Both mutants displayed a series of metabolic and transcriptional alterations, especially in the lipid, flavonoid, and terpenoid pathways. Levels of several diterpenoid phytoalexins were substantially increased in d10 and d14, together with the induction of terpenoid gene cluster and the corresponding upstream transcription factor WRKY45, an established determinant of plant immunity. The fact that WRKY45 is a target of IPA1, which acted as a downstream transcription factor of SL signaling, suggests that SLs contribute to plant defense through WRKY45 and phytoalexins. Moreover, our data indicated that SLs may modulate rice metabolism through a vast number of clustered or tandemly duplicated genes. Our work revealed a central role of SLs in rice metabolism. Meanwhile, integrative analysis of the metabolome and transcriptome also suggested that SLs may contribute to metabolite-associated growth and defense.Plants have evolved many metabolites to meet the demands of growth and adaptation. Although strigolactones (SLs) play vital roles in controlling plant architecture, their function in regulating plant metabolism remains elusive. Here we report the integrative metabolomic and transcriptomic analyses of two rice SL mutants,  Since the first proto-life forms emerged on Earth some four billion years ago, a myriad of metabolites has been produced in a series of organisms to meet the demands for energy, growth and development, and for adaptation to the environment. Plants, which are evolutionarily forced to adapt to a constantly changing environment due to their sessile lifestyle, have been estimated to produce approximately 0.1 to 1 million phytochemicals . VariousThe wide array of plant metabolites represents a world of rich biological complexity and significance ,5. FlavoWRKY45, which is essential for rice resistance to various biotic and abiotic stresses [Various evolutionary routes to biochemical innovation have been adopted in plants, one of which is the use of biosynthetic gene clusters ,20,21,22stresses ,31,32, hstresses .Arabidopsis [MAX1 in rice, three of which (denoted as CYP711A2-4) are tandem duplicates on the first chromosome [CYP711A2 and CYP711A3  share convergent functions in the conversion of carlactone to carlactonoic acid, while CYP711A4/Os1500 is nonfunctional due to a premature stop codon [CYP711A2 to phosphate deficiency is also distinct from that of CYP711A3 [P450s on chromosome 1 also respond to the variation in SL content between indica and japonica, two major subspecies of rice [Ideal Plant Architecture 1 (IPA1), which encodes a SQUAMOSA promoter binding protein-like (SPL) transcription factor downstream of SL signaling [IPA1 contributes to balanced growth and immunity in rice [In addition, gene duplication and divergence also contribute to the evolution of plant metabolic diversity, as evidenced by evolutionary metabolomics ,34,35,36bidopsis ,40. Therromosome . CYP711Aop codon . AdditioCYP711A3 . Moreove of rice . This inignaling . IPA1 co in rice , which h in rice .d10 and d14. Compared to the wild-type (WT) plants, a series of metabolic and transcriptional alternations were identified in the two mutants, especially in the lipid, flavonoid, and terpenoid pathways. The defective SL pathway also led to the altered accumulation of different kinds of diterpenoid phytoalexins. Notably, WRKY45 and the biosynthetic gene cluster of diterpenoids under its control displayed altered transcript levels in d10 and d14, suggesting that SLs may contribute to plant immunity through WRKY45 and phytoalexins. Moreover, we also found that SLs regulate metabolism via genes with evolutionary signatures.A myriad of efforts has been made to illustrate the function of SLs in overall plant architecture . Despited10 and d14 [To capture a wide range of metabolic responses to SLs in rice, a previously established and developed widely targeted metabolomics method  was used and d14 . The lead10 and d14. In total, 183 and 129 metabolites accumulated at relatively higher and lower levels, respectively, in d10 leaves than in WT leaves  were identified (fold change > 1.5 times). First, we compared the metabolomic data of the WT with those of T leaves . Furtherolamines . For theectively . Metabol for d10 . Next, wd lipids .To obtain a molecular interpretation of the metabolic responses in the SL mutants, Illumina RNA-sequencing-based transcriptome profiling was conducted. A total of approximately 264 million clean reads were derived, with an average of 97% that could be mapped to the reference genome of rice . RNA reaD10 is significantly induced in the two mutants  and PIN1b (LOC_Os02g50960), displayed elevated transcript abundance in the two mutants   were identified, considering a two-fold expression change as the cut-off with a  in d14) . A combi as well .d10 and d14. We found that ten pathways were significantly enriched in d10 vs. WT and d14 vs. WT, including three shared by d10 and d14, namely, biosynthesis of secondary metabolites, diterpenoid biosynthesis, and amino sugar and nucleotide sugar metabolism , which is an important component in WRKY45-mediated resistance [japonica type WRKY45-1. Along with the increased transcript abundance of WRKY45 in d10 and d14, the expression levels of ST1 in the SL mutants were reduced to approximately 25% of that in WT, albeit with a low statistical significance  analysis was performed with the DEGs in tabolism A. To invtabolism B. Furtheh MapMan C. Consis and d14 D. There e oryzae . The japsistance . The SL ificance D. This oWRKY45-1 . Therefod10 and d14 displayed enhanced accumulation of diterpenoid phytoalexins, including phytocassane D, phytocassane E, and oryzalexin C. This is consistent with the observation that genes related to secondary metabolites were enriched in the MapMan mapping onto the biotic stress pathways. This finding also supports the view that SLs may contribute to plant defense.Of the 45 structurally identified or annotated terpenoids, 10 were identified as DAMs in both of the mutants, and 60% of these DAMs were up-regulated A. NotablCPS2, KSL5, and KSL6 were induced significantly in d10 and d14, with a more than 3.2-fold change in expression level in each genotype compared with the level in WT  involved in the synthesis of diterpenoids [CPS2, three KSLs (KSL5–7), and six P450s (two from the CYP71Z family and four from the CYP76M family). The clustered P450s have been characterized to be co-regulated with CPS2 and KSL7, which is a feature of gene clusters [CPS2 cluster. To address this issue, we analyzed the expression pattern of the P450s, with the exception of CYP76M8, due to its relatively low abundance (mean FPKM less than one in each sample). CYP71Z6 was expressed at ~2.2-fold higher levels in the SL mutants than in the WT. The expression level of CYP76M7 in d10 and d14 was reduced to less than half of that in WT , which consists of CPS4, KSL4, a dehydrogenase gene (MAS), and two P450s belonging to the CYP99A family [MAS and CYP99A members from the CPS4 cluster, even though there was no significant difference in the expression of CPS4 or KSL4 in the SL mutants and WT  was approximately 8% and 38%, respectively, of that in WT leaves  [PEAMT2 [To explore the potential molecular basis of SL-regulated lipid metabolism, transcriptomic data were then further analyzed. In total, we identified four DEGs that may be involved in the glycerophospholipid pathway. Two genes encoding PLD were found to be induced in epressed . Serine (PEAMTs) . We also [PEAMT2 , that waWhile our understanding of the association between SLs and various end phenotypes and the underlying mechanisms is growing, the role of SLs in metabolism and the way in which SLs contribute to adaptation via metabolites remain unknown. Herein, we report a combined analysis with transcriptomic and metabolomic data of rice mutants of the SL biosynthetic and signal transduction pathways. In this work, we discovered a series of metabolic responses to SLs in both primary and secondary metabolism, including in lipid, flavonoid, and terpenoid metabolism. Follow-up analysis of transcriptomic data showed the potential involvement of SLs in defensive reactions in rice. Furthermore, we found that SLs target clustered or tandemly duplicated genes to regulate GA-associated metabolism.d10 and d14. The flavonoid pathway is apparently repressed in the two mutants. That is, the metabolic flux distribution is controlled by SLs, which may occur through an upstream switch. Glycerophospholipid-related metabolites accounted for the majority of the lipids with altered abundance in the mutants. It has been documented that phosphate deficiency leads to a reduced level of glycerophospholipids, as well as induced the accumulation of SLs [d10 and d14 could be a reflection of SL-mediated adaptation to phosphate deficiency.Based on the improved widely targeted metabolome strategy, 794 metabolites were detected in our work. In brief, lipids, terpenoids, and flavonoids display strong yet distinct changes in SL mutants. The majority of lipids and terpenoids with significant changes exhibited increased accumulation in n of SLs . In addin of SLs ,61. The WRKY45, which is critical for plant defense [WRKY45-mediated defense, namely, ST1 [d10 and d14. This observation is reminiscent of the study of WRKY45-1 overexpression lines, which also repress ST1 and exhibit pathogen susceptibility. That is, SLs are likely to contribute to defensive reactions via WRKY45. This hypothesis is supported by further evidence from a study on IPA1, which has been identified as a key transcription factor downstream of SLs [Magnaporthe oryzae (M. oryzae) activates the phosphorylation of IPA1, which can then bind to the WRKY45 promoter and subsequently activate WRKY45 expression. Consequently, phosphorylated IPA1 enhances the rice defense against M. oryzae [IPA1 and WRKY45.Based on the transcriptomic and metabolomic data, we reasonably inferred a potential role of SLs in plant defense. Pieces of evidence underpinning this hypothesis arise from the observation that a series of genes, which are predicted to be of importance in defensive reactions, exhibited changed transcript abundances in the SL mutants. Notably,  defense ,31,32, wely, ST1 , was repm of SLs . Infecti. oryzae . In othed10 and d14. These compounds are considered to be pivotal in plant immunity and are induced by microbial infections. Consistent with the metabolic responses of diterpenoid phytoalexins, biosynthetic genes, including CPS2, KSL5, KSL6, and CYP71Z6, were also activated in the mutants. This indicates the involvement of SLs in plant defense, considering the established function of phytoalexins in plant resistance to diseases. Notably, WRKY45 has been shown to regulate a series of biosynthetic genes of diterpenoid phytoalexins [WRKY45 is activated in d10 and d14, subsequently inducing the expression of biosynthetic genes and the accumulation of phytoalexins. Wang et al. have revealed that GR244DO treatment induces WRKY38, which plays a role in defense responses in Arabidopsis [d10 and d14, which is the opposite of the hypothesis that SLs contribute to enhanced immunity via IPA1 and WRKY45.Focusing on the terpenoid pathway, we found that diterpenoid phytoalexins accumulated at relatively high levels in oalexins , includibidopsis . That isMAX1 in rice [WRKY45-mediated clustered genes in the diterpenoid pathway. Moreover, we found that duplicated genes of GA biosynthesis divergently responded to defects in the SL pathway, providing additional evidence for the potential involvement of SLs in evolutionary metabolism.Metabolic and functional diversification occurred during land plant evolution, benefiting from gene duplication and divergence . It has  in rice . The evo in rice ,40,64,65 in rice . Herein,Arabidopsis [D14 has been proven to be positively controlled by GAs via histone H3 lysine 27 trimethylation [Our work also provides new insights into the crosstalk of SLs with GAs, mutants of which in various cereals led to the green revolution . GAs andbidopsis . Ito et bidopsis . Most rehylation . By and IPA1, WRKY45, and phytochemicals. In addition, we also identified the epistatic effect of SLs on GA biosynthesis, through which SLs may contribute to plant growth. The integrative analysis of the metabolome and transcriptome also suggested that SL-mediated metabolism may be a critical strategy evolved by plants to modulate growth and defense.In conclusion, our work revealed an essential role of SLs in rice metabolism, coordinately modulating lipid, flavonoid, and terpenoid pathways. Meanwhile, we provide new insights into the involvement of SLs in rice immunity, which may occur through D10 and D14 were created in our previous work [Mutants of ous work . The ricous work .Samples for metabolite profiling were collected from seedlings grown in hydroponic culture for one month. The second and third upper leaves from three individual plants per line were harvested, frozen in liquid nitrogen, and combined as one biological replicate of each sample for metabolite extraction. Three biological replicates were collected from each genotype.Leaves for RNA extraction were collected from plants identical to those used in the metabolomic analysis. Each sample was harvested from three individual plants and frozen in liquid nitrogen.http://www.anpel.com.cn/), the metabolites in the mixture were quantified by the MRM method of LC-MS 8060  [−1 lidocaine) for normalization and then log 2 transformed the values to further improve the normalization.We used a grinder  at 30 Hz for 1.5 min to grind samples that were freeze-dried in a vacuum. Then, 100 mg of powder was weighed, and 70% methanol aqueous solution was added at 0.1 mg/mL. The sample mixture was extracted by ultrasonication at 40 Hz for 10 min. After centrifugation and filtration  ,71,72, sTotal RNA was extracted with a TRIzol reagent  according to the protocol provided by the manufacturer. We determined the integrity of the total RNA with a 2100 Bioanalyzer , which is quantified using a NanoDrop . Then, we construct the sequencing library using RNA samples with high-quality . We purified polyA mRNA from total RNA using oligo-dT-attached magnetic beads. The purified polyA mRNA was then subjected to a fragmentation buffer. Taking these short fragments as templates, the first-strand cDNA was synthesized using reverse transcriptase and random primers, followed by second-strand cDNA synthesis. Then, the synthesized cDNA was subjected to end repair, phosphorylation, and “A” base addition according to the library construction protocol. Then, sequencing adapters were added to both sides of the cDNA fragments. After PCR amplification of the cDNA fragments, the 150 to 250 bp target fragments were cleaned up. Then, we performed paired-end sequencing on an Illumina HiSeq X Ten platform .http://rice.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/all.dir/all.con) using Hisat2 v2.1.0. The conversion of the mapping output files from SAM to BAM format and the sorting by positions were performed using SAMTOOLS v1.9. StringTie v1.3.4 was used to determine the FPKM values and read counts by using the script preDE.py. DEG analysis was performed with count tables in R v3.6.2 using DEseq2, and genes with a p-value < 0.05 and FC > 2 were classified as DEGs.To remove low-quality bases and sequencing adapters, the raw data were first processed by FASTP v0.19.4 with default settings. Subsequently, the clean data were mapped to the rice reference genome (https://david.ncifcrf.gov/). For further pathway analysis of DEGs, MapMan v3.6.0 (http://mapman.gabipd.org/web/guest) was used to visualize stress-related and overview changes. We summarized an overview pathway based on MapMan results and then mapped the different expression genes to the overview pathways with MapMan software.The KEGG analysis was conducted with DEGs by using the David website . Ubiquitin (LOC_Os09g39500) was used as an internal control in qRT-PCR. The relative expression of the DEGs was calculated with the 2−ΔΔCT method. The qRT-PCR assay was carried out using two biological replicates with two technical replicates.The relative expression of the DEGs was validated by qRT-PCR. The primers of the nominated genes were designed using Oligo7 software  or previhttps://www.megasoftware.net/) was used for alignment with protein sequences, and the maximum likelihood method was used to construct phylogenetic trees. After obtaining the newick file, the file was imported into the EvolView online tool (https://www.evolgenius.info//evolview/) for processing and beautification.ClustalW in MEGA7 (RNA sequence data that support the findings of this study have been deposited under SRA BioProject accession number PRJNA622884.


Patients frequently experience a weight gain after organ transplantation. This increase in weight is the result of multiple factors, and is usually intensified by glucocorticoids and immunosuppressive drugs. It can also delay graft function and cause serious health problems. The objective of this study was to study the obesity as well as its causes and consequences in kidney transplant patients. The sample population consisted of 282 renal transplant patients, 170 men and 112 women, 18-74 years of age, who were monitored over a period of five years. For the purposes of our research, the patients were divided into two groups: (1) normal weight 18.5 ≤ BMI <25; (2) overweight 25 ≤ BMI ≤30. The association between BMI as an independent variable and graft survival was determined by means of a Cox regression analysis. Overweight patients were characterized by a higher comorbidity prevalence. In the Cox multivariate analysis, the initial BMI, evaluated as a continuous variable continued to be an independent predictor of delayed graft function and chronic nephropathy. This study evaluated the BMI as a continuous value instead of a categorical value. In conclusion, our results suggest that an increase in BMI without categorical variation can be an independent risk factor for graft loss. Consequently, obesity prevention for renal transplant patients should include dietary counseling and management, moderate physical activity, and steroid minimization. Obesity is a serious risk factor for deteriorated kidney function in healthy people as well as for those with chronic kidney disease (CKD) Weight gain frequently occurs after a kidney transplant Furthermore, this weight increment contributes to an adverse cardiovascular risk profile and could be implicated in the pathogenesis of graft dysfunction. The accumulation of abdominal fat can increase the inflammatory mediators produced by adipocytes, such as tumor necrosis factor alpha and interleukin-6, which contribute to interstitial and glomerular fibrosis. Obesity is associated with an increased rate of glomerular filtration, which over time eventually leads to glomerulosclerosis and the subsequent loss of kidney function. Moreover, hypertension caused by the activation of the sympathetic nervous system and renin-angiotensin system can also accelerate the loss of kidney function in obese patients Consequently, post-transplant weight gain is associated with hypertension, diabetes, dyslipidemia, and ischemic heart disease The sample population consisted of 282 kidney transplant patients, 170 men and 112 women, 18-74 years of age, who were being treated at the kidney transplant consultation of the Virgen de las Nieves University Hospital in Granada (Spain). The subjects were not randomly selected and their participation in the study was determined by the fact that they periodically visited the consultation for monitoring and check-ups. The patients were monitored over a period of five years. Exclusion criteria were (i) multi-organ transplantation, (ii) previous renal transplantation, (iii) patients <18 yr of age, (iv) obesity (BMI > 30), and (v) underweight (BMI < 18.5). BMI was calculated using weight and height at the time of transplantation. We excluded the obese and under-weight, as each circumstance is known to be a major risk for transplantation with associated confounding considerations The immunosuppression regimen was the following: steroids (Ste) associated with mycophenolate mofetil (MMF), Tacrolimus (Tac), CD25, Cyclosporin A, and Thymoglobulin (TMG). During the first four weeks, the patients received 20 mg/day of prednisone, administered in a single morning dose, which was progressively reduced until reaching a maintenance dose of 5-10 mg/day in the third month. The CsA dose, divided into two, was adjusted to maintain trough levels of 150-240 ng/ml during the first six months after transplant, and after that, trough levels of 100-150 ng/ml. The Tacrolimus dose, also divided into two, was adjusted to maintain levels of 10-15 ng/mL during the first three post-transplantation months, after which it was tapered down to 5-8 ng/ml. The MMF dose, divided into two, oscillated between 1-2 gr/day.2. The patients were divided into two groups: (1) normal weight 18.5 ≤ BMI <25; (2) overweight 25 ≤ BMI ≤30.In total, four combinations of immunosuppressive drugs were studied. The patients were divided into four groups, each of which received one of the following combinations of drugs: Group 1 [Ste + MMF + Tac + CD25]; Group 2 [Ste + MMF + CsA + CD25]; Group 3: [St + MMF + Tac + TMG]; Group 4 [Ste + MMf + Tac]. In addition, anthropometric measurements of weight and height were performed at 6, 12, 24, 36, 48 and 60 months with a balance/stadiometer (Perperson 113481). Weight was measured in kilograms and height in centimeters. The body mass index was calculated with the following formula: BMI = weight (kg)/[height (m)]On their release from the hospital, the patients were instructed to consume 1.4-1.5 g/kg/day of diet protein  during the first three months after the kidney transplant. Accordingly, they were told that lipids should not constitute more than 30% of the total diet, and that simple sugars should be avoided. After three months, the patients should reduce protein consumption to 1 g/kg/day.All eligible patients gave informed consent to participate in the study, which was approved by the Human Ethics Committee of our Hospital.p < 0.05 was considered statistically significant. Continuous variables were compared using the Mann-Whitney U test, whereas categorical variables were compared using Fisher's exact test (Table 1).Statistical analysis was carried out using the IBM SPSS Statics 20 software package. All values were expressed as frequencies, percentages, and means ± standard deviation (SD). A value of Tables 4). Analysis of variance (ANOVA) was used to evaluate the differences between weight and immunosuppression treatment (Table 2). All data were expressed as means ± standard deviation (X ± SD). A value of p < 0.05 was considered statistically significant.To evaluate overweight as a risk factor of kidney deterioration, a Cox regression was used with a confidence interval of 95%. The variables in the analysis were the following: graft recipient age, gender, BMI, dialysis time, kidney donor age, chronic allograft nephropathy, acute rejection, delayed graft function, diabetes, and hypertension  and BMI  significantly increased from the first to the fifth year after transplant. Table 1 shows the characteristics of the patient and graft after his/her classification as normal weight or overweight. As reflected in the table, recipient age and kidney donor age are significantly greater in the overweight group as compared to the normal weight group. In contrast, there are no significant differences regarding dialysis time and gender.The mean age of the sample population was 44.09 ± 14.04 years; mean weight was 66.24 ± 13.77 Kg; and mean BMI was 25 ± 4.62 Kg/mIn relation to the characteristics of the graft, the results showed a significantly high prevalence of chronic graft nephropathy, diabetes and delayed graft function. The same thing occurs with hypertension, number of HLA incompatibilities, and acute rejection though the differences are not statistically significant. The cold ischemia time was similar in both groups of patients.Table 2).In regard to the immunosuppressant treatments, all groups experienced an average weight gain of 5.5 kg from the first month after transplant until the end of the first year. After that, their weight continued to steadily increase during the following post-transplant years until reaching an average gain of 6.8 kg at the end of the fifth year. This increment was particularly striking in Group 3 in which the five-year weight gain was 14 kg as well as in Group 4, in which the gain was 9 kg (Table 3).The variables that showed a significant correlation between overweight and post-transplant outcome in the univariate analysis were the following: graft recipient age, BMI, kidney donor age, chronic graft nephropathy, and diabetes (Table 4).The results of the Cox multivariate analysis indicated a significant correlation between overweight and graft recipient age, kidney donor age, and initial BMI, which were evaluated as continuous variables, and delayed kidney function and chronic graft nephropathy, which were evaluated as categorical variables. Graft rejection, hypertension, and diabetes were also included in the Cox regression model . This is a serious problem since during the post-transplant period, obesity is an important risk factor for graft survival as well as for the development of cardiovascular diseases, hypertension, diabetes, and dyslipidemias. Moreover it is a frequent cause of morbidity and mortality in these patients This study analyzed 282 kidney transplant patients to determine the causes and consequences of obesity and its prevalence. The results obtained showed a high prevalence of overweight patients, which increased from the first to the fifth year after the transplant. As can be observed, there was a significant gain in weight and BMI after the graft  and a BMI> 30 (obese). We then evaluated whether the BMI, expressed as a continuous value, was a risk factor in the different categories. Our results showed that the BMI was significantly associated with chronic nephropathy and delayed graft function, including the patients within the same BMI category .Moreover, the multivariate as well as the univariate analysis reflected a correlation between negative graft outcome and age, gender, BMI, delayed graft function, chronic nephropathy, acute rejection, hypertension, and diabetes. Furthermore, in the multivariate analysis with continuous BMI values, it was evident that the BMI was significantly associated with graft failure, independently of the co-variables, delayed graft function and chronic nephropathy, both of which are also implicated in a negative graft outcome Weight gain after transplant is very common. In fact, various authors even affirm that it is universal This situation is a serious problem for clinical work and practice, given the potential consequences and difficulty in achieving and maintaining normal weight, The etiopathogenesis of obesity in renal transplants is multifactorial, and many of the variables that affect the transplant are similar to those that affect the population in general  Steroids are a key factor in weight gain because steroid ingestion leads to metabolic effects, redistribution of body fat, water retention, and above all, an increased appetite The prevention of weight gain after transplantation is managed by a multidisciplinary team with psychological support, who focus on helping patients to improve their dietary habits. More research is needed to evaluate how new immunosuppressive agents, steroid minimization strategies, and steroid-free immunosuppression will affect post-transplant outcomes in the obese populations. Some patients have opted for bariatric surgery. Despite the relatively small number of cases, the results are promising 2 and the weight 6.6 Kg. Consequently, obesity prevention for renal transplant patients should include dietary counseling and management, moderate physical activity, and steroid minimization.In conclusion, our study found that there was a high prevalence of post-transplant overweight and obesity during the first post-transplant year. The BMI was evaluated as a continuous value instead of as a categorical value. Our results indicated that an increment in BMI, without categorical variation, can be an independent risk factor for graft loss. After the first year, the BMI of the patients increased 2.5 Kg/m


N = 409), two subdimensions of COVID-19 conspiracy beliefs were associated with negative attitudes toward vaccine science. These results were partly replicated and extended in a pre-registered study 2 (N = 396). Moreover, we found that COVID-19 conspiracy beliefs , as well as a conspiracy mentality  negatively predicted participants’ intentions to be vaccinated against COVID-19 in the future. Lastly, conspiracy beliefs predicted support for chloroquine as a treatment for COVID-19. Interestingly, none of the conspiracy beliefs referred to the dangers of the vaccines. Implications for the pandemic and potential responses are discussed.Many conspiracy theories appeared along with the COVID-19 pandemic. Since it is documented that conspiracy theories negatively affect vaccination intentions, these beliefs might become a crucial matter in the near future. We conducted two cross-sectional studies examining the relationship between COVID-19 conspiracy beliefs, vaccine attitudes, and the intention to be vaccinated against COVID-19 when a vaccine becomes available. We also examined how these beliefs predicted support for a controversial medical treatment, namely, chloroquine. In an exploratory study 1 ( Conspiracy theories can be defined as “attempts to explain the ultimate causes of significant social and political events and circumstances with claims of secret plots by two or more powerful actors” , p. 4. TConspiracy beliefs may also influence the course of a crisis that initially favored their appearance. Indeed, conspiracy beliefs have consequences, notably in the health domain . For exaIn this research, we sought to replicate the aforementioned relationship between conspiracy beliefs, rejection of vaccination, and support for alternative treatments, in the context of the COVID-19 pandemic. Indeed, previous studies were conducted before the sanitary crisis that the world is currently experiencing. Replicating past results is essential for at least two reasons. Firstly, replication is necessary to establish the validity of frequentist statistical inferences , which iReplication efforts should be encouraged even more given the magnitude of the stakes. In the context of the COVID-19 pandemic, conspiracy beliefs may foster distrust toward health authorities and their recommendations, which could potentially impede efforts to put an end to the pandemic. In the short term, respect for containment behavior guidelines  is crucial to limit the spread of the pandemic we are currently experiencing, because the development of a treatment (including a vaccine) could take months . HoweverIn this research, we examine how the endorsement of various COVID-19 conspiracy beliefs is predictive of two vaccine-related outcomes: attitude toward vaccination science (Studies 1 and 2), conceptually replicating a research by Note that in both studies, we referred to COVID-19 (the disease) and not to SARS-CoV-2 (the virus that causes the disease), to be in line with the terminology of the French media coverage of the pandemic and therefore avoid misunderstandings.https://osf.io/3qyf4/?view_only=c2aa291fb1604b73aef9057cfc41980e.In Study 1, our goal was to explore the relationship between COVID-19 conspiracy beliefs and attitudes toward vaccines science. Data, materials in French (with English translation) and analyses are available on the OSF repository at the following address: Mage = 28.4, SD = 11.4, min = 18, max = 72, see the N = 250 requested to achieve correlations stability  to March 27. In total, 609 participants participated in the survey. Two hundred participants were removed from the data for not completing the questionnaire, for failing the attention or seriousness checks, or for being under 18 years old. The final sample was constituted of 409 participant  to Strongly Agree (coded 5).r = 0.53, p < 0.001 . Given the wide variety of COVID-19 conspiracy theories , we desiWe translated into French the 5-items scale (1 reverse coded) developed by Participants reported their age, gender (M/F/Other), geographic location, and political orientation on a scale ranging from 1 (far left) to 9 (far right), with the possibility to tick “other.”95% CI, t = −10.36, p < 0.001, and “ingroup” conspiracy beliefs, β = −0.44, 95% CI, t = −8.71, p < 0.001. Finally, we tested a model integrating both dimensions as predictors, with outgroup COVID-19 conspiracy beliefs introduced at step 2, and ingroup COVID-19 conspiracy beliefs introduced at step 3  would be (1) negative predictors of both pro-vaccination attitudes and vaccination intention, and (2) positive predictors of pro-chloroquine attitudes. Lastly, we included conspiracy mentality as an exploratory measure.Whereas Study 1 was exploratory, Study 2 aimed at testing a set of pre-registered hypothesesMage = 26.1, SD = 10.3, min = 18, max = 70, see the r = 0.16 with two tailed tests.The study was disseminated online among undergraduate students from Rennes 2 and Lille Universities who were awarded course credit for answering. It was also shared by authors on social media in order to diversify the sample, from April 17 to April 25. In total, 469 participants participated in the study, out of which 396 remained  to Strongly Agree (coded 5). Measures of attitudes and conspiracy beliefs about chloroquine, as well as vaccination intention, were pretested for internal reliability and ceiling and floor effects in an online preliminary study . The two factors structure found in Study 1 yielded a satisfactory fit , and the dimensions returned satisfactory internal reliability . The confirmatory factor analysis of the scale yielded an acceptable fit for a single factor structure, suggesting that the items captured a single construct . Note that when carrying out an exploratory factor analysis on ingroup, outgroup, and chloroquine conspiracy beliefs altogether, the three postulated dimensions were observed see .The general propensity to endorse conspiracy theories was measured with a validated French translation of the Conspiracy Mentality Questionnaire CMQ; . It is aWe created a 5-item scale (2 reverse-coded) to measure participants’ attitudes toward chloroquine medical treatment for COVID-19 . Items were preceded by a paragraph introducing the question of chloroquine . The confirmatory factor analysis of the scale yielded a satisfactory fit for a single factor structure .We used the full 5-item scale  developed by We adapted the single item used by Participants reported their age, gender (M/F/other), and political orientation on a scale ranging from 1 (far left) to 9 (far right), with the possibility to tick “other.” They also reported their level of education on a multiple choice question .Descriptive statistics and correlations between measured variables are displayed in We also tested the extent to which conspiracy mentality, rather than belief in specific conspiracy theories, predicted the three outcomes (see Step 2 (CMQ) in M = 4.72; SD = 1.80). Strikingly, 22% of the sample (N = 87) answered below the median point (4), and among them 29 (7.3% of the sample) reported that they would “refuse vaccination without hesitation.”We might add that, contrary to our expectations, we did not find a strong ceiling effect for the vaccination intention scale  have stronger relationships to vaccines science attitudes and vaccination intention than conspiracy beliefs about the ingroup (French government and industries). This might be explained both by the foreign origin of the pandemic  and distrust toward multinational pharmaceutical companies . It would surely be of interest to further investigate factors explaining the difference between ingroup and outgroup conspiracy beliefs.Rather concerning is the fact that in our sample, more than one participant out of five leaned toward refusal of the hypothetical COVID-19 vaccine, even though it was described as having been approved by the health authorities. This is congruent with data showing that the French population is extremely distrustful of vaccines . If a COLastly, we wish to emphasize that none of the conspiracy beliefs or conspiracy mentality items referred to the dangers of vaccines. Hence, a wide range of conspiracy beliefs seems to be associated with a distrust of vaccines. This is congruent with the idea that conspiracy beliefs are underpinned by a generic belief system, which is characterized by negative attitudes toward powerful groups . It therOur research has limits. Firstly, the cross-sectional design we used does not allow for inference to be drawn regarding causality. Although in line with previous research, we suspect that conspiracy beliefs may fuel negative attitudes toward vaccination , one couSecondly, some unmeasured factors may influence negative attitude toward vaccination and vaccination intention, such as concern about drug companies profiteering from vaccines , distrusThirdly, the phrasing of our vaccination intention measure may explain the high level of vaccination hesitancy in our sample. Indeed, people may need more information and guarantees about the success of medical trials and possible side-effects before stating their intention to use such a new vaccine.Lastly, our samples were not representative of the French population, with an overrepresentation of female, southern-located, educated, and left-wing participants. Moreover, online surveys do not reach the population that has no access to the internet  can be found in the article/The university of the leading authors does not have institutional review boards for psychology or social science research. We thus applied the 1964 Helsinki Declaration and its later amendments (2001), the ethical principles of the French Code of Ethics for Psychologists (2012), and the American Psychological Association Ethical Principles of Psychologists and Code of Conduct (2017). Participants were informed about the purpose of the study in a cover letter and were assured that their data would remain confidential. Participants had to give explicit written consent to access the study.PB conceptualized this research project, contributed to the methodology development, data collection, data curation, statistical analysis, original draft, and did the project administration and supervision. KN contributed to the methodology, data curation, statistical analysis, and original draft. SD contributed to the methodology development, data collection, and original draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The heme protein myeloperoxidase (MPO) is a major constituent of neutrophils. As a key mediator of the innate immune system, neutrophils are rapidly recruited to inflammatory sites, where they recognize, phagocytose, and inactivate foreign microorganisms. In the newly formed phagosomes, MPO is involved in the creation and maintenance of an alkaline milieu, which is optimal in combatting microbes. Myeloperoxidase is also a key component in neutrophil extracellular traps. These helpful properties are contrasted by the release of MPO and other neutrophil constituents from necrotic cells or as a result of frustrated phagocytosis. Although MPO is inactivated by the plasma protein ceruloplasmin, it can interact with negatively charged components of serum and the extracellular matrix. In cardiovascular diseases and many other disease scenarios, active MPO and MPO-modified targets are present in atherosclerotic lesions and other disease-specific locations. This implies an involvement of neutrophils, MPO, and other neutrophil products in pathogenesis mechanisms. This review critically reflects on the beneficial and harmful functions of MPO against the background of immune response. In humans and higher animals, protection against different threats that affect the homeostasis of host’s tissues is ensured by a coordinated action of the immune system in close association with activation of components of the acute phase, complement, coagulation, and contact systems ,2. An imWith these activities, immune response is primarily directed to diminish the degree of tissue destruction and to restore homeostatic conditions. Otherwise, immune cells themselves release numerous hazardous substances during their activation. These agents are predominantly used to inactivate, kill, and destroy pathogenic microorganisms. However, they can also damage cell material, cause necrotic cell death, and induce the release of antigens in the host. Altogether, the immune system plays a dual role with respect to cell and tissue damage . On the This general dual activity of the immune system is also reflected in the functional responses of many cells activated after their recruitment to inflammatory loci. Polymorphonuclear leukocytes (PMNs), also known as neutrophils, are key mediator cells of innate immunity. These cells are rapidly recruited to inflammatory sites. The infiltration of PMNs into infected and injured tissue is mediated by adhesion molecules, cytokines, chemotactic agents, and components of the extracellular matrix ,4. In thThe heme protein myeloperoxidase (MPO) is a major active component in activated human neutrophils ,7. It isIn this review, the major focus is on the characteristics of neutrophil-derived MPO. The main properties of MPO and physiological consequences of MPO activation are summarized during immune response. This concerns the role of MPO in neutrophil functions during inactivation and killing of ingested microbes and fungi as well as the potential contribution of released MPO in the modification of proteins in disease progression. In sum, this heme protein plays also a dual role in immune response with both helpful and devastating properties. A hypothesis about the switch from the protecting to harmful functions of MPO, neutrophils, and general immune functions is presented too.To discuss the role of MPO in immune response, it is first necessary to consider major characteristics of this enzyme. As there are numerous comprehensive reviews about structural, biochemical, and redox properties of MPO ,7,16,17,Human myeloperoxidase is a cationic, dimeric protein with a ferric protoporphyrin IX (heme) in each subunit. In MPO, both heme groups are structurally and functionally identical . Each heIn each subunit of MPO, the heme is deeply buried into the protein bulk and connected with the surface by a narrow substrate channel. At the entrance to the distal cavity, there is a conserved hydrophobic region among mammalian heme peroxidases. Aromatic molecules and some other substrates can get close enough to the heme to allow an electron to transfer between the heme and substrates .3+). During activation, other heme states can result known as Compound I, Compound II, and Compound III (•+Por) [2•−) is attached to ferric heme iron. This compound can also be presented as a resonance structure where dioxygen is bound to ferrous heme iron [In resting MPO, the heme iron is in the ferric state (Feound III . Compoun (•+Por) . In Compeme iron .E’°) for the interconversion of MPO states halide ion concentrations, it has been calculated that Clof blood . In secrroxidase ,29,30, Mroxidase ,32.Hypochlorous acid as a myeloperoxidase product: In numerous reports, the formation of HOCl and chlorinated products by MPO was measured in the presence of taurine at pH 7.4 [t pH 7.4 ,35,36,37t pH 7.4 ,39,40. Mt pH 7.4 . Taurinet pH 7.4 ,43.In the cytoplasm of PMNs, the taurine concentration is 22–26 mM . In plas−/HOSCN is more restricted and mainly concerns the oxidation of sulfhydryls [2O2-halide system in the presence of physiological concentrations of chloride and bromide at pH values higher than 7 [Numerous substrates are targets for free HOCl ,49, and fhydryls . As a refhydryls ,53. Morer than 7 ,55.Peroxidase cycle: Compound I is also able to oxidize numerous small substrates in a one-electron reaction under the formation of a substrate radical and reduction to Compound II. Important substrates are urate, tyrosine, tryptophan, sulfhydryls, nitric oxide, polyphenols, nitrite, xenobiotics, hydrogen peroxide, xenobiotics, and others [d others ,58,59,60The reduction of Compound II to resting MPO is more restricted due to a lower standard reduction potential for the couple Compound II/resting MPO (0.97 V at pH 7) in contrast to the couple Compound I/Compound II (1.35 V at pH 7) ). In tisThe reaction sequence resting MPO→Compound I→Compound II→resting MPO comprises the peroxidase cycle see . In the Other reaction cycles: Compound III is formed in a reaction of ferric MPO with a superoxide anion radical with a rate constant of 2 × 106 M−1s−1 at pH 7.4 [2•− at this pH [2•−, which precedes with a rate of 1.3 × 105 M−1s−1 at pH 7.4, resting MPO is recovered under the formation of H2O2 and O2 [t pH 7.4 . This ra this pH . In a re2 and O2 . Compoun2•−-dependent reactions of MPO become more likely, as the rate constant of spontaneous dismutation of O2•− decreases steadily with increasing pH. These values are 6 × 104 M−1s−1 at pH 8, 6 × 103 M−1s−1 at pH 9, and 6 × 102 M−1s−1 at pH 10 [2•−-driven reaction sequence, only resting MPO and Compound III are involved  by the oxidation of cytoplasmic NADPH, transfer of electrons through the phagosomal membrane, and one-electron reduction of O2 in the phagosomal space. Dismutation of O2•− yields hydrogen peroxide and dioxygen. The enhanced formation of O2•−, opening of ion channels, and altered ion fluxes through the phagosomal membrane contribute to a rapid increase in pH in newly formed phagosomes [Components of fused granules participate in the creation of a special milieu in phagosomes, which facilitates the inactivation of microorganisms . At the agosomes ,93.Candida albicans [Originally, it was assumed that the phagosomal pH increases rapidly to about 7.8–8.0 (during the first three minutes), and decreases gradually to 7.0 (after 10–15 min) and later to 6.0 (after about 1 h) ,95. The albicans ,97. Concalbicans .The significance of NADPH oxidase in an increase in phagosomal pH is demonstrated by the use of the NADPH oxidase inhibitor diphenylene iodonium (DPI) or by the application of NADPH oxidase-deficient neutrophils. Under these conditions, the phagosomal pH drops down to about 6.3 and slightly lower ,97.In newly formed phagosomes, an alkaline milieu is important for the rapid and efficient inactivation of phagocytosed microorganisms. Numerous bactericidal constituents of azurophilic and specific granules have a pH optimum around 8–9, such as elastase, cathepsin G, proteinase 3, and lysozyme ,90. NeutIn azurophilic granules of resting neutrophils, MPO as well as other cationic protein components are probably inactivated by sequestration with negatively charged proteoglycans and the presence of low pH ,99. Thes+ ions in reactions of the formed O2•− is responsible for this pH increase. During the early phase of phagocytosis, discharged MPO can likely act as a weak superoxide dismutase, considerably enhancing the removal of O2•−. In one dismutase cycle, two protons are consumed.Immediately after phagosome formation, the pH rises to alkaline values as a result of NADPH oxidase activation and compensatory ion fluxes ,96. The 2•− promotes also its reaction with nitrogen monoxide (NO), an agent that is freely diffusible. The reaction product of O2•− and NO is peroxynitrite (ONOO−). The protonated form of ONOO−, peroxynitrous acid ONOOH, is known to convert resting MPO directly into Compound II [2•− efficiently recovers resting MPO from Compound II [Moreover, the high yield and stability of Opound II . As O2•−pound II , peroxynpound II .2+ [Superoxide anion radicals also interact with sulfur–iron-clusters under the release of Fe2+ ,102. Thi2+ .− under formation of −OSCN in the halogenation cycle or the oxidation of O2•− to O2 in the peroxidase cycle.At alkaline pH values, MPO is far from the optimum for efficient halogenating and peroxidase activities. Under these conditions, MPO is unable to oxidize chloride at sufficient rate. However, it cannot be excluded that hydrogen peroxide favors Compound I formation and the reaction of Compound I with traces of SCN2•− is diminished, and the halogenating and peroxidase activity of MPO activity rises slowly. It remains unsolved whether acidic pH values can be achieved in phagosomes of neutrophils.Later, the phagosomal pH decreases step by step, reaching neutral values. It remains unsolved whether some phagosomes become leaky during phagocytosis with changes of pH to cytoplasmic level. Under these conditions, the dominance of O2•−-dependent reactions of MPO. An enhanced catalase activity of MPO was also proposed in the pH region 9–10 [2O2 by the sequential conversion: Compound III→ferrous MPO→Compound II→Compound III [2O2-driven conversion of Compound II to Compound III is the rate-determining step, this cycle can explain the observed accumulation of Compound II at alkaline pH [We are far from a thorough understanding of the role of MPO during the early phase of phagocytosis in PMNs. The main reason for this is the lack of sufficient data about MPO reactions in the pH region 8–10, typical of early phagosomal pH values. It is quite evident that under these conditions, most reactions of halogenation and peroxidase cycles do not work or only work to a limited degree. Otherwise, superoxide anion radicals are stable enough due to the low rate of spontaneous dismutation and can drive now Oion 9–10 . Howeveround III . As the aline pH .Staphylococcus aureus by neutrophils [2•−-driven MPO reactions under alkaline conditions. Hydrocyanic acid (HCN) forms a complex with resting MPO, whereby HCN is deprotonated by the interaction with distal histidine [pKa value of 9.2 for HCN [2•−, which can compete with HCN for ferric MPO, it is questionable whether KCN is able to inhibit MPO at alkaline pH values in the phagosome. Azide, another MPO inhibitor, caused a significant decrease in phagosomal pH [The application of different inhibitors revealed cooperative effects of superoxide anion radicals and a myeloperoxidase-dependent pathway in the killing of trophils . Despitetrophils , the auttrophils , which iistidine . This bi for HCN . Considesomal pH  indicati2O2-halide system and inhibition of killing by MPO inhibitors or by the application of neutrophils from MPO-deficient individuals [In vitro experiments demonstrate the killing of microbes by the MPO-Hividuals ,110,111.ividuals ,113. UndOther data support an involvement of the halogenating activity of MPO in the termination of PMN serine proteases in phagosomes and protection of pericellular tissues from unwanted reactions. For example, the MPO product HOCl is known to inactivate elastase, cathepsin G, proteinase 3, and matrix metalloproteinase 9 .2•−. Later, MPO can probably contribute with its halogenation and peroxidase activity to the termination of phagocytotic activities and protection of surrounding tissues from uncontrolled proteolysis.In summary, in the early phase of phagocytosis in neutrophils, MPO apparently helps to intensify and prolong the duration of an alkaline milieu by activities mainly based on the consumption of OIn activated neutrophils, a broad range of active agents ensures protection against different types of microbes and fungi. There are multiple deactivation and killing pathways and a redundancy exists between these pathways.Candida albicans reported [For example, in humans, total and subtotal MPO-deficiency occurs with an abundance of 1 case on 2000–4000 individuals ,116. As reported ,119,120.Unlike humans, serious complications were reported in investigations of different disease models using MPO-knockout mice ,122,123.In MPO-knockout mice, no MPO protein is present. Human MPO deficiency is mainly assessed by diminished or missing peroxidase activity , a propeCandida albicans, the pH reaches with 8.5 a lower mean maximum value and decreases faster than in human cells [2•− and can utilize more H2O2.Moreover, human and murine neutrophils differ considerably in their properties . Mouse nan cells . It remaThese data support a higher degree of redundancy in deactivation and killing pathways in human neutrophils than in murine ones.Activated neutrophils are known to undergo apoptosis ,131. TypIn dying PMNs, so-called neutrophil extracellular traps (NETs) are formed and released. These traps represent a network of extracellular DNA-derived fibers, to which MPO, elastase, cathepsin G, gelatinase, lactoferrin, calprotectin and some others are attached ,136. ExtMyeloperoxidase is required for NETs formation, as PMNs from patients completely deficient in MPO cannot form traps . HydrogeBesides necrotic cell death, aggressive agents from activated PMNs can also be released into the surrounding medium as a result of an incomplete, the so-called frustrated, phagocytosis ,145. In At inflammatory sites, tissue-resident and monocyte-derived macrophages engulf and digest waste material and undergoing cells including apoptotic and necrotic neutrophils. It has been assumed that macrophages can modulate their activity depending on the dominance of apoptotic and necrotic cell material ,148,149.An important aspect of the interaction between apoptotic PMNs and macrophages is the rapid recognition of apoptotic cells by macrophages. Their delayed phagocytosis by macrophages can result in secondary necrotic processes ,154,155.2•− [2•− in these cells and limits, thus, the consumption of H+ by O2•−-driven reactions.Interestingly, in classically activated macrophages (type 1 macrophages) from human origin, the phagosomal pH rises to about 8.5 within five minutes, displays some oscillations of alkalinization, and remains nearly unchanged or returns to neutral values during the next 25 min ,156. Thi2•− ,72 diminThe presence of MPO on surface epitopes of non-vital neutrophils and other cells implies the question of whether attached MPO plays a role following phagocytosis of undergoing cells by macrophages. At present, a clear answer on this subject cannot be given. Probably, MPO can play a similar role in alkalinization of the phagosome in classically activated macrophages, as assumed in phagocytosing neutrophils. The strong pH decrease in phagosomes of alternatively activated macrophages would, however, favor better conditions for the chlorinating and peroxidase activity of MPO.Myeloperoxidase can be released from PMNs at inflammatory sites as a result of incomplete phagocytosis, cell necrosis, or as component of NETs. The cationic nature of MPO favors an interaction with numerous negatively charged serum proteins, endothelial cells, and components of the extracellular matrix. The activation of MPO by hydrogen peroxide causes halogenation and oxidation reactions in critical residues of the attached targets. On the basis of these interrelations, an involvement of MPO in several pathologies is discussed ,161,162.1-antiproteinase  [1 [Myeloperoxidase is known to interact with numerous plasma proteins. Critical residues in albumin ,164,165,trypsin) ,168,169,psin) [1 ,171, andpsin) [1 ,173 are Myeloperoxidase binds to heparin/heparan-containing epitopes of endothelial membranes . ComplexIn the extracellular matrix and coronary smooth muscle cells, MPO is known to bind to collagen IV and fibronectin ,178. OthHowever, the incubation of purified MPO with plasma revealed ceruloplasmin as the major binding target for MPO ,183. In 2+ and Cu+ [3+ and the transfer to the iron-binding protein transferrin [The copper-containing ceruloplasmin is a serum protein with multiple anti-inflammatory and antioxidant functions. It oxidizes Fe and Cu+ . Cerulop and Cu+ ,186. Itsnsferrin .The complex formation between MPO and ceruloplasmin strongly inhibits the halogenating and peroxidase activity of MPO ,189,190.Atherosclerotic plaques limit vessel lumen, disturb blood circulation, and cause serious health problems for the affected patients. In plaque formation, lipoproteins, endothelial cells, immune cells, smooth muscle cells, and others are involved ,197,198.In atherosclerotic lesions, active MPO was detected ,202. ChlIt remains unclear whether the interaction of MPO with lipoproteins and endothelium components is the initial reason for plaque formation, or whether the presence of MPO and MPO products in plaques results from subsequent processes like the infiltration of PMNs into inflamed areas.− or cyanide oxidation by MPO leads to the carbamylation of lysine residues in proteins, a process that has been associated with foam cell formation [In addition, several further MPO-related mechanisms are assumed to play a potential role in plaque development. Myeloperoxidase-derived HOCl is known to oxidize low-density lipoproteins and convert them into a pro-atherogenic species ,206. Theormation ,210. Myeormation ,212. Anoormation ,214. It There are several reports specifying a relationship between enhanced MPO plasma levels and the development of cardio-vascular problems ,216,217.Rupture of atherosclerotic plaques is a major reason for acute cardiovascular disease . Among cAntineutrophil cytoplasmic antibodies (ANCA) are involved in ANCA-associated vasculitis, which represents a small vessel vasculitis. There are mainly antibodies against myeloperoxidase and, to a lesser extent, against proteinase 3 . Both prThe development of MPO-ANCA and other ANCA is poorly understood . Among tOnce formed, ANCA can recognize over-activated neutrophils, which are attached to inflamed vessel wall areas. These attacks further promote the inflammatory process, leading to a necrotizing vasculitis. Glomerulonephritis, vasculitis of the upper and lower respiratory tract, and diabetic retinopathy are prominent forms of ANCA-associated vasculitis ,225,226.1-antiproteinase is discussed [1-antiproteinase is limited to inactivate elastase and proteinase 3 at inflammatory sites and a partial protease-mediated hydrolysis of ceruloplasmin may occur. Partially proteolyzed ceruloplasmin is unable to inhibit MPO [In the pathogenesis of different types of vasculitis, a relationship between MPO and αiscussed ,227. Myeiscussed ,167. As ibit MPO . CollectIn many diseases, the infiltration of neutrophils into inflamed tissue regions is closely associated with increased MPO levels. Active MPO was detected in tissue sections of patients with pancreatitis , in periMany disease scenarios are accompanied by chronic inflammatory processes, in which the inflammation is only insufficiently terminated. In inflamed tissues, there is a mixture of unperturbed tissue regions with necrotic areas, and regions where a de novo reconstitution of tissues occurs. Generally, it is impossible to give a clear description of chronically affected tissues as the appearance of these regions varies enormously. There are also flashes of the active inflammation with silent phases.In the termination of inflammation, a switch occurs from a pro-inflammatory phase to resolution of inflammation followed by restoration of tissue homeostasis ,246,247.In human blood and tissues, numerous mechanisms and strategies exist to protect biological material at inflammatory sites from damage caused by destroying agents released from activated neutrophils, other immune cells, and damaged tissue cells. Known examples for the latter agents are free hemoglobin, and free myoglobin, which are released from red blood cells during intravascular hemolysis ,250, andIntact cells and tissues are generally protected by the presence of molecules, which deactivate cytotoxic agents by binding, inactivation, or degradation. Examples of these protective interactions are given in 1-antiproteinase, α1-antichymotrypsin, and haptoglobin), which can be up-regulated to a certain degree during the course of inflammation [Some of the listed protecting species are acute phase proteins (such as ceruloplasmin, αammation ,271. OthReleased myeloperoxidase is inactivated by complex formation with ceruloplasmin ,189,190,As shown in 1-antiproteinase deserves special attention. This acute phase protein is produced in the liver and protects the lungs and other tissues from neutrophil elastase [1-antiproteinase deficiency may develop chronic obstructive pulmonary disease, chronic liver disease, skin inflammations, ANCA-related vasculitis, glomerulonephritis, and Bowel disease [Of the antagonizing agents against neutrophil products, αelastase ,274. Ind disease ,276,277. disease .During the resolution of inflammation, transient immunosuppression is important to allow final apoptosis of immune cells, to restore the normal tissue homeostasis, to terminate all inflammatory processes, and to induce, if necessary, de novo formation of tissues ,278. ManStrong deviations of normal tissue homeostasis can result in sepsis. According to the last consensus definition in 2016 , sepsis Immunosuppression impacts PMN functions in septic patients. These cells show a delayed apoptosis and diminished chemotactic mobility ,286. WitIn sepsis, tissue-damaging processes can vary widely and depend largely on the state of individual protective mechanisms ,290. BesIn septic shock patients, plasma levels of MPO–DNA complexes, which result from neutrophil extracellular traps, are significantly enhanced in contrast to healthy volunteers and closely related to the severity of organ dysfunction . PatientThe fate of myeloperoxidase reflects the general role of neutrophils in immune response. On the one hand, neutrophils and neutrophil constituents are highly essential to successfully combat foreign bacteria and other pathogens. In the phagosomes of PMNs, myeloperoxidase is involved in the creation and maintenance of an alkaline milieu, which is optimal for the activity of serine proteases and other granule components in the deactivation and killing of microbes. Halogenation and peroxidative activities of MPO are apparently involved in later phases of phagocytosis of pathogens and can play a role in the control and termination of phagocytic activities in neutrophils, and maybe also in macrophages, after the ingestion of undergoing PMNs. In addition, MPO is a mandatory element for the formation of neutrophil extracellular traps.On the other hand, the release of PMNs constituents during frustrated phagocytosis or from necrotic PMNs can damage intact host tissues. Myeloperoxidase as a major constituent of PMNs contributing to this damage by its close association with negatively charged components of plasma and extracellular matrix and by the chemical modification of these targets. The acute phase protein ceruloplasmin binds and inactivates MPO. The ability of MPO to damage molecules and tissues can be enhanced at inflammatory sites by the massive release of MPO from PMNs and the imbalance between proteases and anti-proteinases. In several disease scenarios and sepsis, the involvement of MPO in the pathological process is discussed. It remains unknown whether MPO contributes to basic mechanisms of disease induction or whether the increased MPO level in affected tissues results from downstream effects due to the recruitment of neutrophils to inflammatory sites.In sum, MPO exhibits, like neutrophils and many other components of the immune system, both a protective and harmful role in the maintenance and disturbance of tissue homeostasis.


The primary endpoint was the clinical response of the Disease Activity Score assessing 28 joints with C-reactive protein (DAS28-CRP) differences in the changes from baseline to 54 weeks between MTX+IGU and IGU groups. Secondary endpoints, such as the clinical response, retention rate, and safety, were evaluated. (3) Results: The DAS28-CRP difference in the changes between the two groups were −0.2. DAS28-CRP were significantly reduced from the baseline in the MTX+IGU and IGU groups . The retention rates were 71.4% in the MTX+IGU groups and 59.2% in the IGU groups (p = 0.16). Adverse events were observed in a total of 6 (17.1%) MTX+IGU patients and 20 (28.2%) IGU patients (p = 0.21). (4) Conclusions: IGU therapy may be a useful treatment option for patients who cannot be treated with MTX.(1) Background: We evaluated the clinical response of iguratimod (IGU) in patients with rheumatoid arthritis (RA) being treated with or without methotrexate (MTX) over 54 weeks. (2) Methods: 106 patients with RA undergoing IGU were retrospectively observed. RA patients were divided into those treated with MTX+IGU ( Rheumatoid arthritis (RA) is a well-known autoimmune disease which causes arthritis and is characterized by destruction of cartilage, bone, and tendon. Methotrexate (MTX) remains the anchor drug for the treatment of RA. MTX has contributed to improvement of various clinical symptoms, as well as disease control . In 2016Iguratimod (IGU) is a csDMARD that has been prescribed in daily medical practice for RA patients in Japan since 2012. The therapeutic effects of IGU are derived from its action of suppressing production of inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, IL-6, IL-8, and IL-17 ,5,6,7, sn = 35), and those treated with IGU without MTX , while patients were permitted to continue the other csDMARDs which had been already taken before this study. The observation period of this study was 54 weeks. The baseline data was obtained when the IGU treatment was initiated. Patients who had been treated with MTX before they started IGU therapy were assigned to the MTX+IGU group. To evaluate the appropriate therapeutic effect of MTX, in the MTX+IGU group, patients discontinued MTX within 24 weeks after addition of IGU were excluded. Patients who were not treated with MTX during the observation period were assigned to the IGU group. In the IGU group, patients who started MTX after initiating IGU therapy were excluded. Concomitant oral glucocorticoids (stable dose ≤7.5 mg of prednisolone (PSL)), SASP, bucillamine (BUC), or tacrolimus (TAC)) were permitted in both groups. The doses of PSL, SASP, BUC, and TAC could be adjusted at the discretion of the physician in charge.This study was conducted at a single center, Kindai University School of Medicine in Osaka, Japan. The study included 106 Japanese patients diagnosed with RA who were treated with IGU during the period from 2014 to 2017. RA was diagnosed according to the 2010 American College of Rheumatology (ACR) criteria and the EULAR classification criteria . PatientIGU was orally administered to the RA patients in both groups at the dose of 25 mg for the first 4 weeks and 50 mg thereafter. MTX was orally administered to the MTX+IGU group at a dose of 2–16 mg/week, and the dosages were determined also at the physician’s discretion.This study was conducted according to the principles expressed in the Helsinki Declaration of 1983 and was approved by the Research Ethics Committee of Kindai University School of Medicine. This study was registered at the Research Ethics Committee of Kindai University School of Medicine (registration no. 30-061) and University Hospital Medical Information Network . Written informed consent for their data to be used was obtained from all patients.The 106 patients’ characteristics were recorded at baseline including the age, sex, disease duration, current therapy, and the anti-cyclic citrullinated peptide antibody (ACPA) titer. At baseline, 24 weeks, and 54 weeks, the recorded clinical data included the 28 swollen and tender joint counts (SJC and TJC) and a patient visual analogue scale (Pt-VAS) in which 0  =  best and 100  =  worst. The following laboratory data were assessed at baseline, 24 weeks, and 54 weeks: C-reactive protein (CRP), the erythrocyte sedimentation rate (ESR), matrix metalloproteinase-3 (MMP-3), and rheumatoid factor (RF).The effectiveness parameters included the DAS28-CRP, TJC, SJC, and Pt-VAS, and the laboratory parameters of CRP (mg/dL), ESR (mm/h), MMP-3 (ng/mL), and RF (IU/mL). The differences in the changes from baseline in absolute value were measured and compared between the MTX+IGU and IGU groups.With respect to the DAS28-CRP disease activity categories , we diviWe analyzed the IGU retention of the MTX+IGU and IGU groups by using the Kaplan–Meier method for up to 48 months from 2014 to 2018. A log-rank test was performed for the comparison of the MTX+IGU and IGU groups. For RA treatment, the patient’s age and the presence of interstitial pneumonia are important factors in deciding whether to choose MTX; therefore, to reduce the bias associated with age and the presence of interstitial pneumonia, we adjusted the age of both groups and excluded patients with interstitial pneumonia.The treating physicians evaluated the physical examination findings, clinical symptoms, laboratory results, and chest radiographs. A physical examination and the laboratory tests were evaluated at each patient visit during the treatment period. Chest radiographs were taken when respiratory symptoms were present. If IGU was discontinued, the physician in charge recorded the reason for discontinuation and made any adjustments to the treatment based on his/her assessment.p-values < 0.05 were considered significant. The missing data were excluded from the analysis without supplementing the data with estimated or calculated values.Statistical analyses were performed using GraphPad prism software  and SAS 9.4 . The primary endpoint was the clinical response of the DAS28-CRP differences in the changes from baseline to 54 weeks between MTX+IGU and IGU groups. The secondary endpoints were effectiveness between the MTX+IGU and IGU groups that were made on clinical responses , retention rate, and safety. For the clinical responses, the differences of delta values between two treatment groups were derived, where the delta values were defined as the differences between measurements at 24 weeks and baseline and between 54 weeks and baseline. For the time-to-retention, Kaplan–Meier curves were estimated. In addition, as age was considered as a confounder in this retrospective study, for both clinical responses and time-to-retention, statistical analyses to adjust for age were performed using the inverse-probability-weighting (IPW) method , where tp = 0.02). There were no significant differences between the two groups in the disease duration of RA, clinical disease activity , or the positive percentage and titers of RF or ACPA. In the MTX+IGU group, the mean dose of MTX was 7.8 ± 0.5 mg/week. There were no significant differences between the patient groups in the percentage of users of PSL, SASP, BUC, or TAC.The baseline characteristics of the IGU group and MTX+IGU group are summarized in We analyzed the changes from the patients’ baseline values in the clinical assessment. p = 0.52) with no significant difference. For other clinical parameters, there were also no significant differences in the amount of change from baseline in the clinical data between the two groups at 24 weeks or at 54 weeks. Since there was a significant difference in age between the two groups at baseline, we performed a statistical adjustment for age, and the results of the amount of change from baseline in the clinical parameters between the two groups after age adjustment are shown in The differences in the absolute change from baseline in each clinical parameter at 24 and 54 weeks in both groups are listed in In the evaluation of remission based on the DAS28 criteria at months 0, 24, and 54, as shown in In the MTX+IGU group, all patients used MTX in combination until 24 weeks and 6 patients discontinued MTX between 24 and 54 weeks. At baseline, the MTX dosage was 7.8 ± 3.2 mg/week in the MTX+IGU group. At 24 and 54 weeks, the MTX dosages were 7.2 ± 3.0 and 6.1 ± 4.2 mg/week, respectively. The MTX dose did not differ significantly during the 54 weeks of the study period.p = 0.16, hazard ratio (HR) 0.62, p = 0.11, HR 0.58, p = 0.34, HR 0.72, p = 0.27, HR 0.68, p = 0.21). Liver dysfunction was the most common adverse event leading to IGU discontinuation in the IGU group and was noted in 12 patients (16.9%). In the MTX+IGU group, there were no patients whose IGU was discontinued due to liver dysfunction. There was a significant difference in the rate of liver dysfunction between the two groups (p = 0.01).The reasons for IGU discontinuation are shown in After the age adjustment, similar results were obtained considering the age differences between the groups . Among the 12 patients with liver dysfunction in the IGU group, six patients had either aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) values that were >100 IU/L. In addition, 16.9% of patients in the IGU group had liver dysfunction requiring IGU discontinuation. This hepatic dysfunction was temporary and was almost eliminated by discontinuing the IGU.There have been several reports on the therapeutic effects of the combination of IGU and MTX ,19. OkamThe results of our present retrospective analyses of 106 patients with RA revealed that both the IGU and MTX+IGU treatment groups showed a significant decrease in DAS28-CRP at 54 weeks compared to the baseline, with no significant difference between the two groups. Differences in baseline disease activity may be related to population selection bias, but there was no significant difference in disease activity between the two groups. Recent studies have reported that low baseline DAS28-CRP levels are predictors of remission and low disease activity achievement in IGU treatment ,22. ThesIn a post-marketing study, Mimori et al. reported that the retention rate of IGU-treated patients was 56.3% at 52 weeks . OkamuraIt has been reported that liver dysfunction was an adverse reaction characteristic of IGU therapy ,24,25. MHerein, the MTX+IGU group included patients with MTX-IR, for whom it is difficult to reduce PSL and csDMARDs doses or discontinue these agents, and/or difficult to increase the MTX dose sufficiently. The present IGU group included patients who could not be treated with MTX, such as those with interstitial pneumonia (IP) or renal dysfunction.Our results demonstrated a good therapeutic effect and a good retention rate of IGU with or without MTX treatment. However, there was a significant difference in baseline age between the IGU and MTX+IGU groups. In general, MTX may not be used to treat elderly patients due to their reduced renal function, which may explain why the present IGU patients were older. Similar results were obtained in our IGU and MTX+IGU groups after the adjustment for age.n = 106), which may lead to low statistical power and accuracy of the analysis. In addition, there was a difference in the number of patients in the MTX+IGU and IGU groups. Third, there was a lot of missing data in our study. An increase in missing data leads to a decrease in sample size and analysis accuracy, resulting in bias. Missing data arose due to discontinuation of therapeutic drugs, and the decision was left to each physician. Since this study is a retrospective study, there were no preset treatment discontinuation criteria. Discontinuation of treatment at the discretion of each physician could also lead to bias. Thus, a randomized control trial is indispensable to establish the effects of IGU treatment with and without MTX.Our study has some limitations. First, the lack of randomization and blinding may have resulted in bias due to indication and selection of the patients. In this study, the adjustment of medications, including csDMARDs and PSL, was determined by each physician, and there was no uniformed protocol. However, gender, disease duration, seropositivity, and disease activity may also be variables that affect the efficacy and retention of RA drugs, and our adjustment for confounding factors may be inadequate. Second, the study population was rather small (IGU therapy was not inferior to the clinical efficacy and retention rate of MTX+IGU therapy. IGU therapy may be a useful treatment option for patients who cannot be treated with MTX.


Coronavirus disease 2019 (COVID-19), the global pandemic that has spread throughout the world, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given the limited scientific evidence on the manifestations and potential impact of this virus on pregnancy, we decided to report this case.The patient was a 38 year-old Iranian woman with a triplet pregnancy and a history of primary infertility, as well as hypothyroidism and gestational diabetes. She was hospitalized at 29 weeks and 2 days gestational age due to elevated liver enzymes, and finally, based on a probable diagnosis of gestational cholestasis, she was treated with ursodeoxycholic acid. On the first day of hospitalization, sonography was performed, which showed that biophysical scores and amniotic fluid were normal in all three fetuses, with normal Doppler findings in two fetuses and increased umbilical artery resistance (pulsatility index [PI] > 95%) in one fetus. On day 4 of hospitalization, she developed fever, cough and myalgia, and her COVID-19 test was positive. Despite mild maternal symptoms, exacerbated placental insufficiency occurred in two of the fetuses leading to the rapid development of absent umbilical artery end-diastolic flow. Finally, 6 days later, the patient underwent cesarean section due to rapid exacerbation of placental insufficiency and declining biophysical score in two of the fetuses. Nasopharyngeal swab COVID-19 tests were negative for the first and third babies and positive for the second baby. The first and third babies died 3 and 13 days after birth, respectively, due to collapsed white lung and sepsis. The second baby was discharged in good general condition. The mother was discharged 3 days after cesarean section. She had no fever at the time of discharge and was also in good general condition.This was a complicated triplet pregnancy, in which, after maternal infection with COVID-19, despite mild maternal symptoms, exacerbated placental insufficiency occurred in two of the fetuses, and the third fetus had a positive COVID-19 test after birth. Therefore, in cases of pregnancy with COVID-19 infection, in addition to managing the mother, it seems that physicians would be wise to also give special attention to the possibility of acute placental insufficiency and subsequent fetal hypoxia, and also the probability of vertical transmission. Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that has spread throughout the world. Unfortunately, there is still limited scientific evidence on the manifestations and potential impact of this virus on pregnancy. In this article, we aim to report the maternal and fetal effects of the virus on a complicated triplet pregnancy.A 38 year-old Iranian woman with a triplet (three chorionic and three amniotic) pregnancy was hospitalized at 29 weeks and 2 days gestational age due to one-time high blood pressure at 140/90mmHg and elevated liver enzymes. A week before admission, she had been hospitalized in another hospital for about 5 days to evaluate the cause of increased liver enzymes. She had a 2-year history of primary infertility and had become pregnant by ovulation induction, and also had a 5-year history of hypothyroidism, which was euthyroid during pregnancy. She had also been diagnosed with gestational diabetes a month before admission, which was being treated with 16 units of insulin daily (6 units Levemir and 10 units NovoRapid), so her blood glucose levels were maintained in a normal range, and glycated hemoglobin concentration was 5.6 %. She was at risk for preeclampsia due to her advanced age and triplet pregnancy, so aspirin was administered. On the other hand, due to hospitalization for more than 3 days, she was at increased risk of deep vein thrombosis and was treated with enoxaparin. Additionally, given the possibility of iatrogenic preterm delivery, she had received a course of betamethasone for fetal lung maturation.Two weeks before hospitalization, liver enzymes increased to four times the normal levels: alanine amino transferase (ALT) 218 units per liter (U/L) and aspartate amino transferase (AST) 283 U/L. After a thorough evaluation and ruling out preeclampsia, and due to a probable diagnosis of gestational cholestasis, she was treated with ursodeoxycholic acid (300 mg twice a day) from 1 week before hospitalization. Lab tests at admission included the following: ALT = 94 U/L, AST = 57 U/L, total bilirubin = 0.7, direct bilirubin = 0.1, and lactate dehydrogenase (LDH) = 276 U/L. Other tests including white blood cell count, hemoglobin, platelet, serum creatinine, and urinalysis were in the normal range. She underwent 24-hour Holter blood pressure monitoring, and among all measurements, only 18.8% of systolic and 15.6% of diastolic blood pressure exceeded the set limit of 140 and 90 mmHg, respectively. Echocardiographic findings were quite normal, and 24-hour urine protein was also reported in the normal range. On the second day of hospitalization, an ultrasound exam was performed, which showed that biophysical scores and amniotic fluid were normal in all three fetuses. One of the fetuses had increased umbilical artery resistance (pulsatility index [PI] > 95%) and estimated weight below 5%, but umbilical cord and middle cerebral artery findings were normal in the other two fetuses. On the fourth day of hospitalization, the patient developed fever and cough. The following day, due to the persistent fever and cough and also an onset of myalgia, real-time reverse transcriptase polymerase chain reaction (RT-PCR) was conducted on nasopharyngeal swabs for SARS-CoV-2 nucleic acid. All steps including sample collection, processing and laboratory testing were based on World Health Organization (WHO) guidelines. Chest X-ray and computed tomography scan were not performed due to the patient’s lack of consent.The result of RT-PCR was positive for the SARS-CoV-2 virus. However, the patient’s clinical symptoms were mild. She had a mild fever, with maximum temperature of 38.3 °C. She had no complaint of shortness of breath, diarrhea, nausea or vomiting, her respiratory rate was 18–20 per minute, and oxygen saturation was above 95% at all times. On the same day that she developed clinical symptoms, an ultrasound exam was performed: the fetus who already had increased umbilical artery resistance showed an exacerbated condition involving absent umbilical artery end-diastolic flow, and another fetus showed umbilical artery resistance (PI > 95%), but umbilical cord and middle cerebral artery findings were normal in the third fetus, and biophysical scores and amniotic fluid were normal in all three fetuses. Based on these findings, the patient underwent serial ultrasound exams, and unfortunately, exacerbated umbilical flow resistance in the second fetus also progressed to absent umbilical artery end-diastolic flow (Fig. Two of the babies, weighing 1250 and 1320 g, each received three doses and the other baby received two doses of surfactant. All three newborns developed clinical symptoms of sepsis and pulmonary hemorrhage. The primary results of the COVID-19 test were negative for all three newborns. Because of the poor general conditions of the newborns, and considering the false-negative probability of the initial test, the COVID-19 test was repeated immediately after receiving the first test results, and the result was positive for the baby who weighed 1600 g and also had better umbilical cord and placental circulation before birth. Unfortunately, we did not examine the umbilical cord blood and amniotic fluid samples for the virus, so we cannot conclusively link the COVID-19 RT-PCR positive test in this fetus to vertical transmission, but because the babies were completely isolated and had no suspected exposure during the period between the two tests, the possibility of vertical transmission cannot be ignored.The baby who weighed 1320 g died 3 days after birth with collapsed white lung and sepsis. The baby who weighed 1250 g also had symptoms of sepsis and died 13 days after birth. The baby who weighed 1600 g and had a positive COVID-19 test eventually recovered and was discharged in good general condition 3 weeks after birth.We report the case of a woman with a triplet pregnancy who was infected with COVID-19. Despite the presence of some comorbidity, she had only mild symptoms of COVID-19 infection, but rapid and progressive placental insufficiency occurred simultaneously with the peak maternal infectious disease symptoms. A COVID-19 test was positive in one baby, who was discharged in good condition 3 weeks after birth, and negative in two other babies, who died 3 and 13 days after birth.A recently published study identified maternal age and underlying diseases as risk factors for severity of COVID-19 symptoms , 3. In oAvailable data about COVID-19 vertical transmission is still contradictory. At the beginning of the pandemic, no vertical transmission was reported ; howeverDelayed positive tests in newborns have also been reported in other studies , 6–8. InOur case was a triplet pregnancy. Most previous studies are in singleton pregnancies, although there are also some reports of twin pregnancies –12. Some have hypothesized that maternal respiratory failure and hypoxia may transiently reduce uterine placental blood flow , 14, butThere are still insufficient data about COVID-19 in pregnancy. Our case was a complicated triplet pregnancy; however, COVID-19 symptoms in the mother were mild. Given the rapid and progressive placental insufficiency after COVID-19 infection, it seems prudent that in cases of pregnancy with COVID-19 infection, in addition to assessing and managing the mother, special attention should be given to the possibility of acute placental insufficiency and subsequent fetal hypoxia. The possibility of vertical transmission should also be considered.