To determine whether patients who receive corneas from the same donor have similar risks of endothelial failure and rejection.Patients with Fuchs endothelial dystrophy (FED) and pseudophakic bullous keratopathy (PBK) who received their first corneal transplant between 1999 and 2016 were analysed. Patients receiving corneas from donors who donated both corneas for the same indication were defined as ‘paired’. Gray’s test was used to compare the cumulative incidence of endothelial failure and rejection within 5 years post-transplant for ‘paired’ and ‘unpaired’ groups. Cox regression models were fitted to determine whether there was an association between recorded donor characteristics , age and sex and endothelial graft failure and rejection.10 838 patients were analysed of whom 1536 (14%) were paired. The unpaired group comprised 1837 (69%) recipients of single corneal donors and 7465 (69%) donors who donated both corneas for another indication. ECD was lower for unpaired single cornea donors (p<0.01). There was no significant difference in endothelial graft failure or rejection between paired and unpaired groups for FED  or PBK  nor for donor ECD, age, sex and paired donation after adjusting for transplant factors .The absence of a significant difference in graft outcome for corneal transplants for FED and PBK between paired and unpaired donors may reflect a homogeneous donor pool in the UK. Pairs of donor eyes from the same donor have similar endothelial cell densities and is associated with similar post corneal transplant central corneal thickness.We found similar endothelial graft rejection and failure in recipients of corneas from paired and unpaired donors.The borderline significant donor effect in Fuchs endothelial dystrophy patients for endothelial graft survival would suggest that there may be donor factors which have not been identified. This requires investigation.The cornea differs from other tissues or organs in that in its healthy state it is a relatively privileged site for transplantation due to the absence of blood and lymphatic vessels 3 5Both donor and recipient factors play a role in graft rejection and failure. Many of the recipient risk factors associated with rejection are well recognised, including young recipient age, number of previous transplants, vascularisation, previous rejection episodes and the indication and type of transplant.8–6810.1136/bmjophth-2021-000965.supp1Supplementary dataBrightbill and Kaufman drew attention to investigating the outcomes of penetrating keratoplasty (PK) between recipients of paired donor eyes.In this study, the demographics and endothelial outcomes for ‘paired’ corneal transplants were compared with ‘unpaired’ single corneal donor transplants and from donors who donated both corneas but for an alternative indication or regraft. An association between cause of failure and type of rejection for each patient of paired donor transplants was also investigated. The potential similarity in outcomes between transplant recipients of the same donor as well as potential differences between single corneal donors was of interest. It is unclear if corneas from donors where only one eye is suitable for transplant, differ in quality from corneas from donors where both eyes are issued for transplantation and whether this has a bearing on graft outcome. Patients with FED and PBK patients were selected because rejection and failure is due to endothelial failure and in particular, patients with FED are a relatively homogeneous group with few risk factors. Additionally, other donor factors were explored such as donor age, ECD and gender mismatch to determine whether these factors were associated with an increased hazard of endothelial graft failure and rejection. As well as recorded donor factors, an unknown donor effect was fitted to account for unexplained donor to donor variation and for any correlation between pairs of corneas from the same donor.Data were obtained from the UK Transplant Registry managed by NHS Blood & Transplant (NHSBT). NHSBT collect data on all corneal transplants performed in the UK. Completion of a transplant surgery record and follow-up forms at 1, 2 and 5 years post-transplant is required for all surgeons undertaking corneal transplantation in the UK.2.Data from all adult patients (aged 16 or above) with an indication of FED or PBK who received their first corneal transplant using NHSBT supplied corneas from adult (aged 16 or above) donors, between 1 April 1999 and 31 March 2016 in the UK were included. The cohort was also restricted to patients who received a PK or endothelial keratoplasty (EK) with an ECD greater than 2200 cells/mmOnly patients receiving corneas from donors who had donated both corneas for a first transplant for the same ocular disease were defined as ‘paired’ transplants (group A) in the analysis. ‘Unpaired’ transplants were defined as patients receiving a cornea from donors who had donated a single cornea  as well as patients receiving a cornea from donors who had donated two corneas where one cornea was transplanted for a re-graft or for another ocular disease .Endothelial graft failure: graft failure due to endothelial decompensation, primary graft failure or recurrence of the original disease. In addition to patients who had not experienced graft failure by the last follow-up, patients who experienced graft failure from any non-endothelial cause were censored at the time of graft failure.Endothelial rejection: The first episode of endothelial rejection. In addition to patients who had not experienced rejection by the last follow-up, patients who experienced rejection from any non-endothelial cause were censored at the time of rejection.Demographic data were reported as numbers, percentages and were compared using Fisher’s exact test. The causes of failure or rejection were compared for each pair of transplanted corneas for both FED and PBK combined. The cumulative incidence of endothelial graft failure and rejection were described for the paired and unpaired groups using the Aalen-Johansen estimatorCause-specific Cox regression analysis was used to determine whether there was an association between donor factors and endothelial graft failure and endothelial rejection within 5 years post-transplant, by indication for transplant. The effect of unknown donor factors was accounted for by including a random donor effect in each model. This also allowed for correlation between outcomes for two recipients of corneas from the same donor. A sensitivity analysis was performed to validate the results by repeating the same analysis using both paired and unpaired transplants. The donor characteristics of ECD, donor age and donor-recipient sex match were included in each model. Other donor morbidity such as diabetes, or medications were not available. The recipient and transplant characteristics considered for inclusion in each model were: recipient age, sex and high risk recipient , graft type, risk of glaucoma at the time of transplant, as well as post-operative factors: cataract surgery, elective removal of all sutures, selective suture adjustment or removal, loose or broken suture, glaucoma medication, other immunosuppressants and wound leak complications. Postoperative risk factors were included as time dependent variables.In all models, for each recipient and transplant characteristic considered for inclusion, a value of p≤0.1 based on the likelihood ratio test (comparing models with and without the risk factor) was deemed sufficient evidence for the risk factor to be included in each given model. If there were <15% of data missing, a complete-case analysis was used for modelling.Patients and public were not involved in the design, conduct, analysis or writing of the manuscriptData from 11 381 patients were included who had undergone a first corneal transplant for FED or PBK and met all the cohort criteria during the period of interest. A total of 543 (5%) were excluded from the analysis due to a lack of follow-up data. Of the 10 838 patients with follow-up data, 1536 (14%) patients received a cornea from a donor who had donated both corneas to two patients with the same indication for transplantation (group A paired). The unpaired transplants consisted of 9302 (86%) patients who received corneas either from single cornea donors  or from donors who donated one cornea for FED or PBK patients and the other cornea was used for either a regraft or for another ocular disease . Characteristics at the time of transplant for paired and unpaired transplants are compared in For many of the characteristics, there was some evidence of a statistical difference between the cohorts. Donors were younger in the unpaired double cornea donor cohort for PBK (p<0.01) and for FED (p<0.01) compared with the paired, and unpaired single cornea donor cohorts. ECD was lower for the unpaired single cornea donor cohort for PBK (p<0.01) and FED (p<0.01). The unpaired double cornea donor cohort had a slightly higher proportion of male to female transplants for FED (p=0.04) compared with the other two cohorts. Overall, however, the paired and unpaired cohorts were of sufficient similarity to conclude that the paired cohort was representative of the entire first corneal transplant cohort.In cases where both recipients experienced graft failure, endothelial decompensation was the most common cause of failure (N=7) and endothelial rejection was the most common cause of rejection (N=4). Across all causes of failure and rejection, the numbers of cases were very small. For this reason, a formal statistical test comparing causes of failure and rejection was not performed. Overall, the results indicated that there was not a relationship between the causes of graft failure or the causes of graft rejection between paired cornea donor recipients. There were 56 primary graft failures in the paired cohort , 17 were10.1136/bmjophth-2021-000965.supp2Supplementary dataThere was no evidence of a difference in the incidence of endothelial graft failure between paired, unpaired single cornea donor or unpaired double cornea donor cohorts in either FED or PBK   for unpa10.1136/bmjophth-2021-000965.supp4Supplementary dataThere was no evidence of a donor effect associated with 5-year endothelial graft survival when adjusting for transplant and post-transplant risks for PBK or FED  . SimilarOverall, none of the donor factors that were included in the PBK and FED models, donor age, ECD and donor–recipient sex match, were associated with endothelial rejection nor endothelial survival up to 5 years post-transplant.The quality of the donor tissue is an important factor in corneal graft survival. As such, standards are set so that the donor tissue issued by eye banks meets certain quality assurance standards such as above a minimum ECD, endothelial cell quality, and corneal clarity. In the UK, the donor pool is very homogeneous, with a predominance of older white male donors with 51% of donors 70 years or more  and 63% male .2.ECD is the most critical donor factor in determining graft survival,et al reported evidence of an adverse effect of donor age of 80 years or older on graft survival following PK in over 24 000 grafts.et al in a cohort of 895 eyes with 15 years of follow-up found that grafts from donors aged 80 years and older were at a higher risk of failure.32 65There is conflicting evidence among studies regarding the influence of donor factors on graft outcome, particularly where the boundaries for donor factors should be, such as donor age, postmortem times. The current available evidence, therefore, is presented in et al reported a 40% reduced hazard in FED patients graft survival for female donors to female patients matched transplants compared with H-Y mismatched group .et al.Donor and recipient mismatch for either gender or H-Y have been found to have an effect on graft outcome according to the type of transplant and indication. Hopkinson Our study has several limitations, it largely consisted of donors, aged 61 year and over with the oldest donor 104 years. The intention, however, was to treat elderly FED and PBK patients meaning the median age match (recipient age–donor age) was 1 year but with an IQR of 7–8 years. Donor information collected was limited to age, sex and ECD and we do not have information on other donor characteristics and co-morbidity such as diabetes. Furthermore, some characteristics were not comparable in the paired and unpaired cohorts although to a small extent and in some parts of the analysis. The availability, however, of the UK Transplant Registry offers a unique opportunity to explore donor factors in the UK, which smaller studies or those relying on voluntary data returns may not be able to address. The accuracy, however, of a given analysis can depend on data completeness. In our complete-case study, only 5% were excluded due to incomplete follow-up data.Overall, we did not identify donor factors that were associated with endothelial rejection and endothelial survival; paired and unpaired transplants had very similar outcomes. There was, however, a borderline significant donor effect in FED patients for endothelial graft survival and would suggest that there may be donor factors which have not been identified and requires further investigation. With regard to the correlation between paired donor transplants, the number of cases with the same cause of failure or rejection was very small so there was no similarity in each transplant outcome. The general lack of difference in endothelial outcomes between paired and unpaired transplants for FED and PBK may reflect a homogeneous donor pool in the UK.


Systemic pentavalent antimonials, mainly meglumine antimoniate, continue to be the first-choice drugs for treatment of cutaneous leishmaniasis (CL) despite their toxicity, difficulty of administration and high cost. In the search for therapeutic alternatives, combining two treatment interventions has emerged as a potential alternative to either reduce the use of antimonials with the associated toxicities, or to increase efficacy. Here, we report the results of a recently completed trial assessing the efficacy and safety of a combination of thermotherapy (TT) plus a short course of miltefosine (MLT) for the treatment of uncomplicated CL in Colombia and Peru.A multicenter, randomized, evaluator-blinded, phase II, controled clinical trial was conducted. Adult volunteers with a parasitologically confirmed diagnosis of uncomplicated CL were randomly allocated to receive either a single session of TT or a combination of TT plus a short course of MLT (3 weeks). Therapeutic response outcomes and safety were assessed.130 subjects were included in the study, of whom 64 were randomly assigned to the TT arm and 66 to the TT + MLT arm. Cure at 3 months’ follow-up was achieved in 57.8% (n = 37) and 80.3% (n = 53) in the TT and TT + MLT groups, respectively, in the intention to treat analysis. The TT + MLT regimen was better that TT alone (p = 0.0055). The presence of vesicles at the site of heat application was the most common adverse event reported associated with the use of TT; while vomiting (31.8%) and elevation of liver enzymes (28.8%) were the most frequent adverse events reported associated with the use of MLT.The combination of TT plus a short course of MLT was shown to be significantly better than TT alone for the treatment of uncomplicated CL in the New World.NCT02687971.Registered in clinicaltrials.gov  Leishmania parasites transmitted by sandflies. It is a complex and neglected disease. Generally, it manifests as ulcers, nodules, or other lesions, mainly on the face and extremities. An estimated 0.7–1 million new cases occur annually in around 88 countries in South and Central America, Africa, Europe, the Middle East, and Asia, with the appearance of new outbreaks in different regions of the world.Cutaneous leishmaniasis is a disease caused by For more than 70 years, pentavalent antimonials have been the first line treatment, despite their toxicity, varying efficacy and need of administration of painful injections for 20 days or more.There is limited availability of effective and safe treatment alternatives and the strength of scientific evidence on the efficacy and safety of the different schemes is variable, limiting the control of the disease from a therapeutic approach. Hence the need for effective, safe, low-cost treatment alternatives with non-invasive routes of administration.This article reports the results of a clinical trial aiming to evaluate a combined therapy with two therapeutic alternatives (miltefosine and thermotherapy) approved for the treatment of cutaneous leishmaniasis, comparing it with thermotherapy alone. In this study, the combined therapy was significantly better than the monotherapy in managing cutaneous leishmaniasis. Leishmania, with L. tropica in the Old World and L. braziliensis in the New World being the most important species, given the severity and duration of the disease and the difficulty of treating them [Of the different clinical forms of leishmaniasis, cutaneous leishmaniasis (CL) is the most frequent. An estimated of 0.7–1 million new cases from around 88 countries are estimated to occur annually . CL is cing them ,4. Typicing them .L. panamensis and, to a lesser extent, by L. braziliensis. Meanwhile, in Peru, the predominant species are L. braziliensis and L. peruviana [Between 2001 and 2019, a total of 1,028,054 new cases of CL or mucocutaneous leishmaniasis (MCL) in the Americas were reported to the Pan American Health Organization (PAHO). In 2019, of the 41,617 new cases of CL and MCL, 75% were reported by Brazil , Colombia , Peru , Nicaragua  and Bolivia [v/kg/day for 20 days, continue to be the first-choice drugs to treat CL despite their toxicity, difficulty of administration and high cost. Alternative treatment regimens, including miltefosine, pentamidine, isethionate, amphotericin B and topical therapies, such as intralesional antimonials, heat therapy or cryotherapy, are less commonly used [Pentavalent antimonials, mainly meglumine antimoniate at doses of 20 mg Sbnly used ,8.Combining two treatment interventions has emerged as a potential alternative to either reduce the use of antimonials and their associated toxicities or to increase efficacy. The most used combination so far is the association of intralesional antimonials with liquid nitrogen, which in the Old World has shown to be more effective than using either intervention alone –11. In tL. braziliensis, L. panamensis and L. guyanensis only. In studies conducted in the Americas, the effectiveness of oral miltefosine varies from 90% when evaluated in infections caused by L. panamensis to <60% for CL caused by L. braziliensis and L. mexicana [Thermotherapy, especially generated via radio frequency waves (ThermoMed) has been widely tested for CL in both Old and New Worlds. The procedure and the device were approved for commercialization by the United States Food and Drug Administration (FDA) for the treatment of CL in 2007. Thermotherapy was also included in the list of recommended treatments for uncomplicated CL cases by WHO in 2010  and by Pmexicana ,20. Althmexicana .Here, we report the results of a recently completed phase II, randomized multicenter trial assessing the efficacy and safety of a combination of thermotherapy (TT) plus a short course of miltefosine (MLT) for the treatment of uncomplicated CL in Colombia and Peru.In Colombia, the protocol was approved by the Research Ethics Committee of the IPS Universitaria, by the Ethics Committee of the Military Hospital of the Colombian Army and by the National Regulatory Authority (Instituto de Vigilancia de Medicamentos y Alimentos—INVIMA); in Peru the protocol was approved by the Universidad Peruana Cayetano Heredia Ethics Committee and by the Peruvian National Institute of Health. The study was carried out according to international norms of good clinical practice. Before entry into the study, investigators obtained written informed consent from all participants. The trial was conducted following Good Clinical Practice standards.https://clinicaltrials.gov/ct2/show/NCT02687971).This is a randomized, multicenter, evaluator-blinded, phase II, clinical trial, registered at clinicaltrials.gov NCT02687971  not located on the ear, face, close to mucosal membranes, joints, or on a location that in the opinion of the principal investigator would make the application of TT difficult. Subjects were excluded for the following reasons: females with a positive serum pregnancy test, breast-feeding or of childbearing potential who do not agree to take appropriate contraception during the treatment period and until day 90 after the onset of treatment; clinically significant medical problems as determined by history or laboratory studies; previous use of antileishmanial drugs (within 8 weeks); or abnormal laboratory values at baseline  / aspartate aminotransferase (AST) 3 times above normal range).Leishmania by polymerase chain reaction (PCR) following previously published protocols [Leishmania species identification, as previously described [Proof of infection was documented either through microscopic identification of amastigotes in stained lesion tissue, the demonstration of motile promastigotes in aspirate cultures, or demonstration of rotocols ,22. In Pescribed .Subjects were randomly allocated to receive TT  or TT  plus MLT 2.5 mg/kg/day for 21 days.The 21-day course for MLT was chosen based on the results of a Phase II trial conducted in Colombia showing that a mean dose of miltefosine of 133 mg/day for 20 days resulted in a cure rate of 82%  and of aThermotherapy administration: local heat was generated using a device manufactured by Thermo-Med Technologies, Inc. . The lesions were cleansed, moistened for at least 10 minutes with normal saline and then anesthetized with 2% intralesional lidocaine before administration of thermotherapy. Heat was applied locally with a handset that includes two electrodes which were placed on the lesion(s) until the entire lesion was covered, including the affected areas around the ulcer. Patients were instructed to use clorixin gel and to dress their lesion after receiving thermotherapy and for up to three days to decrease the effects of burning and the risk of secondary infections.Miltefosine was administered as follows: subjects weighing ≥30 to ≤45kg, received 50 mg twice a day (100 mg /day) whilst subjects above 45kg, received a 50 mg capsule three times per day (150 mg/day). Subjects were instructed to take the miltefosine capsules after meals to avoid gastric problems associated with the drug.Participants assigned to the TT+MLT arm had their liver enzyme and creatinine levels evaluated at baseline and end of treatment (D21), the latter only for participants assigned to the combined treatment arm.Rescue therapy: meglumine antimoniate at doses of 20 mg SbV /kg body weight per day for 20 days was provided free of charge to all subjects who were declared as treatment failures and to those who, for whatever reason, decided to withdraw from the study.All subjects had a follow-up visit 24 hours after TT treatment and thereafter at day 7, 14, 21, 45±5 days, 63±5 days 90±15 days and on day 180±15 days. The following treatment outcomes were applied:Initial cure: complete re-epithelialization of all ulcerated lesions or flattening and/or no signs of induration of the non-ulcerated lesion(s) on day 90.Final cure: initial cure and no relapse by day 180.Relapse: lesion that achieved 100% re-epithelialization/flattening on day 90 that subsequently reopened/indurate by day 180.Failure for ulcerated lesions: <50% re-epithelialization of lesion as compared to day7 by nominal day 63 or <100% re-epithelialization of the lesion(s) area(s) on nominal day 90; or an increase of ≥ 100% of the ulcer area compared to the area at day 7 at any time before day 90.Failure for non-ulcerated lesions: <50% improvement/reduction of induration/flattening of the lesion as compared to day 7 by nominal day 63; persistence of induration of the lesion(s) on nominal day 90; or an increase of ≥ 100% of the induration area compared to the area at day 7 at any time before day 90. Finally, relapse of the lesion any time between day 90 and day10.the proportion of initial clinical cure for each regimen measured at Day 90. Secondary endpoints were the proportion of final cure, the frequency, severity and seriousness of AEs, time to lesion healing and cure rates by Leishmania species, by treatment group.The primary endpoint was defined as Patients were actively followed up and no incentives to come back for follow-up visits were provided.Photos of all lesions were taken at different times. Clinical experts on CL evaluated the lesions. First, a blinded assessment of the lesion was performed on site at day 90 and day 180 visits by a clinician. Second, assessment of the lesion photographs at day 1, day 90 and day 180 visits was performed by an independent DSMB expert. If there was a discrepancy between the assessments performed by the site clinician and the Data Safety Monitoring Board (DSMB) expert, a third expert was asked to review the photos and a final decision was taken based on the majority of votes (two out of three) and used to classify the subject´s outcome.2, where A = longest diameter of ulceration in mm; B = perpendicular to “A” diameter of ulceration in mm and π = 3.14.Considering that TT application can increase the lesion size, the percentage of re-epithelialization/flattening of the lesion(s) was calculated by comparing the size of the ulcer/induration at D7 against the size at the follow-up visit. After cleaning the lesion and removing the crust (if applicable), measurements were taken in two perpendicular directions using an electronic caliper. The area of ulceration was calculated using the area calculation for an ellipse as follows: Area = A/2*B/2*π mmBased on reports, the efficacy of thermotherapy alone in the New World is around 65–70% and, in the absence of information about the efficacy of the proposed combination, a sample size of 118 subjects (59 TT and 59 TT + MLT) was estimated to have an 80% chance of detecting, at a significance level of 5%, an increase in the primary outcome measure from 70% in the TT alone group to 90% in the combination group. Taking into account an estimated 10% loss during follow-up, six more subjects were added, resulting in a sample size of 65 subjects per regimen, or 130 subjects in total.A computer-generated randomization code using a 1:1 allocation ratio was used to assign subjects to either TT or TT + MLT. Randomization was centralized and only the study coordinators had access to the password to generate the treatment allocation.The participant’s baseline characteristics were analyzed by treatment group and the results presented as summary statistics. Intention-to-treat (ITT) and per-protocol (PP) analyses were used to determine the efficacy of treatments. Categorical variables were presented as counts and percentages. Given the distribution of the variables, the Mann-Whitney U test was used for analysis of continuous data. Patients with missing data that made it impossible to determine whether they were clinical cures were considered failures in the ITT analysis. All data were presented separately by treatment group, and by local and systemic AEs. AEs were coded using the most recent version of the Medical Dictionary of Regulatory Activities (MedDRA) preferred terms and grouped by system organ class (SOC) designation. All data were analyzed by treatment group, and by local and systemic AEs.The study was carried out between December 2016 and February 2019. 130 subjects (65 volunteers per research center) were randomly assigned to one of the two treatment arms TT (n = 64) and TT + MLT (n = 66). In total, 91 subjects (37 TT and 54 TT + MLT) completed the study follow-up. One relapse occurred in the TT + MLT group. The description of treatment failures, voluntary withdrawals, and loss to follow-up are shown in L. panamensis was the main species responsible for infection in 30% of the cases. In both treatment groups, the majority of participants presented a single lesion , and the majority of lesions were ulcerated: 88.2% and 96.9% for the TT and TT + MLT groups, respectively  and 2 (3.0%) in the TT and TT+MLT groups, respectively, were lost during the follow up period, whilst 9 (6.9%) developed a new lesion (TT n = 6 and TT + MLT n = 3) and were, therefore, removed from the study, declared as failures and administered rescue treatment .The mean time for the lesions to heal was significantly lower (p = 0.02) in the TT + MLT group  than in the TT alone group  .Leishmania, we observed that subjects with lesions due to L. braziliensis and/or L. peruviana responded better to TT + MLT (20/27 = 74.1%) than to TT alone (9/21 = 42.8%), although treatment response was lower than in subjects with lesions due to L. panamensis in both treatment groups  (Although the numbers were low and the study was not powered to find differences in cure rates between different species of 0 = 60%) .Ninety two percent of subjects in the TT group and 100% in the TT+MLT group reported one or more AE . PresencOf the subjects included in the TT + MLT group, 56.1% (n = 37) reported having missed at least one dose of MLT, including one (1.5%) for AST/ALT elevation and n = 10 (15.2%) due mostly to gastrointestinal AEs associated with the medication.During the study period, 3 serious AE were reported: an episode of urolitiasis in a subject in the TT group, and hypoglycemia and a skin lesion due to a road traffic accident in two subjects in the TT + MLT group; none of these were considered related to the study treatments.In this first trial of a combination of one application of local heat plus 3 weeks of oral miltefosine, all study participants received their treatment according to which group they were assigned at the time of randomization. The study demonstrated that a single application of local thermotherapy in combination with 3 weeks of oral miltefosine is safe and delivers a higher cure rate than TT alone. Similarly, the time to heal for lesions treated with the combination was shorter than for lesions treated with TT alone.Various combinations of systemic and topical treatments have been tested for CL with the aim of increasing the efficacy of local therapies and reducing the AE associated with systemic treatment alone, by using a reduced dose. Until now, the most frequently used combination was intralesional injection of antimonials alternating with local applications of liquid nitrogen. This combination has been shown in the Old World to be more effective that either treatment alone . Other cThe advantages offered by this combination are that: a) it uses two approaches that are currently recommended for individual use for which there is good information regarding their efficacy and safety when used alone; b) the use of a local and a systemic treatment would hypothetically have an additive effect, since systemic treatment would eliminate circulating or remaining parasites located in the periphery of the lesion, which might be the cause of relapse, that local treatment fails to remove ; c) it oOur results confirmed the superiority of the TT + MLT combination over TT alone for the treatment of uncomplicated CL cases in NW. In the ITT analysis, initial cure was observed in 37 out of 64 (57.8%) and 53 out 66 (80.3%) subjects treated with TT alone or the TT + MLT combination, respectively (p = 0.0055). In the PP analysis, initial cure was achieved by 37 out of 56 (66.1%) and 53 out of 62 (85.5%) subjects in the TT and TT + MLT groups, respectively (p = 0.013). Lesions in subjects treated with the combination healed faster than those in subjects treated with TT alone, 61.2 vs 74.1 days (p = 0.02).L. panamensis or L. braziliensis, as was the case for the subjects included in this study [The cure rate observed in the TT alone group was lower than that found in a pooled analysis of eight studies from the Old and New Worlds, which reported an efficacy of 73.2% (95% CI = 69.6–76.7%), however, it is similar to the 64% (95% IC 56–72) cure rate at 3 months reported from Colombia when treating subjects with lesions due to is study ,18,29. Iis study .L. panamensis or L. braziliensis, the ITT efficacy of miltefosine was 58.6% (85/145 patients) [A limitation of the present study is the lack of a miltefosine only group in the study design, hence it is not possible to determine with precision how much the addition of TT to a 3-week course of miltefosine increased efficacy. Efficacy results from different studies in the New World using miltefosine at a dose of 2.5mg/kg/day for 28 days vary from 53% to 94% ,31. ThesTo our knowledge only one study was conducted during the early clinical development of miltefosine for CL that used a 3-week regimen. In this study, the per-protocol cure rate of subjects who received a dosage of 50–100 mg/day for 3 weeks was 66%, as compared to 94% in subjects who received 133–150 mg/day for 3–4 weeks . ShorterL. braziliensis strains from different geographical areas. In fact, it has been postulated that some strains of L. braziliensis may have a reduced capacity to internalize miltefosine from the extracellular medium [The disparity in cure rates with miltefosine found in the different clinical trials is probably due to the intrinsic sensitivity to miltefosine of r medium .For thermotherapy, the most frequently reported AE are consistent with those described in other studies, such as pain at the application site, edema and the presence of vesicles –19,36.In this study, the presence of vesicles at the application site was reported in a significantly higher percentage of subjects compared to previously published studies. This finding can, however, be explained by the safety visit that was carried out 24 hours after the thermotherapy session, which allowed more accurate recording of this AE. Equally, the AEs reported in subjects who received miltefosine were similar to those previously reported  and occurred in approximately the same proportion of subjects treated with four weeks of miltefosine ,37–40. SAlthough there have been many advances in the development of new oral treatments for leishmaniasis, most of these efforts have been directed to VL. While patients with CL may also benefit from these efforts, completing the clinical development and registration of any of the potential new treatment candidates under development will require several more years, meaning that currently available treatments for CL probably represent the entire therapeutic arsenal for the coming five to ten years.We believe there is an opportunity to improve the current treatment recommendations by using a combination of single treatments that are already recommended by WHO and PAHO, but not yet included in many countries’ CL treatment guidelines. This approach could rapidly generate information about the safety and efficacy, and potentially reduce the use of antimonials in patients with uncomplicated CL  which represent approximately 60% of all CL cases worldwide. Based on the encouraging results of the present study, we are now planning to conduct a phase III study aiming to determine if this TT + MLT combination is non-inferior to the current recommended first line treatments, meglumine antimoniate  or miltefosine monotherapy , for uncomplicated CL cases in the New World.the combination of thermotherapy plus a short course of miltefosine showed to be significantly better than thermotherapy alone for the treatment of uncomplicated CL in the New World. In the short-mid- term and until a new, safer and more efficacious treatment become available, the use of combined therapies, using already tested and recommended monotherapies, seems a promising option for patients with uncomplicated CL.S1 Checklist(DOC)Click here for additional data file.


Herein, we demonstrate a case study to efficiently find the herbal medicine(s) with potent hCYP3A4 inhibition in vitro and to accurately assess the potential HDIs risk in vivo. Following screening over 100 herbal medicines, the Chinese herb Styrax was found with the most potent hCYP3A4 inhibition in HLMs. In vitro assays demonstrated that Styrax could potently inhibit mammalian CYP3A in liver and intestinal microsomes from both humans and rats. In vivo pharmacokinetic assays showed that Styrax  significantly elevated the plasma exposure of two CYP3A-substrate drugs (midazolam and felodipine) when midazolam or felodipine was administered orally. By contrast, the plasma exposure of either midazolam or felodipine was hardly affected by Styrax (i.g.) when the victim drug was administered intravenously. Further investigations demonstrated that seven pentacyclic triterpenoid acids (PTAs) in Styrax were key substances responsible for CYP3A inhibition, while these PTAs could be exposed to intestinal tract at relatively high exposure levels but their exposure levels in rat plasma and liver were extremely low. These findings well explained why Styrax (i.g.) could elevate the plasma exposure of victim drugs only when these agents were orally administrated. Collectively, our findings demonstrate that Styrax can modulate the pharmacokinetic behavior of CYP3A-substrate drugs via inhibiting intestinal CYP3A, which is very helpful for the clinical pharmacologists to better assess the HDIs triggered by Styrax or Styrax-related herbal products.Human cytochrome P450 3A4 (hCYP3A4) is a predominant enzyme to trigger clinically relevant drug/herb-drug interactions (DDIs or HDIs). Although a number of herbal medicines have been found with strong anti-hCYP3A4 effects  It has s (DDIs) . TherefoRecently, our group and colleagues developed a novel fluorescence-based high-throughput assay for assessing the inhibitory effects of compounds/herbal extracts on human CYP3A4, which offers a powerful platform for investigating CYP3A4-related HDIs or DDIs . With thin vivo. Therefore, this study aims to solve two key questions: 1) Can Styrax change the pharmacokinetic behavior of co-administrated CYP3A-substrate drugs and trigger HDIs in vivo; 2) Styrax triggers clinically relevant HDIs via inhibiting either hepatic CYP3A4 or intestinal CYP3A4 or both.The major constituents in Styrax have been reported, in which cinnamic acids, phenylpropanoids and pentacyclic triterpenoid acids (PTAs) are major constituents in this herb, while the PTAs in Styrax are reported as naturally occurring CYP3A inhibitors . HoweverTo better solve these two questions, two CYP3A-substrate drugs (midazolam and felodipine) were selected as victim drugs in this work, while two administrated ways  were used to study the pharmacokinetic interactions between Styrax and two CYP3A-substrate drugs (midazolam and felodipine). Briefly, the inhibitory effects of Styrax against CYP3A-catalyzed midazolam 1′-hydroxylation were carefully assessed in liver and intestinal microsomes from both humans and rats. After then, the pharmacokinetic interactions between Styrax  and CYP3A-substrate drugs (midazolam and felodipine) by two administrated ways  were assessed in rats. Furthermore, the inhibitory effects of the PTAs in Styrax on CYP3A were assessed, while the tissue distributions of seven major PTAs in Styrax (the key substances responsible for CYP3A inhibition) were investigated carefully.+ were purchased from Sigma-Aldrich . MgCl2, 0.9% NaCl, and sodium carboxymethyl cellulose (CMC-Na) were provided by Sinopharm Chemical Reagent . The soybean oil was supplied by COFCO Fulinmen Food Marketing Co., Ltd. (China). Midazolam was from J&K Scientific . 1′-Hydroxymidazolam was provided by Shanghai Macklin Biochemical Co., Ltd. . Felodipine was purchased from Shanghai Yuanye Biotechnology Co., Ltd. . Osalmide was ordered from Meilun Bio. Tech . N-ethyl-1,8-naphthalimide (NEN) and N-ethyl-4-hydroxyl-1,8-naphthalimide (NEHN) were synthesized by using the previously reported protocol  was used throughout. LC grade methanol, acetonitrile and formic acid were supplied by Fisher Scientific Co., Ltd. . Other reagents were of the highest grade commercially available. The purities of all compounds used in this study were greater than 98%.The standard extract of Styrax was acquired by Shanghai Hutchison Pharmaceuticals . More than 100 herbal medicines granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd. . Lansoprazole was provided by Hairong . D-glucose-6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PDH) and β-NADPprotocol . Betulin2, and HLMs were added to the 96-well plate in turn, then the samples were vortexed and pre-incubated for 3 min at 37 °C. The oxidative reactions were initiated by adding β-NADP+ to yield a final volume of 200 μl, then the formation rates of NEHN (hydroxylated metabolite of NEN) were continuously monitored for 30 min at 37 °C using a 96-well fluorescence microplate reader . The percentage of organic solvent in all incubation is not over 1%. Please refer to the supplementary material  were added to the EP tube in turn and vortexed, then the samples were pre-incubated for 3 min at 37 °C. Next, the reactions were initiated by adding β-NADP+ to yield a final volume of 200 μl. Following incubation for 30 min at 37 °C, and 200 μl ice-cold acetonitrile containing internal standard  was subsequently added to quench the oxidative reaction. The mixture was then centrifuged at 20,000 ×g, 4 °C for 20 min, and the supernatant was quantified by LC-MS/MS analysis as described in The inhibitory effects of Styrax or its constituents against CYP3A were assayed by using midazolam as the substrate . Brieflygroup 1: Styrax + midazolam ;group 2: soybean oil + midazolam ;group 3: Styrax + midazolam (intravenous);group 4: soybean oil + midazolam (intravenous);group 5: Styrax + felodipine ;group 6: soybean oil + felodipine ;group 7: Styrax + felodipine (intravenous);group 8: soybean oil + felodipine (intravenous);Sprague-Dawley rats (weighing 200–220 g) were purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.  2018-0006). The animal experiments were carried out in Experimental Animal Center of Shanghai University of Traditional Chinese Medicine  and conducted in accordance with the State Committee of Science and Technology of China. In brief, forty male rats were housed under controlled environmental conditions  for 1 week with free access to food and water. The rats were fasted overnight before dosing but were free to drink water, and provided food after finishing the study. Styrax was suspended in soybean oil. Oral midazolam/felodipine was suspended in 0.5% CMC-Na, and intravenous midazolam/felodipine was suspended in 0.9% NaCl. Styrax or soybean oil was administered orally at a dose of 100 mg/kg. The oral dose of midazolam is 20 mg/kg and the intravenous dose is 5 mg/kg. The oral dose of felodipine is 10 mg/kg and the intravenous dose is 2 mg/kg. The dose of Styrax in rats was referred to the daily recommended oral dose in humans. The dose of midazolam and felodipine in rats was referred to the doses in published literature . Forty rStyrax  or soybean oil  was administered orally. After 30 min, midazolam/felodipine was administered orally and intravenously. For oral midazolam groups, blood samples were collected at 5, 10, 20, 30, 60 min, 2, 3, 4, 6, 9 h. For intravenous midazolam groups, blood samples were collected at 2, 5, 10, 20, 30, 60 min, 2, 3, 4, 6, 9 h. For oral felodipine groups, blood samples were collected at 10, 30, 60 min, 3, 4, 6, 8, 10, 24 h. For intravenous felodipine groups, blood samples were collected at 2, 5, 10, 15, 30, 45, 60 min, 4, 6, 10 h. Then blood samples were centrifuged at 8,000 rpm, 4 °C for 10 min, the supernatant was stored at −80 °C until analysis . 50 μl pNine Male Sprague-Dawley rats (250–300 g) were purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd.  and were fed in Experimental Animal Center of SHUTCM. All animals were housed under controlled environmental conditions  for 1 week with free access to food and water. The rats were fasted overnight before dosing but were free to drink water. Nine rats were randomly divided into three groups . Styrax (100 mg/kg) was administered orally, then nine rats were anesthetized by pentobarbital after administrating 1, 2, and 4 h, respectively. Blood samples were collected from the abdominal aorta, and the tissues  were removed rapidly and placed on ice. After then, the blood samples were centrifuged at 8,000 rpm, 4 °C for 10 min, and the supernatant was stored at −80 °C until analysis.Firstly, each tissue  was accurately weighed after sucking the water on tissue surface. Each tissue was put into an EP tube and then cut (3-5 times) by a scissor. After then, PBS (pH 7.4) and magnetic beads were added into EP tube, grind the tissue by a tissue grinder. The tissue grinding fluid was centrifuged at 9,000 g, 4 °C for 20 min. 50 μl tissue supernatant was diluted by adding 250 μl LC grade acetonitrile. The protein was precipitated by centrifuging at 20,000 ×g, 4 °C for 30 min and the supernatant was collected to determine the protein concentration by a BCA Protein Quantification Kit. After then, the concentrations of seven PTAs were determined by using UHPLC-Q-Orbitrap HRMS analysis. Mass spectrometry parameters were described in 50) was determined as the concentration of Styrax/PTAs at the catalytic activity of CYP3A was inhibited by 50% compared to the negative control (solvent only). In this study, all assays were tested in triplicate, while the data were shown as mean ± standard deviation (SD). IC50 values were fitted by GraphPad Prism 6.0 . Meanwhile, the following formula was used to calculate the residual activities of CYP3A:The dose-inhibition curves of Styrax/PTAs against CYP3A were depicted by using different dosages of Styrax/PTAs, while the half maximal inhibition concentration  = /the Peak Area or Fluorescence value of the metabolite in negative control (solvent only) × 100%.Firstly, with the help of fluorescence-based high-throughput CYP3A4 inhibition assay, the inhibitory potentials of more than 100 herbal medicines against CYP3A4-catalyzed NEN 4-hydroxylation were assayed in HLMs by using a single dose . As shown in 50 values of 3.72 μg/ml, 2.84 μg/ml, 2.90 μg/ml and 1.67 μg/ml, for HLMs, HIMs, RLMs, RIMs, respectively. These findings demonstrated that Styrax could potently inhibit mammalian CYP3A in liver and intestinal microsomes from both human and rat in vitro, with similar inhibition tendency and potency. However, it is still unclear that Styrax can inhibit hepatic CYP3A or intestinal CYP3A in vivo. Therefore, the in vivo pharmacokinetic interactions between Styrax and midazolam were carefully investigated by using two administrated ways (i.v. and i.g.).Next, the inhibitory effects of Styrax against mammalian CYP3A were validated by using a substrate-drug (midazolam) in different tissue preparations. To this end, the inhibitory effects of Styrax against CYP3A-catalyzed midazolam 1′-hydroxylation were carefully investigated in different enzyme sources including HLMs, HIMs, RLMs, and RIMs. As shown in in vivo effects of Styrax on the pharmacokinetics of midazolam (i.v. and i.g.) were carefully investigated in rats, by using soybean oil (without Styrax) as control groups. The blood concentration-time profiles of midazolam with two administrated ways  were shown in (0-inf) of midazolam (i.g.) was increased 2.04-fold (from 211.23 ng/mL·h to 431.20 ng/mL·h), while the Cmax value of midazolam (i.g.) in rats plasma was increased 2.28-fold (from 129.16 ng/ml to 294.05 ng/ml). Moreover, the metabolic half-life (t1/2) of midazolam (i.g.) was prolonged 2.11-fold (from 2.14 to 4.52 h). These findings clearly suggested that Styrax strongly elevated the plasma exposure of midazolam when midazolam was co-administered orally. Considering that both intestinal CYP3A and hepatic CYP3A participate in midazolam metabolism when this agent is administrated orally, while Styrax (i.g.) cannot influence hepatic metabolism of midazolam, suggesting that Styrax may modulate the in vivo pharmacokinetic behavior of midazolam (i.g.) via inhibiting intestinal CYP3A but not hepatic CYP3A.Next, the via two administrated ways (i.v. and i.g.). As shown in (0-inf)  and Cmax  of felodipine when felodipine was administered orally in combination with Styrax (i.g.) in rats. These findings further suggest that Styrax can modulate the pharmacokinetic behavior of orally administrated CYP3A-substrate drugs (midazolam and felodipine) by inhibiting intestinal CYP3A but not hepatic CYP3A in vivo.To further confirm that Styrax could influence the pharmacokinetic behavior of orally administrated agent, felodipine  was used as a victim drug to assess the pharmacokinetic interactions between Styrax  and felodipine Next, the chemical constituents in Styrax were analyzed by using UHPLC-Q-Exactive Orbitrap HRMS. The total ion chromatograms (TICs) of Styrax were shown in 50 values of 19.39, 32.20, 14.83, 7.74, 0.64, 2.51, and 0.99 μM, for maslinic acid, corosolic acid, oleanolic acid, betulinic acid, epibetulinic acid, betulonic acid and oleanonic acid, respectively. Meanwhile, all tested PTAs dose-dependently inhibited CYP3A4-catalyzed NEN 4-hydroxylation, with the IC50 values of 3.44, 41.93, 1.10, 4.11, 0.47, 0.96, and 0.80 μM, for maslinic acid, corosolic acid, oleanolic acid, betulinic acid, epibetulinic acid, betulonic acid and oleanonic acid, respectively. These results suggest that the PTAs in Styrax are key substances responsible for CYP3A inhibition, while epibetulinic acid, betulonic acid and oleanonic acid display potent CYP3A inhibitory effects.A previous study revealed that the PTAs in Styrax displayed potent inhibition on CYP3A-catalyzed testosterone 6β-hydroxylation . In this50 value of oleanonic acid against CYP3A4. These results suggested that the major PTAs in Styrax could be distributed in intestinal tract at relatively high exposure levels but their plasma and liver exposure in rats was extremely low. These findings well explain why Styrax (i.g.) significantly elevates the plasma exposure of CYP3A-substrate drugs only when these agents are orally administrated, and why Styrax cannot modulate the pharmacokinetic behavior of CYP3A-substrate drugs when these agents administered intravenously.Next, the tissue distribution  of seven PTAs from Styrax was determined in rats after single oral dose of Styrax (100 mg/kg). As shown in via acting on various therapeutic targets or prolonging the half-lives of active compounds in either Chinese medicines or Western medicines  and molecular mechanisms involved in the pharmacokinetic HDIs triggered by Styrax.As one of the most abundant drug-metabolizing enzymes (DMEs) in human liver and intestine, CYP3A4 plays a crucial role in the metabolic clearance and detoxification of a wide range of Western medicines and DDIs . In thisand DDIs . It is wand DDIs . In thesin vitro assays demonstrated that Styrax and its major constituents (seven PTAs) potently inhibited CYP3A-catalyzed oxidative metabolism of various substrates. In vivo assays showed that Styrax only affected the pharmacokinetic behavior of oral victim CYP3A-substrate drugs (midazolam and felodipine), which prompted us to carefully evaluate the tissue distribution of Styrax/its active constituents (seven PTAs) after oral administration of this herbal extract in rats. These results clearly showed that seven PTAs from Styrax could be exposed to the intestinal tract  at relatively high levels in rats after single oral dose of Styrax (100 mg/kg), but these PTAs were hardly exposed to the liver. The extremely low exposure of these PTAs from Styrax to mammal liver could be attributed to the poor oral bioavailability and the first-pass metabolism of PTAs  and paxlovid (nirmatrelvir and ritonavir) (in vivo. Considering that hepatic CYP3A plays an important role in endogenous metabolism (such as bile acids and hormones) and the metabolic clearance of toxins, potent inhibition of hepatic CYP3A may lead to serious consequences  (It should be noted that some CYP3A4 potent inhibitors (such as ritonavir) have been approved by FDA in combination with CYP3A4-substrate drugs to slow down the metabolic clearance of some important CYP3A4-substrate drugs tonavir) . Thus, tabolism) . In thisin vitro and to accurately assess the potential HDIs risk in vivo. With the help of fluorescence-based high-throughput hCYP3A4 inhibition assay, the Chinese herb Styrax was found with the most potent hCYP3A4 inhibition in HLMs. In vivo pharmacokinetic assays demonstrated that Styrax  only affected the pharmacokinetic behavior of orally administrated CYP3A-substrate drugs (midazolam and felodipine). Further investigations demonstrated that seven PTAs in Styrax were key substances responsible for CYP3A inhibition, while these PTAs could be exposed to intestinal tract at relatively high exposure levels but the exposure levels of these PTAs in rat plasma and liver were extremely low. Collectively, our findings showed that the Chinese herb Styrax (i.g.) could significantly elevate the plasma exposure of orally administrated CYP3A-substrate drugs but hardly modulated the pharmacokinetic profiles of intravenously administrated CYP3A-substrate drugs, which could be attributed to the high exposure levels of the PTAs in Styrax in intestinal tract and the extremely low exposure levels in rat liver. All these findings clearly suggest that the tissue exposure of some key constituents in herbal medicines strongly affect their in vivo inhibitory effects on CYP3A4, which are very helpful for the clinical pharmacologists to better understand the pharmacokinetic HDIs triggered by herbal products that contains naturally occurring CYP3A4 inhibitors.In summary, we demonstrate a case study to efficiently find the herbal medicine(s) with potent hCYP3A4 inhibitory effect


ABCC2, HNF4α, SLCO1A2 and SLC22A6  were measured. Bi- and multivariate analyses were performed for single ARV and classes. We included 259 participants providing 405 paired plasma and CSF samples. CSF/plasma ratios (CPR) showed an increase for NRTIs and nevirapine with low penetrations for the majority of ARVs. At bi-variate analysis, several associations, including the effect of BBBi , age (zidovudine and darunavir), and high CSF neopterin (NRTIs and border-line for PIs) were suggested. An association was found between genetic variants and integrase strand transfer (ABCC2 and HNF4α), non-nucleoside reverse transcriptase inhibitors (SLCO1A2), and protease inhibitors (SLC22A6). At multivariate analysis age, gender, BMI, and altered BBB were independent predictors of nucleoside reverse transcriptase CSF concentrations; age (for protease inhibitors) and body mass index and altered BBB (integrase strand transfer inhibitors) were also associated with ARV CSF exposure. We describe factors associated with CSF concentrations, showing that demographic, BBB integrity and, partially, genetic factors may be predictors of drug passage in the central nervous system.Neurocognitive impairments are common in people living with HIV. Some conditions, such as chronic inflammation, astrocyte infection and an impaired blood–brain barrier (BBBi), along with host genetic variants in transporter genes, may affect antiretroviral (ARV) exposure in the cerebrospinal fluid (CSF). The aim of this study was to evaluate ARV CSF penetration according to compartmental inflammation, BBB permeability and single-nucleotide polymorphisms (SNPs) in drug transporter encoding genes. CSF neopterin (ELISA), plasma and CSF ARV concentrations (HPLC) and host genetic variants in  The most recently available UNAIDS data show that approximately 38 million people were living with HIV (PLWH) in 2019 .Although it is a very complex and multifactorial disease, several risk factors have been identified, including sustained immune activation that causes  structural damage and enhanced permeability of the blood–brain barrier (BBB), poor ART penetration within the central nervous system (CNS) with potential low-level residual viral replication, and a selection of drug resistance, antiretroviral neurotoxicity, patient comorbidities (including vascular disorders), and host genetics ,4,5. OthMeasuring drug exposure in the central nervous system is highly complex: most of the studies included experimental animals or ex vivo measuring. In vivo techniques using radiological and nuclear assays are ongoing, but so far, cerebrospinal fluid (CSF) concentrations have been used as a proxy of brain tissue exposure. Although recent data in preclinical species suggest that CSF underestimates ART brain tissue concentrations, it may provide helpful insights into the determinants of this process .Few data are available in the literature concerning inflammation, genetic, and BBB-related factors affecting antiretroviral CSF and plasma concentrations. For these reasons, the aim of this study was to measure and characterize ART CSF penetration according to several variables including compartmental inflammation, BBB permeability and single-nucleotide polymorphisms (SNPs) in drug transporter encoding genes.PLWH receiving antiretroviral therapy enrolled in the PRODIN study  were included. The study was a prospective study that included HIV-positive patients undergoing lumbar puncture for clinical reasons: demographic, clinical, therapeutic variables were collected and serum, genetic and CSF biomarkers were measured. All participants signed a written informed consent form.Blood samples were collected in lithium-heparin tubes, centrifuged at 3000 rpm for 10 min at 4 °C to obtain plasma, and then stored at −20 °C; CSF was stored at −20 °C.Albumin levels were measured in serum and CSF through Immunoturbidimetric methods . CSAR, intended as a ratio between CSF albumin (mg/L)/serum albumin (g/L), was employed to evaluate BBB function: the definition of BBB damage was derived from age-adjusted Reibergrams   and cobicistat  . All plaABCB1 (encoding the P-glycoprotein) 3435 C > T (rs1045642), ABCB1 1236 C > T (rs1128503), ABCB1 2677 G > T (rs2032582), ABCC2 (encoding the multidrug resistance protein 2)-24 G > A (rs717620), ABCG2 (encoding the ATP binding cassette subfamily G member 2) 421 C > A (rs2231142), ABCG2 1194 + 928 C > A (rs13120400), HNF4a (encoding the hepatocyte nuclear factor 4 a) 975 C > G (rs1884613), SLCO1A2 (encoding the solute carrier organic anion transporter family member 1A2) 38 A > G (rs10841795), SLCO1A2 516 A > C (rs11568563) and SLC22A6 (encoding the solute carrier family 22 member 6) 453 G > A (rs4149170).Genomic DNA was isolated from blood samples . Genotypes were assessed through a real-time PCR allelic discrimination system . Investigated gene SNPs were Patients’ demographic and clinical data have been collected and described as follows: categorical variables were described as frequency and percentage, while numerical ones as the median and interquartile (IQR) range.p value < 0.05). Non-parametric tests of Kruskal–Wallis (for more than two groups) or Mann–Whitney (for two groups) have been used in order to identify differences in continuous variables among different groups of patients, due to the non-normal distribution of data.Statistical analyses have been focused on investigating the strength of the association between CSF and plasma drug concentrations by calculating the Spearman coefficient and considering the level of statistical significance .n = 185; 71.4%) with European ancestry  based on self-reporting (no region level was available); the median age was 48 years (IQR—inter quartile range 42–55) and median BMI was 22.6 kg/m2 (IQR 20–25.1). The number of prescribed drugs ranged from 1 to 5, with the large majority of patients (n = 180) taking three antiretrovirals. The median CD4 and nadir CD4 cell count were, respectively, 336 (IQR 147–590) and 99 (IQR 25–211) cells/µL. Among the study participants, 47.5% and 56.4% showed plasma and CSF viral loads lower than 50 copies/mL. Markers indicating an altered BBB were found in 19.7% of participants (n = 51).We included 259 participants. The majority of patients were male  between plasma and CSF concentrations , but theThe CSF/plasma ratio (CPR) was calculated for each drug and expressed as a percentage: ABC and AZT showed the highest CSF-to-plasma ratios (CPR) , followed by 3TC, FTC and NVP . All the other drugs showed very low CPRs .p = 0.006) and RAL . Inflammation seemed to have an impact on drug concentrations: NRTIs (p = 0.008); in particular, TDF (p = 0.038) and FTC (p < 0.001) showed higher CSF concentrations in patients with altered neopterin levels . Age was directly associated with CSF NRTI exposure  with AZT  and 3TC  being higher in the CSF of elderly participants; however, DRV , ATV and RAL  also showed a similar trend. Only for PIs, the CPR results were slightly associated with age .We then assessed the effect of several variables on CSF concentrations and CPRs using bivariate and multivariate analysis. In participants with impaired BBB, the following drugs showed significantly higher CSF concentrations: FTC  genes that encode for MRP2 and HNF4α, two proteins involved in drug transport and regulation, respectively.Pharmacogenetic analyses  showed tABCC2 -24 AA carriers showed significantly lower (p = 0.017) INSTI CSF concentrations, whereas HNF4α 975 CC genotype carriers had significantly higher (p = 0.005) INSTI’s CPR. NNRTI concentrations were affected by SLCO1A2 polymorphisms: SLCO1A2 38 AA carriers showed significantly higher (p = 0.021) NNRTI CSF concentrations. Finally, PI’s CPR could have been influenced by the SLC22A6 453 variant: genotype GG carriers had significantly lower (p = 0.047) PI CPRs. Considering single drugs, genetic polymorphisms’ influence was analyzed . This confirms what was found by Ene et al., who suggested that NRTIs as a class have the advantage of good CSF concentration . A studyConcerning inflammation, NRTI CSF exposure was major in individuals with neuroinflammation, intended as neopterin levels higher than 1.5 ng/mL. Probably, this could be due to tight junction disruption caused by neuroinflammation ,22, and In particular, inflammatory mediators, through transcriptional and post-transcriptional mechanisms, lead to enzyme and transporter inhibition. Consequently, inflammation has an effect on pharmacokinetic parameters of most of the drugs which are metabolized and transported by these proteins and for which TDM is recommended, in order to improve the personalization of drug treatment for each patient .Age was also associated with an increase in CSF NRTI concentrations, and DRV, RAL and ATV also showed a similar trend, but without statistical significance; this could be attributable to physiological ageing, leading to a more permeable BBB. Advancing age is characterized by different organ impairment: in detail, a reduction in renal and hepatic function could lead to pharmacokinetic changes, including an increase in the volume of distribution of lipid soluble drugs, thus prolongating the elimination half-life; on the other hand, pharmacodynamic changes could involve altered (often increased) sensitivity to several classes of drugs .Reduced homeostatic ability affects different regulatory systems in different subjects, thus explaining, at least partly, the increased interindividual variability occurring as people get older. Important pharmacokinetic and pharmacodynamic changes occur with advancing age. This is one of the few studies showing an effect of host genetics on CSF penetrations of ARV .ABCC2 24 C > T affects ABCC2 transcriptional regulation [HNF4α 975 C > G genetic variant is present upstream of the HNF4α promoter gene, and it has no known function [HNF4α 975 CC genotype correlated with increased EFV exposure [SLC1A2 38 T > C genetic polymorphism in exon 1 causes an isoleucine to threonine amino acid change, increasing the uptake of estrone sulfate and methotrexate [SLC22A6 453 G > A, RAL concentrations resulted in not being influenced by this genetic variant [gulation : particugulation . Studiesgulation . In addigulation . In thisfunction ,31. Receexposure . Moreoveexposure . SLC1A2 otrexate . In our  variant ; moreoveThis study investigated the role of different factors influencing CSF and plasma ARV concentrations.In conclusion, considering the complexity of drug-related side effects or viral resistances, it could be beneficial to use an integrated approach, combining both clinical screening, TDM and pharmacogenetic analyses; this combined method could be useful to improve tailored medicine in order to better manage these kinds of patients.


This review highlights the key role of fatty acid desaturases in the synthesis of naturally occurring, more common and not unsaturated fatty acids. The three major classes of fatty acid desaturases, such as acyl‐lipid, acyl‐acyl carrier protein and acyl‐coenzyme A, are described in detail, with particular attention to the cellular localisation, the structure, the substrate and product specificity and the expression and regulation of desaturase genes. The review also gives an insight into the biocatalytic reaction of fatty acid desaturation by covering the general and more class‐specific mechanistic studies around the synthesis of unsaturated fatty acids Finally, we conclude the review by looking at the numerous novel applications for desaturases in order to meet the very high demand for polyunsaturated fatty acids, taking into account the opportunity for the development of new, more efficient, easily reproducible, sustainable bioengineering advances in the field. FAs are a source of a large variety of lipid signalling molecules and they are involved in numerous biological pathways such as cell adaptation and survival, modulation of ion channels, endocytosis and exocytosis, pathogen defence and cell signalling.2FA desaturases are enzymes able to perform dehydrogenation reactions by converting a single bond between two carbon atoms (C—C) to a double bond (C=C) on the acyl chain of a FA molecule in a stereospecific manner.cis‐UFAs, whose configuration confers higher fluidity by forming kinks within the FA chain, compared to the trans‐UFAs, which instead give a compact and rigid structure.The biochemical relevance of the reactions of FAs desaturation is linked to the fundamental role in the regulation of lipid membrane homeostasis. Desaturases are able to insert the appropriate number of double bonds in the acyl chains of FA molecules, according to the level of fluidity required in the membrane for processes such as cell adaptation, activation of various biochemical mechanisms and membrane‐bound protein functions. The control over the lipid bilayer fluidity is particularly determined by the naturally higher level of b5‐like system, sometimes also used by some acyl‐lipid desaturases.These three classes can be distinguished in terms of electron carrier system used, solubility and substrate specificity. Thus, in terms of the oxidation reaction, acyl‐lipid and acyl‐ACP desaturases have been shown to use ferredoxin as electron donors, compared to acyl‐CoA desaturases, which utilise the participation of a cytochrome 3Euglena gracilis.cis configuration, between the C‐9 and the C‐10 of the FA acyl chain forming a ∆9‐oleate 18:1 UFA.Ricinus communisCoriandrum sativum, Pelargonium hortorum, Thunbergia alata.R. communis has revealed a wide range of important information about the enzyme structure and insights into the binding modes and biocatalytic reaction, which have not been fully exploited yet.Acyl‐ACP enzymes are a class of soluble desaturases, which have been found exclusively in higher plants' plastids, where the de novo biosynthesis of PUFAs takes place Figure .17 The2+‐Fe2+), in a symmetrical manner: one of the iron centres coordinates with a glutamate residue (Glu105) in a bidentate fashion, and with an N atom, belonging to a histidine residue (His146). .9,12‐18:2, Δ6,9,12‐18:3 and Δ6,9,12,15‐18:4.Synechocystis, Synechococcus, Anabaena variabilis.Arabidopsis thaliana.B. cereus acyl‐lipid desaturase, shows the presence of four transmembrane domains (TM 1‐4), two peripheral helices (P1‐2), cytosolic N‐ and C‐ termini and highly conserved histidine boxes.Acyl‐lipid desaturases are a class of membrane‐bound desaturases, which are found in the membranes of the endoplasmic reticulum in higher plants and cyanobacteria Figure , where tA key tyrosine residue (Tyr40) in the first transmembrane domain was found to be fundamental for the desaturases to anchor and accurately orientate in the lipid bilayer.59‐18:1‐ACP, and incorporated into the glycerolipids of the chloroplast and endoplasmic reticulum membranes, acyl‐lipid desaturases are able to insert a second and a third double bond, synthesising the ω‐6‐18:2 and ω‐3‐18:3.9‐18:1, by using ∆9‐acyl‐CoA desaturases, but they lack ω‐3 and ∆12‐desaturases for the biosynthesis of EPUFAs.Acyl‐CoA desaturases are integral membrane proteins found in both eukaryotes and prokaryotes Figure . They arcis configuration.Substrate recognition seems to be determined by hydrophobic and electrostatic interaction with polar amino acid residues.6syn dehydrogenation, giving an olefinic product and an iron‐bound molecule of water. , UFAs with a double bond in a trans configuration, or characterised by the presence of functional groups such as hydroxyl, epoxy or cyclopropyl, halogen or acetylenic groups on their acyl chain.Vernonia galamensis.Calendula officinalis, Momordica charantia and Vernicia fordii.cis‐ethylenic double bond, have been described in plants, animals and primitive low organisms, such as taxoleic acid , pinolenic acid , sciadonic acid  and juniperonic acid .A. leveillei and defined as Acyl‐CoA‐dependent desaturases class, because of their structural similarity with ∆9‐stearoyl‐CoA‐desaturase.b5 fusion ω3‐desaturase has been discovered in Chlamydomonas reinhardtii, which is able to produce C20 NMI‐PUFAs, instead of the more typical methylene interrupted arachidonic acid.The desaturases that have been described so far are responsible for the biosynthesis of commonly occurring UFAs, which are methylene‐interrupted FAs with double bonds in the 8Medicago truncatula genes for FAD3 (fatty acid desaturase) and FAD7 desaturases where an increase in the gene encoding for these desaturases was detected when the plants were grown at a lower temperature.Arachis hypogaea, the exposure to a high concentration of NaCl has resulted in a reduced expression of the genes encoding for ω3‐desaturases, and consequently, a lower level of α‐linolenic acid was also detected.A. thaliana, post‐translational modifications have also been identified to have an effect on the biosynthesis of UFAs. It has been shown that when either the 5′‐UTR or N‐terminal regions of the desaturase gene sequences were enhanced in the number of copies, the expression of the desaturases would consequently increase.A. thaliana, the increase of the temperature can activate the opposite mechanism on the C‐terminal of the desaturase gene sequence, with a consequent reduction of production of UFAs.A. thaliana has shown that the gene expression of plants desaturases may also be regulated according to concentration and tissue‐specificity of exposure to chemicals and phytopheromones and consequently suggesting that desaturases are involved in plant signal transduction for important mechanisms of cell adaptation.S. cerevisiae has led to reduced activity of stearoyl‐CoA desaturases and that the same feedback regulation for gene expression has been also found in animals.B. subtilis.Microorganisms, plants and animals are able to finely regulate the biosynthesis of UFAs in response to chemical and physical changes within the cell and from the external environment.Through this description, it is clear how biosynthesis of UFAs catalysed by desaturases is a very complex and finely regulated process. This accounts for a coordinated system of feedback regulation of transcription in all organisms, that allows for the maintenance of cell membrane homeostasis and therefore biological functions.9Mortierella alpina; S. cerevisiae and E. coli have been used as heterologous systems to express and characterise S. putrefaciens desaturases for EPA synthesis,I. galbana,T. aureum and M. alpina,H. polymorpha.I. galbana, ∆8 from E. gracilis, ∆5 desaturases from M. alpina and more genes encoding ω‐3 desaturases were cloned into A. thaliana to increase the yield of PUFA products.P. vialii and ∆6 and ∆12 desaturases from M. alpina.C. elegans ω‐3 desaturases, have been studied and used for clinical treatments of colon and breast cancer, skin damage, reproduction, intestine diseases and neuronal apoptosis.The broader knowledge around the structure–activity relationship for FA desaturases, the mode of action, the substrate specificity and selectivity and the variability of products constitute a solid base to develop new biotechnological advances in the synthesis of UFAs.10The biosynthesis of UFAs is an essential pathway from prokaryotes to eukaryotes, from microorganisms to plants and animals: it is the way in which all organisms are able to control the lipid membrane homeostasis and a numerous series of biological cell functions. In order to maintain a balance in the pool of UFAs in the cell, a large variety of desaturases are involved in catalysing the insertion of a certain number of double bonds on a FA molecule in a stereospecific and selective manner. Desaturases have been shown to be a remarkable group of enzymes for their structure–function binomial relationship, to display their catalytic activity to synthesise their products, with chemical structure diversity, from the most common UFAs to unusual FAs. The expression of these enzymes is finely modulated by feedback control of transcriptional regulation of their genes from environmental factors. This structural, mechanistic and biochemical information have been gathered after decades of investigation around FA desaturases, which have led to a broader understanding of the importance and functions of the enzymes and their products. Thus, cloning and identification of a large variety of desaturases in a broad number of different organisms have given insights into the PUFA biosynthetic pathway and their importance in many physiological processes. Therefore, it has opened a modern path for lipidomic research to try and achieve one of the biggest tasks in the field: finding alternative sources of PUFAs to meet the high demand for this essential biomolecule in pharmaceutical, nutraceutical and food industries. The challenge has already started with the use of biotechnological tools to develop promising PUFA cell bio‐factories. The process is still open to a large number of improvements that can be achieved in the near future by always bringing new knowledge in the field around the structures and mechanism of desaturase, and by looking at the development of new, more efficient, easily reproducible, sustainable bioengineering advances.ACPacyl carrier protein, 1CoAcoenzyme A, 1EPUFAessential polyunsaturated fatty acid, 3FAD 3fatty acid desaturase 3, 21FAsFatty acids, 3NMI‐PUFAsnon‐methylene‐interrupted PUFAs, 20PUFAspolyunsaturated fatty acids, 1RIPpathway of RNA polymerase I, 23SCAPsterol cleavage activation protein, 23SREBPsterol regulatory element‐binding pathway, 23UFAsunsaturated fatty acids, 1VLC‐PUFAvery long‐chain polyunsaturated fatty acids, 3


A recombinant strain  was constructed using an electrotransformed complementary plasmid. Expression of the alr-VP4 fusion protein from pPG-alr-VP4/Δalr W56 was detected using Western blotting. Mice orally immunized with pPG-alr-VP4/Δalr W56 exhibited high levels of serum IgG and mucosal secretory immunoglobulin A (SIgA), which exhibited neutralizing effects against PoRV. Cytokines levels in serum detected using ELISA, indicated that the recombinant strain induced an immune response dominated by Th2 cells. Our data suggest that pPG-alr-VP4/Δalr W56, an antibiotic-resistance-free LAB, provides a safer vaccine strategy against PoRV infection.Porcine rotavirus (PoRV) mainly causes acute diarrhea in piglets under eight weeks of age and has potentially high morbidity and mortality rates. As vaccine carriers for oral immunization, lactic acid bacteria (LAB) are an ideal strategy for blocking PoRV infections. However, the difficulty in knocking out specific genes, inserting foreign genes, and the residues of antibiotic selection markers are major challenges for the oral vaccination of LAB. In this study, the target gene, alanine racemase ( Porcine rotavirus (PoRV) causes diarrhea in piglets, leading to substantial economic losses in pig farming . The lesSalmonella, Escherichia coli, and vaccinia virus, have been used to express and transmit foreign antigens in live vectors. Among these live vectors of oral vaccines, it was a major challenge that the antigen inserted into the live vector was potentially denatured and inactivated, leading to fever and diarrhea in the gut milieu [There is no effective treatment for PoRV infection, and vaccination is the primary method of control. Currently, PoRV commercial vaccines, such as attenuated  and subut milieu ,12,13.alr gene encoding alanine racemase food-grade selective marker has been widely used in biological genetic engineering [alr gene can catalyze the transformation of L-Alanine to D-Alanine, and D-Alanine is an important element in the bacterial cell wall biosynthesis of peptidoglycan. Because the alr gene is stably expressed in the genome of LAB without causing drug resistance, it can be used as a candidate marker for auxotrophy in food-grade LAB. Upon knockout of the genomic alr gene, alr-deficient LAB can grow normally only when D-Alanine is added externally or transferred into plasmids expressing alr-deficient complementary genes [Our previous studies suggest that lactic acid bacteria (LAB) are promising carriers for expressing and delivering exogenous antigens in oral vaccines ,15,16. Cineering ,21. The ry genes .alr) deficient LAB using the CRISPR-Cas9D10A gene editing system and an alr gene complementary expression plasmid to insert the gene of the major protective antigen VP4 of PoRV. Finally, we obtained a genetically engineered pPG-alr-VP4/Δalr W56 strain that expressed the alr protein and VP4 protective antigens of PoRV. To provide a reference for developing an effective vaccine against PoRV, the immune effect of the recombinant strain was evaluated by oral immunization of the mice.In this study, we constructed alanine racemase .The boratory  used in boratory . Epithelalr-HASup/HASdown) targeting alr sites were designed , and fixed overnight at 4 °C. After washing three times with 0.1 mol/L, pH = 7.2 phosphate buffer, the bacterial pellet was dehydrated by adding 50%, 70% and 90% ethanol solutions at 4 °C for 10 min. The bacterial pellet was harvested, dehydrated with 100% ethanol, and subjected to a displacement reaction. The obtained samples were first frozen and then dried and placed in a scanning electron microscope for observation after being coated.The L. casei W56 and L. casei Δalr W56 were adjusted to about 1 × 108 CFU/mL (OD600 = 1.0 ± 0.02) and inoculated into MRS liquid medium at a ratio of 1:100. The two strains were cultured at pH = 5.85 , 2.0, 3.0 and 4.5, respectively, at 37 °C for 3 h. After doubling dilution with PBS, they were spread on MRS solid agar plates. The survival rate of the two strains was calculated after culturing at 37 °C for 24 h; 0.1% and 0.3% pig bile salts were added to L. casei W56 and L. casei Δalr W56 culture medium treated in the same way as above, and cultured at 37 °C for 3 h. After doubling the dilution with PBS, they were spread on MRS solid agar plates. The survival rate of the two strains was calculated after culturing at 37 °C for 24 h.The viable counts of L. casei W56 and L. casei Δalr W56 single colonies were inoculated into different biochemical reaction tubes. After culturing at 32 °C for 10 h, the results were judged according to Bergey’s Manual of Systermatic Bacteriology.alr gene was amplified with His-Tag, SacI, and KpnI restriction endonuclease sites, and the VP4 gene was amplified with FLAG tags, KpnI, and ApaI restriction endonuclease sites. Then the above two gene segments were co-connected to the E. coli-LAB shuttle vector pPG-T7g10-PPT to obtain the complementary plasmid pPG-alr-VP4  at a dilution of 1:2000 as the primary antibody and HRP-conjugated goat anti-mouse IgG antibody at a dilution of 1:5000 (Sigma) as the secondary antibody. The expression of target protein bands was visualized using a chemiluminescence imaging system with ECL Plus Western blotting detection reagents .To determine whether the alr-VP4 protein was expressed and assess its stability, the bacterial strains L. casei Δalr W56, pPG-alr-VP4/Δalr W56, and pPG/Δalr W56 were activated by streaking. Single colonies were picked and inoculated into MRS liquid medium with or without 10 µg/mL chloramphenicol and 200 µg/mL D-Alanine and cultured at 37 °C until the OD600 was approximately 1.0. The bacteria were washed once with PBS. After centrifugation, the bacteria were resuspended in PBS, and the concentration was adjusted to 1010 CFU/mL. The three groups mentioned above of bacterial liquid and PBS were orally inoculated into mice each time for three consecutive days, once every two weeks, for a total of three times. The number of mice and the immunization doses in the immunization program were shown in Four-week-old female BALB/c mice were obtained from Liaoning Changsheng Biotechnology Co., Ltd. . Strains 2-PBS (50 mmol/L), and the supernatant was collected after centrifugation (dilution not required). Mouse serum was diluted 10-fold to detect IgG antibodies and cytokine levels . All samples were analyzed using the ELISA commercial kit .Genital tract mucus, intestinal mucus, nasal fluid, and fecal extracts were treated as previously described to detect SIgA antibodies . In brie450.Polystyrene microtiter plates were coated with PoRV as an antigen or PBS as a negative control and incubated overnight at 4 °C (0.1 mL per well). After blocking for two hours with 5% skim milk at 37 °C, the samples were added in triplicate and co-incubated with the antigen for one hour at 37 °C. HRP-conjugated goat anti-mouse IgG or IgA antibody at a dilution of 1:5000 (Sigma) was incubated for one hour at 37 °C. After washing, the color was developed using tetramethylbenzidine (Sigma), and the absorbance was measured at OD2 TCID50/mL)) was added to each culture well and co-incubated with the diluted samples for one hour at 37 °C. MA104 cells were seeded in 96-well culture plates and cultured at 37 °C for 18 h before infection. The cells were then infected with the coincubation mixture for two hours at 37 °C. The supernatant was removed, 0.1 mL of cell maintenance fluid was added to each well, and the TCID50 values were determined after incubation at 37 °C for 36 h. Neutralization titers were expressed as the reciprocal of the highest dilution of serum that showed at least a 50% reduction in the number of infected cells as compared with the negative control serum.Mouse serum and intestinal mucus were collected on day 42 and sterilized by filtration to detect IgG and SIgA, respectively. After inactivation at 56 °C for 30 min, all samples were serially diluted two-fold to 1:256, and 0.1 mL of the diluted samples were added to 96-well cell culture plates. PoRV , p < 0.05 indicated statistical significance (*), p < 0.01 indicated high significance (**), p < 0.001 indicated extremely high significance (***).Calculations were performed using the GraphPad Prism software . All data are expressed as the mean ± standard deviation (x ± s). All animal experiments were approved by the Ethical Committee for Animal Experimentation of the Northeast Agricultural University, Harbin, China.alr gene was knocked out in the L. casei W56 genome using the CRISPR-Cas9D10A nickase-based plasmid pLCNICK-alr  . These results indicated that the use of CRISPR-Cas9D10A nickase-based plasmid pLCNICK-alr could be capable of mediating rapid and efficient chromosomal deletions of the alr site.The NICK-alr A to obtaR primer  showed t2763 bp) B, and DNΔalr W56 was constructed via the electrotransformation of the complementary plasmid pPG-alr-VP4  was serially inoculated 50 times to evaluate its stability. The 10th, 20th, 30th, 40th, and 50th generations of recombinant strain plasmids were extracted, and the proteins were lysed. The inherited stability was tested using PCR and Western blotting. All recombinant strains amplified the alr-VP4 sequence 1968 bp , we collected samples from the mice at different time points after oral immunization. A schematic of the oral immunization program and sampling schedule is shown in Δalr W56 to elicit mucosal and systemic immunity was evaluated by determining the specific IgG and SIgA antibodies, respectively. No significant changes were observed in serum levels of anti-PoRV-specific IgG and SIgA antibodies before immunization. The results demonstrated that the SIgA antibody levels in the genital tract fluid  expressing the PoRV-VP4 antigen effectively elicited an anti-PoRV local and systemic immune response in vivo.Furthermore, anti-PoRV IgG antibody levels in mouse serum increased on the seventh day after the first oral immunization with pPG-alr-VP4/Δalr W56 F. The IgΔalr W56 was 1:51.4, which was more robust than that in mice orally administered PBS (1:2), L. casei Δalr W56 (1:2) and pPG/Δalr W56 (1:2). The intestinal mucosa SIgA antibody in mice that received pPG-alr-VP4/Δalr W56 also possessed stronger anti-PoRV neutralizing activity (1:22.9) than in mice orally administered PBS (1:2), L. casei Δalr W56 (1:2), and pPG/Δalr W56 (1:2). This result proves that the recombinant strain pPG-alr-VP4/Δalr W56 can induce effective neutralizing antibodies in mice under the immunization program in this study, and reveals that the auxotrophic Lactobacillus Δalr W56 are feasibile as antigen delivery carriers to construct oral immunization vaccines.Based on the detection results of specific IgG and SIgA antibody titers against PoRV collected at different time points, we detected the neutralizing activity of the serum IgG antibody and intestinal mucosa SIgA antibody on day 42 after the initial immunization. As shown in Δalr W56 in mice after oral immunization and found that higher levels of the Th1-associated cytokine IFN-γ and Th2-associated cytokine IL-4 were induced compared to those in the control groups   A. In ordl groups B. Moreovl groups C. These L. casei is an ideal candidate for use in oral vaccines [L. casei strain expressing the PoRV VP4 gene and LTB mucosal adjuvant. Oral immunization of mice significantly induced the production of serum IgG and mucosal SIgA antibodies, which had a significant neutralization effect against PoRV infection. Yin et al. constructed recombinant LAB without antibiotic selection markers and constitutively stably expressed the PoRV-neutralizing epitope VP4 protein, which immunized mice and also showed a good immune protection effect [L. casei can maintain the balance of intestinal flora and promote the digestion and absorption of intestinal nutrients as well as enhance immune regulation [PoRVs are the primary cause of acute diarrhea in piglets, leading to substantial economic losses in pig farming. However, there is currently no effective treatment for the inhibition of PoRV infection . Vaccinavaccines ,28,30. Qvaccines  construcn effect . In summgulation ,34,35,36L. casei as the vector for the oral vaccine in this study. Developing a food-grade selection marker without antibiotic resistance genes in food-grade microorganisms is vital for constructing genetically modified LAB products that meet biosafety standards. The construction of a food-grade nutritionally-deficient screening marker has an effective application in a non-resistance screening system [Lactobacillus reuteri. Guo et al. [L. casei but also demonstrated that the Cas9D10A-Nickase system is an effective tool to overcome DSB-induced bacterial lethality.Overcoming drug resistance caused by resistance genes and manual control of the screening of recombinant strains were crucial for selecting g system . Howeverg system ,40,41,42g system  used Caso et al.  combinedo et al. ,46. To oL. casei W56, thereby knocking out the alr gene, and the auxotrophic strain L. casei Δalr W56 can be stably passaged without mutation reversion. Simultaneously, we constructed a LAB expression plasmid, pPG-alr-VP4, complementary to L. casei Δalr W56. We verified that L. casei Δalr W56 could resume normal growth by exogenous supplementation with the alr-missing gene, indicating that the host bacteria and plasmids achieve functional complementarity. We also explored the expression of the PoRV protective antigen VP4 using the complementary plasmid. The results showed that the recombinant L. casei pPG-alr-VP4/Δalr W56 expressing VP4 protein can induce mice to produce effective mucosal and systemic immune responses against PoRV. This indicates that this gene editing system can effectively carry out precise editing of the Lactobacillus genome, and the complementary plasmids can also achieve the immune function of recombinant strains by expressing foreign antigens.In this study, we used the CRISPR-Cas9D10A gene editing system to cut the single-stranded genomic DNA of Δalr W56 can effectively induce specific serum IgG and mucosal SIgA antibodies and have neutralizing activity against PoRV. The SIgA-mediated protection of mucosal immunity in small intestinal epithelial cells plays an essential role in preventing PoRV infection [As PoRV mainly infects small intestinal epithelial cells after passing through the mucosal layer, the mucosal immune system is important in the prevention and control of PoRV infection. Our results indicated that oral administration of pPG-alr-VP4/nfection . Serum Infection . Generalnfection ,49,50. IΔalr W56 strain could colonize the intestine, and then stimulate the intestinal mucosal immune system to produce specific SIgA. When the sensitized lymphocytes of the local mucosa enter the blood circulation, they will gradually differentiate and mature. Under the mediation of different receptors, they can reach other mucosal sites and induce the special SIgA for activation of the immune system, this phenomenon is known as the common mucosal immune system (CMIS) [Δalr W56 can provide effective immunity against PoRV infection by inducing high levels of mucosal SIgA and serum IgG antibodies.In this study, after oral immunization in mice, the pPG-alr-VP4/m (CMIS) ,52. Thusm (CMIS) . TherefoΔalr W56, cytokine levels were determined after the oral administration of the vaccine. IFN-γ and IL-2 are Th1-associated cytokines, which contribute to antibody production and participate in the cellular immune response, T lymphocyte proliferation and induce cytotoxicity [When stimulated by foreign antigens, the body responds with Th1-type cellular immune responses and Th2-type humoral immune responses in mammals. The degree of immunogenicity that induces an effective immune response in the body is an important indicator for estimating the efficacy of an oral vaccine. In this study, to comprehensively evaluate the immune response induced by pPG-alr-VP4/toxicity . IL-4 antoxicity . In addialr gene in the genome of L. casei using the CRISPR-Cas9D10A gene editing system and constructed a complementary plasmid expressing alr-VP4 simultaneously. Construction of the recombinant strain  used food-grade selective markers instead of traditional resistance gene markers to realize the artificial operation of the strain and has completely independent intellectual property rights. We subsequently demonstrated that the recombinant strain could efficiently induce specific local mucosal immunity, systemic humoral immunity, and cellular immunity via oral immunization in mice, suggesting that it is a promising strategy for developing an oral vaccine against PoRV infection.In this study, we knocked out the