatments had a strong positive effect on the VB cross‐sectional area of ABR6, whereas Bd21 responded negatively. Thus, MT resulted in a significant (ea Table . After Wts Table .P ≤ .05) increased the area of the inner VB  and the outer VB  compared with control plants , outer (P = .171), and total number of VB (P = .140) in response to treatments. No significant differences in inner (P = .959), outer (P = .142), and total number of VB (P = .194) in ABR6 plants were found  and ABR6 (P = .223), the cell walls of these same cells were significantly (P ≤ .05) thicker, especially after WT with an increase of 19% and 14% in Bd21 and ABR6, respectively, compared with controls. Together, these results show that mechanical stimulation by wind or MT leads to altered cellular anatomy of Brachypodium stems with notable genotypic differences in terms of treatment effects, in particular for VB area.Although cross‐sectional cell size, measured for the first four rows of cells above the bundle sheath , showed 3.4As a first step to examine if the treatments impacted cell wall‐related characteristics, stem cross sections were stained with phloroglucinol for lignin. The staining was clearly more intense for stem sections of both accessions after WT and MT as compared with control stem sections Figure a. DifferP ≤ .05) after both treatments in both accessions compared with control plants  differences in the content of both phenolics in Bd21 and ABR6 . FA content decreased slightly after both treatments in ABR6. In Bd21, MT also induced a decrease in FA content (11% decrease). However, WT resulted in an 11% increase compared with Bd21 control plants  and three (IN3) from fully mature stems of Brachypodium plants exposed to the various treatments were evaluated with a 3‐point bending test. The obtained values for Young's modulus for Bd21 controls were similar to those previously reported for senesced Brachypodium Bd21 stem segments  differences in the enzymatic release of the three main cell wall sugars after both treatments for the two accessions  reduction was observed in ABR6 after WT (10% reduction) and WT  is consistent with findings from dicots after wind stress  in stem cell wall lignin content upon mechanical stimulation, particularly in the interfascicular tissue and cortex. These findings suggest a correlation between the increase in lignin and increased Young's moduli of internodes. Lignin associated structural reinforcement may result in increased tensile strength  for both genotypes.Figure S4A Comparison of LM19 immuno‐localisation results between treatments in ABR6.Figure S4B Comparison of JIM7 immuno‐localisation results between treatments in Bd21.Figure S4C Comparison of LM5 immuno‐localisation results between treatments in Bd21.Figure S4D Comparison of LM5 immuno‐localisation results between treatments in ABR6.Figure S4E Comparison of LM13 immuno‐localisation results between treatments in Bd21.Figure S5 PME activity in leaves of Bd21 and ABR6 after the three treatments .Figure S6 Mechanical properties of fresh stem material.Figure S7 Total sugar release from enzymatic hydrolysis of stem cell wall material after the treatments.Table S1 Internode length after the three treatments  in Bd21 and ABR6.Methods S1 Detailed description of methods.Methods S2 Fixation, embedding, sectioning, and immuno‐localisation of Brachypodium stems.Click here for additional data file.


Oreochromis niloticus and water collected from the polluted Yamuna River, Agra, India. The heavy metals in water, recorded as follows: Fe > Mn > Zn > Cu > Ni > Cr > Cd and all were found to be above the prescribed limits. According to metal pollution index, exposed muscle (49.86), kidney (47.68) and liver (45.26) have been recorded to have higher bioaccumulation. The blood biochemical analysis of exposed O. niloticus indicated significant increase in activities of aspartate aminotransferase (+ 343.5%), alkaline phosphatase (+ 673.6%), alanine aminotransferase (+ 309.1%), and creatinine (+ 494.3%) over the reference. However, a significant decrease in albumin (A): globulins (G) ratio (− 87.86%) was observed. Similarly, the exposed fish also showed significant increase in total leucocyte count (+ 121%), differential leucocyte count, respiratory burst (+ 1175%), and nitric oxide synthase (+ 420%). The histological examination of liver and kidney showed tissue injury. Moreover, micronuclei (0.95%), kidney shaped nuclei (1.2%), and lobed nuclei (0.6%) along with DNA damage in the form of mean tail length in the liver (20.7 µm) and kidney (16.5 µm) was observed in the exposed O. niloticus. Potential health risk assessments based on estimated daily intake, target hazard quotient, hazard index, and target cancer risk indicated health risks associated with the consumption of these contaminated fishes. In conclusion, the present study showed that exposure to heavy metals contaminated water can alter immunological response; induce histopathological alterations and DNA damage in the studied fish. The consumption of this contaminated water or fish could have serious impact on human health.The current study was carried out on dominant fish  Heavy metals are discharged into the environment after used in industries, factories, domestic wastes and leakage from the dump sites3. These metals reach to the environment such as air, water, soil and food in different forms. In spite of their low concentrations in the different surface waters, they show high bioaccumulation in fishes and other fauna and flora of water bodies4. Hence, fish is good experimental model to assess the adverse effects of heavy metals. Turan et al.5 took Clarias gariepinus as a bioindicator of Orontes River, Turkey to highlight the degraded quality of water and adverse effects of heavy metals  in terms of bioaccumulation, oxidative stress and genotoxicity. Ahmed et al.6 studied the bioaccumulation of heavy metals  in different species of fish Apocryptes bato, Pampus chinensis, Liza parsia, Mugil cephalus, Hyporhamphus limbatus, and Tenualosa toil, since they all are consumed by locals of Bangladeshi population and reported six-times higher non-carcinogenic risk in children. Maurya et al.7 studied bioaccumulation and human health risk due to Zn, Pb, Cu, Cd, and Cr pollution in Cirrhinus mrigala, Cirrhinus reba, Catla catla, Labeo rohita, Crossocheilus latius, Clupisoma garua, and Mystus tengara collected from Ganga river basin.Pollutants  are released into the environment by anthropogenic activities. Humans use water bodies as sink for all their waste disposals, due to which quality of aquatic ecosystem is degrading day by day. This results in to less diversity of aquatic species and low quality of food from these resourcesOreochromis niloticus (nile tilapia) and studied several important enzymes of liver and kidney such as aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT) and creatinine (CK). These enzymes get released into the circulation as a result of some pathologies8. Their innate immunity which is considered as the main defense system of fish was also studied9. Few authors also considered, fishes as the important model for non-specific/innate immunity and ecotoxicology10. Immuno-competence is indispensable for the maintenance of the complete health of living being and is immensely attuned to toxins, like heavy metals11. Researchers also suggested that damage of innate immunity may be more remarkable in fish compared to mammals9. Respiratory burst assay is as an indicator of the innate immune system which releases cytokines and inflammatory response in fish. During respiratory burst process, the phagocytes and leukocytes of pathogens increase their oxygen consumption with the help of NADPH oxidase enzymes and generate various reactive oxygen specie such as singlet oxygen (1O2), superoxide anion radical (O2), hydroxyl radical (OH−), and hydrogen peroxide (H2O2) to be used as a defense mechanism against toxic agents12. In addition, the nitric oxide synthase (NOS), the total leukocyte count (TLC), the differential leukocyte count (DLC), and the globulin concentrations are also served as good immunological index12. The heavy metals are known to cause histopathology and genotoxicity. Histopathology of the target organs such as liver and kidney could give clear picture of toxicity of the heavy metals. Among genotoxicity assays micronuclei and single cell gel electrophoresis (SCGE) are the rapid and more reliable tools for the ecotoxicological evaluations14. Besides, some populations, chiefly those who take large amounts of fishes, can accumulate concentrations which are deleterious to their health as well as their children15. The present study aims to discuss the toxicities of heavy metals on the marker enzymes, immunotoxicity, histopathology and genotoxicity in freshwater tilapia fish, Oreochromis niloticus. The human health risk assessment such as estimated daily intake (EDI), non-carcinogenic (THQ) and carcinogenic risk (TR) and hazard index (HI) are also calculated.The present investigation chose most abundant fish O. niloticus) such as temperature (27.5 °C), pH (7.4) and D.O (6.3 mg/L) were found to be within ideal standards of Bureau of Indian Standards (BIS)16 and WHO (adapted from UNEPGEMS)17. On the other hand, the heavy metal proportion was in the order of Fe (48 mg/L) > Mn (19 mg/L) > Zn (13 mg/L) > Ni (11.9 mg/L) > Cu (7 mg/L) > Cr (3.5 mg/L) and all were found to be beyond the prescribed standards of BIS and WHO17. At reference site, the temperature (27 °C), pH (7.5), D.O (6.5 mg/L) were also found to be ideal. Furthermore, the heavy metals concentration were within the prescribed limits and were present as follows: Fe (5.2 mg/L) > Zn (3.9 mg/L) > Cu (3.6 mg/L) > Mn (0.02 mg/L). While Ni and Cr were found to be below the detection limits of atomic absorption spectrophotometer. The bioaccumulation was studied in muscle, liver, and kidney of O. niloticus , ALT (+ 309.1%), AST (+ 343.5%) and CK (+ 494.3%) activities were found to be comparatively higher in exposed O. niloticus as compared to reference fish  and excessive globulin (+ 157.22) amount was observed in the exposed fish compared to reference O. niloticus, consequently leading to lower A:G ratio in exposed O. niloticus (0.25) than reference O. niloticus (2.06)  Table . Corrobo31. Leucocytes represent a major component of body’s defense against foreign bodies, infections, and toxins. A significant increase in TLC (+ 121.08) level was observed as compared to reference O. niloticus , lymphocytes (+ 112.12%), eosinophils (+ 394.11%) and monocytes (+ 70.73) were found to be significantly higher when compared to the reference fish  in exposed O. niloticus than reference fish indicates heightened phagocytic activity to counter the toxic agents (Table 36. A significant higher level of NOS (+ 420%) was also observed in exposed O. niloticus than reference group  and kidney-shaped nuclei (1.2%) while the least frequent deformity was lobed nuclei (0.6%) as compared to reference fish  respectively . The percent change over reference in liver and kidney were + 475% and + 251% respectively. Furthermore, it shows liver as the target organ for genotoxicity. Besides, this also clearly indicates that these metals bind to the DNA and damage it. This can also be co-related with the generation of reactive oxygen species due to heavy metals which cause oxidative stress and in turn leading to lesions in DNA. Some studies also reported the higher DNA damage in different fish species such as C. punctatus, O. niloticus, C. gariepinus, L. cephalus due to the exposure of heavy metals45. It has been reported that heavy metals come in contact with DNA and form adducts by intercalating or covalently binding with the DNA molecule. These metals generate ROS which could also lead to single and double strand breaks47.The SCGE assay is fast and sensitive technique for assessing the genotoxicity in terms of DNA damage caused by environmental pollutants. DNA plays crucial role in various life processes because it carries genetic information. Figure https://www.epa.gov/risk_assessment/health-risk.htm). In the present investigation parameters used for human health risk assessment were EDI, THQ, HI, and TR via consumption of fish O. niloticus . In this study, EDI calculations showed multi-fold higher values than the reference concentration. THQ values were highest for Cr (4190.78 × 10–6) followed by Cu > Ni > Zn > Fe > Mn for both adult male and female persons. Furthermore, higher HI was recorded for females (8485.68 × 10–6) than males (7443.54 × 10–6) /RfD was equal to or less than the its RfD51. If it is > 1–5 times the RfD then low risk, if > 5–10 times the RfD then moderate risk, but if > 10 times the RfD then the higher risk. Here, for all the heavy metals this ratio was found to be more than 20 times and up to thousand times higher, pointing possible health hazard associated to the public. Since, fish fillet contain all the inspected heavy metals, so hazard index (HI) must be calculated which is numerically equal to addition of all the THQs. If HI value is greater than 1, means it is an alarm for public health safety53. Fortunately, HI values do not raise concern in the present investigation. As per USEPA, criteria Cr and Ni are among the potent carcinogens list54. In the current piece of work, both male and females were found to be more susceptible to carcinogenic risk posed by Cr than Ni 51 categorizes TR as follows, if TR ≤ 10−6, then risk is low; if 10−4 to 10−3 then moderate; if 10−3 to 10−1 then high; if ≥ 10−1 then very high. Likewise, THQ, the TR is also not a specific evaluation of expected cancers. Rather, the upper limit of the likelihood that the individuals may have cancer sometimes in his/her lifetime if the subsequent exposure to that chemical take place51.As stated by United State Environmental Protection Agency (USEPA), the human health risk assessment is the process to estimate the nature and probability of harmful health effects of chemicals in contaminated environmental media, now or in future to humans  were captured from Yamuna river  [27° 11′2.59″N and 78°1′47.58″E]. Another group of fish (n = 18) were collected from reference water body, Agra, which has no visible point and nonpoint source of pollution. Body length and weight of both groups (polluted and reference) of fish’s were 13.9 ± 1.6 cm, 48 ± 1.4 g and 15.4 ± 2.2 cm and 56 ± 1.5 g respectively. Muscle, liver, and kidney of both groups were acid digested as per the method described by Ref.3 for measurement of heavy metals . Metal pollution index (MPI) was calculated as below mentioned in the equation45:1, Cm2 ….. up to Cmn are the concentrations of different metals in the sample. Heavy metals in water were determined by HNO3 acid digestion method via Atomic Absorption Spectrometer55. Moreover, dissolved oxygen, temperature, conductivity, and pH were measured with the help of digital meters.Fishes were anesthetized in MS-222 (10%), and blood samples were obtained by cardiac puncture for enzymatic assays such as alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK). The said enzymatic analysis was performed by ready to use kits  following instructions of the manufacturer.56 with slight modifications and has globulin concentrations after albumin reduction from the total proteins. Albumin to globulin (A:G) ratio was also computed.Albumin protein was assayed spectrophotometrically with the help of commercial kit . Total protein was determined as per the method described by Bradford3 mm−3) was counted with the help of hemocytometer. Blood smear was made and stained with giemsa for DLC counting.TLC  and were studied for histological changes in exposed tissue as compared to the reference.Liver and kidney of both groups of fish were removed and after washing in normal saline, fixed in Bouin's fluid as specified by Javed et al.14. Smeared slides were fixed in 100% methanol, dried, and the stained with giemsa for around 10 min. To calculate the frequencies of different nuclear abnormalities, 1000 erythrocytes were scored from both groups.Blood smear was made to study micronuclei (MN) and other nuclear abnormalities in erythrocytes like lobed nuclei, kidney shaped nuclei by the method mentioned in Ahmad and Ahmad57 with some modification. Liver and kidney slides were scored by using Komet 5.5 imaging system attached with Olympus fluorescent microscope (CX41). DNA damage in terms of migrated tail lengths were measured as the distance between the center of the head and the end of migrated tail in µm.DNA damage was measured by single cell gel electrophoresis (SCGE). The SCGE was performed under alkaline condition following the protocol of Singh et al.−3 kg/day), Bw is an average body weight for Indian adult male and female are 57 kg and 50 kg respectively58.59.https://countryeconomy.com/demography/life-expectancy/india). RfD is reference dose of metals (mg/kg/day) (USEPA54) HI was calculated by addition of all THQs (USEPA59.−1. ATc is averaging time for carcinogens (365 days/year × 67 years), because average life expectancy for Indians .TR reveals the carcinogenic risk, dimensionless and was determined as per USEPA region III risk-based concentration table54 so, TR values for Cr and Ni only was calculated  Ingested dose of pollutant is equal to the absorbed dose60.(b) Cooking has no effect on pollutantsAll the parameters were analyzed in duplicates and values are reported as mean ± standard error mean. Statistical analysis of the data had been performed with Student’s t-test, two-way ANOVA and Duncan’s Multiple Range Test (DMRT) with the help of SPSS software.All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. It was permitted by Ministry of Environment and Forests, Government of India under registration no. 714/02/a/CPCSEA issued and approved by the institutional ethical committee of Department of Biochemistry, Aligarh Muslim University, Aligarh, India.


Breast asymmetry can be congenital or developmental, however a tumorous growth may be the cause of this condition after puberty. A 19-year-old female presented with a slowly developing breast asymmetry pre-operatively diagnosed as Pseudoangiomatous Stromal Hyperplasia (PASH). The patient underwent tumour excision with breast gland remodelling. Postoperative course was uneventful. Breast asymmetry is characterised by differences in the size, shape or position of the breasts . It may A 19-year-old female reported breast asymmetry slowly developing since the beginning of breast development at the age of 13. During a 6-year period, the left breast gradually increased in size and was misdiagnosed as a developmental asymmetry by her paediatrician. Besides aesthetic and emotional problems, the patient had no pain in the breasts or showed any discharge from the nipple.Upon physical inspection, the left breast appeared larger but with symmetrical inframammary folds (IMF) and there were no signs of a breast malignancy such as dermal pitting, flaking or erythema, or presence of a sunken or inverted nipple .Left vs right breast measurements were: Sternal to nipple distance 27.5 vs 21 cm, IMF to nipple distance 12 vs 9.5 cm, NAC diameters 7 vs 4 cm. Breast volume was calculated with the BREAST-V application, was 693 cc for the left and 336 cc for the right breast [No palpable masses were identified on the right breast, while on the left breast a large firm mass was presently occupying all quadrants. Endocrinology consultation was negative since blood analysis revealed prolactin levels 24.8 ng/ml at time 0, 21.3 ng/ml at 15′ and 18.3 ng/ml at 30′ (range 2–29 ng/ml), TSH was 1.670 μIU/ml (range 0.4–4 μIU/ml), growth hormone 0.31 ng/ml (1–14 ng/ml) and the patient had a normal menstruation cycle. Ultrasound showed a solid nodular mass of at least 10 cm diameter with compacted glandular characteristics, contrast-enhanced MRI imaging confirmed a coarse formation of 90 × 70 mm, capsulated, with uneven uptake after contrast enhancer, likely a benign lesion, BRADS 3. Core needle biopsy (CNB) was carried out and resulted in the diagnosis of Pseudoangiomatous Stromal Hyperplasia (PASH).Under general anaesthesia, the patient underwent removal of the tumour with remodelling of the left breast by means of a Wise pattern skin reduction approach. The tumorous mass appeared well encapsulated and was easily dissected from the surrounding tissues . It was PASH, first described by Vuitch et al. , is a coIn most cases, PASH is an incidental histologic finding, although considered a rare condition, its prevalence is not well defined due to conflicting literature reports. Ibrahim et al.  demonstrIn this case, the PASH was of such size that it caused symptomatic breast asymmetry which was initially misdiagnosed as developmental breast asymmetry. This illustrates that a clinician should be aware that breast asymmetry might be caused by more uncommon conditions even when initial patient history and anamnesis would suggest otherwise. Therefore, in these patients, careful clinical examination and radiological examinations should always be performed for a correct diagnosis and preoperative planning that leads to tumour removal and achievement of aesthetically pleasant symmetrical results.


Dysregulation of circRNAs has been reported to be functionally associated with chronic obstructive pulmonary disease (COPD). The present investigation elucidated the potential role of CircRNA0001859 in regulating chronic obstructive pulmonary disease acute (COPD) and Acute Exacerbation of COPD (AECOPD).Mice model of COPD was established to screen and verify the dysregulated expression of CircRNA0001859. Fluorescence in situ hybridization (FISH) and quantitative real-time PCR (qRT-PCR) were carried out to detect the expression of CircRNA0001859. 38 stable COPD patients, 24 AECOPD patients, 57 COPD with lung cancer patients and 28 healthy person with age and sex matched to total patients were used for the present investigation.circRNA0001859 was downregulated in the lung tissue of mice after the three kinds of treatments (Cigarette smoke (CS)/NK alone or CS + NNK) for inducing COPD. FISH assay verified the downregulation of circRNA0001859 both in the mice lung and human bronchial epithelial cell of COPD model. Furthermore,, the level of circRNA0001859 was also downregulated in the peripheral blood of COPD and lung cancer patients. CircRNA0001859 might act as a diagnostic and prognostic biomarker for the treatment of in COPD and AECOPD with Are under the receiver operating characteristic curve (ROC curve) (AUC) of 0.7433 and 0.8717, respectively.We explored a novel circRNA0001859, which might act as a potential therapeutic biomarker for the treatment of COPD and AECOPD. Chronic obstructive pulmonary disease (COPD), a heterogeneous disorder is estimated to be the third leading cause of chronic morbidity and mortality worldwide . COPD isCircular RNAs (CircRNAs) are evolutionarily conserved noncoding RNAs; have unique closed loop structure formed by reverse splicing and subsequent removal of 5′ cap and 3′ poly-A tail. They have higher stability than the linear RNAs as they are not digested by RNase R exonuclease . CircRNACircRNAs have been proved to be involved in respiratory diseases, such as lung cancer acute respiratory distress syndrome, pulmonary hypertension pulmonary tuberculosis and silicosis , 14. AlsIn the present investigation, we have explored the potential role of a novel circRNA, circ0001859 in COPD and AECOPD. Our results have demonstrated that circ0001859 might act as a potential biomarker for diagnostic and therapeutic target to treat COPD and AECOPD.This study was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University, China. We have taken written informed consent from participants prior to their enrollment in the study.Animals, Diet and Study Groups 28 healthy persons, 38 stable COPD patients, 24 AECOPD patients,57 COPD complicating lung cancer patients were recruited from the First Affiliated Hospital, College of Medicine, Zhejiang University between December 1, 2014 to March 31, 2016. These groups were further classified into the following :1. Healthy controls (HCs): HCs were selected with age, gender, and body mass index (BMI) matched to total COPD patients. HCs with any other co-existing diseases, such as infection, lung diseases, renal or hepatic dysfunction and history of severe infection, solid tumor, hematological diseases, and autoimmune diseases were excluded from this study.2. AECOPD patients: The inclusion criteria of AECOPD patients were: age > 40 years; diagnosed with COPD according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria, USA; increased dyspnea and decreased performance in daily activities, altered amount and color of sputum, increased coughing, pyrexia and/or altered mental status.The exclusion criteria were: Patients with complicated asthma, lung cancer, or other relevant lung diseases; history of severe infection, malignant tumors, and autoimmune diseases were excluded.3. Stable COPD patients: The inclusion criteria of stable COPD patients were as follows: age > 40 years; diagnosed with COPD according to GOLD criteria; without acute exacerbation in the last 6 months. The exclusion criteria were similar to AECOPD patients.4. Lung cancer patients complicated with COPD: lung cancer patients complicated with COPD symptoms were those pathologically confirmed and diagnosed as lung cancer, meantime, diagnosed as COPD before.Cigarette was combusted through a 50 mL syringe apparatus. CSE was obtained by combustion of two filter less cigarettes per trial and subsequently bubbled via bubbling the smoke of through 10 mL of Dulbecco’s Modified Eagle Medium (DMEM) for 2 min. The solution was neutralized with 1 M NaOH (pH 7.4) and then sterilized through a glass ball filter of pore size, 0.22 μm. The smoked medium was considered 100% CSE and diluted with Roswell Park Memorial Institute (RPMI 1640) medium  to obtain the desired concentration .2 and saturated humidity.Human bronchial epithelial cell line (BEAS-2B) was purchased by American Type Culture Collection (ATCC), USA. BEAS-2B cells were cultured in RPMI 1640 medium  containing 10% fetal bovine serum  supplemented with100 mg/L streptomycin, and 100 U/mL penicillin. The cells were incubated at 37 °C under 5% COA total of 40 adult male C57BL/6 mice  were obtained from the Animal Center, of Zhejiang University and randomly divided into 4 groups. Each group has 10 mice. They were housed at 24 ± 1 °C with 50% relative humidity under 12 h light/dark cycle and were allowed to standard mouse food and water in environmentally controlled pathogen-free conditions during all the experiments. All protocols were approved by the Animal Experimental Ethics Committee, of Zhejiang University. The mice in cigarette smoke (CS) group were exposed to the smoke of four cigarettes without filter via a TE-10 smoking machine as previously described . The mice were placed in a 60 × 57 × 100 cm fume box and exposed to CS four time per day with 30 min intervals. Mice were exposed to the smoke 5 days/week for 14 weeks . The micThe levels of cytokines, including malondialdehyde (MDA), glutathione (GSH), Superoxide dismutase (SOD), Interleukin 10 (IL-10), Interleukin 6 (IL-6), Tumor necrosis factor alpha (TNF-a) in the bronchoalveolar lavage fluid, or cell supernatant were measured with the ELISA kits  according to the manufacturer’s instructions. Three independent biological repeats were used for each analysis. All samples were stored at − 80 °C before conducting the experiment.Bronchoalveolar lavage fluid experiment was performed on the left lung after ligating the right lung. Repeated lavage was performed with 0.4 mL phosphate-buffered saline and fluid was collected thrice. The recovery rate was above 80%. The lavage fluid was collected in 1.5 mL tubes (placed on ice). Absolute cell counts were determined using a MEK-7222 k automatic hematology analyzer . For the ELISA assay, the BALF solution was mixed well and centrifuged . The supernatant was aspirated into 1.5 mL tubes for the further research.PLY3211 lung function analysis system  was applied for lung function measurement of all the mice used in the present study before sacrifice. Mice were anesthetized with pentobarbital (40 mg/kg), tracheostomized, and then put into the body chamber of the system. The average breathing frequency was set at 150 breaths/min. Parameters of pulmonary function such as FEV0.1, Cydn and Raw were measured.The lung tissues were resected and fixed in 10% formalin for 24 h. Then, these tissues were transferred to 75% ethanol, embedded and sectioned into 5 μm thickness. The sections on glass slides were dried at 60 °C for 15 min, and then deparaffinized and stained with hematoxylin and eosin (HE) by incubating the tissue sections in Harris hematoxylin  followed by serial eosin . The sections were subjected to graded ethanol steps and finally, neutral gum sealing piece was performed.Peripheral blood samples of healthy controls and patients were collected and stored at room temperature for few minutes. Subsequently, samples were centrifuged at 3000 rpm, 4 °C for 15 min and stored at − 80 °C for further experiments. RNA from serum, HBE cells and mouse lung tissues was extracted using a TRIzol Plus RNA purification kit . The quality of RNA samples and small RNA fraction was checked via the Nanodrop spectrophotometer . The primer were as follows: circRNA0001859 forward: GCCATTGTAGAAGCTGGTGG, reverse: AGCTGCACTAGAGTCCCAAG; U6 forward:CGCTTCACGA ATTTGCGTGTCAT, reverse: GCTTCGGCAGCACATATACTAAAAT.The sections were inactivated with 3% hydrogen peroxide for 10 min at room temperature. The sections were blocked with 5% bovine serum albumin (BSA) for 20 min and incubated with anti-ki67 polyclonal antibody  at 4 °C overnight. Then, the sections were incubated with the Horseradish Peroxide (HRP)-conjugated secondary antibody at room temperature for 2 h. Then, the targets were visualized by diaminobenzidine  and counterstained with hematoxylin. Images of stained lung tissue sections were captured with an inverted fluorescence microscope  and analyzed with Image Pro Plus 6.0 software .After RNA extraction, the concentration of RNA was determined using NanoDrop system. Afterwards, the samples were then labeled using the miRCURY™ Hy3™/Hy5™ Power Labeling Kit  and hybridized with the miRCURY™ LNA MicroRNA Arrays . Following the washing steps, the slides were scanned using the Axon GenePix 4000B microarray scanner .RNA extraction were performed with TRIzol reagent , precipitated with isopropanol, washed with 75% ethanol and dissolved in RNase free water. Reverse transcription were performed with 1 μg RNA using the cDNA transcription kit . 20 ng cDNA was used for qRT-PCR to validate the expression of relative mRNA expressions; it was determined by using SYBR green mix . The relative gene expression was evaluated based on the 2–ΔΔCt method.5 cells were seed onto autoclaved glass slides and cultured for 24 h. After fixing with 4% paraformaldehyde for 20 min followed by permeabilization with 0.5% Triton X-100 for 10 min, the cells were cultured at 37 °C overnight. Finally, the slides were incubated with DAPI to stain cell nucleus and observed them under a fluorescence microscope .Alexa Fluor 555-labeled circRNA0001859 probes were designed and synthesized by RiboBio . FISH experiment was carried out with a FISH Kit . 1 × 10P < 0.05 was considered as statistical significance. Three independent biological repeats were used for each analysis.SPSS 20.0 software  were used to analyze all data for statistical significance. All the data are presented as the means ± SD. Unpaired and paired t-tests were used for the statistical analysis. A receiver operating characteristi ccurve (ROC) was drawn to assess diagnostic accuracy. Spearman’s correlation analysis was used to evaluate the correlation between circRNA0001859 and risk factors. P < .001) , 38 COPD, 24 AECOPD and 57 COPD patients with lung cancer were took part in the present study  treatment lasting for 14 weeks. Notably, tobacco carcinogen, NNK was used to establish the transformation from COPD to lung cancer (TFCLC) model for investigating the potential role of circRNA0001859 in TFCLC. Firstly, we verified the sensitivity of our model system using several experiments. HE staining showed that CS/NNK alone treatments for 1 month significantly increase the alveolar volume of mice. Increase in the rupture of large, alveolar septum and infiltration of inflammatory cell, indicated that CS/NNK alone might induce early COPD models. CS + NNK treatment of mice for 1 month dramatically increased the alveolar volume, severe alveolar fracture and invasion of more inflammatory cells Fig. a-c. FinaAs circRNA0001859 was significantly down-regulated in the lung of mice COPD therefore, we speculated whether it was dysregulated in human COPD or not. We treated human bronchial epithelial with CS/NNK or CSE + NNK to mimic human COPD in vitro; higher release of inflammatory cytokines, such as IL6 and TNF-α Fig. a, b. qRTCircRNA0001859 was downregulated in serum obtained from in COPD, AECOPD and COPD patients with lung cancer and mice serum CS or NNK alone and CS + NNK treatments.CircRNAs are more stable than linear RNAs and therefore can be used as stable prognostic and diagnostic markers. The dysregulated expression of circRNA0001859 in mouse and bronchial epithelial cell model of COPD suggested us whether this circRNA expression was altered in serum. Next, we evaluated the level of circRNA0001859 of serum obtained from HC, COPD, AECOPD and COPD complicating lung cancer patients. Interestingly, circRNA0001859 was downregulated in the serum from in COPD, AECOPD and COPD patients with lung cancer as compared to the healthy controls Fig. a. FurtheProportions of serum circRNA0001859 in HCs, stable COPD and AECOPD.We analyzed the correlation between circRNA0001859 expression and the factor of lung function FEV1% in COPD and AECOPD patients. The results indicated a positive correlation between circRNA0001859 expression and FEV1% both in the COPD and AECOPD patients Fig. a, b. On A Number of biomacromolecules have been dig out that has the possibility to be the diagnostic molecular such as miR-125b, miR-146a/b, mir-190a-5p and miR-210 –23. Howe4 (leukotriene B4) and IL (nterleukin)-8 [It was demonstrated that CS exposure alters systemic mechanistic pathways, such as oxidative stress, metabolic alterations, autonomic nervous system dysfunctions and hormonal changes . Nicotineukin)-8 .In the present work, we succesfully established the COPD and AECOPD model. With microarray, FISH and qRT-PCR analyses, we found the dysregulated expression of circ0001859 in COPD. Considering the potential effect of circRNA0001859 in COPD progression, we speculated whether circRNA0001859 could be a prognostic molecule. The markers derived from lung tissue are not routinely available for clinical disease monitoring, whereas blood is readily accessible. Thus, we evaluated the level of circRNA0001859 in the blood of mice and human. Consistently, we found that circRNA0001859 is decreased in the blood of COPD mice and patients compare to normal and healthy controls. In addition, the level of circRNA0001859 was lower in CS + NNK treated mice in comparison with that in CS treatment alone group. These findings indicated the critical role of circRNA0001859 in diagnose of the COPD.Previously, using functional analysis, a total of 2132 circRNAs and 2734 mRNAs are dysregulated in COPD patients as compared to the normal controls; the ten most upregulated circRNAs in COPD patients are has_circ_0007294, has_circ_0004903, has_circ_0003003, has_circ_000157, has_circ_0001853, has_circ_0043926, has_circ_0002136, has_circ_0026466, has_circ_0051433, has_circ_0000364 and the ten most downregulated circRNAs are has_circ_000074, has_circ_0025453, has_circ_0056158, has_circ_0075043, has_circ_0077995, has_circ_0051785, has_circ_0031660, has_circ_0001744, has_circ_0043144, has_circ_0064900 [Here, the normal bronchial epithelial cells and exposed them to cigarette smoke and other chemicals. These cells may behave differently from bronchial epithelial cells obtained from COPD patients. It is the limitation of this study.In conclusion, we have investigated the potential role of a novel circRNA, circ0001859 in regulating COPD and AECOPD. Three treatment groups, CS or NK alone and CS + NNK were given to human and mice lung cells. Interestingly, circ0001859 was found to be downregulated under all the three treatment groups and under CS + NNK, circ0001859 was more downregulated then the CS or NNK alone. Our investigation has indicated that circ0001859 might act as a potential prognostic and diagnostic biomarkers for treating COPD and AECOPD.


Direct‐acting antiviral agents (DAAs) for hepatitis C virus (HCV) infection have resulted in high rates of sustained virologic response (SVR) following 8 to 24 weeks of treatment. However, difficult‐to‐cure/cirrhotic patients typically require a longer treatment duration and less is known regarding the long‐term durability of SVR or effect on liver disease progression; to assess this, the IMPACT study followed patients for a 3‐year period after end of treatment.The Phase II, open‐label, nonrandomized IMPACT study assessed the efficacy, safety, and pharmacokinetics of the combination of three DAAs  in HCV genotype 1/4‐infected, treatment‐naïve/‐experienced cirrhotic patients with portal hypertension or decompensated liver disease. Patients from a single site in the United States were assigned to one of two groups by Child–Pugh (CP) score: CP A, CP score less than 7 and evidence of portal hypertension; CP B, CP score of 7 to 9. All patients received simeprevir 150 mg, daclatasvir 60 mg, and sofosbuvir 400 mg once‐daily for 12 weeks between September 2014 and August 2015. All 40 patients included in the study  achieved SVR 12 and 24 weeks after end of treatment, and the combination was well tolerated.All patients who reached the 3‐year follow‐up timepoint maintained SVR . CP scores and Model for End‐stage Liver Disease scores remained relatively stable, and mean FibroScan and FibroTest scores declined. No new safety signals were identified.http://ClinicalTrials.gov number: NCT02262728).In the IMPACT study, virologic response to simeprevir, sofosbuvir, and daclatasvir was durable over 3 years ( AEadverse eventCPChild–PughDAAdirect‐acting antiviral agentEOTend of treatmentGTgenotypeHCVhepatitis C virusITTintent‐to‐treatMELDModel for End‐stage Liver DiseaseQDonce dailyRAVresistance‐associated variantSAEserious adverse eventSVRsustained virologic responseSVR12sustained virologic response 12 weeks after end of treatment1In 2015, it was estimated that 71 million individuals worldwide had chronic hepatitis C virus (HCV) infection.Current guidelines recommend the use of interferon‐free combinations of direct‐acting antiviral agents (DAAs) for the treatment of HCV infection.Simeprevir, sofosbuvir, and daclatasvir are DAAs with non‐overlapping resistance profiles, different mechanisms of action, and different metabolic pathways that target chronic HCV infection.http://clinicaltrials.gov number: NCT02262728) was the first to assess the combination of simeprevir, sofosbuvir, and daclatasvir for 12 weeks in HCV GT1‐ or 4‐infected treatment‐naïve or ‐experienced patients with Child–Pugh (CP A) cirrhosis with portal hypertension, or decompensated liver disease (CP B), with a planned 5‐year follow‐up period.The Phase II IMPACT study , daclatasvir 60 mg QD, and sofosbuvir 400 mg QD for 12 weeks. For all patients, a posttreatment follow‐up phase was scheduled for a total period of 5 years, during which patients attended follow‐up visits every 6 months. As mentioned previously, the follow‐up period was subsequently shortened to 3 years.The study included both treatment‐naïve and interferon‐based (± ribavirin) HCV treatment‐experienced patients of at least 18 years of age with chronic HCV GT1‐ or 4‐infection and cirrhosis (defined as a FibroScan® score >14.5 kPa at screening). Liver disease was classified by CP score; CP A, score less than 7 with documented portal hypertension; CP B, score 7 to 9.2.2During the long‐term follow‐up period of the study, efficacy , safety, change in Model for End‐stage Liver Disease (MELD), CP, FibroScan, and FibroTest  scores were assessed.2.2.1Blood samples were collected at predefined time points during the long‐term follow‐up period, at the 1‐, 1.5‐, 2‐, 2.5‐, and 3‐year follow‐up visits, and plasma was subsequently isolated. RNA extraction and quantification of HCV RNA was performed in a central laboratory using the COBAS® AmpliPrep/COBAS® TaqMan® HCV Quantitative Test v2.0 . Further details of this methodology have been reported previously2.2.2Liver disease status was monitored by the assessment of CP, MELD, FibroTest, and FibroScan scores, which were assessed at screening or baseline and during follow‐up.2.2.3During the long‐term follow‐up phase, reporting of adverse events (AEs) was limited to all serious AEs (SAEs) only.2.3The objectives of the long‐term follow‐up phase were to assess the durability of SVR in the IMPACT study and the effect of treatment on liver disease progression.2.4Since this was an exploratory analysis within an exploratory study, no formal sample size calculation was performed; however, it was considered that a total sample size of 40 patients would be sufficient to explore the primary and secondary objectives, as reported previously.2.5The study was approved by IntegReview IRB, a regional Institutional Review Board in Austin, Texas, and met the principles of the Declaration of Helsinki. All patients provided written informed consent.33.1In total, 74 patients were screened for the IMPACT study. All of the 40 patients enrolled in the treatment phase  entered the long‐term follow‐up phase , with HCV GT1a infection (65% [26/40]), and a median age of 58.5 years. In the CP A group (n = 19), portal hypertension was diagnosed by the presence of upper gastrointestinal (GI) varices in all patients. A mean baseline MELD score of 10.1 was reported for the CP B group (n = 21), and 95% of these patients (20/21) had clinical features of decompensation . A full description of the patient characteristics has been reported previously.3.3All patients remaining in the study at the 3‐year follow‐up visit (15/15 in the CP A group and 18/18 in the CP B group) had maintained their SVR, and no late viral relapse was observed Figure . Figure Individual MELD scores decreased from baseline to the 3‐year follow‐up visit in 18/32 56%) patients Figure . The med6% patien3.4During the treatment phase, there were no deaths or AEs that led to treatment discontinuation.4The Phase II IMPACT study assessed the combination of simeprevir, sofosbuvir, and daclatasvir for 12 weeks in HCV GT1‐ or 4‐infected, treatment‐naïve, or ‐experienced patients with portal hypertension or decompensated liver disease. These three DAAs have different mechanisms of action and metabolic pathways, with non‐overlapping resistance profiles.During the long‐term follow‐up phase of this study, the sponsor decided not to continue their HCV clinical development program. This decision was not driven by a safety concern. Irrespective of this decision, the 3‐year follow‐up data analyzed here, provides insight into durability of response in patients with decompensated liver disease and, therefore, it was considered to be of significant relevance to the field.The SVR results from the final visit of the IMPACT study, 3 years after EOT, revealed that all patients remaining in the study (15/15 in the CP A group and 18/18 in the CP B group) maintained SVR. Despite the fact that simeprevir is no longer marketed, these results are encouraging because they suggest that combining DAAs with different mechanisms of action can lead to the achievement of long‐term SVR in difficult‐to‐cure patients.There have been few reports regarding the long‐term durability of SVR to DAA treatment. A recent study by Kozbial et al of 551 patients with advanced fibrosis, decompensated or compensated cirrhosis and SVR after interferon, and ribavirin‐free DAA therapy found that eradication of HCV was durable irrespective of the DAA combination used.The results of the IMPACT study also provide insight into the long‐term effects of such treatment regimens on liver function. The CP scores for the majority of patients in the CP A and CP B groups remained stable; where CP scores differed from baseline at the 3‐year follow‐up visit, more patients' CP scores improved rather than worsened by the 3‐year time point. In both CP A and CP B groups, mean change from baseline in MELD scores remained stable from baseline to the 3‐year time point, while individual and mean FibroScan and mean FibroTest scores generally decreased from baseline in both CP A and CP B groups. Individual FibroScan improvements in liver stiffness were most pronounced in those patients with the highest stiffness scores at baseline. This may be due to the regression of liver fibrosis or reductions in inflammation associated with SVR.In this study, four patients developed de novo HCC following SVR. A recent review article summarizing the results of 11 studies examining the incidence of HCC following DAA treatment reported a de novo incidence rate of 0% to 7.4%, with the authors commenting that their review does not suggest that there is a higher rate of de novo HCC occurrence or recurrence after DAA therapy.This was an exploratory study, and as such, there were several limitations, including the open‐label design and that the study was conducted at a single center. There was also a relatively small sample size, which only included patients with CP A and B stage of liver disease. In addition, the posttreatment follow‐up was shortened to 3 years, following the discontinuation of the sponsor's HCV clinical development program. At the time this study was conducted, the MELD score was used as a measure of change in liver function as it was the most suitable measurement for patients with CP A and B. The recently reported Albumin‐Bilirubin gradesIn conclusion, HCV eradication by triple DAA therapy provided durable SVR and a good clinical prognosis in HCV‐infected patients. To further assess the long‐term clinical benefit of achieving SVR in patients with advanced liver disease, studies involving greater patient numbers and longer durations of follow‐up would be required.Janssen Research & Development funded this study; contributed to the study design; participated in the collection, analysis, and interpretation of data; and participated in the preparation and approval to submit this manuscript.E.L. has attended Advisory Committees or Review Panels for AbbVie, Achillion Pharmaceuticals, Bristol‐Myers Squibb, Enanta Pharmaceuticals, Gilead Sciences, Janssen, Merck & Co., Novartis, Regulus, Santaris Pharmaceuticals, and Theravance; has received grant/research support from AbbVie, Achillion Pharmaceuticals, Bristol‐Myers Squibb, Enanta Pharmaceuticals, Gilead Sciences, GlaxoSmithKline, Janssen, Merck & Co., Roche, Salix, Santaris Pharmaceuticals, Tacere, and Theravance; and has performed speaking and teaching for AbbVie, Bristol‐Myers Squibb, Gilead Sciences, and Janssen. F.P. has attended Advisory Committees or Review Panels for Abbott/AbbVie, Achillion Pharmaceuticals, Boehringer Ingelheim, Bristol‐Myers Squibb, Genentech, Gilead Sciences, Idenix, Inhibitex, Janssen, Merck & Co., Novartis, Pfizer, Salix, and Vertex and has received grant/research support from AbbVie, Achillion Pharmaceuticals, Anadys, Boehringer Ingelheim, Bristol‐Myers Squibb, Genentech, Gilead Sciences, Idenix, Merck & Co., Novartis, Pharmasset, Salix, Tibotec/Janssen, and Vertex. J.A.G. has attended Advisory Committees or Review Panels for AbbVie, Bristol‐Myers Squibb, Gilead Sciences, Janssen, and Merck & Co. and has received grant/research support from AbbVie, Bristol‐Myers Squibb, Gilead Sciences, Merck & Co., and Tibotec/Janssen. M.B., G.B., A.V., L.V., and M.G. are employees of Janssen Pharmaceutica NV and shareholders of Johnson & Johnson. V.V.E. is an employee of Janssen Pharmaceutica NV. P.V.R. and D.L. are employees of Janssen Research & Development and shareholders of Johnson & Johnson. With the exception of Janssen Research & Development, for which contributions are detailed in the “Funding” section, all other listed companies had no involvement in the study design; collection, analysis, and interpretation of data; or preparation and approval to submit this manuscript.Conceptualization: Maria Beumont, Greet Beets, Ann Vandevoorde, Pieter Van Remoortere, Leen Vijgen, Veerle Van Eygen, and Mohamed GamilFormal Analysis: Eric Lawitz, Fred Poordad, Julio A. Gutierrez, Maria Beumont, Greet Beets, Ann Vandevoorde, Pieter Van Remoortere, Donghan Luo, Leen Vijgen, Veerle Van Eygen, and Mohamed GamilInvestigation: Eric Lawitz, Fred Poordad, Julio A. Gutierrez, Maria Beumont, Greet Beets, Ann Vandevoorde, Pieter Van Remoortere, Leen Vijgen, Veerle Van Eygen, and Mohamed GamilMethodology: Maria Beumont, Greet Beets, Ann Vandevoorde, Pieter Van Remoortere, Leen Vijgen, Veerle Van Eygen, and Mohamed GamilWriting ‐ Reviewing & Editing: Eric Lawitz, Fred Poordad, Julio A. Gutierrez, Maria Beumont, Greet Beets, Ann Vandevoorde, Pieter Van Remoortere, Donghan Luo, Leen Vijgen, Veerle Van Eygen, and Mohamed GamilAll authors have read and approved the final version of the manuscript.Dr Lawitz had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.Dr Lawitz affirms that this manuscript is an honest, accurate, and transparent account of the study being reported, that no important aspects of the study have been omitted, and that any discrepancies from the study as planned  have been explained.https://www.janssen.com/clinical-trials/transparency. As noted on this site, investigators and physicians can request access to the clinical study report and participant‐level data if these data will be used for scientific research that will advance medical knowledge and public health. Data can be requested immediately following publication, with no end date. Data requests will be evaluated by an independent review panel—the Yale Open Data Access (YODA) Project—and can be submitted at http://yoda.yale.edu.The data sharing policy of Janssen Pharmaceutical Companies of Johnson & Johnson is available at


The patient responded well to steroids, but required a combination of 4 anti‐seizure drugs to control his seizure activity.2A 14‐year‐old boy was admitted to our emergency department (ED) in status epilepticus. Four weeks previously, he was seen at a neurology department because of generalized convulsive status epilepticus (GCSE), myoclonus, and hallucinations. The seizure frequency was about once 10 days, and the onset duration varied from 20 minutes to 60 minutes. He was hospitalized, but no seizures occurred during the hospitalization. Magnetic resonance imaging (MRI) of the brain revealed no obvious abnormalities. Electroencephalogram (EEG) monitoring for 24 hours indicated no epilepsy discharge waves. He was discharged on sodium valproate for seizure control. Twenty‐four hours before admission to our ED, he was again seen at the other hospital for GCSE. He had experienced 4 episodes, with an onset duration that varied from 10 minutes to 20 minutes. Sodium valproate, carbamazepine, and diazepam were administrated; however, the status epilepticus was not suspended. He was thus transferred to our ED. This study was approved by the institutional review board (IRB) of the First People's Hospital of Lianyungang City. Written informed consent was obtained from the patient. Written informed consent was obtained from the patient's father for the publication of any potentially identifiable images or data included in this article.On examination in our ED, he was alert and his general examination was without abnormalities. Neurological evaluation only showed a bilateral Babinski sign, and he appeared to have normal cognitive development. He appeared to be of normal weight and height for his age, and his nutritional status appeared sufficient. He had no history of recent infections or flu‐like symptoms, injury, toxicosis, or academic achievement decline. He had a history of syncope several years prior, but the details of the event could not be obtained.Routine laboratory tests, including a complete blood count (CBC), erythrocyte sedimentation rate, platelet count, tests of kidney  and liver function , and protein, lactate, and ammonia levels were normal. Urine toxicology was unremarkable. However, his glucose was slightly below the lower limit of normal . Testing for specific antibodies associated with autoimmune encephalitis including N‐methyl‐D‐aspartate (NMDA) receptor antibody, voltage‐gated potassium channel (VGKC) antibody, anti‐leucine‐rich glioma‐inactivated (LGI1) antibody, anti‐contactin‐associated protein‐like 2 (Caspr2) antibody, anti‐Hu antibody, and anti‐CRMP5/CV2 antibody was negative.Thyroid function tests revealed a low thyroid‐stimulating hormone (TSH) level , but normal free triiodothyronine  and free thyroxin  levels. However, serum anti‐thyroid antibody levels were elevated. His anti‐TPO antibody level was 76.7 mIU/L (reference range: 0.25‐34 mIU/L), and his anti‐TG antibody level was 237.1 mIU/L (reference range: 0‐4 mIU/L).Cerebral computed tomography (CT) showed no signs of increased intracranial pressure, hemorrhage, or a space‐occupying lesion. MRI, including T1, T2, and diffusion‐weighted imaging (DWI) with apparent diffusion coefficient (ADC) mapping, and fluid‐attenuated inversion recovery (FLAIR) sequences, and contrast‐enhanced angiography of the brain also revealed no abnormalities, especially no signs of encephalitis. EEG showed a marked slowing of the background rhythm, suggestive of encephalopathy, and spike waves corresponding with seizure activity  refers to continued clinical or electrographic seizures after an adequate initial benzodiazepine dose, followed by a second‐line antiepileptic drug (AED). Non‐drug treatments can be considered for RSE, such as surgery, and neuromodulation treatment. The purpose of epilepsy surgery is to complete resection of an epileptogenic lesion to control seizures.4Hashimoto's encephalopathy is a rare disease that should be considered in patients with new‐onset seizures and RSE, and mental status changes when standard investigations are negative. Patients typically respond well to intravenous steroids and anti‐seizure drugs.All authors disclose that they have no conflicts of interest.


Atopic dermatitis (AD) is a chronic and recurrent inflammatory skin disease, affecting up to 10% to 20% of children and 3% of adults. Although allergen sensitization, skin barrier abnormalities and type 2 immune responses are involved, the exact molecular pathogenesis of AD remains unclear. MicroRNAs (miRNAs) are short (19‐25 nucleotides) single‐stranded RNA molecules that regulate gene expression at post‐transcriptional level and are implicated in the pathogenesis of many inflammatory and immunological skin disorders. This systematic review sought to summarize our current understanding regarding the role of miRNAs in AD development. We searched articles indexed in PubMed (MEDLINE) and Web of Science databases using Medical Subject Heading (MeSH) or Title/Abstract words (‘microRNA/miRNA’ and ‘atopic dermatitis/eczema’) from inception through January 2020. Observational studies revealed dysregulation of miRNAs, including miR‐143, miR‐146a, miR‐151a, miR‐155 and miR‐223, in AD patients. Experimental studies confirmed their functions in regulating keratinocyte proliferation/apoptosis, cytokine signalling and nuclear factor‐κB‐dependent inflammatory responses, together with T helper 17 and regulatory T cell activities. Altogether, this systematic review brings together contemporary findings on how deregulation of miRNAs contributes to AD. In addition, we discuss the potential use of miRNAs as diagnostic biomarkers and therapeutic targets in AD.22.1We searched articles indexed in PubMed (MEDLINE) and Web of Science databases using Medical Subject Heading (MeSH) or Title/Abstract words (‘microRNA/miRNA’ and ‘atopic dermatitis/eczema’) from inception through 9th January 2020. Although there was no initial limitation imposed on language during the search, only English‐based literature and non‐English studies with available English abstracts were further considered. We included any original study in which the role of miRNAs in AD was examined in relation to pathogenesis, diagnosis, prognosis and treatment with meeting abstracts and reviews excluded. The searching process was conducted independently by two investigators. Experts in the field of miRNAs or dermatology were involved in the literature analysis.2.2The present study is a systemic literature review. We do not involve human beings or experimental subjects in this study, and no any identifiable private information is collected.3A total of 73 items from PubMed and 117 items from Web of Science were found based on the search criteria, among which 25 original studies investigating miRNAs in AD were finally included in this systematic review. The papers excluded were either conference abstracts, not original articles, not directly related to AD, or lacking in evidence of dysregulation of the studied miRNA(s) in human AD patients  in mouse epidermal keratinocytes in an experimental model of AD induced by the vitamin D3 analogue MC903.3.33.3.1Vaher et al reported that miR‐10a‐5p was up‐regulated in both non‐lesional and lesional skin of patients with AD as compared to healthy control skin.3.3.2Gu et al demonstrated that miR‐29b was up‐regulated in lesional skin and sera from AD patients as compared with healthy individuals.3.3.3Previous studies showed that miR‐124 was involved in inflammatory reaction. In addition, it was demonstrated to directly target nuclear factor (NF)‐κB in B‐cell lymphoma.3.3.4IL‐13, an important T helper 2 cytokine, has been demonstrated to impair normal epidermal barrier function. In this capacity, IL‐13 plays a critical role in many allergic diseases, such as asthma and AD.3.3.5Previous studies showed that miR‐146a was an anti‐inflammatory miRNA with a compensatory up‐regulation in psoriasis.3.3.6MiR‐151a belongs to the miR‐28 family. Its host gene, focal adhesion kinase (FAK), is located on chromosome 8q. Chen et al showed that miR‐151a was involved in the pathogenesis of AD by regulating IL‐12 receptor β2 (IL12RB2), a subunit of IL‐12 receptor.3.3.7MiR‐155 is implicated in the regulation of innate and adaptive immune responses. In particular, miR‐155 is necessary for the differentiation of T helper type 17 (Th17) cells in autoimmune diseases.miR‐155 gene might contribute to AD susceptibility. Sääf et al reported that the expression of BIC gene which encodes the precursor of miR‐155 was increased in AD skin as compared with healthy controls.BIC/miR‐155 gene were found to be associated with AD (P < .05). Nevertheless, such associations were not statistically significant after correction for multiple testing.Genetic variations in 3.3.8MiR‐223 expression is predominantly found in neutrophils, monocytes and eosinophils and is associated with tobacco smoking.4Through profiling and functional studies, the roles of miRNAs in AD are just emerging. Mechanistic investigations have linked miRNA dysregulation to aberrant skin barrier function, cytokine signalling and NF‐κB‐dependent inflammatory responses, together with Th17 and Treg activities Table . NeverthThe above‐mentioned biological processes regulated by miRNAs have important therapeutic implications. For instances, barrier‐restoring therapies have aroused interest in AD treatment research.The literature has ample examples illustrating the use of miRNAs as novel diagnostic biomarkers.The authors declare no competing financial interests.Xin Yu, Meifang Wang, Linfeng Li, Matthew TV Chan and William KK Wu contributed to research conception, designed the study and wrote the manuscript.


P = .02) and B‐symptoms (P = .001). B‐symptoms significantly decreased progression free survival (PFS)  and lymphoma specific survival (LSS) . Data showed that B‐symptoms might be a more reliable prognostic indicator than substage B in canine nodal diffuse large B‐cell lymphoma. Prospective studies assessing B‐symptoms in a larger cohort of patients and in other common lymphoma types are warranted. The abstract was presented at the fourth meeting of the European Canine Lymphoma Network Group in Lugano, 22 June 2019 and published in the proceeding of the meeting on the page 26.In humans B‐symptoms refer to systemic symptoms of lymphoma such as fever, weight loss, and night sweats and influence the prognosis of patients. In canine lymphoma, substage B is used to describe any clinical sign observed. Aim of the retrospective study was to compare the prognostic value of substage B with B‐symptoms to predict treatment response and survival in canine nodal diffuse large B‐cell lymphoma. Affected dogs treated with CHOP chemotherapy between 2008 and 2019 were included. B‐symptoms were defined by weight loss greater than 10% of normal weight, fever and the occurrence of unexplained resting tachypnoea substituted human night sweats. Substage B was defined as any symptoms but lymphadenopathy. Fifty‐five cases were included. B‐symptoms were present in 20/55 (36%) and substage B in 40/55 (74%) patients. No significant associations between B‐symptoms or substage B and weight, sex, breed, WHO stage and lymphoma grade were found. Treatment response was negatively associated with both substage B ( Approximately 50% of all newly diagnosed cases and more than 80% of aggressive lymphomas are DLBCL.The CHOP  chemotherapy protocol has been the mainstay of therapy for several decades.One of the most widely reported prognostic indicators is lymphoma substage, which has been correlated with complete treatment response (CTR), progression‐free survival (PFS) and lymphoma‐specific survival (LSS) in several studies.In human oncology, presence of B‐symptoms is used to refer to systemic clinical signs of lymphoma as fever, weight loss, and night sweats and this classification influences prognosis.Aim of our retrospective study was to compare the ability of substage B vs the presence of B‐symptoms to predict treatment response and survival in canine nodal DLBCL (nDLBCL). We hypothesized that the presence of B‐symptoms is superior to substage B in predicting outcome.22.1Previously treatment‐naïve dogs diagnosed with nDLBCL at the Vetmeduni Vienna (Austria) and the Veterinary University Brno (Czech Republic) between 2008 and 2019 were included in this retrospective study. Fifty‐five dogs from 113 patients with diagnosed lymphoma met the inclusion criteria: (a) confirmed nDLBCL by histopathology and immunohistochemistry, (b) no prior oncologic treatment including corticoids, (c) a standardized first‐line chemotherapy CHOP , and (d) a standardized clinical staging at the time of the initial diagnosis .2.2Medical record data were reviewed and patient characteristics , history, clinical symptoms, results of lymphoma staging, classification and grading, CTR and survival (PFS and LSS) were recorded. LSS was calculated from the date of diagnosis to the date of death related to the lymphoma. PFS was calculated from the date of diagnosis to the date of disease progression or recurrence. Follow‐up information was obtained from medical records and by phone conversations with referring veterinarians and pet owners. Since 2016 a lymphoma‐related questionnaire was used to assess history and signalment of patients. Dogs with primary extranodal or disseminated DLBCL were excluded from the study. Extranodal DLBCL was defined as lymphoma arising primarily outside of lymph nodes and spleen. Disseminated DLBCL was characterized as lymphoma involving broad spectrum of nodal and extranodal sites without evidence of primary origin.2.3Medical records were explored for staging data including assessment of tumour extent  classification.Ultrasonographic changes in homogeneity as well as echogenicity of liver and spleen were considered positive for lymphoma despite published literature.Cytological samples of liver and spleen were considered as positive for lymphoma involvement if more than ≥5% of nucleated cells consisted of large lymphocytes.2.4Substage A was defined as the absence of clinical signs but peripheral lymphadenopathy and substage B as presenting with any clinical signs of illness including gastrointestinal, constitutional, respiratory, neurologic, metabolic and nutritional symptoms.2.5WHO classification was performed from prospectively collected (n = 14) and archived samples (n = 41) in all included patients. Histopathology samples were reviewed by one experienced veterinary pathologist (A.F.B.) who was blinded to follow‐up data, to avoid observer bias. A peripheral enlarged lymph node was surgically removed, formalin‐fixed and paraffin embedded, stained with haematoxylin and eosin, and examined by the veterinary pathologist. For immunohistochemistry, a polyclonal antibody against CD3 (Dako A 0452), a monoclonal antibody against CD79a , and in a part of the cases a polyclonal antibody against CD20 (Spring Bioscience E 2560) were used on paraffin‐embedded sections. DLBCL was classified according to published adapted veterinary WHO classification for lymphoid neoplasia.2.6All dogs were treated with a first‐line CHOP chemotherapy, consisting of cyclophosphamide , doxorubicin , vincristine  and prednisolone (week 1‐5).2.7>30% but <100% reduction in size of all measurable disease; stable disease (SD) <30% reduction in size of all measurable disease with no change in size or <20% increase in size of all measurable disease; and progressive disease (PD), >20% increase in size of all measurable disease or the appearance of any new lesion.Treatment response was evaluated by using the following VCOG criteria for response evaluation in peripheral nodal lymphoma in dogs: complete remission (CR), 100% reduction in size of all measurable disease; partial remission (PR), 2.8Statistical analysis was performed with the statistical software package SPSS v.20 for Windows  and the statistics add in software for Microsoft Excel Analyse‐it .t‐test for normally distributed data and the Mann‐Whitney U‐test for non‐normally distributed or categorical data. Pearson r correlation was used to compare the relationship between linear‐related variables, and Spearman rank correlation between nonparametric variables.Statistical analysis included the PFS was calculated as the interval between initiation of treatment and disease progression or relapse, whereas LSS was measured as the interval between the day of primary diagnosis and lymphoma‐related death. Dogs lost to follow‐up or dead for lymphoma‐unrelated causes, as well as those still in complete remission at the end of the study, were censored from survival analysis. CTR was defined as the achievement of complete remission during and at the end of induction chemotherapy.P‐value <.05 was considered to be significant. Variables found to display statistical significance at the 5% level were included in a multiple analysis using multiple Cox proportional hazard analyses method.Survival curves were analysed with the Kaplan ‐ Meier method. Survival times are presented as median (range). Differences between Kaplan ‐ Meier curves were assessed by the log‐rank test. For all statistical analyses, a 3Fifty‐two dogs were included into this study. The median body weight of the dogs was 28 kg (range: 3‐55 kg) and the median age at the time of the initial examination of 8.9 years range: 3‐15 years) . Bone marrow aspiration was performed in nine cases (16%) and revealed positive results in all cases. Splenic infiltration was recorded in 27 dogs (49%). The liver was considered infiltrated in 11 dogs (20%), as documented by sonographic changes and/or confirmative cytology. Peripheral blood was involved in 10 dogs. In addition to these, 14 (25%) dogs had also extranodal involvement other than peripheral blood, bone marrow or liver, lung infiltrates were most frequently recorded (n = 9) followed by intraocular (n = 3), dermal (n = 2), central nervous system (n = 1) and urinary bladder (n = 1) lesions. Based on the WHO staging system one dog (2%) had stage II, 10 (18%) stage III, 27 (49%) stage IV and 17 (31%) stage V disease  defined as centroblasts and/or immunoblasts, with scant eosinophilic cytoplasm, round nucleus with multiple nucleoli (centroblasts) or a single prominent central nucleolus (immunoblasts). The mitotic activity of these cells was variable. There were 12 grade I, 23 grade II and 20 grade III cases (Table All included dogs were treated with first‐line chemotherapy (CHOP). Thirty‐five (64%) dogs completed the planned treatment schedule in CR and three (5%) died/were euthanized due to PD. In the remaining dogs n = 17, 31%) based on a progression during first‐line treatment, a rescue chemotherapy was started: seven were treated with a combination of dexamethasone, melphalan, D‐actinomycin and cytarabine (DMAC), four with a combination of lomustine, vincristine, procarbasine and prednisolone (LOPP), three with lomustine monotherapy, two with rabacfosadine and one with metronomic chlorambucil  were treated with rescue chemotherapy because of recurrence: 14 dogs received again a CHOP‐based protocol, whereas six patients were treated with DMAC, three with LOPP, two with lomustine monotherapy and one with rabacfosadine.Median PFS was 188 days (range: 15‐1544 days) and median LSS was 259 days (range: 29‐1645 days). At the end of the study data analysis closure, nine dogs were alive, three lost from follow‐up, 36 dead because of lymphoma and seven from causes other than DLBCL. Other causes of death were renal (n = 2), cardiac (n = 2) failure and secondary neoplasia (n = 3). The median follow‐up period was 257 days (range: 25‐1634 days).P < .001 for both) (Table The presence of B‐symptoms was significantly associated with shorter PFS and LSS (h) Table . Median h) Table . Median P = .18) and LSS (P = .23) (Table The presence of substage B was not associated with shorter PFS (SS P = .2 Table 33) Table . Median 3 Table .P < .001), but not substage B (P = .18), WHO stage (P = .7) and WHO grade (P = .89) remained statistically significant variables for PFS in univariate analysis. The univariate Cox regression analysis confirmed that the presence of B‐symptoms (P < .001), but not substage B (P = .23), WHO stage (P = .28), WHO grade (P = .9) remained significant prognostic factors for LSS. The presence of B‐symptoms (P < .001) and substage B (P = .02), but not WHO stage (P = .7) and WHO grade (P = .89) remained statistically significant variables for CTR in univariate analysis. The presence of B‐symptoms remained an independent prognostic indicator for PFS (P = .001), LSS (P = .001), and CTR (P = .001) in multiple regression analysis  making its influence on presented results unlikely. Nevertheless, the respective relevance of these three signs within B‐symptoms alone and their combinations shall be further investigated in prospective studies with canine nDLBCL. We will suggest to set a timeframe for tachypnoea at rest for at least 3 days.Strengths of the study are the long‐term follow‐up of a well‐defined group of dogs with nDLBCL treated with a standardized first‐line chemotherapy and low number of patients lost from the follow‐up.In conclusion, this study demonstrated that B‐symptoms may be a better prognostic indicator than substage B in canine nDLBCL.B‐symptoms remained an independent predictor of survival in the multiple regression analysis. Human studies proved that B‐symptoms are associated with increased concentrations of acute phase proteins and some proinflammatory cytokines.The authors declare no potential conflict of interest.


Respiratory syncytial virus (RSV) is a major cause of hospital admission for acute lower respiratory tract infection in young children.We aimed to identify risk factors for hospitalized RSV disease and its severe outcomes.We conducted a retrospective cohort study analyzing data of a ICD‐10‐code‐based hospital surveillance for severe acute respiratory infections (SARI). Using univariable and multivariable logistic regression analysis, we assessed age‐group, gender, season, and underlying medical conditions as possible risk factors for RSV and its severe outcomes including ICU admission, application of ventilator support, and death, respectively.Of the 413 552 patients hospitalized with SARI in the database, 8761 were diagnosed with RSV from week 01/2009 to 20/2018 with 97% (8521) aged <5 years. Among children aged <5 years, age‐groups 0‐5 months  and 6 months‐1 year , and underlying respiratory and cardiovascular disorders specific to the perinatal period  were risk factors for being diagnosed with RSV. Age‐group 0‐5 months , low birth weight , preterm newborn , underlying respiratory and cardiovascular disorders specific to the perinatal period , congenital malformation of the heart , congenital malformation of the great vessels , congenital defect originating in perinatal period , cardiovascular disease , neurological disorders , blood disease , and liver disease  contributed to ICU admission in RSV cases.Using ICD‐10‐based surveillance data allows to identify risk factors for hospitalized RSV disease and its severe outcomes, and quantify the risk in different age‐groups. In infants and young children, RSV is the most common cause of acute lower respiratory tract infection (ALRI) and a major cause of hospital admission for ALRI. Worldwide, about 33 million RSV‐associated ALRI episodes occurred in children younger than 5 years, and about 3 million were severe enough to necessitate hospital admission in 2015.Currently, there are no licensed vaccines against RSV; only passive immunization with palivizumab is available for young children at high risk.Risk groups of severe RSV will benefit most from the RSV immunization once RSV vaccines become available. So far, some studies indicated that besides some socio‐demographic and environmental factors including being male, household crowding, and passive smoking, underlying medical conditions such as prematurity, congenital heart disease, and chronic pulmonary and cardiovascular diseases were associated with RSV disease.The development of RSV vaccination strategies and the evaluation of the effects of RSV vaccination in the future rely on timely epidemiological data and long‐term observation of epidemiological situation of severe RSV and risk factors for severe RSV through national and other large‐scale RSV surveillance systems. The Robert Koch Institute (RKI) established an International Statistical Classification of Diseases, 10th revision (ICD‐10)‐code‐based surveillance system for severe ARI (ICOSARI) in cooperation with a private hospital network in Germany in 2015. Data of respiratory hospitalizations have been collected through the ICOSARI.The aim of the present study was to identify risk factors, in particular underlying medical conditions as risk factors, for hospitalized RSV disease and its severe outcomes based on the ICD‐10‐code‐based surveillance data.1.1We performed a retrospective cohort study based on secondary data analysis of the ICOSARI database. The hospital network of ICOSARI includes 45 original sentinel hospitals, and additional 42 hospitals joined the hospital network after 2013. In 2015, 84 sentinel hospitals of ICOSARI located in 13 of the 16 federal states of Germany, covered 4.3% hospitals, and accounted for 5.9% of hospitalized patients in Germany. Since 2015, digital data of hospitalizations with any of respiratory ICD‐10 code (chapter X: J00‐J99)The ICOSARI system was approved by the RKI and HELIOS Kliniken GmbH data protection authorities. As ICOSARI involved no interventions and the analysis was based on anonymized data only, no ethical clearance was required for this study.We used the following case definitions:Severe ARI (SARI) case: any patient hospitalized with any of the ARI ICD‐10 codes J09‐J22  as primary or secondary discharge diagnosisRSV case: SARI case diagnosed with any of the RSV‐specific ICD‐10 codes  as primary or secondary discharge diagnosis.ICU‐admitted RSV case: RSV case ever admitted to an ICU during the hospital stay.Ventilated RSV case: ICU‐admitted RSV case ever required ventilator support during the hospital stay.Deceased RSV case: RSV case died in hospital.ICU admission, application of ventilator support, and death were considered markers of severe outcomes of hospitalized RSV disease.Due to the possible inconsistent recording practices on ICU admission and application of ventilator support in the sentinel hospitals , data on ICU admission and application of ventilator support from the original sentinel hospitals before 2013 and from the additional sentinel hospitals before 2015 were excluded from our data evaluation.The underlying medical conditions evaluated in our study were neonatal disorders , extremely low birth weight <1000 g), low birth weight (1000‐2499 g), extreme immaturity of newborn (<28 weeks), preterm newborn (28‐37 weeks), respiratory and cardiovascular disorder specific to the perinatal period , congenital disorders , other comorbidities , chronic pulmonary disease (excl. asthma and COPD), diabetes, cardiovascular disease, neurological disorders, blood disease , renal failure, liver disease, tuberculosis, cancer, HIV/AIDS), and pregnancy. The underlying medical conditions could be primary or secondary discharge diagnoses. The specific ICD‐10 codes of the medical conditions chosen for our data evaluation are listed in Table 000 g, loFor descriptive data analysis, we described the number of total RSV cases, ICU‐admitted RSV cases, ventilated RSV cases, and deceased RSV cases by age‐group , gender, calendar week, and underlying medical condition, respectively. The mean and median length of hospital stay, ICU stay and ventilation, and discharge mode were investigated, respectively.P value of <.05 was considered statistically significant. Only variables indicating significant associations with RSV or any markers of severe outcomes of RSV in univariable logistic regression models were kept in multivariable logistic regression models, respectively.We carried out univariable and multivariable logistic regression analyses to assess age‐group, gender, season, and underlying medical conditions as possible risk factors for being diagnosed with RSV among SARI cases, for ICU admission, application of ventilator support, and death among RSV cases, respectively. The univariable and multivariable logistic regression analyses were stratified in two age‐groups <5 years and ≥5 years. Underlying neonatal disorders were only evaluated as possible risk factors in the age‐group <5 years. Normally, relative risk (RR) should be calculated to measure the association between the exposure and the outcome in a cohort study. However, the odds ratios (OR) provides a reasonable approximation of the RR if the outcome is rare and occurs in less than 10% of the unexposed population.We used Stata (version 15) and Microsoft Excel 2010 for the data analyses.22.1A total of 413 552 SARI cases were identified from week 01/2009 to 20/2018. More than half  of them were male, and 64% (263 133) were ≥65 years old  RSV cases were identified. More than half  of them were male. Most RSV cases were children aged <5 years . The mean and median length of hospital stay were 6 and 5 days, respectively. Most  RSV cases were discharged home, 2% 136) transferred to other facilities, and 0.3% (25) died in hospital. Of the RSV cases, 8228 (94%) received RSV‐specific ICD‐10 codes as primary discharge diagnosis . Of the total RSV cases, 6773 were from the original sentinel hospitals after 2013 or from the additional sentinel hospitals after 2015  ICU‐admitted RSV cases were identified. More than half 262, 53%) of them were male, and 89% (438) were aged <5 years. The mean and median length of ICU stay were 9 and 5 days, respectively. During the ICU stay, 38% (185) required ventilator support. Most  ICU‐admitted RSV cases were discharged home, 6% (28) transferred to other facilities, and 3% (15) died in hospital  were male, and 81% (149) were aged <5 years. The mean and median ventilation length were 211 and 112 hours, respectively. Most  ventilated RSV cases were discharged home, 10% (18) transferred to other facilities, and 8% (15) died in hospital Figure .2.5Of the 25 deceased RSV cases, more than half  were female, and nearly half  were ≥65 years old Table . The mea2.6An overview of the number of total RSV cases and RSV cases with severe outcomes by calendar week is shown from week 01/2015 to 20/2018. During the study period, the most severe season was 2016/17 Figure .2.7The proportion of total RSV cases with different underlying medical conditions varied from 0% to 3.5%. Respiratory and cardiovascular disorder specific to the perinatal period  and cardiovascular disease  were the most common underlying medical conditions. They both were also the most common underlying medical conditions in the ICU‐admitted  and ventilated RSV cases . Cardiovascular disease  and blood disease  were the most common underlying medical conditions in deceased RSV cases. RSV cases with underlying sickle‐cell disorder (2), tuberculosis (2), and pregnant RSV cases  were rare. No RSV case was diagnosed with HIV/AIDS ; being female; the seasons 2012/13, 2014/15, 2016/17, and 2017/18 (reference season: 2013/14); and underlying low birth weight, preterm newborn, and respiratory and cardiovascular disorder specific to the perinatal period were significantly associated with an increased risk of being diagnosed with RSV among SARI cases. Underlying congenital malformation of the heart and great vessels, congenital defect originating in perinatal period, cystic fibrosis, asthma, neurological disorders, blood disease, and cancer were significantly associated with a lower risk of being diagnosed with RSV. Age‐group 0‐5 months was significantly associated with ICU admission, whereas age‐group 2‐4 years was significantly associated with death among RSV cases. Gender and season were not associated with any severe outcomes of RSV. Most underlying medical conditions were significantly associated with severe outcomes of RSV.In the age‐group ≥5 years, the age‐group 5‐64 years; the seasons 2012/13, 2014/15, 2016/17, and 2017/18; and underlying congenital defect originating in perinatal period, cystic fibrosis, vitamin D deficiency, asthma, and chronic pulmonary disease were significantly associated with an increased risk of being diagnosed with RSV among SARI cases. Underlying COPD, cardiovascular disease, and renal failure were significantly associated with a lower risk of being diagnosed with RSV. The age‐group ≥65 years was associated with death among RSV cases. Gender and season were not associated with any severe outcomes of RSV. Some medical conditions were significantly associated with severe outcomes of RSV.2.9Among SARI cases aged <5 years, the age‐groups 0‐5 months  and 6 months‐1 year ; being female ; the seasons 2012/13 , 2014/15 , 2015/16 , 2016/17 , and 2017/18 ; and underlying respiratory and cardiovascular disorder specific to the perinatal period  were significantly associated with an increased risk of being diagnosed with RSV. Underlying congenital malformation of the heart  and great vessels , congenital defect originating in perinatal period , asthma , neurological disorders , and blood disease  were significantly associated with a lower risk of being diagnosed with RSV , underlying low birth weight (OR: 6.77. 95% CI: 1.28‐35.71), preterm newborn , respiratory and cardiovascular disorder specific to the perinatal period , congenital malformation of the heart  and great vessels , congenital defect origination in perinatal period , cardiovascular disease , neurological disorders , blood disease , and liver disease  were significantly associated with ICU admission , preterm newborn , respiratory and cardiovascular disorder specific to the perinatal period , congenital malformation of the heart , congenital defect originating in perinatal period , cystic fibrosis , cardiovascular disease , neurological disorders , blood disease , and liver disease  were significantly associated with application of ventilator support , cardiovascular disease , neurological disorders , blood disease , and liver disease  were significantly associated with an increased risk of death ; the seasons 2012/13 , 2014/15 , 2016/17 , and 2017/18 ; and underlying congenital defect originating in perinatal period , cystic fibrosis , vitamin D deficiency , and chronic pulmonary disease  were significantly associated with an increased risk of being diagnosed with RSV , application of ventilator support , and death . Underlying renal failure  was significantly associated with ICU admission. The age‐group ≥65 years  was significantly associated with an increased risk of death  and RSV fatality rate (0%‐5%) vary in different studies.9% and RSThe majority of RSV cases were aged <5 years in our study. Thus, we investigated risk factors separately in two age‐groups <5 and ≥5 years. To avoid confounding effects, we included in the multivariable models variables with significant association with RSV or its severe outcomes in the univariable models.In the multivariable models for the age‐group <5 years, among SARI cases, children in the first months of life were significantly more likely to be diagnosed with RSV, and among RSV cases, they were more likely to be admitted to ICU. Our findings are concordant with the majority of reports that young age is a risk factor for hospitalization due to RSVLow birth weight and prematurity are widely recognized as important risk factors for RSV.In the multivariable models for the age‐group ≥5 years, older adults were significantly unlikely to be diagnosed with RSV. However, they were significantly more likely to have severe outcome of death if they were diagnosed with RSV. This finding can be explained that RSV similar to seasonal influenza can cause severe respiratory complications in older adults, resulting in respiratory failure and high mortality.Underlying Down syndrome was reported as a risk factor for RSV in some studies,Our study has some limitations. Based on the hospitalization data alone, information on socio‐demographic factors except age and gender as well as environmental factors for RSV could not be captured. Thus, socio‐demographic and environmental factors and underlying medical conditions could not be evaluated in one model, which limited a comprehensive understanding of the risk factors for RSV and its severe outcomes. Further, no data were available to identify any children who had received palivizumab which would reduce the strength of association with risk factors. ICOSARI is a ICD‐10‐based syndromic surveillance system. Virological data of the ICOSARI network were not available. As the coding behavior of physicians may vary based on use of laboratory diagnostics and level of coding awareness, some true cases may be missed or wrongly included due to miscoding which could lead to information bias. RSV is also a common pathogen of ARI in older adults. In our study, among those aged ≥65 years, the proportion of RSV cases in SARI cases was lower than that reported in the literature.4Using ICD‐10‐based surveillance data allows to identify risk factors for being diagnosed with RSV and severe outcomes of RSV in hospital, to quantify the risk in different age‐groups, and to monitor the risk routinely. Our findings will contribute to the development of a baseline for evaluating RSV vaccination strategies and RSV vaccination impact in the future, in particular on the target groups, and help reducing burden of RSV disease and its severe outcomes.Further studies regarding risk factors for RSV are needed with the focus on the underlying medical conditions with inconsistent findings compared with the literature with consideration of socio‐demographic and environmental factors.None.Wei Cai, Silke Buda, Walter Haas, Siddhivinayak Hirve, and Wenqing Zhang were involved in designing the study. Silke Buda and Ekkehard Schuler participated in the collection of ICOSARI data. Wei Cai analyzed the data and drafted the manuscript. All authors reviewed and approved the final manuscript.