Under-and extra-immunization of tetanus boosters are important issues to consider in reducing the burden of vaccine-preventable disease in adults. The present study aimed to analyze the trend of vaccination coverage (VC) and risk factors associated with extra-immunization of tetanus during an 8-year period using a national-scale cohort database. Taiwan’s one-million representative research database, the Longitudinal Health Insurance Database (LHID2005) was used. A total of 771,443 adults aged between 20 and 79 years were enrolled and followed from 1 January 2006 to 31 December 2013. VC at the beginning was as low as 35.1%, declining gradually and dropping to 33.9% at the end of follow-up. While a total of 303,480 tetanus boosters were used during the study period, more than half (55.5%) of these boosters were considered as extra-immunized. Both individual characteristics and visit characteristics were strongly associated with extra-immunization. Males, young and older adults, and those with a higher number of comorbidities were more likely to receive extra-immunization boosters, especially when they had more severe symptoms, visited an emergency room, or visited a hospital with lower accreditation levels located in a less urbanized area. This information could enhance implementation of evidence-based programs for tetanus boosters. Tetanus-related death is a rare but increasing concern among adult population nowadays while recent surveys have shown that immunity to tetanus continues to wane among adults, making tetanus a continuing threat with increasing age in adults ,2,3. RouExtra-immunization against tetanus, that is, administering more than the required amount of regular booster or at a shortened interval regardless of previous vaccine status, has caused both safety and economic concerns even though continuous efforts have been made to improve vaccination coverage among adults . AlthougThe present study aimed to analyze the change in vaccination coverage (VC) and risk factors associated with extra-immunization of tetanus during an 8-year period using a national-scale cohort database. The National Health Insurance Research Database (NHIRD) of Taiwan, covering the entire population of 23 million insurants and accumulated since 1997, provided a unique opportunity to observe the long-term change in the immunization status of tetanus of the whole cohort [Since 1995 Taiwan’s Government has implemented a National Health Insurance (NHI) program, which provides unrestricted access to medical care and universal health insurance for all residents in Taiwan. The NHI program has comprehensively enrolled 99% of the Taiwanese population and contracted with 97% of the providers of healthcare services in Taiwan. The data was named NHIRD and has been released to the public for research purposes after a vigorous encryption de-identification and anonymization process. The NHIRD contains comprehensive information on the insured subjects, including gender, date of birth, dates of clinical visits (both preventive services and emergent visits), the International Classification of Diseases (Ninth Revision) Clinical Modification (ICD-9-CM) codes of diagnoses, details of prescriptions, expenditure amounts, and characteristics of health providers, etc.This study was initiated after approval from the Institutional Review Board of the National Yang-Ming University Hospital, Taiwan (NYMUH IRB No. 2014A020) and the National Yang-Ming University, Taiwan. The Institutional Review Board waived the requirement for written informed consent from each of the patients involved since all identifying personal information in NHIRD is encrypted.This was a population-based retrospective cohort study. A representative set of NHIRD called the Longitudinal Health Insurance Database (LHID) was used as the study cohort. The LHID2005 contains one million people who were sampled from the whole population  registered in Taiwan at the end of 2005. The LHID2005 comprises anonymous, comprehensive and representative data on the health care of the entire Taiwan population and the medical history of each enrolled individual can be traced back to 1995, which provides an extraordinarily comprehensive immunization history over at least ten years.We enrolled all adults aged between 20 and 79 years on 1 January 2006, when follow up of the cohort was initiated. For each selected individual, we extracted his/her history of tetanus vaccination (number of doses reimbursed and age at delivery) and followed up until 31 December 2013, the end of the study, or the date out-of-cohort (death or emigration) depending on which came first. We were also able to trace each subject’s prior vaccination history for up to 10 years before enrollment  using LHID2005.In the current study, we defined tetanus boosters as inclusive of all tetanus toxoid-containing vaccines, including tetanus toxoid (TT) vaccine, tetanus and diphtheria (Td) vaccine and tetanus, diphtheria, and pertussis (Tdap) vaccine administered to recipients aged 20 years and over. Annual administration rates of tetanus boosters were calculated by dividing the total doses of administered tetanus boosters by the number of the target population.The participants were classified into either of the following groups according to their immunity status against tetanus: “live with effective immunity’” and “live without effective immunity”. We assumed that participants gained effective tetanus immunity, (that is they became live with effective immunity), immediately following the date they received a tetanus booster. According to previous large serology studies, we also assumed that immunity waned after ten years equally for every tetanus booster recipients ,21. As aVaccination coverage (VC) was calculated by dividing the number of adults live with effective tetanus immunity by the number of the target population and 95% confidence intervals were also estimated.Tetanus boosters were categorized as either an effective booster or an extra-immunized booster, according to the recipient’s tetanus immunity. A tetanus booster was considered extra-immunized if the recipient already had effective tetanus immunity from a recent booster within the recommended interval (10 years with a 3-month overlap). To further assess risk factors associated with extra-immunization, recipients’ demographics as well as characteristics of visits for tetanus boosters were further analyzed. Subjects’ co-morbidities were categorized according to Elixhauser’s co-morbidity model by the presence of either diagnostic codes in the outpatient records or discharge codes in the database within two years before the date of the visit . The sevAll of the data were linked using the SQL server 2017  and analyzed by Stata software . The annual administration rate of tetanus boosters and VC were calculated to demonstrate the long-term change of VC in the study cohort. A logistic regression model was used to assess risk factors associated with extra-immunization. Adjusted odds ratio (AOR) for extra-immunization for each factor was estimated by controlling other factors in the model. The predicted probability of receiving extra-immunized tetanus boosters was calculated for individuals of each characteristic as they were average on all other characteristics. The base probability was calculated for individuals who were average on all characteristics. The probability of extra-immunization and the difference from the base probability of extra-immunization, both expressed in percentage, were calculated to quantify the effect of levels of each factor. A two-tailed level of 0.05 was considered statistically significant.The study cohort enrolled 771,443 adults aged between 20 and 79 years and the entire cohort was followed up from 1 January 2006 to 31 December 2013. Participants were completely observed throughout the study period with a minimal loss .During the 8-year follow up, the entire cohort received a total of 303,480 doses of tetanus booster with an administrative rate at 39.3%. Only one out of twenty adults received a tetanus booster every year, the annual administration rates decreased gradually from 5.4% to 4.8%. Moreover, more than half  of these boosters were considered as extra-immunized, which are excessively administered to recipients who already have effective tetanus immunity from recent boosters within the past ten years. As a result, the VC declined slightly with aging of the entire cohort and it finally dropped to 33.9% at the end of follow up, 1.2% lower than the beginning of the study .To further assess the risk factors for extra-immunization boosters, we analyzed 303,480 visits for tetanus boosters made by 214,847 recipients during the observation period. Extra-immunization boosters represented a considerable portion of total visits . Half of the recipients  were extra-immunized with only a few recipients administered with more than 2 boosters during the study period. A logistic regression model showed that both individual characteristics and visit characteristics were significantly associated with extra-immunization boosters; males, young and older adults, and those with an increased number of comorbidities were more likely to receive extra-immunization boosters especially when they had more severe symptoms, visited an emergency room, or visited a hospital with lower accreditation levels located in a less urbanized area .There is a tendency for extra-immunization boosters to be frequently used in urgent situations. The emergency room was the place most prone to give extra-immunization compared to out-patient-departments (the probability of extra-immunization for an ER visit was 57.9% vs. 53.4% for an OPD visit). Moreover, the probability of extra-immunization during the most urgent visits was 4% higher than non-urgent visits (immediate resuscitation visits were 58.7% vs. 54.7% for non-urgent visits) .It appears that hospital-related factors are linked to extra-immunization. Visits to academic medical centers and metropolitan hospitals are less likely to result in extra-immunization compared to local hospital and clinics. The degree of urbanization of the location of the hospital may decrease the probability of extra-immunization (most urbanization 49.7% vs. least urbanization 62.3%) .Both under-immunization and extra-immunization are a continuing threat to adults and the preventive healthcare system. While one’s effective immunity against tetanus usually wanes every ten years if there is no routine decennial tetanus boosters, one could also easily be extra-immunized because of the lack of an accurate immunization history. Epidemiologic information from large-scale observational studies is needed. The current study uniquely followed the dynamic change in effective immunity against tetanus (in terms of VC) of 771,000 adults aged between 20 and 79 years for eight years. We found that the effective immunity of the entire cohort degraded gradually despite the ready use of tetanus boosters at the point of wound care. Moreover, a large portion of tetanus boosters were extra-immunized. Both individual characteristics and visit characteristics were strongly associated with extra-immunization.Our study provided convincing support for detailed tetanus booster programs for adults. Relying solely on tetanus boosters at the point-of-care is not sufficient to counter waning tetanus immunity. In our study on the change in VC, we showed that even though Taiwan has readily accessible and affordable medical services, their contribution to protection against tetanus is still minimal; furthermore, more than half of the administered boosters were extra-immunized. Therefore, we suggest that future immunization strategies should be stratified by age and gender. The current study showed a clear age and gender gap in the administration of tetanus boosters. This discrepancy may be explained by patients’ misconceptions about immunization recommendations as well as attitudes towards vaccinations . TherefoUnderstanding the factors that contribute to extra-immunization is crucial in reducing its incidence. To our knowledge, this is the first study to investigate the occurrence and factors for extra-immunization using a large and comprehensive database. It had been reported that there are higher rates of extra-immunization in vulnerable and at-risk populations in the US, including those in underprivileged racial/ethnic minority groups . In the Centralized immunization registries could facilitate tetanus immunization for adults. Immunization history from patients can often be misleading because of incorrect memory . In TaiwA claim or reimbursement database, could be an effective alternative to resource-consuming surveys that are commonly used in traditional immunization programs. Our study represents a novel approach for reusing routinely collected data. Since it is impossible to measure serum protective tetanus antitoxin level for the whole population, by assuming the efficacy of tetanus according to previous seroepidemiology studies, we were able to closely observe the vaccination coverage trends through the use of vaccine reimbursement data. This new method represents an innovative approach to provide timely, updated, reliable immunization information that enables program managers to do real-time monitoring, investigate potential problems and take appropriate remedial action to improve performance.Our results provide compelling evidence for long-term observation of the trends in tetanus effective immunity of an entire cohort. Additionally, our study offers an understanding of incidence and risk factors for extra-immunization. Both would enhance implementation of evidence-based programs for tetanus boosters. However, some limitations are worth noting. First, the current study assumed that effective immunity against tetanus wanes after ten years equally for all particpants in the cohort. This would underestimate vaccination coverage but overestimate extra-immunization in young adults and middle-aged adults. A more sophisticated mathematical model should be considered for accurate estimation. Second, detailed clinical notes are not available in our database. Thus, in the present study we were not able to determine the appropriateness of tetanus boosters for poor titers, immuno-compromised patients or severe wounds. As a result, extra-immunization could be slightly overestimated. Third, some persons may purchase their vaccine without claiming reimbursement. In Taiwan’s national health insurance scheme, everyone has equal and full access to wound care as well as to vaccination services. Thus, this issue is minimized in the current study. Fourth, errors/duplications in record keeping  involving vaccination dates or doses may underestimate extra-immunization rates. A field study of immunization status among such minority/underprivileged groups is warranted.The current study provides compelling evidence about the trend of effective immunity and risk factors for extra-immunization by following a representative cohort. Vaccination coverage declines gradually without a universal immunization program. A high percentage of extra-immunization tetanus boosters were noted. Both individual characteristics and visit characteristics were strongly associated with extra-immunization. This information would enhance the implementation of evidence-based programs for tetanus boosters.


Choline is critical for infant development and mother’s milk is the sole source of choline for fully breastfed infants until six months of age. Human milk choline consists to 85% of water-soluble forms of choline including free choline (FC), phosphocholine (PhosC), and glycerophosphocholine (GPC). Donor milk requires safe handling procedures such as cold storage and pasteurization. However, the stability of water-soluble forms of choline during these processes is not known. The objectives of this research were to determine the effect of storage and pasteurization on milk choline concentration, and the diurnal intra- and inter-individual variability of water-soluble choline forms. Milk samples were collected from healthy women who were fully breastfeeding a full-term, singleton infant <6 months. Milk total water-soluble forms of choline, PhosC, and GPC concentrations did not change during storage at room temperature for up to 4 h. Individual and total water-soluble forms of choline concentrations did not change after storage for 24 h in the refrigerator or for up to one week in the household freezer. Holder pasteurization decreased PhosC and GPC, and thereby total water-soluble choline form concentrations by <5%. We did not observe diurnal variations in PhosC and total water-soluble forms of choline concentrations, but significant differences in FC and GPC concentrations across five sampling time points throughout one day. In conclusion, these outcomes contribute new knowledge for the derivation of evidence-informed guidelines for the handling and storage of expressed human milk as well as the development of optimized milk collection and storage protocols for research studies. S-adenosylmethionine, which is the main methyl donor involved in creatine and phosphatidylcholine synthesis, and DNA methylation, among other biochemical reactions [Choline is an essential nutrient with crucial roles in brain function, neurodevelopment, and growth ,2. Bioloeactions . Cholineeactions .Exclusive breastfeeding ad libitum is the recommended feeding practice for the first six months of life, with continued breastfeeding up to two years of age . Human mThe practice of human milk pumping and storage for later use is on the rise both at home and in clinical settings to accommodate various situations when feeding human milk at the breast is not possible, e.g., mothers returning to work; insufficient volume of mother’s own milk, and need for donor milk ,15. CurrWater-soluble forms of choline concentrations in human milk may vary within and between women. Higher maternal dietary choline intake increases milk concentrations of water-soluble, yet not fat-soluble, forms of choline, as shown in a 12-week dose-response feeding study and a supplementation trial from 18 gestational weeks to 90 days postpartum ,13. No cThe objectives of this research were to determine the effect of cold storage and pasteurization on the concentrations of water-soluble forms of choline in human milk, and to determine the intra- and inter-individual variability of water-soluble forms of choline concentrations in human milk within a day. Water-soluble forms of choline seem stable during short-term cold storage and most forms remained unchanged after four hours of storage at room temperature and six months of storage at ultra-low freezing temperatures, as employed in research settings. Holder pasteurization significantly impacted milk concentrations of water-soluble forms of choline, but to a small extent that is outweighed by the microbiological safety benefits of pasteurization. We observed intra-individual variability of individual but not total water-soluble forms of choline throughout one day, and recommend standardized sampling protocols for research studies.The research consisted of two cross-sectional studies, i.e., the stability and variability study, as well as the secondary analysis of bio-banked milk samples for the pasteurization study. For all studies, healthy women who were exclusively breastfeeding a healthy, full-term and singleton infant <6 months of age were eligible to participate. Exclusion criteria included: suffering from diabetes mellitus Type 1 or 2, or any chronic disease involving fat metabolism, taking routine medications known to influence fat metabolism, or consumption of more than 1 alcoholic drink per day.A convenience sample of 6 mothers of 2- to 6-month old infants was included in this study. Women were recruited through active and passive recruitment methods in the Greater Vancouver area in 2014. Signed informed consent was obtained prior to enrolment. Information on maternal and infant age, sociodemographic status, supplement use, and general infant health, was collected using a self-administered questionnaire. Ethical approval was granted by the University of British Columbia and the British Columbia Children’s and Women’s (C&W) Hospital Research Ethics Board (H12-03191).Women provided a fresh complete milk expression collected in the morning (between 9:00–10:00 a.m.) using a commercial pump (Medela); samples were immediately transferred on ice to the lab to be aliquoted and analyzed. One milk aliquot was analyzed immediately, with the rest of the samples being stored at different temperatures for different durations see , prior tMid-feed human milk samples (milk collected after breastfeeding or pumping milk for approximately 3 min) from mothers of 1-month old infants were used in the pasteurization study; these bio-banked samples were derived from a completed, randomized controlled trial studying the effect of DHA supplementation during pregnancy on the cognitive and visual outcomes on infants . The stuTwenty women were enrolled in this cross-sectional study. Recruitment and enrolment procedures were similar to those described for the stability study. Sociodemographic, health and supplement use data were collected using the same self-administered questionnaire as in the stability study. Ethical approval was granted by the University of British Columbia and the British Columbia Children’s and Women’s Hospital Research Ethics Board (H12-03191).Each participant provided 5 mid-feed milk samples at different times throughout one day. For feasibility purposes, the five different time points were flexible as follows: before breakfast, before lunch, 45–60 min after lunch, 45–60 min after dinner, and before bedtime. Participants were instructed to place the vials in their home freezers immediately after pumping. Frozen samples were transferred to the lab on ice the day after collection and stored at −80 °C for 2 weeks, when choline analysis was completed.g at 4 °C for 10 min, transferred to an autosampler vial and mixed with acetonitrile with 0.1% formic acid in dilutions of 1:5. The inter-assay and intra-assay coefficient of variation (CV) based on 5 replicates were as follows: For FC, 5.5% and 4.1%; for PhosC, 6.4% and 5.2%; and for GPC 9.5% and 2.3%; respectively.Concentrations of water-soluble forms of choline in milk were determined using isotope dilution liquid chromatography tandem mass spectrometry as previously described . In briep values < 0.05.Participant characteristics are presented using descriptive statistics. Normality of data distribution was assessed using Shapiro–Wilk test. Differences in milk concentrations of water-soluble forms of choline under different storage conditions were determined using the related-samples Friedman test, followed by Wilcoxon signed-ranks test as post-hoc analysis and adjusted using the Bonferroni correction. Differences in water-soluble forms of choline concentrations after pasteurization were determined using the Wilcoxon signed-ranks test. Intra-individual variability in water-soluble forms of choline concentrations was determined using the related-samples Friedman test, followed by Wilcoxon signed-ranks test as post-hoc analysis and adjusted using the Bonferroni correction. Analyses were performed using the IBM SPSS statistics software . Level of significance was set at The characteristics of women included in each study are summarized in The concentration of total water-soluble forms of choline, PhosC, and GPC did not significantly change during storage at room temperature for up to 4 h . CompareWe observed no changes in total water-soluble forms of choline and PhosC concentrations independent of condition and duration of cold storage, including in the refrigerator at 4 °C for 24 h, in the freezer at −20 °C for up to 1 week, and in the ultra-low freezer at −80 °C for up to 6 months . The conTotal water-soluble forms of choline concentrations significantly decreased by approximately 5% after Holder pasteurization in 33 milk samples . This dep = 0.027) and a 12.2% decrease in GPC from T1 to T3 (p = 0.027).Total water-soluble forms of choline and PhosC concentrations did not vary significantly within a woman during the day based on analysis of five milk samples per mother . HoweverAdditionally, as shown in In this study, we report three findings on the stability of water-soluble forms of choline in expressed human milk: First, storing human milk for 3 or 4 h at room temperature significantly increases milk FC concentration. Second, thawing human milk after 6 months of freezing at −80 °C significantly increases FC and GPC concentrations. Third, Holder pasteurization decreases PhosC and GPC concentrations, and thereby lowers total concentration of water-soluble forms of choline. Additionally, we report that total concentration of water-soluble forms of choline does not underlie diurnal variations within a woman; however, significant diurnal variations in FC and GPC concentrations may occur among some, but not all, women. These findings contribute to the literature being harnessed to develop evidence-informed guidelines for the handling and storage of expressed human milk as well as the development of optimized milk collection and storage protocols for research studies.Milk expression for in-home use or human milk banking have become increasingly recognized as a first best alternative to direct breastfeeding. While ensuring microbiological safety of expressed milk remains a top priority, maintaining milk’s bioactive and nutritional quality is an important weighing factor in developing milk handling and storage guidelines. The impact of storage and pasteurization on the immunological properties, digestive enzymes, antioxidant capacity, and macro- and micronutrient composition in human milk has been recently reviewed ,27,28,29S-adenosylmethionine [The increase in FC concentration may be explained by the enzymatic breakdown of the lipid-soluble choline compounds phosphatidylcholine and sphingomyelin. The presence of these phospholipids within the milk fat globule membrane, a complex tri-layer of proteins and lipids, poses an analytical challenge for their accurate quantification . In addithionine . Additiothionine . To dateThe cold storage conditions we tested are relevant to home and clinical use  and to storage conditions in research settings (ultra-low freeze storage at −80 °C). Our findings suggest that short-term storage of milk aliquots in the refrigerator (at 4 °C) for 1 day and in the home freezer (at −20 °C) for up to one week and long-term storage at −80 °C for up to 6 months does not alter the total concentration of water-soluble forms of choline in expressed human milk samples. However, milk FC and GPC concentrations increased in samples that were thawed after six months of ultra-low freeze storage . The increase in FC and GPC concentrations after prolonged freezer storage may be explained by the breakdown of lipid-soluble forms of choline, similar to the increase in FC concentration in milk stored at room temperature for 4 h. Similar changes were observed in serum samples that were inappropriately processed and stored, with increasing concentration of total water-soluble choline concentration . We wereTo our knowledge, this study is the first to test the effect of pasteurization on water-soluble forms of choline concentrations in human milk. We showed that Holder pasteurization lowered milk PhosC and GPC concentrations by about 5%, and thereby total water-soluble forms of choline concentration by 3%. Limitation of our study was the use of previously frozen milk samples, for 5–9 years at −80 °C, which is not consistent with clinical practice of pooling human milk and it undergoing pasteurization. We used biobanked milk samples because of low milk volumes collected in the stability and variability study, as well as to reduce participant burden. Additionally, for this preliminary study, we were mainly focusing on the comparison of choline concentrations between before and after pasteurization. We acknowledge that the long-term storage at −80 °C may have affected the milk concentrations of the water-soluble forms of choline; however, the total water-soluble choline concentration in the pre-pasteurized samples was similar to that of the fresh milk samples in the stability study , as well as compared to those reported in the literature ,24,32. TConsidering the crucial benefits of pasteurization for microbiological safety, we evaluate the <5% decrease in milk concentration of water-soluble forms of choline as minor yet recommend the confirmation of our findings in a second and larger study. The impact of pasteurization on lipid-soluble choline compounds should also be investigated in future studies. If confirmed, our finding of a 5% decrease should reassure clinicians that the benefits of providing pasteurized human milk continue to outweigh the risk of nutrient losses. Indeed, despite previous reports of a decrease in several nutrients, including folate, vitamin C and B6, due to pasteurization ,25,26, tIn regards to the variability of water-soluble forms of choline concentration in expressed human milk, we observed diurnal changes in milk FC and GPC concentrations, but not in PhosC and total water-soluble choline forms. The total concentration of water-soluble forms of choline in expressed human milk we found was similar to the concentrations previously reported by our team for Canadian lactating women ,32 and bIn conclusion, we provide new information on the stability of water-soluble forms of choline concentration that will help in the development of evidence-based guidelines for the safe handling and storage of expressed milk samples. Because breast milk is the recommended sole source of nutrients, including that of choline, for infants under the age of 6 months, the handling of expressed milk needs to address microbiological safety as well as nutrient-protective needs. Further research is warranted on the effect of acute versus long-term dietary choline intake on the composition of choline forms in human milk as well as the metabolic and functional significance of individual choline forms on infant growth and development.


Systemic sclerosis (SSc) is a rare autoimmune disease, which is potentially lethal. The physiopathology of the disease is still incompletely elucidated although the role of fibroblasts, endothelial cells (ECs), immune cells. and the environment  has been demonstrated. This is an intractable disease with an urgent need to provide better therapeutic options to patients. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach thanks to the number of trophic and pleiotropic properties they exert. Among these, MSCs display anti-fibrotic, angiogenic, and immunomodulatory capacities that might be of interest in the treatment of SSc by acting on different processes that are dysregulated in the disease. In the recent years, the therapeutic effectiveness of MSCs has been demonstrated in different preclinical animal models and is being investigated in phase I clinical trials. Both allogenic and autologous transplantation of MSCs isolated from bone marrow or adipose tissue is being evaluated. The rationale for using allogenic MSCs in SSc, as well as in other autoimmune diseases, is based on the possibility that autologous MSCs might be altered in these diseases. In SSc, reports from the literature are controversial. Nevertheless, the role of the oxidative environment and of the crosstalk with neighboring cells (fibroblasts and ECs) on the functional properties of MSCs has been reported. Here, we review the preclinical and clinical data reporting the interest of MSC-based treatment in SSc and question the use of autologous or allogeneic MSCs in perspective of clinical applications. Systemic sclerosis (SSc) is a rare autoimmune disease, which affects most frequently middle-age patients with a prevalence ranging from 100 to 300 per million depending on the country. The pathophysiology of SSc is still not completely understood even though three main axes of dysfunction are reported: fibrosis, vascular activation and immune abnormalities. The disease is characterized by vascular damage and diffuse fibrosis, which mainly affects skin and lung tissues but heart and digestive tract could also be involved . One of There is no curative treatment to date. Only symptomatic treatments are commonly proposed to patients to alleviate pain and improve function. Novel therapeutic strategies are being envisaged among which mesenchymal stem cells (MSCs)-based therapy, which is currently under evaluation in the clinics. The first clinical trial has evaluated the safety of MSCs-containing autologous stromal vascular fraction (SVF)  but the in vitro. However, recent lineage-tracing experiments indicate that in vivo, pericytes do not contribute to other cell lineages  . In addilineages . These rMSCs are defined by 3 criteria, as proposed by the International Society for Cell Therapy: (1) plastic adherence, (2) expression of the cell surface markers CD73, CD90, CD105, and lack of expression of the hematopoietic markers CD11b or CD14, CD19 or CD79α, CD34, CD45, HLA-DR, and (3) capacity of differentiation into adipocytes, chondrocytes, and osteoblasts . These eThanks to this pleiotropic activity, the therapeutic efficacy of MSCs has been investigated in different pathological conditions, from inflammatory diseases to acute or degenerative diseases. So many different applications as rheumatic diseases , stroke Before the initiation of clinical trials, the interest of using MSCs in the treatment of SSc has been evaluated in different preclinical models. Three models have been used: the model of bleomycin-induced pulmonary fibrosis, the model of hypochlorous acid (HOCl)-induced SSc and the tight skin (Tsk1/+) mouse model of SSc.The model of bleomycin-induced fibrosis is the most widely used model to replicate scleroderma, or dermal or pulmonary fibrosis in mice or rats. According to the mode of administration, fibrosis can be induced in skin when bleomycin is injected daily sub-dermally or in lung after a single intra-tracheal instillation. Fibrosis in lung, skin, and internal organs may be induced via the use of osmotic pumps that deliver a constant amount of bleomycin over a number of days , 24. It The therapeutic potential of MSCs has been first demonstrated in the mouse model of lung fibrosis. One intravenous injection of 500,000 allogenic BM-MSCs on the day of bleomycin instillation was shown to reduce collagen content and inflammation in the lungs . At day The therapeutic effect of BM-MSCs has been also confirmed in rat models of bleomycin-induced lung fibrosis. One intravenous injection of five millions of BM-MSCs resulted in decreased levels of TGFβ1, platelet-derived growth factor-A (PDGF-A), PDGF-B, insulin growth factor-1 (IGF1), and in lower collagen content in lungs . AnotherIn a dermal fibrosis model induced by daily subcutaneous injection of bleomycin for 4 weeks, syngeneic BM-MSCs were injected subcutaneously into the lesion skin every day . TreatmeThe impact of MSCs from different species or tissues on treatment efficacy has been evaluated in the mouse model of bleomycin-induced lung fibrosis. Human UC-MSCs injected systemically 24 h after bleomycin instillation in SCID mice were reported to reduce inflammation markers, collagen content, and inhibit expression of TGFβ, interferon-γ (IFNγ), tumor necrosis factor-α (TNFα) . In this6cells/kg) or high (5.6 × 106cells/kg) doses of human BM-MSCs at day 7 and 15 after fibrosis induction did not lead to improvement of lung function or rescue of damage tissue  with murine or rat BM-MSCs on the day of bleomycin instillation resulted in a significant reduction of fibrotic changes that was not seen when BM-MSCs-based treatment was delayed (day 7) , 36. Sime tissue . Injectie tissue . In thise tissue . Injectie tissue . In addie tissue .The age of the donors may be another parameter that might change the therapeutic potential of MSCs. One study compared ASCs from aged (>22 months) and young (4 months) mice . At day For more a decade since 2003, the skin or lung bleomycin-induced model of local fibrosis was the single model used to evaluate the therapeutic effect of MSCs. However, it does not reproduce the main characteristics of the diffuse human SSc. With respect to the systemic nature of the disease, relevant models of systemic fibrosis have to be used to better evaluate the efficacy of MSCs in models closer to human SSc.vintegrin, TNFα, and miR-199-3p in lung and skin wounds. These results therefore support the hypothesis that ASCs may prevent systemic fibrosis and enhance wound healing but rely on the use of aged mice that are expensive and difficult to get.A study relied on the use of an established aged (18–22-month-old) mouse model of bleomycin-induced lung fibrosis to test the hypothesis that fibrosis may develop simultaneously in multiple organs and notably affects lung, skin and wound healing. Mice developed irreversible lung and skin fibrosis as well as delayed wound closure at day 21 . In this5 BM-MSCs. We also compared different sources of cells, i.e., allogenic, xenogeneic, and syngeneic BM-MSCs as well as xenogeneic ASCs, which seemed to be the most potent as compared to BM-MSCs  mouse occurred as a spontaneous mutation in fibrillin-1, which controls the biologically active levels of TGFβ through binding to the latent TGFβ binding proteins (LTBP) 1 and 4 . It has Altogether, the beneficial effect of MSCs-based treatment has been proved in the different models of local or systemic scleroderma Table . Some clThe capacity of MSCs to differentiate into several cell types of musculoskeletal tissues was the first property that has attracted the attention of researchers and clinicians . Since tSystemic administration of MSCs results in their homing in lungs and cell engraftment is increased in lungs from bleomycin-induced mice, in particular in areas of injury where they adopt an epithelial cell morphology and display anti-oxidative role , 48. Of in vivo administration or increasing their trophic action. Indeed, preconditioning of BM-MSCs before administration was shown to enhance their therapeutic efficacy. Namely, oncostatin M- or hypoxia-preconditioned BM-MSCs improved pulmonary respiratory function thanks to decreased inflammatory and fibrotic markers as compared to unconditioned BM-MSCs and this effect was mediated at least through the upregulation of HGF  have been identified in murine models of lung fibrosis , 51. Hown of HGF , 53. Pren of HGF . In orden of HGF . Similarn of HGF . Thioredn of HGF . Indeed,Early studies evaluating the phenotypic and functional characteristics of MSCs from SSc patients reported no major difference as compared with MSCs from healthy donors. Indeed, BM-MSCs from two patients with SSc have been compared to those of healthy donors 10 years ago . No majoBy contrast, other studies have reported some differences. As an example, MSCs isolated from the skin of scleroderma patients expressed more PDGF-R than those from healthy donors, resulting in the production of higher levels of ROS . NeverthA unique study has identified altered functions in SSc ASCs . No alteIn recent decades, fat tissue grafting has been used to treat skin atrophy or fibrosis thanks to its biocompatibility and property of filling but also to its high content in multipotent stem or progenitor cells with regenerative potential. The first report on the implantation of fat in hands of patients who presented Raynaud's phenomenon was published in 2014 . Among t7 intravenous administration of allogeneic haploidentical-related donor BM-MSCs. No adverse events were reported and 3 months after treatment, a significant decrease in the patient's painful ulcerations was measured. Vascular improvement in the blood circulation of hands and fingers was noticed but level of Scl-70 autoantibodies was not decreased. In 2011, the same team reported four supplementary cases of allogeneic BM-MSC systemic injection (0.22 to 1.8 × 106 BM-MSCs/kg bodyweight) (The first patient who received BM-MSCs in the treatment of progressive diffuse SSc has been reported in 2008 . A youngyweight) . Here ag5 ASCs/mL HA was done in a single area for each patient, either the face or the arm chosen with the patient consent. The procedure improved considerably skin fibrosis for four out of six patients and moderately for one patient. All patients showed arrest of local disease progression (100%), four of them presented regression of dyschromia (67%), five patients increased skin softening (83%), four patients showed better sensitivity (67%), and one patient reported erythema reduction (17%). The study demonstrated that ASCs could be successfully implanted locally in patients with severe disease, thus representing a good, feasible, and efficient cell-based soft-tissue augmentation strategy. Of interest, the same team thereafter compared autologous fat and ASC transplantation to evaluate clinical improvement of mouth opening in two groups of five patients  solution has been first tested in 2013 . Infiltrpatients . Both prThe results of the first open phase I clinical trial evaluating the interest of MSCs, and more precisely of MSC-containing SVF, on 12 SSc patients with severe functional hand handicap were published in 2015 . Injectiwww.clinicaltrials.gov). One trial has been completed but no result is available. Only one of those is recruiting in France since 2014. This is a phase I/II trial which assesses the effect of one intravenous injection of intrafamilial allogenic MSCs on 20 patients. The trial is still enrolling patients and data are not yet available. Another phase I/II clinical trial has started in November 2017. It evaluates the therapeutic effect of allogeneic BM-MSCs after injection in intramuscular areas of affected limbs in 20 scleroderma patients with digital ulcers and is recruiting (NCT03211793)  .In summary, data from case reports and clinical trials are scare and need to be taken with cautious in terms of efficacy Table . One queMany studies have now established the beneficial effect of the administration of BM-MSCs, ASCs or MSCs from other tissue sources in different preclinical models characterized by local or systemic fibrosis. Some evidence of safety and efficacy of MSC-containing SVF or culture expanded MSCs has been described from the clinics but efficacy needs to be further proved in phase II clinical trials that are ongoing. Both autologous and allogeneic MSCs from BM or adipose tissue are being assessed but the risk that the functional properties of MSCs isolated from SSc patients are altered is under debate. Contradictory results are reported in the literature but a number of reports discuss the reduction of the number of clonogenic cells, proliferative rate, differentiation, and angiogenic potentials. MSCs from SSc patients display a more mature and myofibroblast-like phenotype, probably related to microenvironmental signals dysregulated during the disease. They express higher levels of TβRII and TGFβ, which is released into the extracellular medium where it can act in an autocrine or paracrine manner. Moreover, the crosstalk between MSCs and ECs contribute to the altered expression of different molecules involved in angiogenesis, inducing a switch of perivascular MSCs toward a myofibroblast population, further supporting the fibrotic process. The finding that MSCs from SSc patients constitutively overexpress mediators involved in the fibrotic and angiogenic processes might indicate that MSCs are altered by the environment secondary to the onset of the disease or, that they might participate to the physiopathology of the disease. With respect to the use of autologous MSCs for clinical applications, further investigation on their functional properties is likely needed.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We describe a framework for creating a time-resolved group average template of the developing brain using advanced multi-shell high angular resolution diffusion imaging data, for use in group voxel or fixel-wise analysis, atlas-building, and related applications. This relies on the recently proposed multi-shell multi-tissue constrained spherical deconvolution (MSMT-CSD) technique. We decompose the signal into one isotropic component and two anisotropic components, with response functions estimated from cerebrospinal fluid and white matter in the youngest and oldest participant groups, respectively. We build an orientationally-resolved template of those tissue components from data acquired from 113 babies between 33 and 44 weeks postmenstrual age, imaged as part of the Developing Human Connectome Project. These data were split into weekly groups, and registered to the corresponding group average templates using a previously-proposed non-linear diffeomorphic registration framework, designed to align orientation density functions (ODF). This framework was extended to allow the use of the multiple contrasts provided by the multi-tissue decomposition, and shown to provide superior alignment. Finally, the weekly templates were registered to the same common template to facilitate investigations into the evolution of the different components as a function of age. The resulting multi-tissue atlas provides insights into brain development and accompanying changes in microstructure, and forms the basis for future longitudinal investigations into healthy and pathological white matter maturation. The preMRI has become the modality of choice for studying the developing brain in a non-invasive way. During the neonatal period, the human brain increases in size rapidly  and cereAcquisition, processing and analysis of neonatal diffusion data is challenging due to this modality's sensitivity to motion and the comparatively low signal to noise ratio, especially in neonates with relatively high overall free water content. Recent developments in diffusion MRI acquisition strategies now allow the routine collection of large amounts of data . This ma1.1Prior work in neonatal group-analysis of diffusion data aims to establish and improve inter-subject correspondence, increase sensitivity and robustness to large shape and intensity changes during development, increase specificity to tissue classes and white matter (WM) fibre tracts, and to build models with interpretable parameters with respect to biological tissue properties. A comprehensive review of diffusion models and analysis methods is beyond the scope of this manuscript, see  for a reNeonatal diffusion MRI studies often involve: the statistical analysis of aggregate values from manually drawn regions of interest , atlas-bDiffusion tensor imaging has been used widely to study white matter and brain development . HoweverRecently, the availability of multi-shell HARDI data has allowed the use of higher order diffusion models in the developing brain . There aresponse function derived from WM and orientation distribution function (ODF)-based groupwise registration  atlases have typically been created using the diffusion tensor model . HARDI a1.3A group-average template allows the spatial alignment of information across the cohort and facilitates subsequent group-level and subject-level analysis and processing . In geneTemporal patterns in foetal and neonatal brain MRI have been modelled based on image intensity, on deformations, or combinations thereof. In , a tempoFollowing registration, a template representative of the cohort can be created by (possibly weighted) averaging of the transformed images or via sparse patch-based image fusion techniques. Sparse template creation techniques can reduce blurring due to anatomical variability or misalignment but possibly introduce inconsistencies between adjacent or distant patches. Shi et al. use group sparsity constrained patch-based representations to recover anatomical detail beyond that achievable with image registration . An exte1.4In this work, we describe a framework for building an unbiased, time- and orientation-resolved group average template of WM maturation based on advanced diffusion MRI methods, in a cohort of neonates scanned over a range of ages, during which large changes in brain volume, shape and contrast occur. We apply this framework on high-quality multi-shell HARDI data acquired as part of The Developing Human Connectome Project (dHCP)response functions to describe the signal ‘signature’ for each different tissue component. The image registration is driven based on two such tissue components  framework  and requmponents , 2017b, Our approach contributes to different aspects of the neonatal analysis pipeline. We use a rich, multi-component, orientation-resolved representation that captures the majority of the HARDI signal and can be used to infer fibre-specific information. To characterise the WM maturation in a fibre-resolved manner, we utilise a data-driven representation instead of a biophysically motivated diffusion model.http://brain-development.org/brain-atlases/.To demonstrate this multi-component decomposition and multi-channel registration, we create a population-specific atlas of the developing brain using state-of-the-art HARDI data sampled over four diffusion weighting shells from the dHCP. To our knowledge, this is the highest quality in-vivo neonatal HARDI atlas to date, offering an unprecedented time-span, angular and spatial resolution. Our framework is compatible with any commonly used analysis framework, in particular fixel-based analysis. Hence, our proposed framework allows fibre-resolved investigations of normal and abnormal brain development. The atlas and related data will be available from 22.1The cohort used for this atlas consists of 113 babies scanned as part of the dHCP. From all subjects available, subjects with known clinical abnormalities  and lesiWe chose to include subjects from the healthy appearing dHCP cohort in this work, and did so in a way that strives to minimise any bias of the weekly templates on the basis of gender, age since birth and anatomy. To ensure comparable anatomical variability across age, we selected 9 to 11 subjects per weekly template. If more datasets were available for a template, we ranked them using the following criteria and chose only the 11 best samples.The images were grouped so that the number of motion artefact free volumes per subject is maximised while minimising both the deviation from normal (age and gender-matched) birth weight and the age since birth. To better balance age range and gender-bias, subjects were assigned to up to two time points. See figures A.13 for plots of cohort age, weight and quality measures.2.2The multi-shell high angular resolution diffusion single-shot spin-echo echo-planar images were acquired on a Philips 3T Achieva scanner using a dedicated neonatal head coil  with a m2.3topup (eddy (N4 (The preprocessing of the data consists of: (i) removal of motion-corrupted volumes using a deep neural network classifier ; (ii) Matopup  (FSL) anup (eddy  (FSL); aeddy (N4  (ANTs).bet (Brain masks were generated using a combination of bet  (FSL) an2.4response functions to be defined, each of which characterises the signal for the corresponding tissue component within each b-value shell, along with its angular dependence. Response functions can be either estimated from the data , to minimise any potential influence on the estimated response functions. Any residual bias fields were subsequently corrected following MSMT-CSD by ensuring that the summed density of the three components has an average value of mtnormalise command available as part of MRtrix3  compared to registration using the native WM component only.We calculated the deformation fields for each subject by registering each subject's CSF component and single native WM component with equal weights to the respective two component version of the template, and subsequently applied those warps to the subject's three component decomposition to create the final three component atlas.2.7excluding the current image (leave-one-out) to ensure faster convergence.We created unbiased weekly templates by iteratively averaging the respective registered images to the corresponding group average template for that week. The templates were created in 28 stages, with increasing degrees of freedom for the transformation and with increasing spatial and angular resolution. More specifically, these stages consisted of six rigid followed by six affine registration stages, each with decreasing voxel sizes from 3.3 mm to 1 mm and increasing angular resolution . The two ODF images are jointly scaled so that they sum on average to First, we estimated CSF and WM response functions for each subject independently, following an initial coarse bias field correction. Those are used to deconvolve the signal into two components which are then subsequently corrected using a more fine-grained bias field correction using e-groups .mtnormalise. This ensures that ODF amplitudes are comparable across all subjects irrespective of their age.Following the normalisation of the two subject-specific response functions, we average the normalised CSF response functions of all subjects and the normalised WM response functions of the youngest and oldest groups of the cohort and use those to deconvolve the initial bias field corrected dMRI images. We, therefore, use the same three response functions for all subjects. The final three component decomposition is bias-field corrected and normalised using 2.9We selected 17 regions of interest in white and grey matter structures. The areas included five regions in the CC from the genu to the splenium, along with further regions in the posterior and anterior limb of the internal capsule, the cingulum, external capsule, fornix, head of the caudate, middle cerebellar peduncles, optic radiation, putamen, superior cerebellar peduncles, thalamus and cortical GM (see figure A.15). In these regions of interest, the mean and standard deviation of the component densities were calculated for each weekly template to extract time-resolved developmental patterns.Each ROI was defined by S.J.C. and M.P. on the jointly aligned group average template using the average FA and average component volume fraction maps and a consensus was reached. Furthermore, we consulted the diffusion tensor brain atlas  for refe33.1The appropriateness of the model was also investigated by looking at the residuals of each MSMT-CSD fit across the age range, as shown in 3.2The component volume fraction maps in elopment . In our ages see . In contages see .Fig. 7DiIn general, the transition from young to mature-appearing WM occurs from central to peripheral, caudal to cranial and posterior to anterior. The slices shown in In the cortex of younger subjects, we observe clear radial organisation  which re4response functions in CSF and in WM in the extremes of the age-range to model temporal changes in signal characteristics as changing relative volume fractions.The diffusion tensor model has been used extensively to study brain maturation  but currGroup-level and longitudinal analysis of these decomposition patterns requires spatio-temporal alignment. We adapted an existing registration approach, which allows the groupwise alignment of ODFs, to neonatal data. Registration is driven by two signal components, taking advantage of the information in both channels without biasing the alignment of the 3-component model due to spatial variability in maturation patterns. This template creation strategy does not explicitly model temporal consistency but could be extended to incorporate temporal shape and intensity constraints similar to atlases discussed in section Our approach allows a group-level and longitudinal analysis of white matter regions or tracts of interest in the neonatal period using components that are derived from the data and not modelled after biological tissue properties. While it does not offer the level of biological specificity that more explicit microstructure models might provide, it relies on very few assumptions, which we believe is a strong advantage given that the microstructure changes occurring over this period are complex and likely to differ substantially from a priori expectations.4.1Histology studies have reported spatially varying onset and progression of myelination . MyelinaWe investigate regional differences of WM maturation patterns similarly to earlier studies which used diffusion-weighted imaging , diffusiThe middle and superior cerebellar peduncles see  exhibit peduncle .We observe that in the posterior limb of the internal capsule, the relative fraction of the ter term , whereaster term .In comparison, the external capsule, the fornix, the cingulum and the optic radiations do not myelinate before term . We also1-and T2-weighted imaging  and more mature (44.1 weeks PMA) appearing anisotropic components. Furthermore, we were able to distinguish fibre populations within the same voxel with distinct time courses. This framework provides a basis for longitudinal investigations into healthy and pathological brain maturation.


Pseudomonas aeruginosa (MDRP) among hospitalized adult patients with Gram-negative infections.We developed a clinical bedside tool to simultaneously estimate the probabilities of third-generation cephalosporin-resistant Enterobacteriaceae (3GC-R), carbapenem-resistant Enterobacteriaceae (CRE), and multidrug-resistant N = 6). Models were converted to a singular user-friendly interface to estimate the probabilities of a patient having an infection due to 3GC-R, CRE, or MDRP when ≥ 1 risk factor was present.Data were obtained from a retrospective observational study of the Premier Hospital that included hospitalized adult patients with a complicated urinary tract infection (cUTI), complicated intra-abdominal infection (cIAI), hospital-acquired/ventilator-associated pneumonia (HAP/VAP), or bloodstream infection (BSI) due to Gram-negative bacteria between 2011 and 2015. Risk factors for 3GC-R, CRE, and MDRP were ascertained by multivariate logistic regression, and separate models were developed for patients with community-acquired versus hospital-acquired infections for each resistance phenotype . Resistant infection rates were 1.90% for CRE, 12.09% for 3GC-R, and 3.91% for MDRP. A greater percentage of the resistant infections were community-acquired relative to hospital-acquired . The most important predictors of having an 3GC-R, CRE or MDRP infection were prior number of antibiotics; infection site; infection during the previous 3 months; and hospital prevalence of 3GC-R, CRE, or MDRP. To enable application of the six predictive multivariate logistic regression models to real-world clinical practice, we developed a user-friendly interface that estimates the risk of 3GC-R, CRE, and MDRP simultaneously in a given patient with a Gram-negative infection based on their risk .We developed a clinical prediction tool to estimate the probabilities of 3GC-R, CRE, and MDRP among hospitalized adult patients with confirmed community- and hospital-acquired Gram-negative infections. Our predictive model has been implemented as a user-friendly bedside tool for use by clinicians/healthcare professionals to predict the probability of resistant infections in individual patients, to guide early appropriate therapy.The online version of this article (10.1186/s12879-019-4363-y) contains supplementary material, which is available to authorized users. Pseudomonas aeruginosa (MDRP) [The prevalence of highly resistant Gram-negative infections has increased dramatically over the past decade among hospitalized patients throughout the United States  and the a (MDRP) , 2.Patients with infections due to 3GC-R, CRE, or MDRP have limited treatment options and are highly vulnerable to receiving inappropriate empiric or delayed appropriate therapy because clinicians frequently fail to recognize these infections prior to culture and susceptibility reporting. The deleterious consequences of delayed appropriate therapy are well described . Not onlAs a mechanism to promote appropriate antibiotic usage within a healthcare institution, the World Health Organization recommends that healthcare institutions create tools and implement policies informed by real-world data to increase the probability of patients receiving early appropriate therapy . To enabTo address this unmet need, the goal of this study was to develop a user-friendly clinical tool that simultaneously estimates the likelihood of having an infection due to 3GC-R, CRE, or MDRP when one or more risk factors are present among patients with infections due to Gram-negative bacteria. First, we sought to describe the risk factors for 3GC-R, CRE, and MDRP among patients with community- and hospital-acquired Gram-negative infections using data from a large cohort of hospitalized patients across multiple facilities. Prediction models were developed from these risk factor analyses to estimate the probability of having a 3GC-R, CRE, or MDRP infection when one or more risk factors were present in a given patient. The prediction models were then used to create a user-friendly clinical instrument for use at the bedside.In summary, our prediction tool was developed to help clinicians identify hospitalized adult patients with Gram-negative bacteria who would benefit the most from tailored empiric treatment regimens in the critical period when a Gram-negative pathogen is identified on a Gram stain or with a rapid diagnostic test and antibiotic susceptibility results are not yet available. With this information, physicians can make more informed empirical antibiotic selections, and thereby increase the likelihood of timely appropriate antibiotic therapy. Studies have shown that the critical window between infection onset and delivery of appropriate antibiotics is 48–72 h –19. Our Input data for the development of the models were from a retrospective observational study of the Premier Hospital database, which contains coding and billing information for approximately 50 million admissions from more than 500 acute-care hospitals and is the largest hospital-based database in the United States. The study was limited to the approximately 160 institutions that contributed microbiology data for the entire study period of January 1, 2011, to October 1, 2015 [Patients included in the study population were adults (≥18 years) who had ≥1 admission to a hospital with evidence of a complicated urinary tract infection (cUTI), complicated intra-abdominal infection (cIAI), hospital-acquired or ventilator-associated pneumonia (HAP/VAP), or bloodstream infection (BSI)  diagnosis and procedure codes for each cohort  . PatientHospital-level characteristics included the type of facility , setting , geographic area , geographic division , and number of beds . Prevalence of resistant Gram-negative infections  in the hospital facility was included and defined by the median prevalence value across all hospitals.2 or Fisher’s exact test was performed for comparisons between two categorical variables; Student t or Mann-Whitney U test was used for comparisons between dichotomous and a continuous variable. Tests for trends were used to assess for the presence of a relationship between cumulative number of prior antibiotic exposures with Gram-negative activity and presence of resistant pathogen.The first step of analysis was to summarize the study population by standard descriptive statistics  to compare patients with resistant and nonresistant infections. Pearson χP < 0.20 were included as potential predictors at model entry in each LASSO logistic regression analysis. Variables with P < 0.1 were retained in the final models. Model performance was evaluated using the model lift among the top 10% of scored subjects in the test data. The model lift was defined as the probability of a positive case given a top 10% score divided by the probability of a positive case in the overall sample. A higher lift indicated a stronger association between the predicted score and the outcome. The predicted and observed likelihood of each resistant phenotype in each model was examined for any discordance.Because of the large number of predictors identified, least absolute shrinkage and selection operator (LASSO) logistic regression, or L1-regularized logistic regression, was used to select the set of predictors that best predicted the outcome by evaluating each coefficient simultaneously. Separate models for each resistance phenotype were conducted for hospital-acquired and community-acquired infections because they represent different patient populations with potentially different sets of risk factors for resistant infections. For the LASSO logistic regression analyses, the study sample was randomly split into the training set (70% of the study sample) and the test set (30% of the study sample). The training set was used to construct the LASSO logistic regression model. Cross-validation based on area under the curve (AUC) of receiver operating curves on training data was used to determine the best LASSO logistic regression model. Variables found to be predictive in the univariate analysis at Six models were developed: three for patients with hospital-acquired 3GC-R, CRE, and MDRP infections and three for community-acquired 3GC-R, CRE, and MDRP infections. A clinical prediction tool implemented using Microsoft Excel™  was developed to provide a convenient interface to the statistical models for use at the patient bedside. Infection, hospital, and patient characteristics for an individual patient are input into the tool, which then displays the probability of a resistant infection in tabular and graphical forms. The final tool contains the hospital- and community-specific models for each category of resistant pathogens and is available from Additional file A total of 124,068 patients contributed to the dataset during the study period. Baseline event counts in the training and test datasets are shown in Additional file Detailed results of the bivariate risk factor analyses are shown in Additional file Among hospital-acquired infections, the most important predictor for all three resistant phenotypes was the number of antibiotics received during the current hospital admission. Infection type was predictive of the presence of resistant infection: cUTI was not a predictor of resistance, cIAI and BSI were predictive of CRE and 3GC-R only, and HAP/VAP predicted the presence of all three resistant phenotypes. Among the infection types examined, only HAP/VAP was a predictor of MDRP. Lesser predictors of CRE and 3GC-R infection, in addition to hospital prevalence, admitting source, and hospital admission during the previous 6 months , were patient stay in the ICU and diabetes with complications. Prior infection in the last 3 months was predictive of MDRP infection, as were chronic pulmonary disease and paraplegia/hemiplegia. Comorbidities such as cancer, cerebrovascular disease, congestive heart failure, and mild liver disease predicted the hospital acquisition of 3GC-R and to a lesser extent CRE, but not MDRP . The MDRP models had the highest AUC and the highest lift (indicating a stronger association between predicted score and outcome) for both hospital- and community-acquired infections. Because of the importance of prior antibiotic use, the hospital-acquired models had higher AUC and lift values than the corresponding community-acquired models  antibiotic treatment for an individual patient according to the full context of patient characteristics and circumstances.Consisting of four worksheets, the full tool and a detailed user guide are available at Additional file One of the fundamental pillars of antimicrobial stewardship is ensuring that patients with life-threatening infections receive early, appropriate antimicrobial therapy. Despite the longstanding recognition of the positive benefits of “getting it right the first time,” delayed appropriate therapy rates for patients with serious Gram-negative infections are still reported to be > 30% in several publications . To faciAdvances in rapid diagnostics have shortened the lag time between infection onset to pathogen identification from days to hours and have had positive effects on clinical outcomes when paired with robust antimicrobial stewardship programs . AlthougWhen developing the clinical tool, we also believed it was important to develop two different models to differentiate the two distinct hospitalized patient populations with Gram-negative infections because risk factors or strength of their association can vary by population. The first focused on patients who presented to the hospital ; the second centered on patients who developed their infection during their hospitalization . Interestingly, patients with community-acquired Gram-negative infections had a higher baseline risk of having resistant Gram-negative infections relative to those with hospital-acquired infections in this study, highlighting the need for the development of two separate clinical prediction models. Another distinguishing feature of this clinical prediction model was the inclusion of the prevalence of each resistant phenotype of interest in the hospital where the patient developed the infection. It is well established that a patient’s risk of having an antibiotic-resistant infection is driven in part by the bacteria present in the healthcare institution. Therefore, we believe it was important to consider it as a covariate in the clinical model-building phase. Finally, we believe that our model also rectifies issues related to autocorrelation, in which a patient could theoretically be counted twice if exposed to more than one type of infection. This exposure could potentially occur on different sites or on different days, leading to potentially different exposures. Because we considered the site of infection in model development, the risk of autocorrelation was minimized, confirming the quality of our model.Although there were some distinguishing aspects to our clinical prediction tool, our model development approach was similar to previous studies –27. UsinThe information from the multivariate logistic regression analyses was then used to develop models to estimate the likelihood of having these infections when one or more risk factors were present in hospitalized adult patients with Gram-negative infections. The major advantage of using logistic regression to develop clinical prediction rules is the functionality of the final models. In addition to identifying variables that are independently associated with stronger odds of having the outcome of interest, the final logistic regression models are mathematic equations that can be used to predict the probability of antimicrobial resistance based on the combination of significant risk factors present in a given individual with an infection , 36. Ada. Finally, additional prediction methods such as neural networks, random forest, and SuperLearner, which allow for the incorporation of several algorithms simultaneously to deliver the strongest prediction model, may improve the prediction modeling observed in this study [Several things should be considered when interpreting these findings. As the data used for the development of our models was from a database, our study is subject to the limitations associated with retrospective observations studies, and the ICD-9 codes may not be 100% accurate. As with all electronic health databases, there may be errors of omission and/or commission in coding. Because our operational definitions were based on information within the database, study measures may be less accurate than those based on medical record review or data gathered prospectively. Because the Premier Hospital database lacks information on healthcare utilization outside of Premier facilities, we did not include prior receipt of antibiotics in the community-acquired model. We did not consider prior colonization with a resistant pathogen as part of these analyses as only clinical culture data were available in the database. We also cannot exclude the possibility of patient-to-patient transmission of the resistant strains, which may weaken the association between acquisition of resistant pathogens and the identified risk factors. Another limitation was that the tool was not validated using an external dataset; a validation study should be performedis study –39. DespBased on a large retrospective observational study, we developed six separate models for the prediction of hospital- and community-acquired infections due to 3GC-R, CRE, and MDRP among hospitalized adult patients with Gram-negative infections. The performance of our models is superior to or comparable with the performance of similar published models because of (1) the large number of patients and institutions contributing data, (2) the number and diversity of potential predictors considered, and (3) the inclusion of antibiotic resistance rates in the included hospitals. Our predictive model was implemented as a user-friendly bedside tool for use by physicians or healthcare professionals to predict the probability of resistant infection in an individual patient to expedite and direct initial antibiotic therapy and improve outcomes among hospitalized adult patients with Gram-negative infections in the critical period when a Gram-negative pathogen is identified on a Gram-stain or with a rapid diagnostic test and antibiotic susceptibility results are not yet available.Additional file 1:contains the Clinical Bedside Tool. (XLSM 162 kb)Additional file 2:contains Appendices A, B, and C, Tables S1, S2, and S3. (DOCX 51 kb)


Palmoplantar keratoderma (PPK) is a defect in cornification that is characterized by progressive hyperkeratosis of palms and soles. Many phenotypes are linked with PPK, making exome-based diagnosis increasingly efficient. In this report, we identified tyrosinemia type II on whole-exome sequencing in a 7-year-old Syrian refugee that presented with PPK. Dietary therapy helped improve her overall symptoms. PPK can be either acquired through environmental exposure or infection or may be the consequence of an inherited disorder  1]. Man. Man1]. 1.1A 7-year-old Syrian refugee presented to the genodermatosis clinic following several years of progressive painful and yellowish thickening of the palms and soles, localized mostly to pressure points . She alsTAT , consistent with tyrosinemia type II. This variant was not observed in approximately 6500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project, and rarely reported, only at the heterozygous state, in ExAC or GnomAD, in keeping with not being a benign variant. This substitution occurs at a position that is conserved across all species, and not likely to impact protein structure  [Her parents are first cousins and otherwise healthy. Her brother shared similar clinical features. Thus, the possibility of an underlying autosomal recessive condition was considered. Genetic testing for PPK using Exome/Slice (phenotype directed exome testing) showed a novel homozygous likely pathogenic missense variant in exon 3 of tructure . This vaN-acetyltyrosine and 4-tyramine. She was started on dietary restriction of phenylalanine and tyrosine with clinical improvement of her symptoms  with normal phenylalanine levels. Urine organic acid showed the presence of tyrosine metabolites, including L-form of 4-hydroxyphenyllactate, 4-hydroxyphenylacetate, and 4-hydroxyphenylpyruvate, and small quantities of symptoms .276600), previously referred to as oculocutaneous tyrosinemia or Richner–Hanhart syndrome [The clinical and laboratory findings in this patient are consistent with a diagnosis of Tyrosinemia type II  [p-hydroxyphenylpyruvate in the liver. The inability to properly deaminate tyrosine in the cytosol of hepatocytes via TAT results in the release into circulation and subsequent hypertyrosinemia. Excess tyrosine is metabolized by other enzymes, resulting in additional metabolites used to support the diagnosis of tyrosinemia. Functional mitochondrial tyrosine aminotransferase in tissues without the full tyrosine metabolic pathway produces the 4-hydroxyphenylketones detected in urine. Additionally, acetylation or oxidation produces N-acetyltyrosine and 4-tyramine when tyrosine concentrations are high. In this case, the patient presented with typical symptoms of tyrosinemia type II, but the diagnosis was made through genetic testing. Treatment using protein restriction and supplementation of amino acids without phenylalanine and tyrosine have been shown to improve symptoms [Tyrosinemia type II is is caused by disease causing variants in the #613018) , which e#613018) . This ensymptoms .The patient was initiated on dietary restriction of phenylalanine and tyrosine with minor improvement in symptoms following 3 months of dietary changes , arrows Written informed consent was obtained from the parents of the patient for the publication of this case report and any potentially-identifying information/images.


Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the commonest cause of X-linked and recessive retinitis pigmentosa (RP), accounting for 10%–20% of all cases of RP. The phenotype is one of the most severe amongst all causes of RP, characteristic for its early onset and rapid progression to blindness in young people. At present there is no cure for RPGR-related retinal disease. Recently, however, there have been important advances in RPGR research from bench to bedside that increased our understanding of RPGR function and led to the development of potential therapies, including the progress of adeno-associated viral (AAV)-mediated gene replacement therapy into clinical trials. This manuscript discusses the advances in molecular research, which have connected the RPGR protein with an important post-translational modification, known as glutamylation, that is essential for its optimal function as a key regulator of photoreceptor ciliary transport. In addition, we review key pre-clinical research that addressed challenges encountered during development of therapeutic vectors caused by high infidelity of the RPGR genomic sequence. Finally, we discuss the structure of three current phase I/II clinical trials based on three AAV vectors and RPGR sequences and link the rationale behind the use of the different vectors back to the bench research that led to their development.Mutations affecting the  Retinitis pigmentosa GTPase regulator (RPGR) gene accounts for over 70% of X-linked RP cases whereas less common forms of the disease are caused by retinitis pigmentosa 2 (RP2) and 23 (RP23 or OFD1) genes [Inherited retinal diseases, most of which are retinitis pigmentosa (RP), affect 1 in 4000 people worldwide. The hallmark of this heterogeneous group of disorders is premature degeneration of rod and cone photoreceptors that leads to early vision loss. RP can be inherited as an autosomal recessive, dominant, X-linked, oligogenic, or mitochondrial trait. X-linked RP is one of the most severe forms of retinal degeneration and it accounts for 10%–20% of all RP cases . To date1) genes .RPGR-related X-linked RP is characterised by severe disease in males with early onset and rapidly progressing sight loss that leads to legal blindness commonly by the fourth decade of life [RPGR disease are typically asymptomatic with a characteristic phenotype that manifests as a radial streak pattern originating from the fovea [ of life . The clahe fovea ,7. Rarelhe fovea .RPGR. Several treatment options have been under investigation and with the emergence of novel gene-based therapies for inherited retinal disease, this seems the most logical strategy to develop for the RPGR disease. Due to its severe phenotype, relatively high incidence and the fact that more commonly mutated genes such as ABCA4 or USH2A are too large to be packaged into AAV vectors, the RPGR disease has drawn significant interest amongst scientific and clinical research communities over the last years. However, due to the inherent instability in the retina-specific RPGRORF15 isoform sequence [RPGR vector has been very challenging. In attempts to improve the sequence stability and fidelity several approaches have been explored including codon optimisation [RPGR gene structure and its evolutionary conservation that has led to an improved understanding of protein’s molecular function and mechanisms implicated in the pathogenesis of RPRG-related retinal dystrophy. The pre-clinical development of gene therapy vectors that has resulted in their progression into three phase I/II clinical trials is covered in detail, including discussion on three different RPGR cDNA sequences used in the trials.At present, there is no approved treatment for retinitis pigmentosa caused by mutations in sequence ,10,11,12misation ,14,15, wRPGR gene is located on the short arm of the X-chromosome (Xp21.1). The gene exhibits a complex expression pattern with 10 alternatively spliced isoforms, five of which are protein coding [Ex1-19 isoform. In humans, the RPGREx1-19 isoform contains 19 exons and expresses a full-length messenger RNA transcript of 2448 bp, which generates an 815 amino acid sequence that forms ~90 kDa protein in a variety of tissues [ORF15 variant [ORF15 isoform is 3459 bp, encoding a 1152 amino acid sequence which forms a ~200 kDa protein. As with the widely expressed variant, amino acids 54–367 (exons 3–10) form a regulator of chromosome condensation 1 (RCC1)-like domain. The alternative ORF15 exon consists of a highly repetitive purine-rich sequence coding for multiple acidic glutamate-glycine repeats. This is followed by a C-terminal tail region rich in basic amino acid residues, called the basic domain.The human n coding . The fir tissues . Since t variant ,18. ThisEx1-19 and RPGRORF15 are predominantly localised to the photoreceptor connecting cilia [The reason for this complex expression pattern of the RPGR protein remains largely unknown, but may be related to the functional role of its splice isoforms in various cell types. The RPGR protein is widely expressed in vertebrate tissue including eye, brain, lung, testis and kidney. In the eye, the two major isoforms, RPGRng cilia . The conThe RCC1-like domain, present in both major splice forms, adopts a seven-bladed β-propeller structure and it is strongly conserved across evolution, in vertebrates and invertebrates . This doORF15, interacts with at least two proteins, a chaperone protein nucleophosmin and a scaffold protein whirlin [The retina-specific ORF15 domain is also highly evolutionarily conserved across varied species, indicating a functional importance . However whirlin . NeitherEX1-19 variant is still expressed in these cells. Hence in-frame deletions in the ORF15 region lead to progressive loss of function as the deletion length increases [The function of the repetitive glutamine-glycine-rich domain itself has been difficult to establish due to its variable length and relatively poor conservation at the individual amino acid level, although the overall charge and repeat structure length remain conserved in vertebrates. However, recent evidence shows that this intrinsically disordered region is heavily glutamylated , a post-ncreases .Molecular mechanisms and pathogenesis of RPGR-related X-linked RP have been under investigation for several decades. The drive to better understand the disease process comes from the high incidence with mutations in the gene encoding RPGR accounting at least 70% of X-linked RP and up to 20% of all RP cases ,4. MoreoEx1-19 has yet to show a firm association with any human disease. The RPGR-related phenotype seems to be confined to the retina and several studies have established an essential role for RPGRORF15 in photoreceptor function and survival [ORF15 result in abnormal protein transport across the connecting cilium, which can lead to photoreceptor cell death [Despite ubiquitous expression of the constitutive RPGR variant in ciliated cells throughout the body, the RPGRsurvival ,11. Genell death ,30,31. Hll death ,33. Thesll death . Similarll death .RPGR account for ~70% of cases of X-linked RP and have been identified across exons 1–15, yet up to 60% of mutations occur in the ORF15 region [RPGR. Surprisingly, no disease-causing mutations have been reported in exons 16–19 [Mutations in 5 region ,30. The ns 16–19 .ORF15 function. It remains intriguing that despite its ubiquitous expression, the RPGREx1-19 is unable to compensate for the loss of function of RPGRORF15 in the retina to rescue the phenotype. It is possible that the alternative splicing in the retina could favour the RPGRORF15 variant, so the majority of transcripts will be the RPGRORF15 isoform, with few constitutive variants available to compensate. One study failed to identify the constitutive transcript in the retina [ORF15 becomes the predominant isoform [ORF15 in the photoreceptors, perhaps it is not so surprising that the constitutive variant cannot offer the same functional benefit as the RPGRORF15 variant.The most common mutations are small deletions that lead to frameshifts followed by nonsense mutations . Within e retina , which s isoform . NotablyThe diagnosis of RPGR-related retinal dystrophy is made on the basis of presenting symptoms and retinal signs seen on clinical examination and various imaging modalities. In addition, study of family history showing X-linked inheritance  and genetic testing identifying the pathogenic mutation are important in confirming the diagnosis. In cases of uncertain diagnosis and unequivocal genetic test results we have adopted several important steps, which are discussed below, in order to minimise the risk of establishing an incorrect diagnosis, and administering the patient with an incorrect gene if recruited into a gene therapy clinical trial.RPGR-related retinal dystrophy is associated with a very heterogeneous phenotype that ranges from pan-retinal rod-cone to predominant cone dystrophy . The pheThe RPGR phenotype  has beenRPGR mutations also show high phenotypic variability [RPGR gene therapy as discussed below.Female carriers of iability   is usually a robust approach in determining pathogenic variants in RP. However, the ORF15 region of RPGR is not normally sequenced with NGS methods and is currently only performed upon specific request. Moreover, sequencing of the ORF15 region in riations . The strSeveral non-gene based treatment approaches have been investigated for the preservation of vision in X-linked RP including a nutritional supplement, docosahexaenoic acid  and a ciORF15 (3.5 kb) is within the AAV carrying capacity and the relatively high prevalence and disease severity have justified development of this therapy. However, the repetitive sequence of ORF15 not only makes it a hotspot for mutations but also creates challenges for therapeutic vector production. Attempts to generate AAV vectors for RPGR gene-supplementation strategies have been thwarted by the poor sequence stability of the ORF15 region and transgene production has struggled to control spontaneous mutations and maintain the complete sequence [ORF15 coding sequence (the purine-rich region was generated first and then ligated to the rest of the DNA sequence) [ORF15RPGR vector and evaluated in the Rpgr-KO mouse. However, despite improved stability, some vector preparations were still ridden with micro-deletions that led to expression of alternatively spliced truncated forms of the RPGR protein that was mislocalised to photoreceptor inner segments and only a partial rescue of the phenotype in treated mice. The truncated forms of the protein were further investigated for their ability to rescue the RPGR phenotype in the Rpgr-KO mouse [ORF15RPGR were tested. The long form demonstrated significant improvement in the disease phenotype, whilst the short form failed to localise correctly in the photoreceptors and showed no functional rescue of the phenotype. Importantly, as discussed above, large deletions in the ORF15 region can affect the glutamylation of the protein and lead to impaired function. Indeed, a follow-up study by the same group tested these truncated vectors [Emerging gene-based therapy using the AAV vector is currently the most promising therapeutic strategy for RPGR X-linked RP. The size of the coding sequence of RPGRsequence ,53,54,55sequence , driven sequence  thus posKO mouse . The sho vectors  for theiORF15 to stabilise the sequence, remove cryptic splice sites and increase expression levels from the therapeutic transgene [ORF15 vector was shown to offer therapeutic rescue in two mouse models of X-linked RP (-/yRpgr and Rd9). This vector is now being used in a Phase I/II/III gene therapy clinical trial in humans (NCT03116113). In addition, the codon optimised form of the RPGR vector used in the canine studies [RPGR gene therapy vector [RPGR. The codon optimized construct was validated in vitro in pull-down experiments and in a murine model, demonstrating production of functional RPGR protein.To circumvent these issues, the research team of Fischer and colleagues (2017) generated a full-length, human, codon-optimised version of RGPRransgene . This enNCT031161. In addiy vector . The strRPGR coding sequence (ORF15 cDNA sequence (AAV2/8.hRK.ORF15coRPGR). The second trial sponsored by Meira GTx (NCT03252847) is using a wild-type AAV2/5 capsid with a truncated, non-codon optimised RPGR sequence under control of the human rhodopsin kinase promoter (AAV2/5.hRK.ORF15RPGR). The third trial conducted by Applied Genetic Technologies Corporation (NCT03316560) is using mutated AAV2 capsids  mutations) packaged with full-length, codon optimised human ORF15RPGR sequence also driven by the rhodopsin kinase promoter (AAV2tYF.GRK1.ORF15coRPGR).The results of the pre-clinical studies described above support the use of AAV-based gene therapy for RPGR-related X-linked RP in humans, in the early to mid-stage of the disease. Ideally, patients with moderately reduced visual acuity and constricted visual fields, but a preserved central ellipsoid zone, should be recruited into gene therapy trials for best expected therapeutic benefits. Interestingly, development of RPGR therapy from bench to bedside has resulted in setting-up of three multi-centre dose-escalation gene-therapy clinical trials  trials are very similar and encode the full-length human wild-type RPGRinked RP  and was RPGR gene through cryptic splice sites that create multiple isoforms poses challenges in elucidating its function. However, mounting evidence suggests that retina-specific RPGRORF15 is unique to vertebrates and plays a crucial role in regulating protein trafficking between inner and outer segments as well as in microtubular organisation. Importantly, RPGRORF15 contains a characteristic repetitive purine-rich region that is highly glutamylated and only the glutamylated RPGRORF15 is fully functional. Thus, any mutations that reduce the glutamylation process adversely affect RPGR protein function. In addition, the ORF15 region created challenges for the researches interested in developing RPGR gene-based therapies as the repetitive region made it unstable and prone to mutations. The current approach in developing a codon-optimised version of the RGPRORF15 to stabilise the sequence, remove cryptic splice sites and increase expression levels from the therapeutic transgene is now being used in humans, following proof-of-concept studies in murine and canine models of X-linked RP. This approach has allowed the rapid progression towards the first in-human gene therapy trial (NCT03116113) for X-linked RP, which began in March 2017. In parallel, two additional independent research consortia have been developing gene therapies for the RPGR disease. With recent approval of gene replacement therapy Luxturna, for the treatment of RPE65-related retinal disease, the precedence for approval of future gene-based therapies has been set and results of the RPGR early phase clinical trials are awaited with great expectation.X-linked RPGR-related RP is a heterogenous group of disorders with no clear genotype–phenotype correlation. Both rod-cone and cone-rod retinal dystrophies are seen with relatively early onset and rapid progression to blindness that is related to mutations that cause loss of function of this key photoreceptor protein. The complex expression pattern of the