Neurocognitive impairment remains an important HIV-associated comorbidity despite combination antiretroviral therapy (ART). Since the advent of ART, the spectrum of HIV-associated neurocognitive disorder (HAND) has shifted from the most severe form to milder forms. Independent replication of HIV in the central nervous system despite ART, so-called cerebrospinal fluid (CSF) escape is now recognised in the context of individuals with a reconstituted immune system. This review describes the global prevalence and clinical spectrum of CSF escape, it role in the pathogenesis of HAND and current advances in the diagnosis and management. It highlights gaps in knowledge in sub-Saharan Africa where the HIV burden is greatest and discusses the implications for this region in the context of the global HIV treatment scale up. Globally, 36.7 million people are living with HIV with 21 million of these on antiretroviral (ART) [10 of the plasma VL and CSF escape is defined as any VL in the CSF above the limit of detection of the assay used  when the VL in the plasma is undetectable by the same assay [A contentious issue is whether independently replicating HIV in the brain parenchyma and the proxy for this—the presence of HIV virus in the cerebrospinal fluid (CSF), is responsible for neurocognitive impairment (NCI) in the post-ART era. The terms commonly used are CSF discordance and CSF escape. The definitions of these terms are based on the quantification of CSF HIV RNA in paired CSF and plasma specimens see . CSF disme assay ,4,5. Altme assay , the cauIn this review, we describe (1) the prevalence of HAND globally, (2) the clinical spectrum of CSF escape/discordance (3) the contribution CSF escape/discordance to HIV-associated neurocognitive disorder (4) critically review the role of CSF HIV RNA and the evolution of HIV drug resistance in the pathogenesis of CSF escape/discordance, (5) appraise the current state of clinical biomarkers in the diagnosis of HAND and CSF escape/discordance and (6) review progress with management.It is worth noting that the majority of research on HAND has been done in Western settings where Subtype B HIV-1 predominates but little is known about HAND in sub-Saharan Africa (SSA) where subtypes C, A, D, G and AG predominate. We aim to identify the research gaps globally but highlight the implications in SSA where the burden of HIV is greatest.It is clear that since the advent of ART, the prevalence of the most severe form of HAND–HIV associated dementia (HAD), has markedly reduced . The MulThere is evidence to suggest that HIV-1 subtype plays a role in neurocognitive dysfunction. Neurocognitive impairment has been associated with CRF_02AG when compared with subtype G in Nigeria . In UganThere is controversy around the actual prevalence of HAND because based on these criteria, the prevalence of HAND is sufficiently high to warrant questions about diagnostic overestimation ,24.In the pre-ART era, HIV encephalitis was common and associated with high CSF viral load . Since tA retrospective longitudinal study of neurological symptomatic cases from a clinic in Boston found a prevalence of CSF escape of 6% (12/200) . A retroThe spectrum of clinical manifestation of CSF escape/discordance in the ART era ranges from asymptomatic to coma and death. There have been several cases reports and case series describing patients with chronic HIV on ART with symptomatic CSF escape/discordance ,36,37,38CSF HIV RNA is thought to be a proxy for independent replication of HIV in the brain parenchyma as it correlates significantly with brain HIV RNA ,39. HIV The pathophysiological process behind asymptomatic and symptomatic CSF escape/discordance appear to be distinct . In asym+ lymphocytes [+ predominance [In symptomatic CSF escape, there is neuroinflammation characteristic of a meningoencephalitis, with cellular CSF predominated by CD8phocytes ,50. Braiominance . This maominance ,35,36,37There is evidence to suggest that CSF escape/discordance in neurosymptomatic individuals or participants having a lumbar puncture for clinical reasons is associated with a longer duration of ART ,30, low CSF escape has also been described in patients on PI monotherapy . HoweverIndependent replication in CSF and blood compartments have been shown ,64,65,66Reported MRI changes in neurosymptomatic CSF escape/discordance are consistent with diffuse white matter signal abnormality (DWMSA) in the dentate nuclei, brainstem, capsular and subcortical white matter ,31,36,69QALB) , a marker of endothelial tight joint permeability is elevated and directly correlates with NFL [There are interesting developments in biomarkers of neuropathology and their application to HIV CNS disease . CSF HIVwith NFL .Amongst ART treated, neurocognitively unimpaired persons, for most part, CSF neopterin usually decreases to normal levels ,74,75. Np < 0.0001) [The published data on biomarkers in CSF escape/discordance are limited. However, case series have reported evidence of immune activation with elevated neopterin in CSF but not plasma in both symptomatic  and asym 0.0001) .ART as well as being neuroprotective , improveOne published study used 1 g of intravenous methylprednisolone for 5 days followed by a tapering dose of oral prednisolone to treat neurosymptomatic CSF escape/discordance with mixed results . AlthougIn the era of ART, the neuropathogenesis of HAND is not simply due to viral replication and neuroinflammation but with multifactorial contribution including the legacy of neuronal injury, HIV-induced metabolic changes, neurotoxicity of ARTs and poor drug penetration ,86. OtheA cross-sectional study of the NNTC cohort of post-mortem brain tissue showed that the presence of brain HIV RNA did not correlate well with HAND diagnosed in the absence of features associated with HIV-replication, that is, HIV encephalitis or microglial nodule encephalitis. However, a higher brain HIV RNA level correlated with worse performance on NP tests . This sup = 0.002) and the presence of CSF escape at first or second visit was associated with a decline in neurocognitive performance compare to those without CSF escape (p = 0.02) [CSF escape/discordance is reported in patients with HAND. The detection of HIV-1 in CSF even at low-level (<50 copies/mL) using single copy assay was 12.7% in a CHARTER subgroup study . In this = 0.02) . In Mukh = 0.02) .Although the precise contribution of CSF escape/discordance to HAND is unclear, independent replication of HIV-1 in the brain likely contributes to neurocognitive decline particularly in the context of drug resistance. As CSF escape/discordance is a potentially modifiable risk factor for neurocognitive decline, there does appear to be a role for detecting, quantifying and sequencing CSF HIV RNA in HAND diagnosis and management ,85,88. TDiagnosing HAND has challenges in this setting. It requires a battery of NP tests, that require a specific skill set to deliver and sometimes specialised equipment. These tests were developed in resource-rich settings  and untiThe biological causes of HAND in ART treated patients in SSA are not well understood however there is evidence to suggest that HIV-1 subtype may play a role ,11. MostThere are currently no published data on CSF escape/discordance from SSA. Based on the current knowledge of risk factors for CSF escape/discordance, which include long duration on ART, low nadir CD4 count, LLV, history of plasma viral blips and frequent drug interruptions, together with the rise in the prevalence of pretreatment drug resistance in RLS , it is hFurther research should aim to determine the prevalence, the clinical spectrum and pathophysiology in symptomatic and asymptomatic patients, the genetic variability of compartmentalised viruses, the evolution of drug resistance in this setting, as well as the role of HIV-1 subtypes in the pathogenesis of CSF escape/discordance. Prospective follow up studies are required to determine if asymptomatic persons with CSF escape will progress to symptomatic CSF escape.No studies in SSA have systematically evaluated plasma or CSF biomarkers in ART treated person with CSF escape/discordance. Investigating biomarkers may pave the way to developing useful, non-invasive diagnostic tests for assessing and managing patients with neurological involvement of HIV-1.The overwhelming view is that in the era of cART, the neuropathogenesis of HAND is not simply due to viral replication and consequent neuroinflammation but a multifactorial aetiology including the legacy of neuronal injury, HIV-induced metabolic changes, neurotoxicity of ARTs, poor drug penetration, drugs and alcohol and liver disease. CSF escape/discordance does however appear to have an independent role in NCI. Furthermore, the presence of ongoing replication in the CNS may be a barrier to HIV-1 eradication and the goal of achieving a cure.


Hair restoration surgery is one of the most commonly performed cosmetic surgery procedure in men. The main aim of the study was to know the effect of follicular unit extraction (FUE) follicular unit extraction (FUE) on donor area in terms of hair mass/density.2 each were marked. The first box was marked in the midline and the 2nd and 3rd boxes were marked about 3 cm from the midline. Another two boxes, each of 1 cm2 were also marked at the distance of 3 cm. Two boxes of 1 cm2 were marked on either side. The extraction was performed using 0.9 mm punch. The number of extracted hair were counted.Ten male patients undergoing hair restoration by FUE were included. In each patient, ten boxes of 1 cm2. The extracted hair count was 54.85 hair per cm2 which was about 35.44% of the total donor density (range: 28.9-42.8%). The graft to hair ratio in the extracted follicular units was 1:2.3 (range: 1:1.65-1:2.75).The mean age of the patients was 31.7 years. The average hair count in the donor area was 154.76 hair per cmAs the donor density varies, the FUE should be performed with caution. The strip surgery, also known as follicular unit transplantation (FUT) or follicular unit strip surgery (FUSS). The second kind of surgery, FUE, involves the harvesting of follicular units from the donor area with the help of manual machines/instruments, motorized machines or robots. Over the last decade, FUE has gained more popularity due to certain commercial, technical and other factors.1Hair restoration surgery is one of the most commonly performed cosmetic surgery procedure in men. More than sixty percent of the male population experiences some degree of hair loss at some stage of their lives.2 The pros and cons of each procedure depend on the patient and surgical expertise of the surgeon.No study to date is available which could prove the superiority of one technique over the other. Both techniques are technically demanding, require thorough knowledge of the hair restoration process and the surgical expertise to carry out the procedure. Each have certain complications. The FUE technique is more popularized due to the fact of lack of linear scar but the total surface area of the scars is many times larger than the surface area of the scar of a strip surgery. Similarly, FUE technique allows harvesting of the follicular units from a larger area which is not possible with the strip surgery.3 The density of the given area commonly keeps on decreasing with the advancing age. There is no study mentioned in the literature which determines the effect of FUE on the donor area. The main aim of the study was to know the effect of FUE on donor area in terms of hair mass/density. As the hair loss is not static, the final phenotype is determined by the genes. The male hormones and the therapies used to alter the hair loss process.2 each were marked. The first box (A) was marked in the midline at the level of external occipital protuberance  were marked about 3 cm from the midline. Another two boxes (D and E), each of 1 cm2 were also marked at the distance of 3 cm from the outer border of the box B and C. About 3cm below the box A, another box of 1 cm2 was marked (F). Two boxes of 1cm2 each were marked on either side (G and I on one side and H and J on the other side) of the box F as described earlier with an intervening distance of 3 cm. All of these boxes were photographed using a macro lens. The hairs were counted manually in each of these boxes. The extraction was performed using 0.9 mm punch. The number of extracted hair were counted for each box in each patient. The boxes were photographed postoperatively. The 22 was included . The mean age of the patients was 31.7 years. The average hair count in the donor area was 154.76 hair per cm2 . The graft to hair ratio in the extracted follicular units was 1:2.3 (range: 1:1.65-1:2.75)  .4-6Removing hair from the donor involves the expertise on the part of the surgeon. Over the last decade, there has been increased trend in FUE procedure by the physicians. Those patients who want to have short hair or who want to avoid linear scar, now can undergo hair restoration procedure. The donor area contains hair clustered into ‘follicular units’. The defining term for donor area remains the ‘hair density’. The donor density varies according to the age and race. For example, hair density in Caucasians is different from Asian or Africa.7 Whereas FUE reduces the overall density by ‘cherry-picking’. FUE is not a straight forward or simple procedure. It has a steep learning curve, need a physical stamina, patience, excellent hand-eyes coordination etc. It also requires diagnostic skill, aesthetic insight and sensitivity and expertize to deal with rare unexpected complications. Various side effects of FUE have been mentioned in the literature and this list is increasing every year due to larger number of procedures performed by novice surgeons. These side-effects include visible scarring, skin necrosis, cyst formation, neuralgia, hiccups etc.8-10Both FUE and FUT reduce the donor area but the way of execution is different. FUT decreases the height of donor area, a part of it gets re-stretched by ‘stretch-back’ phenomenon.11 There are multiple factors which predict the success of the hair restoration surgery. The patient’s age, type of baldness, family history, environmental factors all play a vital role. As the hair loss is an ongoing process which never halts at any age, decrease in density in the donor area is also seen.12,13Although various surgeons’ experiences have been presented in the literature and conference with excellent results but no study is yet there to show the long term results after FUE procedure. The most important point in FUE surgery is the number of hair which is removed from the donor area. The transection rate plays an important role in the outcome of a hair restoration surgery by FUE.14 All the studies done on FUE describe the removal/extraction of grafts or number of hair. None of these describes the effect on donor hair. Harvesting more than 3000 grafts in no doubt a great effort on the part of the surgeon but the question of hair density left behind and transection remains there. In a study by Avram using robotic FUE, the overall transection rate was 6.6 with a wide range from 0.4% to 32.1%.15 The more the number of grafts harvested, the more is the transection rate. On the other hand, a transection rate of 1.59% is mentioned in the study by Pathomvanich et al.16 A few techniques, e.g., use of Haber’s dissector and scissors tissue dissection, may have even lower transection rates in FUT surgery.16,17In the study by Bernstein, the average graft to hair ratio was 1:2.72.11Most of the published data on FUE indicate the inclusion of small areas in the studies. The authors are of the views that there are certain key limitation points which are not included. One of the most important point omitted is the ‘fatigue’ factor while harvesting larger number of FUs . Another equally important factor is the angle of hair exit. The angles become more acute on sides and below superior nuchal line which results in higher rate of transection. Similarly, hair angles also vary in different races, e.g., the chances of transection are higher in African than Caucasians. The authors also assume the transection rate mentioned in the many studies may not depict the real picture due to these important factors. The hidden transection rate is also another factor.18Harvesting more grafts means removal of FUs beyond the limits of ‘safe donor area’ which can be a potentially ‘danger zone’ or strictly an ‘unsafe zone’ to harvest. This unsafe/danger zone may be affected as a result of ongoing aging hairless and male pattern baldness which would result in slightly-scars of FUE if f it is done in a young patient. Most of the studies indicate that patients undergoing FUE are less than 35 years. Another important point is the definition of ‘safe donor area’ which should be revised. The authors are conducting a study to review the ‘donor area’ in the male population above the age of 50 years. Thus the key point in FUE remain the donor area (safe zone or danger zone or unsafe zone) and the number of extracted hair. In younger patients with advanced baldness (type V or more), the FUs are harvested from the non-safe areas by FUE.19 The removal of 50% donor density means the FUE procedure has resulted near the level of ‘watershed line’ for ‘visibly detecting the hair loss’. A 2nd session of FUE procedure of even 20% will reduce the overall donor density to about 70%, which will be obvious to the naked eye. The ongoing aging hair loss will also be added and would cause gross baldness and the visibility of the scarring of FUE. Therefore, the number of grafts removed in FUE is not important, rather the number of hair removed are of the utmost importance. The patients having 20–30% of the actual density in the donor area would look odd as the donor density will be low showing the scalp. In the current study, about 35% of the donor hair were extracted. In a study by Beehner conducted to compare the results of FUE and FUT, the survival rate of FUs by FUT was 86% as compared to 61.4% by FUE after 14 months postoperatively.20Many surgeons suggest that about 50% of the donor density can be harvested.21 The ethical issue may arise if they would not refer to a colleague who can perform both FUT and FUE. Moreover, the surgeons may be replaced eventually, after sometime, by the technicians and robots etc. as it is more cost-effective. In order to do hair restoration surgery for advanced baldness, the maximization of grafts harvesting should be done. This procedure is advertised as a ‘profitable turnkey’ model for new revenue streams to the physician’s practice. The extra role of ‘technicians’ or ‘non-physicians’ in various steps of hair restoration seems improper and illegal.21Many factors play a role in the ultimate survival including, storage solution and its temperature, out of body time, desiccation/dehydration etc. The boom of FUE is resulting mainly because of the advertisement done by the FUE machines marketing companies and mainly doctors trying not to learn the surgical techniques. As described by Rassman, a new breed of hair restorative surgeons is coming in the market who are trained only for FUE and probably have no knowledge about the ‘strip harvest surgery’ and cannot offer the treatment to a patient if needed.The so called ‘Turkey-phenomenon’ where one physician, not necessarily the surgeon, supervises the various simultaneous surgeries done by the technicians. In an uncontrolled FUE environment, the danger of donor depletion also remains there. Criticism on FUE sometimes is taken as if the critique is against FUE whereas FUE is actually a great addition in the armamentarium of a hair restorative surgeon. The only point is its judicious use in the patients. A state of the art about two decades age, is not acceptable now. The decision should be tailor-made. st session and not more than 10-20% in 2nd session; (ii) FUE sessions should be combined with FUT to maximize the number of grafts/hair in a given session; (iii) FUE should be done carefully during the learning curve and initially should be done under supervision; (iv) When the final procedure is done, the scar of the strip surgery should be repaired by FUE from scalp or body or by micropigmentation; and (v) FUE should not be perfumed in very young patients and if needed, only grafts from the safe zone should be harvested. Family history should be kept in mind. Therefore, we can conclude that as the donor density varies, the FUE should be performed with caution.From the study, there are a few recommendations for FUE proposed by the authors. (i) FUE should be limited to less than 35% of total hair density in 1The authors declare no conflict of interest.


Pinctada margaritifera. For this, eight uniform grafting experiments were designed using two hatchery-produced pearl oyster families as donors, which were followed through time, between 7 and 30 months in age. For each age category, 20 donors were studied for each culture site giving a total of 2400 grafted oysters. Several phenotypic measurements were made: 1) donor family growth performance from shell size records, 2) pearl size and corresponding quality traits, and 3) expression of some genes related to biomineralization processes on both the mantle graft and on pearl sac tissues. Results showed that: 1) donor age has an impact on pearl size, with grafts coming from the youngest donors yielding the biggest pearls; and 2) grafts from donors between 12 and 18 months in age produced pearls of the highest quality , a result supported by an analysis where the level of expression for a panel of genes associated with biomineralization was greatest in donors within the 12 to 18 months age group. These results indicate that donors aged between 12 and 18 months have high potential for biomineralisation and nacre deposition, and likely produce larger and higher quality cultured pearls than older donors.Ageing is defined as the progressive decline in tissue and organ functions over time. This study aims to evaluate the ageing effect on cultured pearl quality phenotypes  in the graft-recipient animal model:  The process of ageing affects living organisms, from single cell yeasts to multi cellular animals and plants. Most evolutionary biologists define ageing as the age-dependent or age-progressive decline in tissue function over time –10. In aPinctada spp or Pteria spp (Family: Pteriidae) [Pinctada margaritifera, mainly from French Polynesia, with minor productions in the Cook Islands, Fiji, the Marshall Islands and Micronesia, is valued for its ability to produce high quality colored pearls. From a genetic point of view, grafting associates two tissues from different individuals, the recipient and donor (two distinct genomes) that create a genetic chimera with each genome maintaining its own genetic identity throughout the grafted organism [P. margaritifera, the gonad of a “recipient” oyster is surgically implanted with a spherical shell bead nucleus (made from the shell of a freshwater mussel from the Mississippi River) alongside a small graft (~4mm2) of mantle tissue cut from a donor oyster. This small piece of donor mantle tissue is called saibo. The saibo grows around the nucleus and becomes a “pearl sac”, which secretes successive nacre layers on the nucleus. This process results in the formation of a cultured pearl in about 15–20 months. As only small pieces of saibo are required for grafts, a single donor oyster can provide material for multiple recipients. Once a pearl has been harvested, it is then classified based on several factors that, in combination, will determine its quality and therefore its market value [Since the development of artificial pearl cultured techniques in the early 1900’s, the production of nucleated culture marine pearls has become a significant industry throughout Southeast Asia, northern Australia and Pacific Island nations [eriidae) , with aneriidae) . The blaorganism . To initPinctada margaritifera cultured pearls are still produced using wild populations due to the abundance of natural oyster resources and, until recently, the challenges associated with controlling the reproduction and early life stages in culture. Rearing of the species over its entire life cycle, including artificial breeding, became possible through the domestication of P. margaritifera. This advance has allowed oyster age to be controlled, in contrast to current Polynesian pearl aquaculture methods which rely on wild spat collection where collected individuals are of undetermined age. To date, studies that examined pearl quality traits mainly focused on the genetics of the donor oyster. The influence of the donor on pearl quality traits has been definitively demonstrated using reciprocal xenografts between P. maxima and P. margaritifera in which donors were found to have a significant effect on both color and surface complexion [mplexion . Studiesmplexion  or familmplexion  effect omplexion –29. A remplexion  and founmplexion . No studP. margaritifera, on the culture pearl phenotypes and on the potential for biomineralization. To do this, we conducted grafting experiments using donors from a single biparental family at 12, 18, 24 and 30 months of age, and assessed resulting pearl quality from each cohort. To determine whether our results were reproducible (and to assess even younger donors), we conducted a second series of experiments in a separate location, in which donors from a second unrelated family were used as grafts at 7, 12, 18 and 24 months of age. Several phenotypic measurements were made: 1) the shell biometric growth parameters of the donor families, 2) the cultured pearl size and quality traits, and 3) the expression of eight gene encoding proteins involved in the biomineralization process in both the mantle graft of the donor and in the final pearl sac tissue. In the end, this study contributes to the optimization of the graft process by identifying the ideal age for the donor oyster which then improves pearl quality traits in hatchery pearl culture systems.This study investigated the age effect (independent from shell size) of hatchery-produced donor The pearl oysters used in the current study are marine-cultured animals, and all of the experiments on pearl oysters were conducted following the institutional and national guidelines. The authority who issued the permission for pearl oysters transfers is: "Direction des Ressources Marines et Minières". No endangered or protected species is involved in the present study, and no specific permission is required for the location of the culture experiment, as it is in public maritime area.2. The site of culture is located at Avaturu village (Gauguin’s pearl farm).Rangiroa atoll is located in the north-western part of the Tuamotu Archipelago , about 350 km northeast of Tahiti. The atoll has a flattened elliptic shape, is 80 km in length and ranges from 5 to 32 km in width. The lagoon surface is 1446 km2. The island is the central and largest of the archipelago and has a large lagoon 24 km in diameter containing reefs. The culture site was located at Regahiga Pearl Farm.Mangareva Island is located in the Gambier Archipelago , about 1590 km southeast of Tahiti. The island surface area is 27 kmP. margaritifera families were produced at the Ifremer hatchery facilities , using wild female and male broodstock from Rangiroa Island  for F_RGI family, and Mangareva Island  for F_GMR family. Spawning was induced by thermal shock [Two bi-parental al shock . Artifical shock . After aCTN from the two families, used as donors, were randomly selected and transferred (at six months old) by plane to Rangiroa atoll for F_RGI family and Mangareva Island for F_GMR family to allow the oysters to adapt (1 month) and grow in local environmental conditions before they were randomly selected for the grafting procedure. These pearl oysters (attached to CTN) were regularly cleaned by high pressure sea water sprayDonors from the F_RGI family were used in grafting experiments atthe Rangiroa atoll culture site at 12, 18, 24 and 30 months old (every 6 months). Twenty donors were randomly selected each time. F_GMR family was used in grafting experiments at Mangareva island culture site at 7, 12, 18 and 24 months old. Twenty donors were also randomly selected each time. All grafts were performed under standard production conditions by a single expert from each pearl farm, Gauguin’s pearl Farm (Rangiroa) and the Regahiga Pearl Farm (Gambier), to minimize the grafter effect on pearl quality phenotypes . The recPearl phenotype categories were recorded to characterize the quality of the pearl :Shape was characterized in two ways: the presence / absence of circle/s  and the shape category .Color was evaluated through the darkness level  and the visually perceived color category, which is conferred by bodycolour pigments and secondary colors (overtone). 5 major color categories were detected including green, grey, peacock, yellow and one named “other” including white, blue and aubergine pearls.rebuts).Cultured pearl grade was determined for each sample according to the official A–D Tahitian classification  from the most to least valuable quality: A, B, C, D and Rejects  were determined separately so that they could be studied independently. To ensure homogeneity in parameter assessment, all evaluations were made visually by the same operators.Pif-177, MSI60, Perline), calcite-related genes  and Shematrin9 (only for pearl sac tissues)) and one gene implicated in both layers (Nacrein) (P. margaritifera tissue: SAGE (SAGES: AGCCTAGTGTGGGGGTTGG/ SAGER: ACAGCGATGTACCCATTTCC)  [In order to assess expression levels of known biomineralisation genes in donor tissue of different ages, we sampled graft tissues (3 to 5 pieces per donor) during the graft operation and pearl sac tissues during the harvest. In order to minimize the mixture of recipient tissues, the pearl sacs were excised from host oysters by removing the outer layers with a surgical blade until a thin (< 0.5 mm) layer of tissue surrounding the pearls remained, and immediately transferred and preserved into 2.0 ml tubes with RNAlater”. Samples were preserved in RNAlater and stored at –80°C for subsequent RNA extraction to evaluate relative gene expression of aragonite-related genes (Nacrein) . Two gend REF in  and GAPDCAACTTC) . The rel-1 and continuous fluorescence measurement. All measurements were made in duplicate and all analyses were based on the Ct values of the PCR products. We allowed a difference of less than 0.5 ct between our two replicates. If the difference is superior to 0.5 ct, the qPCR reaction was repeated, and the sample was removed if congruent ct values were once again not obtained.After removing the RNAlater by pipetting and absorption, total cellular RNA was extracted from either the individual graft tissue or pearl sac samples, using TRIzol reagent (Life Technologies) according to the manufacturer’s recommendations. RNA was quantified using a NanoDrop ND-1000 spectrophotometer  and the quality of the RNA was checked to exclude degradation using an agilent 2100 bioanalyser. The RIN values were between 6.50 and 7.40 corresponding to a sufficient quality for quantitative real-time PCR analysis. Total RNA for each individual was then treated with DNAse I using a DNA-free Kit (Ambion). First strand cDNA was synthesized from 500 ng total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and a mix of poly (dT) and random hexamer primers. Real-Time PCR amplifications were carried out on a Roche Light Cycler 480. A no-RT control was screened by qPCR using a housekeeper gene to ensure there was no DNA contamination. The amplification reaction contained 5 μL LC 480 SYBR Green I Master Mix (Roche), 4 μL cDNA template, and 1 μL of primer (1μM), in a final volume of 10 μL. Each run included a positive cDNA and a blank control for each primer pair. The run protocol was as follows: initial denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 60 s. Lastly, the amplicon melting temperature curve was analyzed using a melting curve program: 45–95°C with a heating rate of 0.1°C s(target)ΔCt target (control—sample) / E(ref)ΔCt ref (control—sample) [The relative expression ratio (R) of a target gene was calculated based on E and the CP deviation of an unknown sample versus a “control”, and expressed in comparison to a reference gene as follows: R = E—sample) . Here, t—sample) . PCR effPrior to the grafting operation, shell height, width and thickness of the 200 donor oysters were measured using Vernier calipers . At 18 mThe normality of the data distribution and homogeneity of variance were tested for pearl size, donor oyster biometric parameters and relative gene expression ratio using the Shapiro-Wilk test and Bartlett’s test. When necessary, transformations were used to adjust data to the distribution (logarithm or square roots).2 tests.Group donor age was treated as a fixed variable. Firstly, an ANOVA test was performed to test age of donor effect on donor shell biometric parameters, cultured pearl weight, thickness, graft and pearl sac relative gene expression ratio. If the overall test was significant, a Dunn procedure with a Bonferroni correction for multiple tests was performed among all pairs of age groups. Qualitative classes based on cultured pearl surface defects, lustre, grade, darkness and circles were re-encoded to give quantitative scores that would enable the mean value of age group to be obtained for each criterion, thus allowing them to be ranked. Scores from 0 to 4 were attributed to the different classes from the least to the most valuable . For each criterion, Kruskall-Wallis tests were then applied to compare the age and donor groups. For the cultured pearl “color categories” and shape categories, donors and times of harvest effect were compared using χp values were lower than 0.05. Statistical analyses were performed using R software (version 3.2.1)[In all cases, the differences were considered statistically significant when on 3.2.1).A total of 1086 pearls from the 8 experimental grafts were analyzed (1086 pearls from 2400 grafted oysters). We studied the impact of the age of donor oyster on the family shell growth, pearl size, pearl quality traits and relative gene expression in the graft and pearl sac tissue.Results of pearl size in Rangiroa and Gambier for each donor age category are illustrated in In Rangiroa, pearls from experiments RGI12, RGI18 and RGI24 were significantly thicker  and heavier  than pearls from experiment RGI30. Pearls from experiment RGI18 showed the greatest average nacre thickness (1.52 ± 0.39 mm) and weight (1.21 ± 0.38 g) and experiment RGI30 showed the thinnest (1.27 ± 0.39 mm) and lightest (0.98 ± 0.39 g), representing a difference of 17% nacre deposit and 23% nacre weight.In Gambier, pearls from experiments GMR7, GMR12 were significantly thicker  and heavier  than pearls from experiments GMR18 and GMR24 (p < 0.0001). Moreover pearls from GMR18 were significantly thicker and heavier than pearls from GMR24 . Pearls from experiment GMR7 showed the greatest average nacre thickness (1.51 ± 0.47) and weight (0.80 ± 0.33) and GMR24 showed the thinnest (1.19 ± 0.40 mm) and lightest (0.60 ± 0.26) pearls corresponding to a difference of 21% nacre deposit and 25% nacre weight.p = 0.01 and p = 0.001, respectively). Cultured pearls from GMR24 and RGI24 presented the “best” surface quality, with 40% and 51%, respectively of cultured pearls having less than 5 defects against GMR7 (24%) and RGI30 (22%). A large proportion of cultured pearls from GMR12 (48%) and RGI30 (48%) had up to 10 defects.An age effect of high significance was detected for a number of cultured pearl surface defects in Gambier and Rangiroa . Cultured pearls from GMR18 and RGI24 recorded the highest level of lustre pearl, but also the same amount of pearl without any lustre (A significant age effect was recorded for “lustre” in Gambier and Rangiroa (y lustre .p < 0.0001). The pearls from GMR7 and RGI30 recorded the highest level of D-R pearls with a proportion of 49% and 57%, respectively, while GMR18 and RGI24 recorded the highest level of A-B pearls with 35% and 46%, respectively against 17% for GMR7 and 17% for RGI30. Cultured pearl quality trait distributions from each experimental graft are shown in Data analysis showed a significant age effect on cultured pearl grade (p < 0.0001). Pearls from GMR7 experiment were more circled than in other experiments with 62% of circled pearls compared to 31% of circled pearls, on average, in other experiments.A low significant age effect was detected for shape categories in Rangiroa (p = 0.05) and Gambier (p = 0.03). No significant difference was recorded between the four different donor age experiments in Rangiroa for the absence or presence of circles, but a significant effect was recorded in Gambier . The different color proportions produced by the different experiments are illustrated in A significant age effect was recorded for “color categories” .Concerning the graft tissue, the relative gene expression ratio for the seven candidates varied at Rangiroa and were significantly different among the three donor ages . PIF, MSI60, PERL1 and CALC1 relative gene expression ratio were significantly higher for the 12 month old donors , compared to the 24 month old donors which had a relative gene expression ratio inferior to 0.6 for the four genes (p < 0.0001).At Gambier, the relative expressions of 7 genes that encode for the biomineralization matrix proteins in the graft were significantly different between the donor age categories (p < 0.0001). PIF177, MSI60, PERL1, CALC1, ASP and PRISM14 relative gene expression ratio were significantly higher for GMR18 compared with GMR24 which had a relative gene expression ratio inferior to 0.6 for all genes .Among the eight candidate genes studied in the pearl sac at Gambier, only the expression of SHEM5 was significantly different in the pearl sac between the two donor age categories (p = 0.0002). SHEM5 and ASP had higher relative gene expression ratio in pearl sac from younger donor tissue .i.e., only 24 month old donor oysters at (RGI24 and GMR24) were significantly different between Rangiroa and Gambier sites for shell height  and shell thickness . The growth curves show the maximum slope  for donors younger than 18 months old at both Rangiroa and Gambier.At the time of grafting, the donor oyster width, height and thickness were recorded to provide average growth measurements for the two families. Results for shell growth with respect to donor age and culture site are described in P. margaritifera. This study is also the first to examine the impact of pearl donor oysters aged less than 2 years on pearl quality traits. We found that donor age impacts the size of the pearl, pearl grade and surface quality. Donor age also impacts the relative gene expression ratio of aragonite-related genes , calcite-related genes  as well as the gene implicated in both layers (Nacrein) for graft tissue. For pearl sac tissues only calcite-related genes  were impacted by donor age.This study is the first to evaluate the impact of pearl oyster donor age on pearl size (including nacre weight and thickness) and on the expression levels for a panel of eight genes involved in shell biomineralisation in P margaritifera undergoes rapid shell growth until it reaches 18 months and then the rate slowly begins to decrease as it begins to invest more energy into reproduction [-1 during the second year of the life cycle and decreased with the age of the pearl oyster, reaching a mean value of 3.1μm.d-1 during the fourth year of the life cycle and confirming that growth rate in shell or tissue is directly related to oyster age.The major result of this study is that donor age has an impact on pearl size. In fact we found that pearls from the oldest donors, 30 months in Rangiroa and 24 months in Gambier, yielded the lightest and the thinnest pearls. The ageing of the donor’s mantle cells clearly alter the quantity of nacreous deposit. Grafts originating from young donors produced bigger pearls than grafts produced by older donors (more than 24 months). This result may be related to the donor shell’s growth curve where maximum growth occurs before 18 months. As a general rule, growth rates are directly related to bivalve age. oduction . Pouvreaoduction  also conoduction . This phoduction . In Pouvoduction , incremeP. margaritifera [Age for donor and recipient oysters is a parameter that is important to consider. A donor aged between 12 and 18 months seems to be the ideal candidate for a maximum nacre deposition. In French Polynesia, donor oysters are currently sourcedfrom wild spat collection and used in operations after 24 months of age. Our results suggest that better results would be obtained by using younger oysters. The results were consistent with the graft biomineralisation analysis where in GMR30, the oldest donor oysters showed the lowest level of expression for the aragonite candidate genes . PIF was previously found to be positively correlated with shell deposition rates in ritifera  and thusritifera . Among tritifera –26. Our ritifera ,30,44. Rritifera  where 2-P. margaritifera if donor age is optimized. Donor age (between 12 and 18 months) with high potential for nacre deposition and high biomineralization potential will increase cultured pearl size and quality.In the present study we can conclude that using donors that are too young (<12 months) or too old (>24 months) decreases the quality of the pearls produced . This study demonstrates that donor age influences the pearl phenotype and that there is potential to improve pearl size and quality in S1 Table(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(XLSX)Click here for additional data file.


A prospective study was conducted to evaluate the clinical performance of ReCell vs meshed split-thickness skin grafts  for the treatment of deep partial-thickness burns. Effectiveness measures were assessed to 1 year for both ASCS and Control treatment sites and donor sites, including the incidence of healing, scarring, and pain. At 4 weeks, 98% of the ASCS-treated sites were healed compared with 100% of the Controls. Pain and assessments of scarring at the treatment sites were reported to be similar between groups. Significant differences were observed between ReCell and Control donor sites. The mean ReCell donor area was approximately 40 times smaller than that of the Control (P < .0001), and after 1 week, significantly more ReCell donor sites were healed than Controls (P = .04). Over the first 16 weeks, patients reported significantly less pain at the ReCell donor sites compared with Controls (P ≤ .05 at each time point). Long-term patients reported higher satisfaction with ReCell donor site outcomes compared with the Controls. This study provides evidence that the treatment of deep partial-thickness burns with ASCS results in comparable healing, with significantly reduced donor site size and pain and improved appearance relative to STSG.Early excision and autografting are standard care for deeper burns. However, donor sites are a source of significant morbidity. To address this, the ReCell Although the use of STSG is considered standard treatment, grafting is associated with significant pain, pruritus, infection, dyschromia, dyspigmentation, delayed healing, and hypertrophic scarring.5 Furthermore, in large TBSA injuries, donor site availability is a limitation for rapid wound closure using traditional skin grafting techniques.Each year, nearly 500,000 Americans suffer from acute thermal burn injuries requiring medical treatment, resulting in approximately 40,000 hospitalizations and 3400 deaths.6–11 The realization that a treatment in which minimal split-thickness donor skin could be used to achieve definitive closure for burn wounds in a clinically advantageous time frame led to the investigation of strategies to harness the healing potential of the patient’s own skin.The clinical benefits of earlier intervention for burn wounds are well recognized and include increased survival, reduced hospital length of stay, decreased hypertrophic scarring, decreased pain duration, and reduced infection-related complications.® Autologous Skin Cell Harvesting Device  was designed for point-of-care processing of a small split-thickness skin sample to produce an autologous skin cell suspension (ASCS). Using minimal donor skin with an effective treatment area expansion ratio of up to 80:1, the ASCS induces rapid epidermal regeneration and re-epithelialization. The ReCell device consists of a stand-alone, battery operated unit, a proprietary enzyme solution, buffer solution, sterile surgical instruments, and spray applicators to be used at the point-of-care, with no culturing processes involved in the procedure.The ReCell12 ReCell-generated ASCS has been used to treat a wide variety of wound conditions such as burns,14 STSG donor sites,15 chronic wounds,17 hypopigmented scars,18 vitiligo,20 and large congenital melanotic nevus.21 A randomized study conducted by Gravante et al demonstrated that deep partial-thickness burns treated with ASCS produced using the ReCell device showed similar results to standard skin grafting, with the use of significantly less donor skin. Additionally, subjects treated with ASCS reported experiencing significantly less pain than patients treated with meshed STSG.13Preclinical data demonstrate that ASCS contains a mixed population of skin cells found at the dermoepidermal junction of predominantly keratinocyte, fibroblast, and melanocyte phenotypes.In burn wounds, the ReCell device is intended to generate ASCS that retains the known performance attributes of a STSG, while minimizing donor site morbidity. The aims of this study were to demonstrate similar definitive wound closure results using ASCS when compared with STSG and demonstrate improved donor site healing with the ReCell device.This was a multicenter, prospective, within-patient controlled, randomized clinical trial conducted under a U.S. Food and Drug Administration (FDA) Investigational Device Exemption (NCT01138917). For each patient, two similar areas within a burn injury were treated according to random assignment, resulting in a control wound treated with a 2:1 meshed STSG and a ReCell wound treated with ASCS. Donor skin for each treatment was harvested from separate but similar uninjured areas. All study wounds were photographically documented throughout the 52-week trial. Before study initiation, the protocol was approved by the U.S. FDA, the U.S. Army Medical Research and Materiel Command Human Research Protection Office (USAMRMC HRPO), and Institutional Review Boards (IRBs) at each individual site.2 was required, allowing for treatment of two separate or contiguous 100 to 320 cm2 areas. Exclusion criteria for the study included the following: burn injuries caused by chemicals, electricity, and/or radioactive substances; burn injuries <1% or >20% TBSA; treatment areas involving the head, face, neck, hands, feet, genitalia, or over joints; presence of pre-existing local or systemic infections; antibiotic treatment for >48 hours before autografting for other than prophylactic reasons; hypersensitivity to trypsin; a pre-existing chronic condition that in the opinion of the investigator might interfere with wound healing ; inability to follow the protocol; medications that could interfere with wound healing ; other concurrent conditions that in the opinion of the investigator might compromise patient safety or study objectives; and pregnant or breast-feeding women or women who wished to become pregnant during the length of study participation.Patients ranging in age from 18 to 65 years were eligible for study enrollment if they presented with an acute, deep partial-thickness thermal burn from 1% to 20% TBSA that required autografting for definitive closure. A minimum burn injury treatment area of 200 to 640 cmWithin-subject allocation of treatments to selected burn wounds was performed at random, using a predetermined random assignment of treatments. Following excision and confirmation of deep partial-thickness depth, and before randomization, the surgeon marked each area as site “A” or site “B.” Wound marking was performed using a sterile marker, allowing easy identification of the proposed treatment sites. An envelope was opened indicating treatment allocation.2. Before processing donor skin with the ReCell device, the skin was trimmed down to no greater than 4 cm2 (average 3.9 cm2) so that it would cover up to 320 cm2 or up to 1:80 expansion ratio.For the Control wound donor site, an autograft was harvested from a preselected, noninjured skin site according to standard of care (SOC), typically at a thickness of 0.008 to 0.010” but no less than 0.006”. For the ReCell wound, a thin split-thickness skin sample was harvested at a second donor site discrete from the Control wound donor site such that the two wounds could be dressed and assessed in isolation. The ReCell wound donor site was harvested at a depth of 0.006 to 0.008” with the average size of 4.7 cmThe treatment sites were excised to remove all nonviable tissue, and hemostasis was achieved per SOC. The surgeon verified that the wound was deep partial-thickness and contained viable dermis throughout the wound base.Treatment using the ReCell device was performed in accordance with the manufacturer’s instructions. Briefly, the split-thickness skin sample was transferred to the warmed proprietary enzyme solution for a period of 15 to 20 minutes to promote breakdown of cell-cell and cell-matrix adhesions, including those at the dermoepidermal junction. The skin sample was then removed from the enzyme, placed on the sterile tray of the device, and tested to determine whether the epidermis could be freely separated from the dermal tissue. When the tissue layers could be freely separated, the skin was rinsed in buffer solution and returned to the sterile tray of the device, dermal side down. Both the dermal and epidermal layers were vigorously scraped with a scalpel blade to completely disaggregate the skin sample. Following complete disaggregation, the cells were suspended in a buffer solution, filtered, drawn into the provided application syringe, and applied over the area randomized to receive ASCS treatment.The harvested STSG was meshed using a skin meshing system of choice at a ratio of 2:1. The prepared 2:1 meshed STSG was maintained in saline moistened gauze until placement on the prepared wound bed. The autograft was secured in place using sutures or staples at the surgeon’s discretion.™ Clear Wound Dressing, Covidien, Minneapolis, MN). A secondary dressing of Xeroform™ Petrolatum Gauze Dressing  was placed over the Telfa Clear primary dressing. Additional padding of gauze and a crepe bandage were used at the surgeon’s discretion for exudate and protection. The Telfa Clear primary dressing remained in place for a minimum of 6 to 8 days and was not manipulated until the first postoperative study visit unless medically necessary. Secondary dressings were replaced as needed. Telfa Clear was also used as the primary dressing for donor sites, with secondary dressing selection at the discretion of the surgeon. Most frequently, Xeroform was used as a secondary dressing for donor sites.After applying the ASCS and the 2:1 meshed autograft, both treatment sites were covered with a nonadherent, low-absorbent, small pore dressing (TelfaPrimary effectiveness endpoints were 1) the incidence of wound closure  of the treated sites at 4 weeks and 2) the incidence of complete donor site healing at 1 week , as determined by the surgeon. Percent re-epithelialization of the treatment sites was assessed using standardized planimetry/tracing procedures. The tracings were uploaded to a central reading facility  for calculation of percent re-epithelialization.Secondary endpoints included pain, visual appearance, and scarring. Pain and visual appearance were assessed using visual analog scales . For both the treatment and donor sites, pain was assessed at weeks 1, 2, 3, 4, 8, and 16, and visual appearance was assessed at weeks 16, 24, and 52. Investigators used the Vancouver Scar Scale (VSS) to evaluate scarring at weeks 16, 24, and 52 for both the treatment and donor sites.All adverse events were summarized and tabulated by treatment, system organ class, and preferred term. Adverse events were also evaluated by severity and if they were device-related adverse events. Additionally, descriptive statistics were provided for the following safety variables including infection at recipient sites, infection at donor sites, and graft loss at recipient site.The study was designed to investigate the clinical performance of ASCS relative to Control STSG, for the treatment of deep partial-thickness burn injuries. Coprimary effectiveness objectives were to test noninferiority of the incidence of ASCS-treated site wound closure at 4 weeks when compared with that of the Control, and the superiority of the incidence of ReCell donor site healing at 1 week when compared with that of the Control.πT − πS, Δ1) is 0.000, the proportion discordant (η = π10 – π01) is 0.100, and the proportion of both yes (π11) is 0.750 for a population of 90 subjects. Results are based on 1000 simulations using the Newcombe–Wilson score method to construct the confidence interval.22For the null hypothesis to be rejected and noninferiority to be established, the lower limit of the observed one-sided 97.5% confidence interval was expected to exceed −0.100 (a prespecified 10% noninferiority margin) with 81% power when the expected difference (The noninferiority margin of 10% was determined based on input from experienced burn surgeons and considered two factors: 1) it is normal for there to be minor variation in the rate of healing even for the same patient and 2) the clinical benefit that is realized at the donor site  provides balance for acceptance of a 10% noninferiority margin for recipient site healing.In the comparison of the coprimary endpoint of superiority of donor site wound healing between the ReCell donor site and the STSG donor site, it was assumed that at least 95% of the ReCell donor sites would be healed at 1 week, whereas 40% of the STSG donor sites would remain unhealed. For these assumptions, a sample size of 42 pairs will have 95% power to detect a difference in proportions of 0.350 when the proportion of discordant pairs is expected to be 0.500 and the method of analysis is a McNemar’s test of equality of paired proportions with a 0.050 two-sided significance level.Thus, a recruitment target was established aiming to yield 90 evaluable subjects such that the noninferiority analysis is sufficiently powered.Study populations  were defThe statistical analysis of the data obtained from the study was conducted by an independent third-party  using SAS Version 9.3. Continuous variables were summarized using descriptive statistics, specifically the mean, median, standard deviation, minimum, and maximum. Categorical variables were summarized by frequencies and percentages.Between May 2010 and August 2014, 133 patients were assessed for eligibility across 12 burn centers within the United States, and 101 patients were enrolled in the study. The mean patient age was 39.5 ± 13.1 years, and 84% of the patients were male, whereas the majority of burns (77%) were due to fire/flame-related injuries . SubjectOf the 101 patients enrolled (ITT), 87 subjects completed the 52-week follow-up without major protocol deviations (PP). The analysis population for incidence of definitive closure of the treatment sites (MPP) consisted of 83 subjects, with four subjects being excluded from the PP population who had SSD applied at the ASCS-treated sites following ASCS application .2 and control treatment area 165 cm2 .The two treatment areas were comparable with respect to anatomic location and size, with the ASCS treatment area averaging 168 cm 165 cm2 . The aveBoth treatments were clinically effective in healing >97% of the treated burns by week 4. The incidence of definitive closure, as defined as ≥95% re-epithelialization, was 97.6% (81/83) for the ReCell-generated ASCS treated site and 100% (83/83) for the control STSG–treated site . The difAdditionally, subject-reported pain at the treatment site during the first 16 weeks was not significantly different between the ASCS and control sites . SimilarP = .04, P < .001) with an odds ratio of 4.3. Additionally, this superiority persisted and subjects reported a statistically significant, and clinically meaningful, reduction in pain at the ReCell donor site compared with the Control donor site through the week-8 visit , including treatment and donor sites, were mild in nature . However, for 2 of 3 subjects, hypertrophic scarring was reported at the Control site and other nonstudy wounds as well. Therefore, the relatedness of the scarring to the study device is questionable. Overall, two subjects underwent subsequent intervention for graft loss at the ReCell site (one regrafting procedure and one debridement with redressing). In both cases, the wounds were healed at 4 weeks and remained healed.P = .25). At the treatment sites, a greater number of total AEs occurred with the ReCell treatment than the Control treatment .For the ReCell and Control donor sites, there were no differences in the incidence of AEs  and measured 121 cm2. A 90.25-cm2 STSG was harvested from the left anterior thigh at 0.010” thick, meshed 2:1, and applied to site A (control). A 4-cm2 skin sample was harvested from the medial thigh at 0.007” thick and processed using the ReCell device, with ASCS being applied to Site B. Telfa Clear was used to cover both of the treated sites and donor sites, followed by Xeroform and gauze dressings. One week following intervention, both the ASCS site and the control site were healed. At week 52, the color and pigment of the ASCS site matched surrounding skin, whereas the control site was mildly mismatched in color and pigment. The overall VSS for the control site was 2, whereas the ASCS-treated site was 0.A 62-year-old white male sustained an 8.5% TBSA flame burn inclusive of an injury to the left forearm . The bur® Autologous Cell Harvesting Device is a safe and effective alternative to conventional meshed autografting for the treatment of deep partial-thickness burns. It was demonstrated that, at 4 weeks, in a patient population receiving proper wound care, the incidence of healing was comparable between the treatment groups (97.6% for ASCS and 100% for Control), with no differences in pain, subject satisfaction with appearance, or scarring outcomes.In this study, it was demonstrated that the use of ASCS generated from the ReCellAdditionally, ReCell donor sites demonstrated increased healing, decreased pain, improved subject satisfaction with appearance, and less scarring. It was demonstrated that the requirement for harvesting of donor skin may be 1/80th the size of the burn injury area treated when using the ReCell device, thus reducing the skin required for coverage by 97.5% relative to 2:1 meshed STSG. Furthermore, as shown in this study, the use of ReCell additionally decreases the donor site depth compared with standard grafting, likely contributing to the reduced pain and improved healing outcomes.Using ReCell, the reduction of donor skin requirements gives rise to significant clinical benefits, offering surgeons a new strategy for the treatment of burn injuries. This is particularly true for patients having limited donor tissue availability, as well as for patients in whom the creation of larger donor sites may lead to significant morbidity. Additionally, for indeterminate depth injuries where skin grafting is intentionally delayed until it is certain that a graft is needed for definitive closure, the use of ReCell may be a solution for earlier intervention, as morbidity and complications associated with donor sites are reduced.2 in burns of 1% to 20% TBSA and excluded application to joints, hands, feet, genitalia, and faces. Although this study excluded these patients and treatment regions, previously published studies using ReCell demonstrate successful use in deep partial-thickness injuries for patients with varying TBSA and across various anatomic locations.24 In addition, ReCell treatment to the donor sites has previously been shown to significantly improve donor site healing.15 Therefore, based on the collective data, the authors conclude that the results demonstrated in this study may be extrapolated for the treatment of all deep-partial thickness thermal burns.This study provides definitive evidence for the safe and effective use of ReCell in the treatment of deep partial-thickness burns. As part of the study design to mitigate the variability across groups, treatment was limited to an area of 320 cmFurther implications from this study include the importance of understanding the learning curve clinicians must overcome in using innovative cellular products. During this study, adverse events occurred that were not attributable to the ASCS application but were due to the failure of proper after care. As the cellular suspension contains disaggregated skin cells, it is critical that the environment is optimized to allow for the regeneration and maturation into a robust epidermal layer. Thus, following ASCS application, cytotoxic agents must be avoided and dressings and garments should be used to protect the newly formed skin.Overall, the study demonstrates that ASCS generated by the ReCell device is a viable alternative to STSG for the treatment of deep partial-thickness thermal burns. Definitive wound closure of ASCS-treated sites was comparable to sites treated with 2:1 meshed autograft. Patients benefitted from significantly improved incidences of donor site healing and reduced pain and morbidity of the associated smaller donor sites. Reducing the amount of donor skin required to achieve complete and definitive burn wound closure opens the possibility for treating extensive and complex burns sooner than the current SOC. These findings have potential implications for a paradigm shift in the approach used to achieve rapid and permanent closure of burn injuries. Furthermore, achieving definitive closure using less skin compared with standard autografting has the potential to decrease the number of surgical procedures required to achieve wound closure as well as reducing hospital length of stay, thus decreasing the overall costs related to the treatment of burn injuries.This study was sponsored by Avita Medical, the Biomedical Advanced Research and Development Authority (BARDA), and the Department of the Army, AFIRM 1 Contract #W81XWH-08-2-0032 . The study results and conclusions do not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred.


This paper explores whether egg donation could still be ethically justified if in vitro gametogenesis (IVG) became reliable and safe. In order to do this, issues and concerns that might inform a patient’s reasoning in choosing to use donor eggs instead of IVG are explored and assessed. It is concluded that egg donation would only be ethically justified in a narrow range of special cases given the  availability of IVG treatment and, further, that egg donation could itself be replaced by donation through IVG techniques. Two possible criticisms of this position are then considered: Ones based on respect for patient wishes, and on loss of donor benefit. It is concluded that whilst neither argument constitutes a strong enough reason to continue with programmes of egg donation, egg‐sharing programmes could still be permitted come the advent of IVG; these could then provide a morally acceptable source of “natural” donor eggs. This paper explores issues and concerns that might inform a recipient’s reasoning in choosing to use donor eggs instead of eggs produced through IVG . This section concludes with the assertion that egg donation can only be ethically justified in specific, serious cases given the  availability of IVG treatment, and further, that even then egg donation as we know it today could be replaced by donation through IVG techniques. Following this, two possible replies to this conclusion—respect for patient wishes, and the loss of donor benefit—are explored.One might be inclined to wonder at this point whether there could indeed be instances where a fertility patient would still prefer to use donated eggs, even where IVG was available and cost‐effective, and had been shown to be safe. This is not unthinkable, and it is an important question to ask. There is of course a practical aspect to this, for if there were no demand for egg donation then both public and private healthcare organizations would need to respond accordingly. But this possibility raises ethical questions as well, as indicated by the question above—the answer to which would of course inform whether it would be ethically acceptable to continue to pursue egg donation once IVG has reached a point where it is safe, effective and cost‐effective. Further, the question and corresponding discussion also provide a new context in which broader questions around both IVG and egg donation can be explored. The assumption in the literature seems to be that IVG will eliminate the need for gamete donation if it is found to be safe, effective, inexpensive and ethically acceptable. As such, there has been no consideration of whether there might in fact be demand for gamete donation to continue in parallel to provision of IVG, or to what the ethical implications of this would be in the context of egg donation in particular (given the invasiveness of the process and the risks and harms involved). Some of the reasons why people might prefer donated eggs to the use of IVG are explored in the broader literature—in particular the view that the latter intervention is “unnatural,” and concerns regarding the psychosocial impact that being born through IVG might have on the resulting child. These are provided as possible criticisms of IVG more broadly, and while this will be true of their use here, the context in which they are being discussed in this paper  IVG becomes a feasible alternative. This is achieved by exploring reasons why would‐be recipients might opt for the use of donor eggs over having IVG (even at the expense of genetic relatedness), before arguing that the majority of these are not sufficient to justify egg donation.2.1One reason why people might decide to opt to use donor eggs as opposed to IVG is to avoid that which they perceive to be “unnatural”; for as Testa and Harris note, some people view the “natural” as being “superior to the artificial or synthetic.”per se is often very harmful; disease, mutations, pestilence, floods, hurricanes, fire, landslides and the like can cause massive loss of human life and do terrible damage to the environment.”unnatural is practically or morally better than the natural, but rather that there is no intrinsic moral difference between the two. As John Stuart Mill writes: “The scheme of Nature, regarded in its whole extent, cannot have had, for its sole or even principal object, the good of human or other sentient beings. What good it brings to them is mostly the result of their own exertions.”However, as many writers point out, “natural” does not always mean “better”—practically or morally.Furthermore, it is important to note that “the unnatural” is ubiquitous in the world today; indeed, even farm produce cannot be considered completely natural, given the selective breeding, cloning and even induced mutation that has occurred over the centuries.more natural  than another “unnatural” intervention. So then a critic might argue that egg harvesting and IVF may well be unnatural but less so than IVG interventions and so should be preferred. However, as already noted, the natural could at best be said to be morally neutral and so the notion that one intervention might be considered to be more natural  than another is not morally relevant in any case.This said, one might argue that there exists a sliding scale of “naturalness”—that something that is perceived as “unnatural” could nevertheless be seen as So then the “naturalness” argument fails both due to its faulty premise , and because it is incoherent. That IVG can be said to be “unnatural” is not a reason for moral objection to the endeavour, and it is certainly not a reason to favour egg donation, especially given that it too involves “unnatural” processes. As Testa and Harris write: Natural reproduction may be more enjoyable than synthetic reproduction, or cheaper, or safer or more private, these are reasons to prefer it. But there is no reason to prefer a natural process simply because it is natural.2.2Another reason for preferring donor eggs arises in cases where people want their child to inherit certain beneficial genetic traits from a gamete donor. Parallels could be drawn here with the use of the so‐called “genius sperm bank” However, the “genius sperm bank” example above is relevantly different from egg donation, because egg donation is far more harmful, risky and medicalized than sperm donation. Furthermore, Bourne and colleagues2.3Some people might opt for donor eggs rather than IVG because of concerns about the psychosocial impact that being the product of IVG interventions might have on the resulting child.did know of her origins: “problems might also arise even if the child does not know how she came to be born, if the parents’ or family’s knowledge that MRT were used changes how they relate to her.”One possible way of dealing with this concern is offered by Mertes and Pennings, who suggest that parents could simply refrain from telling their children that they were the product of IVG if they were concerned about the impact that the knowledge could have—especially as, unlike in the case of egg donation, both social parents would pass any genetic paternity or maternity test. Making the comparison with IVF, they write: “There is a general consensus that there are no strong reasons why a child should be informed that it is the result of in vitro fertilization.”people born through IVG techniques, whilst perceptions of the intervention itself will likely play a role here, media representation will again be an important factor both in informing opinion and in normalizing the use of the technology and the existence of people born from it. As Scully (again with reference to MRT) writes:If identity is created in part through cultural narratives, and can be damaged in a morally significant way by the lack of a ‘good story’, then ensuring that such good stories are available to MRT children and their families becomes a collective moral responsibility... it may become necessary to establish a form of systematic monitoring to follow how the media and other social institutions discuss MRT children and families, and engage with these agencies to counter potentially hurtful, harmful or limiting identity stories with more nuanced ones based on accurate empirical knowledge.It is this normalization that will be key, and education will play a crucial role in this endeavour, be that education in an institutional sense  or through popular media. Referring to studies into public perceptions of medical interventions, Gilbert and colleagues note that: “mass media influences patient education, comprehension, and understanding of health issues. In that respect, content and stereotypes disseminated through mass media influence prospective patients’ hopes and expectations.”Language will also be important; talk of “artificial” or “synthetic” gametes is not only inaccurateSo whilst concerns regarding the psychosocial impact of being born of IVG are reasonable, they may well be assuaged through public education—especially by way of the media and through the avoidance of certain provocative (and indeed misleading) terms such as “artificial” or “synthetic” gametes. Through these methods, public2.4Concerns regarding heritable illnesses or conditions might inform a recipient’s decision to opt for using donor eggs as opposed to having IVG—for instance, where the intended parent has a predisposition to some hereditary condition. This would constitute an ethically justifiable reason for pursuing egg donation. It could be countered that preimplantation genetic diagnosis (PGD) should be pursued first in order to try to identify affected embryos and prevent their transfer; however, one might reply that PGD is imperfect and might not successfully identify all conditions all of the time. However, further research into PGDRelated to this, there may be cases where a recipient would opt to use donor eggs where there is a risk of mitochondrial disorder in resulting offspring. As Smajdor and Cutas note: “[w]omen with mitochondrial mutations would still need to derive their mitochondrial DNA from other sources.”2.5Given the cases listed above, it seems that the only instances of egg donation which could be morally justified are those where mitochondrial transfer would be required, or where there are concerns regarding heritable illnesses or conditions that cannot be dealt with using PGD . This being said, perhaps egg donation (as we know it today) could be replaced with IVG techniques. So if a donated egg was required  then the would‐be egg donor could instead donate skin cells (for instance) from which eggs could be derived, as opposed to undergoing the invasive interventions currently used in egg donation and the related harms and risks.Of course it could be the case that the recipient of the donated egg (created through IVG techniques) would have concerns about “naturalness”, or of what to tell the resulting children with regards to their origins. However, as stated above, such concerns are not sufficient to justify the use of egg donation (as it is today) in any other circumstances, and so we might be unwilling to make an exception here as well. Although, as I will discuss in the following section, there may yet be alternative options available for those who would still prefer the use of “natural” donor eggs.3respecting their wishes in this matter . The second moves the focus from would‐be egg recipients to egg donors, and considers the argument that the donors themselves would be disadvantaged should IVG mark the end of egg donation as we know it today.This section considers two potential criticisms of the conclusions drawn in the previous section. The first suggests that we should offer the alternative of donor eggs to recipients as a way of 3.1One could argue that it would be wrong to rule out the use of egg donation as we know it today as we should strive to accommodate and respect the wishes of patients (and would‐be recipients) where possible; parallels can be drawn with Jehovah’s Witnesses here. The Jehovah’s Witnesses’ interpretation of the Bible leads them to forbid their members to receive blood transfusions, so members often refuse the intervention. As a result, some surgeons endeavour to use specialist “bloodless surgery” techniques in order to be able to treat these people whilst still respecting their views and wishes.This parity argument has some grounding in the broader literature; for instance, Juth and Lynøe write that there are no morally relevant differences between bloodless surgery and the uncommon intervention of hymen restoration,First, in the case of bloodless surgery, this involves costs such as financial costs, time costs and pressure on resources; however, whilst the use of donated eggs involves these costs as well, it also involves the further cost of direct harms to an individual: The donor.Second, a further difference between these cases is that of the risk of societal isolation and cultural shaming—for if a Jehovah’s Witness accepts a blood transfusion, in addition to religious concerns about what this might mean for the patient’s soul, there is a risk that the individual in question would be shunned and cast out from their societal group . It could be argued that this gives further weight to the patient’s decision to opt for bloodless surgery. When discussing cases where the recipient desires to use non‐IVG donor eggs, she is unlikely to face the same consequences. This said, it is unclear how some social and indeed religious groups might react to IVG technology when it comes into common use; it could well be that some religions ban use of the technology by its members under penalty of shunning or shaming—much like Jehovah’s Witnesses and blood transfusions. If this does become the case then there would be a far stronger argument for parity on these grounds.emergencies in which bloodless surgeries are required (and the physician’s conscientious objection negated), but the same cannot be said of the preference for donated eggs, use of which would constitute only a quality‐of‐life intervention, not a life‐or‐death one.It is also interesting to note that surgeons can choose not to treat patients who refuse blood transfusions (if it is not an emergency). As the Royal College of Surgeons explains: “Surgeons have the right to choose not to treat patients if they feel that the restrictions placed on them by the refusal of blood products are contrary to their values as a doctor.”In any case, fertility patients would have their refusal to use IVG‐produced eggs respected, as “[a]ll patients in the UK with mental capacity have the absolute legal and ethical right to refuse treatment or any aspect of treatment.”3.2donors would be disadvantaged if IVG were to spell the end of egg donation, since they themselves can be said to benefit from donating: Be that financially ; in either case the woman in question could be offered the choice of donating excess eggs to offset the cost of her IVF treatment (as is currently done).With regards to the more intangible benefits of donation, there are two responses. First, egg donation even as it is today might not be entirely unjustifiable. As I concede above, egg‐sharing schemes could be justified even where IVG interventions are available. Also, known donor donation could possibly be considered justifiable as well —for if the egg donor in question knew the reasons behind the donation (even if those reasons have been deemed here as insufficient to justify the intervention) but was still willing to donate for these reasons, then that would be her own fully informed decision.4This paper forms part of a broader ongoing debate about the replacement of current biomedical interventions with emerging technologies—in particular the use of stem‐cell‐derived forms of organic objects such as organs and gametes. It explores whether, and under what circumstances, egg donation would be ethically justifiable should IVG technology for reproductive purposes become inexpensive, effective and safe. Section 2 concludes that avoiding risk of disease provides the only reasonable justification for using donor eggs and, even then, IVG‐based egg donation should be preferred. Section 3 adds the caveat that non‐IVG egg donation could also be ethically justifiable when the eggs have been obtained through egg‐sharing schemes; this is because the donor in such cases would have undergone the risks and harms involved in egg retrieval in any case for her own medical benefit. This also applies to other cases where the donor has not undergone any further risks and harms; for example, the donation of eggs left over following reproductive treatment.The author declares no conflict of interest.


Proteinuria after kidney transplantation portends a worse graft survival. However the magnitude of proteinuria related to patient and graft survival and its correlation with donor and recipient characteristics are poorly explored.This study investigated the impact of post transplant proteinuria in the first year in 1127 kidney transplants analyzing the impact of different donor ages. Proteinuria cut off was set at 0.5 g/day.> 0.5 g/day correlated with poor graft and patient outcome in all donor age groups. In addition, 6-month-1-year proteinuria increase was significantly associated with graft outcome, especially with donors > 60 years old . 1-year graft function (eGFR < or ≥ 44 ml/min) had similar impact to proteinuria (≥ 0.5 g/day) on graft failure (Hazard Ratio 2.77 vs Hazard Ratio 2.46). Low-grade proteinuria (0.2–0.5 g/day) demonstrated a trend for worse graft survival with increasing donor age. Also in kidney-paired analysis proteinuria ≥0.5 effect was more significant with donors > 50 years old (Odd Ratio 2.3).Transplants with proteinuria Post-transplant proteinuria was increasingly harmful with older donor age. Proteinuria ≥0.5 g/day correlates with worse outcomes in all transplanted patients. Prognostic value of proteinuria and eGFR for graft and patient survival was comparable and these two variables remain significant risk factors even in a multivariate model that take into consideration the most important clinical variables . Over the past decades, increase of donor pool by using elderly donors has been largely adopted to reduce kidney transplant (KT) waiting list . “Old foProteinuria is known to be an independent risk factor for cardiovascular disease and mortality in native kidneys as well as an indicator of renal damage and a predictor of allograft loss after kidney transplantation , 6. SeveEven if older donor age is mentioned as risk factor for development of post-KT proteinuria , 13, the. Amer and colleagues analyzed 1-year post KT proteinuria finding an Hazard Ratio (HR) for graft loss of 2.15 (CI 95% 0.68–6.8), associated with proteinuria between 150 and 500 mg/day and an HR of 5.11 (CI 95% 1.4–19.2) with higher level of proteinuria [Halimi and coworkers showed a strong correlation of 1- and 3-month proteinuria (per every 0.1 g/day increase) with graft loss . Amer anIn a recent study Naesens et al. found a strong direct correlation between 1-year post KT proteinuria and graft loss irrespectively from graft histology, but only for proteinuria values higher than 1 g/day (HR 2.17) . In the To the best of our knowledge, only one available study is specifically focused on proteinuria impact on transplants from Extended Criteria Donors, although on a relatively small sample size and without comparison with standard donors .Aim of this study was to analyze the impact of different degrees of 1-year proteinuria on patient and graft survival in kidney transplants from different donor ages and to evaluate the correlation between proteinuria and donor or recipient risk factors with graft loss. A secondary aim is to evaluate proteinuria as risk factor for kidney graft survival in multimodal models taking into account renal function and other main clinical variables.We performed a retrospective observational cohort study including all deceased donor grafts performed at Turin University Renal Transplant Center “A. Vercellone” from January 2003 to December 2013. The study was submitted and approved by the local ethics committee . To exclude confounding factors and homogenize study populations we excluded multi-organ grafts analyzing the remaining 1127 consecutive kidney transplants (KT).15. CNI levels were targeted on the basis of patients’ characteristics, KT time and transplant eras; for the vast majority of patients, target plasmatic tacrolimus levels were: 10–15 ng/ml in the first two months, 8–10 ng/ml up to the sixth month, 5–8 ng/ml up to the first two years. Steroid was tapered to 5 mg/day within 45–60 days and subsequently withdrawn in selected patients. Micofenolate dose reduction and discontinuation were performed when appropriate after hospital discharge. Mammalian target of Rapamycin-inhibitors (mTORi), combined or not with low doses CNI, were used in selected cases  with a switch generally after the sixth post-transplant month.Immunosuppressive regimen differed according to different transplantation eras and different populations. Briefly, the most adopted schedules were: induction with steroid boluses, rapidly tapered to 20 mg/day of oral prednisone and two doses of anti-CD25 antibody . Subjects at high immunological risk  were treated with antithymocyte globulin . Maintenance therapy was generally based initially on a triple-drug protocol. Calcineurin inhibitor (CNI), either tacrolimus (80% of cases) or Cyclosporine, was associated to Mofetil Micofenolate /Micophenolic Acid  or Azathioprine and prednisone. In KT from Extended Criteria Donors, CNI administration was, in most cases, delayed until serum creatinine reached 2.5 mg/dl to reduce the impact of nephrotoxicity, as we previously publishedThe recipients’ follow-up was performed with scheduled clinical visits for the entire follow-up and hospital admissions when major complications occurred. Data were collected from patients’ individual charts: creatinine and proteinuria (in 24-h urine collections) were assessed at discharge, at 3, 6 months and at 1, 2 and 5 years after transplantation. Renal allograft function (eGFR) was estimated by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. Pre-transplant donor biopsies were performed on the basis of a multidimensional assessment including macroscopic appearance, renal function, donor comorbidities and echographic characteristics [Post-transplant renal biopsies were performed for cause . A single group of trained pathologists, through the whole study period, analyzed all pre-transplant and post-transplant kidney biopsies. Follow-up ended on November 2017. Karpinsky score was used to define suitability for single or dual KT or refusal of organs.Patients were divided in 3 groups according to donor age: group A (< 50 years), group B (50–69 years), group C ≥ 70 years.. The outcomes were analyzed for patients with at least 1 year of follow-up according to 1-year post-KT extent. To eliminate confounding factors, as native kidneys proteinuria, we mainly explored proteinuria values after the sixth post-KT month; when comparing 6-month and 1-year proteinuria (1-year PTO), the latter showed a better correlation with death censored graft survival (DCGS) with an AUC 0.64 vs 0.59 . On the basis of median value of proteinuria in our cohort we further analyze low-grade proteinuria (between 0.2 g/day and 0.5 g/day). Similarly,eGFR value at 1-year post-KT showed the best correlation with DCGS (AUC 0.74) ; primary outcomes were evaluated in this subset as in general cohort.“Declaration of Istanbul on Organ Trafficking and Transplant Tourism”.The study was submitted and approved by the local ethics committee  and was performed in adherence with the last version of the Helsinki Declaration. All patients allowed to have their clinical data collected for study purposes by written consent. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the X2 or, for small samples, with Fisher’s exact test. The distribution of continuous variables was analyzed with Kolmogorov-Smirnov test. Continuous variables were described as mean ± standard deviation when normal and median with 25°-75° percentile when non-normal distributed. Mann-Whitney, Kruskal-Wallis, t-test or variance analysis with Bonferroni post hoc test were used when appropriate to analyze difference between groups.Discrete data were described as percentages and analyzed with Pearson’s Receiving operating characteristic (ROC) curve and under the curve area (UCA) were used to illustrate the diagnostic ability of parameters chosen. We used UCA to determine the proteinuria time point with the strong association with DCGS.Cumulative graft and patient survival were analyzed by Kaplan-Meier (KM) curves.p-value < 0.05 as entry criterion and p-value > 0.10 as removal criterion.We fitted univariate model for main clinically chosen covariates  infection) and we identified predictors as significant at level α = 0.05 (log rank test). Then we fitted a multivariate model with univariate predictors using stepwise with All statistical analyses were performed using Spss . Significance level for all tests was set at α <  0.05.Our analysis included 1127 kidney recipients, transplanted at Turin University Renal Transplant Center “A. Vercellone” between January 2003 and December 2013. The selected population was classified by donor age: group A (less than 50 years old) including 339 patients, group B (50–69 years old) including 496 patients and group C  including 292 patients. Main donor, recipient and transplant-associated characteristics are shown in Table p = 0.03).Assuming 0.5 g/day as proteinuria cut-off, the association of 1-year PTO with DCGS and graft survival was present for all donor age classes Table ; the impp = 0.09) Fig. As we noticed that median value of proteinuria in our population was nearly 0.2 g/day, we explored the impact of low grade proteinuria (0.2–0.5 g/day) compared with proteinuria < 0.2 g/day in the whole cohort and in different donor ages. In the low grade proteinuria group univariate analysis did not show any significant association of 1-year PTO with patient and graft survival and DCGS at any donor age. Yet, a definite (not significant) trend was evident for donors ≥70 years, regarding graft and DCGS , together with various factors .In order to investigate whether other donor factors could be related with post-KT proteinuria, Karpinsky score was evaluated when pre-implantation biopsies were available . Moreover we analyzed distribution of total Karpinsky score in different one-year proteinuria groups (< or ≥ 0.5 g/day) without finding significant differences , while a higher glomerulosclerosis score showed a good correlation with a higher 1-year proteinuria (p = 0.04). Nevertheless, total Karpinsky score as well as glomerulosclerosis score were not associated with DCGS differences.In particular, regarding histology, we analyzed the distribution of total Karpinsky score in recipients of single KT and in different donor age groups finding a significant difference : in 44.0% of patients proteinuria increased between these 2 time points while in 56.0% it remained stable or decreased. Median positive variation of proteinuria was 0.12 g/day  while median negative variation was 0.05 g/die (22.4% of patients had a decrease of proteinuria ≥0.1 g/day). Positive/negative variation values were comparable for different donor age groups.p = 0.71)  Fig. . On the Again, this correlation was stronger when graft and DCGS were considered in recipients of elderly donors Fig. . The difn = 558) we identified 0.1 g/day as 6mo-1 yr proteinuria cut-off with the best association with DCGS (UCA 0.68).Including only subjects with 6mo-1 yr proteinuria stable or increasing (p <  0.01). That significant difference was confirmed irrespectively of donor age.Patients with increase in 6mo-1 yr proteinuria higher than 0.1 g/day had lower DCGS if compared with patients with lower increase . In the remaining patients, proteinuria impact (≥ 0.5 g/day) was not associated with significant variation in DCGS in the different age classes in univariate analysis (data not shown).To test the role of a potential confounding factor, the same analysis was performed excluding patients mTor inhibitors treated at one year post transplantation (Early adverse events (during first post KT year) were evaluated in the different proteinuria subpopulations Table . As expeTo compare proteinuria and creatinine impact on graft survival, 1-year eGFR was also evaluated. 1-year eGFR was strongly associated to patient, graft and DCGS   were analyzed.p <  0.01. This impact was not relevant in recipient of donors < 50 years while was greater with donors ≥50 years Fig. Also In this case a great impact on DCGS was found: DCGS was respectively 86.6% for recipients with proteinuria < 0.5 g/day and 51.9% for the twin kidney with proteinuria > 0.5 g/day; For the general population, the risk of adverse outcomes  increases with higher levels of albuminuria so that it was included in 2012 Kidney Disease Improving Global Outcomes guidelines as key marker for chronic kidney disease (CKD) .Numerous studies in patients with diabetic and non-diabetic renal diseases confirm that marked albuminuria (> 300 mg/day) is associated with a faster rate of CKD progression. On the contrary, moderate-level albuminuria (150–300 mg/day) is not a reliable surrogate marker for CKD progression in intervention clinical trials because reduction of albuminuria can be linked to both improving and worsening CKD progression , 18.The deleterious impact of proteinuria, at relatively early post-KT time, on long-term outcome was clearly demonstrated in several previous studies , 9, 14. Evaluation of risk factors related to post-KT proteinuria was attempted in several studies finding a multitude of different donor-related, recipient related or transplant related factors including, among others, delayed graft function, greater body mass index at transplant, older donor age, greater HLA mismatch, tacrolimus use and antihypertensive use , 20.Beyond the causes, however, the intrinsic risk of developing proteinuria at a relatively early stage (first post-KT year) should be addressed as a key risk factor for transplant outcome .Our study demonstrated that occurrence of proteinuria,(≥ 0.5 g/day), at the first post-transplant year, was significantly linked to graft survival and patient survival in the whole population under study. For the first time in literature, to the best of our knowledge, the impact of proteinuria on KTs from different donor age classes was analyzed, demonstrating a synergic effect of proteinuria ≥0.5 g/day and donor age ≥ 70 years on DCGS.Very low grade proteinuria (between 0.2 and 0.5 g/day) was not related to outcome. Nevertheless, we demonstrated an association  between DCGS and low-grade PTO (≥ 0.2 <  0.5 g/day) only in kidneys from donors older than 70 years old. Fig. .Older donor kidneys seemed more sensitive to proteinuric injuries in comparison with younger ones with very relevant differences of DCGS in different donor age classes with same proteinuria. This is also highlighted by the fact that any variation of proteinuria between 6 months and 1 year post-KT portends a worse graft outcome when donor age was ≥60 years Fig. .With ageing, kidney undergoes through processes that lead to reduced functional reserve and also to a lower renal reserve response to higher functional requests (e.g. protein load) due to a reduced capacity of renal autoregulation –25. ThesPredisposition to proteinuria development in our population appeared to be certainly related to both donor and recipient characteristics  but, as it is demonstrated by paired kidney analysis, early post-KT events  contribute to determine kidney fate and prognosis. As shown in Table It is well known that kidney allograft function in a stable condition  is an important predictor of graft failure , 32. OneAmong the other clinical variables, CMV viremia post-transplant resulted as an independent predictor of DCGS in Cox multivariate analysis (HR 2.1), as mentioned in previous studies , 35. As Need for surrogate endpoints to enhance trial feasibility has been outlined by a recent review where proteinuria has been defined as a prognostic biomarker .In the current scenario, the majority of available organs are represented by “suboptimal” donors (formerly known as ECD or with a Kidney Donor Profile Index greater than 85% according to the recent USA definition). Our study demonstrated prognostic significance of proteinuria, in particular with this kind of donors.The link among proteinuria, donor age and subsequent higher proteinuria-mediated damage in older donors is an important issue of our study. Several mechanisms were advocated for proteinuria mediated tissue damage such as intratubular complement activation , 38, intAs mentioned in previous studies and confirmed by our data, the evidence suggesting a benefit for ACEi/ARB use in transplant recipients is still lacking. They showed that the use of these agents was often associated with a significant reduction in proteinuria and eGFR without a concurrent improvement of patient or allograft survival as it does in non-transplant settings such as in diabetic nephropathy , 44. ThiIt is well known that Mtor-inhibitors may induce proteinuria by targeting glomerular podocytes . This isOur study has some strenghts and some limitations. Strenghts of the study are related to homogeneity of the population, characterized by a wide range of data coming from over a thousand of KTs performed with the same team of surgeons, nephrologists and pathologists. Patients were centrally followed in the long term with all data recorded in patients’ charts.Another strength, in our opinion, is the validation of prognostic impact of proteinuria in a subset of paired kidneys, thus limiting undetermined donor-derived confounding factors .We acknowledge that a limitation of this study is the absence of protocol graft biopsies for center policy; however, this limitation reduces its importance when we consider that proteinuria impact was shown by some authors to be independent from the underlying renal allograft histology .Other limitations are: absence of routinely donor specific antibody evaluation in the first year, which was available only in a minority of patients, so that we did not evaluate our population under this aspect; moreover lack of qualitative differentiation of urinary protein, considering that tubular or glomerular proteinuria could have different impact on graft outcome, as underlined in previous studies , 48, 49.If it is unlikely that a single noninvasive biomarker will yield a high predictive performance for graft loss, it is however auspicable a correlation of post-KT proteinuria trajectories with clinical events to guide clinical measures. Clinicians often do not know how to handle early low-grade proteinuria given the fact that it is often considered as aspecific, possibly due to native kidneys residual function (in the early period). Our study clearly evidence that proteinuria is always deleterious to transplant outcome even at early follow up time point. Based on our results we suggest that, in the context of elderly donors and in the absence of acknowledged effective pharmacological tools, when other causes of proteinuria are excluded , close monitoring of proteinuria should be repeatedly performed. Dealing with immunologic “low-risk” patients, where utility of protocol biopsy is still debated, the presence of proteinuria in the first year at a relatively low extent (0.5 g/day), even in the absence of donor specific antibodies, should suggest a careful evaluation of patients leading to for-cause biopsy. Utility of protocol biopsy in patients with non-standard donor, even in the absence of risk factors, should be ascertained by further studies.