Analysis of adjacent tooth resistance is essential in wisdom teeth extraction to prevent adjacent tooth trauma, however it lacks adequate attention nowadays. This study aims at suggesting special extraction methods based on adjacent tooth resistance analysis for prevention of adjacent tooth damage. In this study, 136 complicated mandibular third molars extracted using piezosurgery were reviewed and classified based on the adjacent teeth resistances shown in orthopantomogram (OPG) during their mesio-distal rotations: degree I refers to teeth with no adjacent teeth resistance; degree II refers to teeth with resistance released after mesial-half crown sectioning; degree III refers to teeth which still had resistance after mesial-half crown sectioning. With the use of surgical simulations using cone beam computerized tomography (CBCT) reconstruction, all teeth in degree I were designed to rotate mesio-distally; 86.36%(38/44) teeth in degree II were designed to rotate mesio-distally after mesio-half crown sectioning; 69.09%(36/55) teeth in degree III were designed to rotate bucco-lingually. All teeth were extracted successfully, and only one adjacent tooth was subluxated due to the incomplete bone removal. Our study suggested that in order to prevent adjacent teeth trauma, complete bone removal is of importance, and impacted teeth with higher adjacent teeth trauma risks should consider bucco-lingual rotations. Risk of adjacent tooth trauma in mandibular third molar extraction refers to the possibility of adjacent tooth damage (looseness or dislocation)1The prevention measures for this complication include bone removal and tooth sectioning45This is a retrospective study. This study followed the Declaration of Helsinki on medical protocol and ethics and the regional Ethical Review Board of Shanghai Ninth People’s Hospital approved the study. All patients were informed about surgical purpose, surgical protocol, recovery period, possible complications and signed a consent form.st 2012 to May 1st 2015, 113 cases with 136 complicated mandibular third molars extracted using piezosurgery by the same surgeon (Chi Y) were reviewed. The surgeon was a chief physician with 30-year clinical experience. The inclusion criteria were the complicated mandibular third molars which had adjacent teeth. The complicated mandibular third molarsFrom May 1Based on the positions (Pell & Gregory’s classification), there were 25, 37 and 74 teeth in position A, B and C. Based on angulations (winter’s classification)All teeth were categorized into 3 types according to the adjacent teeth resistances during the mesio-distal rotations of the impacted teeth after complete bone removal .Risk degree I: no adjacent tooth resistance .Risk degree II: mild adjacent tooth resistance  .Risk degree III: high resistance existed  .Surgical simulation was performed in each case with the following steps: (1) the CBCT data were transferred into mimic software  to reconstruct and differentiate the wisdom tooth, its adjacent tooth, the mandible, as well as the inferior alveolar canal; (2) the osteotomy lines were designed to remove adequate volume of bone; (3) the wisdom tooth was designed to rotate with or without tooth sectioning.The surgery in each case was performed based on the preoperative digital simulation. The piezosurgery device was applied in both bone removal and tooth sectioning.Evaluation criteria include the success rate, the adjacent teeth mobility and pulp vitalities. Mobility assessment: degree I refers to the one with bucco-lingual mobility; degree II refers to the one with both bucco-lingual and mesio-distal mobilities; degree III refers to the one with bucco-lingual, mesio-distal and vertical mobilities. Pulp vitality testing  was only chosen for the adjacent teeth with vital pulps: the tested tooth was regarded abnormal when its measured value was different from the normal tooth (the difference should be > 10).The general data of the patients are listed in The surgical procedure of each tooth was in accordance with the preoperative digital simulation. All wisdom teeth were extracted successfully with the exception of one adjacent tooth which was subluxated. All adjacent teeth were undamaged with normal morbility and/or normal pulp vitality.According to our study, the complicated mandibular third molars were inclined to have higher adjacent teeth trauma risk . This highlights the importance of paying more attention to this complication. The obstruction in the mesio-distal rotation path of the wisdom teeth is commonly accepted as adjacent teeth resistance, which is the most critical factor related to the risk degree. Based on this, we designed a risk degree classification to categorize complicated wisdom teeth. Using whether the wisdom teeth could rotate mesio-distally as a whole or with mesio-half crown sectioning as dividing basis, the classification has the surgical guidance value as well.Piezosurgery is recommended in complicated impacted teeth extractions nowadays4The methods to prevent adjacent teeth trauma include the removal of bone and tooth sectioning. With the exception of certain vertical impactions, teeth with incomplete bone removal will have the trend of forward moving3The extent of the bone removal, as well as the tooth sectioning could be designed preoperatively with the help of the digital surgical simulation. Digital surgical simulation was performed based on CBCT reconstruction with the advantages of adjustable transparency and flexible movements of different anatomic structuresBy reviewing the extractions, two methods were concluded in this study: the mesio-distal and buccal-lingual rotations. Tooth sectioning could assist either of them. Mesio-distal rotation includes crown and root rotation. The former refers to rotation in the sagittal plane using the apical region as the rotation center, while the latter refers to rotation in the sagittal plane using the contact area with adjacent tooth as the rotation center. Bucco-lingual rotation includes crown and root rotations in the horizontal plane with the same criteria in mesio-distal rotation.Teeth in risk degree I are mainly those mesially/vertically impacted in positions A/B, which could be best extracted with mesio-distal rotations of the crowns. Teeth with risk degree II mainly consist of those mesially/horizontally impacted in positions A/B. Most teeth in risk degree II could be extracted with mesio-distal rotations with mesio-half crown sectioning . Some horizontally impacted teeth in degree II could be extracted with bucco-lingual rotations . Most of the teeth in risk degree III were horizontally/invertedly impacted in positions B/C, both having high adjacent teeth and bone resistances. Previous studies have reported three surgical methods to extract those teeth, including coronectomy151618In this study, the designed methods can contribute to the successful extraction of complicated wisdom teeth. One adjacent tooth was subluxated during the mesio-distal rotation of the impacted third molar  after the incomplete bone removal. Immediate fixation was done. Pulp vitality (1 year of follow-up) and tooth mobility (3 months of follow-up) were normal. The adjacent tooth trauma occurred because of the incomplete bone removal, which led to the forward movement of the wisdom teeth. The experience of this case shows the significance of complete bone removal.This study has its limitation due to its retrospective character. In this study, the surgical design and digital simulation, as well as the surgeon’s skill contribute to the satisfied outcome. For other inexperienced surgeons, the extraction methods should be implemented with more precise design.The extraction methods dictated by the adjacent teeth resistances prevent adjacent teeth trauma. Digital simulation based on CBCT reconstruction has the surgical guidance value. Complete bone removal is of importance, and teeth with higher adjacent tooth trauma risks should consider bucco-lingual rotations.How to cite this article: Ye, Z.-X. et al. Adjacent tooth trauma in complicated mandibular third molar surgery: Risk degree classification and digital surgical simulation. Sci. Rep.6, 39126; doi: 10.1038/srep39126 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Epidemiological studies from Sub-Saharan Africa indicate that allergies are on the rise in this region especially in urban compared to rural areas. This increase has been linked to improved hygiene, lifestyle changes, and lower exposure to pathogens in childhood. Reduced exposure to parasitic worm (helminth) infections and allergy outcomes has been the focus of a number of population studies over the years. Paradoxically, there are parallels in the immune responses to helminths and to allergies. Both conditions are associated with elevated levels of immunoglobulin E, high numbers of T helper 2 cells, eosinophils, and mast cells. These immune parallels have meant that the diagnosis of allergies in parts of the world where helminths are endemic can be hampered. The aim of this review is to examine observations from population studies conducted in Sub-Saharan Africa that demonstrate how helminth infections influence the parameters used to diagnose allergy outcomes in this region. We explore specifically how helminth infections hinder the in vitro diagnosis of allergic sensitization, influence the clinical manifestations of allergy, and also the effect of anthelmintic treatment on allergy outcomes. Advancing our understanding of how helminths influence allergy diagnosis is imperative for the development of improved tools to assess, diagnose, and treat allergic disorders in both helminth-endemic and non-endemic countries worldwide. Time trends indicate a sharp rise in the incidence of allergic disease worldwide especially among children . This inHelminth infections remain highly prevalent in tropical areas with over 1.5 billion people worldwide thought to be chronically infected with at least one soil-transmitted helminth . MoreoveDespite the lack of geographical overlap in areas of the world with the highest burden of helminth infection and places most affected by allergic disease, strong parallels exist between these two conditions on the immunological level. Both are associated with the induction of strong T helper 2 (Th2) immune responses leading to elevated levels of immunoglobulin E (IgE) as well as the presence of eosinophils, mast cells, and Th2 cytokines , 24. TheIn this review, findings from population studies conducted in Sub-Saharan Africa that illustrate how helminth infections influence the parameters used to diagnose allergy outcomes are examined. We explore specifically how helminth infections hinder the in vitro diagnosis of allergic sensitization and influence the clinical manifestations of allergic disease.The clinical diagnosis of IgE-mediated allergy in many countries is based primarily on a physical examination, history of symptoms, and the determination of allergic sensitization •. AlthouAllergic sensitization—the production of serum-specific IgE against harmless antigens known as allergens—is a well-established factor in the development of allergic disease  and crucIn vitro assessment of allergic sensitization can be determined by the measurement of allergen-specific IgE by assays such as an enzyme-linked immunosorbent assay (ELISA), radioallergosorbent test (RAST), ImmunoCAP assay or more recently, microarray allergen assay .Skin prick testing is used to assess allergic sensitization to a panel of environmental or food allergens as well as to negative and positive controls. A skin prick test (SPT) involves applying an allergen in liquid form to the skin of a patient, piercing the drop with a lancet and determining whether a reaction occurs within 15 to 20 min following application . If the Ascaris lumbricoides had 28 times higher levels of total IgE [In the late 1960s, S.G.O. Johansson and colleagues published one of the first studies linking the then newly characterized IgE antibody to helminth infections. Their investigation found that a group of pre-school children in Ethiopia had up to 20 times higher levels of total IgE compared to Swedish children and that a sub-group of Ethiopian children infected with the helminth otal IgE •. Even iotal IgE •.Subsequently, several investigations from Sub-Saharan Africa have observed raised levels of allergen-specific IgE related to helminth infection that did not translate into allergy symptoms •, 32, 33Since the early 1980s, two forms of cross-reactivity linked to allergy have been described: cross-reactivity due to proteins and cross-reactivity due to the sugar structures on glycoproteins known as cross-reactive carbohydrate determinants (CCDs).Ascaris and cockroach or mite, is not always significant [Cross-reactivity between plant-derived proteins is well-characterized especially in relation to food allergy . Althougnificant .Ascaris lumbricoides and homologues from house dust mite or cockroach, there are very few studies in this area from Sub-Saharan Africa. However, published literature provides indications that IgE cross-reactivity between homologous proteins may explain some observations made in studies from Africa over the years. For example, in urban black adolescents in Cape Town, South Africa, a positive association was seen between specific IgE to Ascaris antigen and SPT reactivity to aeroallergens (house dust mite and cockroach) [Ascaris such as tropomyosin or GST and their corresponding homologues in house dust mite and cockroach.Although a number of population studies from South America have investigated IgE cross-reactivity between proteins from helminths such as ckroach) . AlthougAscaris, other helminths have been implicated in IgE cross-reactivity involving proteins. For example, cross-reactivity between the filarial nematode Onchocerca volvulus tropomyosin (OvTrop) and house dust mite tropomyosin (Der p 10) has been shown [Aside from en shown . While tN-acetylglucosamine and a β--linked xylose on a core mannose [Cross-reactive carbohydrate determinants (CCDs) on insect and plant glycoproteins are carbohydrate components that have been linked to IgE cross-reactivity . The mos mannose . Aalbers mannose . A subse mannose .S. haematobium infection was also positively linked to peanut IgE sensitization as well as a strong correlation seen between IgE to CCDs and IgE to whole peanut extract. Inhibition assays in a subset of children showed that S. haematobium soluble egg antigen and the CCD marker bromelain were strong inhibitors of IgE binding to peanut extract [The role of helminths in carbohydrate cross-reactivity was illustrated in an investigation in Ghana among 1604 school-children in which raised levels of IgE against whole peanut extract were observed that did not translate into peanut allergy symptoms •. In tha extract •. IgE to extract .Although IgE to CCDs is generally thought to be biologically inactive, the clinical relevance of IgE to some CCDs has been reported in a study that found that about one third of CCD-positive sera from individuals with tomato allergy had biologically relevant CCD-specific IgE . MoreoveIgE cross-reactivity between helminth antigens and environmental or food allergens demonstrates the limited diagnostic value of assessing IgE reactivity to whole allergen extracts in populations from helminth-endemic areas. Whole extracts are made up of multiple allergenic and non-allergenic components which often lack standardization in their allergen content . In receS. haematobium infection, CRD in a subset showed that despite these high levels, very low responses to recombinant peanut allergens Ara h 1, 2, and 3 were seen [Among Ghanaian schoolchildren with elevated IgE to whole peanut extract that was strongly associated with ere seen . Althougere seen . While 4ere seen . Both thA. lumbricoides, Strongyloides stercoralis, and hookworm, involve the migration of the larval stage through the lungs which may result in respiratory symptoms often associated with asthma such as wheeze and dyspnoea (shortness of breath) [Research has shown that in some individuals who have low exposure to helminths such as travelers or recent migrants to a helminth-endemic area, robust inflammatory allergic responses can develop to helminths . In addi breath) •.A. lumbricoides infection and symptoms of asthma such as wheeze seen in a number of population studies in Sub-Saharan Africa. For example, in South Africa, an investigation found that A. lumbricoides infection among urban and rural children was associated with increased odds of a marker of airway hyperresponsiveness: exercise-induced bronchoconstriction [A. lumbricoides infection, and reported wheeze among participants greater than 5 years of age [Th2-mediated responses to migrating larvae during helminth infection provides one explanation for positive associations between triction . More res of age . It is is of age .Onchocerca volvulus infection, and in this form, microfilariae induce a state of immune hyporesponsiveness in the host which ensures the survival of the parasite through the regulation of strong Th2 immune responses [Migrating larvae are also a feature of other helminth infections such as onchocerciasis where larvae known as microfilariae migrate to the skin, eyes, and organs . Onchoceesponses . The moresponses . In bothesponses .Onchocercavolvus and all those tested were found to be positive for onchocerciasis based on serology for the marker of active disease IgG4 or skin snip test [The effects of the reversal of immune hyporesponsiveness associated with chronic onchocerciasis have been illustrated by a small retrospective study of 27 Ethiopian immigrants in Israel who presented with pruritic skin disease simulating atopic dermatitis •. In thanip test •. Interenip test •. They anip test •.Toxocara spp. can also induce Th2 inflammatory responses in the airways or the skin [Anisakis. Marine mammals are definitive hosts of Anisakis while fish and cephalopods (octopuses/squids/cuttlefish) are intermediate hosts for its larval stages [Anisakis by consumption of the larval stage of the parasite in undercooked/raw fish or cephalopods [Anisakis infection can include gastro-allergic reactions as well as anaphylaxis while exposure through the respiratory tract or skin can result in anaphylaxis, rhinoconjunctivitis, asthma, and dermatitis [Anisakis [Anisakis infective larval stage (L3) induced strong Th2 immune responses in mice. A later study by the same group found that following epicutaneous exposure to Anisakis antigens in mice, the Th2 cytokine interleukin (IL)-13 played an important part in protein contact dermatitis while another Th2 cytokine IL-4 was key in driving systemic anaphylaxis [Aside from migrating larval stages, transient helminth infections where humans are not the definitive host like the skin . This hal stages . Humans halopods . Symptomrmatitis . NieuwenAnisakis . Their iphylaxis .Anisakis infection in humans in other parts of Sub-Saharan Africa. It is possible that this is as a result of under-reporting as many cases of the infection go undiagnosed [There are no other published studies on iagnosed .Chronic helminth infections induce immune regulatory networks in the host which are characterized by regulatory T cells, regulatory B cells, and alternatively activated macrophages . An antiRemoval of helminths by chemotherapy can reverse immune hyporesponsiveness to allergens associated with chronic helminth infections. Although observations have varied, several longitudinal studies conducted in helminth-endemic areas worldwide have observed a positive association between anthelmintic treatment and allergen skin reactivity –77. HoweSchistosoma infection and meben-dazole for intestinal helminths) or a placebo every 3 months and skin prick tested every 6 months for a 30 month follow-up period [In Gabon, a randomized, controlled trial was conducted in which 317 children aged 5–13 years living in a helminth-endemic area were given anthelmintic treatment  for soil transmitted helminths among pregnant women was strongly associated with an increased risk of doctor-diagnosed infantile eczema in their children •. Praziqnegative •. The trnegative •. Subseqnegative . By 5 yenegative .S. mansoni infection (47.2%) [Recently, Stein and colleagues examined the burden of helminth infections among 126 immigrants from Ethiopia newly arrived in Israel and related these to allergy outcomes •. The mo (47.2%) •. Among  (47.2%) •. Less t (47.2%) •. Both cStudies from helminth-endemic areas in Sub-Saharan Africa on IgE cross-reactivity between helminth antigens and allergens have shown that the specificity and diagnostic accuracy of in vitro testing of allergy can be greatly improved by the use of recombinant allergens for the evaluation of IgE responses to allergens. Future investigations in this region could greatly benefit from the use of CRD.Although allergy studies among Ethiopian immigrants in Israel have had small sample sizes, they provide pertinent examples of how changing environments due to intercontinental migration from a helminth-endemic region to a high income country can lead to clinical manifestations of allergy symptoms that are closely linked to helminth infections. With more global migration and movement of people, such observations have implications for improving allergy diagnosis not only in helminth-endemic areas but in other parts of the world.Research from Sub-Saharan Africa has shown that novel allergens exist that are relevant to this region, particularly in relation to food allergy . AdditioUnderstanding the relationship between helminth infections and allergies has advanced our knowledge of how environmental factors that influence pathogenic exposures can hamper the diagnosis of allergy outcomes. Future investigations must build on our understanding of these links to generate new tools to diagnose, treat and prevent allergies not only in high income countries but also in the developing world where allergies are emerging as conditions of public health importance.


To evaluate the perinatal effects of a prenatal therapy based on intra-amniotic nutritional supplementation in a rabbit model of intrauterine growth restriction (IUGR).IUGR was surgically induced in pregnant rabbits at gestational day 25 by ligating 40–50% of uteroplacental vessels of each gestational sac. At the same time, modified-parenteral nutrition solution  was injected into the amniotic sac of nearly half of the IUGR fetuses (IUGR-T group n = 106), whereas sham injections were performed in the rest of fetuses (IUGR group n = 118). A control group without IUGR induction but sham injection was also included (n = 115). Five days after the ligation procedure, a cesarean section was performed to evaluate fetal cardiac function, survival and birth weight.Survival was significantly improved in the IUGR fetuses that were treated with intra-amniotic nutritional supplementation as compared to non-treated IUGR animals , whereas, birth weight  and fetal cardiac function were similar among the IUGR groups.Intra-amniotic injection of a modified-parenteral nutrient solution appears to be a promising therapy for reducing mortality among IUGR. These results provide an opportunity to develop new intra-amniotic nutritional strategies to reach the fetus by bypassing the placental insufficiency. Intrauterine growth restriction (IUGR) is generally defined as a significant reduction in fetal growth rate resulting in a birth weight in the lowest 10th percentile. It affects 7–10% of all pregnancies , suboptiPlacental insufficiency is the most common cause of IUGR where the nutrient transport to the fetus is compromised . To dateIn this study, we used a rabbit model of placental insufficiency to test the hypothesis that intra-amniotic nutrient delivery would improve the perinatal outcome of IUGR fetuses, by analyzing survival, birth weight and fetal cardiac remodeling.The study has been reported according to the ARRIVE guidelines for repoth day of gestation (full term is approximately 31 days). Dams were housed in separate cages on a reversed 12/12 h light cycle, with free access to water and standard chow. At 25th day of gestation, IUGR was induced surgically by uteroplacental vessel ligation and intra-amniotic injections were performed. At 30th day of gestation,an abdominal incision was made and the uterine horns were exteriorized to perform fetal echocardiography in a subgroup of fetuses. Subsequently, fetuses were delivered by cesarean section. Experimental design and timeline are shown in Thirty-eight time-mated 24 months old New Zealand White pregnant rabbits were provided by a certified breeder at 18On gestational day 25, an abdominal midline laparotomy was performed and both uterine horns were exteriorized under endovenous anesthesia of Ketamine  and Xylazine . Gestational sacs of both horns were counted and numbered, and each fetus was identified according to the fetal position within the bicornuate uterus. Prior to surgery, each uterine horn was randomly allocated to a group  based on a computer generated randomization number sequence. As each dam has two uterine horns, in order to obtain three experimental groups , horns of each dam was assigned to a paired combination of these groups, resulting in three combinations . Based on the ligation and nutrient injection, the experimental groups were created: Control , IUGR  and IUGR-T . IUGR was surgically induced by ligation of 40–50% of the uteroplacental vessels of the assigned gestational sacs . In addiAt 30 days of pregnancy, an abdominal midline laparotomy was performed and uterine horns exteriorized under endovenous anesthesia of Ketamine  10 mg/kg and Xylazine  3mg/kg. After that, fetal echocardiography was performed in a subgroup of fetuses using vivid q ultrasound equipment  with i12L-RS linear transducer, placed directly on each exteriorized gestational sac. Cardiac area, thoracic area, ventricular base-to-apex length and transverse diameters, and septal myocardial wall thickness were measured in end-diastole from a 2D image. Then, cardio-thoracic ratio was calculated by dividing cardiac area per thoracic area. Ventricular sphericity indexes were calculated as base-to-apex length divided by transverse diameter. Heart rate was also measured using Doppler applied on the left outflow tract.Immediately after echocardiography, all live and stillborn fetuses were obtained by uterine horn incision and weighted. Dams were sacrificed by endovenous overdose of sodium pentobarbital (200 mg/kg), immediately after fetal extraction. All living newborns were sacrificed by immediate decapitation. Survival rate was determined by the ratio of live fetuses at the time of the cesarean section to all viable fetuses at the time of the ligature procedure. Intestine samples were collected after delivery for subsequent analysis.http://rsbweb.nih.gov/ij) in order to evaluate intestinal structure.After sacrificing the fetuses, one-centimeter tissue sample was collected from the proximal small intestine and fixed with 4% paraformaldehyde in PBS for 24 h at 4°C. Fixed intestinal samples were embedded in paraffin to obtain 5μm sections to be stained with hematoxylin and eosin. Histology images were acquired using a microscope  and software . Quantification of intestine diameter, villus height and muscular and sub-mucosal layer thickness was performed using Image J software  from 38 dams. Of these 339 fetuses, 201 were alive at the day 30 of cesarean section . The mean litter size was 11.4 ± 2.3.Non-treated IUGR fetuses presented a significantly lower survival rate (IUGR 44% vs. control 71% p = 0.003) and lower birth weight as compared to controls Figs  and 3. Hth centile of normally distributed weight at birth [An analysis performed for a subgroup of fetuses that weighed less than 30 grams  rFetal echocardiography revealed similar cardiac alterations in both IUGR and IUGR-T fetuses with larger hearts, thicker myocardial walls and a more spherical left ventricle as compared to controls .Regardless of the absence of any positive change in fetal cardiac functions and birth weight among IUGR-T treated fetuses, the notable improvement in the survival rate in this group versus IUGR fetuses may suggest that the nutritional supplementation administrated was able to reach circulation in IUGR fetuses, very likely through swallowing and intestinal absorption. Actually, performed histological analyses of the small intestine  provide Our results support intra-amniotic injection of nutrients as a promising therapy for reducing mortality among IUGR, regardless of no apparent effects on birth weight. These results open opportunities for intra-amniotic nutritional strategies to reach the fetus bypassing the placenta.The striking finding of our study is the improvement of survival rate in IUGR fetuses receiving intra-amniotic nutritional supplementation. Our results demonstrate that intra-amniotic injection of a modified-parenteral nutrition notably reduces mortality in an animal model of placental insufficiency. In contrast, intra-amniotic nutrition was not able to ameliorate birth weight or fetal cardiac adaptation. Several studies in the early 90s also attempted to supply nutrients in the amniotic cavity with dissimilar results. Mulvihill et al. demonstrated a positive effect on fetal growth with similar mortality by 5-days intra-amniotic continuous infusion of bovine amniotic fluid or dextrose plus amino acids in rabbits ,28. In cUterine horn position seems to be a relevant factor for birthweight in rabbit model. Bautista and colleagues  demonstrThe present study also showed that IUGR induction by uteroplacental vessels ligation had a negative impact on the gut structure that seems to be ameliorated by intra-amniotic injection of nutrients. Similarly, previous studies exhibited improved small intestine growth in IUGR animals by esophageal infusions of nutrients to fetal rabbit and fetal sheep ,29, demoWe acknowledge gender issues as one potential limitation for our study as sex of the animals could not be determined at the time of birth, therefore it was not possible to analyze differences in survival rate and birth weight by gender. As it was stated in the study of Tarrade et al., sexual In conclusion, our study demonstrates that intra-amniotic nutrient supplementation increases survival rates of IUGR fetuses remarkably, particularly among those more severe IUGR animals. The use of a mixed nutritional solution containing essential carbohydrates, amino acids and electrolytes seems as an appropriate approach for reducing the mortality in IUGR. The findings from our study could be considered as a potential advance to fetal intervention of IUGR. This would raise the possibility of therapeutic strategies to improve survival particularly in those more severely restricted fetuses. Future studies are warranted to evaluate different fetal nutritional supplementation in IUGR outcomes in order to find the optimal mode, dose and timing of administration and nutritional composition.


The risks associated with unruptured intracranial aneurysms can be neurologically debilitating and even fatal. Evaluation of these aneurysms is critical for determining what type of intervention is warranted, if at all. Cerebral angiography has long been the gold standard in the evaluation of intracranial aneurysms. However, this diagnostic modality is accompanied by several risks that are made clear to the patient before they consent to the procedure. These risks include the possibility of stroke, groin hematomas, contrast-induced anaphylaxis, contrast nephropathy, and catheter-associated infections. Dynamic CT angiography (4D-CTA) has been studied as an assessment tool for cerebral vasculopathies such as stroke, arteriovenous malformations, and aneurysms. It has been shown that 4D-CTA has the advantage of being less invasive and has a shorter examination time than cerebral angiography. In this article, we present a rare case of a giant calcified aneurysm and compare the findings of a cerebral angiogram and a 4D-CTA study. Approximately two to five percent of the general population have an intracranial aneurysm . RuptureDynamic computed tomography angiography 4D-CTA) has been studied as an assessment tool for cerebral vasculopathies such as stroke, arteriovenous malformations, and aneurysms. The 4D-CTA modality combines the noninvasive nature of conventional computed tomography angiography (CTA) with the dynamic capability of conventional angiography to characterize neuro-vascular structures and illustrate their flow dynamics through continuous volumetric computed tomography (CT) acquisition D-CTA has. RecentlA 73-year-old male presented to an outside hospital complaining of dizziness and vertigo with occasional jerking of his right lower extremity while walking. Past medical history includes diabetes mellitus, hyperlipidemia, congestive heart failure, gastroesophageal reflux disease, and valvular heart disease. The patient has no history of drug or alcohol use, but was previously a smoker. The patient was later transferred to our university hospital and was admitted to the neurosurgery service for further evaluation of his symptoms. Upon presentation, the patient’s physical and neurological exam was unremarkable and the patient had a Glasgow Coma Score of 15.The patient was transferred to our hospital with a non-contrast head CT study done at the initial outside facility. The image showed a giant calcified left middle cerebral artery (MCA) mass upon admission to our academic medical center. A diagnostic cerebral angiogram and 4D-CTA study were ordered for further work-up and evaluation of aneurysmal blood flow. The angiogram showed no intracranial aneurysm opacification Figure . AnalysiThe risks associated with a giant unruptured aneurysm warrant a thorough work-up that elucidates several characteristics such as size, calcification, blood flow, and thrombosis. These are used to help assess rupture risk and the postoperative outcome. Neurosurgeons, interventional neurologists, and neuroradiologists rely heavily on imaging modalities to determine whether a particular intervention such as surgical clipping or endovascular coiling should be indicated for a patient. In a single-institution study comparing patients with unruptured intracranial aneurysms who underwent either coiling or clipping, Bhatia et al. showed that calcification was a sole marker of adverse clinical outcomes in the case of an unruptured aneurysm, whereas size alone was not. Moreover, poor postoperative outcomes were more likely by about eight-fold in the presence of aneurysmal calcification. They concluded that calcification of the neck may uniquely complicate the surgical procedure of clip placement requiring consideration of other methods such as the use of hypothermic circulatory arrest which is not typically desirable ,7.In this case, the diagnostic work-up of the patient allowed for comparing cerebral angiography and 4D-CTA imaging, techniques that have been widely used in the setting of diagnosing intracranial aneurysms. Both imaging modalities were used to assess rupture risk, and it was reassuring to discover that both yielded similar findings and ultimately showed no aneurysmal blood flow likely due to a completely thrombosed lumen. The angiogram showed no contrast filling of the calcified aneurysm, while the 4D-CTA showed no contrast opacification within the non-calcified component of the aneurysm.The perioperative risks of giant calcified intracranial aneurysms necessitate advanced, yet minimally invasive diagnostic testing that can precisely guide care providers in the treatment of these patients. Recently, Vanrossomme et al. proposed 4D-CTA as an imaging modality that could assess aneurysmal wall motion as a potential predictor of rupture risk . This neIn the presence of a giant calcified aneurysm, 4D-CTA showed similar findings to that of a cerebral angiogram. Given its increasing accessibility, potential to provide new rupture risk measures, and advantages over cerebral angiography, 4D-CTA is emerging as a favorable imaging modality in the diagnosis and follow-up of patients with giant calcified intracranial aneurysms.


The progressive organization of immune effectors into functional ectopic lymphoid structures, named tertiary lymphoid organs (TLO), has been observed in many conditions in which target antigens fail to be eliminated by the immune system. Not surprisingly, TLO have been recurrently identified in chronically rejected allografts. Although significant progress has been made over the last decades in understanding the molecular mechanisms involved in TLO development (a process named lymphoid neogenesis), the role of intragraft TLO (if any) in chronic rejection remains elusive. The prevailing dogma is that TLO contribute to graft rejection by generating and propagating local humoral and cellular alloimmune responses. However, TLO have been recently observed in long-term accepting allografts, suggesting that they might also be able to regulate alloimmune responses. In this review, we discuss our current understanding of how TLO are induced and propose a unified model in which TLO can play deleterious or regulatory roles and therefore actively modulate the kinetics of chronic rejection. In France alone, the personal, societal, and economic consequences of vital organ failure have a cost of more than €70 billion a year .Vital organ failure is a life-threatening condition where a vital organ  does not perform its expected function. Recent lifestyle changes in developed countries, and the increased incidence of chronic diseases such as hypertension, obesity, and diabetes, have set the stage for accelerated risk for, and the occurrence of, vital organ failure. As a result, vital organ failure is currently recognized as the leading cause of debility and premature death worldwide  therapeutic option.In clinical transplantation, the donor is from the same species but genetically different. Consequently, the immune system of the recipient inevitably recognizes the antigenic determinants  that differ between the recipient and the donor, particularly the highly polymorphic molecules from the major histocompatibility complex  in humans. The alloimmune response that develops against the donor-specific HLA molecules is responsible for tissue damage, which leads to the failure of the transplanted organ, a process named “rejection.”In the absence of a clinically applicable protocol able to induce the specific tolerance of the allogenic transplant by the recipient’s immune system , 2, the Rejected grafts are characterized by interstitial infiltration of cellular effectors, mainly T cells and macrophages, but also dendritic cells, NK cells, B cells, and plasma cells.In contrast with acute rejection, where infiltrates exhibit no particular spatial organization, during chronic rejection immune cells tend to organize themselves in structures that morphologically resemble the secondary lymphoid organs.Analyzing all sorts of human kidney grafts removed for terminal chronic rejection, we and others showed that in the majority of chronically rejected grafts the immune cells were grouped, conferring a nodular organization to the infiltrate , 7. ThesThe structural organization of immune effectors observed in chronically rejected renal grafts Figure  does notBecause the microarchitecture of organized immune infiltrates is highly reminiscent of that of secondary lymphoid organs, these lymphoid structures have been named TLO.Primary immune responses are initiated in secondary lymphoid organs, which are located at strategic sites where antigens are most likely to be encountered.The development of secondary lymphoid organs, a process named lymphoid organogenesis, is initiated during embryogenesis independently of antigen recognition at predetermined sites as a result of complex interactions between hematopoietic, mesenchymal, and endothelial cells , 19. LymThe induction phase depends on lymphoid-tissue inducer cells, which arise in the fetal liver. Under the influence of TRANCE  or IL-7  lymphoid-tissue inducer cells express membrane-bound lymphotoxin: a heterotrimer containing lymphotoxin α and lymphotoxin β that allow lymphoid-tissue inducer cells to interact with the lymphotoxin β receptor (LTβR) of stromal cells. Signaling through the LTβR initiates NFκB signaling in stromal cells, which promotes the production of homeostatic chemokines , 19.Homeostatic chemokines are crucial for the organization phase. CXCL13 recruits circulating B cells to what becomes the B cell area of lymphoid tissues, and the T zone chemokines (CCL19 and CCL21) attract T and dendritic cells to shape the T cell area , 19. TheChronic rejection provides optimal conditions for studying the molecular mechanisms involved in the development of TLO Figure . Indeed,pos B cell population expressing IgD and Bcl-2 were similar to primary follicles, while nodules with a core of CD20posIgDnegBcl-2neg B cells, highly expressing Bcl-6 that had pushed aside the CD20posIgDposBcl-2pos B cells, resembled secondary follicles, i.e., germinal centers  involved in the formation and the maintenance of canonical secondary lymphoid organs   , 19. Thesection) . These r1β2-expressing lymphoid-tissue inducer cells and lymphotoxin-β receptor-expressing stromal organizer cells er cells , 30. In er cells .It is conceivable that instead of conflicting with each other, these different works reveal the fact that several pathways can promote the initiation of TLO depending on the initiating events. This hypothesis was recently substantiated by the demonstration that the development of bronchus-associated lymphoid tissue was triggered by different pathways according to the pathogen responsible for lung inflammation .While significant progress has been made in the identification of the molecular mechanisms that participate to the development of TLO, the endogenous signals capable of inhibiting the lymphoid neogenesis are far more elusive. Through evaluation of synovial tissues from rheumatoid arthritis patients it has been recently reported that low interleukin-27 (IL-27) expression corresponds with an increased incidence of TLO and gene signatures associated with their development and activity. The presence of synovial TLO was also noted in mice deficient in the IL-27 receptor after the onset of inflammatory arthritis . IL-27 mpos Tregs) in both a murine experimental model and human samples  they have an antigen-specific receptor (B cell receptor), which when engaged by surface-tethered antigens leads to the formation of an immunological synapse that coordinates cell signaling events and promotes antigen uptake for presentation on MHC class II molecules , even whin vitro generated effector B cells that produced either TH1- or TH2-type cytokines were shown to promote the activation and differentiation of naïve T cells into effector TH1 and TH2 cells, respectively (in vivo. In a murine model of Toxoplasma gondii infection, TNF production by B cells was shown to be required for the generation of an optimal TH1 cell protective response (H. polygyrus was shown to depend on IL-2-producing B cells (In addition to presenting antigen, B cells can also enhance T cell-mediated immune responses through the secretion of cytokines and chemokines. Studies from the group of Frances Lund  have shoectively . The impresponse . In anot B cells . The exaSince grafts in which TLO were harboring germinal center reactions had a shorter life expectancy Figure , we haveThe absence of an unequivocal deleterious role for B cell clusters has led to the conclusion that these structures could be like “fish in a sunken ship,” i.e., although fish are frequently seen in a sunken boat, they play no role in the process responsible for the shipwreck.An alternative explanation could reconcile these apparently conflicting results. As discussed above, the proportion of B cells that infiltrate chronically rejected kidney grafts does not correlate with the functionality of intragraft TLO . The attAnother layer of complexity has recently been brought into the picture by experimental evidence that certain B cell subsets are endowed with an immune regulatory role . For insTransplanted organ expresses donor-specific alloantigens, which stimulate a recipient’s immune system. Prevention of acute rejection of the graft is achieved using a combination of non-specific immunosuppressive drugs that can only partially block the alloimmune effectors. The residual enduring alloimmune response promotes immune injuries known as chronic rejection, the main cause of late allograft loss. As in other chronic immune diseases, immune effectors within chronically rejected allografts progressively organize into functional TLO that display the same microarchitecture as secondary lymphoid organs, a process known as lymphoid neogenesis. Because biopsy-based studies have reached conflicting conclusions regarding the pathological significance of these TLO, it has been proposed that the presence of TLO in rejected grafts is a non-specific response to local inflammation-induced production of chemokines. While that can indeed sometimes be the case, it should not be excluded that under appropriate conditions, lymphoid neogenesis turns non-functional TLO into ectopic germinal centers, in which a local aggressive humoral immune response can be elicited. Alternatively, functional TLO can also regulate immune responses and slow down the destruction process.Therefore, we propose that TLO be considered as active players, able to modulate the kinetics of the natural history of chronic rejection. Future works will determine if the versatility of TLO can be manipulated to design innovative therapeutic interventions that would improve graft life expectancy.All the authors listed have made 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.


Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat.Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset  is an important neglected tropical disease that has caused severe epidemics and epizootics throughout Africa and the Arabian Peninsula. Severe outbreaks have involved tens of thousands of both human and livestock cases for which no effective, commercially available human vaccines are available. Vaccine candidates have been developed based on the complete deletion of two known RVF virus virulence factors, the NSs and NSm genes. These vaccines were previously demonstrated to be protective in rats, mice, and sheep. In this study, we expand upon those results and evaluate the vaccine candidates in a non-human primate model for RVF. The animals received a single dose of vaccine that led to the development of a robust immune response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated animals. All vaccinated animals that were subsequently challenged with RVF virus were protected against viremia and liver disease. These results demonstrate that the vaccines are safe and effective in non-human primates, which provides the impetus for further development of these candidates for use in humans. Phenuiviridae, genus Phlebovirus) was first isolated in 1930 in East Africa [Culex, Mansonia and Anopheles mosquitoes appear to be the principal vectors for humans [Culex pipiens) reinforces the feasibility of accidental or intentional import of RVFV from endemic regions with subsequent maintenance in nascent vector and host populations [Rift Valley fever virus  or by cr humans –7. Humanr humans . The sprulations . The emeulations  and the ulations . FurtherClearly, RVFV is an important threat to human and animal health for which no specific treatment currently exists. Several RVFV vaccines have been developed , but curThe rRVFV vaccine candidates containing the insertion of the enhanced green fluorescent protein and the precise deletion of the NSs gene alone (ΔNSs:GFP rRVFV) or the NSs/NSm genes in combination (ΔNSs:GFP-ΔNSm rRVFV) were described by Bird et al. to be highly attenuated, immunogenic, and efficacious in the rat lethal disease model . FurtherCallithrix jacchus). Marmosets were more susceptible to RVFV than rhesus macaques and experienced higher rates of morbidity, mortality, and viremia and marked aberrations in hematological and chemistry values. Depending on the route of exposure, these animals exhibited acute-onset hepatitis, delayed-onset encephalitis, and hemorrhagic disease, which are dominant features of severe human RVF [Rhesus macaques historically have been used to evaluate potential vaccines and therapeutics for RVFV . We prevuman RVF . An addiuman RVF , 40. Cynuman RVF , 40. ColHere, we demonstrate that both the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates were found to be generally safe and immunogenic in a marmoset model of RVF. The vaccinated marmosets exhibited no signs of clinical illness post-vaccination and post-challenge and developed strong neutralizing antibody titers. Our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this important public health threat.This work was supported by an approved USAMRIID IACUC animal research protocol (AP-10-066). Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. Approved USAMRIID animal research protocols undergo an annual review every year. Animals are cared for by a large staff of highly qualified veterinarians, veterinary technicians, and animal caretakers. All personnel caring for and working with animals at USAMRIID have substantial training to ensure only the highest quality animal care and use. Humane endpoints were used during all studies and marmosets were humanely euthanized when moribund according to an endpoint score sheet.Construction of the rRVFV has been previously described , 26, 35 Callithrix jacchus), 1 to 3 years old and ranging in weight from 257 to 398g were obtained from World Wide Primates  35 days post-vaccination. This dose was chosen based on our previous model development study [For the study design, two groups of six RVFV seronegative marmosets were inoculated subcutaneously with 5 lognd sheep , 25. Allnt study . FollowiWhole blood was added to an EDTA tube  for complete blood count (CBC) determination using a Hemavet instrument  according to manufacturer’s instructions. Clinical chemistry analyses were performed by addition of whole blood to a lithium heparin tube (Sarstedt) using the comprehensive diagnostic panel analyzed on a Vetscan instrument  according to manufacturer’s instructions. Normal ranges in the chemistry and hematology results of healthy marmosets  were useFull necropsies and histological examination were performed by a board-certified veterinary pathologist. The following tissues were collected during necropsy: Axillary, inguinal, submandibular, mesenteric and tracheobronchial lymph node; submandibular salivary gland; haired skin; brachial plexus; sciatic nerve; skeletal muscle; bone marrow (femur); eyes; brain; pituitary gland; spleen; adrenal gland; kidney; liver; stomach; duodenum; pancreas; jejunum; ileum; cecum; colon; testis/ovary; prostate gland/uterus; urinary bladder; tongue; tonsil; trachea; esophagus; thyroid gland; lung; thymus; and heart. All collected tissues were immersion-fixed in 10% neutral buffered formalin for at least 21 days. The tissues were trimmed and processed according to standard protocol . HistoloAnti-RVFV total IgG ELISA was performed essentially as described previously , with thRepeated measures ANOVA was used to compare chemistry, viremia, weight, temperature, and antibody response over time and between groups. All analyses were conducted using GraphPad Prism 7.00 .10 PFU of the ΔNSs or ΔNSs/NSm rRVFV while five marmosets served as the sham-vaccinated controls. All animals were monitored for weight loss, survival, and clinical signs of infection and blood samples were collected for virological, hematological, immunological, and chemical analyses. All of the vaccinated and sham-vaccinated marmosets survived and no animals exhibited clinical signs of illness, experienced significant weight loss (Marmosets (n = 6/group) were inoculated subcutaneously with 5 logght loss , S1 Fig ght loss , S2 Fig 10 PFU/mL was detected on day 2 post-challenge.Viral RNA was detected on day 2 post-vaccination by qRT-PCR in 4/6 animals that received ΔNSs rRVFV and 5/6 animals that received ΔNSs-ΔNSm rRVFV , S3 Fig.In our previous model development study we observed marked aberrations in hematological and chemistry values from marmosets exposed to RVFV. In particular, the liver enzyme ALT was significantly increased when marmosets were exposed to RVFV subcutaneously. We therefore collected blood samples post-vaccination and post-challenge for hematological and chemical analyses. Overall, no significant change in the hematology and clinical chemistry values of the vaccinated animals was observed post-vaccination or post-challenge. For example, the ALT levels  in vacciOD values were low , which is most likely due to the secondary antibody being raised against rhesus macaques and not marmosets (a marmoset specific antibody does not exist). The neutralizing antibody and RVFV IgG titers increased for all animals post-challenge.All vaccinated animals developed neutralizing antibodies by day 14 post-vaccination . These tThe tissues were tested for viral RNA by qRT-PCR and all tissues from the vaccinated animals were negative except for the spleen and axillary lymph node of one animal receiving the rZH501-ΔNSs vaccine and the skeletal muscle and cerebrum of one animal receiving the rZH501-ΔNSs-ΔNSm vaccine . All of In summary, both the ΔNSs and ΔNSs-ΔNSm vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated marmosets exhibited no signs of clinical illness post-vaccination and post-challenge and developed strong neutralizing antibody titers. Additionally, minimal viremia as detected by qRT-PCR was observed post-vaccination and no viral RNA was identified in the serum of vaccinated animals post-challenge.RVFV significantly impacts livestock and human health making it a good target for a one-health prevention approach through animal vaccination. Livestock vaccination during non-epidemic periods or as an early countermeasure against early outbreaks could eliminate one of the main sources of human infection and limit the scope of epidemics. However, previous RVFV outbreaks are generally recognized only after human cases are diagnosed , 50. AddCurrently, there is no fully licensed vaccine for veterinary or human use available in non-endemic countries. In endemic countries, there is no clear guidance for livestock vaccinations to prevent RVF outbreaks. Furthermore, previous veterinary vaccines for RVF has been plagued with numerous concerns such as high manufacturing costs, a poorly defined genetic identity, poor efficacy, no capacity to differentiate vaccinated from naturally infected livestock, and the risk of vaccination in pregnant animals due to associated teratogenesis and abortion , 51, 52.Bird et al. developed rationally designed vaccine candidates based on the complete deletion of two known RVFV virulence factors, the NSs and NSm genes , 25. The10 PFU of virus and exhibited no signs of clinical illness, experienced no significant weight loss, or temperature changes post-vaccination and post-challenge. Additionally, minimal viral RNA was observed post-vaccination and no viral RNA was identified in the serum of vaccinated animals post-challenge. No significant change in the hematology and clinical chemistry values of the vaccinated animals was observed post-vaccination or post-challenge. In contrast, the liver enzyme ALT was significantly increased in sham-vaccinated control animals suggesting that the vaccinated animals were protected from RVFV-induced liver disease. Collectively, these results demonstrate the general safety of these vaccine candidates in NHPs. However, more extensive safety testing such as an assessment of neurovirulence would be necessary for advance development efforts. This would be especially important for the ΔNSs vaccine candidate which was shown to cause a uniform fatal encephalitis after intranasal, but not subcutaneous exposure in C57BL/6 mice [We demonstrate that both the ΔNSs rRVFV and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated marmosets received 5 logL/6 mice . A separL/6 mice . These sin vivo virus replication and less stimulation of the antiviral immune response. However, we did detect similar levels of viral RNA in the blood on day 2 post-vaccination for both the single and double deletion viruses. It is possible that differences in the kinetics or magnitude of virus replication occur between the single and double deletion viruses that we didn’t detect with the current study design. However, even with the slight reduction in antibody titers all animals were completely protected by both vaccine candidates. Since the double-genetic deletions of the entire RVFV NSs and NSm genes does not significantly decrease overall vaccine efficacy, it makes sense to pursue this as the lead candidate for licensure. The ΔNSs-ΔNSm rRVFV is likely safer due to multiple attenuating lesions leading to a reduced possibility of reversion to full virulence.The immunogenicity of the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates was noteworthy. All vaccinated animals developed high neutralizing antibody titers by day 14 post-vaccination, which peaked by day 21 post-vaccination. Antibody titers were slightly higher for animals receiving the ΔNSs rRVFV than animals vaccinated with the double-deletion ΔNSs-ΔNSm rRVFV, but this difference was not found to be statistically significant. The ΔNSs rRVFV may be slightly more immunogenic because of the single deletion in a known RVFV virulence factor compared to ΔNSs-ΔNSm rRVFV, which has two gene deletions. We would expect that the ΔNSs-ΔNSm rRVFV would be more attenuated presumably due to reduced 80 of 1:237 [80 values of ≥1:640 [80 ranged from 1:6,400 to 1:8,267 on day 21 post-vaccination, indicating that the level of neutralizing antibody was substantially higher to that demonstrated in earlier studies of RVFV vaccines in NHP models or in human volunteers. However, it is difficult to directly compare antibody titers between various studies for the aforementioned reasons.It is difficult to directly compare antibody titers as an indication of protective immunity to those of previous studies with other RVFV vaccines because of the differences in the candidates/approach, species level differences in immunity, and timing for assessing the response. However, a retrospective study of human volunteers (n = 598) receiving a three-dose regimen  of inactivated TSI-GSD-200 vaccine reported that subjects developed a mean PRNTof 1:237 . The livf ≥1:640 , 56. In 10 PFU/mL) was chosen based on our previous model development effort, which indicated that we would likely see 50% mortality with the sham-vaccinated control animals. Surprisingly, no mortality was observed for sham-vaccinated animals, which may be a result of the age of the animals used in the current study which ranged from 1 to 3 years old. The age of the animals used in our previous model development effort were 2 to 11 years old with the majority of the animals being older (between 8–11 years old). In fact, the animals that succumbed to RVFV by subcutaneous exposure were 10–11 years old [The virulent virus challenge dose used in this study Click here for additional data file.S1 FigIndividual weights of marmosets post-vaccination (top) and post-challenge (bottom). Percent change in baseline of weights of marmosets post-vaccination (top) with rZH501-ΔNSs (n = 6), rZH501-ΔNSs-ΔNSm (n = 6), or sham inoculated controls (n = 5) and post-challenge (bottom) with 6 log10 PFU of the virulent strain ZH501. The symbols represent the mean value and the error bars represent the standard error of the mean.(TIF)Click here for additional data file.S2 FigIndividual temperatures of marmosets post-vaccination (top) and post-challenge (bottom). Percent change in baseline of the temperature of marmosets post-vaccination (top) with rZH501-ΔNSs (n = 6), rZH501-ΔNSs-ΔNSm (n = 6), or sham inoculated controls (n = 5) and post-challenge (bottom) with 6 log10 PFU of the virulent strain ZH501. The symbols represent the mean value and the error bars represent the standard error of the mean.(TIF)Click here for additional data file.S3 FigIndividual viremia determined by qRT-PCR in marmosets post-vaccination (top) and post-challenge (bottom). RNA detected by qRT-PCR in marmosets post-vaccination (top) with rZH501-ΔNSs (n = 6), rZH501-ΔNSs-ΔNSm (n = 6), or sham inoculated controls (n = 5) and post-challenge (bottom) with 6 log10 PFU of the virulent strain ZH501. The symbols represent the mean value and the error bars represent the standard error of the mean. Because of the difficulty viewing the results on day 2 PI, the animal ID’s with RNA detected are in bold text in the legend. The dashed line represents the assay LOD. PFUe, plaque-forming unit equivalent.(TIF)Click here for additional data file.


DNA methylation on cytosine residues is known to affect gene expression and is potentially responsible for the phenotypic variations among different crop cultivars. Here, we present the whole-genome DNA methylation profiles and assess the potential effects of single nucleotide polymorphisms (SNPs) for two mungbean cultivars, Sunhwanogdu (VC1973A) and Kyunggijaerae#5 (V2984). By measuring the DNA methylation levels in leaf tissue with the bisulfite sequencing (BSseq) approach, we show both the frequencies of the various types of DNA methylation and the distribution of weighted gene methylation levels. SNPs that cause nucleotide changes from/to CHH – where C is cytosine and H is any other nucleotide – were found to affect DNA methylation status in VC1973A and V2984. In order to better understand the correlation between gene expression and DNA methylation levels, we surveyed gene expression in leaf tissues of VC1973A and V2984 using RNAseq. Transcript expressions of paralogous genes were controlled by DNA methylation within the VC1973A genome. Moreover, genes that were differentially expressed between the two cultivars showed distinct DNA methylation patterns. Our mungbean genome-wide methylation profiles will be valuable resources for understanding the phenotypic variations between different cultivars, as well as for molecular breeding. Vigna radiata [L.]) is a self-pollinated diploid plant with 11 chromosomes (2n = 22), which taxonomically, belongs to the Phaseoleae tribe and the Fabaceae family. It is an important legume crop that is widely cultivated in Asia and serves both as a cash crop and as an important source of nutrition. The mungbean can be used in several ways; the seeds, sprouts, and young pods are all consumed as sources of protein, amino acids, vitamins, and minerals, and its by-products are also used as green manure and feedMungbean With the recent advances in sequencing technologies, along with the availability of fine-tuned chemistries, obtaining the whole-genome DNA methylation profile is feasible via Illumina Hiseq2000 on three biological replicates of leaf mRNA samples from both cultivars at the V1 growth stage, in order to understand the effect of DNA methylation on gene expression 2pression . A totalArabidopsis thalianaGlycine maxA. thaliana and G. max, which have mCG as the predominant sites3Phaseolus and VignaThe VC1973A methylome contains 7,804,417 methylated CGs , 9,092,603 mCHGs , and 20,106,381 mCHHs  . The totTo understand the distribution of DNA methylation around genes, we calculated the weighted methylation levelsA. thaliana and G. max. That is, a slight increase was observed at transcription start sites and in the middle of gene coding regions, and a steep increase was found in the upstream and downstream regions  were rarely expressed compared to the low methylated (<10%) genes. Additionally, the genes that were methylated >5% by mCHH showed low expression levels . OverallAs an ancient whole-genome duplication (WGD) in mungbean genome occurred nearly 59 million years ago (MYA)P-value < 0.01). It is possible that single nucleotide polymorphisms (SNPs) between the two cultivars affect DNA methylation levels by changing the cytosine context. Therefore, we surveyed the SNPs found in VC1973A versus V2984, using previously generated resequencing dataWe next compared the DNA methylation states of VC1973A and V2984. Of the commonly called 114,679,466 cytosine contexts, we found 85,832 sites that are differentially methylated  suggesting that DNA methylation on possible downstream regulatory region may suppress the expression of this gene in VC1973A  between the two accessions, we compared gene expression levels from the RNAseq data using Cuffdiff software VC1973A . Moreove VC1973A . These Dvia known pathways in the CG, CHG, and CHH contexts is a type of epigenetic gene regulationDNA methylation that occurs Arabidopsis and soybean3Here, we found that in the mungbean genome, the overall proportion of mCHH DNA methylation was the highest compared to the other possible methylation sites examined. This is quite different than what has been previously reported in the model plants, The gene content within a given genome can be increased by several types of duplication events, such as whole-genome duplication, segmental duplication, and tandem duplicationThe dependency of DNA methylation on genetic variants, such as those resulting from SNPs, allowed us to classify the epialleles into three major groups: obligate epialleles, pure epialleles, and facilitated epiallelesde novo DNA methylation, and the consequent phenotypic variations within the mungbean germplasm.Our BSseq analysis of the mungbean genome has expanded the understanding of genome-wide gene family regulation by DNA methylation. The notable feature of abundant mCHH traces in mungbean, and the frequent variations between cultivars as obligate epialleles, will also provide information that may prove valuable towards the goal of unraveling the role of To identify methylated genomic regions, we implemented bisulfite sequencing, or BSseq. Young leaves from VC1973A and V2984 were collected in triplicate at the third growth stage. DNA was extracted using the cetyltrimethyl ammonium bromide method (Gelvin and Schilperoot 1995), bisulfite treatment was performed, and DNA sequencing libraries were constructed for both VC1973A and V2984 samples. Briefly, input gDNA (5 μg) was fragmented by Covaris shearing . The fragments were blunt-ended and phosphorylated, and a single ‘A’ nucleotide was added to the 3’ ends in preparation for ligation to a methylated adapter that has a single-base ‘T’ overhang, using the TruSeq DNA library preparation kit provided by Illumina . The ligation products were then purified and size-selected by agarose gel electrophoresis. Size-selected DNA was subsequently bisulfite-treated twice and purified using procedures that were adapted from the EpiTect Bisulfite Kit . The bisulfite-treated DNA was subsequently PCR-amplified to enrich for fragments that have adapters on both ends. The final purified product was then quantified using qPCR, according to the qPCR Quantification Protocol Guide and qualified using the Agilent Technologies 2100 Bioanalyzer . BSseq libraries were then sequenced using the HiSeq™ 2000 platform  for 101 cycles.We extracted RNA from three replicates of each VC1973A and V2984 leaf sample that was analyzed by BSseq (described above). RNA samples were validated using a 2100 bioanalyzer RNA 6000 NANO chip, and DNA samples were validated with Picogreen . We selected high quality DNA and RNA pairs for each cultivar in triplicate. RNAseq libraries were constructed using the Illumina TruSeq Kit , following the manufacturer’s guidelines.The sodium bisulfite non-conversion rate was calculated as percentage of methylcytosines aligned with the unmethylated chloroplast genome (Mungbean chloroplast reference [KPS1 = VC1973A doi:10.1093/dnares/dsp025]). Reads produced from bisulfite sequencing were supplied to Bismark softwareFor the correction of methylation count, we used the mungbean chloroplast genome as negative control for the DNA methylation as it is known to highly unmethylated. Based on the read mapping status on the chloroplast genome, we could calculate the error rate of bisulfite sequencing method as 0.001114. Applying this error rate on the counts of methylated and unmethylated read supports on every cytosine contexts, we could determine the methylation status using binomial test. P values from the binomial test were converted into Q value for correct the significance levelFisher’s exact test shows significance  from the contingency table of the methylated and unmethylated counts of Sunhwanogdu and Kyunggijaerae 5 within each candidate DMR.To retrieve DMRs, we retrieved the counts of the corrected methylated read supports within 200 bases window along with the chromosomes allowing 150 bases overlap. The windows containing more than 5 methylated reads were collected to define the candidate DMRs. This operation was done for both Sunhwanogdu (VC1973A) and Kyunggijaerae#5 (V2984). The final DMRs were determined if With data >90% of the methylation level and supporting depth >10, paralogous genes were identified based on peptide homology using the NCBI blastp program . The paralogous gene pairs were then clustered using MCScanXHow to cite this article: Kang, Y. J. et al. Genome-wide DNA methylation profile in mungbean. Sci. Rep.7, 40503; doi: 10.1038/srep40503 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Immuno-oncology not only refers to the multifaceted relationship between our immune system and a developing cancer but also includes therapeutic approaches that harness the body’s immune system to fight cancer. The recognition that metabolic reprogramming governs immunity was a key finding with important implications for immuno-oncology. In this review, we want to explore how activation and differentiation-induced metabolic reprogramming affects the mevalonate pathway for cholesterol biosynthesis in immune and cancer cells. Glycolysis-fueled mevalonate metabolism is a critical pathway in immune effector cells, which may, however, be shared by cancer stem cells, complicating the development of therapeutic strategies. Additional engagement of fatty acidy oxidation, as it occurs in regulatory immune cells as well as in certain tumor types, may influence mevalonate pathway activity. Transcellular mevalonate metabolism may play an as yet unanticipated role in the crosstalk between the various cell types and may add another level of complexity. In humans, a subset of γδ T cells is specifically adapted to perform surveillance of mevalonate pathway dysregulation. While the mevalonate pathway remains an important target in immuno-oncology, in terms of personalized medicine, it may be the type or stage of a malignant disease that determines whether mevalonate metabolism requires training or attenuation. Immune cell activation is associated with shifts in cellular metabolism , 2. In cM1 macrophages, classically activated by the Th1 cytokine interferon-γ (IFN-γ) plus lipopolysaccharide (LPS), are myeloid effector cells, which are characterized by the expression of high levels of pro-inflammatory cytokines, reactive nitrogen and oxygen intermediates, promotion of Th1 response, and strong microbicidal and tumoricidal activity . Like ThM1 macrophages and DCs engage glycolysis-fueled lipogenesis to expand cellular compartments such as the endoplasmic reticulum and the Golgi as well as to prepare the entire secretory machinery for effector responses . For thiMyelopoiesis is driven by colony-stimulating factors including granulocyte/macrophage-colony-stimulating factor (GM-CSF) and M-CSF, which are important for the development and function of monocytes and macrophages. In murine myelopiesis, M-CSF stimulation activated mTORC1 and mTORC1-driven glycolysis initiated a transcriptional program involving activation of the protooncogene Myc , which iGranulocyte/macrophage-colony-stimulating factor plays a critical role in promoting glycolysis-fueled mevalonate metabolism  in M1 maAlthough the maintenance of immature antigen-presenting DCs is facilitated by fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) in a steady state , maturatreg) cells  cells  and toleg) cells  also opeg) cells . The rolmpetence . These fvia excessive protein prenylation   Figure  were shode novo pathway of FAS. As a consequence, arachidonic acid-pulsed monocytes acquire inflammatory phenotype and function  but may increase to >30% during periods of starvation. In addition, unusually high concentrations of extracellular isoprenols (>10 μM) may result in a relative contribution of up to 50% . MoreoveIsopentenyl diphosphate (IPP) can also be exported to the extracellular space. In DCs, the cholesterol efflux transporter ATP-binding cassette transporter A1 (ABCA1) has recently been shown to also mediate the efflux of IPP . Since cConsistent with a role of extracellular IPP in immune surveillance, the ecto-ATPase CD39 has recently been shown to also dephosphorylate and inactivate IPP and other mevalonate-derived phosphoantigens, thus limiting the duration and strength of phosphoantigen-induced γδ T cell responses . AdditioAlong the same line, add-back experiments, in which mevalonate metabolism of immune –46 and cC. albicans cell wall , which catalyzes the cytosolic formation of acyl carnitines at the outer mitochondrial membrane for mitochondrial import and subsequent oxidation of FAs . The etoin vivo . Unfortuin vitro and in a xenotransplantation model of human CML (Although c-Abl-specific tyrosine kinase inhibitors (TKIs) substantially extend the survival of patients with chronic myeloid leukemia (CML), TKIs fail to eliminate leukemic stem cells resulting in minimal residual disease. Recently, primitive CML cells were shown to rely on upregulated OXPHOS for their survival, and intriguingly, combination treatment with the TKI imatinib and tigecycline, an antibiotic that inhibits mitochondrial protein translation, selectively eradicated leukemic stem cells both uman CML .As outlined above in the context of regulatory immune cells, additional engagement of FAO may divert acetyl-CoA from mevalonate metabolism Figures  and 2A. It is now becoming increasingly clear that mevalonate metabolism governs immune surveillance. However, cancer cells and in particular cancer stem cells may likewise depend on this metabolic pathway. Such a similarity in metabolic orientation between tumor cells and immune effector cells infiltrating the tumor microenvironment inevitably leads to a competition for the nutrients, metabolites, and oxygen that are required for fueling mevalonate metabolism and may ultimately even turn into a struggle for survival. While pharmacological inhibition of mevalonate metabolism in tumor cells may attenuate growth and proliferation, tonic flux through the mevalonate pathway in innate immune cells such as macrophages may contribute to trained immunity.reg cells and M2 macrophages) acquire regulatory function and tumor cells may undergo metastasis. Inhibition of FAO therefore appears to be desirable either as the primary therapeutic approach or as an adjuvant preceding cancer immunotherapy. In addition, the limitation of mevalonate pathway activity resulting from enhanced FAO might increase the sensitivity of tumor cells and regulatory immune cells to statins. Future personalized cancer medicine should include the assessment of the metabolic status of the patients’ tumor in order to develop the appropriate therapeutic strategies. Sequential therapy regimens might start with inhibitors of mevalonate metabolism and FAO to directly block tumor cells as well as regulatory immune cells, followed by immunotherapies that induce trained immunity in innate immune cells via mevalonate pathway stimulation.The additional engagement of FAO as it has been described for breast and prostate cancer cells may limit the availability of acetyl-CoA for mevalonate generation and metabolism. As a consequence, immune cells (TAll authors listed have made substantial contributions to text and figures and have approved the manuscript for submission.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.


Current iterations of the hygiene hypothesis suggest an adaptive role for helminth parasites in shaping the proper maturation of the immune system. However, aspects of this hypothesis are based on assumptions that may not fully account for realities about human helminth infections. Such realities include evidence of causal associations between helminth infections and asthma or inflammatory bowel disease as well as the fact that helminth infections remain widespread in the United States, especially among populations at greatest risk for inflammatory and autoimmune diseases. Beginning in the late 20th century, there was an increase in the reported cases of inflammatory and autoimmune diseases worldwide, such as asthma, inflammatory bowel disease (IBD), food allergies, and multiple sclerosis (MS) . This phWe argue here that parasitic helminth infections have not been demonstrated as necessary for proper host immune maturation in either humans or in animal models. Several animal studies and select human studies have demonstrated beneficial anti-inflammatory responses to autoimmune and inflammatory diseases when treated with helminths. However, a number of other studies have noted helminths can exacerbate and even induce inflammatory conditions. Without significant evidence to support their therapeutic potential, we express concern over the safety of administrating living parasites with known human morbidity. We instead suggest directing future studies towards the identification and trial of key helminth-derived immunomodulatory molecules that might provide a safer and better mechanistically understood therapy .Necator americanus), whipworms (Trichuris trichiura), roundworms (Ascaris lumbricoides), and schistosomes (mostly Schistosoma haematobium and Schistosoma mansoni) cause chronic, debilitating diseases—which include iron deficiency anemia, asthma, vitamin deficiency, abdominal pain, colitis, and dysentery—leading to growth failure and impaired cognitive development . Wh. Wh11]. Asthma and Loeffler’s pneumonitisAscaris lumbricoidesToxocara canisToxocara catiInflammatory bowel disease: enteritis and colitisSchistosoma mansoniTrichuris trichiuraStrongyloides stercoralisA. lumbricoides infection) is a risk factor for the development of asthma phenotypes in endemic regions ) and intestinal schistosomiasis (a disease infecting over 250 million people that results from infection by S. mansoni) are major causes of pathology of the small and large intestine [T. trichiura attaches to the colon, where it can induce acute and chronic colitis, Trichuris dysentery syndrome (TDS), and rectal prolapse, especially in heavy infections [Trichuriasis  homologue [T. muris infections and human ulcerative colitis, and blocking of ectopic MUC5AC expression was identified as a likely contributor in the effectiveness of oral tacrolimus therapy in patients with refractory ulcerative colitis [T. muris-infected colonic tissue histologically resembles established mouse models of IBD, with a defective epithelial barrier and a dominant Th1 immune infiltrate, leading to impaired intestinal mucosa homeostasis [h1 immune infiltration, leading to the chronic colonic pathology in both trichuriasis and IBD [Trichuris-induced colitis and IBD, in 2013, Levision and colleagues identified through quantitative trait loci mapping several key overlapping genes expressed during human Crohn’s disease and Trichuris-induced colitis [T. muris-infected mice [T. muris-infected mice showed direct suppression of critical Th2 cytokines, IL-4 and IL-13, necessary for worm expulsion [Trichuris-induced colitis and both Crohn’s disease and ulcerative colitis warrants further investigation of Trichuris-induced colitis as an environmental driver of IBD worldwide. There is a need to conduct further epidemiological studies in order to determine the attributable risk of colitis in low- and middle-income countries to trichuriasis.As opposed to the Tomologue ,37. MUC5 colitis ,39. Furteostasis . Resisti and IBD ,42. Expa colitis . Interleted mice –46. Impoxpulsion . More rexpulsion . The cliHeligmosomoides polygyrus bakeri, which is hypothesized to recruit Tregs to control colitis, in part through secretion of IL-10. Additional studies have shown other, phylogenetically distinct helminthic species—including Hymenolepis diminuta (cestode), S. mansoni (trematode), and Litomosoides sigmodontis (nematode)—are capable of reducing inflammation in the gut, predominately through IL-10 secretion[Despite the severe negative consequences of parasitic infections that are highlighted above, some studies have presented a counterview of generalized human helminth infections as potentially protective against the development of autoimmune and inflammatory diseases. This protection is hypothesized to be due to their known immunomodulatory properties within a host. Helminths can maintain host evasion for years through an array of mechanisms that down-regulate host innate and adaptive responses. These include immunomodulatory proteins that contain host-related glycans and lipids that direct cytokine mimicry and interference, nonprotein signature molecules that result in immunosuppressive host cytokine release, and direct interference of antigen presentation. The presence of T regulatory cells (Treg) along with immune modulatory cytokines IL-10, TGF-β, and, at basal levels, IL-18, promote helminth survival while simultaneously quelling the host inflammatory response ,48. One secretion.Lancet Infectious Diseases review questioning helminth therapy versus elimination by Wammes and colleagues highlights this question by identifying the discrepancy between findings in murine models and human studies [We are left with two seemingly contradictory sets of findings regarding the role of helminth parasites in human inflammatory disease and atopy. A recent  studies . Even se studies .A. lumbricoides, T. trichiura, and Strongyloides stercoralis) in the United States [Toxocara canis and Toxocara cati respectively—is considered to be the most common helminth infection in the US [Toxocara infection [Toxocara spp. elicit Loeffler’s type pathology during their larval migrations through the human lungs, causing an asthma phenotype [Toxocara had decreased FEV1 when additional population-based factors such as age, sex, and body mass index were controlled [Adding to the confusion of these findings is an erroneous assumption on which a component of the hygiene hypothesis is sometimes based: namely, that helminth infections have largely disappeared in high-income countries. A 2011 systematic review by Starr and Montgomery identified a now 30-year gap since the last high-quality epidemiological study on soil-transmitted helminths . It was further suggested that hookworm challenge infections might exert such effects by reversing microbial dysbiosis [For IBD and celiac disease, therapy has focused on using the pig whipworm n beings ,63. In tn beings . Two subon rates ,66. A 20 therapy . Althougal hosts . Additioal hosts . These cn humans ,70. For patients . A seconreatment . More reysbiosis . Althougysbiosis .T. suis egg was administered to 5 MS patients during a three-month period and showed a mild reduction in the number of new gadolinium-enhancing lesions, but there were no changes in neurological symptoms, nor was there a placebo-controlled comparison [Helminth therapy has been tried as a treatment for MS after the observation was made that 12 patients with MS and concomitant natural intestinal helminth infection in Argentina had a reduction in the number of relapses and lower MRI activity in comparison to uninfected MS patients . Buildinmparison .The failed therapeutic potential of helminth treatment in humans has been surprising to some scientists. As highlighted above, there have been several animal studies that demonstrate an immunosuppressive role of helminths. Further, human epidemiological studies have suggested a protective role for helminths against autoimmune and inflammatory conditions. However, many of these studies identified delayed or diminished development of allergic or autoimmune responses when helminth treatment was used prophylactically, with few studies that demonstrate a clear therapeutic advantage in already established conditions . FurtherThe rationale for iatrogenic administration of helminths to treat inflammatory diseases has occasionally been based on specious assumptions. Significantly, the assumptions that helminths infections are negligible in the US and other Western nations and that helminths have intrinsic anti-inflammatory properties in humans. We do not argue that helminths evolved with an extraordinary ability to manipulate host immune responses, but their role in relation to humans is not one of symbiosis, as is seen with the bacterial microbiome, but is one of clear parasitism. Given the known risks of live helminth inoculations, research highlighting helminth immunomodulatory properties should direct future research towards identifying helminth-derived molecules of therapeutic potential. This approach is likely to result in a more viable therapeutic window. Further, it’s important not to diminish the role of helminths in inciting and exacerbating inflammatory diseases globally, including in wealthy countries such as the US.A recently emerged attractive alternative hypothesis to explain the rise of inflammatory diseases is a “biome depletion” theory. This suggests inflammatory disease may be due to a loss of species diversity or alteration of composition of the commensal microbiome within the human body . InteresThe immense conflicting data regarding the benefits versus harms of live helminths as a therapeutic modality to date warrants further questioning of the utility of additional human clinical trials. Therefore, directing future research and trials towards helminth-derived immunomodulatory molecules allows for safer and better-described therapies that could alleviate the suffering from autoimmune conditions without the commensurate risk of a parasite infection. Indeed, the aim of experimental animal models should be to develop novel treatments that mimic the effects of helminths without requiring the presence of parasites in the host.There is overwhelming evidence that demonstrates the clinical and economic ramifications of helminths in endemic settings . Therefo