The current interest in recombinant factor VIII (rFVIII) products stems from the fact that they offer a technological solution to prolonging the half-life of and reducing the risk of formation of alloantibodies (inhibitors) against FVIII in treated patients with hemophilia A (HA). The Italian health care system has authorized the use of a wide range of rFVIII concentrates of the first, second, and third generation, as well as new innovative rFVIII preparates with an extended half-life (EHL) . Some of these products have unique pharmacodynamic and pharmacokinetic (PK) profiles, including an EHL. The first-generation full-length rFVIII (FL-rFVIII), octocog alfa (Recombinate® Baxter/BIOVIIIx), although the oldest rFVIII product, has several desirable features. Third-generation products include two modified octocog alfa molecules  as well as the B domain-deleted rFVIII (BDD-rFVIII) moroctocog alfa (ReFacto®-Pfizer). The B domain-truncated (BDT-rFVIII) turoctocog alfa , the BDD-rFVIII simoctocog alfa , the single-chain BDT-rVIII lonoctocog alfa , and the BDD-rFVIIIFc efmoroctocog alfa  are new, innovative products. Simoctocog alfa, because its peculiarities, is considered a fourth-generation rFVIII concentrate. Turoctocog alfa, simoctocog alfa, and lonoctocog alfa have a high affinity for von Willebrand factor (vWF) that reduces renal clearance and prolongs the half-life of rFVIII. Efmoroctocog alfa, a first-in-class rFVIII-Fc fusion protein (rFVIIIFc), has a half-life 1.5–1.8 times longer than that of conventional plasma-derived FVIII (pd-rFVIII) and other rFVIII products. Clinical studies have evaluated the efficacy, safety, and inhibitor development of all these innovative concentrates in both previously treated (PTPs) and untreated patients (PUPs). This review considers the rFVIII products that are indicated for the treatment of patients with severe HA, focusing on those that are commercially available in Italy. Their PK characteristics, immunogenicity, and clinical benefits are discussed and compared. The treatment of HA has dramatically improved since the 1990s, when the outbreak of blood-borne infections, mainly due to the human immunodeficiency and hepatitis viruses, caused high rates of death and co-morbidities in many patients (www.EUHASS.org) has carried out safety monitoring of substitution therapies for patients with hemophilia  step and nanofiltration were employed in the second and third generations, and chromatographic purification techniques were also performed) and furthermore the progressive removal of human and animal proteins from the final formulation. The different generations of rFVIII products reflect the evolution of the methods used in their manufacture. First-generation rFVIII production methods include the use of animal proteins in the cell culture medium and the addition of human serum albumin to stabilize the recombinant product; the culture medium of second-generation rFVIII contains human- or animal-derived proteins but not human albumin; finally, the most recent, third-generation rFVIII products have no added human- or animal-derived proteins at all.For patients with severe HA, prophylaxis with FVIII concentrates is the main treatment regimen to reduce joint bleeding and other types of hemorrhage, thereby improving the health-related quality of life (HRQoL) of both patients and their caregivers , 4. MoreHA prophylaxis usually consists of intravenous FVIII injections every other day or three times per week due to the short half-life of FVIII of approximately 12 h . HoweverThis review summarizes the characteristics of the rFVIII products currently available in Italy for the treatment of severe HA . The potRecombinate®  is the only one of two first-generation rFVIII concentrates that is still commercially available in Italy. It is derived from a conditioned medium of chinese hamster ovary (CHO) cell cultures transfected with cDNAs for FVIII –10. HumaBeginning in 1990, a multicenter, multinational, prospective clinical trial of Recombinate® was conducted on PUPs with severe/moderate HA (baseline FVIII ≤2%) in order to evaluate the safety and efficacy of the product, including the development of inhibitors . Of the Of the 72 patients, 22 (30.5%) developed rFVIII inhibitors: a low titer ( ≤5 BU) was measured in 13 (59%) patients and a high titer (>5 BU) in 9 (40.9%) patients. In 12 of the 22 patients (54.5%), the inhibitors became undetectable, including in 11 patients in the low-titer group. At the end of the study, nine of the 72 patients (12.5%) still had a high titer of inhibitors.Ewenstein et al., in a prospective pharmacovigilance study that considered the incidence of inhibitor development worldwide in patients treated with Recombinate®, demonstrated that the overall percentage was 11.9%  for PUPs and 0.123% (CI: 0.030–0.512%) for PTPs. The incidence of high-titer inhibitors (>5 BU) was 5.96% (CI: 3.00–11.8%) for PUPs and 0.0554% (CI: 0.0113–0.271%) for PTPs  of a longer mean half-life, as determined at months 18 and 24 , who participated for a median of 3.7 (1.0–5.7) years with the aim of comparing the PK of rFVIII with that of pd-FVIII. The safety and long-term home-treatment efficacy of rFVIII was also assessed . At stud8 and 24 .P < 0.05). Of the five patients who developed inhibitors (12.5%), three were in the prophylaxis group and developed inhibitors after 24–48 exposure days (EDs). The remaining two patients, who were in the on-demand group, developed inhibitors at 20 and 2 EDs. No patient suffered from life- or limb-threatening bleeding or from hemorrhage that required hospitalization.The Evaluation Study on Prophylaxis: a Randomized Italian Trial (ESPRIT) compared prophylaxis with on-demand treatment with the aim of preventing joint bleeding and joint damage in children with severe HA . Forty oIn 2003, a plasma/albumin-free formulation of octocog alfa containing sucrose was introduced as the first third-generation rFVIII originating from its precursor Recombinate®. The adapted recombinant system used in the production of the rFVIII molecule includes culture medium free of human- or animal-derived additives. The CHO cell line co-expressing FL-FVIII and vWF has not undergone any further genetic manipulations, and the cDNA sequences encoding the recombinant proteins are identical to those used to obtain Recombinate®. Processing includes an S/D step, a viral inactivation that preserves the structural and functional integrity of the rFVIII molecule but inactivates lipid-enveloped infectious agents . FollowiThe PK of Advate® has been evaluated in adults and children based on FVIII activity measurements in blood samples obtained up to 48 h following each infusion. The mean half-life values in adults and children differ as a function of patient age , 4.Advate® has been evaluated in 11 clinical trials in PTPs and in one trial in PUPs with severe to moderate HA . The five completed clinical trials have demonstrated the safety and efficacy of Advate® in adult and pediatric PTPs, for both prophylactic and on-demand treatment regimens and for perioperative hemostatic coverage  17)..17).A three-part study investigated the use of Advate® in 10- to 65-year-old PTPs without previous or detectable inhibitors. Fifty-six patients were randomized to Part 1 of the study, comparing the PK of Advate® with that of Recombinate®. Patients with severe or moderate HA (baseline FVIII ≤2%) received an initial infusion (50 ± 5 IU/kg). Bioequivalence between the older and newer products in terms of PK was demonstrated. Part 2 of the study evaluated the efficacy, safety, and immunogenicity of Advate® in 108 patients (baseline FVIII ≤2%) who followed a standard fixed prophylactic regimen over a period of at least 75 EDs. Of the 510 bleeding episodes that occurred during the study, 93% were resolved with 1–2 infusions of Advate®, with an excellent/good hemostatic response rate of 86%. Part 3 was a double-blind, randomized, cross-over comparison of the PK of pilot-scale-prepared Advate® with commercial-scale-prepared Advate®. The bioequivalence of the products was demonstrated . The lonIn a clinical trial that enrolled 55 PUPs (defined as having had up to three exposures to an FVIII product at the time of enrollment), 16 (29.1%) patients who received Advate® developed inhibitors: seven (12.7%) had high titers (>5 BU) and nine (16.3%) had low titers. Inhibitors were detected at a median of 13 EDs (min-max: 6–26 EDs)  is a bioequivalent albumin-free rFVIII produced from the second-generation product ReFacto®. It is manufactured using CHO cells grown in a chemically defined serum- and albumin-free cell culture medium that, per the definition of a third-generation product, contains no materials derived from human or animal sources. ReFacto AF differs from ReFacto® in that a synthetic peptide affinity ligand (TN8.2) replaces the murine monoclonal antibody used in affinity chromatography and a 35-nm viral filtration step is included in the purification process . SucroseTwo studies evaluated the properties of Refacto AF® in 204 PTPs with severe/moderate HA . The objP(t≤50) = 0.33, [95% CI: 0.25–0.43]), including 15 (14.5%) with a high titer  = 0.16, [95% CI: 0.10–0.25]). The incidence of inhibitors was comparable to that in previously published studies of PUPs  National Haemophilia Database identified a consecutive cohort of 103 PUPs with severe HA treated with ReFacto AF® . The stu of PUPs , 25. AntKovaltry®  is an unmodified Fl-rFVIII with an amino acid sequence identical to that of sucrose-formulated rFVIII  but developed using innovative techniques . KovaltrThe PK of Kovaltry® was investigated in PTPs (0–61 years of age) with severe HA who were administered a dose of 50 IU/kg . KovaltrLEOPOLD KIDS (part A) was a multi-center, open-label, uncontrolled study of Kovaltry® in pediatric PTPs . The PK, efficacy, and safety of routine prophylaxis and the perioperative management of bleeding were investigated. The primary efficacy variable was the ABR that occurred during routine prophylaxis within 48 h of the previous infusion. ABR during prophylaxis, independent of the time of infusion, was also analyzed. The prophylactic regimen was 25–50 IU/kg at frequencies of either two times per week, three times per week or every other day, adapted to the patient's need by the investigator. In LEOPOLD KIDS part A, Kovaltry® values for the maximum concentration and AUC tended to be lower in children than in adults. LEOPOLD-KIDS part B, a study in PUPs, is ongoing .In all of the completed LEOPOLD studies, Kovaltry® was shown to be efficacious, whether for prophylaxis, on-demand treatment, or perioperative hemostasis. The effective prophylactic dose, defined as achieving a low ABR, was 20–40 IU/kg, given two or three times per week. The incidence of treatment-related adverse events was <7% across all LEOPOLD studies, and no PTPs developed inhibitors during the completed primary studies .NovoEight®  is produced from CHO cell lines without the use of human or animal plasma. Turoctocog alfa includes two amino acid chains, a heavy chain and a light chain, joined by non-covalent bonds. This activated glycoprotein has the same molecular structure as human FVIII and is subjected to post-translational changes that are similar to those of the plasma-derived molecule. The Tyr1680  sulfation site, important for vWF binding, is fully sulfated in the turoctocog alfa molecule. The high affinity with vWF helps to reduce the renal clearance of the product and prolong its half-life. Sucrose, sodium chloride, L-histidine, polysorbate 80, L-methionine, calcium chloride dihydrate, sodium hydroxide, and hydrochloric acid are the excipients. Viral inactivation is ensured by detergent inactivation and a 20-nm nano-filtration step.Three multicenter, open-label, uncontrolled studies were conducted to evaluate the safety and efficacy of turoctocog alfa in the prevention and treatment of bleeding in PTPs with severe HA (FVIII ≤1%). The studies included 213 patients: 150 adolescent and adult patients >12 years of age without inhibitors and with ≥150 EDs, and 63 pediatric patients <12 years of age without inhibitors and with ≥50 EDs. Of these 213 patients, 187 participated in the extension safety study. Treatment with turoctocog alfa was shown to be safe and effective for bleeding treatment and for prophylaxis in 14 patients undergoing a total of 14 surgical interventions, including 13 major and 1 minor operation; hemostasis was achieved in all surgical interventions, and no failure was reported.The efficacy and safety of turoctocog alfa were evaluated in the guardian 1 trial . Its priA multicenter, multinational, open-label, sequential dosing PK study comparing turoctocog alfa with Advate® was conducted in 23 patients with severe HA . The resGuardian 3 evaluated the safety and efficacy of turoctocog alfa in pediatric patients 0–11 years of age with severe HA (FVIII <1%). Patients with >50 EDs to any FVIII product were included in the study . The priA secondary endpoint in the guardian 1 and 3 studies was HRQoL related to the use of turoctocog alfa. Those results showed a fairly stable HRQoL for patients already treated with prophylaxis . The greAll patients in the guardian 1 and 3 studies or in phase 1 PK trials were eligible for participation in the extension study, guardian 2, which assessed the efficacy and safety of turoctocog alfa in prophylaxis or on-demand treatment in PTPs of all ages. The primary safety endpoint was the frequency of FVIII inhibitor development. Efficacy endpoints included ABR during prophylaxis, hemostatic response in the treatment of bleeds, and the number of injections required to treat bleeds. The results showed that the extended use of turoctocog alfa was safe and effective in the prevention and treatment of bleeding episodes in patients of all ages .TM 4 will assess the efficacy and safety of turoctocog alfa in the prevention and treatment of bleeds in treatment-naïve HA patients  is a BDD-rFVIII produced in genetically modified human embryonic kidney cells, rhFVIII, and so is defined as a fourth-generation rFVIII product , 38. It Three studies  were carried out in 135 PTPs ≥12 years (74 adult and 61 pediatric patients) with severe HA treated with simoctocog alfa for bleeding episodes or the prevention of bleeding during surgery , 40. In GENA 08 evaluated 32 patients age ≥18 years who were placed on standard prophylaxis with simoctocog alfa for the prevention or treatment of hemorrhagic events. Hemostatic prophylaxis during surgery was also determined . In patiGENA-03 evaluated the efficacy of simoctocog alfa in the treatment of bleeding episodes in 59 children 2–12 years of age. Resolution in 81% of the bleeding episodes was achieved with one or two injections . The effProspective non-registrative trials, such as the extension studies GENA-11 (extension of GENA-01) and GENA-13 (extension of GENA-03) and the new study GENA-09 and its extension GENA-04, have also been carried out . The PK GENA-21 (NuPreviq), a prospective, multicenter, open-label, phase III study evaluating PK-guided prophylaxis vs on-demand treatment with simoctocog alfa in 66 adult PTPs with severe HA, was recently completed. Personalized prophylaxis with simoctocog alfa was shown to be a more convenient treatment in the 58% of patients who were able to extend the injection intervals from three times weekly to twice weekly or less. In addition to less factor consumption, a lower ABR was recorded in this group .To assess the efficacy of simoctocog alfa with respect to surgical prophylaxis, an integrated analysis of five studies  was carried out due to the limited number of PTPs enrolled in each study. Overall, 19 patients underwent 34 surgical procedures (20 minor and 14 major). Drug efficacy was rated as excellent or good in 100% of the minor and 92% of the major operations. None of the patients developed inhibitors.Finally, the GENA-05 (NuProtect) trial evaluated the immunogenicity, safety, and efficacy of simoctocog alfa in PUPs with severe HA . Data frLonoctocog Alfa  is a unique, single-chain rVIII molecule. It has a truncated B-domain and heavy and light chains covalently linked to form a stable and homogeneous compound. The molecule binds with high affinity to vWF, which results in its increased stability and protection from degradation. Lonoctocog alfa is produced by recombinant DNA technology in CHO cells. L-histidine, calcium chloride, sodium chloride, sucrose, and polysorbate 80 are the excipients. Safety from infective agents is ensured by chromatography, S/D treatment, and serial nanofiltration.Lonoctocog alfa was shown to be effective for prophylaxis, treatment, and the surgical management of bleeding episodes in PTPs with severe HA. Two prospective, non-comparative, multicenter trials (AFFINITY program) were carried out, one in children and the other in adolescents/adults. Eligible patients had been treated with an FVIII product for 50–150 EDs. The co-primary efficacy endpoints were the treatment success rate (excellent or good) and ABR in routine prophylaxis, which were compared with on-demand treatment . The thrpost-hoc sub-analysis, the vWF antigen level for both products was shown to have a positive effect on half-life and a negative effect on clearance. This finding suggests that stronger binding to vWF improves the PK profile of FVIII concentrates. In pediatric PTPs, the half-life was shorter and the clearance longer than in adolescents and adults  than that of Recombinate®, and clearance was 28–31% lower. In a d adults   , 49. In Efmoroctocog alfa  is a first-in-class fusion protein consisting of a single molecule of human BDD-rFVIII covalently linked to the dimeric Fc domain of human immunoglobulin G1 (rFVIII-Fc). The excipients used are sucrose, sodium chloride, L-histidine, calcium chloride dihydrate, and polysorbate 20. The half-life of efmoroctocog alfa is about 1.5 times longer than that of conventional pd-FVIII and of rFVIII products.The efficacy of efmoroctocog alfa in prophylaxis, treatment of acute hemorrhage, and perioperative management has been investigated in two open-label, non-comparative, multinational phase III trials enrolling PTPs ≥12 (A-LONG) or <12 (Kids A-LONG) years of age who had severe HA. Adults and adolescents were eligible for A-LONG if they had been treated with any FVIII product for ≥150 EDs and had a history of ≥12 bleeding events in the 12 months prior to the study. Children were eligible to enter Kids A-LONG if they had been treated with any FVIII product for ≥50 EDs . These pivotal phase III trials also showed that the routine prophylactic administration of efmoroctocog alfa in PTPs  was well tolerated, with no evidence of increased immunogenicity. In addition, efmoroctocog alfa was effective in preventing bleeding episodes, including in target joints. Prophylaxis with efmoroctocog alfa was also associated with maintained or increased physical activity in the majority of patients, including those with target joints at baseline. Recommended long-term prophylaxis consists of intravenous injections of efmoroctocog alfa every 3–5 days .n = 21); the rate of high-titer inhibitors was 14.7% (n = 10). High-titer inhibitors developed after up to 11 EDs   , 55. EfmData on inhibitor frequency in PTPs and PUPs using all of the rFVIII products are reported in Following the outbreak in the 1980s of blood-borne infectious diseases (particularly HIV and HBV/HCV infections) in hemophilia patients who received pd-FVIII products that had not been subjected to virus inactivation or removal treatments, the manufacturers of those products introduced virus sterilization steps into their production methods and increased the purity of the preparations via DNA genetic engineering, thus greatly improving safety. All of the rFVIII products currently available in Italy have been tested in well-conducted clinical trials in which they were shown to be effective and safe. However, the ever-increasing purity of the molecule has made these preparations more susceptible to inhibitor development than was the case with pd-FVIII concentrates. Consequently, there is currently an urgent need to minimize the risk of inhibitor development linked to replacement treatment/prophylaxis with rFVIII products. This is especially the case for high-titer inhibitors, which, according to clinical studies carried out worldwide, mainly develop in PUPs after ≥ 25/50 EDs.The first-generation rFVIII molecules Recombinate® and Kogenate® were introduced in Italy in the 1990s. Recombinate® is currently distributed in Italy by BIOVIIIx. The characteristic of the product that accounts for its continued use is its stabilization by human serum albumin and polyethylene glycol (PEG 3350). The covalent linkage of polyethylene glycol (PEGylation) to FVIII increases the molecular weight and size of the protein by surrounding it with a hydrophilic cloud. This molecular change is thought to have a protective effect, reducing the product's susceptibility to proteolytic activity and degradation. PEGylation also changes the surface charge of the protein, which may interfere with its clearance, as the half-life of PEGylated rFVIII is longer than that of non-PEGylated FL-rFVIII . In addiAdvate® , a third-generation, human-protein-free FL-rFVIII, is derived from Recombinate® and has similar efficacy, safety, and immunogenicity in PTPs and PUPs with severe HA. However, Advate® and other rFVIII products differ from Recombinate® in that their excipients include polysorbate 80.The BDD-rFVIII ReFacto AF® is an innovative structural modification of FVIII derived from ReFacto®. Its efficacy is equivalent to that of FL-rFVIII products. The immunogenicity of ReFacto AF® in PUPs was evaluated in a study conducted by UKHCDO. The results showed a slightly high incidence of high-titer inhibitors (14.5%) in PUPs.Kovaltry®, a derivative of Kogenate FS®, is an unmodified FL-rFVIII produced using a genetically engineered BHK cell line. Its post-translational modifications are similar to those of endogenous FVIII, including glycosylation and tyrosine sulfation, which account for the high affinity of Kogenate FS® for vWF, in turn slightly prolonging its half-life. The results from the Leopold study confirmed the efficacy and safety of Kovaltry® for twice/thrice weekly prophylaxis, on-demand treatment, and the control of perioperative bleeding in patients of all ages. Moreover, at least within the completed primary studies, no FVIII inhibitors developed in PTPs. Anti-HSP70 antibodies were detected in some patients but were without any clinical effect.Turoctocog alfa-rFVIII (Novoeight®) was the first BDT-rFVIII. Its high affinity for vWF accounts for its slightly longer half-life. The guardian study of a large population of PTPs with severe HA demonstrated that turoctocog alfa is effective and safe in preventing and treating bleeding, is well tolerated, and is not associated with unexpected safety issues or FVIII inhibitor development. A PK study comparing turoctocog alfa with Advate® showed that the primary PK parameters of the two products were comparable.Of particular interest is the BDD-rFVIII simoctocog alfa (Nuwiq®), the first product obtained from a human embryonic kidney cell line. The GENA study and its extensions demonstrated that simoctocog alfa was a good/excellent rFVIII in adult and pediatric PTPs, whether for prophylaxis, on-demand treatment, or intraoperative bleeding prevention. Definitive data on inhibitor development in a large population of PTPs enrolled in various prospective trials, as well as preliminary data from the NuProtect study in PUPs, suggest a low inhibitor rate in patients treated with simoctocog alfa.Lonoctocog alfa (Afstyla®) is a unique single-chain rFVIII. Its safety, efficacy, and PK profile were evaluated in two open-label studies, one in children and the other in adolescents/adult populations. The results of those studies showed good/excellent hemostatic efficacy both in the control of episodic hemorrhagic events and in bleeding prophylaxis. In addition, the adolescent/adults study demonstrated hemostatic efficacy during the perioperative management of bleeding in patients undergoing surgical procedures. The high affinity of lonoctocog alfa for vWF results in its stabilization and reduced clearance of rFVIII molecules. Data on inhibitor incidence in PUPs from the ongoing CSL627 UNDERSCORE 3001 extension study are not yet available.Efmoroctocog alfa (Elocta®), a BDD-human r-FVIII covalently linked to the Fc domain of human IgG1, is the only EHL rFVIII commercially available in Italy. The half-life of efmoroctocog alfa is 1.5–1.8 times longer than that of other, conventional rFVIII products . Data from pivotal phase III studies  and an extension study (ASPIRE) demonstrated the long-term effectiveness of efmoroctocog alfa for the treatment of acute bleeding episodes, perioperative management and routine prophylaxis in PTPs with severe HA.Routine prophylaxis consisting of intravenous injections every 3–5 days is recommended, although once weekly may be suitable for some patients. Neither inhibitor development nor severe adverse events have been reported. Preliminary data from an interim analysis of the PUPs A-LONG ongoing study now available would confirm the hemostatic efficacy of efmoroctocog alfa. The cumulative incidence of inhibitor development is 30.9% while the rate of high-titer inhibitor development is 14.7%. Efmoroctocog alfa used prophylactically is well-tolerated and efficacious, with a very low median ABR overall and no spontaneous joint bleeding.Currently, the National Health Care System in Italy guarantees the availability of rFVIII products for the prophylaxis and episodic treatment of patients with severe HA. Third-generation rFVIII concentrates represent a wide range of high-quality products that offer efficacy and safety in all therapeutic regimens. Today, with the availability of EHL-rFVIII, the quality of life of both patients and caregivers is improving. Nonetheless, the first-generation rFVIII Recombinate®, far from being obsolete, continues to be effective and safe. The development of inhibitors, the most serious complication of replacement therapy, seems to be of very limited importance in PTPs who have switched to third-generation rFVIII products. In clinical studies, all of these newly developed concentrates have demonstrated efficacy and safety; nevertheless, they do not seem to be less immunogenic in PUPs than other rFVIII products. Despite the structural and functional improvements of new rFVIII concentrates, the incidence of high-titer inhibitors in PUPs remains a very significant and still unsolved problem.All authors wrote and revised the final version of the manuscript.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.


Recently, an alcohol predictor was developed using DNA methylation at 144 CpG sites (DNAm-Alc) as a biomarker for improved clinical or epidemiologic assessment of alcohol-related ill health. We validate the performance and characterise the drivers of this DNAm-Alc for the first time in independent populations.N = 1049 parents from the Avon Longitudinal Study of Parents and Children (ALSPAC) Accessible Resource for Integrated Epigenomic Studies (ARIES) at midlife, we found DNAm-Alc explained 7.6% of the variation in alcohol intake, roughly half of what had been reported previously, and interestingly explained a larger 9.8% of Alcohol Use Disorders Identification Test (AUDIT) score, a scale of alcohol use disorder. Explanatory capacity in participants from the offspring generation of ARIES measured during adolescence was much lower. However, DNAm-Alc explained 14.3% of the variation in replication using the Head and Neck 5000 (HN5000) clinical cohort that had higher average alcohol consumption. To investigate whether this relationship was being driven by genetic and/or earlier environment confounding, we examined how earlier versus concurrent DNAm-Alc measures predicted AUDIT scores. In both ARIES parental and offspring generations, we observed associations between AUDIT and concurrent, but not earlier DNAm-Alc, suggesting independence from genetic and stable environmental contributions.In The stronger relationship between DNAm-Alcs and AUDIT in parents at midlife compared to adolescents despite similar levels of consumption suggests that DNAm-Alc likely reflects long-term patterns of alcohol abuse. Such biomarkers may have potential applications for biomonitoring and risk prediction, especially in cases where reporting bias is a concern. Alcohol use and misuse are responsible for a large proportion of the global burden of disease ; howeverObjective biomarkers of alcohol intake are useful tools for epidemiology and public health because they have the potential to improve exposure assessment, especially when reporting bias may be a major issue, and inform screening and risk prediction strategies. Currently, alcohol biomarkers exist for exposure assessment over short-term windows  or at high doses  . What isDNA methylation, currently the best characterised epigenetic modification, which is involved in regulating gene expression, has been found to be altered in relation to several environmental and biological factors –9. PatteLiu et al. developed four new blood DNA methylation-based alcohol models (DNAm-Alcs) as biomarkers of alcohol intake, using a large population sample (the CHARGE consortium ). Of theHere and for the first time, we aim to assess the out-of-sample performance of the DNAm-Alcs by using data at multiple time points from an independent population, the subset of participants from the Avon Longitudinal Study of Parents and Children (ALSPAC) included in the Accessible Resource for Integrated Epigenomic Studies (ARIES), and further replication using the Head and Neck 5000 (HN5000) clinical cohort. Repeated measures of alcohol use and blood DNA methylation in two generations of the ARIES study and at different points in the life course, including DNA methylation profiles collected prior to own alcohol exposure, were used to address our aims. Our first aim was to test the performance of the DNAm-Alcs in ARIES and HN5000. Second, we aimed to further investigate the performance of DNAm-Alcs in ARIES at predicting measures of alcohol use disorders (AUDIT scores) in different groups of individuals , which is designed to estimate the extent to which an individual engages in harmful patterns of alcohol consumption. Lastly, http://www.bristol.ac.uk/alspac/researchers/our-data/).The Avon Longitudinal Study of Parents and Children (ALSPAC) is a population-based birth cohort drawn from the South West of England. Pregnant women resident in Avon, UK, with expected dates of delivery from 1 April 1991 to 31 December 1992 were invited to take part in the study. The initial number of pregnancies enrolled is 14,541 . Of these initial pregnancies, there was a total of 14,676 foetuses, resulting in 14,062 live births and 13,988 children who were alive at 1 year of age. Detailed information on the mothers and their offspring has since been regularly collected , 21. Thehttp://www.ariesepigenomics.org.uk/) [N = 1049, mean age = 50.2 ± 5.4 SD) and the offspring generation at adolescence  with DNA methylation, alcohol and covariate information available  and in cord blood at birth (N = 438) and childhood  in the offspring, were included in some analyses.Blood from 1018 mother-child pairs (children at three time points and their mothers at two time points) were selected for analysis as part of the Accessible Resource for Integrative Epigenomic Studies  . ARIES cThe Head and Neck 5000 (HN5000) clinical cohort study, a clinical cohort study of individuals with head and neck cancer, was used as an additional independent replication population. Full details of the study methods and overall population are described in detail elsewhere , 24. BriN = 281, mean age = 58.4 ± 9.9 SD)At baseline, participants were asked to complete a series of questionnaires and information on diagnosis, treatment and co-morbidity was recorded on a short data capture form. Diagnoses were coded using the International Classification of Diseases (ICD) version 10  and clinAlcohol intake and AUDIT score  were avaParticipants were also asked to report the quantity of their typical alcohol consumption where ‘one drink referred to ½pint of beer/cider, a small (125 ml) glass of wine or a single (25 ml) measure of spirit’, each of which is roughly equivalent to one UK alcohol unit (8 g of ethanol). Alcohol intake was then calculated by multiplying participant drinking frequency by the typical quantity consumed and converting from UK units/week to grammes/day. In all statistical models, alcohol intake was log-transformed (log (grammes/day +1)) to coerce its distribution to approximate normality. For categorical analyses, drinkers were grouped according to the following thresholds of alcohol intake: ‘non-drinkers’ reported no alcohol consumption (intake grammes/day = 0), ‘light drinkers’ consumed < 0 g/day ⩽ 14 for women and < 0 g/day ⩽ 28 in men, ‘at risk drinkers’ consumed < 14 g/day < 28 in women and < 28 g/day < 42 in men, ‘heavy drinkers’ ⩾ 28 g/day in women and ⩾ 42 g/day in men. These categorical thresholds, which are the same as those used by Liu et al., were selected in order to maximise comparability between our results.Alcohol use disorder identification test (AUDIT) scores were taken from the same time points as the measures of alcohol intake, cross-sectionally to DNAm at midlife in the ARIES parental generation and in adolescence in the offspring. AUDIT includes responses to 10 questions that evaluate participant alcohol misuse in the past year, producing scores that range from 0 to 40. Self-reported non-drinkers are included in this scale, receiving a score of 0.In HN5000, detailed information on alcohol history was obtained at baseline via the self-reported questionnaire. Participants were asked about their current drinking status and their use of alcohol prior to receiving their head and neck cancer diagnosis. Respondents were asked to report their average weekly alcohol consumption of a range of beverage types  before they were diagnosed with cancer. From these measures, average intake of alcohol consumption in units per week was derived, which we subsequently converted to log (grammes/day +1).meffil package [p value ≥ 0.01, those with sex or genotype mismatches) were excluded from further analysis, and probes containing < 95% of signals detectable above background signal  were also removed. Functional normalisation [In ARIES, cord blood and peripheral blood samples  were collected according to standard procedures. DNA extraction, wet laboratory preparation and DNA methylation measurement by Illumina Infinium HumanMethylation450K (HM450) BeadChip were performed as part of the ARIES project, as previously described , 28. Bri package . Sampleslisation  was perfmeffil package [In HN5000, blood samples were processed and frozen upon receipt and stored at − 80 °C. Genome-wide DNA methylation data was generated in participants using Infinium MethylationEPIC BeadChip (EPIC array)  at the Bristol Bioresource Laboratories. Following extraction, DNA was bisulphite-converted using the Zymo EZ DNA MethylationTM kit  and genome-wide methylation status of over 850,000 CpG sites was measured using the EPIC array according to the manufacturer’s protocol using an Illumina iScan. All data pre-processing and normalisation was again performed using the  package , which eFor the ARIES parental generation at midlife, offspring generation in both childhood and adolescence, and HN5000 participants, DNA methylation levels were adjusted for age, sex, body mass index (BMI) and Houseman estimate white blood cell (WBC) counts by residualizing on these factors by ordinary least squares (OLS) regression prior to calculation of DNAm-Alcs , 32. LevWe computed DNA methylation-derived alcohol scores (DNAm-Alcs) using coefficients made available from the lasso models estimated by Liu and colleagues , predictdnamalci R package .DNA methylation levels were residualised from the linear effects of the covariates described above prior to computing DNAm-Alc. We provide software for producing all four DNAm-Alc estimates using our freely available R2 or the percent of the variance in the outcome variable explained. For binary outcome measures, receiver operating characteristic (ROC) curves were generated and area under the curve (AUC) was calculated using the pROC package [R2 in order to account for the discrepancy in model degrees of freedom.Overall performance of DNAm-Alc scores in predicting continuous measures, including alcohol intake and AUDIT score, was assessed by  package  to evaluWe further performed sensitivity analyses to check the robustness of our results to the impact of smoking and the inclusion of non-drinkers. To this end, we ran cross-sectional models using DNAm values that had been residualised using concurrent smoking as an additional covariate, and excluding participants identified as self-reported non-drinkers.R2 from the OLS model was again used to compare models with different numbers of covariates.As an additional comparison, we re-fit linear models with the same sets of CpGs used in each DNAm-Alc, as well as age, sex and BMI as explanatory variables. This provided an inflated assessment of prediction due to over-fitting, as has been described in detail by Hattab et al. , but allAll statistical analysis was performed in R version 3.3.1 .N = 1049 ARIES participants at midlife, using DNA methylation levels independent of age, sex and BMI  versus low AUDIT score categories in ROC analysis produced AUCs that ranged from 0.67 with the 5 CpG DNAm-Alc to 0.80 for the 78 CpG score in ARIES participants at midlife Fig. . These ATo determine whether the relationship observed between DNAm-Alc and AUDIT using cross-sectional data could be explained by genetic confounding, we compared the predictive performance of DNAm-Alc based on methylation of blood drawn earlier in life, at a period of expected low or no alcohol use, to the predictive performance of DNAm-Alc based on methylation of blood drawn later in the same participants, at the same time as completion of the AUDIT questionnaire. If DNAm-Alc from blood drawn earlier in life is shown not to be associated with later AUDIT, this would then rule out both genetic and earlier environment confounding of the cross-sectional DNAm-Alc–AUDIT relationship. A schematic flow-chart describing this analysis is provided in Fig. N = 518 participants with DNAm-Alc at available at both time points and AUDIT at midlife (Table p = 0.33), albeit with a slightly smaller maximum (21 versus 26). We found no evidence for a relationship between DNAm-Alc during pregnancy and AUDIT at midlife, both when considered as a single independent variable  and when adjusting for DNAm-Alc at midlife . Conversely, we observed a strong relationship between DNAm-Alc at midlife and AUDIT  even when accounting for pregnancy DNAm-Alc .In the ARIES mothers, we compared the effects of the 144 CpG DNAm-Alc measured during pregnancy to those of DNAm-Alc measured cross-sectionally to AUDIT, 21.1 years later on average during midlife in n = 601) and at birth (n = 438). Again, mean AUDIT in each of these subsamples did not differ markedly from the full distribution . AUDIT in adolescence appeared to be independent of DNAm-Alc in childhood (p = 0.88), including after adjusting for adolescent DNAm-Alc (p = 0.44). However, the cross-sectional relationship between DNAm-Alc and AUDIT in adolescence, while small in total magnitude (adjusted R2 = 1.2), persisted with similar effect size when adjusting for childhood DNAm-Alc (β = 0.94 compared to β = 0.99). While DNAm-Alc at birth showed some suggestive evidence of a relationship with AUDIT in adolescence when considered alone , this effect attenuated when adjusted for cross-sectional DNAm-Alc . We considered whether the suggestive relationship between DNAm-Alc levels at birth and AUDIT in adolescence could have been driven by maternal drinking during in utero development, but this did not appear to be the case in follow-up analysis that added examination of maternal DNAm-Alc during pregnancy and which failed to show effects independent of DNAm-Alc at birth . Further, when we reproduced the validation approach of Liu et al. in the same ARIES sample, we again observed performance metrics similar in magnitude to their initial findings , suggesting that the performance decrease we observed when applying the independently estimated coefficients to ARIES was due to the elimination of effects due to over-fitting. Importantly, we have facilitated future application of the correct, independently estimated Liu et al. DNAm-Alc coefficients through our freely distributed R software package, dnamalci .Compared to the variance of alcohol intake explained by the DNAm-Alcs in Liu et al.’s initial report , we obseWhile the DNAm-Alc showed limited explanatory capacity in ARIES, its performance was markedly improved in our HN5000 replication population, achieving 14% of variation explained  versus low AUDIT individuals, at midlife where we had also seen the strongest relationship in continuous analysis. The two best-performing DNAm-Alcs in this regard, the 78 and 144 CpG biomarkers, had AUCs of 0.80 and 0.79 which is in a range comparable to that reported by Liu et al. for intake categories. The ability to discriminate categories of alcohol was again improved when replicating in HN5000 compared to ARIES, reaching a maximum AUC = 0.77 for the 144 CpG score at identifying heavy from non-drinkers. As mentioned previously, this population differed from AREIS in several ways, including having a greater number of participants in the higher intake categories (e.g. N = 153 in the heavy intake group) which may have contributed to this improved performance.We also observed reduced capacity of the DNAm-Alcs to predict categories of drinkers by alcohol intake in ARIES compared to the estimates by Liu et al. Fig. . In factOur assessment of the timing of the relationship between alcohol use and the DNAm-Alcs, the third aim of our study, observed that these scores captured an epigenetic response largely independent of confounding from genetic or environmental predisposition to alcohol dependency. This was most clearly observed in the ARIES mothers, who had a strong relationship between DNAm-Alc and AUDIT when both were measured in participants at the same time but for whom almost no relationship was observed for DNAm-Alcs calculated from DNAm measured roughly 20 years previously, at a time when alcohol consumption was much lower during the women’s index pregnancy , helped identify the duration of exposure potentially reflected by these biomarkers. The DNAm-Alcs appeared to reflect more than just concurrent intake over a short-term window, like that captured by a breathalyser. The correlation observed between DNAm-Alcs and intake/AUDIT in ARIES adolescents with comparable levels of alcohol use to adults over a shorter time frame was much smaller than that of ARIES parents at midlife with longer histories of alcohol use Table . This suThe results of our current study should be interpreted in the context of the following limitations: (1) Tissue specificity—DNA methylation measured in peripheral blood WBCs was used both for the original development of the DNAm-Alcs and our current validation. Blood may not be the tissue most directly relevant for assessing the impacts of alcohol abuse . However, alcohol is absorbed by peripheral-blood and is relevant to the direct assessment of alcohol exposure. Further, blood-based biomarkers may be more clinically useful, less invasive and more readily accessible than liver or brain tissue. (2) 5-Hydroxymethylcytosine (5hmC)—Our study was limited to DNA methylation measured as 5-methylcytosine (5mc) at the 144 CpGs in the DNAm-Alcs developed by Liu and colleagues. As such, we were not able to determine whether 5hmC may have been acting as an intermediate or additional regulator of any of the biological processes described. However, a recent study suggests that the amount in blood samples will be quite small: 5hmC was detected at only < 8% CpG sites whereas 5-mc was detected at > 80% . (3) MeaOur study also had notable strengths, including access to repeated DNAm measurements in participants drawn from a prospective, population-based, longitudinal birth cohort. These repeated observations, some of which were collected at time points prior to or during limited alcohol use, were unique in structure and allowed assessment of the role of genetic confounding that would not have been possible with many other study designs.While we find that the DNAm-Alcs do not always predict alcohol intake as well as initially hoped, our revised evaluation in a large population-based sample demonstrates that they do still capture non-trivial amounts of information about the biological impacts related to alcohol use. Importantly, our results using AUDIT score rather than intake alone suggest that these biomarkers may explain additional variation in alcohol use disorder beyond alcohol consumption. Although DNAm signals often correlate strongly with local polymorphisms, our analysis in repeated measures from two separate generations was able to find strong evidence that DNAm-Alcs are largely independent of genetic confounding. The stronger relationship we observed between DNAm-Alcs and AUDIT in parents at midlife compared to offspring in adolescence despite similar levels of cross-sectional consumption suggests that DNAm-Alc may reflect the biological impact of alcohol use over time. This was echoed in our findings from HN5000 which showed the strongest DNAm-Alc—intake relationship and was the population with the greatest amount of consumption. Overall, we confirm Liu et al.’s initial conclusion that DNAm-Alcs may have clinical utility as a test for detecting heavy alcohol consumption, with some evidence that such markers track long-term patterns of alcohol use rather than recent intake.Additional file 1. R2 between DNAm-Alcs and measures of alcohol intake (log(g/day +1)) and AUDIT score, in ARIES parents at midlife and offspring at adolescence with adjustment of DNAm-Alcs for concurrent smoking.Additional file 2. R2 between DNAm-Alcs and alcohol intake (log(g/day +1)) in ARIES parents at midlife and offspring at adolescence, excluding self-reported non-drinkers.Additional file 3. R2 between DNAm-Alcs and alcohol intake (log(g/day +1)) and AUDIT score in HN5000.Additional file 4. Adjusted R2 from models regressing measures of alcohol use, alcohol intake (g/d), in ARIES parents at midlife and offspring at adolescence on the methylation β-values of CpGs used in constructing DNAm-Alcs, as was the modelling approach presented in Liu et al. 2016.Additional file 5. Area under the curve for prediction of ‘at risk’ category of drinkers by DNAm-Alcs.Additional file 6. Area under the curve for prediction of binary alcohol categories by DNAm-Alcs in HN5000.Additional file 7. Estimates of the effects of DNAm-Alc in ARIES offspring at birth and in mothers during pregnancy on offspring AUDIT at adolescence considered both in separate single-predictor models and simultaneously.


Rituximab is a promising steroid sparing agent used in the treatment of moderate to severe pemphigus vulgaris. Its exact place in the algorithm of pemphigus treatment, vis-à-vis other, conventional adjuvant therapy (CAT) is not known.To describe and compare disease course outcomes and morbidity among patients with moderate to severe pemphigus who received rituximab therapy (RT) in addition to prednisone and CAT, versus those who were treated with prednisone and CAT alone.A 16-year retrospective case control study was designed with adult patients who were seen at the Duke University Dermatology Immunodermatology clinic from 1999–2015, who had a diagnosis of pemphigus vulgaris, and required prednisone and at least 1 systemic CAT. All patients had at least 6 months follow up from the initial visit. Interventions included RT, systemic CAT, and prednisone. The main outcome measured was prednisone intake. Secondary outcomes were complete remission (CR) and partial remission (PR).40 patients were included in the study. All initially received prednisone and at least 1 systemic CAT. 13/40 eventually went on to receive RT, while 27/40 remained on CAT (CAT-only). Patients in the RT group, pre-RT, had a median prednisone intake of 658.57 mg/month. Rituximab treatment significantly reduced this to 177.22 mg/month (p = 0.002). Median prednisone intake of the CAT-only group was 141.33 mg/month. This was significantly less than Pre-RT (p = 0.01) and on par with Post-RT intake (p = 0.58). 54% of patients in the RT group and 64% of those in the CAT-only group achieved CR. All patients in the RT group and 96% of those in the CAT-only group achieved at least PR.32.5% of our patients with moderate to severe pemphigus vulgaris failed prednisone and traditional CAT treatment and required rituximab therapy. Rituximab reduced the monthly prednisone intake in these patients by 73%. This suggests that a subset of patients with moderate to severe pemphigus may benefit from early institution of rituximab therapy. Rituximab significantly reduces the monthly prednisone requirement among CAT-resistant pemphigus vulgaris patients to levels on par with CAT-responsive patients. It has , .Treatment of moderate to severe forms of pemphigus vulgaris has relied on the use of steroid sparing agents, but theRituximab, a humanized anti-CD20 antibody, has shown great promise in small, non-comparative retrospective studies, non-comparative clinical trials –11, and Herein, we retrospectively compare outcomes among patients with moderate to severe pemphigus vulgaris who received rituximab versus other, more traditional, conventional adjuvant therapy (CAT) at a single institution. In addition, because all patients who received rituximab also received these other adjuvant therapies prior to rituximab therapy (RT), we compare outcomes in these patients before and after their first rituximab dose.We conducted a single-centre retrospective, case-control study of adult (> = 18 years of age at baseline) patients diagnosed with moderate to severe pemphigus vulgaris, who required systemic corticosteroid therapy plus systemic adjuvant therapy, and who were seen at Duke University from May 1999 through February 2015. This study was approved by the Duke University Institutional Review Board, with waiver of consent.Patients were identified using the Duke Enterprise Data Unified Content Explorer (DEDUCE). Patients were included if they had a clinical diagnosis of pemphigus vulgaris, a direct immunofluorescence (DIF) from perilesional skin that demonstrated keratinocyte cell surface IgG, and at least 6 months of documented clinic follow-up. In addition, patients who received rituximab had to have at least 6 months follow up from their first rituximab infusion.A total of 68 adult patients with pemphigus vulgaris were identified on initial screening. Among these, 46 patients met the inclusion criteria for age and follow up duration. However, two patients received topical treatment only, and 4 patients received prednisone but did not require systemic adjuvant therapy and were therefore excluded. A total of 40 patients were included in the study. All received oral prednisone and conventional adjuvant therapy (CAT). Thirteen (13) or 32% of these patients went on to receive rituximab (RT group). Twenty-seven (27) or 67.5% of these patients remained on CAT (CAT-only group).Patient demographics, disease duration, and treatment data were collected. All patients received oral prednisone and CAT consisting of mycophenolate mofetil, azathioprine, cyclophosphamide, adalimumab and infliximab. Patients were divided into two groups: (1) those who went on to receive rituximab at any point in the treatment (RT group) and (2) those who did not receive rituximab (CAT-only).Pemphigus activity was calculated retrospectively using the Pemphigus Disease Activity Index (PDAI) Disease activity score–16. BecaBaseline was defined as the first patient presentation to the clinic, regardless of whether they had previously been diagnosed and treated for pemphigus vulgaris. Initial rituximab (IR) treatment was defined as the time at first infusion of rituximab. Among patients who received RT, monthly prednisone intake and serum marker titres were calculated separately based on phase of RT: (1) from baseline to IR (pre-RT), and (2) from IR onwards (post-RT).Pre-RT and post-RT monthly prednisone intake were compared using Wilcoxon signed-rank test. Monthly prednisone intake of CAT-only patients were compared separately to pre-RT and post-RT prednisone intake of patients in the RT group using crude and adjusted linear regression, controlling for age and gender. Due to non-normal distribution, monthly prednisone intake was log-transformed prior to inclusion in regression models. To describe trends in cumulative prednisone intake, cumulative prednisone intake at each documented follow-up was plotted and the linear slope calculated. T-test was used to compare cumulative prednisone dose at different time points after baseline.Time to PR/CR, CR or discontinuation of prednisone were compared between the two groups using unadjusted Cox proportional hazards regression. For time dependent outcomes, IR was considered time zero for the RT group. For the CAT-only group, time zero was either the initiation of CAT, or baseline, if the patients were already on CAT during their initial presentation to clinic.Thirteen out of the 40 patients, or 32% of the patients included in this study required RT in spite of initial treatment with prednisone and CAT. Twelve of the 13 patients were already on oral prednisone and eight were already on CAT prior to baseline. The median time from baseline to rituximab therapy was 19.6 months . Median All 13 patients who received rituximab also received at least one other adjuvant therapy prior to rituximab treatment: mycophenolate mofetil, azathioprine, dapsone or infliximab. The median time from baseline to IR was 19.6 months (range 0.7 to 89.3). Each cycle of rituximab treatment was 1 gm/infusion x 2 cycles, 14 days apart. Sixty-nine percent (9/13) of patients who received rituximab received more than one treatment cycle. Five (5) patients received 2 cycles, 2 patients received 3 cycles, 1 received 4, and another 6 cycles. The gap between rituximab cycles was at least 12 months, although one patient had a six-month gap between his first and second cycle.Eleven patients in the RT group had at least six months follow up between their baseline or initial clinic visit and IR. Among these patients, the median PDAI score at IR was half of the median PDAI at baseline , but only 177.2 mg (IQR 97.3–309.5) post-RT  visits per patient year was higher in the pre-RT phase. Among RT patients in their pre-RT phase, there were 51 episodes of infectious events requiring either oral or IV antibiotic therapy, 16 hospital admissions and 9 ER visits over 332 patient months . These patients had a drop in their PDAI score from their initial visit to the time they received rituximab, likely due to prednisone. Their anti-Dsg3 titres, on the other hand, were increasing, although we did not have enough patients in our study to achieve statistical significance. Those in the RT group also had a median disease duration 1.8 times longer before their initial visit to our specialty dermatology clinic. Since retrospective data suggests that those who receive rituximab earlier in the course of their disease may respond more quickly , the impMonthly prednisone requirement distinguishes this CAT-resistant, RT group from those who stayed on CAT alone (CAT-only). The monthly prednisone requirement in the pre-RT group was almost four times that of the CAT-only group in both crude (p = 0.02) and adjusted (p = 0.01) regression models. Based on our estimates at the 3-month time point, our RT patients were accumulating around 911 mg of prednisone per month (around 30 mg per day) in their pre-RT phase vs. 445 mg per month (around 14.8 mg per day) in the CAT-only group. Rituximab significantly decreased the monthly prednisone intake to match levels in CAT-sensitive patients (p = 0.58), even after adjusting for age and gender (p = 0.61). Although on the surface, the likelihood of remission was not significantly different between the two groups, RT patients had more severe disease, as reflected by their increasing requirement for prednisone and anti-Dsg3 titers in spite of prednisone and CAT therapy. This suggests that a subset of patients with moderate to severe pemphigus would benefit maximally from early institution of rituximab, and this subset can be identified by requirement for significant prednisone (30 mg per day) and increasing anti-Dsg titers.The pre-RT incidence of infections, hospitalizations and ED visits was also markedly higher in the RT group compared to CAT-only patients, and improved after institution of RT. This suggests that a subset of patients with moderate to severe pemphigus would benefit maximally from early institution of rituximab. Our data suggests that this subset can be identified after 3 months or less of CAT therapy by the requirement for 30 mg of prednisone or more and increasing anti DSG titers.In summary, rituximab may facilitate remission by a different, more efficient mechanism compared to CAT as it decreased prednisone intake dramatically and significantly in the RT group despite their more severe disease. These patients, who comprised 33% of our pemphigus cohort, may present to clinic with similar demographics and PDAI scores as those who respond to conventional adjuvant therapy. This study proposes the use of monthly prednisone intake and anti-Dsg3 titre trends as quantitative metrics that can be measured within the first three months of therapy to identify the patient subset that may benefit most from early rituximab therapy. For these CAT non-responders, early identification and intervention can decrease the duration of active disease, total prednisone intake and associated comorbidities, and adverse events including hospitalizations and systemic infections. The retrospective nature of this study and small sample size given the rarity of this disease warrant prospective validation with larger comparative prospective studies comparing CAT-only therapy with rituximab therapy. In addition, the use of 10 mg/day of prednisone or lower to define minimal therapy may limit our estimation of prednisone-associated adverse effects, as even low dose corticosteroid therapy can be associated with increased blood pressure, Cushingoid changes, and weight gain, among othersIt is possible that even CAT responders may also benefit from earlier rituximab intervention in terms of fewer infections, hospitalizations, lab draws, and clinic visits. Further, other outcome measures such as quality of life and cost need to be further studied in prospective studies and may suggest additional benefit of rituximab therapy in both CAT-responsive and CAT-resistant patients. It is important to recognize these two subsets of CAT-responsive and CAT-resistant patients in designing these clinical trials to elucidate rituximab-specific benefits.


Background: Increased death due to the opioid epidemic in the United States has necessitated the development of new strategies to treat addiction. Monoclonal antibodies and antidrug vaccines provide a tool that both aids addiction management and reduces the potential for overdose. Dual drug vaccines formulated by successive conjugation or by mixture have certain drawbacks. The current study examines an approach for combatting the dangers of fentanyl-laced heroin, by using a hapten with one epitope that has domains for both fentanyl and heroin.Results: We evaluated a series of nine vaccines developed from chemically contiguous haptens composed of both heroin- and fentanyl-like domains. Analysis of the results obtained by SPR and ELISA revealed trends in antibody affinity and titers for heroin and fentanyl based on epitope size and linker location. In antinociception studies, the best performing vaccines offered comparable protection against heroin as our benchmark heroin vaccine, but exhibited attenuated protection against fentanyl compared to our fentanyl vaccine.Conclusion: After thorough investigation of this strategy, we have identified key considerations for the development of a chemically contiguous heroin–fentanyl vaccine. Importantly, this is the first report of such a strategy in the opioid–drug–vaccine field. In 2016, the Centers for Disease Control (CDC) estimated that approximately 20.4% of the U.S. population  was suffering from chronic pain [®), a competitive antagonist of µ-opioid receptors. Other treatments for opioid substance abuse focus on mitigating cravings and managing long-term addiction. However, these treatments have an inherent risk for continued abuse. Combatting the complex nature of addiction requires a multifaceted strategy – several elements of the opioid crisis that need to be addressed include, but are not limited to, new non-addictive opioid drugs, innovative treatments to prevent relapse, and preventing and/or reversing overdose.Respiratory depression is a common side effect of opioid overdose and can lead to serious complications, including death. The current treatment for overdose is naloxone  mixing two individual vaccines together (admixture vaccine) –10; or 2As a starting point for our exploration of chemically contiguous drug–hapten conjugates, we envisioned generating a series of structurally diverse heroin–fentanyl haptens with a focus on linker placement and bond distance between each drug’s chemical connections in order to probe the effect of the drug-epitope structure on eliciting an immune response. The second consideration in our chemically contiguous hapten design was the location of the linker to the carrier protein. We chose three arrangements: 1) extending the linker off the phenylamide ring of fentanyl; 2) extending the linker from the phenethyl ring of fentanyl; or 3) anchoring the linker in between the two drug-like portions of our singular epitope. Again, HF-3, HF-5, and HF-8 could share an intermediate, while the fentanyl-like domains of HF-1, HF-2, HF-3, and HF-7  was synthesized according to literature procedure and purified by recrystallization [1 with tert-butyl bromoacetate and subsequent deprotection with trifluoroacetic acid yielded the N-glycine derivative of norheroin, 16. This critical intermediate was used to construct HF-3, HF-5, and HF-8 butanoic acid was accessed via protection and deprotection of the amine with a phthaloyl group . Reductive amination of 4 with commercially available phenethylpiperidin-4-one furnished intermediate 5 in good yield  hydrate followed by sodium periodate to obtain aldehyde 7 butanoic acid, an alkene was installed with a Suzuki reaction, which in turn underwent ozonolysis to give 21, shown in 21 to give 24, completing the fentanyl-like domain . However, increasing the conjugation to a 1 mg scale caused the protein to precipitate immediately upon addition of HF-1 in 9:1 DMF/H2O solution, eliminating the possibility of conjugation. To mitigate this precipitation problem, glycerol was added to the soluble KLH or BSA solution  were performed with activated HF-4, had solubility issues upon addition to carrier protein and therefore needed higher glycerol content to ameliorate protein precipitation and conjugation issues. The linker placement at the phenethyl position greatly improved conjugation compared to the other phenylamide group; HF-4 had a hapten density of 21.1, drastically higher than HF-2 (hapten density: 12.6) and HF-3 (hapten density: 8.3). Gratifyingly, we observed copy numbers on BSA for every synthesized hapten. Immunoconjugates were then prepared with keyhole limpet hemocyanin (KLH) for murine immunization studies.Another trend observed for the chemically contiguous haptens was the increased amount of time needed to activate the carboxylic acids. Typically, fentanyl and heroin haptens reach optimal threshold activation (above 80%) within thirty minutes to several hours. However, these haptens required a minimum of four to twelve hours for at least 50% activation. In addition, bulkier haptens, including The nine haptens were formulated with Alhydrogel  and murine CpG ODN as previously described ,17. BrieFemale BALB/cByJ mice were immunized and bled using the protocol detailed in our previous study, except this study incorporated an extra boost at week 7, bleed at week 8, and antinociception at week 9 . The vac50 affinity continued to improve up to week 8. Individual conjugate vaccines had the highest level of midpoint titers of all groups as well as the best affinity values  had affinity only toward one of the two drugs, not both as initially anticipated. Also of note was that, while HF-4 and HF-6 had excellent affinity toward 6-AM, this did not translate to antibody-fentanyl affinity, which was 40 and 70-fold poorer. As expected this poor affinity to fentanyl was recapitulated as a nonstarter in behavior as seen in the tail flick antinociception assay . Week 8 titers show a slight correlation, although it is was not found to be significant. Moreover, nearly all of the vaccines produced very low titers and affinity to fentanyl compared to fentanyl-KLH vaccine (Figures S8 and S9, We also sought to determine if there was any predictive correlation between the in vitro SPR and ELISA results, and the behavioral results. To this end, nociception EDt titers , we saw HF-7 conjugate vaccine did show promising results as its heroin response was on par with Her-hapten immune response and it provided some efficacy against the fentanyl in heroin mixture. As linker-carrier protein regiochemical placement seemed more controlling for a balanced immune response against both drugs, it will be of interest to explore linker-carrier-protein display off the 3 or 6-position of heroin, thereby more prominently displaying the fentanyl moiety. These studies will be reported in due course.Our chemically contiguous hapten campaign was initiated to investigate the possibility of taking two structurally different opioids, heroin and fentanyl, and combining them into a singular, unique opioid hapten that, when formulated with an adjuvant, would present us with a vaccine capable of generating antibodies to both drugs. Based on this idea, a series of nine vaccines were prepared that enabled us to detect subtle influences on antibody response to each drug. In sum, we found little traction with each hapten’s vaccine efficacy from the standpoint of the chemical connection between the two drugs. Interestingly, immune response seemed to be more influenced by linker-carrier protein regiochemical placement. Although none of the nine haptens outperformed the individual conjugate vaccines against their assigned drug, the 1H NMR, 13C NMR, and high-resolution mass-spectrometry signals.Supporting information features SPR and ELISA experimental results, and additional experimental data. It also includes detailed procedures for all reported intermediates and final compounds, as well as their File 1Supporting experimental results.File 21–53.Experimental procedures for compounds File 31–53.NMR spectra of compounds


Before and after each SCTC, four tests  and a 5-km cross-country run (endurance) were performed. Seasonal data were continuously recorded during all SCTCs. Body mass decreased significantly (p<0.05) in all groups following SCTCs. These training-cycles induced a significant increase (p<0.05) in the 70-seconds push-ups, pull-ups and sit-ups and a decrease (p<0.01) in the 5-km cross-country running time among all trainees. The main effect of the season was present in all tests (p<0.01). With regard to the percentage of changes, the results from the 70-seconds push-up, pull-up and sit-up tests were significantly higher in winter and spring (p<0.01) compared with the two other seasons, while 5-km cross-country performance improvements were significantly higher (p<0.01) in spring and summer, compared to the two other seasons. In summary,14-week of SCTCs improved upper-body strength resistance and running endurance performance in the commandos. Improvements in strength resistance performance were greater during cool weather (winter and spring), while improvements in running endurance performance were higher during hotter (spring and summer) seasons.The purpose of the present investigation was to follow-up the effect of specific commandos’ training-cycles (SCTCs) on upper-body strength resistance and running endurance performance, as well as determine whether variation in seasonal parameters has any effect on physical performance. Fourteen SCTCs were held over eight years, involving 466 participants. Participants were assigned to four subgroups according to their distribution over the seasons: summer ( A commaThe goal of special forces training is to reach or maintain the physical performance level required for more demanding occupational requirements or deployment standards. In sports training, total training load, nutrition and recovery are typically planned in an individual way in order to optimize training adaptations and minimize training-related injuries and overtraining. All SFSs receive the same program and experience the same conditions in their training , which iMilitary training and operations consist of tasks that can be completed through combined strength and endurance training . The anaNumerous factors, such as age, genetics, diet, social and psychological variables, and fitness level, have been reported to influence endurance capacity and strengthen the endurance of well-trained athletes. Several studies have demonstrated a strong link between these factors and external temperature and humidity . HoweverIn hot environmental temperature, the challenge of maintaining heat balance is much greater. The physiological changes during exercise in a hot environment include an increase in skin blood flow, higher heart rate to support metabolism , vasodilIt appears that an internal temperature, particularly that of the higher upper-nerve centers, close to 40°C is also the origin of early fatigue, as confirmed by the results of electroencephalography . Hence wAt the time of writing, there are no studies available that have compared and quantified the effects of weather conditions on physical performance during SFS training. Therefore, the aim of this study was to (i) examine the effects of specific commandos’ training-cycles (SCTCs) which included physical fitness, technical and tactical training courses, on physical performance of SFSs and (ii) determine whether SCTCs gains could be affected by seasonal variations in weather conditions.Participants reviewed and signed consent forms approved by the local Ethics Committee for Human Research (ECHR) of the National Guard Commandos School committee of Tunisia: “Commandos scientific staff” composed of officers and experts and in accordance with the ethical standards of the 1964 Helsinki Declaration. The study protocol described in this manuscript was reviewed and approved by this committee and all participants were volunteered and could decline participation in this research with no consequences for their status in the training program.n = 124), autumn (n = 145), winter (n = 52) and spring (n = 145). It should be noted that four out of the 17 training-cycles of this study were removed, as they took place in-between two seasons.A longitudinal repeated cross-sectional study design was used, involving repeated observations over an eight-year period (from March 2007 to May 2015). This study investigated the effects of an SCTC program on physical performances of commando soldiers, and its variations across the four seasons of the year. The program, which consisted of 14 training-cycles each lasting 14 weeks, ran over eight years and included 466 participants. The training-cycles were grouped into four subgroups  according to their distribution over time, with a tolerance of 2 weeks. Participants were also assigned to four subgroups according to the seasons: summer (Before (P1) and after (P2) the experimental period, all participants completed a testing protocol, including endurance and body-weight resistance exercises, with four tests in total: maximum standard repetitions over 70-seconds for push-ups, pull-ups and sit-ups, and 5-km cross-country tests. Tests were performed with 20 min recovery between tests. However, between-days reliability and sensitivity of each test was assessed.-2) were distributed into 13 training-cycles as described in For this study, 466healthy, trained antiterrorism male soldiers  volunteered to participate to this study. All participants submitted to a health inspection before the testing, and only those with an adequate health status and doctor’s permission were allowed to participate in the study. Inclusion criteria included no contraindications to maximal exercise testing, such as cardiovascular or pulmonary risk conditions, no history of chronic disease, illness, surgeries, hospitalizations, and musculoskeletal or joint injuries during the last six months before the beginning of the experiment. Eligible participants , and the same amount of the total burden of training and living conditions, regardless of age, fitness condition, previous work experience or any other factors . The pronditions . FurtherThe basic phase (28 days) was designed to test and develop the basic military skills, physical and mental endurance, stamina and confidence that a soldier is required to possess in order to successfully accomplish combat missions. The next part (35 days) was designed to develop and assess jungle warfare skills under strictly specific and extreme conditions. The last phase (35 days) was designed to develop and assess urban combat skills, again under strictly specific and extreme conditions . In averBefore the beginning of the study, all participants performed one familiarization session in the week before testing. Data were collected before starting the training-cycle and immediately after the end of the 14-week period. This design was respected over the eight years of the study.www.wunderground.com)Meteorological data  were continuously collected from the National Institute of Tunisian Meteorology’s database and from the official Weather Underground website . Participants performed the tests with commandos’ clothing and shoes but without a bulletproof vest .For each training-cycle, test data were collected at approximately the same time of day (9:00 a.m. to11:00 a.m.), 2h postprandial . Means and standard deviations (SD) were calculated after verifying the normality of distributions using the Shapiro-Wilk procedure. Training-related effects were assessed by two-way analyses of variance with repeated measures (season×time: [4×2]). To help protect against type II errors, power and effect size were estimated using p<0.001) higher in summer (26.6±2.3°C) compared with other seasons. Relative humidity (RH) was greater (p<0.01) in cool weather  than during summer (63.4±13.3%) or spring (63.5±14.9%). The comparison of barometric pressure in-between seasons revealed no significant difference (p = 0.491). Finally, average wind speed was significantly higher during winter than spring (p = 0.39), while the latter was higher (p<0.001) than that for summer and autumn.The means of morning temperatures were significantly (p>0.05). For all tests and body mass, there was a significant effect of time and season   (p<0.05)  . There w = 1.00) . Bonferr(p<0.05) . The posThe main effects analysis revealed the existence of significant differences between all percentage of changes for body mass and the four physical tests used. The Tukey-adjusted post-hoc comparisons Figs –5 showedThe aim of the present study was to investigate the effect of an SCTC program on upper-body strength resistance and running endurance performance (5-km cross-country run), and to determine whether SCTC outcomes could be affected by seasonal variations. The primary finding was that the 14-week SCTC improved upper-body strength resistance and running endurance performance in the trained commandos, independent of the time of year in which the program was held. In addition, seasons significantly affected the percentage of changes in body mass and the performance of all tests. With regard to these percentage changes, improvements in the pull-up, sit-up and push-up tests were lower in autumn compared with other seasons, while improvements in the 5-km cross-country run were higher in spring compared to winter and autumn. The decrease in body mass was greater during summer, compared with autumn and winter.th Ranger Regiment program, body mass decreased by 16%, which was reflected in the changes in body composition, whereby body fat was reduced by 10%. The average energy deficit during the 14 weeks of training was around 35% and the average amount of sleep was only 4 out of 24 h. Some studies have confirmed that intensive military training has an impact on the reduction of body mass and the percentage of fat [On the other hand, we demonstrated that the SCTC program had a significant effect on physical performance and body mass. Body mass decreased significantly for all participants -3.98±1.11%). Similar results have also been reported in several studies using combined endurance and strength training , or spri%. Similae of fat , 28, whie of fat , the resImprovements in muscle resistance and endurance capacity, following combined endurance and strength training , sprint It is noteworthy that our findings indicate that physical performance was significantly different between seasons. It has been well demonstrated that a training-induced increase in performance is more dependent on the balance between environmental temperature and core body temperature, humidity and radiant changes over time . LimitinFurthermore, we only observed an important significant increase in push-up, pull-up and sit-up performance during cool seasons (autumn and spring) compared to the mid-warm season. The percentage of improvements in the pull-up, sit-up and push-up tests was lower in autumn, compared with other seasons, while the percentage of change in the 5-km cross-country test was higher in spring compared to winter and autumn. Only a few studies have reported similar data (percentage of changes) in response to strenuous workouts , while mOther studies have examined the effects of SCTCs, but thiAs recommendation, the results of the present study should be of interest to coaches and other experts working with SFSs. This study has demonstrated that the SCTC program in question may be effective in improving the strength resistance of upper-body muscle and running endurance performance. In addition, as SCTC programs require individualization and periodization as much as possible, based on the planned special forces tasks, as well as the physical fitness and body-mass index of soldiers, they should take into account the relevant environmental conditions if optimal performance and training effects are to be achieved. Indeed, the studied SCTC program resulted in more efficient upper-body strength resistance during winter and spring, and greater running endurance performance during spring and summer. Furthermore, the best performance took place during autumn and spring for strength resistance exercises, and during winter for running endurance efforts. Our findings should help fitness coaches to develop their acclimatization (long-term) and cooling/warm (short-term) strategies in order to optimize the performance and efficiency of their training programs. It would also be appropriate to develop nutritional strategies for greater control over body-mass changes during summer training.In summary, the 14-week SCTC program resulted in increased upper-body strength resistance and running endurance performance in commandos. Although the best cross-country running performances took place in winter, the improvement rate was always greater during spring and summer. For upper-body strength resistance , performance improvements were greater during cool weather (autumn and spring), while improvements after the SCTC program were greater during winter and spring than other seasons. Further research is needed to understand the factors that result in poorer endurance performance during summer and strength resistance efforts during winter.


Dianthin enzymes belong to ribosome-inactivating proteins (RIPs) of type 1, i.e., they only consist of a catalytic domain and do not have a cell binding moiety. Dianthin-30 is very similar to saporin-S3 and saporin-S6, two RIPs often used to design targeted toxins for tumor therapy and already tested in some clinical trials. Nevertheless, dianthin enzymes also exhibit differences to saporin with regard to structure, efficacy, toxicity, immunogenicity and production by heterologous expression. Some of the distinctions might make dianthin more suitable for targeted tumor therapies than other RIPs. The present review provides an overview of the history of dianthin discovery and illuminates its structure, function and role in targeted toxins. It further discusses the option to increase the efficacy of dianthin by endosomal escape enhancers. Dianthus caryophyllus L.) and sweet William (Dianthus barbatus L.). It was first described by Stirpe and colleagues ..2 fragme.011 nM .P. dioica L. leaves, saporin-L1, saporin-S6 or ricin A-chain with regard to enzymatic activity  ..N-terminG. paniculata L. on the efficacy of dianthin-30-EGF and other fusion toxins [50 of 0.45 nM for dianthin-30-EGF and 57 nM for saporin-S3-EGF while in the presence of SA1641, the IC50 was tremendously decreased to less than 1 fM corresponding to a more than million-fold enhancement in efficacy. In contrast, ricin A-chain-EGF was only enhanced 16-fold and an enzymatically active P. aeruginosa exotoxin fragment fused to EGF was not enhanced at all, indicating that intracellular routing to late endosomes is important for the endosomal escape enhancer effect [S. officinalis L., on chemical conjugates of dianthin-30 with the antibodies cetuximab, panitumumab and trastuzumab [50 values of 0.0053 nM for dianthin-30-cetuximab and 0.0015 nM for dianthin-30-panitumumab, both tested on EGF receptor overexpressing colorectal carcinoma HCT-116 cells, and 0.023 nM for dianthin-30-trastuzumab on HER2 overexpressing mammary gland ductal carcinoma BT-474 cells , indicating that SO1861 is suitable to enhance the endosomal escape of RIPs but not of small molecules [Weng and colleagues tested the effect of the endosomal escape enhancer saponin SA1641 isolated from n toxins . Withoutr effect  (Table 5stuzumab . The thr 0.02 nM  (Table 5olecules . Furtherolecules ,94.In order to demonstrate the potential of dianthin for targeted tumor therapies, successful application in mouse tumor models would be valuable. In animals, in addition, other aspects such as distribution in the body, liberation, absorption, metabolization and excretion influence efficacy and kinetics of targeted toxins and endosomal escape enhancers, and other aspects such as immunogenicity come into play. At the moment, there are only few studies available. Endosomal escape enhancers that are injected subcutaneously into the neck are rapidly distributed in the body. Already after 10 min, the highest concentration in the kidneys is reached and all other organs showed the peak concentration after 10 to 30 min . ApproxiHis-tagged dianthin-30 genetically fused to EGF was tested in combination with SO1861 in a nude mouse model with subcutaneous HCT-116 tumors . A tumorDianthin-30 and dianthin-32 are the best investigated dianthin enzymes with dianthin-30 being the lead in the last decade. Three types of activity measurement can be distinguished, direct detection of the enzymatic activity by quantitation of released adenine from various substrates, determination of protein synthesis inhibition in a cell free system, and quantitation of cell viability in cell culture assays. The activity for different RIPs does typically not correlate between the three methods as different properties of the molecule affect the efficacy. Dianthin is a highly active enzyme but as a type 1 RIP, it does not possess a cell binding domain and therefore exhibits low cytotoxicity. This makes dianthin an attractive candidate for targeted tumor therapies as the toxin can be chemically coupled or genetically fused to a ligand that recognizes tumor-specific receptors. Regrettably, most of the targeted dianthin molecules that have been bound to the target receptor are degraded in lysosomes or recycled back to the cell surface after internalization. Endosomal escape enhancers such as particular glycosylated triterpenoids can substantially enhance the release into the cytosol and thus increase the cytotoxicity for target cells by a thousand- up to more than a million-fold. Systematic cell culture experiments provided evidence that dianthin appears to be suitable for targeted tumor therapies. Dianthin can be expressed with higher yield as saporin and is presumably less immunogenic. First studies in mice with targeted dianthin in the presence of endosomal escape enhancers demonstrated the vast potential of this plant enzyme.


The bonds formed between the enzyme-substrate complex, enzyme-cadmium and differences in interactions in presence of cadmium has been studied in detail. The present study should help in understanding the modeled structure of rice-nitrate reductase in 3-D which may in turn guide enzyme related studies in silico. The present study also provides an insight as to how cadmium interacts with nitrate reductase to alter the enzyme activity.Cadmium is a well known toxic heavy metal, which has various detrimental effects on plant system. In plants an important enzyme involved in the production of nitric oxide, nitrate reductase, is also affected by cadmium toxicity. According to many studies cadmium has an inhibitory effect on nitrate reductase activity. Similar effect of cadmium was found in our study where an inhibitory effect of cadmium on nitrate reductase activity was noted. However, the mechanism behind this inhibition has not been explored. With the help of homology, 3-D structure of rice-nitrate reductase is modeled in this study. Its binding with nitrate, nitrite and cadmium metal  We have also studied the interaction of OsNR with nitrite. A detailed study of interactions between enzyme-substrate complex and cadmium and the alterations in the bonds formed in presence of cadmium is carried out in silico. The bonds formed between the enzyme-substrate complex, enzyme-cadmium and differences in interactions in presence of cadmium has been studied in detail in silico. The present study should help in understanding the modeled structure of rice-NR in 3-D which may in turn direct enzyme related studies in silico. The present study also provides an insight as to how cadmium interacts with NR to alter the enzyme activity.Nitrate reductase NR EC 1.7.1.1-3) is a molybdoenzyme involved in the production of nitric oxide in the plant system. NR contains molybdenum as cofactor (Moco) and comes under the category of enzymes that take part in two-electron transfer reactions in all carbon, nitrogen, and sulfur cycles Hille, . All euk.7.1.1-3 cv. HUR 3022 were surface sterilized with 0.1% sodium hypochlorite solution and imbibed in water for 24 h. Seeds were germinated in petriplates for 3 days, seedlings were transferred and raised in sand cultures for next 7 days in plastic pots saturated with either Hoagland nutrient solution 2. The choice of 50 μM Cd(NO3)2 in this study was based upon the seed viability test performed under increasing concentrations of Cd(NO3)2 and reported earlier by our lab  as described earlier , and 30-mM KNO3 were added to each tube. Samples were vacuum-infiltrated for 5 min and incubated in a shaking water bath at 25°C for 30 min in the dark. After incubation, 1 mL aliquot from each sample was transferred to a new tube, followed by the addition of 1 mL of sulphanilamide (1% w/v in 1.5 M HCl) and 1 mL N-(1-naphthyl)-ethylenediaminedihydrochloride (0.02% w/v in 0.2 M HCl). The samples were incubated at room temperature for 30 min. The absorbance at 540 nm was measured with a spectrophotometer.NR activity was measured according to Jin et al. . BrieflyAll computational studies were carried out on Intel dual core based Microsoft Windows XP professional workstation.CAA33817.2) was retrieved from the protein sequence database hosted in the National Center for Biotechnology Information (NCBI)A search for rice NR at NCBI database revealed the presence of 48 entries. Amino acid sequence of NR from rice . Each of these molecules were retrieved in sdf.file format from PubChem and converted to pdb.file format using Molegro softwareThe 3-D structure of organic compounds heme (CID:53627695), flavin adenine dinucleotide (CID: 643975), cadmium (CID:23973), Moco; Molybdenum cofactor (CID: 23304237), NADPH (CID: 52945042), Nitrate (CID: 943) and Nitrite (CID:946) were retrieved at PubChem database  to construct the model. Homology modeling depends on the identification of one or more known protein structures, which share ~30% homology with the query sequence. The basis of this computational technique is that two proteins with similar sequences would adopt similar tertiary structures  OsNR protein backbone with heme cofactor containing Fe at its center, FAD, NADPH and Moco to obtain OsNR (ii) OsNR with nitrate and nitrite to obtain [OsNR-nitrate] and [OsNR-nitrite] complex (iii) [OsNR] complex with cadmium (Cd) metal (iv) [OsNR-nitrate] complex with Cd (v) [OsNR-nitrite] complex with Cd.The three dimensional structure of OsNR obtained above was used for docking studies. Stepwise protein-ligand dockings were performed using Molegro Virtual DockerDockings were viewed at Molegro Molecular Viewer and Discovery Studio viewer and useful conclusions drawn after analysis at Pymolt-test.The data were analyzed by a simple one-way variance analysis (ANOVA) and significant differences were compared by Figure 916 amino acid residues were present in the protein sequence of rice NR obtained from NCBI. This sequence showed 89% sequence similarity with 1CNF and 43% with 2BIH.The secondary structure analysis of OsNR was done at PROCHECK and showed that the structure contains 10 sheets, 3 beta-alpha units, 14 beta hairpins, 10 beta bulges, 42 strands, 25 helixes, 12 helix-helix turns, 105 beta turns and 10 gamma turns  and three amino acid residues common with that of nitrite binding . These results suggest that binding sites of Cd, nitrate and nitrite are in vicinity in the Moco containing subunit of the OsNR.The [OsNR-Nitrate-Cd] complex is shown in Figure Nitrate binding shows one common amino acid residue with its original binding site while interacting alone (Asp330). While Cd has no common amino acid residues with its original binding site Table .The [OsNR-Nitrite-Cd] complex is seen as Figure (Oryza sativa L.) is a worldwide significant crop with extensive studies on effects of different stresses on its growth and development. In the wet-lab studies it was observed that exogenous application of cadmium (Cd) caused decreased NR activity in rice seedlings as compared to control plants. With the knowledge of complete rice genome  in [OsNR-nitrate-Cd]. Whereas Cd has no common amino acid residue with its original binding site. It can be inferred that in [OsNR-nitrate-Cd] complex, nitrate is binding near its original binding position while interacting alone with OsNR. This may result in a low enzyme activity due to lack of proper binding of the substrate nitrate. Cd, which is binding to an entirely different site in [OsNR-nitrate-Cd] complex as compared to its original binding site in [OsNR-Cd] complex, may also bring some conformational changes in the enzyme structure thus resulting in a decreased enzyme activity. Also in the binding of OsNR with both nitrite and Cd it was found that both nitrite and Cd were binding on the Moco domain. So it might be assumed that both of them are competing with each other for the active site, hence reducing the activity of the enzyme. There are also some changes in the amino acid residues of nitrite and Cd binding in [OsNR-nitrite-Cd] complex as compared to their residues in OsNR-nitrite and OsNR-Cd complexes. In presence of both nitrate and Cd with OsNR, nitrate shifted from its original position which might lead to a decreased NR activity as the substrate is not binding to the active site of the molecule. Cd, binding to an entirely new position in OsNR-nitrate-Cd docking as compared to its original position in OsNR-Cd complex, might have caused some structural changes in the enzyme. Both these occurrences might result in an altered or decreased NR activity. Competition between both Cd and nitrite for the same binding site in [OsNR-nitrite-Cd] complex might also lead to a decrease in NR activity. These results may establish a possible correlation between decrease in NR activity and Cd toxicity in plants for the first time.Nitric oxide, a signaling molecule, is of prime importance inside the plant system. It plays role in many plant processes such as programmed cell death, stomatal closure, growth, germination, signaling, biotic and abiotic stress response (Wendehenne, in silico. Docking of OsNR with nitrate and nitrite resulted in several strong H-bonds. Met473 and Ser530 were found to be common binding residues for both nitrate and nitrite suggesting thereby that nitrite may act as a competitive inhibitor of NR. Amino acid residues: Arg197, Arg529, Asp330, Met471, Met473, Ser530, Thr531 were playing important role in binding of Cd to OsNR. Seven amino acids: Arg-197, Arg- 529, Asp-330, Met-471, Met-473, Ser- 530, Thr-531 were found to be common in binding of Cd and nitrate with OsNR. Amino acid residues Asn331, Met473 and Ser530 were found to be common in nitrite and Cd binding. It can probably be concluded that Cd is binding to a similar site in OsNR as that of nitrate and nitrite. In presence of both nitrate and Cd with OsNR, nitrate shifted from its original position which might lead to a decreased NR activity as the substrate is not binding to the active site of the molecule. It is the first report in rice which well correlates with the wet-lab results where activity of NR decreased under Cd-stress perhaps due to altered binding dynamics involving different amino acid residues.Interactions involved with the binding of cadmium with rice NR in presence and absence of nitrate or nitrite was analyzed PS and IS have performed all the wet lab and computational studies. PS, IS, and KS have prepared and edited the manuscript jointly.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.