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The skin is the first protective barrier of our body. Wound healing is therefore an essential mechanism. However, this phenomenon can be impaired when wounds are too large or chronic, for example, in diabetes. Interestingly, adult skin heals with scars, whereas foetuses present scarless regeneration. The objective of this review is to highlight the difference in healing pathways between foetal and adult skin and to present the recent therapeutic strategies envisaged using foetal properties in the clinic. The main features that distinguish foetal wound healing from adult wound healing are less tissue inflammation, faster reepithelialisation, and less contraction of the neodermis, allowing foetal tissues to regenerate. Recently, new therapies in regenerative medicine have been introduced using these foetal properties. For the first time, our team has developed CICAFAST, an innovative dressing composed of foetal keratinocytes and fibroblasts, which has been tested on a skin graft donor site in a clinical Phase 1/2 trial.

BackgroundWound repair is one of the most complex biological processes of human life. Allogeneic cell-based engineered skin substitutes provide off-the-shelf temporary wound coverage and act as biologically active dressings, releasing growth factors, cytokines and extracellular matrix components essential for proper wound healing. However, they are susceptible to immune rejection and this is their major weakness.Thanks to their low immunogenicity and high effectiveness in regeneration, fetal skin cells represent an attractive alternative to the commonly used autologous and allogeneic skin grafts.Methods/designWe developed a new dressing comprising a collagen matrix seeded with a specific ratio of active fetal fibroblasts and keratinocytes. These produce a variety of healing growth factors and cytokines which will increase the speed of wound healing and induce an immunotolerant state, with a slight inflammatory reaction and a reduction in pain.The objective of this study is to demonstrate that the use of this biological dressing for wound healing at the split-thickness skin graft (STSG) donor site, reduces the time to healing, decreases other co-morbidities, such as pain, and improves the appearance of the scar.This investigation will be conducted as part of a randomized study comparing our new biological dressing with a conventional treatment in a single patient, thus avoiding the factors that may influence the healing of a graft donor site.DiscussionThis clinical trial should enable the development of a new strategy for STSG donor-wound healing based on a regenerative dressing. The pain experienced in the first few days of STSG healing is well known due to the exposure of sensory nerve endings. Reducing this pain will also reduce analgesic drug intake and the duration of sick leave.Our biological dressing will meet the essential need of surgeons to “re-crop” from existing donor sites, e.g., for thermal-burn patients. By accelerating healing, improving the appearance of the scar and reducing pain, we hope to improve the conditions of treatment for skin grafts.Trial registrationClinicalTrials.gov, ID:NCT03334656. Registered on 7 November 2017.

Background: The DYNAMIC study is based on three properties of tetracyclines. (1) Tetracyclines are known to chelate zinc from matrix metalloproteinases. It is possible their chelating activity may help inhibit COVID-19 infection by limiting its ability to replicate in the host. (2) As seen with dengue virus, tetracyclines may also be able to inhibit the replication of positive polarity single-stranded RNA viruses, such as COVID-19. (3) Tetracyclines are also modulators of innate immunity (anti-inflammatory activity), a property that has been used to treat inflammatory skin diseases for many years. They could therefore participate in limiting the cytokine storm induced by COVID-19. Moreover, the lipophilic nature of tetracyclines and their strong pulmonary penetration could allow them to inhibit viral replication at this level. Among the tetracyclines, doxycycline has three advantages: its long safety history (side effects are uncommon with no notable risks), its short treatment duration and its low cost.Methods: The trial will involve 330 patients who are positive for SARS-CoV-2 infection and have one or more risk factors for worsening the disease. These patients will be included as outpatients for early treatment of illness. For logistical reasons and in order to be able to standardise the study as much as possible, recruitment will take place in 6 hospital departments covering the whole of France. For 14 days they will be given either 200mg of doxycycline a day or placebo. Our hypothesis is a considerable reduction in the number of patients hospitalised due to COVID-19 thanks to the treatment of doxycycline.Discussion: This study could have an impact on the overcrowding of patients with COVID-19 at the hospital which is one of the major world-wide problems of this pandemic. This treatment would therefore contribute to supporting the deconfinement strategy by blocking the viral infection early and reducing the infectious period.Trial Registration: On ClinicalTrials.gov, registration number NCT04371952, first published on 30 April 2020.

Background Inflammatory Bowel Diseases (IBD) affect psychological, family, social and professional dimensions of patients’ life, leading to disability which is essential to quantify as part of Patient-Reported Outcomes (PROs) newly included in the targets to reach in IBD patients. Up to now, the IBD-Disability Index (IBD-DI) was the only validated tool to assess disability, but it is not appropriate for use in clinical practice. The IBD Disk was developed, a shortened and self-administered tool, adapted from the IBD-DI, in order to give immediate representation of patient-reported disability. However, the IBD Disk has not been validated yet in clinical practice. The aims of the VALIDate study are to validate this tool in a large population of IBD patients and to compare it to the already validated IBD-DI. Methods The VALIDate study is an ongoing multicentric prospective cohort study launched in April 2018 in 3 French University Hospitals (Nantes, Rennes, Angers), with an objective to reach a sample of 400 patients over a period inclusion of 6 months. Each patient will fill in the two questionnaires IBD Disk and IBD-DI at baseline, then between 3 and 12 months later, during a follow-up visit. Clinical and socio-demographic data will also be collected. During these two consultations, gastroenterologists and patients will evaluate disease activity thanks to a semi-quantitative 4-grade scale, named respectively PGA (Physician Global Assessment) and PtGA (Patient Global Assessment). This cohort will allow to evaluate the validity of the IBD Disk with respect to the IBD-DI in order to generalize its use for clinical practice. Other psychometric criteria of the IBD Disk will also be analysed as its reliability or its discriminant capacity. Close attention will nonetheless be needed to minimize the number of lost to follow-up patients between baseline and follow-up. Discussion The VALIDate study is the study designed to validate the IBD Disk, a visual tool easily useable in daily practice to assess disability in IBD patients. The results of this trial should enable the diffusion of this tool. Trial registration The trial is registered in ClinicalTrials.Gov with registration number NCT03590639. First posted: July 18, 2018.

Background and Aims Inflammatory bowel diseases [IBD] are disabling disorders. The IBD-Disability Index [IBD-DI] was developed for quantifying disability in IBD patients but is difficult to use. The IBD-Disk is a visual adaptation of the IBD-DI. It has not been validated yet. The main objectives were to validate the IBD-Disk and to assess the clinical factors associated with a change in the score and its variability over time. Methods From May 2018 to July 2019, IBD patients from three university-affiliated hospitals responded twice to both IBD-Disk and IBD-DI at 3–12 month intervals. Validation included concurrent validity, reproducibility, and internal consistency. Mean IBD-Disk scores were compared according to clinical factors. Variability was assessed by comparing scores between baseline and follow-up visits. Results A total of 447 patients [71% Crohn’s disease, 28% ulcerative colitis] were included in the analysis at baseline and 265 at follow-up. There was a good correlation between IBD-Disk and IBD-DI [r = 0.75, p Conclusions This study validated the IBD-Disk in a large cohort of IBD patients, demonstrating that it is a valid and reliable tool for quantifying disability for both CD and UC.

Background The epiretinal membrane (ERM) is a degenerative condition associated with age, which can cause loss of vision and/or metamorphopsia. The treatment of symptomatic ERM involves surgical removal including a vitrectomy followed by peeling of the ERM using a microforceps. As the internal limiting membrane (ILM) is adherent to the ERM, it is sometimes removed with it (spontaneous peeling). If ILM remains in place, it can be removed to reduce ERM recurrence. However, it is important to clarify the safety of ILM peeling, while it increases surgical risks and cause histological disorganization of the retina that can lead to microscotomas, may be responsible for definitive visual discomfort. Methods PEELING is a prospective, randomized, controlled, single-blind, and multicentered trial with two parallel arms. This study investigates the benefit/risk ratio of active ILM peeling among individuals undergoing ERM surgery without spontaneous ILM peeling. Randomization is done in the operating room after ERM removal if ILM remains in place. After randomization, the two groups—“active peeling of the ILM” and “no peeling of the ILM”—are compared during a total of three follow-up visits scheduled at month 1, month 6, and month 12. Primary endpoint is the difference in microscotomas before surgery and 6 months after surgery. Patients with spontaneous peeling are not randomized and are included in the ancillary study with the same follow-up visits and the same examinations as the principal study. Relevant inclusion criteria involve individuals aged > 18 years living with idiopathic symptomatic ERM, including pseudophakic patients with transparent posterior capsule or open capsule or lensed patients with age-related cataracts. The calculated sample size corresponds to 53 randomized eyes (one eye/patient) per arm that means 106 randomized eyes (106 randomized patients) in total and a maximum of 222 included patients (116 spontaneous peeling). Discussion ILM peeling is often practiced in ERM surgery to reduce ERM recurrence. It does not impair postoperative visual acuity, but it increases the surgical risks and causes anatomical damages. If active ILM peeling is significantly associated with more microscotomas, it may contraindicate the ILM peeling during primitive idiopathic ERM surgery. Trial registration ClinicalTrials.gov, NCT02146144. Registered on 22 May 2014. Recruitment is still ongoing.

Background: The DYNAMIC study is based on three properties of tetracyclines. (1) Tetracyclines are known to chelate zinc from metalloproteases (MMPs). It is possible that their chelating activity may help to inhibit COVID-19 infection by limiting its ability to replicate in the host. (2) As seen with dengue virus, tetracyclines may also be able to inhibit the replication of positive polarity single-stranded RNA viruses, such as COVID-19. (3) In addition, tetracyclines are modulators of innate immunity (anti-inflammatory activity), a property that has been used to treat inflammatory skin diseases for many years. They could therefore participate in limiting the cytokine storm induced by COVID-19. The lipophilic nature of tetracyclines and their strong pulmonary penetration could allow them to inhibit viral replication at this level. Among tetracyclines, the advantages of doxycycline are triple: its long history of safety (infrequent side effects with no notable risks), the short duration of treatment and its low cost.Methods: By estimating the rate of patients presenting pulmonary signs requiring hospitalisation in at-risk patients infected with COVID-19 at 25%, we hypothesise that on doxycycline, this rate would decrease to 12%. The main objective involves 2 embedded hypotheses tested successively in case of rejection of the previous one: (i) Decrease the rate of patients requiring hospitalisation, (ii) Decrease the use of mechanical ventilatory assistance.Discussion: This study could have an impact on the management of COVID-19 risk factor patientsupstream of hospitals by general practitioners. These patients,if kept at home under experimental treatment, would participate in reducing the risk of dissemination of SAR-CoV-2in the population.Thus, this treatment would contribute to supporting the deconfinement strategy through blocking the viral infection early and reducing the contagious period.Trial Registration: On ClinicalTrials.gov, registration number NCT04371952, first published on 30 April 2020.

Background: Acne vulgaris of adult females has increased over the past 10 years; it affects currently 20 to 30% of adult females. The physiopathology of adult female acne is distinguished from that of teenagers essentially by two factors: hormonal and inflammatory. On a therapeutic plan, in female acne, four types of systemic treatment approved in this indication include: cyclines (leading to bacterial resistance); zinc salts (less effective than cyclines); and antiandrogens (risks of phlebitis). The last alternative is represented by isotretinoin, but its use in women of childbearing potential is discouraged because of the teratogen risks.In this context, spironolactone could represent an interesting alternative. It blocks the 5-alpha-reductase receptors at the sebaceous gland and inhibits LH production at the pituitary level. It has no isotretinoin constraints and does not lead to bacterial resistance. Currently, a very few studies have been performed on a limited number of patients. They showed that at low doses (lower than 200 mg/day), spironolactone can be effective against acne.In that context, it seemed clearly interesting to perform the first double-blind randomized study of spironolactone vs cyclines, which remains the moderate acne reference treatment, and to demonstrate the superiority of spironolactone’s efficacy in order to establish it as an alternative to cyclines.Methods: Two hundred female patients will be included. They will have acne vulgaris with at least ten inflammatory lesions and no more than three nodules. After randomization, the patients will be treated by spironolactone or doxycycline for three months and after placebo. The study will be blind for the first six months and open for the last six months. Discussion: The treatment frequently used in female acne is systemic antibiotics with many courses as it is a chronic inflammatory disease. In the context of the recent WHO revelation about the serious, worldwide threat to public health of antibiotic resistance, this trial could give the physician another alternative for adult female acne before using isotretinoin, whose management is more complex. Trial registration: On ClinicalTrials.gov, registration number NCT03334682, first published on 7 November, 2017. Recruitment is still ongoing.

Background Acne vulgaris of adult woman has increased over the past 10 years; it affects currently 20 to 30% of adult women. The physiopathology of adult woman acne is distinguished from the teenager one essentially by 2 factors: hormonal one with a peripheral hyperandrogeny coupled with an hypersensibility of cutaneous androgens receptors and an inflammatory one linked with Cutibacterium acnes. On a therapeutic plan, in female acne, four types of systemic treatment, approved in this indication are: cyclines which are problematic for the bacterial resistance; zinc salts which target only the inflammatory lesions and are less effective as cycline; antiandrogens, with acetate of cyproterone associated with risks of phlebitis and pulmonary embolism and recently meningioma. The last alternative is represented by isotretinoin but the use in women of childbearing potential is binding because of the teratogen risks and in addition, peripheral hyperandrogenia represents an identified risk of relapse. In this context, the spironolactone could represent an interesting alternative. It blocks the 5-alpha-reductase receptors at sebaceous gland and inhibits LH production at the pituitary level. It has not isotretinoin constraints, does not lead to bacterial resistance and targets the peripheral hyperandrogenia. Currently, very few studies have been performed on a weak number of patients. They showed that at low doses (lower than 200mg/day), spironolactone can be effective against acne. Methods: In that context, it seemed clearly interesting to perform the first double-blind randomized study spironolactone vs cyclines, which remains the moderate acne reference treatment and to demonstrate the superiority of spironolactone’s efficacy in order to establish it as an alternative way to cyclines. Discussion: Acne’s treatment frequently used in female acne is systemic antibiotics with many courses as it is a chronic inflammatory disease. In the context of recent WHO revelation about serious, worldwide threat to public health about antibiotic resistance, this trial could permit to give to the physician another alternative in adult female acne before using isotretinoin whose management is more complex. Trial registration : On ClinicalTrials.gov, registration number NCT03334682, first published on 7 November, 2017. Recruitment is still ongoing. Keywords : acne vulgaris, spironolactone, cycline

Background Almost 15% of patients with ulcerative colitis (UC) will require a proctocolectomy with ileal pouch–anal anastomosis (IPAA) as a result of fulminant colitis, dysplasia, cancer, or medical refractory diseases. Around 50% will experience pouchitis, an idiopathic inflammatory condition involving the ileal reservoir, responsible for digestive symptoms, deterioration in quality of life, and disability. Though the majority of initial cases of pouchitis are easily managed with a short course of antibiotics, in about 10% of cases, inflammation of the pouch becomes chronic with very few treatments available. Previous studies have suggested that manipulating the composition of intestinal flora through antibiotics, probiotics, and prebiotics achieved significant results for treating acute episodes of UC-associated pouchitis. However, there is currently no established effective treatment for chronic antibiotic-dependent pouchitis. Fecal microbiota transplantation (FMT) is a novel therapy involving the transfer of normal intestinal flora from a healthy donor to a patient with a medical condition potentially caused by the disrupted homeostasis of intestinal microbiota or dysbiosis. Methods Our project aims to compare the delay of relapse of chronic recurrent pouchitis after FMT versus sham transplantation. Forty-two patients with active recurrent pouchitis after having undergone an IPAA for UC will be enrolled at 12 French centers. The patients who respond to antibiotherapy will be randomized at a ratio of 1:1 to receive either FMT or sham transplantation. Discussion On April 30, 2014, the World Health Organization published an alarming report on antibiotic resistance. Finding an alternative medical treatment to antibiotics in order to prevent relapses of pouchitis is therefore becoming increasingly important given the risk posed by multiresistant bacteria. Moreover, if the results of this study are conclusive, FMT, which is less expensive than biologics, could become a routine treatment in the future. Trial registration ClinicalTrials.gov, NCT03524352. Registered on 14 May 2018.

Background Inflammatory bowel diseases (IBD) are disabling disorders. The IBD-Disability Index (IBD-DI) was developed for quantifying disability in IBD patients but is difficult to use. The IBD-Disk is a shortened and visual adaptation of the IBD-DI. It has not been validated yet. The main objectives were to validate the IBD-Disk in a large cohort of IBD patients and to assess its variability over time. Methods From March 2018 to July 2019, IBD patients from three university-affiliated hospitals responded twice to both IBD-Disk and IBD-DI at 3–12 months intervals (NCT03590639). Validation included concurrent validity, reproducibility, internal consistency, and evaluation of IBD-Disk correlation with IBD activity. Variability was assessed by comparing scores between baseline and follow-up visits. Results A total of 559 patients (73% Crohn’s disease, 27% ulcerative colitis) were included and 389 were followed up (Table 1). There was a good correlation between IBD-Disk and IBD-DI scores (r = 0.75, p < 0.001) (Figure 1). The IBD-Disk was significantly higher in patients with active disease according to Physician/Patient Global Assessment (Figure 2), clinical scores (Figure 3), and biomarkers levels, compared with patients with inactive disease. Reproducibility was excellent (intra-class correlation coefficient = 0.90), as well as internal consistency (Cronbach’s α = 0.89). The IBD-Disk score significantly decreased in patients becoming inactive over time. Conclusion This is the first study to validate the IBD-Disk in a large cohort of IBD patients, demonstrating that it is a valid and reliable tool for quantifying disability in clinical practice. Further studies are warranted to assess its correlation with endoscopic activity, to explore its responsiveness to change, and to evaluate the factors associated with disability.

Background Whether they are injected peri- or intraocularly, corticosteroids are still essential tools in the therapeutic arsenal for treating inflammatory macular oedema. A few years ago, however, only triamcinolone acetonide was available to ophthalmologists. While this compound was initially developed for rheumatological or dermatological use, it has been increasingly deployed in ophthalmology, despite still being off-label. In 2011, the system for delivery of dexamethasone from a biodegradable, injectable implant into the vitreous cavity obtained approval for use in inflammatory macular oedema. While the efficacy and safety of triamcinolone in macular oedema, including inflammatory oedema, have already been studied, there are currently no publications on subconjunctival triamcinolone injections, which are simple, effective and well tolerated. To date, the dexamethasone 700 μg implant has been authorized for the treatment of noninfectious intermediate and posterior uveitis, but there have been no studies to evaluate the efficacy and safety of the different peri- and intraocular strategies, including the treatment of inflammatory macular oedema. Methods This protocol is therefore designed to compare the efficacy and safety of peri- and intraocular corticosteroid injections in the treatment of inflammatory macular oedema. In this ongoing study, 142 patients will be included, and the oedematous eye will be randomised to treatment with either subconjunctival triamcinolone injection or an intravitreal implant containing 700 μg dexamethasone. Follow-up is planned for 6 months with monthly visits. Each visit will include visual acuity measurement, a slit lamp examination, fundoscopy, intraocular pressure measurement, laser flare measurement (if available) and spectral domain optical coherence tomography. Discussion The results of this trial will have a real impact on public health if it is shown that a Kenacort retard® (i.e. triamcinolone) injection costing just €2.84 and performed in the physician’s office (with no additional overhead costs) is at least as effective as the dexamethasone 700 μg implant (Ozurdex®; costing approximately €960 with the injection performed in a dedicated room), with no increased side effects. Trial registration ClinicalTrials.gov, NCT02556424. Registered on 22 September 2015.

Resume L’investigation clinique au CHU de Nantes est realisee au sein du departement investigation, compose d’une coordination et de 24 equipes de recherche clinique reunies sur quatre sites hospitaliers differents. Ces Unites d’Investigation Clinique disposent toutes d’un systeme de management de la qualite mis en place depuis plus de 12 ans, initialement pour les unites d’investigation clinique du centre d’investigation clinique (CIC 1413) de Nantes puis transpose en 2013 aux unites d’investigation clinique emergentes. ObjectifAfin d’evaluer les performances de cette demarche qualite appliquee a toute l’investigation du CHU, le departement investigation s’est engage dans une demarche de certification ISO 9001:2015. Methodes La cellule qualite du departement investigation a suivi une methodologie basee sur des evaluations internes et externes pour realiser un etat des lieux et fixer le retro-planning de la demarche de certification. Resultats Le perimetre de la certification impliquait le departement investigation du CHU (24 unites d’investigation clinique, 150 personnels non medicaux et plus de 500 personnels medicaux). Le client dans notre demarche de certification etait le promoteur d’etude clinique. La demarche a dure 18 mois et comporte des phases de formation et sensibilisation de tout le personnel implique. Le systeme management de la qualite du departement investigation a ete revu et ameliore conformement a la norme. Le departement investigation a obtenu la certification ISO 9001:2015 de toute l’investigation clinique du CHU de Nantes en juin 2017 sans aucune non-conformite. Conclusion Notre travail montre que le departement investigation a reussi a adapter la norme ISO 9001:2015 a la recherche clinique institutionnelle. La certification de l’activite specifique d’investigation clinique est un gage d’organisation optimisee et pertinente au service de la securite des volontaires participant a une recherche clinique, de la qualite scientifique de la recherche et contribue a la satisfaction des promoteurs.

Clinical investigation at the University Hospital of Nantes is carried out within the investigation department, which consists of coordination and 24 clinical research teams gathered at four different hospital sites. The Clinical Investigation Units are all equipped with a Quality Management System. This has been in place for more than 12 years and was initially created for the Clinical Investigation Units of the Clinical Investigation Centre (CIC 1413) in Nantes and then transposed in 2013, to the emerging Clinical Investigation Units.In order to evaluate the performance of this quality approach applied to all UHC investigations, the Investigation Department is committed to obtaining an ISO 9001:2015 certification.The quality division of the investigation department conducted the project according to a methodology, based on internal and external evaluations to determine an inventory and the retro-planning of the certification approach.The scope of the certification involved the investigation department of the University Hospital: 24 clinical investigation units, over 500 medical staff, 150 non-medical staff. In our certification approach, the client was the promoter of the clinical study. The process lasted 18 months and included phases of training and awareness-raising for all the staff involved. The quality management system of the investigation department has been revised and improved in accordance with current regulations. The Investigation Department obtained the ISO 9001:2015 certification for all clinical investigations at the Nantes University Hospital in June 2017 without any non-compliance.Our work shows that the investigation department has successfully adapted the ISO 9001:2015 standard to institutional clinical research. The certification for specific activities in clinical investigation is the guarantee of an optimized and relevant organization for the safety of volunteers, participating in clinical research as well as the scientific quality of the research and contributes to the satisfaction of sponsors.

In the O2– generating flavocytochrome b, the membrane-bound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O2 in the following sequence: NADPHFADheme bO2. Although p-iodonitrotetrazolium (INT) has frequently been used as a probe of the diaphorase activity of the neutrophil flavocytochrome b, the propensity of its radical to interact reversibly with O2 led us to question its specificity. This study was undertaken to reexamine the interaction of INT with the redox components of the neutrophil flavocytochrome b. Two series of inhibitors were used, namely the flavin analog 5-deaza FAD and the heme inhibitors bipyridyl and benzylimidazole. The following results indicate that INT reacts preferentially with the hemes rather than with the FAD redox center of flavocytochrome b and is not therefore a specific probe of the diaphorase activity of flavocytochrome b. First, in anaerobiosis, reduced heme b in activated membranes was reoxidized by INT as efficiently as by O2 even in the presence of concentrations of 5-deaza FAD which fully inhibited the NADPH oxidase activity. Second, the titration curve of dithionite-reduced heme b in neutrophil membranes obtained by oxidation with increasing amounts of INT was strictly superimposable on that of dithionite-reduced hemin. Third, INT competitively inhibited the O2 uptake by the activated NADPH oxidase in a cell-free system. Finally, the heme inhibitor bipyridyl competitively inhibited the reduction of INT in anaerobiosis, and the oxygen uptake in aerobiosis.

The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussière, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussière, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites.

Plasma membranes of neutrophil cells contain the redox component of the O2(-)-generating NADPH oxidase complex, namely a heterodimeric flavocytochrome b consisting of an alpha subunit of 22 kDa and a beta subunit of 85-105 kDa of a glycoprotein nature. The NADPH oxidase is dormant in resting neutrophils. When neutrophils are exposed to a variety of particulate or soluble stimuli, the oxidase becomes activated, due to the assembly on the membrane-bound flavocytochrome b of three cytosolic factors, p47phox, p67phox and Rac 2 (or Rac 1). The effect of phenylarsine oxide (PAO), which reacts specifically with vicinal and neighbouring thiol groups in proteins, was assayed on the NADPH oxidase activity of bovine neutrophils, elicited after activation of the oxidase in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils, GTP[S], ATP and arachidonic acid; the effect of PAO on the oxidase activation itself was measured independently. PAO preferentially inhibited oxidase activation rather than the elicited oxidase activity, and inhibition resulted from binding of PAO to the membrane component of the cell-free system. To determine the PAO-binding protein responsible for the loss of oxidase activation, we used photoaffinity labeling with a tritiated azido derivative of PAO, 4-[N-(4-azido-2-nitrophenyl)amino-[3H]acetamido]phenylarsine oxide, ([3H]azido-PAO). Photoirradiation of plasma membranes from resting neutrophils in the presence of [3H]azido-PAO resulted in the prominent labeling of a protein of 85-105 kDa whose migration on SDS/PAGE coincided with that of the beta subunit of flavocytochrome b as identified by immunoreaction. Upon deglycosylation, the photolabeled band at 85-105 kDa was shifted to 50-60 kDa as was the immunodetected beta subunit. Similar results were obtained with isolated flavocytochrome b in liposomes. Photoaffinity labeling of the beta subunit of the membrane-bound flavocytochrome b or the isolated flavocytochrome b in liposomes resulted in abolition of oxidase activation in the reconstituted cell-free system. Incorporation of [3H]azido-PAO into flavocytochrome b was negligible when photoaffinity labeling was performed on neutrophil membranes that had been previously activated. The results suggest that the beta subunit of flavocytochrome contains two target sites for PAO which are accessible in resting neutrophils, but not in activated neutrophils.

International audience; Carrageenans are sulfated galactans found in the cell walls of numerous red seaweeds (Rhodophyta). They are classified according to the number and the position of sulfate ester groups and the occurrence of 3,6-anhydro-galactose. Although the carrageenan biosynthesis pathway is not fully understood, it is usually accepted that the last step consists of the formation of a 3,6-anhydro ring found in kappa- and iota-carrageenans through the enzymatic conversion of d-galactose-6-sulfate or d-galactose-2,6-disulfate occurring in mu- and nu-carrageenan, respectively. We purified two enzymes, sulfurylase I (65 kD) and sulfurylase II (32 kD), that are able to catalyze the conversion of nu- into iota-carrageenan. We compared their sulfate release rates (i.e. arising from the formation of the anhydro ring) with the viscosity of the solution and demonstrated two distinct modes of action. In addition, we found that some mixtures of sulfurylase I and II lead to the formation of carrageenan solutions with unexpectedly low viscosities. We discuss the implication of these findings for the assembly of a densely aggregated matrix in red algal cell walls.

The levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3-kinase and type I Ins(1,4,5)P3 5-phosphatase. The catalytic domain of Ins(1,4,5)P3 3-kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C-terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3-kinase A in Escherichia coli as a His- and S-tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D-2-deoxy-Ins(1,4,5)P3 was devised. D-2-Deoxy-Ins(1,4,5)P3 and D-3-deoxy-Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D-2-deoxy-Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3-kinase. Therefore, the axial 2-hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3-hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3-hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding.

In the O2- generating flavocytochrome b, the membrane-bound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O2 in the following sequence: NADPH --FAD --heme b --O2. Although p-iodonitrotetrazolium (INT) has frequently been used as a probe of the diaphorase activity of the neutrophil flavocytochrome b, the propensity of its radical to interact reversibly with O2 led us to question its specificity. This study was undertaken to reexamine the interaction of INT with the redox components of the neutrophil flavocytochrome b. Two series of inhibitors were used, namely the flavin analog 5-deaza FAD and the heme inhibitors bipyridyl and benzylimidazole. The following results indicate that INT reacts preferentially with the hemes rather than with the FAD redox center of flavocytochrome b and is not therefore a specific probe of the diaphorase activity of flavocytochrome b. First, in anaerobiosis, reduced heme b in activated membranes was reoxidized by INT as efficiently as by O2 even in the presence of concentrations of 5-deaza FAD which fully inhibited the NADPH oxidase activity. Second, the titration curve of dithionite-reduced heme b in neutrophil membranes obtained by oxidation with increasing amounts of INT was strictly superimposable on that of dithionite-reduced hemin. Third, INT competitively inhibited the O2 uptake by the activated NADPH oxidase in a cell-free system. Finally, the heme inhibitor bipyridyl competitively inhibited the reduction of INT in anaerobiosis, and the oxygen uptake in aerobiosis.

Severe quantitative loss of protein is often observed in high-resolution two-dimensional electrophoresis of membrane proteins, while the resolution is usually not affected. To improve the solubility of proteins in this technique, we tested denaturing cocktails containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and zwitterionic detergents, synthesized for this purpose. Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14–16 carbons proved most efficient, solubilizing previously undetected membrane proteins.