The results showed that abacavir and etravirine disturb oxidative stress. An increase in the percentage of sperms with chromatin damage suggests decreased fertility in rats receiving the studied drugs. Both drugs affected bone formation in growing rats. Additionally, etravirine disturbed vitamin D metabolism.Highly active antiretroviral therapy (HAART) is used in HIV-infected patients. Alongside the prolongation of patients' life, adverse side effects associated with long-term therapy are becoming an increasing problem. Therefore, optimizing of HAART is extremely important. The study is aimed at evaluating the toxicity of abacavir and etravirine in monotherapy on the reproductive system, liver, kidneys, and bones in young, sexually mature, male rats. Thirty-six 8-week-old male Wistar rats randomized into three 12-animal groups received either normal saline (control), abacavir 60 mg/kg (AB group), or etravirine 40 mg/kg (ET group) once daily for 16 weeks. Semen morphology, oxide–redox state parameters  in tissue homogenates , and serum samples were studied. In bones, microcomputed tomography and a four-point bending test were performed. Total sperm count, sperm concentration, motility, and sperm morphology did not differ significantly in AB or ET groups compared to the control. In the flow cytometry of semen, an increased percentage of cells with denatured DNA was noticed for both tested drugs. However, no significant changes of oxide–redox state in testicular homogenates were found, except of increased SOD activity in the AB-receiving group. Additionally, ET significantly altered catalase and GPx in the liver and SOD activity in kidneys. Abacavir decreased catalase in the liver and GSH levels in kidneys. AB caused significant changes to bone microarchitecture  and increased Young's modulus. Etravirine had a greater impact on macrometric parameters of bones . After 4 weeks in the ET group, a lower 1,25-dihydroxyvitamin D According to the World Health Organization (WHO) statistics, there are about 37.9 million people infected with the human immunodeficiency virus (HIV). Despite increased public awareness, many awareness-raising campaigns, and better health education, there are about 1.7 million new HIV cases every year –4. HowevThe proposed mechanism of toxic action of HAART on testes, sperm, bones, liver, kidneys, and other tissues is complex and not fully understood. In hepatotoxicity, e.g., hypersensitivity with acute hepatic necrosis, metabolic-host mediated injury, or mitochondrial toxicity are postulated. Kidney injury may depend on the action on organic anion transporters in proximal tubules and the accumulation of active drugs in this structure. In many tissues, the imbalance of oxidative stress parameters (pro- and antioxidants) with the resultant oxidation of biomolecules is an important trigger mechanism leading to tissue damage and dysfunction –8.Widespread access to antiretroviral therapy has led many perinatally HIV-infected children to reach adolescence and adulthood , 10. TheOsteoporosis is a generalized disease of bones with disorders of bone microarchitecture and low bone mass, leading to increased bone fragility . OsteopoIn HIV patients, the risk of osteoporosis is 3.7 times higher than in the general population . Many stAbacavir can be used in children from 4 months of age  and etrath and 24th week of their lives.Rats are very often used as the animal experimental model for the assessment of the impact of drugs on the male reproductive system –42, boneThe aim of this study was to evaluate the influence of long-term abacavir or etravirine monotherapy on testicular toxicity and bones. As hepato- and nephrotoxicity often limit drug usage, the authors also decided to investigate the effect of these drugs on the liver and kidneys.The study was approved by the Local Ethics Committee for Animal Experiments in Wroclaw at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences . All animal procedures during the study followed the ethical standards and practices of the institution where the study was conducted .Rats were bred and housed at the Animal Laboratory of the Wroclaw Medical University. During the study, animals were housed two per cage with enrichment products  with a 24-hour cycle (controlled 12 hours in the light/12 hours in the dark), at an ambient temperature of 22°C, with ventilation, with free access to water, and standard certified animal feed . The feed contained 0.7% calcium, 0.5% phosphorus, and 600 U.I. vitamin D3/kg.The control group (group C) received a 0.9% saline solution in a volume of 4 ml/kgThe AB group received abacavir at a dose of 60 mg/kg  suspended in a 0.9% saline solution in a volume of 4 ml/kgThe ET group received etravirine at a dose of 40 mg/kg  suspended in a 0.9% saline solution in a volume of 4 ml/kgThirty-six male 8-week-old Wistar rats were randomly divided into three groups (twelve rats each). The groups were organized as follows:According to the recommended 3Rs rule , to miniThe study design is presented in After 4 and 8 weeks of the study, blood samples from the tail vein were taken. Eight hours before their blood was taken, the animals were deprived of the feed. Markers of bone turnover must be assessed after a period of fasting. Additionally, to minimize the impact of calcium and vitamin D-containing food on the evaluation these blood parameters, food deprivation was necessary.After 16 weeks of the study, the rats were anesthetized by intraperitoneal injections of ketamine (60 mg/kg) and xylazine (10 mg/kg), and blood samples were taken via a cardiac puncture. After that, the rats were euthanized by cervical vertebrae dislocation in deep anesthesia. The epididymis, testes, liver, kidneys, and bones (tibia and femur) were dissected immediately and carefully cleaned of surrounding tissues.Spermatozoa were collected by epididymal slicing with a scalpel blade according to the method used by Martinez-Pastor et al. . EpididyThe testes, liver, kidneys, right tibia, and right femur were weighed and measured. Then, the right testis, right liver lobe, and right kidney were stored at -80°C and later homogenized as described below. The right tibia and right femur were evaluated densitometrically and with microcomputed tomography (mCT). Then, the biomechanical properties of the right femur were measured using a four-point bending test. The left testis, left liver lobe, left kidney, and left tibia were stored in 10% neutral buffered formalin for histopathological examination.3 2-D3), parathormone, and aspartate aminotransferase (AST) .Blood samples were centrifuged at 4,000 g at 4°C for 10 minutes in the MPW-350R laboratory centrifuge . The obtained serum was frozen at -80°C until the proper measurements were made. For the serum, the following parameters were assessed using purchased ELISA kits in accordance with the manufacturers' instructions: luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, sex hormone-binding globulin (SHBG), inhibin B, estradiol, thyroid-stimulating hormone (TSH), prolactin, aminoterminal propeptide of type I procollagen (PINP), osteoclast-derived tartrate-resistant acid phosphatase form 5b (TRACP), sclerostin, Dickkopf-related protein 1 (DKK1), osteoprotegerin (OPG), 25-hydroxyvitamin D (25-OH-D), 1,25-dihydroxyvitamin DSerum total calcium, inorganic phosphorus, and creatinine levels were determined in a certified laboratory using an Alinity C instrument with Abbott .The testes, liver, kidneys, right tibia, and right femur were weighed using the RADWAG AS 60/220/C/2 (Poland) electronic weighing scale. Based on the obtained measurements, the testicular index, hepatic index, renal index, tibial index, and femoral index were calculated according to the following formulas:Measurements of bone length were made using the electronic calliper with the 0.01 mm resolution .The epididymis from eight randomly chosen rats in each group was examined. Spermatozoa were collected by epididymal slicing with a scalpel blade according to the method described by Martinez-Pastor et al. . The epiμl) of spermatozoa-rich fluid was placed on a glass slide and covered with a cover slide. The evaluation was performed using the phase-contrast microscope  with a warm stage by two independent researchers, and the mean value was calculated.Subjective motility was determined immediately after removal of the sliced epididymal tissue. A drop (10 Morphology was assessed after staining with Giemsa stain according to the modified Watson method . Spermatμl was suspended in 40 μl of the HTF extender with 50 μl of IDENT Stain (Hamilton Thorne) and incubated for 5 minutes at 37°C before evaluation. The analysis was carried out using IDENT settings  [The computer-assisted analysis of sperm concentration was performed using HTM IVOS ver. 12.2 . The spermatozoa-rich fluid in the amount of 10 ity: 75) .Sperm cell functionality was evaluated using fluorescent staining and flow cytometry analysis. Flow cytometric analyses were performed on the Guava EasyCyte 5  cytometer. The fluorescent probes used in the study were excited by the Argon ion 488 nm laser. Acquisitions were made using GuavaSoft™ 3.1.1 . Nonsperm events were gated out based on scatter properties and not analyzed. A total of 10,000 events were analyzed for each sample. The following features were assessed: membrane integrity, acrosome integrity, mitochondrial activity, lipid peroxidation, apoptosis and membrane lipid disorder, and chromatin status , 56.μl was incubated in the dark for 10 minutes with 5 μl of the SYBR-14 working solution (0.1 μl of SYBR14+ 4.9 μl of TRIS III extender). The analysis was performed after 3 minutes of incubation with 3 μl of PI. The spermatozoa with intact membranes emitted green fluorescence. The cells with red fluorescence were classified as dead [The membrane integrity of rat spermatozoa was assessed using SYBR-14 stain combined with propidium iodide (PI) . The sperm-rich fluid in the amount of 300  as dead .μl of the PNA working solution (1 μg/ml) and incubated for 5 minutes at room temperature in the dark. Before the analysis, the samples were washed, and 3 μl of PI was added [Acrosome integrity was assessed by lectin PNA stain from the Arachis hypogaea Alexa Fluor® 488 conjugate . Diluted samples were mixed with 10 as added .μl was stained with 0.67 μl of the JC-1 stock solution (3 mM stock solution of JC-1 in DMSO). The samples were incubated for 20 minutes at 37°C in the dark. The sperm cells emitting orange fluorescence were classified as having high mitochondrial activity. The spermatozoa emitting green fluorescence were defined as those with low mitochondrial activity [Mitochondrial activity was determined using the JC-1 dye . The spermatozoa-rich fluid in the amount of 500 activity .11-BODIPY581/591 fluorescent lipid probe . One μl of 2 mM C11-BODIPY581/591 in ethanol was added to a diluted sperm-rich fluid and incubated for 30 minutes at 37°C in the dark. Subsequently, centrifugation at 500 × g for 3 minutes was performed, and the sperm pellets were resuspended in 500 μl of the HTF extender. To determine viability, the spermatozoa were stained with PI and incubated for 5 minutes at room temperature. The sperm cells emitting orange fluorescence (nonoxidized state of C11-BODIPY581/591) were defined as live cells without LPO [Lipid peroxidation (LPO) was evaluated by dyeing using the Chout LPO .μM solution in DMSO)  [μl of YO-PRO-1 stain  was added to 1 ml of a diluted spermatozoa-rich fluid (500 μl of HTF and 500 μl of spermatozoa solution). After 10-minute incubation, 3 μl of PI was added before cytometric analysis. The cells showing green fluorescence were classified as YO-PRO-1 positive. The spermatozoa that remained unstained were categorized as living population [Apoptosis and membrane lipid disorder were evaluated with the YO-PRO-1 dye  . One μl pulation .μl) was subjected to brief acid denaturation by adding 200 μl of the lysis solution (Triton X-100 0.1% (v/v), NaCl 0.15 M, HCl 0.08 M, pH 1.4). After 30 seconds, 600 μl of the AO solution  was added. The analysis was performed after 3 minutes of incubation. The spermatozoa with high DNA stainability emitted green fluorescence (HDS). The sperm cells emitting red fluorescence were considered a population of cells characterized by a high DNA fragmentation index (DFI) [Chromatin status was established using the acridine orange dye . The spermatozoa-rich solution (100 ex (DFI) .In testicular homogenates, testosterone, estradiol, and inhibin B concentrations were evaluated using ELISA kits: Rat Testosterone ELISA REF E0259Ra, Bioassay Technology Laboratory; Rat Estradiol ELISA Kit REF E0174Ra, Bioassay Technology Laboratory; Rat Inhibin B ELISA kit REF E0728Ra Bioassay Technology Laboratory; China.μl of the cooled (-20°C) methanol: propanol (1 : 1) mixture was added. The Pro250 homogenizer  was used. After homogenization, the samples were centrifuged for 10 minutes at 15,000 × g, and the supernatant was collected.To 160 mg of testis tissue, 800 2.6.3.Oxide–redox state parameters such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione were assessed in homogenates from the testes, liver, and kidneys. The following kits, Rat Malondialdehyde ELISA Kit E0156Ra, Bioassay Technology Laboratory China; Superoxide Dismutase Assay Kit No 706002, Cayman; Catalase Assay Kit No 707002, Cayman; Glutathione Peroxidase Assay Kit No 703102, Cayman; Glutathione Assay Kit No 703002, Cayman; USA, were used.The MDA concentration was measured in the homogenates that were prepared in the same way as for measurements of reproductive hormones. The oxide–redox state was assessed from the tissues of the liver, kidney, and testes. For all tested parameters, the tissues were prepared in the buffers recommended by the manufacturer at a concentration of 10%. Superoxide dismutase activity was assessed in a tissue solution in the 20 mM HEPES buffer, pH 7.0, supplemented by 1 mM EDTA, 210 mM mannitol, and 70 mM sucrose. Catalase activity was evaluated in the homogenates prepared in 50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA. Glutathione peroxidase activity in the tissues was measured in the 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 1 mM DTT solution. The last parameter—glutathione—was analyzed in the same homogenate solution as the one prepared to evaluate catalase activity. In the latter case, after collecting the supernatant, 0.1 g/ml of MPA was added for 5 minutes at room temperature; then, the samples were centrifuged again at >2,000 g for at least two minutes, and the collected supernatant was stored at -20°C until assayed. The other centrifugation supernatants were stored at -80°C until assayed.Slides were deparaffined and rehydrated, and antigen retrieval was carried out by boiling the sections in EnVision FLEX Target Retrieval Solution pH 9 using a PTLink-20 minutes, 97°C . The visualization of the studied antigen was performed using the EnVision FLEX+, Mouse, High pH System (Dako) according to the manufacturer's instructions. The detection of the minichromosome maintenance 7 protein (MCM-7) antigen  and the glutathione antigen  was conducted for 20 minutes at room temperature. Then, slides were incubated with secondary antibodies conjugated with horseradish peroxidase (EnVision FLEX/HRP-20 minutes at room temperature). Finally, the sections were counterstained with EnVision FLEX Hematoxylin (Dako) dehydrated in graded ethanol concentrations  and in xylene and closed in Dako Mounting Medium (Dako). The primary antibody was diluted in the EnVision FLEX Antibody Diluent background-reducing reagent (Dako).The IHC of MCM-7 sections of testes was evaluated in a blinded way under the BX-41 light microscope . For the evaluation of MCM-7-positive cells, three fields with the highest number of cells showing a positive reaction were selected. The evaluation was performed by counting brown-labeled nuclei at ×400 magnification. The percentage of positive cells in each field was assessed according to the following formula:  × 100%.For the evaluation of the cytoplasmic expression of glutathione levels, the semiquantitative method—immunoreactive score (IRS) according to Remmele and Stegner –59—was aμm thick were cut with a microtome and standard stained with hematoxylin and eosin. The preparations were assessed under the Olympus BX50 light microscope equipped with the Zeiss Axiocam 208 color microscope camera using Labscope.The liver and kidneys were fixed in 10% neutral buffered formalin. The dehydration process was then performed using graduated concentrations of ethanol and ethanol-xylene. The final solution was pure xylene. The tissue was infiltrated with appropriate purified paraffin. From the fixed liver and kidney tissues in paraffin, slices 4 2) [The right tibia and right femur were examined densitometrically. The tests were performed on Hologic DXA equipment  using software for small animals. The scanner was calibrated daily with the phantom supplied by the manufacturer. Bone mineral density (BMD) results were obtained as grams of mineral content per square of bone area (g/cm2) .μm, a voltage of 74 kV at 133 μA, using a 0.5 mm Al filter. 3D structural properties were measured using CTAn . For each long bone (tibia and femur), measures were taken for 2 areas: the cancellous bone and cortical bone.Structural properties were measured using the SkyScan 1172, Bruker® computed microtomograph. Each sample was registered with a resolution of 9 μm, this number of slices corresponds to the region of approximately 3.5 mm in height. The next offset of 100 slices separates the trabecular ROI and cortical ROI. For the measurements, the cortical ROI was defined as 100 slices that correspond to approximately 0.9 mm. Within each ROI, the automatic selection of a specific bone was applied, and then the images were segmented using an adaptive global threshold algorithm. The quantitative analysis  of the cancellous bone structure considered bone volume fraction (BV/TV), bone surface density (BS/TV), specific bone surface (BS/BV), trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp) of trabeculae, structure model index (SMI), connectivity density (Conn.D), total porosity (Po.tot), and degree of anisotropy (DA).Before 3D morphometric analyses, the images were aligned with the major bone axis, and a set of trabecular and cortical regions of interest (ROIs) was selected. The selection of ROIs  was baseFor the cortical bone, the analysis involved the determination of average cortical thickness (Ct.Th), total cross-sectional area inside the periosteal envelope (Tt.Ar), cortical bone area (Ct.Ar), and cortical area fraction (Ct.Ar/Tt.Ar).The histomorphometric examination of the tibia was performed in line with the 2012 update of the standardized nomenclature, symbols, and units for bone histomorphometry .μm thick slides. The slides were stained using the standard hematoxylin and eosin method and scanned using the Hamamatsu NanoZoomer 2.0 histological slide scanner and NDP.scan SQ 1.0. Finally, each sample was exported to a TIFF file.The left tibiae of the rats were fixed in 10% neutral buffered formalin and later decalcified in the 10% neutral buffered EDTA solution. The EDTA solution was changed once after 24 hours. The metaphyseal and epiphyseal regions of the proximal tibia were harvested, embedded in paraffin, and cut into 5 The TIFF file was analyzed with ImageJ 1.52. Every image was briefly segmented, and the total trabecular area (B.Ar) and trabecular perimeter (B.Pm) were measured.The bone volume to tissue volume ratio (BV/TV) was calculated as B.Ar/T.Ar. Next, the bone surface to tissue volume ratio (BS/TV) was calculated as B.Pm/T.Ar×1.2 and the BS/BV ratio as BS/TV×BV/TV. Mean trabecular thickness (Tb. Th) was calculated as 2/BS/BV.Mechanical properties were assessed using a four-point bending test with the MTS 858 MiniBionix machine  . In ordeTo calculate Young's modulus, the cross-section area of the femur sample needs to be measured. The area of each sample was determined at the point where the sample broke by finding the smallest ellipse that outlines the sample and calculating its area. The area was calculated using the measurements of the length of the ellipse's axis with the Zeiss Stereo Discovery V20, Germany stereo microscope.p ≤ 0.05. All results are presented as the median (lower quartile–upper quartile).Due to the lack of a normal distribution of the results for all examined parameters, the statistical analysis was performed with nonparametric tests . The statistical analysis was performed in Statistica v.13. The significance level was On the first day of the study, the control group, the AB group, and the ET group did not differ significantly in body weight. Similarly, no differences in body weight were observed between the groups during the entire study period .The results are presented in The group receiving etravirine for 16 weeks had a significantly greater tibial index, a greater diameter of the tibia, and a lower femur length than in the control group. In the group receiving abacavir at the end of the study, no differences in bone macrometric parameters were found.The levels of reproductive hormones in serum and testicular homogenates are presented in p ≤ 0.05). No significant difference between the abacavir-receiving animals (31.7 (30.3-34.5) %) and the control group was found. The representative photos of the immunohistochemical evaluation are shown in The immunohistochemical examination revealed an increased expression of minichromosome maintenance 7 protein in the ET group compared to the control group (36.4 (35.3-37.8) % vs. 33.7 (32.8-35.5) %, The results are presented in In testicular homogenates, significantly higher SOD activity was noticed only in the AB-receiving group compared to the control rats. Etravirine did not affect SOD activity. Catalase and GPx activities and MDA and GSH levels, as well as the GSH/GSSG ratio in both AB and ET groups, were comparable to the control . In the In liver homogenates, catalase activity significantly decreased in both studied groups (AB and ET) compared to the control group. Additionally, etravirine significantly decreased GPx activity at all time points under consideration .th, 15th and 25th minutes, respectively) were noticed. In the etravirine-receiving group, SOD activity in the kidney was significantly lower  were anatomically normal. Sample images are shown in The results are presented in 3 in rats receiving etravirine was lower than in the control group. After 16 weeks, creatinine levels were lower in the ET group, whereas aspartate aminotransferase activity was significantly higher in the AB group.After 4 weeks of the study, the concentration of 1,25-dihydroxyvitamin DThe results are presented in The histomorphometric parameters analyzed in longitudinal sections of paraffin-embedded tibiae are presented in The bone morphology results vary between the tibia and femur. The results of mCT are presented in The results are given in Abnormalities in testicular and epididymal morphology, sperm morphology and motility, and oxidative stress disturbances caused by HIV infection and HAART therapy may directly affect male fertility. In animal studies, HAART therapy may influence plasma levels of sex hormones, such as testosterone, prolactin, LH, or FSH , 68. HAAIn the current study, the impact of abacavir and etravirine in male rats on reproductive toxicity was evaluated. The paper assessed the hormonal status in serum after 8 and 16 weeks of drug administration and basic morphological parameters of testes and epididymis, as well as semen morphology and viability, oxidative stress parameters, and antioxidant activity.After 8 weeks of etravirine administration, a temporary decrease in serum LH levels was observed. After 16 weeks, serum luteinizing hormone levels were lower in the AB group than in the control group. As there was no significant difference in serum testosterone levels between all the analyzed groups, it cannot be excluded that changes in LH levels observed in this study were associated with the pulsative release of LH .Oxidative stress has been reported to be one of the most important causes of male infertility, but not the only one . The pat2−) to hydrogen peroxide (H2O2) and prevents the formation of highly reactive hydroxyl radicals. Hydrogen peroxide is further directly converted to H2O by catalase or by glutathione peroxidase using the reduced glutathione (GSH) molecule, which is converted to the oxidized glutathione form (GSSG) [Moreover, spermatozoa contain only little cytoplasm, resulting in insufficient cytoplasmic antioxidant defense , 75. Them (GSSG) . MDA is Several in vitro studies suggest that antiretroviral drugs may generate ROS. It was found that various nucleoside reverse transcriptase inhibitors (NRTIs) impair the mitochondrial function in HepG2 cells and alter oxidative stress parameters causing, e.g., an increase in MDA levels (zidovudine and tenofovir) and a decrease in the GSH concentration (stavudine) . EfavireIn the AB and ET groups, increased DNA instability expressed by an elevated percentage of spermatozoa with denatured DNA was found. In the study of testicular homogenates, abacavir was the only drug to significantly increased activity of SOD, which is one of the first in the enzymatic antioxidant pathway. No other changes in prooxidant (MDA) or enzymatic/nonenzymatic antioxidants were noticed in testes, suggesting that the described changes in DNA instability in spermatozoa are not the direct consequence of oxidative stress generation. It must, however, be stated that oxidative stress parameters in the seminal plasma were not assessed in this study and not affected the redox state in testes, not definitely excluding imbalances between pro- and antioxidants in semen.Zini et al. reported that male infertility is associated with poor sperm DNA stability . They alMore pronounced changes in oxidative stress parameters were detected in the liver and kidneys. Etravirine significantly affects SOD activity in kidneys as well as catalase and GPx activities in the liver, suggesting the possible negative impact on the state of antioxidants; still, this does not affect MDA levels—the main prooxidant end product. Similarly, abacavir decreases catalase activity in the liver and GSH levels in rat kidneys without affecting the MDA concentration in both tissues. Both drugs are metabolized in the liver, but different metabolic pathways are important. Abacavir metabolism depends mainly on alcohol dehydrogenase and glucuronidation . EtravirDespite some alterations in the redox state in kidneys, the morphology of AB or ET administration was not affected, as shown in the histological evaluation. Moreover, serum creatinine levels were not significantly changed in the AB group and even were lowered significantly in the ET group, meaning that both drugs are rather not nephrotoxic. The detected mild decrease in creatinine levels in the ET-receiving animals had no clinical significance as the obtained results were within the normal limits for rats for an enzymatic method of plasma creatinine determination .The lack of significant changes in SOD, CAT, and GPx activities and GSH and MDA concentrations in testes between the groups, but the presence of significant differences in the oxide–redox state between the studied and control groups in the liver and kidney, may result from drug pharmacokinetics. Drug concentration in plasma and in other body fluids and tissues depends on absorption, biotransformation, distribution, and excretion . FollowiThe balance between cell proliferation and apoptosis is important for the maintenance of male fertility . In this2 receptors (A2R) [2R agonist inhibited osteoclast differentiation [2R, may shift the balance between bone resorption and bone formation towards osteogenesis. However, in the in vitro study using the osteosarcoma Saos-2 cell line as a model, it was observed that abacavir inhibited osteoblast differentiation and decreased the ability of the cells to form calcium deposits in the extracellular matrix [No effect of abacavir on femoral and tibial BMD was demonstrated in this study. These findings are in line with the observations reported by Stelbrink et al. . Their srs (A2R) . Medeirontiation . Based or matrix .Further studies are needed to explain the effect of abacavir on bone metabolism. Bone strength depends not only on bone mineralisation but also on bone geometry and the shape of bones, the microarchitecture of trabecular bones, turnover, and collagen in the extracellular matrix. Even though Esposito et al. reported that abacavir impairs the mineralisation and synthesis of type I collagen , it did In this study, serum levels of bone turnover markers were measured twice—in week 4 and in week 16. In week 4, there was no difference in serum levels of PINP, TRACP, sclerostin, and Dickkopf-related protein 1 (DKK1) between the groups under analysis. In week 16, serum levels of PINP, TRACP, and sclerostin remained comparable between all groups. Serum DKK1 levels were elevated in the AB group compared to the control group. No difference in serum DKK1 levels between the control group and the ET group was detected. Lower serum levels of PINP and TRACP observed in the study between week 4 and week 16 resulted from an inhibition of bone turnover associated with growth slowing . SclerosIn this study, femoral and tibial BMDs were comparable between the ET and control groups. Additionally, the present study did not demonstrate any effect of etravirine on bone microarchitecture in neither in bone histomorphometry nor bone mCT examination. These findings are consistent with the results presented by other authors , 104, whIn the study using the osteosarcoma Saos-2 cell line as a model, it was observed that the presence of etravirine in the culture medium increased the number of calcium deposits, pointing to an increase in final osteoblast differentiation and an increase in bone formation . In thisth week of this study, a significant decrease in serum 1,25-dihydroxyvitamin D3 levels was measured in the etravirine-receiving group. Decreased vitamin D concentrations and the impairment of vitamin D metabolic pathways by antiretroviral drugs  were found by other authors, which were confirmed by both in vitro and in vivo studies [In the 4 studies , 107. Ef studies , 107. ToAbacavir and etravirine doubled the percentage of sperms with denatured DNA, suggesting decreased fertility in animals receiving these drugs. The results of this study indicate that etravirine may decrease serum vitamin D concentration, which dictates the need to monitor vitamin D levels in etravirine-receiving children. However, no harmful effect of etravirine on bones in growing rats was identified. Abacavir was found to improve bone microarchitecture in growing rats but may affect the liver function.


Selective neuromodulation using designer receptors exclusively activated by designer drugs (DREADDs) has become an increasingly important research tool, as well as an emerging therapeutic approach. However, the safety profile of DREADD expression is unknown. Here, different titers of adeno-associated viral (AAV) vector were administered in an attempt to vary total expression levels of the inhibitory DREADD hM4D(Gi) in excitatory hippocampal neurons. Male Sprague Dawley rats were injected with AAV2/7 encoding DREADD-mCherry, DREADD, or mCherry. Pronounced neuronal loss and neuroinflammatory reactions were observed after transduction with the high titer DREADD AAV, which also resulted in the highest DREADD expression levels. No such effects were observed in the mCherry control group, despite an equally high titer, nor in conditions where lower viral vector titers were injected. In the high titer DREADD conditions, dentate gyrus (DG) evoked potentials were inhibited on clozapine-induced activation of hM4D(Gi), while in low titer conditions DG evoked potentials were enhanced. Recordings of single neuronal activity nevertheless indicated a reduction in spontaneous firing of granule cell layer neurons. Our results indicate that prolonged, high levels of DREADD expression can have neurotoxic effects and that chemogenetic suppression of excitatory hippocampal neurons can paradoxically enhance DG evoked potentials. Designer receptors exclusively activated by designer drugs (DREADDs) are engineered receptors that can be used to selectively modulate specific groups of cells. Especially in neuroscience, DREADDs are widely adopted. However, there is not much known on their safety profile. Here, we assess the effect of different expression levels of the DREADD hM4D(Gi) by varying the titer of the adeno-associated viral (AAV) vector used to transduce specific neurons in the rat’s brain. We found that high expression levels result in strong neuromodulatory effects, but also induce neuronal loss and tissue damage. Less pronounced, non-toxic expression levels paradoxically seem to display opposite neuromodulatory effects at network level.N-oxide, deschloroclozapine, JHU37152, JHU37160 and low doses of clozapine , an inhibitory receptor that activates the Gαi pathway, resulting in decreased neuronal firing and synaptic silencing (Designer receptors exclusively activated by designer drugs (DREADDs) are engineered human muscarinic receptors that are no longer responsive to their original ligand, but can instead be selectively activated by exogenous actuators such as clozapine-lozapine . DREADD ilencing . Especiailencing . Selectiilencing . DREADDsilencing . If a pailencing .In the view of future clinical translation, DREADD gene delivery is obtained through a viral vector carrier, most commonly a recombinant adeno-associated viral (AAV) vector . SelectiHere, we focused on AAV-mediated hM4D(Gi) expression when specifically targeting excitatory (CaMKIIα-expressing) hippocampal neurons, i.e., the granule cells in the dentate gyrus (DG) and the pyramidal cells in the CA regions. This approach represents one of the chemogenetic applications with therapeutic potential, e.g., in epilepsy . The aimad libitum. Before the start of the experimental procedures, animals were handled 10 min/d for 5 d in total to accustom them to human contact.Thirty-six adult male Sprague Dawley rats (Envigo) were used. Animals were treated according to European guidelines (directive 2010/63/EU) and the protocol was approved by the local Ethics Committee on Animal Experiments of Ghent University (ECD 16/31). Animals were kept under environmentally controlled conditions (20–24°C and 20–60% relative humidity) with a fixed 12/12 h light/dark cycle [zeitgeber time (ZT)0 = 9 A.M.]. The animals were group-housed in Type IV cages (Tecniplast) on wood-based bedding (Carfil). Cages were enriched with gnawing woods , paper nesting material , and a cardboard tunnel . Water and food  were provided www.lvvc.be). Vector production and purification was performed as previously described  were injected with AAV vector. An overview of the groups, injected vectors and titers is given in Animals were anesthetized with a mixture of isoflurane  and medical oxygen. After exposure of the skull, two small burr holes were made. Using a 32-gauge needle, a 5-μl Hamilton syringe , and an automated injection system , AAV was injected at two sites in the right hippocampus using these respective stereotactic coordinates: anteroposterior (AP): −3.8 and −5.2 mm to bregma, mediolateral (ML): −2.2 and −5.0 mm to bregma, dorsoventral (DV): −3.2 and −7.0 mm to dura. Per injection site, 3 μl of AAV was injected at a flow rate of 0.3 μl/min. After each injection, the syringe was left in place for an additional 5 min. Afterwards, the skin was closed, rats were subcutaneously injected with the nonsteroidal anti-inflammatory drug meloxicam  and lidocaine  was locally applied to the wound to minimize discomfort. In between injections, syringes were cleaned according to the guidelines of the manufacturer.nD13 = 4) of the animals was also scanned at 21 weeks. The other animals  were scanned five weeks after injection. Anatomical MR images were acquired using a lab animal sized Bruker BioSpin PharmaScan 7T system (Bruker) with a transmit volume coil  and an actively-decoupled rat head surface coil . TurboRARE T2-weighted images  were acquired. Animals were anesthetized as described earlier and equipped with a heating pad and pressure sensor to control body temperature and respiration during scanning.Animals from the D13 group were scanned 3, 5, and 12 weeks after AAV injection. A subset . Depth recording and stimulation electrodes were custom-made by twisting two polyimide-coated stainless-steel wires  around each other. For the stimulation electrode, a distance between tips of 900 μm was maintained.Depth electrodes were implanted stereotaxically  according to the rat brain atlas . The exaAll recorded signals were referenced to an epidural electrode above right frontal cortex. Recording and stimulation electrodes were connected to a headstage with a four-channel unity gain amplifier  through connector pins . A constant-current stimulator  was used for electrical stimulation. EP sweeps were amplified 248 times, digitized at 20 kHz and high-pass filtered at 0.15 Hz. Digitization of signals was performed using a data acquisition device . Acquisition and offline analysis were performed using a MATLAB-based application (The MathWorks).EPs were obtained in response to biphasic charge‐balanced square‐wave paired pulses delivered to the perforant path with a pulse width of 200 μs, intensities ranging from 25 to 1000 μA and an interstimulus interval of 10 s. The conditioning stimulus (EPa) and the test stimulus (EPb) were 10 ms apart. Stimulation intensity for both pulses was increased after every double pulse to obtain an input/output relationship. Per EP recording session, the set of intensities was repeated five times. One baseline recording sessions was performed, followed by subcutaneous injection of vehicle solution  and a second recording 10 min later. Clozapine  was subcutaneously administered, followed by a 15-min waiting period and two more EP sessions, again with 10 min in between.www.open-ephys.org) and an Intan amplifier/filter and digitizer boards (Intan Technologies). The Intan boards digitize the signals in a ± 6.4 mV, 16-bit integer range. The probe was lowered at in the right hippocampus 4.2 mm posterior and 3 mm lateral to λ. Activity was found around 3-mm depth and was estimated as putative DG activity. Around 30 min of baseline activity was recorded, followed by subcutaneous injection of vehicle and clozapine (0.1 mg/kg) and another ∼40 min of recording.In four animals from the DM12 group, unit recording was performed under isoflurane anesthesia. Broadband (1–7500 Hz) electrophysiology signals were acquired at a sample rate of 30 kHz from the DG of the rat, using a 32-channel silicon probe , with the Open Ephys acquisition system  or at a later time point 2–23 weeks after the EP recording . Rats were deeply anaesthetized with an overdose of sodium pentobarbital  and transcardially perfused with PBS followed by 4% paraformaldehyde solution (Merck). The brains were removed and postfixed in paraformaldehyde solution for 24 h, cryoprotected in a 30% sucrose solution, frozen in ice-cold isopentane (Merck) and stored at −80°C until sectioning. Coronal sections (40 μm) were made at the level of the hippocampus using a cryostat .Rats were killed immediately after EP recordings (n = 27), immunofluorescence microscopy was performed on nine slices , an additional vimentin and CD11 b/c staining was performed on two separate slices at the level of the ventral injection site to assess the presence of vimentin+ reactive astrocytes . Three additional animals  were excluded because of insufficient DREADD-mCherry expression at the level of the recording electrode. Exclusion was based on visual assessment of the transduction as indicated by the anti-RFP staining, performed by researchers blinded to the experimental conditions. More specifically, the region at the level of the recording electrode was scored. A score between 0 (no expression) and 5 (very strong expression) was given for the granule cell layer and the hilus. The excluded animals all had a total score of 0. Slope of the field EPSP (fEPSP) was measured by fitting the rising phase of the fEPSP waveform using the least‐squares method. The area of the population spike was defined as the difference between the coastline of the negative peak of the population spike and the tangent connecting the positive peaks before and after the population spike. When multiple population spikes were present, areas were added up. For each animal and each parameter, the normalized values vehicle/baseline and clozapine/baseline were calculated at the stimulation intensity that during baseline recording resulted in a response with population spike area of 75% of the maximal response.Analysis of EP data were performed blinded using MATLAB . One animal was excluded from the analysis, because of hardware problems during the recording (https://spyking-circus.readthedocs.io/en/latest/). A threshold of −6.5 times the median absolute deviation of the signal was used. SpykingCircus was additionally used for spike sorting of single units. Subsequent manual curation of single unit clusters was done using the Phy GUI . Firing frequency of multiunit or single-unit activity was computed in 60-s bins. Baseline and vehicle sessions were averaged and taken together as “baseline.”To extract multiunit activity, the broadband signals were bandpass filtered between 500 and 5000 Hz. Subsequently a thresholding method was used to detect spikes in SpykingCircus .The data analysis protocol was determined a priori based on previously obtained data. Based on a priori sample size calculations, minimal sample size per group was determined to be 5 animals to be able to detect a 10% difference on EP recordings with a power of 80%. Statistical analysis was performed in RStudio with R v4.2; . NormaliF = 3.30, p = 0.047) and dorsal  = 8.27, p = 0.002) hippocampus. There was a significant effect of titer in the ventral  = 16.67, p < 0.001) and dorsal  = 3.54, p = 0.038) hippocampus on the non-injected side. Pairwise comparison showed that expression in the ventral hippocampus of DM13 and M13 was significantly different from expression in DM12 and DM11. For the dorsal hippocampus, expression in group DM13 was significantly different from DM11. At the injected side, there was a significant effect of group in the dorsal hippocampus  = 6.49, p = 0.004). Pairwise comparison showed that expression in the M13 group was significantly different from the DM12 and DM11 group. Although in the injected ventral hippocampus higher intensities were observed with higher titers, effects were not statistically significant  = 1.1, p = 0.362), probably because of the small sample size and the large variance within these groups. In the DM13 group, strong expression was observed throughout the entire injected hippocampus /ml . A control group expressing unfused mCherry  was also included. Expression was visualized by an immunofluorescence staining against the mCherry tag. An example for each group obtained five weeks after AAV injection is given in pocampus D,I. Littpocampus J. In addpocampus E,F. The pocampus K,M. mChepocampus L,N and cpocampus C. mCherrpocampus G, in celpocampus H. Apart F = 33.47, p < 0.001). There was a significant effect of group on the volume of the injected hippocampus  = 38.47, p < 0.001), but not on the volume of the non-injected hippocampus  = 2.43, p = 0.071). Pairwise comparison showed that volume of the injected hippocampus was significantly lower compared with the non-injected hippocampus in the DM13  and D13  group. There was a significant effect of group on the thickness of the cell layers on the injected side  = 35.34, p < 0.001). Pairwise comparison showed that layer thickness was significantly lower in the DM13 group compared with the M13, DM12, and DM11 group .To evaluate the effect of viral vector titer and subsequent DREADD expression levels on neuronal death, the volumes of injected and non-injected hippocampi A,C and tnD13 = 8), and 21 (nD13 = 4) weeks after viral vector transduction in a longitudinal study in the unlabeled DREADD group (D13). The volume of the injected and non-injected hippocampi were determined at different time points after injection  = 63.39, p < 0.001). Statistical analysis showed a significant effect of time in the injected hippocampus  = 147.64, p < 0.001) but not in the non-injected hippocampus  = 0.97, p = 0.447). Pairwise comparison for each time point showed that hippocampal volume was significantly lower for the injected hippocampus compared with the non-injected hippocampus at all time points . There was a significant decrease in hippocampal volume of the injected hippocampus between week 3 and week 5 , week 5 and week 12 , and week 12 and week 21 .To map the development of neuronal loss over time, anatomic T2-weighted MRI scans were performed 3, 5, 12  = 85.24, p < 0.001) and vimentin  = 55.93, p < 0.001) intensities. There was a significant effect of group for the CD11 and vimentin intensities in the injected side  = 32.08, p < 0.001 and F = 35.10, p < 0.001, respectively), but not in the non-injected side  = 0.53, p = 0.67 and F = 2.93, p = 0.06, respectively). Pairwise comparison showed that intensities on the injected side in the DM13 group were significantly different from intensities in the other groups for both markers. The difference between injected and non-injected side was significant in each DREADD-mCherry group for CD11  and vimentin , but larger in the DM13 condition compared with the other groups .Sections obtained five weeks after AAV injection of DREADD-mCherry  and mCherry (M13) expressing animals were stained for the presence of microglia or infiltrative monocytes/neutrophils CD11; F and reaTo evaluate the electrophysiological effects of DREADD-mediated inhibition of hippocampal excitatory neurons, DG EPs were recorded. DG EPs are field potentials evoked by activation of the perforant path, a bundle of axons projecting from the entorhinal cortex to the DG. The EPs have a characteristic pattern composed of a fEPSP, representing glutamatergic neurotransmission between perforant path and DG granule cells, and population spike(s) (PS), reflecting summed action potentials of the granule cells . ActivatF = 40.02, p < 0.001; EPb: F = 16.16, p < 0.001; 2 = 14.65, df = 4, p = 0.005; EPb: χ2 = 16.83, df = 4, p = 0.002; t = −151.77, df = 5, p < 0.001, mean difference of −83 ± 2%; EPb t = −36.21, df = 5, p < 0.001, mean difference of −77 ± 5%) and the DM13 group . Population spike area/fEPSP slope ratio was significantly reduced for EPa and EPb in the D13 group  and for EPa in the DM13 group  compared with the vehicle-treated condition. In the other groups , no significant differences between vehicle and clozapine were observed for fEPSP slope or population spike/fEPSP slope ratio. However, in five out of eight animals of the DM12 group and two out of four animals of the DM11 group, multiple population spikes were observed following hM4D(Gi) activation . In the high titer groups, increased EPb/EPa ratio is partly caused by suppression of the first pulse EPa. In the lower titer groups, the increased paired-pulse relationship is solely caused by an increase in EPb population spike. In four out of ten animals of the DM13 group, EPs could not be recorded despite adequate electrode placement, likely because of extensive hippocampal degeneration as revealed by MRI and postmortem histologic examination  and the second EP  displayed clear hippocampal lesions and elevated levels of microglia or infiltrative monocytes/neutrophils and reactive astrocytes in the injected hippocampus, whereas in DREADD groups displaying less pronounced expression (dilution of the AAV vector titer to E + 12/E + 11 vg/ml) no significant reduction in hippocampal volume was observed. Apart from a minor increase in inflammatory markers, presumably caused by the physical injection itself, inflammation was not observed in these groups. No deleterious effects were observed five weeks after transduction in the brains of animals injected with the mCherry control vector either, despite strong mCherry expression (AAV vector titer of E + 13 vg/ml). The occurrence of neuronal loss was not influenced by the tag fused to the DREADD, since there were no significant differences between conditions with matched titer and volume but without molecular tag. Overall, our results indicate a toxic effect in the hippocampus specifically caused by high levels of expression of the DREADD hM4D(Gi), but not by expression of other proteins nor by introduction of the viral vector itself .Differences in neurotoxicity  could be explained by the membrane-associated nature of the hM4D(Gi) receptor. In microorganisms, overexpression of membrane proteins is frequently associated with high levels of toxicity . In mammSince only for the strongest DREADD-expressing condition, neuronal loss is observed already at early timepoints after transduction, this could explain why DREADD-mediated toxicity following AAV injection remains unreported. Only few rodent studies used equally high viral vector titers in combination with relatively large injected volumes, resulting in similar expression profiles. Furthermore, part of these studies only looked at the outcome at three to four weeks after transduction . Althougin vivo studies, that did not occur in control conditions where green fluorescent protein was expressed  expression following high titer AAV injection, strong inhibitory effects were observed after clozapine-mediated DREADD activation. Excitatory synaptic transmission was significantly decreased, as reflected by the slope of the fEPSP. In animals where a minimal fEPSP could still be recorded after clozapine administration, the intrinsic excitability was significantly suppressed. No significant effects were observed in the mCherry control group, indicating that the observed effects are indeed the result of hM4D(Gi) activation. These findings are consistent with previous reports on the use of hM4D(Gi) .No significant inhibitory effects on EP activity were observed for the groups injected with a lower titer AAV, resulting in more moderate, non-toxic hM4D(Gi) expression. In contrast, a broader fEPSP, larger, and more population spikes and reduced paired-pulse inhibition were seen in the majority of these animals. When looking at a neuronal level, in the dentate granule cell layer spontaneous firing of all neurons was inhibited following DREADD activation. In contrast, a few neurons located deeper in the hilus of the DG  showed an increase in spontaneous firing following clozapine administration. It should, however, be noted that we were not able to distinguish granule cells from interneurons or mossy cells in this limited data set. Nevertheless, the excitatory effects seem paradoxical, as hM4D(Gi) is widely used as a tool to silence neurons . What exOn the one hand, effects could be directly related to differences in titer, resulting in different DREADD expression patterns. There are some reports on similar mixed effects on unit activity following chemogenetic interventions. These effects are hypothesized to be the result of disruption of neuronal activity in a target area, involving disinhibition of some DREADD-negative cells as a result of changes in local microcircuitry or wider networks . AlthougAlternatively, effects can be indirectly linked to titer, because high DREADD expression levels clearly result in hippocampal neurotoxicity, which possibly creates a pathologic environment. Loss of interneurons for example would already disturb the feedback inhibition circuits even without DREADD activation. In addition, mossy cells, highly active glutamatergic neurons that indirectly exert a net inhibitory effect on neighboring granule cells, are also known to be particularly sensitive to toxicity . SubsequOur findings indicate that level of DREADD expression plays an important role on the final outcome of hM4D(Gi) activation, at least for evoked activity. Further research in a larger group of animals, both at single neuron and field potential level, is required to elucidate the precise mechanism of action.We conclude that the titer of the viral vector used to obtain DREADD expression plays a role in the development of hippocampal neurotoxicity, as well as in the expression pattern, which in turn seem to affect the net modulatory effects following DREADD activation. This has possible implications for all DREADD-based research. Based on this study we would recommend to be cautious for deleterious effects of DREADD expression to avoid confounding of results and mention the titer of the AAV used. Especially with regard to therapeutic applications of DREADDs, long-term studies on toxicity are advised. In addition, DREADD function should be assessed electrophysiologically, before inferring conclusions from, e.g., behavioral studies, as DREADD-mediated effects might be dependent on expression pattern.


This study was designed to investigate differences in biochemical parameters between mouse and humans after paraquat (PQ) poisoning and develop a suitable animal model for studying organ damage after PQ poisoning. The prognostic factors of PQ-poisoned patients were further analyzed.  Thirty C57BL/6J mice were randomly divided into five groups , and the mouse model was established by intragastric administration of PQ. Physiological indexes such as the body weight, mental state, and mortality rate were observed. Biochemical parameters were analyzed 24 h after PQ poisoning. We also performed a retrospective analysis of clinical data from 29 patients with PQ poisoning admitted to the Emergency Department of the Affiliated Hospital of Taishan Medical College between April 2016 and February 2018. Biochemical parameters were compared between the mouse model and patients with PQ poisoning. +), carbon dioxide (CO2), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) compared with the nonlethal dose PQ100 and PQ200 groups. The biochemical results of the patients showed that K+ and Cl− levels were significantly reduced in the death group compared to the survival group. Levels of ALT, AST, blood urea nitrogen (BUN), and amylase were higher, and the neutrophil-to-lymphocyte ratio (NLR) was increased in the death group compared with the survival group.  In the PQ poisoning mouse model, the lethal dose group PQ360 showed remarkable increases in serum levels of potassium (K+, Cl−, BUN, ALT, AST, amylase, and NLR can be used to more accurately predict the outcome of patients with PQ poisoning. C57 mice are an appropriate animal model to study liver and kidney functions following PQ exposure. The combination of age, PQ dosage, K Paraquat , also called Yisaoguang (in Chinese Pinyin) and Gramoxone, is a highly effective, nonselective, contact herbicide used widely worldwide. It can be quickly absorbed by the green tissues of plants and quickly withers the foliage. PQ is rapidly deactivated once combined with soil, so it does not affect the roots or perennial rhizomes of plants. This herbicide is extremely poisonous to humans and animals . IntentiA number of predictors for prognostic outcome have been used in patients with PQ poisoning, including plasma PQ concentration , arteriaThis study is aimed at identifying markers for early judgment of PQ poisoning severity to improve diagnosis by conducting a retrospective analysis on clinical data from 29 patients who were diagnosed with oral PQ poisoning. We also evaluated the suitability of an animal model for studying organ damage after PQ poisoning by comparing biochemical parameters between animal model and humans. The results of such studies will be valuable for elucidating the mechanism of PQ poisoning.Thirty adult male C57BL/6J mice (22-25 g) were obtained from the Medical Experimental Animal Center of Weifang Medical College. The animal house temperature was maintained at 22 ± 2°C with a 12 h light/dark cycle. All animals were acclimatized 1 week prior to experiment and had free access to food and water. The studies were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the Weifang Medical University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Weifang Medical University . PQ was purchased from Aladdin .n = 6 each): control group, mice did not receive any substance; sham group, mice were given physiological saline according to their body weight; experimental group 1 (PQ100), intragastric (i.g.) administration of PQ 100 mg/kg; experimental group 2 (PQ200), PQ 200 mg/kg i.g.; and experimental group 3 (PQ360), PQ 360 mg/kg i.g  and stored at -20°C until they were assayed. Various biochemical parameters in serum were measured using an automatic biochemical analyzer  according to the manufacturer's protocols. The biochemical parameters tested include alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (Alb), bilirubin, malondialdehyde (MDA), total organic carbon (TOC), and total cholesterol (TC). The average of three replicates was recorded for each sample. The damage caused by different concentrations of PQ was determined based on biochemical parameter changes.This retrospective study was conducted in accordance with the Declaration of Helsinki and was approved by the Medical Ethic Committee of Taishan Medical University and the associated Institutional Review Board. The study was a retrospective review of the existing data, so informed consent was not required. Written informed consent acknowledging risks related to PQ poisoning and treatments were provided by all patients when they were first admitted to the hospital. Patient records and information were used anonymously.In this study, we retrospectively selected 29 cases diagnosed with PQ poisoning by the Affiliated Hospital of Taishan Medical University between April 2016 and February 2018. There were 15 males and 14 females aged between 14 and 65, with a mean age of 45.52 years old. Volumes of PQ ingested ranged between 5 and 200 ml. All patients were gastrointestinally intoxicated and diagnosed in accordance with the Expert Consensus on Diagnosis and Treatment of Acute PQ Poisoning (2013) by the Chinese College of Emergency Physicians (CCEP). The specific diagnostic criteria were (1) history of PQ ingestion; (2) clinical manifestations: cough, chest distress, and expiratory dyspnea for patients with pulmonary injury, and hematuria and oliguria for patients with renal injury; (3) laboratory assays: PQ detected in blood and urine, hypoxemia and metabolic acidosis detected in blood gas analysis, increases of ALT, AST, bilirubin, creatinine (Cr), and blood urea nitrogen (BUN) on liver and kidney function tests.All patients were given the same treatments: poison elimination , antioxidant treatment (high-dose vitamin C and reduced glutathione), immunosuppressants , and routine anti-inflammatory therapy to protect important organs.Inclusion criteria: (1) patients with acute PQ poisoning through oral intake confirmed on admission, (2) aged ≥ 14 and ≤65, and (3) complete data and clear prognostic outcomes with availability for a follow-up visit 6 months following poisoning.Exclusion criteria: (1) history of ingesting other poisons; (2) history of severe diseases in organs such as the heart, liver, and kidneys that significantly influence the outcomes of PQ poisoning; (3) had received treatment in other hospitals before admission or halted treatment after admission; (4) decline to participate in the study; (5) pregnant or lactating; or (6) comorbid cancer.Two physicians independently collected data on patients with PQ poisoning within 1 h of admission, including urinary PQ concentration, complete blood counts, C-reactive protein (CRP), procalcitonin, liver and kidney function, electrolyte levels, blood glucose, blood coagulation, and myocardial enzymogram. We compared these biochemical parameters with those measured in mice.t-tests if they were normally distributed and with nonparametric tests if nonnormally distributed. P ≤ 0.05 was considered statistically significant for all analyses.GraphPad Prism 8.0  and SPSS 16.0  software packages were used to conduct statistical analyses. One- and two-way analyses of variance (ANOVAs) were used to compare results among multiple groups. The general information and laboratory data  of patients were analyzed with The general state of the control and sham groups showed normal breathing, activity, and behavior; good appetite; and no weight loss. Within 24 hours of PQ exposure, mice in the experimental groups showed abnormalities that mainly manifested as slow movement , unstablThe survival curves in In the first 5 days after PQ treatment, the weight of all experimental group mice was decreased . On day P < 0.05) in serum Na+ compared with the control, sham, and PQ100 groups (P < 0.05). Serum Ca2+ in the PQ200 group was significantly higher compared with the sham group (P < 0.05). Compared with the control and sham groups, there were significant increases in levels of TP, Alb, and GLO in the PQ200 group. Uric acid in the PQ100 group was significantly elevated compared to the control group (P < 0.05). No significant changes in the experimental groups were observed for other indicators such as ALP, serum sialic acid, total bilirubin, direct bilirubin, serum indirect bilirubin, total bile acid, BUN, Cr, or β2-microglobulin  caused a remarkable reduction  and survival  groups. Patients' conditions and laboratory results are shown in According to the outcome, the 29 cases were divided into death (P < 0.01). Patients with lower urinary PQ concentrations also had a higher survival rate (P < 0.01). The two groups differed significantly in age  with the nonlethal dose groups (PQ100 and PQ200) showed that the K00 group .−1 and 5.29 ± 3.05 mmol · ml−1, respectively . + levels were 3.85 ± 0.30 mmol · ml−1 for the survival group, which was significantly higher than 3.45 ± 0.50 mmol · ml−1 in the death group . No significant differences were found between the two groups for serum concentrations of Ca2+, P, and HCO3−. There were also no remarkable differences in prothrombin time, activated partial prothrombin time, international normalized ratio, D-dimer concentration, or fibrinogen levels between the survival and death groups , 0.889 for amylase , 0.883 for urinary PQ concentration , 0.845 for ALT , 0.832 for AST , 0.797 for BUN , 0.779 for age , 0.763 for Cl− , 0.761 for K+ , and 0.737 for NLR   . Except PQ induces oxidative damage and cell death by generating ROS including hydrogen peroxide, superoxide anion, and hydroxyl radicals. Oral PQ ingestion impairs the lung, liver, kidneys, and neural tissues and eventually leads to death due to multiple organ failure . Due to P < 0.05). Compared with the PQ200 and PQ100 groups, both ALT and AST were significantly higher in the PQ360 group . Clinical statistical analysis showed that patients with higher ALT and AST concentrations were more likely to have a poor prognosis [In this study, mice received i.g. PQ to simulate the clinical process of oral poisoning and digestive tract absorption. Liver function assays in patients and mice revealed significant differences in ALT and AST . Patients who died showed significant reductions in Cl− and K+ concentrations compared to those in the survival group. Compared with the PQ200 and PQ100 groups, Cl− concentration was reduced in the PQ360 group . The Cl− concentration trends in the PQ mouse model and patients were consistent, but there was no significant difference in Cl− concentration in mice. We speculate that the reason for this inconsistency is the difference in measurement time. Animal serum electrolyte levels were measured 24 h after PQ ingestion, and K+ significantly increased in the PQ360 group. Conversely, levels were significantly decreased in patients in the death group. It is generally considered that hypokalemia caused by PQ poisoning might be due to four possible mechanisms. (1) Loss of K+ from the kidneys: PQ induces the formation of free oxygen radicals, which contributes to renal tubular necrosis and affects K+ re-absorption [+ and phosphorus via the gastrointestinal tract: blood electrolyte levels were measured for all patients within 1 h of admission, but gastrointestinal tract impairment is a chronic process, so this factor might not be the main cause of hypokalemia. (3) Transfer of extracellular K+ into cells: increased secretion of catecholamines and glucocorticoids and enhanced activity of sodium-potassium pump in response to oxidative stress caused by PQ poisoning will promote K+ entry into cells and lead to decrease serum K+ levels [+ excretion. The increase of K+ in mice is likely due to decreased food and water intake of mice after ingesting PQ, which affects kidney function and prevents K+ excretion. Acute renal failure is a well-recognized complication of PQ poisoning as the compound is primarily metabolized by the kidneys, causing renal injuries such as proximal tubular necrosis [Renal function-related indicators such as Ksorption . (2) Los+ levels . (4) Diunecrosis  and Fancnecrosis . The spe+, Cl−, BUN, ALT, AST, amylase, and NLR can accurately predict the outcome of patients with PQ poisoning. Our results show that different ingested PQ volumes had a statistically significant impact on patient mortality (P < 0.05), which was confirmed by ROC curve analysis. Larger ingested PQ volumes will lead to higher plasma PQ concentrations and poorer prognostic outcomes. We also found that patients with higher urinary PQ concentrations had poorer prognostic outcomes, which is consistent with a report by Deng et al. [P < 0.05) in our results; however, others stated that although BUN was increased in the death group, there were no significant differences between survival and death groups [This study explored and compared prognostic indicators between an animal model and patients. We found that the combination of age, urinary PQ concentrations volume of PQ ingested, Kg et al. . BUN levh groups , 23. SonP < 0.05). However, ROC curve analysis showed that NLR was not a powerful prognostic indicator compared with other measures. When the NLR cut-off value was 15.00, sensitivity was 70.00% and specificity was 94.70% for prediction mortality. This is inconsistent with previous research [P < 0.05), in that patients with higher amylase concentrations had poorer outcomes. This is consistent with the results of two recent studies [As a sensitive infection indicator, NLR is considered as a product of inflammation stress response that can predict patient outcome . PQ can research , 30. Ana studies , 32. A r studies . However studies , so furt+, Cl−, and amylase can more accurately predict the prognosis of patients with PQ poisoning. Our results show that the indicators in mice are not identical to those of humans. There were differences in most biochemical parameters, except for liver function and some renal function indicators. Overall, the C57BL/6J mouse strain is a valuable animal model for research on liver and renal function in subjects with PQ poisoning.In summary, the combination of age; urinary PQ concentration and ingested volume; NLR; liver function indicators ALT and AST; kidney function indicator BUN; and serum levels of K


Clostridium tetani could help to ensure consistent product quality in tetanus vaccine manufacturing, ultimately contributing to reduced animal testing. The aim of this study was to identify RNA signatures related to consistent TeNT production using standard and non-standard culture conditions. Methods: We applied RNA sequencing (RNA-Seq) to study C. tetani gene expression in small-scale batches under several culture conditions. Results: We identified 1381 time-dependent differentially expressed genes (DEGs) reflecting, among others, changes in growth rate and metabolism. Comparing non-standard versus standard culture conditions identified 82 condition-dependent DEGs, most of which were specific for one condition. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene showed significant down-regulation at higher pH levels (pH 7.8), which was confirmed by the quantification data obtained with the recently validated targeted LC-MS/MS approach. Conclusions: Non-standard culture conditions lead to different gene expression responses. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing.Background: Ensuring consistency of tetanus neurotoxin (TeNT) production by  Clostridium tetani neurotoxin (TeNT) is the essential component of (human) tetanus vaccines. The manufacturing process starts with the generation of stable and good manufacturing practice (GMP)-monitored toxin-producing C. tetani seed lots, which are then cultivated for days until the end of their growth phase. At this point, TeNT is harvested from the culture supernatant, purified, and inactivated by the addition of formaldehyde. The resulting toxoid is then mixed with an adjuvant, usually aluminum salts, prior to filling and packaging of the vaccine [The detoxified form of the  vaccine . The finMany quality control assays for batch release have been used for decades and are historically part of Pharmacopeial Monographs from the European Pharmacopoeia (Ph. Eur.) or the United States Pharmacopeia (USP) along with World Health Organization (WHO) guidelines. These batch release methods often rely on animal models. Although animal-based potency and safety assays described within Ph. Eur. vaccine monographs have played a central role in safeguarding the quality of vaccines for decades, there are ongoing international efforts that look for alternative animal-free methods for vaccine quality control. To some extent, these have arisen from public and regulatory concerns about the ethical implications regarding animal testing . The inhOne of the major critical points in the production of clostridial vaccines is the stable and reproducible production of high levels of toxins, as industrial TeNT production is sometimes hampered by low titers and occasional batch failures . QuantitThe goal of this study is to identify RNA signatures related to consistent TeNT production during batch fermentation under manufacturing conditions, with a view to better monitoring the consistency of this manufacturing step.C. tetani cultures were grown using simulated vaccine production processes under different small-scale batch fermentation conditions, comprising standard conditions and modifications thereof ( thereof . A compa thereof . The chotetX) gene expression was consistently high compared to the rest of the transcriptome, and this held for the majority of the bacterial growth conditions. In fact, in seven out of 39 samples the tetX gene had the highest expression level of any of the transcripts monitored. However, when the bacteria were grown either at 36 °C or pH 7.8, the tetX transcriptional activity was somewhat lower  were highly similar (median Spearman’s R = 0.967) and that differences in genome-wide transcriptional profiles generally were more pronounced between time points than across different culture conditions . Therefore, an ANOVA model that included time and culture condition as variables was used in our further analyses.Statistical analysis revealed that 1381 genes were differentially expressed over time . A list The number of DEGs by non-standard culture condition versus standard conditions was 1 for no agitation, 30 for 36 °C, 7 for 32 °C, 50 for pH 7.8, and 0 for increased nitrogen flow . There wtetX (CTC_RS14060), as this gene codes for the neurotoxin used for vaccine manufacturing [tetX was significantly (FDR < 5%) downregulated at pH 7.8 and also, albeit not significantly, at 36 °C , amino acid permease (CTC_RS04695), ethanolamine utilization protein (CTC_RS11165), and five hypothetical proteins . When adjacent clusters were included, we found a set of 20 DEGs for which the overall gene expression changes resembled those of tetX and that were located close together on the chromosome, rather than the plasmid on which the tetX gene is located. Additional BLAST alignment analysis indicated that these 20 genes are part of a prophage locus that corresponds to Clostridium phage phiCTC2A. This locus spans the C. tetani chromosome from positions 1,138,018 to 1,185,037 and contains 62 genes, including four down-regulated genes under both 36 °C and pH 7.8 . A full list of DEGs and their expression profiles is available in A heatmap of the DEG expression changes showed the overall pattern of differences between the various culture conditions . From a acturing . tetX watetX would manifest on a protein level, we utilized the recently described and validated targeted LC-MS/MS methodology [tetX, produced in the culture medium under those different circumstances. The results  and non-standard (non-conforming or stressed) culture conditions. To this end, gene expression analysis was performed on As a method for gene expression analysis, RNA-Seq was used. The main strength of this method is the possibility to simultaneously quantify the number of mRNA transcripts for all genes—and even noncoding transcripts—in the genome. A relative disadvantage is the time needed for library preparation and sequencing, which would not be practical for future in-process monitoring settings. For identifying RNA signatures, the possibility to analyze all genes outweighs this disadvantage, whereas for in-process settings faster methods such as reverse transcription–polymerase chain reaction (RT-PCR) can be used on a small subset of genes.C. tetani genes showed significant (FDR < 5%) expression changes over time, reflecting changes in growth rate and metabolism, among others. The overall aim of this work, however, was not to investigate changes during batch fermentation culture, but rather differences between culture conditions that could be informative parameters for monitoring vaccine manufacturing. Viewed in this way, the degree of regulation was much smaller. For some, but not all, culture conditions, significant DEGs were identified. Most of these DEGs were only significant (FDR < 5%) for one culture condition  down-regulated by the culture conditions pH 7.8 and  36 °C, as well as by time. Despite its differential expression by culture conditions and time, expression of tetX was always high and relatively stable during and between cultures  of the ondition , and theondition  revealedurotoxin . Expresscultures , which si et al. . Licona-tetX expression. The expression of tetX is mainly regulated by the BotR transcription factor , which rom tetX . More repromotor . Howeverpromotor  showed stetX and that were significantly down-regulated by pH 7.8 and/or 36 °C. However, for most of these DEGs, little functional annotation is available, and the magnitude of their down-regulation was smaller than for tetX  as well as regulate toxin gene transcription [C. tetani genome clustered with the transcription of the tetX transcriptional regulator BotR [The DEG heatmap  revealedfor tetX , which mcription ,24. In ftor BotR . It is tC. tetani genome-wide expression regulation. At the culture time points  sampled, tetX expression was consistently high, so it was either expressed from the beginning onwards in our medium, or tetX expression was triggered at a timepoint preceding our earliest sample point. Deviating  culture conditions  led to decreased tetX expression. As tetX has a high expression and sufficiently large dynamic range, it could be useful as an initial transcriptional biomarker for evaluating process consistency and TeNT yields in large-scale batch fermentation cultures. Other DEGs identified in this study might also be useful for assessing the extent of standard and/or non-standard culture conditions or, alternatively, gaining insight into the cellular adaptations (co)occurring during such non-standard culture conditions. Finally, since our findings for tetX under the described conditions were also consistent with the findings at the TeNT protein level, future manufacturing batch monitoring could be based on RNA as well as protein analysis. A choice between RNA- or protein-based batch consistency monitoring will likely depend on technical factors as well as regulatory acceptance. In this respect, it may be noted that both RNA-Seq and LC-MS/MS require specialized equipment and highly trained personnel and therefore other methodologies for determining RNA and/or protein abundance, such as RT-PCR, may be preferential for implementation in vaccine manufacturing processes.Taking into account all the findings described above, it is clear that growth phase and metabolic adaptations are major factors in C. tetani (strain A) batch cultures were grown at Sanofi-Pasteur using simulated vaccine production processes under different small-scale batch fermentation conditions in a 250 mL culture using Ambr 250 High Throughput Vessels , using the medium type as described in [2 flux in headspace) and five non-standard (stress) conditions that differed from standard conditions by changing one of these parameters, namely, no agitation, higher (36 °C) and lower (32 °C) temperature, higher pH (pH 7.8), and an increased N2 flux in the headspace (eadspace . In the A schematic overview of the sampling conditions is given in g). After supernatant removal, the cell pellet was put in suspension with 500 μL of fresh medium. For RNA preservation, 1 mL of RNAprotect Bacteria Reagent  was added. After homogenization, samples were stored at 5 °C for 5 min to 1 h and then stored at −70 °C until RNA extraction. After thawing, 109 cells were collected for RNA extraction. The cell content was not enough for certain samples, and in this case the totality of the sample was used. RNA extraction and purification were performed by using the RiboPure™-Bacteria Kit from Life Technologies  (ref. AM1925) and the RiboPure Bacteria procedure (Part Number 1925M Rev. D). Briefly, the protocol includes the following steps: The RiboPure-Bacteria method disrupts bacterial cell walls by beating cells mixed with RNAWIZ and 0.1 mm zirconia beads in a vortex adapter for 10 min. The lysate is then mixed with chloroform and centrifuged to form three distinct phases; the upper aqueous phase contains RNA. The RNA is then diluted with ethanol and bound to a silica filter. The RNA bound to the filter is washed to remove contaminants, and eluted in a low-ionic-strength solution. Post-elution removal of contaminating genomic DNA was done with Ambion  DNA-free™ Reagents, and removal of the DNase I and divalent cations in the buffer was done using DNase Inactivation Reagent. As with all glass fiber filter purification methods, 5S ribosomal RNAs and tRNAs are not quantitatively recovered using the RiboPure-Bacteria Kit. Prior to storage, RNA QC was performed to determine the concentration and the integrity (RNA integrity number (RIN)). Purified RNA samples were stored at −70 °C and later shipped to RIVM. Total yield per sample varied from 2 to 30 µg of RNA for samples that were used in subsequent steps, with an average of 14 µg. Samples with an RNA yield of 1 µg or less were not used because this amount was insufficient for further analysis.RNA extraction and purification were performed at Sanofi Pasteur. For RNA isolation, 10 mL samples of cell suspension were collected and centrifuged . Additional reagents used were the Ribo-Zero rRNA Removal Kit for Gram-Positive Bacteria , RNA Clean & Concentrator-5 , and the Baseline-ZERO DNase . The library preparation PCR step used 15 cycles. Libraries were pooled and 1.3 mL (1.9 pM) combined with 1% PhiX Control v3  were sequenced using Illumina NextSeq equipment and the NextSeq 500/550 High Output Kit v2.5 (cat.nr. 20024906) and 75 cycles.For the second study, RNA-Seq was performed at BaseClear, using their Illumina-based protocols for bacterial RNA-Seq. A total number of 13 samples was used for RNA-Seq. FASTQ raw data were sent to RIVM via file transfer protocol (FTP) for further analysis.Technical quality control of the RNA sequencing data was performed in a Linux environment using a pipeline developed in-house containing open-source tools such as FastQC  and MultiQC . This quality control step comprised aspects such as basecalling accuracy, GC content, N content, sequence length distribution, and adapter content.C. tetani using Bowtie2 software  at standard settings. Mapping was done using the published reference genome sequence of the E88 strain [tetX. All genome data used were downloaded from NCBI. The E88 genome is practically identical to that of the A strain [Next, NGS reads were trimmed to the first 50 nucleotides and mapped to the genome of 8 strain , which cA strain . Bowtie2https://cran.r-project.org/ accessed on 28 November 2021) and “DESeq2” and “limma” from Bioconductor (https://bioconductor.org/ accessed on 28 November 2021). For the visualization of genome-wide gene expression, data were transformed to transcript per million. For all other visualizations and statistical analysis, a variance stabilizing transformation (vst) was applied. Gene expression values were compared using ANOVA [Normalization and statistical analysis were done in R statistical software using additional R packages “gplots,” “seqinr,” “UpSetR,” and “viridis” from CRAN  and the supernatants filtered through a 0.22 µm filter to ensure no residual bacteria were present. Next, 110 µL of 10 X protease inhibitor mixture  were added to 1 mL of each supernatant, followed by vortexing, storage at ≤−70 °C, shipment to Sciensano, and subsequent storage at temperatures ≤−70 °C until further processing.The initial processing steps of cell suspension aliquots destined for targeted LC-MS/MS protein analysis were performed at Sanofi Pasteur. Samples for these analyses were taken in parallel to culture samples for gene expression analysis. Five mL cell suspensions samples were centrifuged  Tween-20 and underwent the addition of the control peptides (IS), reduction with dithiothreitol, and alkylation of the cysteines by iodoacetamide prior to the addition to a final concentration of 0.1 M ammonium bicarbonate pH 8.0 and 5 µg of trypsin per sample. The digest was incubated overnight at 37 °C and the reaction was stopped by adding 10 µL of a 10% (v/v) formic acid solution, followed by a vacuum concentration step (5 h at 45 °C). The pellet was first incubated at −20 °C for at least two hours prior to reconstitution with 500 µL of 1% (v/v) formic acid solution. After digestion, the peptides were centrifuged at 20,000× g for 5 min and the supernatant was transferred to injection vials. The measurements were performed on a UHPLC device for chromatographic separation followed by detection using tandem mass spectrometry (MS/MS) in multi-reaction mode. For each TeNT quantification experiment, a calibration curve was generated using the stock solution of TeNT for conversion to Lf/mL units. The detailed LC-MS/MS conditions and validation data were described in [The amount of TeNT present in the samples was assessed by means of a targeted LC-MS/MS method that was recently validated for the quantification of TeNT present in bacterial media during vaccine production . Brieflyribed in . All sam2 flux) have not been reported before.TeNT concentrations for standard, no agitation, and 36 °C conditions were previously reported as part of the methodology description and validation ; TeNT da


For patients with rheumatoid arthritis (RA) in China, little is known of how their illness perceptions affect their health-related quality of life (HRQoL). The present study investigated associations between specific illness perceptions due to RA and HRQoL features.For 191 patients with RA, illness perceptions were measured using the Brief Illness Perceptions Questionnaire (BIPQ) comprising 8 domains. HRQoL was determined with the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). Multivariate linear regression analyses were performed.P < 0.001, 95% CI − 7.080 to − 4.077). Positive associations between BIPQ features and HRQoL included personal control  and treatment control . Negative associations with HRQoL were identity  and emotional response .The overall BIPQ of patients with RA was 49.09 ± 11.06. The highest and lowest scores were for concern (9.15 ± 1.81) and personal control (4.30 ± 2.52), respectively. Multivariate stepwise regression analyses showed that the overall BIPQ was significantly negatively associated with each HRQoL feature, and HRQoL total score (β = − 0.343, Patients with RA in China perceive their illness in ways that affect their HRQoL. These results suggest that strategies that target these perceptions may improve the quality of life of these patients. Rheumatoid arthritis (RA) is a chronic disease characterized by symmetric polyarticular arthritis. It is the most common autoimmune disease. Clinical manifestations of RA include joint swelling, pain, and limited function , 2. ProgHealth-related quality of life (HRQoL) may be measured to reveal the physiological, psychological, and social functions of patients with RA , 11, 12.However, to our knowledge, there have been no studies on illness perception and its association with HRQoL for patients with RA in China. The purpose of the present study was to identify those perceptions of illness that influence patients’ quality of life, to better guide clinicians in the management of RA, and improve the ability of patients with RA to self-manage and improve their HRQoL.This cross-sectional study was conducted in an outpatient clinic at Xijing Hospital, Xi’an, Shaanxi, China from March 2017 to December 2017. The Ethics Committee of Xijing Hospital approved the study (KY20140902-5), and all subjects provided written informed consent prior to their participation.The study included patients with RA diagnosed according to the ACR (American College of Rheumatology)/EULAR (European League against Rheumatism) 2010 classification criteria . All theceptions ; the samSociodemographic and clinical data were collected through face-to-face interviews with the patients. Disease activity estimates were based on the Disease Activity Score 28 (DAS28): an index of physician-rated tenderness and swelling scores for 28 joints and an inflammatory biomarker (erythrocyte sedimentation rate or C-reactive protein) CRP as the inflammatory index . IllnessThe Brief Illness Perceptions Questionnaire (BIPQ) assesses an individual’s perceptions and cognitions regarding their disease , 30. TheHRQoL was measured with the Chinese version of the Medical Outcomes Study 36-item Short-Form Health Survey (SF-36). The SF-36 consists of 36 items that measure the following 8 dimensions: physical function (PF); role limitations related to physical problems (RP); bodily pain (BP); general health perception (GH); vitality (VT); social functioning (SF); role limitations due to emotional problems (RE), and mental health (MH). The score of each dimension is converted to a standard score ranging from 0 to 100, with a highest score indicating the best HRQoL . The SF-t-test and one-way analysis of variance were used to analyze inter-group differences with normal distribution.Statistical analyses were performed using Statistical Package for Social Science 18.0  software. The descriptive statistics are presented as the mean and standard deviation for quantitative data, and percentage for count data. The independent samples P < 0.1) for subsequent multivariate regression analyses. Multivariate stepwise regression analysis was used to explore the effect of illness perceptions on HRQoL. After controlling for demographics and disease characteristics, independent variables were entered stepwise into the model. P values < 0.1 were added into the regression, and only P values < 0.05 were considered statistically significant. The total score for quality of life was normally distributed (P = 0.066); the scores of each dimension did not conform to a normal distribution and were analyzed after normalization conversion (P < 0.001).Linear regression analyses were used to test the univariate correlations between the domains of illness perception and HRQoL, and to screen the significant independent variables  were women ; timeline (7.68 ± 2.78); treatment control (7.83 ± 2.3); emotions (7.15 ± 2.9); consequences (6.72 ± 3.02); identity (6.61 ± 2.74); coherence (6.08 ± 2.60); and personal control (4.30 ± 2.52). Concern and timeline received the highest, while personal control was the lowest.P < 0.05, = 0.018, and = 0.039, respectively). When stratified by occupation  and 3 levels of education, each dimension differed significantly among the subgroups except for role limitations due to emotional problems. Significant differences in SF-36 scores based on disease duration were found in all dimensions except general health perception, role limitations due to emotional problems, and mental health. When the population was stratified by DAS28 , each dimension differed significantly among these subgroups  population, the SF-36 scores of the 8 dimensions, from highest to lowest, were social functioning, mental health, physical function, vitality, bodily pain, general health perception, role limitations due to emotional problems, and role limitations related to physical problems. Compared with the younger subgroup (age < 45 y), the older subgroup had significantly lower scores for physical function, vitality, and social functioning  were selected as the independent variables for multivariate stepwise regression . Model 2 included the demographic variables of Model 1, and in addition the clinical characteristics disease duration and DAS28. DAS28 could account for 47.9% of variance of the total SF-36, in addition to the demographic variables . Model 3 included all the variables of Model 2 (demographic variables plus disease duration and DAS28) and the overall BIPQ. . Model 4 incorporated Model 2 and the 8 BIPQ domains, without the overall BIPQ. .Four models were analyzed, with different variables. Model 1 included demographic variables . Age, education, and occupation could account for 17.1% of the variance of the total SF-36 score . The total BIPQ had a significant negative association with each component of the SF-36, and also the total SF-36 (Model 3).In addition, the BIPQ domains personal control, treatment control, identity, and emotional response were associated with HRQoL when the demographic factors and disease characteristics were controlled. The BIPQ domains explained 9.0% of the variance of the total SF-36. Identity and emotional response were negatively associated with HRQoL, however personal control and treatment control were positively associated with HRQoL. Furthermore, consequences were associated with vitality, and coherence with mental health  . This shThe present study also found that patients older than 45 years had significantly lower scores for physical function, vitality, and social functioning compared with younger patients. Patients with a disease duration more than 5 years, and more serious disease activity, had the worst HRQoL. Patients with higher education and engaged in white collar occupations had better HRQoL compared with patients with less education or poorer employment. A literature review reported that increased age was associated with reduced physical function and physical component summary scores for HRQoL . The pre2 = 0.650, F = 174.913, P < 0.001). It must be noted that clinical characteristics accounted for 47.9% of the variance in total HRQoL, in addition to the demographic variables . Although this association is not strong, it obviously has importance. The reason for such discrepancy may be the different study populations and recruitment methods. It has also been reported that sleep impairment is a common clinical condition in patients with RA [Berner et al.  reported with RA , 39, and with RA . Fatigue with RA , These a with RA , 42, andHyphantis et al.  reportedA review regarding illness perceptions concluded that negative emotions can affect the treatment of diseases, limit the physiological functions of patients, and seriously affect the prognosis of diseases. Illness perceptions, depression, anxiety, and quality of life were closely related . IllnessHo et al.  reportedThis study specifically showed that illness perceptions influenced different dimensions of HRQoL in patients with RA. Patients with the worst illness perceptions had the worst HRQoL. Previous research in RA showed that illness perceptions had significant implications for adaptation to illness and notably affected medical disease status, even more so than depression, physical function, or pain . Berner There are some limitations to this study. The cases were from a single hospital and the sample size was relatively small. Longitudinal studies are needed before further conclusions can be drawn. Future studies should add variables such as sleep disturbance, depression, anxiety, and fatigue, and explore how these factors and illness perceptions can affect HRQoL.Illness perceptions were associated with the HRQoL in this study of a patient population with RA in China. Patients with the worst illness perceptions had the worst HRQoL. Illness perceptions are important potential targets to improve the quality of life of patients with RA. Illness perceptions may therefore be a useful basis for future quality of life interventions.