Tagetes spp.) in this study were classified as excluders for cadmium (Cd); however, their leaves also accumulated substantial Cd content. Among the experimental treatments , pig manure resulted in significantly increased growth performance for all marigold cultivars as seen by relative growth rates (119–132.3%) and showed positive effects on leaf anatomy modifications, e.g., thickness of spongy and palisade mesophyll, size of vein area and diameter of xylem cells. This may be due to substantially higher essential nutrient content, e.g., total nitrogen (N) and extractable phosphorus (P), in pig manure that aided all marigold cultivars, particularly the French cultivar which exhibited the highest relative growth rate (132.3%). In the Cd-only treatment, cell disorganization was observed in vascular bundles as well as in palisade and spongy mesophyll, which may have been responsible for the lowest plant growth performance recorded in this study, particularly among the American and Honey cultivars .The marigolds ( Furthermore, the OM content of animal manures  forms strong complexes with Cd resulting in the reduced mobility of Cd in soil and reduced leaching, decreasing both its toxicity and bioavailability. In one study the application of cattle manure to contaminated soil resulted in reduced Cd leaching by approximately 63%3. Many researchers have indicated that leonardite serves as an exceptional substitute for mineral-based fertilizer given that it is rich in humic acid and fulvic acid (50–75%). Application of leonardite (400 kg ha−1) and inorganics  or organic  materials can substantially improve the physicochemical properties of soil, leading to increased crop yield and quality5.Organic amendments improve soil physicochemical factors and immobilize heavy metals in soil via adsorption and isolation of cationic compounds by forming stable complexes. Soil properties affecting the availability of cadmium (Cd) include: Cd adsorption and desorption on iron (Fe) and manganese (Mn) oxides; redox potential (Eh); presence of both calcium carbonate and oxyhydroxides; pH; organic matter (OM); ionic strength; ligand anions; cation exchange capacity (CEC); phosphorus (P); and the proportion of clay minerals and percentage of charged sites occupied by Cd. Control of some of these properties can be employed to reduce Cd phytoavailability and decrease transfer of the metal into the food chain6. Physiological function, such as leaf photosynthesis and respiration, are closely associated with the changes in leaf structure, chemical function and carbon balance; the character of the changes depends on plant species and cultivar, type of root, and environmental factors7. Cadmium is a non-essential element for plants that can be readily taken up by root systems and transported predominantly into the aerial parts of marigolds (Tagetes spp.)8. Elevated Cd concentrations reduce plant growth by limiting rate of photosynthesis, reducing stomatal size in the lower epidermis, and reducing density, mesophyll thickness and intercellular spaces in the mesophyll9. To some extent, heavy metals i.e., zinc (Zn), mercury (Hg), lead (Pb) and Cd may cause changes in trichome length and density on leaf surfaces10.The leaf is the major site of photosynthesis in most vascular plants, and leaf morphology and anatomy play crucial roles in photosynthesis efficiency. Other important leaf traits such as thickness and stomatal density directly influence metal tolerance and sensitivity12. However, the deposition and desorption of heavy metals in leaves is known to vary by species, specific heavy metals content, speciation and certain key environmental factors, e.g., pH, cation exchange capacity (CEC) and organic matter (OM) content13. Comparison of leaf anatomical characteristics among various plant species grown in heavy metal-enriched soil may help identify heavy metal accumulation and patterns of distribution, and in cataloguing the effects of heavy metal toxicity and tolerance. Many plant species exposed to Cd toxicity exhibit alterations in leaf anatomical structures, examples include chickpea (Cicer arientinum L.) and fenugreek (Trigonella foenum-graecum L.)14. Furthermore, many reports indicate that the presence of certain essential nutrients in plant media can affect the modification of leaf anatomical characteristics. For example, Fe deficiency induces more numerous but smaller stomatal apertures in both upper and lower leaf surfaces. Similarly, large quantities of Zn have been found to cause significant reduction in epidermal cell size and area of vascular bundles, and disorganization of spongy parenchyma cells15.Studying anatomical modifications in leaves can help in understanding biological processes in plants regarding heavy metal tolerance and accumulation mechanisms; for instance, elevated heavy metal accumulation in leaves is known to inhibit leaf transpiration rate, resulting in reduced plant growth and yieldsTagetes erecta L. e.g., Honey, American and Sunshine and T. patula L. e.g., French) grown in pot systems in a greenhouse environment.The present study was carried out to investigate the effects of soil amendment applications on Cd accumulation in leaves, in particular the anatomical modifications in leaves of four marigold cultivars , extractable phosphorus (Ext. P), and extractable potassium (Ext. K) contents by approximately 1.5–2.5, 1.1–4.9, and 1–4.7 times, respectively. Highest total N and Ext. P contents were in the Cd-pig treatment (0.5% and 1483.2 mg kg−1). The Cd-cattle treatment also showed increased levels of Ext. P and Ext. K, at 400.2 mg kg−1 and 2444.3 mg kg−1, respectively.Selected physicochemical properties of soils utilized for this study are shown in Table 19. Rehman et al.20 indicated that increased OM content in amendments, applied in different combinations, could increase rice growth when high Cd-contaminated effluent water is applied to soil during planting.Substantial OM contents were measured in the Cd-Leo and Com-Ctrl treatments , whereas lowest OM content was in the Cd-Alon treatment (3.4%). Humic substances originating from organic matter in leonardite can improve the physicochemical properties of soil and stimulate plant growth performancep < 0.05). After plant harvest, Cd-soils showed a slight decrease in Cd content; however, there appears to be no statistically significant difference (p > 0.05). Several researchers have reported that organic soil amendment practices in Cd and Zn co-enriched soils were used to immobilize Cd as stable forms thus reducing mobility and bioavailability in contaminated agricultural soils, thereby markedly safeguarding human health and protecting the local environment2. Furthermore, Cd content decreased slightly to 2.1 times the level in the Com-Ctrl treatment (p < 0.05), which falls within the soil quality criteria for agricultural soil (< 1 mg kg−1)21.Before planting, the spiked Cd resulted in significantly higher Cd contents in Cd-soils when compared with the control soil . The nutrient content of organic amendments such as pig and cattle manure enhance soil N concentrations considerably, which helps induce both plant growth and yield. Therefore, available N from animal manures is considered to have substantial potential and is valuable for optimum plant growth in contaminated soils. Nitrogen is also usually added as a supplement in various organic fertilizers, either alone or in combination with inorganic fertilizers22. The substantial OM content in the Cd-Leo treatment did not exhibit a commensurate influence on plant growth performance in this study. Although soil amendments minimize the detrimental effects of heavy metal exposure to plant cultivars, high levels of heavy metals can decrease plant biomass which might be a consequence of heavy metal stress, oxidative damage and increased membrane permeability24.The order of dry biomass plant tissue quantities was as follows: stems > roots > leaves. Significant differences in dry biomass were recorded from cultivars grown in the Cd-pig treatment across all marigold cultivars (25. They reported that increased biomass of Pelargonium hortorum L.H. Bailey (17.9–26.6%) grown in Pb-contaminated soils was dependent upon application of compost. The descending order of dry biomass production in marigold cultivars for all treatments was as follows: Sunshine > Honey > American > French, whereas the Cd-Catt treatment showed a slightly different pattern: Sunshine > American > Honey > French (Table p < 0.05).Increased plant growth and biomass in Cd-contaminated soils treated with organic amendments were in agreement with the results of Gul et al.. Bailey 7.9–26.6%−1 (Table p < 0.05). Furthermore, all marigold cultivars accumulated lowest levels of Cd in the Cd-Pig treatment. Marigold roots accumulated the highest Cd followed by stems and then leaves, respectively, which is consistent with a previous study8. This plant exhibited excluder potential, that is, the propensity to accumulate Cd in lower concentrations in above-ground parts compared to roots26. Previous research has indicated that compost-amended soils decreased Cd bioavailability by approximately 50% in the hyperaccumulator P. hortorum, whereas compost addition reduced lead (Pb) accumulation in shoots and roots by 33% and 28%, respectively27. Furthermore, the application of organic amendments may result in restricted translocation of soluble Cd from root to shoot of the tested plants. The rather high OM content and soil pH increase Cd retention capacity, and thus decrease Cd solubility and bioavailability in soils27, as seen the remarkably low values in the treatments amended with organic amendments, particularly Cd-Pig and Cd-Leo treatments.Among Cd treatments, all cultivars accumulated high quantities of Cd in whole plants, particularly in the Cd-Alon treatment for the French and Sunshine cultivars, and the Cd-Catt treatment for Honey and American treatments, ranging from 27.6 to 49.1 mg kg−1 Table . However28. Typically, elevated heavy metal content causes a decrease in the diameter and number of xylem cells, which has been reported in similar studies of certain terrestrial plant species, e.g., leaves of Triticum aestivum cv. Ekiz under chromium (Cr) stress29. Such phenomena are somewhat consistent with the present study, as leaf anatomical characteristics in the Cd-Alon treatment for some marigold cultivars showed remarkably smaller major vein areas  and diameter of xylem cells (American and Sunshine cultivars), compared to other treatments.The leaves of four marigold cultivars showed slight anatomical modification in the Com-Ctrl and amended treatments Table . High ac2 and 15.2–17.4 μm, respectively) (p < 0.05), whereas almost all plants in Cd-Alon showed remarkably low values when compared to the other treatments. Plant characteristics showed significant differences in the American and Sunshine cultivars grown in the Cd-Pig and Cd-Alon treatments . Lisa et al.14 found a smaller area of the vascular bundle in chickpea (C. arientinum) treated with Cd solution when compared to untreated plants. Application of organic amendments, particularly pig manure, can lead to increased mesophyll cell size and thickness of spongy parenchyma, sclerenchyma, phloem, and mesophyll in all treatments containing Cd, which can enable a greater capacity for CO2 retention in leaves, resulting in enhanced carbon availability and ultimately higher rates of photosynthesis6. To some extent, leaf mesophyll in the willow tree  stored Cd at higher concentrations when compared to veins28. However, in Arabidopsis helleri ssp. gemmifera Cd was localized in the vascular bundle rather than in the leaf mesophyll30.Large values for major vein area and xylem cell diameter were found in plants cultivated in the Cd-Pig treatment for all marigold cultivars . To some extent, substantial mesophyll thickness in the Cd-Pig treatment may be linked with large palisade parenchyma and spongy parenchyma cells in the mesophyll, as well as with the influence of nutrients in soil31, as seen in Fig. 32.In this study, greater mesophyll thickness was generally found in plants cultivated in the Cd-Pig treatment for all marigold cultivars (235.9–351.2 μm); however, the highest value was in the Cd-Catt treatment for Honey cultivar (371.5 μm) (p < 0.05). Similar results were observed in Sunshine cultivar grown in the Cd-Alon treatment. Furthermore, epidermis thickness on the leaf abaxial side in the Cd-Pig and Cd-Catt treatments for Honey cultivar and in the Cd-Pig treatment for American cultivar showed slight differences in comparison to the Com-Ctrl (p < 0.05). Some evidence indicates that heavy metals present in the growth medium can lead to increased epidermal cell size, which increases quantities of heavy metals absorbed into the epidermal layer and subsequently on the cell wall of the epidermis. This strategy helps plants prevent uptake, translocation and sequestration of heavy metals, thus reducing heavy metal absorption and/or adsorption in mesophyll cells, and increasing rates of photosynthesis11. The significantly elevated levels of heavy metals on the epidermal cell walls, however, can cause deformation and breakdown of chloroplasts and many cell organelles, including cell walls of mesophyll33. Furthermore, an elevated soil Cd content could substantially reduce the photosynthetic rate in the mesophyll of the hybrid Pennisetum grass. However, the toxicity threshold for Cd in plants varies among plant species and within a species among subspecies, ecotypes, cultivars and even tissues9.In this study, French and Sunshine cultivars grown in the Com-Ctrl treatment had significantly lower epidermis thickness on the adaxial side when compared to those grown in the amended treatments  in thylakoids, resulting in decreased plant growth and productivity36.Small major vein areas and diameters of xylem cells were typical of all marigold cultivars grown in the Cd-Alon treatment; these characteristics were clearly visible in the study plants grown in the Cd-Pig treatment across all marigold cultivars. This finding was in contrast with a previous study, as Cd accumulation in vascular bundle of the major vein did not affect xylem vessel diameter in ent Fig. B; althou38. However, the rate of water and solute  uptake and transport from veins across the mesophyll to the point of evaporation from aerial parts, depends on plant genotype, hydraulic capacity, and photosynthetic mechanism39.Venation patterns of the study plants were observed with a light microscope. The marigold cultivars had slightly different densities and numbers of minor veins; specimens of the Sunshine cultivar grown in the Cd-Leo treatment exhibited the highest number. Cultivars were ranked in order of decreasing minor vein density as follows: Sunshine > American > Honey > French; ranking as a function of treatments is: Cd-Leo > Cont > Cd-Catt > Cd-Pig > Cd. Minor veins play a key role in heavy metal mobility, and contaminants primarily assimilate in the leaf mesophyll at the mature stage. High quantities of Cd can be detected in major veins within the leaf mesophyll and mesophyll tissues during the development period, whereas very low Cd accumulation was found primarily in vascular bundles40 hypothesized their utility regarding metal detoxification in plant leaves, as Arabidopsis thaliana (L.) Heynh. accumulated substantial concentrations of Zn in trichomes when compared to other plant organs, which may be considered as a key mechanism for alleviating metal toxicity on plant grown in contaminated soils. The presence of trichomes, and their structure and number on leaf surfaces probably depends on several physicochemical and biological factors, including the presence of heavy metals42.Notably, specialized structures of the epidermis, e.g., trichomes, were found in Honey cultivar grown in Cd-Pig and Cd-Leo treatments as were the American cultivar grown in the Cd-Alon treatment; no such structures developed in any specimens of the French and Sunshine cultivars regardless of treatment type. Increased heavy metal stress can result in enlargement of trichomes, whose structures and size can act as barriers preventing heavy metal adsorption and accumulation via leaf surfaces. However, there is still no clear understanding of the mechanism by which trichomes detoxify heavy metals from leaf surfaces. Ricachenevsky et al.We conclude that organic amendments, particularly pig manure, could reduce the deleterious effects of Cd toxicity and support growth performance of the study plants, as indicated by leaf anatomical changes. The obvious results i.e., cell disorganization in vascular bundles and palisade and spongy mesophyll, were found in the Cd-Alon treatment which contained high quantities of Cd and no organic amendments.T. erecta e.g., Honey, American and Sunshine, and T. patula e.g., French, were purchased from local agricultural shops in Nakhonsawan Province, Thailand. Seeds were sterilized in 10% sodium hypochlorite (NaOCl) for 1 min and then germinated in acid-washed sand with total and extractable Cd levels below detectable limits. All seeds were transferred to a greenhouse at the Nakhonsawan Campus, Mahidol University. Conditions in the greenhouse were temperatures within 27–32 °C, ~ 65% relative humidity and ~ 16,000 lx.Seeds of four marigold cultivars, including ®  also served as control soil; designated as Com-Ctrl. The control soil was the mixture of fermented sugarcane bagasse, sugarcane filter cake, soil and dolomite; thus, soil was friable and suitable for plant cultivation. Cd-enriched soils were spiked with Cd(NO3)2 to attain measured values of 8.1 ± 1.6, 8.3 ± 0.7, 8.3 ± 1.0 and 7.4 ± 1.0 mg Cd kg−1 for Cd-Alon, Cd-Pig, Cd-Catt and Cd-Leo treatments, respectively, whereas the control soil, purchased from an agricultural supplies shop, contained low concentrations of Cd (1.9 ± 0.2 mg Cd kg−1). All pots were saturated with deionized water (DI water) for 4 weeks at 70% water holding capacity. Selected physicochemical properties of the soil samples were analyzed following standard methods e.g., texture, pH, CEC, OM, total nitrogen , extractable phosphorus (Ext. P) and extractable potassium (Ext. K)44.All experiments were carried out according to ethical principles on plant usage. Prior to planting, all soil samples were oven-dried and passed through a 2-mm sieve. Then, each organic amendment (10% w/w) i.e., pig and cattle manure and leonardite, was mixed thoroughly with Cd-enriched soil collected from Mae Sot, Tak Province. Treatments were designated as Cd-Pig, Cd-Catt, and Cd-Leo; a Cd-enriched soil with no amendment was designated as Cd-Alon. Furthermore, commercial soil amended with Osmocote8.Marigold seedlings (~ 10 cm height at 7 days) were transferred to 3.5-kg size plastic pots and placed on greenhouse benches in a randomized complete block design (RCBD). Five replicate pots were used for each treatment, and each replicate comprised a single healthy seedling. The pot treatment systems were designed and modified by the methods of Thongchai et al.® A11 basic mill. Plant materials were digested with concentrated 70% HNO3 and 37% HCl (1:3)43. Cd contents in solution were analyzed using flame atomic absorption spectrophotometry  after filtering with Whatmann® No. 42 filter paper.At 3 months after planting, plants were harvested and oven-dried at 70 °C for 3 days. For each sample, shoots, roots, leaves and whole plant tissues were ground to a fine powder with an IKA® 2710a Montana soil and NIST SRM® 1515 apple leaves) for soil and plant samples were used for the verification of the accuracy of analytical measurements. Percentage recoveries of Cd for the soil and plant materials were in the range of 90–110% of the stated content, whereas the relative standard deviation (RSD) ranged from 1.14 to 4.01%.Two standard reference materials (NIST SRM2) and immediately fixed in FAA  for 48 h as described by Johansen45. The specimens were gradually dehydrated using a graded series of tertiary butyl alcohol (TBA) and then embedded in paraffin wax. Sections were cut at 5 μm with a motorized rotary microtome . The sections were subsequently counterstained with fast green and safranin45. After staining, slides were dehydrated, cleaned with fresh xylene and mounted with Permount® for examination and photographing structural changes of leaf cells under a Leica DM1000 LED microscope with a Leica MC170 HD camera. Measurement of the lumen diameter of xylem cell, mesophyll thickness, major vein area, and epidermal cell size on both adaxial and abaxial sides of the leaf were carried out using the ImageJ program (version 1.52v).At plant maturity the middle portions of the leaves were cut into small pieces  index data were calculated as described by Phusantisampan et al.®  on a Windows-based PC. A one-way ANOVA and least significant difference (LSD) post hoc comparison were used to detect significant differences in the mean values (p < 0.05) and are presented as mean ± standard deviation.Statistical analysis was carried out using SPSS


These findings indicated that acupotomy inhibited osteoclast activity and promoted osteoblast activity to ameliorate hyperactive subchondral bone resorption and cartilage degeneration in immobilization-induced KOA rabbits, which may be mediated by the OPG/RANKL signaling pathway. Taken together, our results indicate that acupotomy may have therapeutic potential in KOA by restoring the balance between bone formation and bone resorption to attenuate subchondral bone lesions.Subchondral bone lesions, as the crucial inducement for accelerating cartilage degeneration, have been considered as the initiating factor and the potential therapeutic target of knee osteoarthritis (KOA). Acupotomy, the biomechanical therapy guided by traditional Chinese meridians theory, alleviates cartilage deterioration by correcting abnormal mechanics. Whether this mechanical effect of acupotomy inhibits KOA subchondral bone lesions is indistinct. This study aimed to investigate the effects of acupotomy on inhibiting subchondral bone resorption and to define the possible mechanism in immobilization-induced KOA rabbits. After KOA modeling, 8 groups of rabbits  received the indicated intervention for 3 weeks. Histological and bone histomorphometry analyses revealed that acupotomy prevented both cartilage surface erosion and subchondral bone loss. Further, acupotomy suppressed osteoclast activity and enhanced osteoblast activity in KOA subchondral bone, showing a significantly decreased expression of tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinases-9 (MMP-9), and cathepsin K (Ctsk) and a significantly increased expression of osteocalcin (OCN); this regulation may be mediated by blocking the decrease in osteoprotegerin (OPG) and the increase in NF- Although cartilage degeneration is the most typical pathological feature of knee osteoarthritis (KOA) ; the criκB receptor activated protein ligand (RANKL) is the critical pathway to regulate bone remodeling [During the course of KOA, persistent abnormal load within the joint leads to subchondral bone microfractures, which initiate bone remodeling . Bone remodeling , 14, whimodeling . OPG canmodeling . Studiesmodeling , 18.Inhibition of subchondral bone resorption as a therapeutic target for KOA has been carried out extensively. Bone loss inhibitors, such as bisphosphonates, are potentially effective modalities for KOA treatment by inhibiting subchondral bone resorption . HoweverAcupotomy is a biomechanical therapy under the guidance of traditional Chinese meridians theory. Acupotome, also referred to as miniscalpel-needle acupuncture, is a fairly recent development in the practice of acupuncture. Acupotome combines the characteristics of acupuncture needle insertion and surgical incision and has a definite clinical effect on KOA. The biomechanical effect of acupotomy is the unique superiority that distinguishes it from other KOA therapies. Preliminary studies –24 have Based on this hypothesis, we established the KOA model rabbits with Videman's extended immobilization method to explore whether acupotomy intervention could alleviate KOA subchondral bone lesions by inhibiting bone resorption, to observe the changes in activity biomarkers of osteoclasts and to evaluate the effect of the OPG/RANKL signaling pathway in subchondral bone.Animal experiments were strictly handled in conformity to the recommendations in the Guidance Suggestions for the Care and Use of Laboratory Animals of Beijing University of Traditional Chinese Medicine . The protocol was approved by the Committee on the Ethics of Animal Experiments of Beijing University of Traditional Chinese Medicine. All operations are carried out under pentobarbital anesthesia to ensure that pain was minimized.Disposable acupuncture needles (specification: 0.25 × 25 mm) were purchased from Beijing Research Taihe Pharmaceutical Co., Ltd. . The HZ series disposable acupotome (specification: 0.4 × 40 mm) was purchased from Beijing Outstanding Huayou Medical Instrument Co., Ltd. . Resin bandages (5#: 150 mm × 1800 mm) were purchased from Hengshui Kangjie Medical Instrument Co., Ltd. . Orthopedic casting tapes (160 series tape: 5.0 cm × 360 cm) were purchased from Suzhou Connect Medical Technology Co., Ltd. . The HANS acupoint nerve stimulator (LH202H) was purchased from Beijing Huawei Industry Development Co., Ltd. .Anti-OCN, anti-MMP-9, and anti-GAPDH antibodies were purchased from Abcam Biotechnology Co. Ltd. (USA). Anti-OPG and anti-RANKL antibodies were purchased from Bioss Biotechnology Co. Ltd. (USA). Horseradish peroxidase- (HRP-) conjugated secondary antibodies were provided by Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. . Bicinchoninic acid (BCA) protein assay kit and Chemiluminescence ECL reagent were provided by Beyotime Biotechnology Co. Ltd. . The mouse polymer method detection kit was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. . TRIzol Reagent and Reverse Transcription kit were provided by Thermo Fisher Biotechnology Co. Ltd. (USA). PCR SYBR Green Master Mix was provided by Promega Biotechnology Co. Ltd. (USA). The commercial TRAP kit was provided by Sigma Biotechnology Co. Ltd. .n = 7; 6w model group, n = 7), blank control group compared with the 4w or 6w Model group with vehicle treatment , KOA induced by immobilization for 4 weeks or 6 weeks with acupotomy treatment , and KOA induced by immobilization for 4 weeks or 6 weeks with electroacupuncture treatment .Fifty-six male New Zealand white rabbits (6 months old) weighing approximately 2.5 kg were provided by the Laboratory Animal Center of Beijing Keyu Technology Co., Ltd. . Rabbits were acclimatized for one week before the experiment began and were given unlimited food and water. Animals were raised in separate cages, with the temperature and humidity maintained at (22 ± 2)°C and 50%–60%, respectively. The rabbits were randomly divided into 8 groups as follows: KOA induced by immobilization for 4 or 6 weeks with vehicle treatment  into the ear marginal vein. After anesthesia was completed, each rabbit was posited supinely on the operating table with the left hind leg exposed. The left hind leg was fixed with the resin bandage from the groin to the toe to maintain the extended position of the knee joint, and additional double layers of orthopedic casting tapes were wrapped to reinforce the fixation; the antibiting bandage was then applied at the outermost layer. During the immobilization period, extremity swelling and mold shedding were observed at all times, adjusting and refixing as necessary. The KOA model was established after effective immobilization for 4 weeks in the 4w model group, the 4w acupotomy group, and the 4w electroacupuncture group, and for 6 weeks in the 6w model group, the 6w acupotomy group, and the 6w electroacupuncture group. Rabbits in the 4w/6w control group did not undergo any operation.After removing the bandages in the 4w/6w acupotomy groups, acupotomy intervention was performed once a week for 3 weeks. We selected four points as the fixed intervention points, located at the tendons of vastus medialis, vastus lateralis, rectus femoris, and biceps femoris. In addition, 1-2 knot nodes of muscle fibers in the extensor and flexor groups around the knee joint were selected according to individual conditions. After routine disinfection, the acupotome was pierced to release these points. The specific operations on each point were as follows. (1) The acupotome was inserted into the tendons by vertical insertion into the skin, and the blade of the acupotome was parallel to the longitudinal axis of the tendons. The local adhesion to the direction of tendons and bone connection was released by longitudinal dredging and transverse stripping, with 1-2 strikes per point; then, pressing the points for several minutes after the acupotome was withdrawn. (2) The acupotome was inserted into the knot nodes of muscle fibers by searching and locating the knot nodes of muscle fibers around the knee joint by palpation. The acupotome was inserted into the skin vertically, and the blade of the acupotome was parallel to the longitudinal axis of the muscle fibers. The local adhesion was released by longitudinal dredging and transverse stripping, with 1-2 strikes per point; then, pressing the points for several minutes after the acupotome was withdrawn.After removing the bandages in the 4w/6w electroacupuncture groups, electroacupuncture intervention was performed every other day for 3 weeks. We selected four acupoints as the needle insertion points, namely, Xuehai (SP10), Liangqiu (ST34), Neixiyan (EX-LE4), and Waixiyan (EX-LE5). After routine disinfection, the needles were inserted into the four acupoints. Electroacupuncture intervention was performed with Han's acupoint nerve stimulator, which connected SP10 and ST34, EX-LE4 and EX-LE5, respectively. Waveforms with dense and dense waves: frequency 2/100 Hz, intensity 3 mA, 15 min each. In the 4w/6w control groups and 4w/6w model groups, only grabbing and fixing were administered. Animals were humanely sacrificed after the indicated intervention.μm thick sections. For histological analysis of cartilage, the sections were stained with safranin O and fast green . The sections were evaluated for pathology of cartilage degeneration with a modified Mankin scoring system [The cartilage-subchondral bone complex samples of the central weight-bearing area of the femur were fixed in 4% paraformaldehyde  for 24 hours at 4°C. All the samples were decalcified in neutral 10% EDTA  for 4 weeks. All samples were embedded in paraffin after dehydration with gradient alcohol and submersion in xylene and paraffin. The paraffin-embedded samples were sectioned from the sagittal position into 5 g system  by a douTo analyze the morphometric parameters of subchondral bone, the sections were stained with hematoxylin and eosin (HE)  using standard protocols. Five fields were taken from each section and photographed under an optical microscope. Bone tissue static parameters including tissue area (T.Ar), trabecular area (Tb.Ar), and trabeculae perimeter (Tb.Pm) were performed using Image pro plus 6.0 (IPP) software. According to the T.Ar, Tb.Ar, and Tb.Pm data, the following parameters were quantified to represent the bone structure: percent of trabecular area (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) using bone histomorphometry .The osteoclast biomarker TRAP was applied to visualize the activity of osteoclasts. The sections were stained with a commercial TRAP kit according to the standard protocols. In general, after dewaxing with xylene and reconstituting with gradient ethanol, the sections were fixed with TRAP fixative solution at 4°C for 90 seconds and then incubated with an AS-BI Buffer, GBC staining solution, and TRAP Buffer mixture at 37°C for 60 min. Nuclei were counterstained for 2 min in hematoxylin solution. Three continuous sections from one sample were stained, and five fields were randomly selected from each section. TRAP-positive stained cells were quantified in subchondral bone regions.μg) of proteins were separated by electrophoresis using 12% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE)  gels and then transferred to 0.45 μm PVDF membranes . Subsequently, the membranes were blocked with 5% nonfat milk for 2 hours at room temperature (RT) and then incubated with primary antibodies against OPG (1 : 500), RANKL (1 : 500), and GAPDH (1 : 1000) on a shaker at 4°C overnight. After washing in TBST, the membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies for 1 hour at RT. Finally, the bands were emerged with chemiluminescence ECL reagent. The grayscale value of the protein bands was analyzed using Image J software .All soft tissues were stripped from the tibia. In the central weight-bearing area of the tibia, the cylindrical cartilage-subchondral bone complex tissues  were intercepted with the circular drill. The cartilage was quickly separated from the subchondral bone at low temperature with a bone rongeur, and then the samples were quickly placed in liquid nitrogen. Total proteins were extracted from the subchondral bone using ultrasonication in RIPA lysis buffer containing 1% protease inhibitor cocktails , and then the protein concentrations were calculated with a bicinchoninic acid (BCA) protein assay kit . Equal quantities  were constructed. The relative expression of the target genes was calculated by the 2Paraffin-embedded samples were prepared to detect the distribution of OPG, RANKL, osteocalcin (OCN) and matrix metalloproteinases-9 (MMP-9) in subchondral bone. Immunohistochemical staining was carried out using standard protocols. Briefly, the sections sustained the heat-induced antigen retrieval in citrate buffer  for 15 min, incubated with anti-OPG, anti-RANKL, anti-OCN, or anti-MMP-9 primary antibodies overnight, incubated with HRP-conjugated secondary antibody for 20 min, and developed using the chromogenic reaction (brown) with DAB  in the dark for 5–8 min. Five fields were randomly selected for each section. The sections stained with OPG and RANKL were counted to determine the number of positively stained cells, and the average optical densities of OCN and MMP-9 were calculated.P < 0.05 and P < 0.01.Statistical analysis was performed with SPSS software  and was presented as the mean ± SD. Multiple comparisons of differences between groups were achieved by the least significant difference (LSD) test, and those among multiple groups were assessed by performing one-way ANOVA. Differences were regarded as statistically significant at P < 0.01) compared with the corresponding 4w/6w control group, while it was significantly decreased (P < 0.01) in the 4w/6w acupotomy and electroacupuncture compared with the corresponding 4w/6w model groups (P < 0.01) in the 4w/6w acupotomy compared with the 4w/6w electroacupuncture groups  compared with the corresponding 4w/6w control group, while Tb.Ar, Tb.Pm, Tb.N, and BV/TV were significantly increased (P < 0.01) and Tb.Sp significantly decreased in the 4w/6w acupotomy and electroacupuncture compared with the corresponding 4w/6w model groups. Moreover, except for Tb.Ar in the 6w acupotomy group, Tb.Ar, Tb.Pm, Tb.N, and BV/TV were markedly increased and Tb.Sp markedly decreased in the 4w/6w acupotomy group (P < 0.01) compared with the corresponding 4w/6w electroacupuncture group  compared with the 4w and 6w control group  than in the corresponding 4w/6w model group, while it was significantly increased  in the 4w/6w acupotomy group compared with the 4w/6w electroacupuncture group  compared with the corresponding 4w/6w control group. It was apparent from the results that the number of TRAP-positive and MMP-9-positive cells increased in a more pronounced manner in the 6w model group, indicating the occurrence of more robust subchondral bone resorption  expression of TRAP and MMP-9 compared with the corresponding 4w/6w model groups. Moreover, the expression of TRAP and MMP-9 in the 4w/6w acupotomy group was markedly lower (P < 0.01) than in the corresponding 4w/6w electroacupuncture group  in the expression of Ctsk mRNA between the 4w/6w acupotomy and electroacupuncture groups  in the 4w/6w model group compared with the corresponding 4w/6w control group, while it was significantly increased (P < 0.01) in the 4w/6w acupotomy and electroacupuncture groups compared with the 4w/6w model group. However, acupotomy intervention further effectively promoted the mRNA expression of OPG (P < 0.01) compared with electroacupuncture intervention (P < 0.01) compared with the corresponding 4w/6w control group, while it was significantly decreased (P < 0.01) in the 4w/6w acupotomy and electroacupuncture groups compared with the 4w/6w model group. Acupotomy intervention further effectively suppressed the mRNA expression of RANKL  compared with electroacupuncture intervention (P < 0.05) between the 4w acupotomy and 4w model group, while there was no difference (P > 0.05) between the 6w acupotomy group and 6w model group. There was no difference (P > 0.05) in OPG protein expression between the 4w/6w electroacupuncture group and the 4w/6w model group. OPG protein expression also did not significantly differ (P > 0.05) in the 4w/6w acupotomy and electroacupuncture groups  in the 4w/6w acupotomy and 6w electroacupuncture groups compared with the 4w/6w model group. Only RANKL protein expression was significantly decreased (P < 0.05) in the 6w acupotomy group compared with the 6w electroacupuncture group (Figures Changes in the expression of OPG and RANKL were quantified using real-time PCR, Western blot and immunohistochemistry. The results of the real-time PCR assay demonstrated that mRNA expression of OPG was significantly decreased (rvention . As expervention . The immrvention . The exp Figures . RANKL p Figures . The imm Figures , indicatSubchondral bone lesions play an indispensable effect in the KOA process, which is thought to be crucial for accelerating cartilage degeneration. Under physiological conditions, the subchondral bone serves as a mechanical support for articular cartilage, which can buffer and disperse mechanical stress from the joint to prevent cartilage injury by localized stress concentration . Continuβ1 [Promoting the repair of damaged cartilage remains the core of KOA therapy. Strategies were proposed to alleviate KOA cartilage degeneration by inhibiting subchondral bone remodeling, including estrogens, bisphosphonates, strontium ranelate, and cathepsin K inhibitors. However, the present clinical efficacy of bone metabolism therapy still has certain limitations. The currently available anti-osteoporotic drugs can improve the radiographic structure of subchondral bone by inhibiting bone resorption to a certain extent, but none of them can retard the progress of KOA in clinical practice . The rolβ1 , the cruβ1 , which iβ1 . Can thiImmobilization of the knee joint is a widely used method for KOA model induction , which hEvidence shows that cartilage surface defects, deletion of Safranin O staining, and disorder of the chondrocyte arrangement, as determined by morphological observation, gradually worsened in KOA cartilage induced by immobilization for 4 and 6 weeks; these symptoms were accompanied by a continuous increase in the Mankin score, indicating the rapid development of KOA. We found that both the acupotomy and electroacupuncture intervention successfully protected against cartilage degeneration and effectively decreased the Mankin score. However, the enhancement of correction of the abnormal mechanics by acupotomy seemed to be more effective in slowing cartilage erosion, and neither intervention could fully reverse the pathological changes in cartilage. These data further support the direct effect of acupotomy and electroacupuncture on promoting KOA cartilage repair .As reported previously in numerous studies, enhanced bone resorption was the most typical pathological characteristic of KOA subchondral bone , which wWhat are the underlying mechanisms of acupotomy intervention in suppressing subchondral bone lesions? The initiation of KOA subchondral bone remodeling is coupled with disruption of the balance between bone resorption and bone formation . OsteoclTo further confirm that acupotomy suppressed bone resorption, expression of the osteoblast-specific gene OCN was examined. Osteoblasts are essential for bone formation, and OCN is indispensable for osteoblast differentiation . The conHow acupotomy intervention regulates osteoclast activity remains to be determined. The maturation, differentiation, and osteoclast activity were mediated by the OPG/RANKL pathway. The actions of OPG and RANKL in damaged subchondral bone have been closely investigated since elevated expression of RANKL and declining expression of OPG have been observed in KOA . StudiesThe present study has the following limitations. First, a positive drug group was not set as the control group for acupotomy, but rather the electroacupuncture group. Based on many reports , 20, bonIn summary, we found that acupotomy inhibited osteoclast activity and promoted osteoblast activity to ameliorate hyperactive subchondral bone resorption and relieve cartilage degeneration in immobilization-induced KOA rabbits, which may be mediated by the OPG/RANKL signaling pathway. The mechanical effect of acupotomy exerts chondroprotective effects by mitigating KOA subchondral bone lesions, which might supply original insights into the treatment of KOA with acupotomy and a novel therapeutic approach in the biomechanical treatment of KOA.


Temporomandibular disorders (TMD) have long been suggested to result from psychological factors. Recent studies, however, tend to consider TMD a chronic psychosomatic illness. The present study was designed to explore the association between TMD and personality profile. The Minnesota Multiphasic Personality Inventory-2-Reconstructed form (MMPI-2-RF) was used to evaluate the association for the first time.A total of 258 subjects participated in this case–control study. TMD cases as detected by the Helkimo index were questioned regarding their personality characteristics and anxiety levels using MMPI-2-RF and Spielberger state and trait anxiety inventory.Patients with TMD scored higher on all personality characteristics except for Aberrant Experiences. The psychological profile of TMD showed no significant difference between theoretical and experimental Ideas of Persecution means. Patients with TMD reported significantly higher mean levels of state and trait anxiety than controls. The most frequently found anxiety levels in TMD cases have been mild state and trait anxiety (77.5% versus 74.4%).Personality characteristic scores were considerably higher in TMD patients. TMD cases detected by Helkimo index manifest both trait and state anxiety as common findings. Chronic pain is a complex phenomenon, involving both psychological and physical aspects. Chronic pain has been studied for a long time using various behavioral and personality inventories . In partThe assumption that psychological factors can contribute to temporomandibular joint disorder (TMD) has been developed during the 1950s . Since tThe American researchers recently found that somatic symptoms are strongly related to TMD onset . In addiThe use of various inventories to measure personality traits in these patients, including earlier forms of the MMPI, has been widely discussed. Orofacial pain has been discussed in terms of older versions of the inventory, that is, the MMPI and MMPI-2 . In thisConscious participation in the study.Adults aged 18 years old or older (80 years at most), according to MMPI-2-RF manual.No systemic disease affecting the lower jaw .No previous trauma to the mandible.No previous orthodontic treatment.No unilateral or bilateral loss of four posterior teeth.A case–control study was conducted to assess the anxiety and personality traits of random outpatients visiting the Golestan University of Medical Sciences (GOUMS). The inclusion criteria were as follows:No exclusion criteria apart from the inclusion criteria was defined. Cases of TMD were detected with Helkimo index . Controls were also selected from GOUMS visitors who did not have TMD and were matched on the basis of their gender and age (5 years). A total of 129 individuals participated in each group. All methods were performed in accordance with the relevant guidelines and regulations. Several variables were used to analyze this study, including the following: sex, age, educational level, personality traits (characteristics) based on the MMPI-2-RF, anxiety based on the STAI, and detection of TMD, based on the Helkimo index.A Helkimo index identifies TMD, maximal mouth opening, jaw deviation, TMJ function, and TMJ/muscle pain. The questionnaire was designed to enable calculation of the Helkimo anamnestic index (Ai) based on subjective feelings and positive or negative answers of subjects regarding the state of their masticatory apparatus. Another component of the questionnaire, the Helkimo clinical dysfunction index (Di) offers an objective functional evaluation of structural and functional disorders of the orofacial complex. The severity of TMD is determined by the sum of the measurements of approximately 25 points. Each individual had a total dysfunction score ranging from 0 to 25 points. A higher score indicates a more acute/serious disorder. Depending on the values obtained, the patients were classified as follows: Di0 – no dysfunction; DiI – mild dysfunction (1–4 points); DiII – moderate dysfunction (5–9 points); DiIII – severe dysfunction (9–25 points) , 15.MMPI-2-RF is a revised instrument comprising 338 items designed to represent the clinically significant items in the MMPI-2. There are six sets of scales in the test, including the Validity, Higher-Order (H-O), Restructured Clinical (RC), Specific Problems, Interest, and Personality Psychopathology Five scales. The H-O scales consist of Emotional/Internalizing Dysfunction (EID), Thought Dysfunction (THD), and Behavioral/Externalizing Dysfunction (BXD). Scales of RC are composed of Demoralization (RCd), Somatic Complaints (RC1), Low Positive Emotions (RC2), Cynicism (RC3), Antisocial Behavior (RC4), Ideas of Persecution (RC6), Dysfunctional Negative Emotions (RC7), Aberrant Experiences (RC8), and Hypomanic Activation (RC9) [The STAI consists of two sets of twenty items, which provide scores that indicate both the level of anxiety the subject has at present (state) and the extent to which the person is prone to experience anxiety (trait). State anxiety items include: “I am tense; I am worried” and “I feel calm; I feel secure.” Trait anxiety items include: “I worry too much over something that really doesn’t matter” and “I am content; I am a steady person.” All items are rated on a 4-point scale . All items are rated on a 4-point scale . The levels of anxiety are interpreted as normal or no anxiety; 10–18, mild to moderate anxiety; 19–29, moderate to severe anxiety; and 30–63, severe anxiety . The samSPSS version 18 was used for describing the means, standard deviation, ranking means, frequency, and percentage. Participants were assessed using MMPI-2-RF and STAI. An independent t-test or non-parametric Mann–Whitney test was used to compare anxiety levels and personal characteristics. A Chi-square test was used to test qualitative variables such as ranking means for anxiety levels. Correlations between anxiety, personality characteristics and TMD clinical dysfunction were examined by Spearman correlation coefficient. All analyses were considered significant at the level of 0.05.A total of 258 individuals participated in the study. Among the subjects were 130 men (50.38%) and 128 women (49.62%). The TMD group consisted of 65 men (50.38%) and 64 women (49.61%). In the healthy control group, the gender distribution was the same. The age range was 21 to 25 years (28.98 ± 7.01). The case group's age was 28.98 ± 6.93 years compared to 28.98 ± 7.25 years in the control group. A non-parametric Mann–Whitney test showed no significant difference between TMD and control group. In terms of education level, 63.8% of control and 71.3% of patient groups had a bachelor's degree or higher. According to the chi-square test, the difference in educational level—including those with less than a bachelor's degree—was non-significant.The proportions of TMD patients with relatively mild, relatively severe, and mild traits of anxiety were 77.5%, 19.4%, and 3.1%, respectively. Similarly, 74.4%, 22.5%, and 3.1% of the TMD cases also had state anxiety that was relatively mild, relatively severe, and mild, respectively. TMD patients showed significantly higher means of both trait and state anxiety compared with controls . Spearman correlation coefficients indicated a significant linear relationship between dysfunction components of TMD and state anxiety . The dysfunction components of TMD and trait anxiety also exhibited a significant linear relationship .In regards to the H-O scales, there was a significant difference between the experimental and theoretical means in TMD case . Experimental means were lower than theoretical means in the present study. In other words, H-O scales were below the expected level or average. Other personality characteristics, with the exception of Persecution Ideas, had experimental means significantly less than theoretical means, as shown in Table Personality features such as EID, BXD, Demoralization, Somatic Complaints, Low Positive Emotions, Antisocial Behavior, Ideas of Persecution, and Dysfunctional Negative Emotions, were more pronounced among TMD groups than those of controls Tables  and 3. SAccording to the Helkimo index, anamnestic evaluations of TMD subjects revealed that 58.91%, 24.8%, and 41.86% of them experienced joint sound, pain, and fatigue  were symptom-free, followed by 18.21% with mild symptoms, and 22.09% with severe symptoms  and its modified version known as the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) include a valid and reliable screening questionnaire for diagnosing most common pain-related TMDs. It is recommended that DC/TMD be used in future studies.Based on the results of this study, there are significant differences between the means of State/Trait anxiety in TMD patients and controls. Trait anxiety and state anxiety are common findings in TMD cases detected by the Helkimo index. TMD patients' personality characteristic scores tend to be significantly higher than those of controls.


Healthcare workers (HCW) have been identified as index cases in disease outbreaks of vaccine-preventable diseases (VPD) in hospitals.We investigated whether Danish paediatric HCW were protected against selected serious VPD.We included 90% of staff members from two paediatric departments. All 555 HCW (496 women) supplied a blood sample for serology and filled in a questionnaire. Antibodies were measured with enzyme immunoassay against measles, mumps, rubella (MMR), varicella zoster, pertussis toxin and diphtheria toxin.Protective levels of IgG were found for measles (90.3%), mumps (86.5%), rubella (92.3%), varicella (98.6%) and diphtheria (80.5%). We found seropositivity for all three MMR components in 421 (75.9%) HCW, lowest in those younger than 36 years (63.3%). Only 28 (5%) HCW had measurable IgG to pertussis. HCW with self-reported immunity defined as previous infection or vaccination, had protective levels of IgG against measles, mumps, rubella and varicella in 87.4–98.8% of cases, not significantly higher than in those not reporting immunity. Previous history of disease had a high positive predictive value (PPV) of 96.8–98.8%. The PPV for previous vaccination ranged from 82.5% to 90.3%. In contrast, negative predictive values of self-reported history of disease and vaccination were remarkably low for all diseases.The immunity gaps found primarily in young HCW indicate a need for a screening and vaccination strategy for this group. Considering the poor correlation between self-reported immunity and seropositivity, efforts should be made to check HCW’s immune status in order to identify those who would benefit from vaccination. Measles, mumps, rubella, varicella, diphtheria and pertussis are highly contagious infectious diseases that can lead to fatal illness. Vaccines for these diseases have been a part of the childhood immunisation programme in Denmark for decades. The diphtheria–tetanus vaccine was introduced in 1950, replaced by the diphtheria–tetanus–pertussis (whole cell) vaccine in 1961, and the diphtheria–tetanus–pertussis–inactivated polio vaccine (DTaP-IPV) in 1997. The measles–mumps–rubella (MMR) vaccine was included in 1987. Two varicella zoster vaccines (Varivax and Varilrix) are available in Denmark, however not as part of the national vaccination programme. Most of the Danish population acquire chickenpox during childhood and the seronegative status in adults is unknown. Despite acceptable vaccination coverage in Denmark (> 90% in recent years for both the first dose of MMR given at 15 months of age and for the second MMR dose given at age 4 years), several cases of measles, often imported from other European countries, have recently been recorded [The MMR vaccine is considered highly effective, but suboptimal long-term immunity and seronegativity in immunised individuals after two doses of MMR vaccine have been reported ,3. StudiPertussis can be life-threatening for infants, and the DTaP vaccine only provides limited protection with vaccine efficacy of 40–85% and a decay over time of antibodies against vaccine proteins . In receMany European countries such as Austria, Finland and Italy recommend vaccination of all HCW, while others like Norway and the United Kingdom recommend vaccination of specific groups, such as paediatricians and neonatal staff . MandatoIn this study, we explored self-reported immunity in paediatric HCW and seroprevalence against the following vaccine-preventable diseases (VPD): measles, mumps, rubella, varicella zoster, pertussis and diphtheria. The study was performed in Denmark, a country that does not have national recommendations for vaccination of HCW, except for hepatitis B in specific groups.From May to August 2019, all HCW employed at two paediatric departments in the greater area of Copenhagen were invited to participate in the project. We defined HCW as nurses, physicians, medical and nursing students, secretaries, dieticians, clowns, cleaning staff and others with direct or indirect contact with patients or access to patient rooms. The departments included were the Departments of Paediatrics and Adolescent Medicine, Rigshospitalet (tertiary care centre) and Hillerød Hospital . To avoid including subjects who had not yet seroconverted or who could have received antibodies through transfusions, HCW who were given vaccinations, blood products or immunoglobulins during the previous 2 months were excluded from participation. HCW with recent infection were included in the study.HCW who volunteered to participate in the study provided a blood sample for serology and filled in a structured questionnaire. The questionnaire collected demographic data including history of infection or vaccination against the following VPD: measles, mumps, rubella, varicella, pertussis and diphtheria. Data were self-reported and registered anonymously. Information was based on recollection and vaccination cards, if available. Self-reported immunity against measles, mumps, rubella and varicella was defined as previous infection or vaccination against the disease. Non-immunity was defined as no history or no knowledge of previous infection or vaccination. Pertussis and diphtheria were not included in these definitions because immunity in these cases wanes after disease or vaccination.Bordetella pertussis toxin at the Department of Clinical Microbiology, Rigshospitalet, Copenhagen using DiaSorin Liaison chemiluminescence immunoassay . The quantitative cut-off value for seronegativity was < 13.5 arbitrary units (AU)/mL for measles, < 9.0 AU/mL for mumps, < 7.0 international units (IU)/mL for rubella and < 50 mIU/mL for varicella. The seropositivity cut-off was ≥ 16.5 AU/mL for measles, ≥ 11.0 AU/mL for mumps, ≥ 10 IU/mL for rubella and ≥ 100 mIU/mL for varicella. Titres in between were defined as equivocal. For pertussis, a titre ≥ 40 IU/mL was defined as indicative of recent contact with B. pertussis [Blood samples were stored at −80 °C until analysis. Sera were tested for IgG antibodies specific for measles, mumps, rubella and varicella zoster viruses, and ertussis .Samples with equivocal and negative serological results for measles, mumps, rubella or varicella using the Liaison assay were reassessed with an enzyme-linked fluorescent assay  at the Department of Clinical Microbiology, Hvidovre University Hospital. The test value for each sample was obtained by calculating the ratio of the relative fluorescence value of each sample to a standard. The quantitative cut-off value for seronegativity was < 0.5 for measles, < 0.35 for mumps and < 0.6 for varicella. The seropositivity cut-off was ≥ 0.7 for measles, ≥ 0.5 for mumps and ≥ 0.9 for varicella. IgG values specific for rubella virus were calculated as IU/mL considering values < 10 IU/mL to be negative and values ≥ 15 IU/mL to be positive. Titres in between were defined as equivocal.Sera tested positive in one of the test systems were considered positive. Equivocal test results were counted as negative for measles, mumps and rubella and positive for varicella as agreed by the European sero-epidemiology network .Corynebacterium diphtheriae toxin was analysed by enzyme-linked immunosorbent assay at Statens Serum Institute, Copenhagen. Titres > 0.1 kIU/L were recorded as protective, 0.01–0.1 kIU/L as limited protective and < 0.01 kIU/L as not protective.IgG against HCW who were seronegative to measles, mumps, rubella, varicella or diphtheria were offered healthcare-provided vaccination in the department. HCW with negative pertussis IgG were informed of the possibility of receiving the DTaP vaccine from their general practitioner as it was not offered free of charge at the hospital.Categorical variables were compared with chi-squared tests or Fisher’s exact test, considering a value of p < 0.05 statistically significant. Logistic regression analysis was applied to investigate factors associated with seronegativity. The following independent variables were assessed in the univariate analysis: sex, age, profession and history of previous vaccination or disease. Variables with a significance level of p < 0.1 in the univariate analysis were entered in the multiple regression model (forward selection). Missing data were left out of the analysis. Positive predictive values (PPV) and negative predictive values (NPV) were calculated for those who remembered their history for disease and vaccination, using the serological test result as gold standard. Subjects answering ‘unsure’ to a history of disease or vaccination were excluded from analysis. Data were analysed using the SPSS software, version 25 for Windows.Written consent was obtained from all participants. The study was approved by the Ethics Committee of the Capital Region of Denmark (H-18057042) and the Data Protection Agency (VD-2019–122).A total of 555 (90%) of 617 HCW employed at the two paediatric departments were included in the study; 303 nurses, 131 physicians, 40 students, 17 dieticians or physiotherapists and 64 non-clinical personnel. Most of the participants, 496 of 555 (89.4%) were women and 49 (8.8%) were younger than 26 years, 158 (28.5%) were 26–35 years, 142 (25.6%) were 36–45 years, 110 19.8%) were 46–55 years, 78 (14.1%) were 56–65 years, and 18 (3.2%) were older than 65 years. Non-participants included 57 (91.9%) women and 40 (64.5%) were nurses. Further characteristics of participants are available in a recent publication focusing on opinions on vaccination of hospital personnel [.8% were The HCW’s self-reported vaccination status and disease history are shown in The percentage of HCW reporting previous disease was high for varicella (87.0%) compared with all other diseases. A history of vaccination was most common for diphtheria (67.9%) compared with approximately half of HCW for MMR and only 2.6% for varicella zoster. Five HCW claimed to have had diphtheria, of whom two were also vaccinated. These were all above 45 years of age.Between 22.1% and 31.6% of HCW were unsure of their vaccination status depending on disease. Similarly, a high proportion of HCW did not know if they had previously been infected, ranging from 10.7% for varicella to 40.9% for pertussis. HCW who recalled both their vaccination status and disease history were 361 of 540 (66.9%) for measles, 322 of 535 (60.2%) for mumps, 342 of 539 (63.5%) for rubella, 378 of 530 (71.3%) for varicella, 309 of 543 (56.9%) for diphtheria and 257 of 539 (47.7%) for pertussis.Self-reported immunity defined as previous vaccination or disease was reported in 441 of 552 (79.9%) for measles, 381 of 546 (69.8%) for mumps, 433 of 551 (78.6%) for rubella and 486 of 553 (87.9%) for varicella, leaving 12.1–30.2% self-reported non-immune. Based on recollection, 238 (42.9%) of all included HCW were potentially susceptible to one or more VPD.Protective levels of IgG were detected for measles, mumps and rubella in 501 (90.3%), 480 (86.5%) and 512 (92.3%) HCW, respectively . HCW youSeropositivity for all three MMR components was found in 421 (75.9%) HCW, depending on age; the oldest age group of 66–75 years had 100% seropositivity compared with 131 of 207 (63.3%) HCW under the age of 36 years. Four HCW did not have detectable antibody levels to any of the measles, mumps or rubella viruses and 130 (23.4%) lacked IgG to one or two components only. Thirty-two of 311 (10.3%) women under the age of 45 years and therefore potentially fertile were seronegative for rubella. Of these, 25 reported previous vaccination and three reported previous rubella infection.Protective IgG levels for varicella zoster virus were found in 547 (98.6%) HCW, of whom 520 were detected by the Liaison assay and an additional 27 by the VIDAS method. Six of the eight seronegative HCW recalled previous varicella infection. Seven HCW denied previous varicella infection and vaccination, all of whom were seropositive. An additional 60 HCW were unsure if they ever had varicella or the vaccine, and 58 of them were seropositive. There were no significant differences between age groups.Diphtheria antibodies > 0.1 IU/mL were present in 447 of 555 (80.5%) HCW. Most of the remaining HCW (n = 105) had antibody levels 0.1–0.01 IU/mL indicating partial protection and only three had levels < 0.01 IU/mL. Seroprevalence was lowest in the oldest age group with only 66.7% considered protected .Altogether, 222 (40%) of the HCW had suboptimal IgG levels towards at least one of the antigens tested: measles, mumps, rubella, varicella or diphtheria. Physicians had the lowest seronegativity of 26.7% compared with 42.3% of nurses and 48.8% of other professions (p < 0.01). There was no difference according to sex or between hospitals.One HCW had anti-pertussis-IgG titre > 100 IU/mL. This employee was vaccinated in 2015 and had no recollection of a previous pertussis infection. Twenty-seven HCW had intermediate titre levels between 40 and 100 IU/mL, four of them recalled previous pertussis infection and 15 reported previous vaccination, however not within the past year. The 27 HCW represented most age groups and 16 were nurses. The remaining 527 HCW had IgG levels < 40 IU/mL.Comparing information of self-reported history of disease or vaccination with serology results, HCW answering ‘yes’ to a history of disease but ‘no’ to history of vaccination had the highest seropositivity to measles, mumps, rubella and varicella  followed by HCW reporting a history of both disease and vaccination .Vaccinated HCW who had no history of disease had seroprevalences ranging from 78.3% for mumps to 100% for varicella. HCW who reported neither a history of disease nor a history of vaccination showed seropositivity rates of 100% for measles, rubella and varicella, and of 76.9% for mumps. Including subjects who were unsure of previous disease or vaccination as non-immune, seroprevalences for self-reported immune vs non-immune HCW were very similar .The odds of being seronegative were higher for HCW under the age of 36 years compared with those older than 55 years, with odds ratios of 22 : 2.98–163.09) for measles, 2.5 (95% CI: 1.18–5.44) for mumps and 4.9 (95% CI: 1.44–16.37) for rubella, a finding that remained significant in the multivariate analysis for measles and rubella  and 4. CSelf-reported history of measles, mumps, rubella or varicella had a high PPV ranging from 96.8% for rubella to 98.8% for varicella . The PPVIn this study, we examined the self-reported and serological immunity to important VPD among 555 paediatric HCW in Denmark. Overall, protective levels of IgG were high for VZV, low for diphtheria and close to 90% for measles, mumps and rubella. A quarter of the HCW did not have IgG against at least one of the MMR components, which is in accordance with findings from Finland, where a quarter 39/157) of paediatric nurses had inadequate immunity to measles, mumps or rubella . Lack of of paediMumps had the highest seronegativity rate of the MMR vaccine components in our study. The mumps component of the vaccine is ca 88% effective after two doses, and protection is estimated to last 10–15 years . OutbreaAs for mumps, rubella antibodies may wane after 12–15 years. However, rubella surveillance data do not indicate that congenital rubella is increasing among vaccinated persons and Denmark achieved rubella elimination status in 2020. Nevertheless, the finding of sero-negative younger women in our study is of concern and occupational health services should contribute to ensure that female HCW stay protected against this VPD.Varicella immunity was high with only eight (1.4%) seronegative HCW. Almost all were immune because of previous infection which may explain why their seroprevalence was higher than that found in vaccinated individuals ,20.The immunological implication of low or missing antibody titres despite vaccination is unclear ,24. In 2B. pertussis, 28 HCW in this study (5%) had recently been infected, where only four recalled symptoms. This is worrying as asymptomatic infection and transmission may occur. In Denmark, pertussis vaccination is given at 3, 5 and 12 months with a booster dose at 5 years. However, pertussis antibodies wane rapidly within years of the vaccination [For single-sample serology, various cut-off values for IgG against pertussis toxin have been proposed . Definincination . Some Eucination , while ocination . In DenmOnly three (0.5%) HCW had undetectable levels of diphtheria antibodies, while 20% were partly protected. The last case of diphtheria in Denmark was seen in 1998, therefore unprotected HCW do not pose a high risk for patients.We found that a history of previous disease was a good predictor of seropositivity to measles, mumps, rubella and varicella. However, the NPV for history of previous disease or vaccination was very poor, which is also observed in other studies ,29. A laEven though the prevalence of seronegativity in our study may seem acceptable, a single nosocomial outbreak may have considerable economic implications. The cost related to a hospital outbreak of 10 measles cases in HCW in Germany was estimated at EUR 700,000, including deficit relating to suspension of the personnel without immunity, the decrease in patients during and in the weeks after the outbreak and serology tests and vaccinations . The besIn this study, MMR booster vaccination was offered to 134 (24.1%) HCW lacking immunity to one or more components of the vaccine based on serology results. If vaccination recommendation had been based on self-reported immunity, 203 (36.6%) HCW would have received an MMR booster vaccination, including 152 subjects seropositive to all MMR components and only 51 of the 134 seronegative HCW. Similarly, if based on self-reported immunity to varicella, 67 (12.1%) HCW would require vaccination compared with only two (0.4%) based on serology and no history of previous chickenpox.A strength of this study is the high response rate of 90%. We are not aware of similar studies involving all HCW categories including non-clinical personnel, students and volunteers from both a tertiary care centre and a general hospital. This makes our findings more generalisable to other paediatric wards in countries with similar vaccination programmes. To increase sensitivity and avoid overestimating the number of unprotected HCW and subsequent unnecessary vaccination, we used two different IgG enzyme immunoassays for antibody testing. The reduction in the number of seronegative HCW by including the VIDAS method was larger than expected. This emphasises the importance of considering more than one test method for seroprevalence studies.This study has some limitations. Firstly, only 10.6% of participants were male and it is therefore difficult to generalise our findings to sex. Secondly, information about previous disease and vaccination was self-reported based on memory and less frequently on vaccination cards or health files. A mandatory electronic vaccination registry was implemented in 2015, but vaccinations given before this period are not all electronic and some respondents could not find proof of previous vaccinations. Opinions among our cohort on vaccination of HCW including influenza are covered elsewhere .This study relied on measurements of humoral immunity to detect host disease susceptibility. A larger number than registered in this study may have been identified as immune if plaque reduction neutralisation (RPN) antibody assays or measurements of cell-mediated immunity had been added ,31. HoweThe immunity status of paediatric HCW from two Danish hospitals was in line with findings from other parts of the world. Immunity gaps were found primarily in young HCW, indicating a need for a screening and vaccination strategy for this group to protect them and to support the elimination goals for measles and rubella. The poor correlation between self-reported immunity and seropositivity emphasises a need for improving vaccine databases and efforts should be made to check immunity status of currently employed HCW to identify those who may benefit from a booster vaccination. Protection of HCW from VPD and reducing the magnitude of future hospital outbreaks calls for national guidelines on vaccination of HCW in Denmark and other countries that have not yet implemented such a strategy.


Clinically relevant postoperative pancreatic fistula (CR-POPF) is a severe complication which may be caused by a perioperative nutrition problem. We aimed to study whether patients with high nutritional risk (NRS2002 score ≥ 5) might benefit from preoperative nutrition support regarding the risk of CR-POPF after open pancreaticoduodenectomy.  Consecutive patients undergoing open pancreaticoduodenectomy with complete record of NRS2002 at two Chinese institutions between 2013 and 2018 were analysed. CR-POPF was diagnosed following the 2016 ISGPS criteria. Nutrition support included oral nutrition supplement and enteral and parenteral nutrition. Clinical and economic outcomes were analysed. P = 0.038). Multivariate analysis showed that preoperative nutrition support was a protective factor for CR-POPF in patients at high risk . Higher albumin and a larger diameter of the main pancreatic duct were found to be other protectors for CR-POPF.  522 cases were included. 135 cases (25.9%) were at high nutritional risk (NRS2002 score ≥ 5), among which 41 cases (30.4%) received preoperative nutrition support. The CR-POPF rate was significantly lower in the preoperative nutrition support group compared with the no nutrition support group  may profit from preoperative nutritional support manifested in the reduction of CR-POPF. The clinically relevant postoperative pancreatic fistula (CR-POPF) is one of the major causes of morbidity after pancreaticoduodenectomy (PD) –3, and mActually, a nutrition care plan contains three steps: nutritional screening, nutrition assessment, and nutrition intervention . NutritiIn China, NRS2002 is the first choice for nutrition screening. The aim of this retrospective study is to analyse whether Chinese patients with nutritional risk screened by NRS2002 may benefit from preoperative nutrition support.Data of consecutive patients undergoing open PD at two university hospitals in China between May 2013 and May 2018 were analysed retrospectively. Approval from the local ethics committee includes the usage and publication of these retrospectively analysed data. Due to the blinded data and retrospective design, written informed consent was not considered necessary by the ethics committee .Baseline variables such as age, sex, body mass index (BMI), ASA classification, jaundice, diabetes, coronary artery disease, hypertension, smoking, and drinking were recorded. Preoperative blood tests contained white blood cell count (WBC), haemoglobin (Hb), albumin (Alb), AST, ALT, total bilirubin (TBil), direct bilirubin (DBil), serum amylase (AMY), APTT, PT, and CA19-9.NRS2002 was used as the screening tool within 24 hours after admission. NRS2002 contains three parts: (1) score for nutritional status , (2) score for severity of disease , and (3) score for age (1 score for patients 70 years or older). The total score ranges from 0 to 7. If the NRS2002 score is more than 3, it means “at nutritional risk” . If the Score for severity of disease in our study was 2 scores in all patients due to the major open abdominal operation. So in the high risk (NRS2002 ≥ 5) group, there are three subgroups of score constitution: (1) subgroup 1 (first type of 5 scores): nutrition impairment = 2, disease severity = 2, and age = 1; (2) subgroup 2 (second type of 5 scores): nutrition impairment = 3, disease severity = 2, and age = 0; (3) subgroup 3 (6 scores): nutrition impairment = 3, disease severity = 2, and age = 1. In order to reduce the selection bias and prevent the imbalance of basal data in the support and no support group, we did comparisons among different subgroups.Nutrition support therapy in this study contained oral nutrition supplement (ONS), enteral nutrition (EN), and parenteral nutrition (PN) implemented for more than seven days which were defined as “nutrition support” and studied in this paper , 19. PN Since this is a retrospective study, and due to the lack of attention of preoperative nutrition support, the application of preoperative nutrition to the patients was not entirely based on the result of NRS2002 but the surgeon's discretion in accordance with the nutrition status, which provided us a chance to do a “real-world” judgement whether preoperative nutritional support was effective. So the patients at nutritional risk could be divided into two groups: preoperative nutrition support group and no support group. The operative procedures and perioperative management are unified in these two hospitals because of long-term collaboration . OperatiWe defined that all postoperative outcomes recorded happened until discharge. Besides CR-POPF, nonfistulous complications like cardiac and cerebrovascular events, postoperative haemorrhage (PPH), biliary fistula, abdominal infection, wound infection, and delayed gastric emptying (DGE) were also analysed. We followed the ISGPS definitions and classifications of PPH and DGE , 23. Post test. A multivariable logistic regression model was used to evaluate the relationship between risk factors and outcomes, which was expressed as an odds ratio (OR) with 95% confidence intervals. According to recent articles, we chose several most commonly used factors to do the logistic regression analysis, including age, BMI, pancreatic duct width, method of pancreaticojejunostomy, and implantation of pancreatic duct stenting . Meanwhile, high albumin was proven to be related to a lower rate of CR-POPF , and a larger diameter of the main pancreatic duct might be another protector for CR-POPF  , = 0.006] .In total, 323 cases (61.9%) were at nutritional risk, among which only 86 cases (26.6%) received preoperative nutrition support. The rate of CR-POPF of patients at nutritional risk (NRS2002 ≥ 3) was 21.4% (69/323). 188 cases were at low nutritional risk (NRS2002 = 3 and 4) with 45 cases receiving preoperative nutrition support. The rate of CR-POPF was 19.7% (37/188). The prevalence of CR-POPF was comparable between nutrition support and no support cohorts in both at-risk patients and at low-risk patients .It was also recommended by ISGPS in 2018 that all patients who undergo pancreatic operation should receive nutritional screening and assessment to determine the nutrition status and the indication of perioperative nutritional care . Nutriti2; Subjective Global Assessment (SGA) Grade C or NRS2002 > 5; and preoperative serum albumin < 30 g/L  [Current published articles mainly focus on postoperative nutritional support, but there are no objective data or guidelines specifically addressing the need for preoperative nutritional support for patients undergoing pancreatic surgery . In 2017unction) . In 2018unction) . HoweverAlbumin was proven to be an independent risk factor of CR-POPF in our study. However, only six cases have albumin below 30 g/L in the high risk group before operation. Due to the small number of cases, further statistical analysis is not possible to determine the cut-off value in our study. But on the other hand, albumin may not be a tool to judge nutritional problems, but it is a parameter of inflammation, which has been recommended by the 2016 American College of Gastroenterology clinical guideline of nutrition therapy in the adult hospitalized patient that “traditional” nutrition indicators such as albumin should be avoided for nutritional assessment .There are several limitations to the present study. Firstly, this is a retrospective study; all we studied were drawn from medical records. Secondly, the sample size is relatively small to further analyse some details and stratification such as age, albumin, and nutrition support route. Thirdly, we might see that except for CR-POPF, postoperative haemorrhage, abdominal infection, postoperative LOS, and total hospital costs did not differ between the preoperative nutrition support and no support groups. We thought it was mainly due to the small sample size and low prevalence of these problems. And we could see a trend in In conclusion, the NRS2002 is simple way to implement a preoperative nutritional assessment before PD in order to prevent the risk of POPF. Patients with high nutritional risk may profit from preoperative nutrition support manifested in decreasing the risk of CR-POPF.


Genetic risk was estimated using polygenic risk scores (PRSs) calculated from the largest available GWAS datasets of PTSD, MDD, and back pain. We then assessed comorbidity and genetic risk influence for developing chronic PTS, DS, and MSP after MVC. Secondary analyses explored whether common social determinants of health ameliorate genetic vulnerability. We found that 6 months after MVC, nearly half 357/781 (46%) of the participants had substantial PTS, DS, and/or MSP, and overlap was common (PTS + MSP (23%), DS + MSP (18%), PTS + DS (12%)). Genetic risk predicted post-MVC outcomes. PTSD-PRSs predicted PTS and DS , MDD-PRSs predicted DS and MSP  and 0.79%, padj < 0.05), and back pain-PRS predicted MSP . Individuals in the highest quintile of PTSD-PRSs had 2.8 and 3.5 times the odds of developing PTS and DS vs. the lowest quintile (95% CI = 1.39–5.75 and 1.58–7.76). Among these high-risk individuals, those living in non-disadvantaged neighborhoods and with college education had 47% (p = 0.048) and 52% (p = 0.04) less risk of developing PTS, and those with high social support had 60% (p = 0.008) less risk of developing DS. Overall, genetic factors influence the risk of APNS after MVC, genetic risk of distinct APNS are overlapping, and specific social determinants greatly augment genetic risk of APNS development after MVC.Posttraumatic stress (PTS), depressive symptoms (DS), and musculoskeletal pain (MSP) are common sequelae of trauma exposure. Although these adverse posttraumatic neuropsychiatric sequelae (APNS) are often studied separately, clinical comorbidity is high. In a cohort of European American motor vehicle collision (MVC) trauma survivors ( Adverse posttraumatic neuropsychiatric sequelae (APNS), such as posttraumatic stress (PTS), depressive symptoms (DS), and musculoskeletal pain (MSP), are common and morbid following traumatic experiences , 2. One Evidence from other settings suggests that shared genetic vulnerability factors may contribute to APNS comorbidity after MVC. For example, results from large genome-wide association studies (GWASs) estimate heritability for these outcomes to be 5–20% –16. PolySocial determinants of health are also known to influence vulnerability to APNS after MVC –24. For In the current study, we used PRS methods to examine genetic vulnerability and shared genetic risk of PTS, DS, and MSP after MVC, using data from a longitudinal observational study of MVC trauma survivors presenting to the ED after MVC and discharged to home after evaluation. We hypothesized that PRS-calculated genetic vulnerability would partially account for clinical comorbidity in APNS development following MVC. In secondary analyses, we assessed the hypothesis that previously identified social determinants of health ameliorate genetic vulnerability.The details of the MVC study have been reported previously . In brieDemographic and crash characteristics  were assessed via patient interview. Information regarding the time of the MVC and the time of ED presentation were abstracted from the medical record.MVC-related PTS symptoms were assessed 6 months following MVC using the Impact of Event Scale: Revised . This 22MVC-related DS was assessed 6 months following MVC using the Center for Epidemiological Studies Depression (CES-D) Scale, a 20-item self-report questionnaire designed to assess symptoms of depression . CES-D sMVC-related overall pain intensity in the past week was assessed via web-based questionnaire or telephone interview 6 months following MVC, using a verbal 0–10 numeric rating scale (NRS). Verbal scores have advantages in acute care settings and verbally administered NRSs have been validated as a substitute for visual analog scales in acute pain measurement in the ED . If partA socioeconomic position (SEP) index, used to assess neighborhood socioeconomic status (SES), was generated for each participant as previously described . In briePerceived social support was assessed using the “significant other” subscale of the multidimensional scale of perceived social support . Four stEducational attainment was obtained via patient self-report during the research interview. Patients were asked to select their educational attainment from seven categories based on their highest grade obtained. Patients with “some college or less” (the median) were compared to patients with a “college education.” . Genotyping of purified DNA was performed using the Axiom Precision Medicine Research Array  at McGill University, Montreal, Canada. Three samples with known genotype (1000 Genomes), 1 blank, and 1 repeat sample were included in each genotyping batch (96 samples), to ensure genotypic accuracy and reliability.χ2-test for allelic frequency. The median sample call rate was 99.9%. Samples were excluded if there was a discrepancy between annotated and genetic sex, chromosomal abnormalities, and kinship coefficients > 0.354 without known relation or sample duplication. Single nucleotide polymorphism (SNP) quality checks included assessments of missing call rate, duplicate discordance, and Mendelian errors. SNPs were filtered for Hardy–Weinberg equilibrium if p < 0.00001 and minor allele frequency (MAF) if MAF < 0.01. A total of 884,652 SNPs were retained  and were used for subsequent genetic imputation. Cleaned genotypes were then imputed to the 1000 Genomes Project phase 3 reference panel [Genetic data cleaning was performed using established pipelines . In briece panel , using tce panel  for pre-n = 23,212 cases and n = 151,447 controls) [n = 246,363 cases and n = 561,190 controls) [n = 119,384 cases and n = 334,478 controls) [GWAS summary statistics from the largest available GWAS analyses of PTSD (ontrols) , depressontrols) , and bacontrols) , were usontrols) .p-value thresholding were used. Imputed base SNPs were filtered with information scores < 0.9 and MAF < 0.01. In addition to the genetic data cleaning, imputed target SNPs were filtered with information scores < 0.9. SNPs in LD were grouped, to not give extra weight to a single marker. The most representative SNP with the smallest p-value was chosen within a given 250 kb window with r2 > 0.1. PRS were generated at incrementally increasing p-value thresholds in the base GWAS and the optimal threshold, explaining the most variance in the target sample, was selected  using summary statistics from three large GWAS .PRS were calculated, evaluated, and plotted using PRSice v2.3.3 . BrieflySociodemographic characteristics were summarized using standard descriptive statistics. A Venn diagram was created to visualize the overlap in PTS, DS, and MSP outcomes 6 months following MVC trauma. Each circle represents a different outcome and overlapping circles indicate participants who had two or more outcomes.ρ2 of 0.12, our study met the minimum sample size of N = 205, which is required to estimate the main effect of PRS on PTS, DS, or MSP using a logistic regression [ρ2 was calculated using a multiple linear regression model where PRS was the outcome and neighborhood SES, social support, educational attainment, age, sex, site, and top ten principal components were predictors. R2 was calculated to be 0.08 in the model with PRS for PTS and 0.12 for the model with PRS for DS. As it is more stringent, 0.12 was used as ρ2 in the sample size calculation. Both the OR per quintile of PRS and Nagelkerke’s pseudo-R2 were calculated to estimate the effect of each PRS. Nagelkerke’s pseudo-R2 of the PRS was calculated as the difference between the Nagelkerke’s pseudo-R2 of the full model that contains PRS along with covariates  and the Nagelkerke’s pseudo-R2 of the null model that contains only covariates. We adjusted for multiple testing using a false discovery rate of 5%, as we had three independent but related outcomes of PTS, DS, and MSP following MVC. Accident severity was not a significant adjuster in patients with available data  of 2, and calculated gression , 60. HerThe PRSs derived from the PTSD GWAS best explained the variance in PTS and DS. Therefore, these PRSs were used for secondary analyses to examine how neighborhood SES, perceived social support, and college education protect against genetic vulnerability to PTS and/or DS. PRS were normally distributed and standardized using R  . The required sample size to achieve an 80% power is N = 156 for a significance level α = 0.05, approximately equal sample size in each group, and the probability of group 1 being 10% and group 2 being 25% (power.prop.test in R). Each test had at least 156 participants.To investigate the effect of each protective factor within subgroups, PRS quintiles for PTS and DS were used to create three risk groups: low (quintile 1), mid (quintiles 2–4), and high (quintile 5), as previous studies show that the greatest protection or risk is at the extremes of polygenic risk . Neighbon = 781) are shown in Table Baseline characteristics of participants included in the current study n = 78 are shown = 424, 54.3%), a substantial subset reported adverse neuropsychiatric symptoms , followed by PTS  and DS . Clinical overlap in these outcomes was common, with 32% (n = 114/357) of individuals reporting two or more adverse neuropsychiatric symptoms. Of this clinical overlap, 23% (n = 85/357) reported both PTS and MSP, 18% (n = 64/357) reported both MSP and DS, and 12% (n = 43/357) reported PTS and DS. Eleven percent (n = 39/357) reported all three outcomes.Although the majority of individuals had recovered 6 months following MVC  and 2.77% of the variance in 6-month DS   variance in 6-month DS and 0.79% variance for the MSP outcome . Finally, the back-pain GWAS explained 1.49% variance in 6-month MSP  but did not explain variance for PTS or DS.PRSs for 6-month PTS, DS, and MSP outcomes were generated using summary statistics from the three largest GWASs of PTSD, MDD, and back pain to date. The PRSs generated from the PTSD GWAS explained 2.21% of the variance in 6-month PTS  and high social support decreased the odds of 6-month DS . Similarly, in the other models, genetic risk increased the odds of 6-month PTS or DS and the three social determinants were associated with reduced odds of 6-month PTS and/or DS.In secondary analyses, we assessed whether three social determinants of health , 24 liv and S3. p = 0.048). Individuals living in a non-disadvantaged neighborhood had 47% less risk of developing PTS compared to individuals living in a disadvantaged neighborhood. In contrast, no difference was observed in the incidence of DS according to neighborhood SES , high genetic risk: (15/118 (12.7%) vs. 12/38 (31.6%), p = 0.008, Fig. We next assessed whether a high level of social support mitigated genetic risk for PTS and DS. For PTS, no statistically significant difference was observed between low and high social support and genetic risk Fig. . For DS,n = 17/180) vs. those with some college or less, 17.5% ; high genetic risk: college education, 11.7% (n = (7/60) vs. some college or less 24.2% ). Among individuals with the highest genetic risk, those with college education had 52% less risk of developing PTS compared to individuals with some college or less. No statistically significant difference due to college education was observed for the low-risk group. We found a statistically significant difference in prevalence of PTS but not DS when comparing individuals with college education vs. those with some college or less in middle and high genetic risk groups  should collect outcome data on multiple APNS outcomes. In addition, these data provide further support for the concept that new medications targeting one of the APNS should be assessed for potential utility in others, in case they target mechanisms or biological pathways common to multiple APNS.Second, clinical outcome data from our cohort demonstrate that PTS, DS, and MSP are common among patients seen in the ED after MVC and discharged to home, and that there is substantial clinical comorbidity between these APNS. Indeed, one-third of the men and women meeting criteria for one of these outcomes also meet criteria for at least one other. This high clinical comorbidity between multiple APNS has important implications for the clinical diagnosis and treatment of trauma survivors , 63. ThiThird, although in the world of research professionals it has been well-demonstrated that biopsychosocial factors, distinct from, e.g., compensation-seeking, are the dominant driver of APNS development after MVC , 65–68, Although we were able to show genetic overlap between comorbid APNS, it is important to note that, similar to other studies , the amoThe GWAS studies from which our PRS scores were derived are small compared to sample sizes for maximal statistical power , 79. As Our finding that modifiable environmental factors interact with genetic risk is consistent with results of other studies examining these outcomes in other settings. Choi et al.  found thOur findings that living in a non-disadvantaged neighborhood mitigates genetic risk for PTS, but not DS, are consistent with previous reports indicating an association between SES and PTS  but not Strengths of the present study include a prospective study design, a single type of trauma exposure, inclusion of both men and women, and additional assessment of psychosocial factors. The focus of the study on White individuals was both a strength and a weakness, in that it increased genetic homogeneity to evaluate study hypotheses with increased power, yet also prevents generalizability of study findings to other groups. Further studies of mixed ancestry groups/diverse groups are necessary. Second, because of our limited sample size, we did not stratify by sex. This might be valuable, as previous studies have shown that APNS such as PTS are more heritable in women than in men . Third, In conclusion, genetic risk can substantially influence an individual’s likelihood of developing PTS, DS, and/or MSP after a MVC trauma. Genetic risk of developing APNS after MVC is overlapping, demonstrating that vulnerability to comorbid APNS outcomes after MVC is partly genetic in origin and specific modifiable environmental risk factors interact with genetic vulnerability, greatly reducing the likelihood of an individual with genetic vulnerability, who is involved in an MVC, develops one or more APNS. Further studies are needed to understand how specific and aggregate genetic risks influence APNS development after MVC, how PRSs can increase accuracy in assessing individual risk for APNS via clinical risk prediction tools , and howSupplementary Info