Seventy percent of patients suffering from birch pollen allergy (BPA) develop a pollen‐related food allergy (prFA), especially to apples, due to a clinically relevant cross‐reactivity between the major allergen in birch Bet v 1 and Mal d 1 in apples. Therefore allergen‐specific immunotherapy with fresh apples (AITA) could be a promising natural treatment of both BPA and prFA.To assess the clinical efficacy of immunotherapy by daily apple consumption for patients with BPA and prFA.4 to Bet v 1 and Mal d 1, conjunctival and oral provocation tests, skin reactivity, and the average daily rhinoconjunctivitis combined symptom and medication score (CSMS) were measured during the peak birch pollen season.A daily defined increasing amount of selected cultivars  was continuously consumed by 16 patients , leading to increased intake of allergen over a period of at least 8 months. Specific IgE and IgGp < .001) and a decreased skin reactivity to apples. Oral allergy syndrome to other birch prFA than apple also decreased (p < .05). Moreover, daily rhinoconjunctivitis CSMS declined by 34% (p < .001), as did conjunctival reactivity to birch pollen extract by 27% (p < .01), while specific IgG4 to Mal d 1 and Bet v 1 increased (p < .01).After 8 months of therapy, patients showed increased tolerance to apples ( p < .001), as did conjunctival reactivity to birch pollen extract by 27% (p < .01), while specific IgG4 to Mal d 1 and Bet v 1 increased (p < .01). In this small pilot trial daily apple consumption improved both birch pollen and related food allergies. If confirmed by a larger controlled trial apples could provide a natural, healthy and cost‐saving causal treatment for BPA.A 70% of patients suffering from birch pollen allergy (BPA) develop a pollen‐related food allergy (prFA), especially to apples. This is due to a strong clinically relevant homology between the major allergen in birch Bet v 1 and Mal d 1 in apples. Therefore a pilot trial using fresh apples for allergen‐specific immunotherapy to birch pollen was performed. After 8 months of therapy, patients showed increased tolerance to apples and other birch pollen‐related food allergens. Moreover, daily rhinoconjunctivitis combined symptom and medication score (CSMS) declined by 34% ( Unfortunately, this hardly ever showed any effect on the concomitant prFA.A key treatment issue would be to find a way for patients to develop permanent tolerance to apples and simultaneously to birch‐pollen. Therefore built on these results, we developed a protocol based on continuously increasing apple consumption starting from small amounts of a low allergenic cultivar and finally reaching high amounts of highly allergenic apples.22.1A detailed description of the concept is provided in a previously published manuscript.2.2Based on the previous results, therapy was carried out as follows: AITA started in September 2018 with fresh harvested low allergenic Red Moon® cultivars. Patients began with the amount tolerated in the OPT. The daily apple portion was chewed carefully for 2 min, leaving some amount sublingually. Afterward, food and water intake had to be avoided for 15 min. If this was well tolerated, the amount of apple was continuously increased (maximum doubled) every 2 weeks until reaching one entire apple. After an average of 6 weeks, Red Moon® was replaced by moderate allergenic Pink Lady® apples for another 6 weeks, one patient receiving Topaz instead. Finally, the patients switched to high allergenic and well storable Golden Delicious apples, which were consumed for the last 5 months. Prior cultivar change, tolerability was again assessed by OPT to find the best suitable starting amount. Apples were handed out every 2 or 3 weeks and had to be stored between 2°C and 8°C until consumption. The median weight of one full fresh apple was analyzed for each cultivar by examining 200 freshly harvested apples . Adverse events were assessed through calls and after each apple delivery. Symptoms  were graded on a 4‐point scale .2.34 for Bet v 1 and Mal d 1 and IgE for Bet v 2 and Bet v 4 were quantified before and after AITA by ImmunoCAP  according to the manufacturer's specifications.Serum concentrations of total IgE, specific IgE, and IgG2.4Twenty‐three different apple cultivars divided into red‐fleshed, old, and new cultivars named Bay 3484 Baya® Marisa (BM), Y103 Kissabel® (RF2), R201 Kissabel® (RF4), RS‐1 Red Moon® (RM), Luresweet Redlove® (RL), Goldparmäne (GP), Kanada Renette (KR), Tiroler Spitzlederer (TS), White Rosemary (WR), White Winter‐Calville (WW), Bonita (BO), Elstar Lb®87/1 (EL), Fuji Zhen® (FJ), Gala Buckeye®(GA), Gloster (GO), Golden Delicious Klon B (GD), Lb 17906 (LB), CIVG198 Modí® (MO), SQ159 Natyra® (NA), Pink Lady® Rosy Glow (PL), Santana (SA), Bay 4210 Sonnenglanz® (SG), Topaz (TO), and birch pollen extract (ALK‐Abelló) were skin tested before and after AIT according to Nothegger et al.2.5OPTs with Red Moon®, Pink Lady®, and Golden Delicious were performed within the AITA every time before a new apple cultivar was consumed and additionally, Golden Delicious was tested before and after AITA, according to Nothegger et al.2.6Conjunctival provocation tests (CPTs) with birch pollen extract , diluted 1:10 with NaCl 0.9%, were performed outside the birch‐pollen season before and after AITA. Symptoms  were recorded on a 4‐point scale , according to Fauquert et al.2.7Cross‐reactivity to other plant foods was assessed before and after AITA by a questionnaire. Patients filled in a yes or no tolerability to the following 26 foods: almond, apricot, blackberry, blueberry, carrot, celery, cherry, coriander, dill, fennel, hazelnut, kiwi, melon, nectarine, parsley, parsnip, peach, pear, peanut, pepper‐green, plum, potato, raspberry, soy, strawberry, and walnut.2.83. Patients were scattered throughout the Tyrol and Southtyrol provinces, but pollen counts in the capital cities were higher in 2018 compared to 2019. For this reason, these results need to be interpreted with caution.The patients birch‐pollen diary in paper form was handed out for daily documentation of rhinoconjunctivitis and medication use  during the birch pollen season ranging from mid‐March to mid‐May. Daily symptom score and daily medication score were recorded according to the EAACI guidelines.2.94) are expressed as median, including ranges. SPT results are shown as median HEP‐Index diameter . Wilcoxon's matched‐pairs signed‐rank test was used to analyze differences before and after AITA. A p value of <.05 was considered significant.All statistical calculations were performed using SPSS 2633.1n = 3), pregnancy (n = 2), and one due to increased side effects. Patients started with an average amount of 62.5 g Red Moon® per day, as illustrated in Figure p < .001) compared with Red Moon®, eating a daily average of only 20 g apple. Golden Delicious was in general much worse tolerated than Pink Lady® or Red Moon®, especially within the first 4 weeks of AIT (weeks 13–16). All patients initially reported to have OAS but became slowly accustomed to the apple. Therefore, the ingested amount was only gradually increased so that 8/17 patients were able to consume a daily amount of three‐quarters to one full apple (198 g) after 10 weeks of therapy with Golden Delicious. In the 10 weeks that followed, daily intake was continuously increased, and symptoms started to decline. Only during the birch‐pollen season (week 28–31), some patients reported to less well tolerate apples. Interestingly, tolerance was better if the apple was eaten in the morning. At the end of AITA in week W32, 13/16 (81%) were able to eat three‐quarter to one full apple without problems, and the remaining 3/16 (19%) tolerated a quarter piece.Twenty‐two patients assessed for AITA had a median age of 48.5 years range, 23–68), 68% were female. During AITA, six withdrew for the following reasons: lack of time  shown in Figure After completion of AITA, patients were asked not to eat apples for at least 3 weeks, to avoid an unspecific desensitization effect. After that, follow‐up examinations started. OPT with Golden Delicious showed an increased significant tolerance of apples (p = .039), for all the therapeutic apples  and especially in Golden Delicious . SPT with birch extract declined by 12% .SPT after AITA also showed a significant decrease in wheal size overall 23 apples tested .CPT after AITA illustrated in Figure p < .001) shown in Figure Moreover, the mean daily combined symptom and medication score (CSMS) during the peak birch‐pollen season in April significantly declined by 34%  to 0.60 kUA/l , p = .013 as did IgG4 specific for Mal d 1 from median 0.12 kUA/l  to 0.38 kUA/l , p = .013). Specific IgE for both Bet v 1 and Mal d 1 did not change significantly.ImmunoCAP results after AITA are illustrated in Figure 3.1.3Before entering the study, most patients  also showed OAS to other fruits, vegetables or nuts. Over 60% displayed OAS after eating cherries (11/16) or raw carrots (10/16), 6/16 (38%) reacted to apricot, hazelnut, kiwi, nectarine, peach, pear, or walnut, <30% to almond, celery, melon, peanut, plum, soybean, or strawberry (1–4/16).p < .01 Wilcoxon rank test). OAS to kiwi, melon and peanut seemed to disappear completely, OAS to cherries improved in 6/11 patients (55%), to carrots in 4/10 patients (40%), to apricot in 4/5 (80%), to hazelnut in 3/5 , to pear in 4/6 (67%), and to plum in 3/4 (75%) of the patients. Tolerance to almonds, celery, nectarine, strawberry, soy, and walnut remained unchanged.After therapy, also OAS to other cross‐reactive plant‐food seemed to decrease aside from the apple allergy  of the patients, they reported that they had to force themselves to eat the daily amount after switching to the high allergenic Golden Delicious. After 4 months, most patients (10/12) reported that they were tired of eating Golden Delicious every day and that they would change the apple cultivar if they could. Nevertheless, all patients were satisfied to eat apples experiencing hardly any or no symptoms with relish after years of not being able to eat apples. Indeed, after completion of the study patients continued to eat daily apples, but regularly changed within higher allergenic cultivars.4Based on previously published findings and following the protocol described,Up to date, birch‐AIT has shown to be largely ineffective in treating prFA.After 8 months' daily consumption of a defined increasing amount of apple with increasing allergen content, 7/16 (52%) of the patients were able to eat on average 138 g more fresh apple, and a further 6/16 (37.5%) displayed no complaints after eating one entire high allergenic Golden Delicious. Tolerance to other cross‐reactive plant foods  also seemed to increase.4 specific for Bet v1 and Mal d 1, indicating a specific immunologic response by reduced release of mediators in mast cells and basophils.Even more interesting is a possible decrease in birch‐pollen induced rhinoconjunctivitis along with a significant reduced CPT‐reactivity with birch‐pollen extract. Moreover, we found an induction of IgGApples have an average weight of 135–228 gAITA was also successful in seven other patients that could not be included in the study due to restricted availability of Red Moon® apples. Hence, these patients were followed outside the study protocol with individually tailored commercially available apple cultivars with increasing allergen content  and showed increased tolerance to apples and decreased BPA symptoms after a minimum of 8 months apple consumption. This indicates that AITA is not only restricted to the cultivars used in this protocol.As with any immunotherapy, it is important to maintain the treatment response. Therefore, patients should continue AITA to achieve permanent and long‐term efficacy with regard to both symptoms of BPA and birch‐prFA. Hence, we advised our patients after reaching the maintenance phase to subsequently consume one entire high allergenic apple  at least twice or three times a week. Moreover, according to the EAACI guidelinesDaily eating of apples was well tolerated and safe and patients remained highly motivated, however, would prefer a greater variety of apples in daily consumption. Therefore, a higher selection and interchange of higher allergenic apple cultivars should be made possible in future subsequent studies. It should be reminded, that patients sensitized against the heat‐stable lipid transfer Mal d 3 protein (>0.35 kU/L) with the potential to induce severe anaphylactic reactions were excluded from this study. The protein also cross‐reacts with homologues proteins in Rosaceae fruits . Whereas the instable Mal d 1 protein causes almost mild symptoms, as the so‐called OAS, in contrast to Mal d 3 that passes the gastrointestinal tract and can provoke more severe anaphylactic reactions.In summary, the AITA protocol used in this study was feasible and effective for birch‐pollen allergic patients with associated OAS to apples. Prior analysis of different apple cultivars' allergenic potential enables to slowly start and increase the daily allergenic amount with a low allergenic apple cultivar followed by subsequent updosing with moderate and high allergenic cultivars until reaching on average one entire high allergenic apple. In this way, a safe and gradual allergen adjustment with low side effects could be achieved. Side effects mainly occurred at the beginning of AITA after apple cultivar change or after increasing the ingested amount.Nonetheless, this study has several limitations. First, it is a small phase 2 pilot study with only 16 patients, second, it is known that the placebo effect of clinical improvement in controlled allergy trials is around 30%, further pollen concentrations were lower in the 2nd year, so effects on the symptom score have to be interpreted with caution.In conclusion, our pilot‐study demonstrated that oral immunotherapy with fresh apples was feasible and safe for the treatment of both BPA and birch prFA. If the effect can be confirmed by a larger controlled phase III trial, daily apple consumption could provide a natural, healthy, and cost‐saving causal treatment for BPA and prFA with high potential for clinical application.The authors declare that there are no conflict of interests.Conception of the study: Klaus Eisendle. Study design and patient selection: Bettina Nothegger, Norbert Reider, Claudia E. Covaciu, Martin Tollinger, Thomas Letschka, and Klaus Eisendle. Laboratory analysis: Valentina Cova, Linda Ahammer, Reiner Eidelpes, Jana Unterhauser, Stefan Platzgummer, Elisabeth Raffeiner, and Martin Tollinger. Acquisition of data and patient care: Bettina Nothegger, Claudia E. Covaciu, Norbert Reider, Thomas Letschka, and Klaus Eisendle. Statistical analysis: Bettina Nothegger and Klaus Eisendle. Writing of the manuscript: Bettina Nothegger, Norbert Reider, Thomas Letschka, and Klaus Eisendle. All the authors have read and approved the manuscript.Supporting information.Click here for additional data file.


The levels of proinflammatory cytokines in the hippocampus were measured by qRT-PCR and ELISA. The results showed that running exercise reversed the decreased sucrose preference of rats with CUS-induced depression. In addition, CUS increased the number of hippocampal microglia and microglial activation in rats, but running exercise attenuated the CUS-induced increases in the number of microglia in the hippocampus and microglial activation in the dentate gyrus (DG) of the hippocampus. Furthermore, CUS significantly increased the hippocampal levels of inflammatory factors, and the increases in inflammatory factors in the hippocampus were suppressed by running exercise. These results suggest that the antidepressant effects of exercise may be mediated by reducing the number of microglia and inhibiting microglial activation and neuroinflammation in the hippocampus.Running exercise has been shown to relieve symptoms of depression, but the mechanisms underlying the antidepressant effects are unclear. Microglia and concomitant dysregulated neuroinflammation play a pivotal role in the pathogenesis of depression. However, the effects of running exercise on hippocampal neuroinflammation and the number and activation of microglia in depression have not been studied. In this study, rats were subjected to chronic unpredictable stress (CUS) for 5 weeks followed by treadmill running for 6 weeks. The depressive-like symptoms of the rats were assessed with a sucrose preference test (SPT). Immunohistochemistry and stereology were performed to quantify the total number of ionized calcium-binding adapter molecule 1 (Iba1) Major depressive disorder (MDD) is a common mental disorder characterized primarily by symptoms of pervasive depressed mood along with anhedonia and feelings of guilt . It is eAlthough the exact etiology and pathogenesis of depression remain unclear, increasing evidence has shown that the inflammatory response is a main factor involved in the development of the disorder , 6. NumeMicroglia are characterized by different states, including a quiescent state in which the cells are ramified or resting and an activated state in which the cells are hyper-ramified or phagocytic . In theiPhysical exercise is an effective nonpharmaceutical treatment for MDD. Various clinical surveys have demonstrated that exercise is effective in improving depressive symptoms in individuals with mild or moderate depression , 30. OurTherefore, we hypothesized that running exercise attenuates depressive symptoms by exerting protective effects against microglial activity and inflammatory cytokines in CUS models. To test this hypothesis, we verified the antidepressant effects of running exercise with a sucrose preference test (SPT). Then we investigated whether running exercise affects these changes using immunohistochemistry and unbiased stereological methods. Finally, we investigated the effects of treadmill exercise on microglial activation and the levels of inflammatory cytokines using immunofluorescence assays, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA).Sixty male Sprague-Dawley rats (6–8 weeks old and weighing 200 ± 20 g) were provided by the Experimental Animal Center of Chongqing Medical University . The rats were housed under a 12-h light/dark cycle at an appropriate temperature (22 ± 2 °C) and provided access to food and water. Before the interventions began, all rats were allowed to adapt to the housing conditions for 2 weeks. During the animal experiment, the animals in each group were treated by the investigators without blinding. All procedures  and the CUS group (n = 37). The control rats were housed at a density of four to five rats per cage, and the CUS rats were housed in individual cages. The two groups of rats were housed in separate rooms and had no contact with each other. The model rats were subjected to different types of stressors, including water or food deprivation, empty bottle exposure, intermittent illumination, cage tilting, restraint, damp bedding exposure, no bedding exposure, cold/hot stress, strobe light exposure, noise exposure, electric foot shock exposure, tail pinching, and cage shaking. Two types of stressors were applied daily, and no stimulus was repeated within 5 days (Table n = 17) and the CUS + running group .The CUS procedure was performed as described in our previous reports , 41. AftIn the CUS + RUN group, the rats ran on a six-lane motorized treadmill for 20 min each day, 5 days per week for 6 weeks Fig. . During During the running exercise period, the rats in the CUS + STD group and the CUS + RUN group were housed at a density of one rat per cage, while the rats in the control group remained housed under normal circumstances with four to five rats per cage.The body weights of the rats were measured during a fixed time frame each week.The SPT was carried out as previously described . Before After 24 h, the weights of the consumed sucrose solution and water were recorded. The percentage of sucrose preference was calculated as sucrose consumption/(sucrose consumption + water consumption) × 100% . The SPTIn the following procedures, all experiments and data analyses were carried out by investigators blinded to the treatment conditions. After the behavioral testing, five rats were randomly selected from each group. The rats were anesthetized with 1% pentobarbital sodium  and transcardially perfused with heparinized saline followed by 4% paraformaldehyde in phosphate-buffered saline . Then the cerebrum of each rat was removed and cut into two hemispheres along the sagittal suture. The brain tissue was postfixed in 4% paraformaldehyde for 24 h and subsequently immersed in a series of sucrose solutions of increasing concentration. The right or left hemisphere was chosen randomly and cut into 50 μm serial sections using a cryostat . Every sixth section containing the hippocampus was sampled in a systematic random manner, and 12 sets of sampled sections were acquired for the following analyses.Two separate sets of serial sections containing the hippocampus were chosen from each group of rats. The free-floating sections were rinsed in PBS with 0.3% Triton X-100  and 0.1% Tween (PBS + T) 6 times for 10 min each. Then, the sections were blocked with 1% fetal bovine serum (FBS), 10% SP 9001-A, 0.1% cold water fish gelatin, and PBS + T for 2 h at 37 °C. The sections were then incubated with a rabbit anti-Iba1 primary antibody  in PBS + T for 2 days at 4 °C. After being rewarmed and rinsed, the sections were incubated with SP 9001-B  for 3 h at 37 °C and followed by SP 9001-C (biotin-HRP-streptavidin) for 2 h at 37 °C. Subsequently, the sections were immersed in a diaminobenzidine solution  for approximately 1 min. Finally, the sections were dehydrated by sequential immersion in a graded ethanol series  and xylene (3 × 10 min).+ cells in the three subfields of the hippocampus were estimated using the optical fractionator method in the stereological system . The total number of Iba1+ cells in each hippocampal subregion was calculated using the following formula:N is the total number of Iba1+ cells in the CA1, CA2/3, and DG in each animal, ∑Q− is the total number of Iba1+ cells actually counted in the specimens, ssf is the section sampling fraction, asf is the area sampling fraction, and hsf is the section height sampling fraction  and a mouse anti-CD68 primary antibody  in PBS + T for 2 days at 4 °C. The sections were washed three times for 10 min at room temperature in PBS + T. For the immunofluorescence staining, the sections were incubated with DyLight 549- and DyLight 488-labeled secondary antibodies  for 2 h at 37 °C. 4’,6-Diamidino-2-phenylindole  was used for the nuclear staining. Fluorescent images were captured at a ×200 magnification under a Zeiss fluorescence microscope . At least ten representative images of each hippocampal subregion were acquired from each rat. The Iba1The levels of the proinflammatory cytokines IL-1β, inducible nitric oxide synthase (iNOS), IL-6, and tumor necrosis factor (TNF)-α are associated with the severity of depression , 45. To −ΔΔCt method.The total RNA was extracted from the hippocampi of the rats using a Total RNA Extraction Kit  according to the manufacturer’s protocol. Complementary DNA (cDNA) synthesis was performed using a PrimeScript RT Reagent Kit with gDNA Eraser  according to the manufacturer’s instructions. The primer sequences and sequence specificity of the primers were designed and tested, respectively, using the National Center for Biotechnology Information (NCBI) Primer-BLAST. The primer sequences are listed in Table The protein expression levels of IL-1β, iNOS, IL-6, and TNF-α in the hippocampus were examined by ELISA. The hippocampi of the rats were isolated on ice and washed with normal saline. Then the tissue samples were homogenized and centrifuged at 12,000 rpm for 15 min. The supernatant was collected, and the total protein concentration was determined using a BCA Protein Assay Kit . Samples containing equivalent amounts of protein were calculated and subpackaged. Subsequently, the protein concentrations of inflammatory cytokines in the hippocampus were determined using ELISA kits  according to the manufacturer’s instructions. The optical density at 450 nm was determined using a microplate reader , and the concentration of these cytokines was calculated according to standard curves.t tests were used for comparisons between two groups. The sample size of each experiment was chosen based on previous experience with the aim of detecting at least a p value <0.05 in the different tests applied. No animals were excluded from the present study.The statistical analyses were performed using SPSS version 23.0 . All data are expressed as the mean ± standard deviation (SD). All data were assessed for normality and homogeneity of variance. The body weight data were analyzed using a repeated-measures analysis of variance (ANOVA). Regarding the remaining data, one-way ANOVA followed by a least significant difference post hoc test was used to compare the results among multiple groups and Student’s p > 0.05, Fig. p < 0.01, Fig. p < 0.01, p < 0.01, respectively; Fig. t = −0.170, p = 0.866, Fig. t = −4.990, p = 0.000, Fig. F = 28.003, p = 0.000). The rats in the CUS + RUN group showed a significantly higher sucrose preference than the rats in the CUS + STD group  = 24.182, p = 0.000; F = 10.510, p = 0.002; F = 7.899, p = 0.006). Specifically, there were markedly more Iba1+ microglia in the CA1, CA2/3, and DG in the CUS + STD group of rats than in the control group of rats . Moreover, after 6 weeks of running exercise, the CUS + RUN group had decreased numbers of Iba1+ microglia in the CA1, CA2/3, and DG compared with the CUS + STD group  was much <50% of the observed interindividual variance (OCV2)  = 6.482, p = 0.012; F = 6.349, p = 0.013; F = 18.676, p = 0.000) and the percentages of Iba1+/CD68+ microglia in Iba1+ cells in the CA1, CA2/3, and DG  = 5.851, p = 0.017; F = 5.935, p = 0.016; F = 15.876, p = 0.000) among the three groups. Specifically, relative to the rats in the control group, the densities of Iba1+/CD68+ microglia and percentages of Iba1+/CD68+ microglia in Iba1+ cells in the CA1, CA2/3, and DG areas were elevated in the CUS + STD group rats , indicating that CUS promoted microglial activation. In addition, the density of Iba1+/CD68+ microglia and percentage of Iba1+/CD68+ microglia in Iba1+ cells in the DG area of the hippocampus in the CUS + RUN group were found to be decreased . However, the densities of Iba1+/CD68+ microglia and percentages of Iba1+/CD68+ microglia in Iba1+ cells in CA1 and CA2/3 of the hippocampus in the CUS + RUN group did not significantly differ from those in the CUS + STD group   = 7.940, p = 0.006; F = 4.248, p = 0.040). Specifically, compared to the control group, the CUS exposure significantly increased the mRNA levels of IL-1β and iNOS in the hippocampus  = 0.810, p = 0.468; F = 0.497, p = 0.621; Fig. The changes in inflammatory cytokines  at the genetic level were detected by qRT-PCR. As shown in Fig.  = 6.725, p = 0.011; F = 10.003, p = 0.003; Fig. p = 0.043 and p = 0.035, respectively). Compared with the CUS + STD group, the protein levels of IL-1β and IL-6 were significantly diminished in the hippocampi of the rats in the CUS + RUN group . In addition, the protein levels of iNOS in the CUS + STD group were significantly increased compared to those in the control group  = 0.136, p = 0.875; Fig. The protein levels of inflammatory cytokines  were measured by ELISA. There were significant differences in the protein levels of IL-1β and IL-6 in the hippocampus among the three groups  induced by CUS are consistent across the three subregions of the hippocampus. Collectively, our present results suggest that microglial number changes in the hippocampus may be involved in the pathogenesis of the depression model, and we are the first to provide evidence that CUS affects the number of microglia in each subregion of the hippocampus. Although most previous studies and the present study found microglial changes in the hippocampi of depression models [Increasing evidence suggests that alterations in the number of microglia are associated with the onset of depression, and the change in the number of total microglia helps determine whether microglia are in the activated or senescent phase , 48. Ibapression –53. In tsed mice , 55. The adopted . Cai et n models , 56, 57,+/CD68+ cells and the percentage of Iba1+/CD68+ cells among Iba1+ cells in the CA1, CA2/3, and DG of the hippocampus, revealing that chronic stress induced microglial activation in the hippocampus. Microglia can be activated during inflammatory conditions that cause deviations from microglial homeostasis, such as stress, stroke, infection, and neurodegenerative diseases [In addition to the changes in the total number of microglia, microglial functional alterations, including microglial activation and concomitant elevations in pro-inflammatory biomarker levels, play crucial roles in the deterioration of depression , 59. Chadiseases . In the diseases . Thus, tdiseases . As IL-1diseases , 67, thi+ microglial populations in the hippocampal CA1, CA2/3, and DG subregions in depression model rats. The current study is the first to provide unbiased and accurate quantitative evidence suggesting that the total number of microglia in the hippocampus is altered in CUS model rats after running exercise. More importantly, our results indicate that hippocampal microglia might be structural targets for the ameliorative effects of running exercise on depression.Running exercise is recognized as an effective therapeutic strategy for depression . Consist+ microglia in the CA1, CA2/3, and DG subregions of the hippocampus in the depression model rats but attenuated the increase in the density of Iba1+/CD68+ microglia only in the DG region of the hippocampus. Impaired functional activity in the hippocampal CA1, CA2/3, and DG subregions has been found to be associated with depressive symptoms [As mentioned above, microglial activation and the levels of inflammatory factors can better reflect the effects of microglia on the neuroinflammatory response than the total number of microglia. In the present study, we found that running exercise attenuated the increases in the number of Iba1symptoms , and thesymptoms . A studysymptoms . In addisymptoms . Intrigusymptoms . Thus, wsymptoms , 78. OurIn conclusion, our study shows that the number and activation of hippocampal microglia along with accompanying neuroinflammation in the hippocampus may be involved in the pathogenesis of depression in model rats. In addition, running exercise reverses these changes in the context of depression, indicating that changes in hippocampal microglia and neuroinflammation might be the structural bases by which running exercise exerts antidepressant effects. Our findings may shed new light on immunomodulatory targets related to the antidepressant effects of exercise and provide a scientific basis for the clinical treatment strategy for depression.Figure legends of supplementary Figure 1Supplementary figure 1


Obesity is an evolutionary, chronic, and relapsing disease that consists of a pathological accumulation of adipose tissue able to increase morbidity for high blood pressure, type 2 diabetes, metabolic syndrome, and obstructive sleep apnea in adults, children, and adolescents. Despite intense research over the last 20 years, obesity remains today a disease with a complex and multifactorial etiology. Recently, long non‐coding RNAs (lncRNAs) are emerging as interesting new regulators as different lncRNAs have been found to play a role in early and late phases of adipogenesis and to be implicated in obesity‐associated complications onset. In this review, we discuss the most recent advances on the role of lncRNAs in adipocyte biology and in obesity‐associated complications. Indeed, more and more researchers are focusing on investigating the underlying roles that these molecular modulators could play. Even if a significant number of evidence is correlation‐based, with lncRNAs being differentially expressed in a specific disease, recent works are now focused on deeply analyzing how lncRNAs can effectively modulate the disease pathogenesis onset and progression. LncRNAs possibly represent new molecular markers useful in the future for both the early diagnosis and a prompt clinical management of patients with obesity. ADINRadipogenic differentiation‐induced ncRNAAdipoQ ASadiponectin antisense RNAADNCRadipocyte differentiation‐associated lncRNAAFatrial fibrillationAngIIangiotensin IIANRILantisense ncRNA in the INK4 LocusAPFautophagy promoting factorASMER‐1 and ASMER‐2adipocyte‐specific metabolic‐related lncRNAsBMIbody mass indexCAIFcardiac autophagy inhibitory factorCARLcardiac apoptosis‐related lncRNACDKN2B‐AS1cyclin‐dependent kinase inhibitor 2B antisense RNA 1Chaercardiac‐hypertrophy‐associated epigenetic regulatorCHDcoronary heart diseasesCHRFcardiac hypertrophy‐related factorCIDECcell death‐inducing DFF45‐like effectorCVDcardiovascular diseasesDGATdiacylgycerolacyltransferaseDNdiabetic nephropathyDNMT1DNA methyl transferase 1GiverGrowth factor‐ and pro‐Inflammatory cytokine‐induced Vascular cell‐Expressed lncRNAhADSCshuman adipose‐derived stem cellsHFDhigh‐fat diethLMRhuman lncRNA metabolic regulatorsHOTAIRHOX transcript antisense RNAHRCRheart‐related circRNAIMFNCRintramuscular fat‐associated lncRNAIRinsulin resistanceLIPCARlong intergenic non‐coding RNA predicting cardiac remodelinglnc‐ORAobesity‐related lncRNAlncRNAlong non‐coding RNAsMALAT1metastasis‐associated lung adenocarcinoma transcript 1MCEmitotic clonal expansion phaseMDRLmitochondrial dynamic‐related lncRNAMeg3maternally expressed gene 3MHRTmyosin heavy chain associated RNA transcriptsMIATmyocardial infarction‐associated transcriptMIR221HGmiR‐221 host geneMIR31HGmiR‐31 host geneMIRT1myocardial infarction‐associated transcript 1NAFLDnonalcoholic fatty liver diseaseOAosteoarthritisPRC2Polycomb Repressor Complex 2SDstandard deviationsSRAsteroid receptor RNA activatorT2Dtype 2 diabetesTINCRtissue differentiation‐inducing non‐protein coding RNAVSMCsvascular smooth muscle cellsWHOWorld Health OrganizationWisperWisp2 super‐enhancer‐associated RNAβlinc1β‐cell long intergenic noncoding RNA1Obesity is defined by the World Health Organization (WHO) as a condition of abnormal or excessive accumulation of body fat that presents a health risk, increasing both morbidity , and even certain forms of cancer)Conventional therapies for patients with obesity, such as lifestyle modifications (diet and exercise) and also pharmacotherapy in adults, remain important but are limited by their results in terms of weight loss and weight loss maintenance at long term, and in the next future the development of new combinatory clinical approaches is needed.2In recent years, the role of RNA is changed, and indeed it is now established knowledge that only 1–2% of the human genome codes for protein.Interestingly, lncRNAs can mediate transcriptional regulation in different ways. Indeed, these molecules can modulate gene expression at multiple levels, ranging from chromatin re‐arrangements to transcriptional regulation or even translational modulation.3Noncoding RNAs are known to play a regulatory role in many developmental contexts, including adipogenesis. Indeed, lncRNAs have been demonstrated to be involved in adipogenesis with subsequent implications for obesity and obesity‐related complications in adults and children.3.1Adipogenesis is the process of adipocytes formation into fat‐containing cells from stem cells or adipocyte precursors. It involves two phases: determination (considered an early stage) and terminal differentiation (late adipogenesis).Early stages of adipogenesis are represented by a mitotic clonal expansion phase (MCE) and by the expression of early regulators such as C/EBPβ and C/EBPδ.The genetic location of lncRNAs could be of crucial relevance in identifying their target genes. Three recently discovered lncRNAs, Gm15051, Tmem189, and Cebpd genomically, locate respectively next to Hoxa1, C/EBPβ, and C/EBPδ in mouse, and their expression levels correlate, suggesting that each of them can positively influence the neighboring gene's expression having the role of transcriptional regulators.3.2As pre‐adipocytes mature into adipocytes, C/EBPβ and δ target the promoters of C/EBPα and PPARγ, master regulators of adipocytes terminal differentiation as they activate genes that are involved in insulin sensitivity, lipogenesis, and lipolysis, with subsequent implications for diseases involving lipid metabolism such as dyslipidemia.This step is critical for late adipocyte differentiation, and indeed numerous lncRNAs have been found to modulate specifically PPARγ Figure , along wPPARγ is not the only player in late adipogenesis, and indeed, lncRNAs can modulate other key targets. Specifically, knockdown of the lncRNA HOXA11‐AS1 can result in the inhibition of adipocyte differentiation through a decrease of C/EBPα, diacylgycerolacyltransferase (DGAT) 2, cell death‐inducing DFF45‐like effector (CIDEC), and perilipin.A specific subclass of lncRNAs, defined as “antisense RNAs,” can modulate the expression of their respective sense gene altering processes in which they are involved. For example, PU.1AS can form a RNA‐duplex with PU1, a molecule that inhibits adipogenesis, hindering its expression and subsequent protein expression with a decreased expression of PPARγ, fatty acid synthase, and adiponectin in mouse.The lncRNA's correlation with adipogenesis can also be negative, as some lncRNAs have been found to be decreased in adipogenesis, such as lnc‐U90926 in murine 3T3‐L1 pre‐adipocytes,Further studies might be needed to clarify specific lncRNA's functions in this process, as controversial evidences are also present. This is the case of maternally expressed gene 3 (Meg3), a novel lncRNA which has been defined as both able to inhibit and promote adipogenesis.3.3−/− mice have a phenotype of resistance to HFD‐induced obesity with decreased fat mass, reduced fatty liver, and improved glucose tolerance.Specific studies correlate lncRNAs with the obese phenotype and obesogenic models. Among them, SRA has been demonstrated to be strictly associated with obesity, as it has been shown that SRAFrom an anatomical point of view, lncRNAs expression can differ in different fat depots, as it is for HOX transcript antisense RNA (HOTAIR) which has been demonstrated to be highly expressed in gluteal‐femoral fat, and mechanical stimulation of this area in human subjects induces exosomal secretion of HOTAIR, which then circulates in the bloodstream resulting in higher serum expression in subjects with obesity and a sedentary lifestyle.4Given the strong implications of lncRNAs in adipogenesis and adipocytes differentiation, it was a natural evolution to study the role of these molecular modulators in obesity and in the related most common complications.4.1Cardiovascular diseases (CVD) include myocardial infarction, CHD, cardiac hypertrophy, heart failure, AF, and atherosclerotic thrombosis.LncRNAs can also target the apoptotic process in cardiomyocytes, which can lead to myocardial infarction. P53 is also implicated in apoptosis modulation, and the lncRNA Meg3 can target p53 and subsequently modulate NF‐κB‐ and ERS‐associated apoptosis in murine ventricular myocytes.Myocardial infarction can indeed influence a differential lncRNAs expression. Specifically, acute myocardial infarction in mice was associated with the upregulation of two lncRNAs named myocardial infarction‐associated transcript 1 (MIRT1) and 2 (MIRT2), which negatively correlated with infarct size and positively correlated with ejection fraction. MIRT1 and MIRT2 modulate the expression of multiple genes known to be involved in processes affecting left ventricular remodeling, such as extracellular matrix turnover, inflammation, fibrosis, and apoptosis.LncRNAs can also influence cardiac hypertrophy and thus aggravate CVD, as cardiac hypertrophy is a crucial hallmark of heart failure.Another form of CVD is AF, which is the most common type of arrhythmia.Numerous lncRNAs have also been found deregulated in CHD, with one recent work highlighting a network of 62 lncRNAs, 332 miRNAs, and 366 mRNA differentially expressed in peripheral blood mononuclear cells (PBMCs) of patients with CHD versus controls.Lastly, lncRNAs are also being investigated for their role in atherosclerotic thrombosis, with multiple recent works focusing especially on this topic.In conclusion, recent evidence indicates the important roles of lncRNAs in the complex regulatory network of CVD, and many of them could be used as novel therapeutic targets and/or biomarkers for early diagnosis or prognosis for CVD. Indeed, current therapies for CVD such as cardiac hypertrophy currently alleviates symptoms, but new genetic analyses could provide new therapeutic targets.4.2Multiple lncRNAs have been found to be upregulated in the plasma of patients with hypertension, such as AK125261, AK098656, and TUG1.A second process that can be modulated by lncRNAs in hypertension is indeed muscular remodeling. Vascular remodeling is an active process that involves changes in cellular growth, apoptosis, migration, inflammation, and production of extracellular matrix proteins. The lncRNA GAS5 can also regulate this process as it can remodel arteries such as the caudal, carotid, renal, and thoracic ones. Indeed, GAS5's knockdown regulate the function of endothelial cells and VSMCs through β‐catenin signaling.Polymorphisms in specific lncRNAs can also induce disease pathology. This is the case of cyclin‐dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B‐AS1), also termed ANRIL and previously mentioned for its implication in CVD, where polymorphisms in its sequence may contribute to higher systolic blood pressure in hypertensive patients.4.3At all ages, the risk of T2D rises with increasing body fat. The prevalence of T2D is 3 to 7 times higher in those who are affected by obesity than in normal‐weight adults. Specifically, T2D is an adult‐onset, non‐ insulin‐dependent type of diabetes and is strictly linked to obesity.Indeed, lncRNAs can be both upregulated and downregulated during disease progression in different cell types Figure . ExpressInterestingly, many lncRNAs have also been reported to be downregulated in patients with T2D. When considering PBMCs screening studies, results showed that multiple lncRNAs were found downregulated. These include LINC00523, LINC00994,Indeed, lncRNAs can modulate the cellular activity of pancreatic β cells. lncRNA‐p3134, found deregulated in human patients and diabetic mice, seems to act as a new signaling molecule that maintains β‐cell mass and enhances insulin synthesis and secretion, and indeed it has been seen that lncRNA‐p3134 can contribute to reverse the insufficient insulin secretion in T2D.4.4Obesity is a major risk factor for the development of chronic kidney disease, through the direct development of nephropathy.Among the other lncRNAs implicated, Rpph1 was found upregulated in mice with DN, regulating also cell proliferation and inflammatory cytokines production in mesangial cells, through a direct interaction with galectin‐3.4.5Obesity can impact tissue types other than the adipose tissue, and indeed it can significantly impact both the musculoskeletal and immune systems, leading to the development of OA.Even so, future works will need to specifically focus on the link between OA, lncRNAs, and obesity. Nanus and co‐authors reported 19 differentially expressed lncRNAs in normal‐weight OA versus non‐OA patient fibroblasts, and these are MALAT1, MIR155HG, SMILR, LINC01426, RP11‐863P13.3, CARMN, RP11‐79H23.3, RP11‐362F19.1, RP11‐290 M5.4, VLDLR‐AS1, RP11‐536 K7.3, HAGLR, LINC01915, RP11‐367F23.2, RP11‐392O17.1, LINC01705, LINC01021, DNAJC27‐AS1, and AF131217.1.4.6Obesity is also linked with the development of hepatic metabolic disease, as nonalcoholic fatty liver disease (NAFLD) and especially its most severe form  present an increased prevalence in patients with obesity (from 3% to 20–40%).Also in this case, RNA sequencing and microarrays allowed the identification of numerous new putative candidates. Indeed, numerous high‐throughput studies were performed in both murine models4.7Obesity is probably the main cause for the development dyslipidemia, which typically consists of increased triglycerides, free fatty acids, apolipoprotein B, and LDL‐C, and decreased HDL‐C.Indeed, lncRNAs can deeply influence lipid homeostasis, but further studies are required in order to determine whether lncRNAs that regulate lipogenesis, lipolysis, β‐oxidation, adipogenesis, and thermogenesis could also become biomarkers for therapies that target dyslipidemias.5Obesity is a complex disease representing a great burden on the health care system, commonly leading to the development of co‐morbidities also in pediatrics. Epigenetics through RNA biology might play a crucial role in elucidating new targetable pathways, and in this context lncRNAs are emerging as interesting new candidate targets and players. Indeed, obesity‐associated lncRNAs play a crucial role in adipose tissue modulation, but their action is not limited to this, as they have been implicated in modulating obesogenic co‐morbidities influencing the cardiovascular system, the immune system, the liver, and even the musculoskeletal system.The authors declare that they have no conflict of interest.Table S1: List of lncRNAs cited in the manuscript listed in alphabetical order with the number of known orthologues and a specific focus on their presence in the most common models Click here for additional data file.


GDF2 (BMP9 gene) nonsense mutations. We studied two pediatric patients homozygous for GDF2 (BMP9 gene) nonsense mutations: one with PAH ; both with facial telangiectases.Disrupted endothelial BMP9/10 signaling may contribute to the pathophysiology of both hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH), yet loss of circulating BMP9 has not been confirmed in individuals with ultra‐rare homozygous Plasma samples were assayed for BMP9 and BMP10 by ELISA. In parallel, serum BMP activity was assayed using an endothelial BRE‐luciferase reporter cell line (HMEC1‐BRE). Proteins were expressed for assessment of secretion and processing.Plasma levels of both BMP9 and BMP10 were undetectable in the two homozygous index cases and this corresponded to low serum‐derived endothelial BMP activity in the patients. Measured BMP9 and BMP10 levels were reduced in the asymptomatic heterozygous p.[Glu279Ter] parents, but serum activity was normal. Although expression studies suggested alternate translation can be initiated at Met57 in the p.[Gln26Ter] mutant, this does not result in secretion of functional BMP9.GDF2 mutations, leading to a loss of circulating BMP9 and BMP10, can cause either pediatric PAH and/or “HHT‐like” telangiectases and PAVMs. Although patients reported to date have manifestations that overlap with those of HHT, none meet the Curaçao criteria for HHT and seem distinct from HHT in terms of the location and appearance of telangiectases, and a tendency for tiny, diffuse PAVMs.Collectively, these data show that homozygous  Ultra‐rare homozygous GDF2 mutations have not previously been shown to lead to loss of circulating BMP9 protein. We show that homozygous GDF2 mutations in two pediatric individuals from two different families, lead to loss of plasma BMP9 and BMP10, resulting in a loss of serum‐derived BMP‐dependent endothelial signaling. The two individuals have different vascular dysplasias: one with pulmonary arterial hypertension and the second with a pulmonary arteriovenous and telangiectasia syndrome that is distinct from hereditary hemorrhagic telangiectasia but may be mistakenly diagnosed as hereditary hemorrhagic telangiectasia. Recognizing that the vascular malformations of HHT did not occur randomly, the consensus clinical diagnostic (Curaçao) criteria were agreed  cause HHT type 1 and HHT type 2, respectively. Mutations in these genes account for up to 96% of all HHT cases  mutations cause a syndrome of combined Juvenile polyposis and HHT , account for 53%–86% of familial PAH cases and 14%–35% of idiopathic cases  and BMP10  missense, truncating, and deletion mutations have been reported in patients with PAH on four separate occasions, bringing the total number of reported cases to 32  and a second patient  who exhibit a phenotype of PAVMs and cutaneous telangiectases, with no evidence of PAH. Although there is some overlap of this disease phenotype with HHT, careful examination of the phenotype suggests this is a distinct “HHT‐like” syndrome.Here we characterize the plasma levels of BMP9 and pBMP10 and serum‐derived endothelial BMP activity in two pediatric patients homozygous for 22.1This study was approved by the following ethical review boards: IRB_00020480 (Utah) and NIHR BioResource for Rare Diseases PAH Project (Sheffield). Informed consent was obtained from all human subjects in this study, though both families specifically refused consent for the inclusion of photographs of external features.2.2After informed consent was given, blood samples were collected into EDTA tubes for plasma preparation and serum separator tubes for serum. Samples were centrifuged within 30 min of collection. The plasma and serum fractions were then removed from their respective tubes and stored at −80°C until assay.2.3® TOP10 Chemically Competent E. coli cells and clones were selected and amplified. Plasmids were purified using PureLink™ HiPure Plasmid Filter Maxiprep Kit (ThermoFisher). The resulting constructs were confirmed by Sanger sequencing using T7 and BGH primers.The plasmid pcDNA3.1/V5‐His‐TOPO/human BMP9‐WT  supplemented with 10% fetal bovine serum  and antibiotic/antimycotic . HMEC‐1 human microvascular endothelial cells stably expressing the BRE‐luciferase reporter (HMEC‐1‐BRE) generated and cultured as described previously  for 6 hr. Cells were lysed with 100 µL OneGlo reagent (Promega), samples were transferred to a white plate, and luciferase activity was measured by a luminescence reader. Samples were measured in duplicate and assays using serum were found to be most sensitive and reliable as plasma was prone to clotting at 2%–3% by volume.2.8HEK‐EBNA cells  were grown to confluence in 6 cm dishes, and then washed with Optimem. Cells were transfected with plasmids (2 µg) and Lipofectamine 2000 (15 µL) according the manufacturer's instructions. After 5 hr, the transfection mix was replaced with 3 ml of serum‐free chemically defined Chinese hamster cell over‐expression media . After 3 days, conditioned media were collected, cleared by centrifugation, aliquoted, and frozen at −80°C. Cell lysates were collected in SDS lysis buffer  supplemented with an EDTA‐free protease inhibitor (Roche). Three independent batches of cells were transfected and harvested. The activity of the Pro:BMP9‐WT control was identical to previously validated Pro:BMP9‐WT  to quantify the protein concentration of each sample. Conditioned media (60 µL) or lysates  were mixed with 5x SDS loading buffer  and boiled at 98°C for 5 min to denature and reduce proteins. Samples were then analyzed by gel electrophoresis and western blotting. For the growth factor domain, samples were blocked in 5% BSA and probed using sc‐514211 . For the prodomain, samples were blocked in 5% non‐fat milk protein (Marvel) and probed using AF3879 . Lysate blots were re‐probed using an antibody toward β‐actin .33.1GDF2 gene as described below:Two individuals from different families were identified as harboring homozygous germ‐line nonsense mutations in the Individual 1:  has pulmonary arterial hypertension (PAH) and multiple spider‐like telangiectases were noted on his face bilaterally during his most recent evaluation at 10‐years of age. A targeted full body exam for cutaneous vascular lesions was otherwise negative. He has no history of even minor recurring epistaxis or other bleeding. An agitated saline contrast echocardiogram was negative for evidence of a pulmonary shunt/PAVM.2, and pulmonary vascular resistance of 4.4 Wood Units. No clinical features of HHT were noted at this time. Given the diagnosis of PAH, the KCNK3 (OMIM 603220), KCNA5 (OMIM 176267), CAV1 (OMIM: 601047), BMPR2, SMAD4, ENG, and ACVRL1 genes were also sequenced, with no pathogenic or suspicious variant detected. The homozygous GDF2 variant was reported as the suspected cause of his PAH  was a 9‐year‐old female who presented with symptoms of a viral respiratory illness, and on admission was noted to be hypoxemic but otherwise did not appear to be acutely unwell. Her mother commented that her daughter's fingers had been “blue tinged” for several weeks. Her saturations on room air were 88%. Heart sounds, electrocardiogram, and chest X‐Ray were all normal and auscultation of her chest was unremarkable; in particular, no bruits could be heard. Hemoglobin level was elevated at 170 g/dL .Multiple spider‐like and linear telangiectases were noted on the cheeks and chin, but not the lips or oral cavity. These telangiectases had first been documented at 3 years of age. On direct questioning she had a history of infrequent epistaxis occurring every few months. Her parents and brother were well although her brother also had a history of infrequent epistaxis. Of note, her parents were consanguineous.Given the cyanosis and telangiectases, a diagnosis of HHT with PAVM was considered and a CT pulmonary angiogram was performed. This showed no evidence of macroscopic PAVMs. The pulmonary artery and right‐sided heart chambers were unremarkable. Given the ongoing hypoxemia, a transthoracic contrast echocardiogram was then performed which demonstrated left atrial opacification after >3 cardiac cycles consistent with an intrapulmonary shunt, while no intra‐cardiac shunt could be visualized. Formal right heart catheterization and pulmonary angiography were therefore performed, revealing multiple small PAVMs in both lower lobes: the right middle lobe and the right upper lobe Figure . There wACVRL1, ENG, and SMAD4 was normal; large deletion/duplication analysis of ACVRL1 and ENG was also normal. The patient was, however, homozygous for a non‐sense variant in the GDF2 gene . Both parents were heterozygous for this variant, which was not present in the brother. Neither parent had any symptoms or clinical signs suggestive of HHT, and PAVMs were excluded by a negative bubble transthoracic echocardiogram in one parent and a negative thoracic CT scan in the other.The diagnosis of HHT was initially suspected, but the atypical nature of the telangiectases and the infrequent nature of epistaxis means that only one of the four Curaçao criteria  were met  levels , we postulated that both homozygous truncating mutations would lead to a complete absence of circulating BMP9. Plasma BMP9 levels were measured in the p.[Gln26Ter];[Gln26Ter] and p.[Glu279Ter];[Glu279Ter] homozygotes, and the p.[Glu279Ter] heterozygous parents by ELISA Figure . As expes Figure .BMP9 and BMP10 are the main mediators of plasma/serum‐dependent activation of the Smad‐responsive BRE‐luciferase reporter in the HMEC1‐BRE reporter line . HEK293 T cell lysates were positive for both the prodomain and GFD from all these vectors, demonstrating that translation can be initiated at Met57 and a truncated protein results  variants have not confirmed that these mutations cause a loss of circulating BMP9. Here, we confirm that plasma BMP9 and pBMP10 levels and serum‐derived endothelial BMP activity are reduced in two individuals harboring homozygous GDF2 mutations. One individual has PAH and the other has PAVMs, with cutaneous telangiectases common to both. Collectively, the cases presented here and previously published reports  and typically occur on the lips and oral cavity, not other aspects of the face. A lack of cutaneous or nasal telangiectases was also noted in the report by Liu et al. of a child with a homozygous GDF2 mutation , who presented with hypoxia and PAVMs  and diffuse, such that they were detectable by angiography, but not CT imaging  mutation  and EU (EP3166628B1) patents entitled: “Therapeutic Use of Bone Morphogenetic Proteins.” All other authors declare that they have no competing interests.Jamie McDonald, Oliver Quarrell, Pinar Bayrak‐Toydemir, Whitney Wooderchak‐Donahue, Robin Condliffe, Carmelo Bernabeu, Paul D Upton carried out conceptualization; Joshua Hodgson, Lidia Ruiz‐Llorente, Oliver Quarrell, Kelechi Ugonna, James Bentham, Rebecca Mason, Jennifer Martin, David Moore, Katie Bergstrom, Pinar Bayrak‐Toydemir, Whitney Wooderchak‐Donahue, Robin Condliffe, Carmelo Bernabeu, Paul D Upton performed data curation; Joshua Hodgson, Lidia Ruiz‐Llorente, Jamie McDonald, Robin Condliffe, Carmelo Bernabeu, Paul D Upton performed formal analysis; Joshua Hodgson, Lidia Ruiz‐Llorente, Robin Condliffe, Carmelo Bernabeu, Paul D Upton performed methodology; Joshua Hodgson, Lidia Ruiz‐Llorente, Jamie McDonald, Oliver Quarrell, David Moore, Katie Bergstrom, Robin Condliffe, Carmelo Bernabeu, Paul D Upton performed validation; Joshua Hodgson, Lidia Ruiz‐Llorente, Jamie McDonald, Oliver Quarrell, Kelechi Ugonna, James Bentham, David Moore, Katie Bergstrom, Pinar Bayrak‐Toydemir, Whitney Wooderchak‐Donahue, Nicholas W Morrell, Robin Condliffe, Carmelo Bernabeu, Paul D Upton carried out investigation; Joshua Hodgson, Lidia Ruiz‐Llorente, Jamie McDonald, Robin Condliffe, Carmelo Bernabeu, Paul D Upton were involved in writing—original draft preparation; Jamie McDonald, Oliver Quarrell, Pinar Bayrak‐Toydemir, Whitney Wooderchak‐Donahue, Nicholas W Morrell, Robin Condliffe, Carmelo Bernabeu, Paul D Upton; Visualization, Jamie McDonald, Nicholas W Morrell, Robin Condliffe, Carmelo Bernabeu, Paul D Upton; Supervision, Nicholas W Morrell, Robin Condliffe, Carmelo Bernabeu, Paul D Upton did writing—review and editing; Rebecca Mason and Jennifer Martin project administration,; Funding acquisition, Nicholas W Morrell, Carmelo Bernabeu, Paul D Upton. All authors have read and agreed to the published version of the manuscript.


DNMT3B − 149 polymorphism, and DNA damage on lung cancer occurrence.Smoking increases DNA methylation and DNA damage, and DNA damage acts as a vital cause of tumor development. The DNA methyltransferase 3B (DNMT3B) enhances promoter activity and methylation of tumor suppressor genes. Tea polyphenols may inhibit DNMT activity. We designed a case-control study to evaluate the combined effects of smoking, green tea consumption, Questionnaires were administered to obtain demographic characteristics, life styles, and family histories of lung cancer from 190 primary lung cancer cases and 380 healthy controls. Genotypes and cellular DNA damage were determined by polymerase chain reaction and comet assay, respectively.DNMT3B − 149 CT genotype, smokers carrying the TT genotype had a greater lung cancer risk . DNA damage levels were divided by the tertile of the healthy controls’ values. Compared to nonsmokers with low DNA damage, smokers with moderate DNA damage  and smokers with high DNA damage  had elevated lung cancer risks. Interaction between smoking and DNA damage significantly affected lung cancer risk.The mean DNA tail moment for lung cancer cases was significantly higher than that for healthy controls. Compared to nonsmokers carrying the DNMT3B − 149 TT genotype, which has higher promoter activity, can increase the lung cancer risk elicited by smoking, and DNA damage may further promote smoking related lung cancer development.Our study suggested that the  Lung cancer is the major cause of death from cancer around the world , 2. A stMethylation modification typically occurs in the gene promoter, especially in the CpG dinucleotide . It has DNMT3B is located on chromosome 20q11.2, which contains a C-to-T transition (rs2424913) in the promoter, − 149 bp from the transcription start site [DNMT3B − 149 polymorphism and specific cancers seldom take into account the effect of environmental factors. Our previous study further revealed that the DNMT3B − 149 TT genotype can increase the lung cancer risk caused by smoking [Specifically, it has been suggested that cigarette smoking arouses DNA methylation , 4, causn rs24249 in the part site . So far, smoking .DNMT3B genotypes can modify the above relationship.Another one of our studies also reported that individuals who never drink green tea have elevated lung cancer risk compared to those who drink at least one cup of green tea per day, and the effect is more pronounced in smokers . Green tDNMT3B − 149 genotypes are related to the level of DNA damage in individuals. We further tested the interactions of smoking, green tea consumption, DNMT3B − 149 genotypes, and individual DNA damage level in the occurrence of lung cancer.In the present study, we are interested in evaluating whether smoking, green tea consumption, and The design and final reports of this study complied with the Helsinki declaration and were approved by the institutional review board of the participating institutions . Informed consent statements were obtained from all participants.From August 2004 to October 2011, a total of 271 lung cancer  patients were recruited from participating institutions in central Taiwan. These hospitals were accessible to patients from all socioeconomic classes. Eligible cases were 20 years of age or older. All patients underwent a series of examinations of pathologic stages by board-certified pathologists. Tumor types and stages were determined according to the World Health Organization classification. Ten patients were not interviewed because of severe illness, 37 patients were not incident cases, and 34 patients were too old (range = 81–92 years) or were without complete questionnaire data. None of the included patients had been exposed to radiotherapy or chemotherapy. The demographic characteristics of excluded patients were comparable with those of included patients, with the exception of age. Among the 190 patients available for matching, cancer types were categorized as follows: 108 (56.8%) patients with adenocarcinoma, 51 (26.9%) with squamous cell carcinoma, and 31 with others . Moreover, 132 (69.5%) patients had an onset age of older than 60 years old, 38 (20.0%) patients had an onset age of between 50 and 59 years, and 20 (10.5%) patients had an onset age of younger than 50 years old.In this study, two controls were individually matched with each lung cancer case by age  and gender. There was no familial relationship among and between cases and controls. They were also selected from the same geographic areas as the lung cancer cases. During the same period of case recruitment, 380 controls were selected randomly from participants with no history of cancer or pulmonary diseases at the time of diagnosis, including tuberculosis, pneumonia, bronchiectasis, pneumoconiosis, pulmonary alveolar pneumonopathy, chronic obstructive pulmonary disease, and asthma. They admitted to the same hospitals for physical check-ups. The primary reason why our potential controls refused to participate in the study was that most subjects were unwilling to take the time for an interview.Epidemiological information was collected from study participants through in-person interviews using a standardized questionnaire including demographic and lifestyle items. Subjects’ cumulative smoking dose was calculated by pack-years, defined as the number of packs of cigarettes smoked daily multiplied by the active smoking years. The same tea leaf can be brewed many times and tea is served in small cups (30–50 ml). A standard cup of tea was defined as 100–120 ml in this study. The period of exposure was assessed from birth to the day when lung cancer was first diagnosed for cases or when the interview was performed for controls. The frequency of green tea consumption was categorized as every day (more than one cup per day), three to four cups per week, one to two cups per week, one to two cups per month, and seldom. The number of cups consumed was assessed from five possible answers (for those who drank tea every day): less than one cup a day, one to two cups a day, three to four cups a day, five to nine cups a day, and ten or more cups a day. The evaluation of green tea consumption was based on a previous study , in whicVenous blood from all participants was collected in heparin tubes, and prepared into plasma, buffy coat and red blood cells. Buffy coat was used to extract genomic DNA by using a Genomic DNA isolation kit .DNMT3B. Primers used for the amplification were 5′-TGC TGT GAC AGG CAG AGC AG-3′ and 5′-GGT AGC CGG GAA CTC CAC GG-3′. The PCR final volume was 50 μl containing DNA (0.5 μl), PCR buffer with 200 ng of primers, 1.5 mM MgCl2, 0.2 mM dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), and 0.1% bovine serum albumin. Amplification conditions were the initial denaturation at 95 °C for 5 min, under 35 cycles of amplification: denaturation at 95 °C for 30 s, annealing at 63 °C for 90 s, elongation at 72 °C for 25 s, and extension at 72 °C for 40 s. The PCR products were digested with BfaI at 16 h at 37 °C. Homozygous CC individuals had product fragments of 208, 126, and 46 bp, while homozygous TT individuals had product fragments of 162, 126, and 46 bp, and heterozygous CT individuals had all four fragments.Polymerase chain reaction (PCR)-restriction fragment length polymorphism was used to distinguish the variation of rs2424913 in 2EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% DMSO) for 1 h at 4 °C. Slides were placed in buffer  for 15 min. Next, the slides were washed three times for 5 min with PBS, moved to an electrophoresis tank, and then stained with 10% ethidium bromide. For each participant, 100 randomly captured comets from slides (25 cells on each of four comet slides) were examined at × 400 magnification using an epifluorescence microscope connected through a black and white camera to an image analysis system . A computerized image analysis system acquired images, integrated intensity profiles, estimated the comet cell components, and evaluated the range of derived variables for each cell. Undamaged cells have an intact nucleus and damaged cells have the appearance of a comet. Tail moment was calculated as the product of the tail length and the DNA fraction in the comet tail to quantify DNA damage. All slides were counted by a reader who was blind to the status of participants.The comet assay was conducted under alkali conditions according to the procedure of Singh et al. . The 10 DNMT3B − 149 T allele in a previous study [Initially, this study estimated four sample sizes. The same type I (α) error of 0.05, type II (β) error of 0.2, and odds ratio (OR) of 1.8 were given. According to the prevalence of smoking status, green tea consumption, us study , and DNAt test for the age variable and a χ2-test or Fisher’s exact test for discrete variables. Hardy-Weinberg equilibrium was performed to test DNMT3B − 149 genotypes for the goodness of fit χ2-test. Because of the positively skewed distribution of the DNA damage level, the Mann-Whitney U test and Kruskal-Wallis test were used to test the level differences for each variable. Mean and median values of the DNA damage level were also presented. Backward stepwise log-linear regression analysis was performed to reduce the full model to a more parsimonious final model, and adjusted OR and a 95% confidence interval (95% CI) were obtained for each variable. Further, likelihood ratio χ2-tests were utilized to test the interaction between two variables with respect to the risk of lung cancer. All tests were two-tailed, and all p values were considered to indicate statistical significance.All data were analyzed using SAS 9.6 software . The normal distribution of the continuous variables was checked by the Kolmogorov-Smirnov test. Subsequently, comparisons between the case and control groups were made using a Student’s DNMT3B − 149 TT genotype carriers .In total, 570 participants were recruited in this study , and the characteristics of this study participants are summarized in Table p < 0.001, Mann-Whitney U test). The tail moment was not associated with various factors in lung cancer cases. We did not observe the correlation between various factors and DNA tail moment in healthy controls, with the exception of smoking habits. In the control group, smokers had lower DNA tail moments than nonsmokers . Similarly, healthy controls with cumulative smoking of more than 40 pack-years and 1–39 pack-years also had significantly lower DNA tail moments than did nonsmokers . Further, we divided the DNA damage levels into high, moderate, and low groups by the tertile of the healthy controls’ values . A borderline significant interaction between smoking status and DNMT3B − 149 genotypes on lung cancer risk was observed (p = 0.06). Smoking status was replaced by cumulative smoking dose in subsequent analysis. A borderline significant interaction between cumulative smoking dose and DNMT3B − 149 genotypes on lung cancer risk was still maintained (p = 0.07). Similarly, nonsmokers with a low level of DNA damage were selected as the reference group. Smokers with a moderate level of DNA damage  and a high level of DNA damage  had significantly increased risks of lung cancer. A significant interaction between smoking status and DNA damage level on lung cancer risk was further observed (p < 0.01). Moreover, the test showed interaction between cumulative smoking and DNA damage level also significantly affected lung cancer risk (p = 0.01).Subsequently, we respectively analyzed the joint effects of smoking status with DNMT3B − 149 genotypes and DNA damage level on lung cancer risk  greater risk of lung cancer. Among those with a high level of DNA damage, green tea drinkers  and non-drinkers  had significantly increased risks of lung cancer. Drinking status was further stratified by consumption duration, and those with a low level of DNA damage and >  10 years of green tea consumption were selected as the reference group. Non-drinkers with a low level of DNA damage had a 2.17-fold (95% CI: 1.28–3.68) greater risk of lung cancer. Among the subjects expressing a high level of DNA damage, those who consumed green tea > 10 years , ≤ 10 years , and non-drinkers  had significantly increased risks of lung cancer. However, no significant interaction between green tea consumption and DNA damage level on lung cancer risk was found.Last, we analyzed the joint effects of green tea consumption, sk Table . HoweverDNMT3B − 149 genotypes on the development of lung cancer were observed. In the current study, DNA damage level for lung cancer cases was significantly higher than that for healthy controls. Significant effects of the interaction between smoking and DNA damage level on lung cancer risk were further revealed.As in our previous studies , 17, indThe comet assay is widely used in studies on genotoxicity testing, but rarely used in cancer epidemiological research . InteresDNMT3B polymorphism is associated with cancer development by increasing the promoter activity of DNMT3B and modulating an aberrant de novo methylation of CpG islands in some TSG [DNMT3B − 149 C to T on DNMT3B expression is still unclear. As expected, the independent effect of DNMT3B − 149 genotypes on the development of lung cancer in Taiwanese cases was observed. A study conducted in a non-Hispanic Caucasian population also showed that DNMT3B − 149 T allele was associated with increased lung cancer risk [It is reasonable to assert that the some TSG , 15. Howcer risk . Howevercer risk . The incDNMT3B − 149 genotypes on lung cancer risk is significant, although the interaction only reached marginal statistical significance. The interaction of smoking and DNA damage level of individuals also significantly affected lung cancer risk, according to the present study. Such epidemiological evidence suggests that smoking elevates lung cancer risk by increasing long-term carcinogen exposure, and simultaneously increases DNA methylation levels, providing a further opportunity to induce cancer. However, as mentioned above, DNA damage was not shown to be associated with various factors in the lung cancer group in this study, and the observed DNA damage can be regarded as the overall effect of unexplored factors. Environmental exposure to exogenous substances may lead to covalent bonding to DNA, which in turn may result in chromosomal variation; this may be the crucial step in chemical carcinogenesis [DNMT3B − 149 TT genotype, which has higher promoter activity, could increase the lung cancer risk elicited by cigarette smoking, and greater DNA damage might further promote smoking related lung cancer development. Further studies are needed to clarify the above speculation.The present study found that the combined effect between smoking and ogenesis . Individogenesis , 22. Accogenesis , 22. BasDNMT3B − 149 genotypes on lung cancer risk. Previously, an animal study showed DNA damage in the lung tissue of rats could be prevented by green tea [Tea polyphenols may prevent mutagenicity and genotoxicity, inhibit tumor initiation, promotion, and cell proliferation, regulate detoxifying enzymes, and trap activated metabolites of carcinogens , 31. Morreen tea . Our obsIn the current study, exposure to cooking fumes was associated with lung cancer risk. It is well-known that oil fumes from stir fry cooking, along with concentration of oil fumes due to poor ventilation, are associated with lung cancer . MoreoveDNMT3B − 149 T allele frequency was 95.4% in our healthy controls, which is similar that found in a Chinese report (97.8%) [DNMT3B − 149 genotypes also fell within Hardy-Weinberg equilibrium in the control group. These findings should confirm the credibility and results of our experimental technology. However, since the frequency of the DNMT3B − 149 CC genotype was small, there may have been false positive results. Additionally, the expressions of DNMT3B were not measured in this study. Therefore, it is necessary to increase the sample size and design more effective methods to confirm our results in the future. Our research may also have been subject to selection bias, because when healthy persons go to hospitals for physical check-ups, they may have healthier behavior. Previously, this study estimated the sample size based on given parameters, including β error of 0.2. Actually, the present study collected 190 lung cancer cases. According to smoking status, green tea consumption, DNMT3B − 149 genotypes, and high DNA damage level, the hazardous exposure prevalence in the control group to the above factors was 31.1, 65.8, 90.8, and 33.9%. The corresponding statistical power was 99.1, 76.8, 14.5, and 83.3%, respectively. Obviously, the statistical power of a single genotype is quite insufficient. After stratified analysis, the statistical power of risk factors for lung cancer risk was also limited due to the small sample size in this study.Although the mechanism of comet formation observed in comet assay has not been fully clarified, it has been widely used to assess DNA damage in cells , 37. How (97.8%) . The freDNMT3B − 149 TT genotype, which has higher promoter activity, could increase the lung cancer risk elicited by cigarette smoking, and greater DNA damage might further promote smoking related lung cancer development.On the whole, this study suggested that the