At delivery, if the cord is not clamped, blood continues to pass from the placenta to the newborn during the first minutes of life, allowing the transfer of 25–35 ml/kg of placental blood to the newborn, depending on gestational age, the timing of cord clamping, the position of the infant at birth, the onset of respiration, and administration of uterotonics to the mother. However, deriving benefits from delayed cord clamping (DCC) are not merely related to placental-to-fetal blood transfusion; establishing spontaneous ventilation before cutting the cord improves venous return to the right heart and pulmonary blood flow, protecting the newborn from the transient low cardiac output, and systemic arterial pressure fluctuations. Recent meta-analyses showed that delayed cord clamping reduces mortality and red blood cell transfusions in preterm newborns and increases iron stores in term newborns. Various authors suggested umbilical cord milking (UCM) as a safe alternative when delayed cord clamping is not feasible. Many scientific societies recommend waiting 30–60 s before clamping the cord for both term and preterm newborns not requiring resuscitation. To improve the uptake of placental transfusion strategies, in 2016 an Italian Task Force for the Management of Umbilical Cord Clamping drafted national recommendations for the management of cord clamping in term and preterm deliveries. The task force performed a detailed review of the literature using the GRADE methodological approach. The document analyzed all clinical scenarios that operators could deal with in the delivery room, including cord blood gas analysis during delayed cord clamping and time to cord clamping in the case of umbilical cord blood banking. The panel intended to promote a more physiological and individualized approach to cord clamping, specifically for the most preterm newborn. A feasible option to implement delayed cord clamping in very preterm deliveries is to move the neonatologist to the mother's bedside to assess the newborn's clinical condition at birth. This option could safely guarantee the first steps of stabilization before clamping the cord and allow DCC in the first 30 s of life, without delaying resuscitation. Contra-indications to placental transfusion strategies are clinical situations that may endanger mother ‘s health and those that may delay immediate newborn's resuscitation when required. Until 1960, delayed umbilical cord clamping (DCC) to promote placental transfusion was common; afterward, cord clamping immediately after birth became standard practice to reduce the risk of postpartum hemorrhage, although without evidence . In the Both techniques, referred in the text as placental transfusion strategies, allow transferring a similar amount of fetal blood, between 25 and 35 ml/kg, from the placenta to the newborn, increasing neonatal volemia, hemoglobin concentration, and blood pressure in the first days of life –6.The amount of blood transferred to the newborn during DCC depends on various factors: the time of cord clamping, the mode of delivery, the position of the newborn, the beginning of spontaneous breathing and respiration, and uterine contractions . The ideMilking the umbilical cord is considered a valid alternative to DCC, as it takes 10–20 s to be performed, allowing rapid neonatal resuscitation when required. It consists of squeezing gently 20–30 cm of umbilical cord three to five times from the placenta to the newborn at a velocity of 10 cm/s , 11. ThiIn the last years, research studies and meta-analysis highlighted the favorable impact of placental transfusion strategies on short-term neonatal outcomes. Both have been associated with a lower incidence of iron deficiency at 3–6 months in term newborns –16. In pThe reported long-term effects of placental transfusions are still scarce; a recent neuro-cognitive follow-up study of term newborns associated DCC to improved fine motor and social domains scores at 4 years of age, particularly in males . UmbilicRecent studies on animal models , 29 showScientific societies and neonatal resuscitation guidelines –34 suggeTherefore, “newborns requiring resuscitation” have been excluded by most research protocols and a significant percentage of infants included in DCC trials were clamped earlier than scheduled, mostly due to clinical concerns about delaying ventilation in non-vigorous infants , 35–38. When stimulation of the newborn occurs before clamping the cord, as suggested by WHO guidelines , most teA feasible option to promote placental transfusion in the most compromised newborns is to provide neonatal resuscitation during DCC. Recently Katheria et al.  randomizUmbilical cord milking could offer an advantage over DCC in preterm newborns who are deemed too unstable to wait for DCC and who are at the highest risk of severe IVH and death. Compared to DCC, UCM have been associated with higher blood pressure in the first day of life . HoweverA recent Italian survey  showed aThe study showed a significant correlation between the implementation of DCC and UCM and the knowledge of related benefits, the availability of obstetric-neonatal guidelines and the engagement across professions within the delivery team.In 2016 the Italian Task Force for the Management of Umbilical Cord Clamping has been constituted to draft recommendations for the management of cord clamping in term and preterm newborns.The aim of this document (first edition) is to provide operators involved in childbirth assistance with an updated consultation tool for optimal management of umbilical cord clamping in term and preterm newborns to standardize placental transfusion strategies in different clinical scenarios. Attention was paid to the application of DCC and UCM in extremely premature infants, according to guidelines for newborn's resuscitation , 32.Preterm deliveries are classically categorized, depending on gestational age at birth, in extremely preterm (<28 weeks), very preterm (<32 weeks), moderate (32–33 + 6/7 weeks), and late preterm (34–36 + 6/7 weeks) birth.Available studies on placental transfusion strategies, however, showed a significant overlap among these categories. For this reason, the panel decided to elaborate recommendations tailored to a unique group of patients that included extreme, very and moderate preterm newborns for whom immediate post-partum medical assistance was expected. Late preterm newborns have been considered separately because, in most cases, they do not necessitate medical interventions at birth, except for the presence of a skilled operator in neonatal resuscitation, as recommended by resuscitation guidelines in 2010 .The working group consists of obstetricians, midwifes, and neonatologists with experience in delivery room resuscitation, neonatal hematology, and cardiology, belonging to the Italian Society of Neonatology (SIN), the Italian Society of Perinatal Medicine (SIMP), and the National Federation of Midwifes (FNCO).The Italian version of the document derives from a detailed review of the English literature until December 2017 and a summary of the recommendations already published on the subject by scientific societies and expert panels. Articles' search was performed in 2 online databases (PubMed and the Cochrane Library).Recommendations have been drawn using the methodological approach proposed by the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) Working Group .After identification and prioritization of the questions to be addressed, articles were screened for further evaluation. GRADE is a consensus process that rates the quality of evidence and strength of recommendations along with values and preferences. The quality of the evidence (or confidence in the estimate of the effect) was categorized as high (where one has high confidence in the estimate of effect as reported in a synthesis of the literature), moderate , low , or very low  , 45.Recommendations were based on the evaluation of the expected benefits and risks connected to placental transfusion practices and the clinical context in which they could be applied. The safety for the mother and the newborn guided the drafting of the recommendations when evidence from the literature was not conclusive. The panel discussed and approved each statement unanimously during quarterly meetings held between 2016 and 2017.The original document has been externally reviewed and accepted by a panel of neonatologists, obstetricians, and midwifes (see Acknowledgment).Quality of evidence and strength of recommendations are summarized in Table p < 0.001) and reduced the prevalence of iron deficiency  at 4 months of age in a population where the prevalence of iron deficient anemia is low.The meta-analysis by McDonald et al.  demonstrThese positive results last up to the end of the first year of life in the case of infants born to mothers with low serum ferritin concentration at delivery , 48. AshThe position (relative to perineum) of the newborn during DCC did not seem to affect the volume of placental transfusion  but a reDelayed clamping has been associated with a slight increase in the need for phototherapy . This result was much debated, as it originates from the inclusion of unpublished data . No differences between early and delayed clamping were found in mortality rate, Apgar score, admission to NICU, respiratory distress, breastfeeding at discharge and up to 6 months. The incidence of asymptomatic polycythemia was similar between DCC and ICC in the review by McDonald et al.  but signDelayed cord clamping improves iron stores in the first months of life in term, vaginally delivered newborns with a high quality of evidence.(strong recommendation) (1A).In vaginally delivered newborns, DCC is recommended for at least 60 s to optimize cardio-pulmonary transition at birth and to promote placental transfusion (strong recommendation) (1B).It is recommended to dry and stimulate non-breathing infants by rubbing the back two to three times to encourage spontaneous breathing before clamping, and to clamp the cord in cases of persistent apnea (weak recommendation) (2B).It is suggested to clamp the umbilical cord by 3 min after delivery to improve iron stores in the first 3–6 months of life, although the optimal clamping time has not been defined (weak recommendation) (2C).Clamping the umbilical cord at 3 min is not mandatory; a longer DCC is suggested  if requested by parents (weak recommendation) (2C).During DCC, newborns may be placed on the mother's abdomen or chest or kept below the perineal plane A recent meta-analysis  showed tZhou et al.  observedAt present, recommendations on DCC in term newborns do not distinguish between vaginal and cesarean deliveries even though no studies explored the effects of DCC in exclusively CD newborns and only a few of them included this category of patients –58.p < 0.001) may have affected results but confirmed the effectiveness of DCC on iron stores in CD term newborns. Recently, Andersson et al.  (2B).It is suggested to delay cord clamping for at least 30 s and up to 60 s after birth to improve iron stores in newborns not requiring resuscitation (strong recommendation) (1B).It is recommended to dry and stimulate non-breathing infants by rubbing the back two to three times to encourage spontaneous breathing before clamping, and to clamp the cord in cases of persistent apnea (weak recommendation) (2D).In the case of DCC longer than 1 min, it is recommended to ensure the presence of a skilled operator in neonatal resuscitation to evaluate the feto-neonatal transition There are only a few studies , 59–62 tCord milking was associated to improved iron stores in the first weeks of life , 60, comErickson-Owens et al.  randomizp < 0.05] and ferritin concentrations at 6 weeks of age , compared with the control group.Upadhyay et al.  enrolled−1 vs. 70.8 ± 39.5 ng/ml, P < 0.001), compared to controls. The efficacy of c-UCM did not vary with the maternal anemia status. In two other studies, DCC (60–90 s) was compared to other placental transfusion strategies  in healthy vaginally-delivered term newborns should not replace a more physiological placental transfusion obtained by DCC.Overall, c-UCM improves iron stores in CD term newborns in the first weeks of life, compared to immediate cord clamping, with a very low quality of evidence.It is not possible to recommend the best milking technique to adopt. The uncut UCM technique may probably result in better transfusion performance but required further evaluation, both in VD and CD term newborns.(weak recommendation) (2C).Umbilical cord milking (both techniques) is not suggested as an equal alternative to delayed cord clamping in VD newborns not requiring resuscitation at birth (weak recommendation) (2C).In CD newborns, if delayed cord clamping is not feasible, umbilical cord milking is suggested as a valid alternative to promote placental transfusion and improve iron stores in the first weeks of life. 0/7–36 6/7 weeks of gestational age) varied from 30 to 180 s after birth . Salae et al.  and mean serum ferritin  at 6 weeks of age compared to ICC. Other studies that included late preterm newborns confirmed that clamping the cord 30–60 s after birth increased blood pressure in the first 24 h after birth and hemoglobin/hematocrit values at different time points, up to 10 weeks of age if their cord were clamped after 30–60 s from birth  (1B).In vaginal and cesarean-delivered infants not breathing at birth, it is recommended to dry and stimulate by rubbing the back two to three times to encourage spontaneous breathing before clamping, and to clamp the cord if the baby continues not to breath (strong recommendation) (1B).It is recommended the presence of personnel with neonatal resuscitation skills to evaluate the newborn in the transition phase (weak recommendation) (2C).It is suggested clamping the cord between 60 and 180 s in vaginally delivered newborns, not requiring resuscitation at birth, to improve hemoglobin concentration and iron stores in the first weeks after birth (weak recommendation).It is suggested placing newborns from vaginal delivery at the perineal level or below for the first 30 s of DCC, then on maternal abdomen (weak recommendation).It is suggested clamping the cord between 30 and 60 s in cesarean-delivered late preterm newborns not requiring resuscitation Only one study that included late preterm infants evaluated the effects of UCM , with thKumar et al.  demonstrNo data are available on the extent of placental transfusion with milking techniques, differentiated by mode of delivery.Umbilical cord milking improves iron stores in the first weeks after birth in late preterm infants, with a very low quality of evidence.Delayed cord clamping and milking were not associated with adverse events both in newborns and in the mother.(weak recommendation).In vaginally and cesarean-delivered late preterm newborns, when DCC is not feasible, UCM is suggested as a valid alternative to improve iron stores Delayed cord clamping in very preterm infants is defined as a clamping that occurs between 30 and 120 s after birth; DCC longer than 120 s has not been tested.The most recent results on the effect of DCC in preterm infants were published in 2017.The Australian APTS trial  that enrThis study did not foresee interventions to promote breathing during DCC and may explain the high protocol violation rate (about ¼ of subjects randomized to DCC received ICC) mostly due to medical concern about the infant. The exclusion of the most critical newborns may have affected the results. Indeed, statistical analysis has not been performed on the effective treatment received by the patient but based on the treatment expected by randomization, which may have introduced an attrition bias, caused by the unequal loss of patients in the DCC group.P < 0.00001). Subgroup analyses showed no differences between randomized groups in Apgar scores, intubation for resuscitation, admission temperature, mechanical ventilation, intraventricular hemorrhage, brain injury, chronic lung disease, patent ductus arteriosus, necrotizing enterocolitis, late-onset sepsis, and retinopathy of prematurity.The second significative study was the meta-analysis by Fogarty et al.  that incThe authors explained the positive impact of DCC in preterm newborns with the reduction of unnecessary and potentially harmful medical interventions in the first hours and days of life that could influence short and long-term outcomes through the activation of the inflammatory cascade and oxidative stress, the increased risk of infections and lengthening of hospitalization.Many authors observed that cord clamping should be individualized and based on clinical conditions, rather than on a pre-defined ideal clamping time –9.Ventilation is the key strategy of neonatal resuscitation  but mostIndeed, first steps of stabilization at birth have been associated with reduced peripartum mortality and mask ventilation , 76; in According to these concepts, the working group proposed a flow chart Figure  for premDelayed cord clamping increased the incidence of polycythemia in preterm newborns [risk difference 3% (95% CI: 2 to 5%)], and the incidence of jaundice (mean difference in peak bilirubin +4 μmol/l) without increasing morbidity or the need for exchange transfusion .Preliminary results of long-term follow-up showed a positive correlation between DCC and improved motor function at 18–22 months of corrected age, compared to ICC .Delayed cord clamping, compared to ICC, in moderate, very and extremely preterm deliveries improved outcomes with a high level of evidence.Milking the umbilical cord in very and extremely premature newborns was associated with a significant decreased risk of intracranial hemorrhage of all grade IVH all grade  and bronchopulmonary dysplasia  in newborns with gestational age < 33 weeks, compared to ICC  and to r–0.83) inWhen UCM was compared to DCC, results are contradictory. Rabe et al.  did not The follow-up study of the cohort by Rabe et al.  showed nIn moderate, very and extreme preterm newborns, especially if cesarean-delivered, cord milking improved short-term outcomes with a moderate level of evidence, and long-term outcomes with very low quality of evidence, without undesirable effects, except for a low risk of symptomatic hyperviscosity . For the(strong recommendation) (1A). First steps of stabilization at the mother's bedside should be provided by the attending neonatologist (strong recommendation) (1A).In vaginal and cesarean-delivered newborns with gestational age < 34 weeks, it is recommended to delay cord clamping for at least 30”. During this period, it is recommended to ensure the maintenance of body temperature, to perform tactile stimulation, to ensure airway patency and possibly their aspiration (strong recommendation) (1A).At 30 s of life, it is recommended to evaluate tone and breathing activity by visual inspection and heart rate by stethoscope: if the newborn is bradycardic (heart rate is < 100 bpm) apnoeic or gasping, it is recommended to clamp the cord and start ventilatory assistance maneuvers according to neonatal resuscitation procedures (strong recommendation) (1A).At 30 s of life, if the heart rate is > 100 bpm and active breathing or efforts to breath are present, it is recommended to clamp the cord at 60 s (weak recommendation) (2C).In vaginally delivered newborns with gestational age between 29 + 0 and 33 + 6 weeks, that do not require ventilatory assistance, the umbilical cord may be clamped at 90–120 s of life (strong recommendation) (1A).The obstetrical-neonatological team should ensure sterility during bedside maneuvers, especially in the operating room (weak recommendation) (2C).It is suggested to place vaginally delivered newborns at or below the perineal plane to promote placental transfusion (strong recommendation) (1B).It is recommended to identify within the obstetric-neonatology team a professional figure responsible for checking and communicating the time elapsed from birth (weak recommendation) (2B).When bed-side neonatological assistance could not be implemented, milking the uncut umbilical cord three to four times before clamping the cord is suggested Rhesus disease and, generally, feto-maternal red blood cell alloimmunization, were excluded from DCC research protocols, as it was likely that delaying clamping may increase the risk for significant hyperbilirubinemia due to the higher amount of opsonized red blood cells (RBC) transfused from the placenta to the newborn that could undergo hemolysis.p = 0.0003), lower incidence of anemia , reduced need for exchange transfusion without increasing the rate of pathologic hyperbilirubinemia. However, the results were not conclusive due to the differences in gestational age (GA), birth weight, mode of delivery and management of jaundice between the two study periods.There is a single retrospective study  that com(weak recommendation).In newborns at risk of anemia due to feto-maternal alloimmunization, it is suggested clamping the cord within 30 s from birth, after the first breaths if these occur before 30 s from birth Antiretroviral therapy during pregnancy together with intrapartum and postnatal prophylaxis can prevent perinatal transmission of HIV-infection from the mother to the newborn , 82, 83.WHO guidelines  suggest Vaginal delivery is considered appropriate for HIV-infected pregnant women who have been maintained on combined antiretroviral therapy and who have viral loads <1,000 copies/ml at or near delivery . Based oThe following recommendations are intended for HIV pregnancies in high-income countries with scheduled cesarean delivery.(weak recommendation) (2C).In newborns from HIV-positive mothers with adequate antiretroviral therapy during pregnancy and HIV-RNA near or at delivery ≤ 1,000 copies/ml it is suggested to delay cord clamping for at least 30 s and up to 60 s to improve iron stores, similarly to what suggested for healthy term and late preterm CD newborns (weak recommendation) (2C).Immediate cord clamping is suggested in all other cases Various studies –88 showeThe higher hemoglobin concentration in the second-born from monochorionic twin has been addressed to the presence of vascular anastomoses allowing either intrapartum inter-twin blood transfusion or placenta-fetal transfusion ; in dichLopriore et al.  retrospeIn twins delivered through cesarean section, no intertwin differences in hemoglobin levels were detected.Very few studies on placental transfusion strategies included dichorionic twin pregnancies, and none included monochorionic twins.Mc Donnel et al.  evaluatep < 0.74). In one twin pregnancy and the triplet one, the hemoglobin difference was <5 g/dl.Kugelman et al.  randomizIn the recent study by Katheria et al. , prematuThe APTS trial  includedThe quality of evidence has been considered low or very low, due to the paucity of data on this subset of patients; however, the panel considered delayed cord clamping in twin pregnancies safe for mother and the child.No studies evaluated the effect of milking in this group of patients.(strong recommendation) (1B).Delayed cord clamping is not recommended in monochorionic twins because the risk of acute intertwin transfusion at birth outweighs the undetermined benefit of delayed cord clamping in this population. (weak recommendation) (2D).In vaginally and cesarean-delivered term or preterm twin newborns from dichorionic pregnancy, it is suggested delaying cord clamping for at least 30 s up to 60 s (strong recommendation) (1B).In vaginally and cesarean-delivered infants not breathing at birth, it is recommended to dry and stimulate by rubbing the back two to three times to encourage spontaneous breathing before clamping, and to clamp the cord if the baby continues not to breath The hemodynamic and hematological improvements associated with DCC could theoretically advantage newborns with CHD. Indeed, the increased blood volume and hematocrit may have a positive effect, especially in cyanotic CHD, through the improvement of tissue oxygenation and increased blood flow at the level of Foramen Ovale and Ductus Arteriosus.There is a single randomized study  that evaBased on the prediction of hemodynamic conditions at birth , CHD couCHD without prediction of hemodynamic instability at birth: interatrial and interventricular septal defects, mild to moderate degree of valvular abnormalities.CHD with a slight risk of hemodynamic instability at birth: obstructions to the right or left efflux with arterial duct dependence. In these conditions, the patency of the arterial duct in the first hours after birth generally allows a physiologic cardiorespiratory and hemodynamic transition at birth.CHD with a high probability of hemodynamic instability at birth could compromise postnatal transition and required immediate resuscitation:- Transposition of great vessels with a restrictive foramen ovale- Hypoplastic left heart syndrome with a restrictive foramen ovale- Total abnormal pulmonary venous return obstructed- Ebstein disease with hydrops- Tetralogy of Fallot with absent pulmonary valve- Heart rhythm disorders with decompensation.(weak recommendation) (2C).DCC between 1 and 2 min is suggested in the case of vaginally delivered CHD newborns (weak recommendation) (2D).It is suggested clamping the cord at 1 min in the case of cesarean-delivered CHD newborns (strong recommendation) (1B).In the case of mild or severe risk of hemodynamic instability, (group 2 and 3), it is recommended to foresee the presence of personnel with resuscitation skills to evaluate the newborn during the transition phase (weak recommendation) (2C).The management of cord clamping in the case of severe CHD (group 3) should be discussed prenatally by a multidisciplinary team  according to the predictable resuscitation needs. Blood gas analysis from the umbilical artery is a tool to assess the metabolic status of the fetus . It provScientific Societies –97 recomWiberg et al.  evaluateDe Paco et al.  found noAll these studies included infants who did not require resuscitation at birth; whether the effect of DCC on arterial cord pH in non-vigorous infants would be similar is an important question requiring further investigations.A possible solution that takes both needs into account (blood gas determination immediately after birth and delayed cord clamping in vigorous infants) is to perform blood gas analysis on the unclamped umbilical cord. Andersson et al.  showed tDi Tommaso et al.  demonstrThe quality of evidence was considered high for the outcome “reliability of cord blood gas analysis during delayed cord clamping.”(strong recommendation) (1A).It is recommended performing umbilical artery gas analysis on double-clamped cord immediately after birth when neonatal asphyxia is expected, to obtain the most objective determination of the neonatal metabolic condition at of birth (strong recommendation) (1B).When cord gas analysis is recommended immediately after birth, but the newborn is vigorous and does not require resuscitation, umbilical artery gas analysis on the unclamped cord is a feasible alternative method Umbilical Cord Blood (UCB) is currently an established source of stem cells, especially in pediatric settings, in very urgent cases and for patients with no HLA matched donor , 103.9 to 1.5 × 109; consequently, discarded units are increased up to over 75% . The timing of cord clamping is the main determinant of volume and number of stem cells collected  .Differently, the National Swedish Cord Blood Bank  demonstrRecent recommendations –113 statScientific societies –112 recoThe following recommendations are intended for altruistic and dedicated umbilical cord blood (UCB) donations. Private cord blood banks are not allowed in Italy. Cord blood may be collected with special permission from the hospital, but units must be stored in foreign cord blood banks; transport and costs are not borne by national health service.(weak recommendation) (2C).In the case of altruistic cord blood donation, it is suggested to clamp the umbilical cord after 60 s and before 120 s after birth (strong recommendation) (1A).In the case of directed donation for at-risk families, with the goal of maximizing the content of hematopoietic progenitors through the volume collected, it is recommended to clamp the cord immediately after birth (strong recommendation) (1B).Health care professionals should give written information to pregnant women and their partners of the benefits of DCC and its impact on cord blood collection and banking Although there are no maternal contraindications to DCC or UCM, there are some emergent conditions (such as massive uterine bleeding) that required immediate cord clamping to safeguard mother's health.Fetal conditions for which DCC and UCM are contra-indicated include all cases of perinatal asphyxia when immediate resuscitation is required; other exclusion criteria include clinical situations for which concerns exist about the possible benefits deriving from a placental transfusion, and results from research studies, when present, are inconclusive.For the following clinical situations, immediate cord clamping is recommended, although based on experts' opinions.Birth asphyxia secondary to hypoxic-ischemic events: placental detachment, cord prolapse, uterine rupture, shoulder dystocia, vasa previa rupture, maternal collapse, amniotic embolism, maternal cardiac arrest.Twin to twin transfusion (TTTS)Newborn from HIV-positive mother (see dedicated paragraph)Fetal hydrops with evidence of fetal hearth decompensationDoubt about the integrity of the umbilical cordCesarean delivery under general anesthesia.Theoretically, when a hypovolemic shock is suspected  c-UCM may be an immediate source of fetal blood to be transfused while initiating resuscitations ‘maneuvers . ResearcIntrauterine growth restriction refers to a fetus with an estimated fetal weight <10th percentile on ultrasound that, because of a pathologic process, has not attained its growth potential . The leaDelayed cord clamping and cord milking have not been tested in this specific population; for this reason, the panel could not issue a recommendation on the subject. Theoretically, both techniques may worsen polycythemia , 81 and A summary of all recommendations is given in Table SG conceived the project, coordinated the work of the panel between 2015 and 2016, wrote the introduction and the material and methods paragraphs, wrote and reviewed the final version of the manuscript. MD and AL wrote the first draft of paragraphs on term newborns, twins, and gas analysis. BP wrote the first draft of paragraphs on late preterms, pregnancy with feto-maternal alloimmunization, HIV-positive pregnancy and contraindications. SF contributed to writing the first draft of the paragraph on congenital heart disease. PS contributed to writing the first draft on cord blood banking. SP contributed to writing the first draft on preterm < 34 weeks and together with SG and BP conceptualize the Flow-chart. All authors approved the final version.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.


The brain is one of the most prominent examples for structural and functional differences between the left and right half of the body. For handedness and language lateralization, the most widely investigated behavioral phenotypes, only a small fraction of phenotypic variance has been explained by molecular genetic studies. Due to environmental factors presumably also playing a role in their ontogenesis and based on first molecular evidence, it has been suggested that functional hemispheric asymmetries are partly under epigenetic control. This review article aims to elucidate the molecular factors underlying hemispheric asymmetries and their association with inner organ asymmetries. While we previously suggested that epigenetic mechanisms might partly account for the missing heritability of handedness, this article extends this idea by suggesting possible alternatives for transgenerational transmission of epigenetic states that do not require germ line epigenetic transmission. This is in line with a multifactorial model of hemispheric asymmetries, integrating genetic, environmental, and epigenetic influencing factors in their ontogenesis. In 1866, German zoologist Ernst Haeckel introduced promorphology – the science of an organism’s external form – and proposed symmetry as a fundamental criterion for classifying organisms . The claThe brain is one of the most striking examples for structural and functional differences between the left and right half of the body. Functional hemispheric asymmetries are found in several aspects of cognition such as memory, emotion, attention, language, and executive functions . This reThe consistency of population-level lateralization in handedness across centuries  and contThe human body is symmetric at the beginning of embryonic development. The first visceral asymmetry is detected when the heart, initially a straight tube, starts to loop at the end of 5 gestational weeks and occupies its typical left-sided position . During Like the visceral organs, the brain develops from an unpaired structure, the neural tube. Initially, it mostly grows ventrally, resulting in the emergence of its main subregions prosencephalon, mesencephalon, and rhombencephalon at the age of 6 gestational weeks. The cerebral hemispheres differentiate toward the end of 8 gestational weeks . The earIn line with structural hemispheric asymmetries already being apparent in human fetal development, prenatal ultrasound studies indicated early signs of motor asymmetries in terms of head turning and arm movements. The majority of human fetuses display a strong preference for turning the head toward the right side. Ultrasound observations of fetuses positioned in the cephalic (head-first) presentation revealed a right side preference between 30 and 38 gestational weeks . While tUsing ultrasound, individual arm movements are detectable at 9 gestational weeks . StartinLanguage lateralization in newborns and infants has mainly been studied in terms of perceptional asymmetries. At the behavioral level, a right ear advantage for dichotically presented syllables has been found in infants  and 4-daThe fact that functional hemispheric asymmetries are already established in the human fetus is in line with a genetic influence . The obsAR) (COMT) (LRRTM1) (PCSK6 gene (proprotein convertase subtilisin/kexin type 6)  encoding for a methyltransferase regulating hemispheric asymmetries in the zebrafish model (The neurogenetics of handedness has been reviewed in detail elsewhere . ShortlyAR) , the cat) (COMT) , the leu(LRRTM1) , and the type 6) . Two recsh model . Overallsh model . Thus, aFOXP2) (KIAA0319/TTRAP/THEM2 locus (GRIN2B) (CCKAR) (PLP1) with language lateralization suggests modulation via the corpus callosum  , the KIAM2 locus , the NMD(GRIN2B)  and the  (CCKAR) . The asscallosum . Overallcallosum  and the 2+ signaling on the edge of the node , another member of the TGF-beta family, left-sided Nodal is transmitted to the lateral plate mesoderm (LPM), an embryonic structure anterior to the node . The Nodsymmetry . Finallyrphology . Researcrphology , while trphology  and funcrphology . Evidencand GDF1 .lefty1 and pitx2c, are expressed in the structure later developing into the left epithalamus. Experimentally manipulated symmetrical expression of Nodal genes as well as the absence of Nodal gene expression does not prevent parapineal migration. However, left- and right-sided parapineal nuclei are equally distributed, and the habenular nucleus is larger on the side containing the parapineal nucleus , regulatory factor X 3 (RFX3), and GLI family zinc finger 3 (GLI3) were also among the top hits. The strongest association in a general population cohort was found for a SNP upstream of the Glypican 3 gene (GPC3), whose disruption in mice causes lung and heart asymmetry defects. Moreover, genes involved in double outlet right ventricle, heterotaxia, and situs inversus  were significantly overrepresented in genes associated with hand skill in both cohorts , a recessive disorder resulting in disruption of motile cilia. The authors found an elevated rate of left-handedness in situs inversus subjects without PCD, but not in those with PCD. Moreover, the typical counter-clockwise bending of the brain (Yakovlevian torque) was reversed in situs inversus subjects, while structural gray and white matter asymmetries and functional language lateralization were not . Knockdown of Lmo4 expression in the right anterior cortex in mice resulted in reduced right-hemispheric neuron number, thinner right- than left-hemispheric axonal projections, and a rightward shift of paw preference . Moreover, these modules were enriched for functional gene groups such as receptor activity in the superior temporal cortex and locomotion in the primary motor cortex. Interestingly, no module showed differential expression levels between the left and right macaque cortex  gene  gene  and affeF2) gene . Howeverlability . A surfelability , 2007. Alability . Based olability  have beelability . However1) to show more or less LG behavior toward their own offspring (F2), respectively  behavior leads to relative hypomethylation of the estrogen receptor alpha (ERα) promoter region in the hypothalamus, resulting in more pronounced production of ERα receptors. In contrast, low LG experience leads to relative ERα promoter hypermethylation and less ERα receptors. The increase or decrease in ERα receptors causes the offspring  or transgenerationally . While both transgenerational and multigenerational stress led to an increase in left paw preference in the F4 male offspring, this shift was more pronounced and significantly different from non-stressed controls in the multigenerationally stressed individuals. Moreover, a preference for the left paw was associated with increased dendritic complexity and spine density in the right parietal cortex  behavior might lead to specific DNA methylation patterns in the offspring (F1), initiating the same behavior toward their children (F2). This in term might result in transmission of DNA methylation patterns to the F2 generation  . MoreoveThis environmental factor affects DNA methylationin utero leads to a stronger cortisol response to the Trier Social Stress Test (TSST) in young adults as compared to control subjects without prenatal stress experience (via epigenetic modification (NR3C1)  axis. For example, the experience of severe stress perience . Similarperience . Severalperience . Recent fication . For exafication . The ass (NR3C1)  has been (NR3C1) . Birth se stress . Interese stress .DNA methylation affects the phenotypeAs mentioned above, the study by PITX2  stimulating the right, but not the left eye, through the semi-translucent eggshell initiating a cascade of molecular events eventually leading to asymmetries at the behavioral level. While epigenetic mechanisms are thought to play a role in this cascade of molecular events, this possibility has not yet been confirmed by empirical testing . In humaper se (The potential mechanism of gene-dependent epigenetic inheritance for familial transmission of handedness raises the question if a similar mechanism is conceivable for language lateralization. However, environmental factors involved in the development of language lateralization have been investigated sparsely, and we found an effect of DNA methylation on cognitive modulation of language lateralization, but there was no effect on language lateralization per se . Thus, fFuture studies should investigate different forms of hemispheric asymmetries and their relations with epigenetic modifications and environmental factors such as birth complications – not only in the individual but also transgenerationally. While epigenetic research in humans remains correlative, animal models allow for experimental manipulation and thus for uncovering causal mechanisms. Knowledge on molecular mechanisms involved in hemispheric asymmetries can benefit from experimental manipulation of prenatal conditions in rats and mice. These animal models also allow for the analysis of behavior, gene expression, and DNA methylation in the left and right hemispheres. Moreover, transgenerational effects can be investigated in more detail in animal models .MAP2 (MAPT and TUBB (In humans, large-scale GWAS are needed to uncover the genetic background of hemispheric asymmetries and their association with visceral asymmetries. Two GWAS on hand preference have recently been performed in the UK Biobank cohort. Significant associations were shown for MAP2  as well and TUBB . Interesand TUBB . Howeverand TUBB . Large sPCSK6 has been associated with strength, but not direction of handedness, suggesting that strength and direction represent different phenotypes affected by distinct molecular pathways (per se, indicated by individual level, but no population level asymmetry when Nodal gene expression is absent or symmetrical (vian visual system, it has been shown that the direction of asymmetry can be reversed by environmental factors, supporting an interplay of genetic, environmental, and potentially epigenetic factors affecting the direction of asymmetry (Future research should also investigate whether an interplay of genetic, environmental, and epigenetic factors contributes to asymmetry at the population level or at the individual level. While individual level asymmetry is characterized by pronounced strength of asymmetry in all individuals, but independent from direction of asymmetry, population level asymmetry is characterized by both strength and direction of asymmetry . For exapathways . This ismetrical . In the symmetry . HoweverIt has to be kept in mind that just as patterns of behavioral lateralization differ between humans and other vertebrates , its undJS wrote the manuscript. OG and SO advised on the manuscript and provided critical feedback.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.


Tetraspanins are exposed at the surface of cellular membranes, which allows for the fixation of cognate antibodies. Developing specific antibodies in conjunction with genetic data would largely contribute to deciphering their biological behavior. In this short review, we summarize the main functions of Tspan8/Co-029 and its role in the biology of tumor cells. Based on data collected from recently reported studies, the possibilities of using antibodies to target Tspan8 in immunotherapy or radioimmunotherapy approaches are also discussed. Tspan8 belongs to the tetraspanin molecular family of surface glycoproteins containing 33 members in humans, which are now referred to as Tspan1–33 . TetraspSome tetraspanins are widely expressed  while others are more restricted . Several clinical observations and experimental data have correlated the expression of some tetraspanins with tumor metastatic potential. CD82 or CD9 expression is generally associated with favorable prognosis in different cancers ,14,15,16Experimental results and clinical trials using monoclonal antibodies (mAbs) suggest that some tetraspanins may be targeted in hematological malignancies and carcinomas . In a siSince antibodies are large proteins, their ability to reach their target may be limited by their diffusion inside different tissues and their components due to certain barriers, such as the hemato-encephalic barrier. However, since the permeability of tumor vessels is usually abnormal, diffusion appears to be increased in such tumor tissues ,28. OnceAccording to current knowledge, the action of tetraspanins relies on their ability to regulate the function of their partner molecules. Even if the detailed molecular basis remains mostly unknown, the binding of mAbs on Tspan8 may result in the inhibition of cell migration, invasion, proliferation and angiogenesis in organized tissues .Unconjugated antibodies mediate ADCC (antibody-dependent cell cytotoxicity) via the activation of accessory cytotoxic cells for killing target cells. Upon fixation on the cell surface, they recruit macrophages or NK cells through their Fc fragment, which can further destroy the target cell. The subclass and glycosylation of the Fc region are important parameters that determine the efficacy of ADCC. Furthermore, the ability of antibodies to mediate ADCC may be optimized through genetic modification of their Fc region.On the contrary, radionuclide- or drug-conjugated antibodies have directly toxic effects in vivo.Depending on the radionuclide used , radionuclide-conjugated antibodies can efficiently treat the target cells and the neighboring cells, which can be of high value in the case of heterogeneous cell expression of the target molecule. In addition, radionuclide-conjugated antibodies can also allow for a combination of imaging and radiotoxicity. This will be detailed in the radionuclide-conjugated Tspan8 antibodies section.When antibodies are conjugated with cytotoxic drugs, an internalization of the antigen/antibody complex is often necessary to allow drug delivery . HoweverAn ideal antigenic target should be expressed at a high intensity on the surface of tumor cells, especially tumor stem cells and not on normal cells. None of the molecules that are currently targeted by mAbs are able to fulfil these criteria and not surprisingly, tetraspanins do not escape this rule.As mentioned above, the tissue distribution of tetraspanins is highly variable. A very restricted distribution of some tetraspanins has already been observed for peripherin/RDS found in the photoreceptors, UPk1a found on the urinary bladder epithelium or CD37 expressed mainly on B lymphoid cells. In contrast, other tetraspanins, such as CD9, CD63, CD81 or CD151, have a very large distribution and may be difficult to target in vivo. Tspan8 is expressed in a limited number of tissues and is mainly found on epithelial cells of the digestive tract. However, it is also located on the epithelial cells of the kidney, prostate and trachea . The secThis important point has been widely studied and previously reviewed. Thus, this will not be detailed in this article. This is mainly realized through the humanization of the molecule but other methods have been previously reported .There are a few proofs of principle that have shown that targeting tetraspanins with antibodies might inhibit tumor growth or even induce partial or complete remission .The effects of the anti-CD9 mAb ALB6 (IgG1) injected intravenously were investigated in a model of human gastric cancer (MKN-28) implanted subcutaneously in nude mice. A reduction of 60–70% in the size of the tumor in the treated group was observed compared to the control IgG-treated mice. At the same time, a significant reduction in cell proliferation and angiogenesis and an increase in apoptotic signals were observed . Since tThere have only been a few studies targeting tetraspanins in humans for therapeutic purposes. A cocktail of antibodies Ba1/2/3 directed respectively toward CD9, CD24 and CD10 to deplete the bone marrow of acute lymphoblastic leukemia patients for autologous bone marrow transplantation was investigated. Even if the specific role of each individual antibody was difficult to evaluate, an absence of toxicity against hematological stem cells was observed in these antibodies .−; T1/2 = 8.02 d; 606 keV). Very encouraging results were obtained in comparison with an anti-CD20 antibody labelled with the same radionuclide ibritumomab tiuxetan , which is administered as a second-line treatment to patients with non-Hodgkin lymphomas (NHLs) that are resistant to chemotherapy DOTA-Ts29.2 in HT29 tumors was higher than that of [111In]DOTA-Ts29.1  to the lysine residues, which was evaluated in studies conducted in vitro and in vivo using a fluorescent tetrazine. We evaluated the best link size between TCO and TS29.2 and observed a higher fluorescent signal with Ts29.2-TCO without a PEG spacer, which can be explained by a higher isomerization rate of TCO to the inactive CCO form . As tetrnjection .177Lu]DOTA-Ts29.2 induced a greater slowing down of tumor growth. The main features in pretargeted radioimmunotherapy PRIT experiments were the reduction of proliferation and increase in apoptosis. As mentioned above, the treatment with nonradioactive antibodies (using 100-times more antibodies than in the [177Lu]DOTA Ts29.2 experiments) also resulted in a slowing down of tumor growth with neither induction of apoptosis nor decrease in angiogenesis. In fact, the nonradioactive antibody should alter the interactions between tumor cells harboring Tspan8 and the microenvironment while its radiolabeled counterpart irradiates all surrounding cells after it attaches to its target antigen. This property should be interesting as it will decrease the number of so-called cancerous stem cells (CSCs) because Tspan8 has been identified on the surface of CSCs in pancreatic tumors [Stoichiometrically compared to its corresponding nonradiolabeled antibodies, [c tumors . RIT hasc tumors . Conversc tumors , leadingc tumors . As mentc tumors . As an eTspan8 expression is restricted and this protein has been described as a significant contributor and potential therapeutic target in several cancer types. Even if secondary effects and immune system involvement cannot be evaluated on tumor-grafted mouse models used for these studies, targeting Tspan8 with radiolabeled antibodies seems to be an effective antitumoral therapy.Tetraspanins may have a broad range of actions in cancers due to their intrinsic membrane localization (cell membrane or exosomes) and high numbers of their interacting molecules ,26. The


HOX genes are highly conserved members of the homeobox superfamily that have a crucial role in determining cellular identity. High grade ovarian cancer is the most lethal gynaecological malignancy. Our understanding of the role of HOX genes in the oncogenesis of ovarian cancer is evolving, and here we review their dysregulated expression patterns, their function in cell survival and invasion, their potential uses as biomarkers, and ways in which HOX genes are being targeted with new and existing drugs. Drosophila melanogaster fruit fly [HOX genes are a member of the homeobox superfamily, with “homeotic” or “HOX” genes coding for transcription factors that were originally recognized for their role in embryogenesis in the ruit fly . There aruit fly .HOX proteins can function as monomers, homodimers, heterodimers or heterotrimers, and the affinity and specificity of their binding to DNA can be modulated by binding proteins of the three amino acid loop extension (TALE) family of co-factors, especially Pre-B cell leukaemia transcription factor (PBX) and myeloid ectopic viral integration site (MEIS) .The roles of HOX genes are wide and varied, including embryonic development and patterning of the anterior to posterior axis, and there is growing evidence that they have a pivotal role in oncogenesis in a number of tumour types, including ovarian cancer ,3. TheirA link between HOX genes and cancer was first evident in haematological malignancies, but since this discovery they have now been implicated in the oncogenesis of a variety of solid organ malignancies with broadly HOX A genes often showing altered expression in breast and ovarian cancers, HOX B genes in colon cancer, HOX C in prostate and lung cancers and HOX D in colon and breast cancers .HOX genes can act as both oncogenes and tumour suppressors but are usually pro-oncogenic in a more supportive role, both at the cellular level, for example by driving cell proliferation and preventing apoptosis, and also at the tumour level, for example by promoting angiogenesis, metastasis and treatment resistance .The mechanisms of HOX gene action are complex and not fully elucidated, and it is not within the scope of this article to give a detailed mechanistic picture. However, broadly, the protein products of HOX genes act as both positive and negative transcription factors that can interfere with various cell signaling pathways, and thus upset the delicate balance of regulatory mechanisms that prevent carcinogenesis . Abate-SHOX genes are expressed in both normal ovarian surface epithelium (OSE) and also in high grade ovarian carcinomas, and their differential expression in these tissues has been investigated in a number of studies ,7,8,9,10In the female reproductive system four HOX genes, HOXA9, HOXA10, HOXA11 and HOXA13 are expressed along the Mullerian duct axis. These tandemly arranged HOX genes are expressed uniformly in embryonic development, although in adults their expression becomes spatially restricted to particular organs. HOXA9 becomes expressed in the fallopian tubes, HOXA10 is expressed in the developing uterus, HOXA11 in the lower uterine segment and cervix and HOXA13 in the upper vagina  showed tA comprehensive analysis of high grade serous (HGS) ovarian cancer specimens in the TCGA database found a dysregulation of HOX proteins in a subgroup of samples termed the “mesenchymal” group, characterized by high expression of HOXB2, B5 and B8 . A furthA small study by Hong et al.  showed 1The very high degree of heterogeneity in ovarian cancer, and especially in HGS carcinoma, goes some way to explaining some of the results and differences between various studies. Common patterns of expression are described below and classified by the likely function of the HOX genes and proteins in this common and very aggressive malignancy.HOX genes can function as both oncogenes and tumour suppressors, and many studies have shown an effect upon cell growth and proliferation. A study by the Naora group  showed oSeveral studies have shown that HOXA10 can increase the growth and invasion of ovarian cancer cells ,19,20, aHOX genes have been shown to have a role in the invasive properties of ovarian cancer cells, especially the HOX A class. This is particularly important, as many patients with ovarian cancer present at a late stage with widely disseminated disease. Seeding of tumour cells onto the peritoneum is a potentially attractive early target for reducing the lethality of ovarian cancer.HOXA4 seems to have a contradictory role in cell invasion. The Auersperg team  found thSimilar reports of the action of HOXA9 have shown that HOXA9 suppression via microRNA-196b  increased the invasiveness of SKOV-3 cells in vitro . AnotherThe Naora team conducted a series of experiments ,26 whereAnalysis of The Cancer Genome Atlas (TCGA) dataset  revealedThe same group showed similar findings with HOXA7; 16/24 patients with moderately differentiated serous ovarian carcinomas were found to have significantly more anti-HOXA7 antibodies compared to healthy controls. However, interestingly, patients with poorly differentiated serous carcinomas did not show serologic reactivity, whilst 68% of patients with benign serous cystadenomas did .The literature shows a great deal of functional redundancy in HOX genes, with many having overlapping or contradictory roles. It has also been difficult to create effective small molecule inhibitors of individual HOX proteins. HOX protein binding to DNA is modulated and enhanced by the binding of co-factors, especially PBX and MEIS. Targeting this interaction is a pragmatic way to affect the function of multiple HOX genes as it is mediated by a highly conserved hexapeptide sequence in HOX proteins . PBX andOur group has developed a cell-penetrating peptide, HXR9, which contains the hexapeptide sequence and acts as a competitive antagonist of HOX/PBX binding. We have tested this drug in two ovarian cancer cell lines, SKOV-3 which shows highly dysregulated HOX expression and OV-90, which does not. HXR9 was able to induce apoptosis and retard tumour growth in vivo for the SKOV-3 cells but not the OV-90 cells . SubsequOnly 2% of the human genome codes for proteins whilst the majority of open reading frames are for non-coding RNA, which can still regulate gene expression and play a critical role in multiple physiological and disease processes. Non-coding RNA can be split into micro RNA (miRNA) and long non coding RNA (lncRNA) depending on their sequence length. The expression and function of lncRNA in ovarian cancer cells versus normal ovarian tissue was examined in a study which showed 2870 lncRNAs were differentially expressed with a “HOX cluster” that was highly dysregulated .Specific lncRNAs have been investigated for their individual roles in ovarian cancer. For example, HOXA11-AS has two exonic variants and the minor allele rs17427875 (A > T) was relatively more common in cases of serous epithelial ovarian cancer . Ectopic expression of HOXA11-AS in cancer cells reduced cell survival, proliferation, migration and invasion in vitro, and reduced tumour growth in mouse models, the T allele more so than the A allele .Another group showed that the lncRNA GAS5 is downregulated in ovarian cancer, and works to inhibit miRNA-196a-5p, which in turn down-regulates HOXA5 that acts as a tumour suppressor in this context. Strategies to decrease HOXA5 expression  increased cell growth and decreased apoptosis both in vitro and in vivo. Lower GAS5 expression in tumors was associated with a larger tumor size and advanced FIGO stage .p = 0.04) [HOX transcript antisense RNA (HOTAIR) is a lncRNA found in the HOXC cluster that regulates the HOXD cluster and has been extensively studied in ovarian cancer. It has been shown to be overexpressed in epithelial ovarian cancers, and multivariate analysis has shown that HOTAIR expression is an independent prognostic factor for overall survival  . In the There have also been reports of a link between HOTAIR and DNA damage response, particularly that induced by platinum chemotherapy, and that HOTAIR could mediate platinum resistance. One study showed increased HOTAIR levels in recurrent platinum resistant ovarian tumours versus primary ovarian cancers  and thatMost ovarian cancers present at an advanced stage when the disease is widely disseminated and likely incurable. Therefore, an early diagnostic biomarker would greatly improve survival. HOX genes are an attractive biomarker as they are often exclusively expressed in cancer cells or highly dysregulated, and expressed at an early stage of the disease. Large studies, including genome wide association studies, have identified HOX genes as playing a key role in ovarian cancer risk . The NaoAn international study by Jacobs et al.  investigHOX genes can also act as prognostic biomarkers to stratify high and low risk patients. A study in clear cell carcinomas  showed tp < 0.001), and that this could reflect a role for these genes in mediating platinum resistance [Individual genes show variability in different cell lines and histological subtypes of ovarian cancer, therefore a gene panel may be a more useful way to examine HOX gene expression. In a study by Michael et al.  a five-gsistance .In addition to gene expression, the epigenetic changes in HOX genes are being studied as biomarkers, particularly methylation status. A large study by Fiegl et al. found that the methylation status of HOXA10 and A11 was the best discriminator between neoplastic and normal tissue, and that HOXA11 methylation status was independently associated with poor outcome . Cheng eHOX genes show a highly dysregulated expression in ovarian cancers of all histological subtypes, with many studies showing either up- or down-regulation of HOX genes. Their functions have not been completely elucidated but they undoubtedly have a critical function in oncogenesis. They have been shown to have an important role in cell survival and proliferation, cell motility, differentiation of tissue-specific lineage and coordinating the immune response to tumour cells. The pathways by which they act are likely to be numerous and require further elucidation. The differential expression of HOX genes may well prove to be a useful diagnostic or prognostic biomarker, especially given their easy availability in tumour tissue, biological fluids such as ascites and circulating tumour cells in blood. Evidence is also accumulating that they could be used as a predictive biomarker of drug response, particularly platinum sensitivity.The significance of HOX genes is underscored by the development of drugs targeted against them, particularly HXR9, which has shown anticancer efficacy both in vitro and in vivo, and also synergy with commonly-used first line chemotherapy agents, such as the platinums.


Here, we describe a novel zebrafish mutant in which a missense mutation in the adam19b gene selectively affects the epibranchial sensory circuits.Little is known about the molecular and cellular mechanisms involved in the formation of the cranial peripheral sensory system in vertebrates. To identify genes involved in the formation of these circuits, we performed a forward genetic screen utilizing a transgenic zebrafish line ( The peripheral sensory nervous system serves as an interface between the internal and external environments and the central nervous system (CNS). The information it carries must be relayed with precision so that appropriate behavioral and/or physiological responses are made. A subset of cranial nerves  containing both motor and sensory axons innervates structures in the head and neck, as well as the non-pelvic viscera. The sensory axons within these nerves originate from four distinct cranial sensory ganglia (CSG): the trigeminal (Vth), the facial (VIIth), the glossopharyngeal (IXth) and the vagal (Xth). These neurons transduce somatosensory, chemosensory and viscerosensory stimuli from peripheral receptors/organs and terminate within discrete regions of the hindbrain in a topographic fashion  that expresses green fluorescent protein (gfp) in sensory neurons of the Vth, VIIth, IXth and Xth cranial ganglia , showed aberrant projections of only the epibranchial axons. Here, we present data showing sl19 is the metalloprotease adam19b and is critical for sensory circuit formation.A powerful approach to identify genes involved in developmental programs is the forward genetic screen. To identify genes utilized in sensory circuit formation, we carried out a forward screen in a transgenic zebrafish line Tg larvae and cDNA synthesized with Superscript III reverse transcriptase . cDNAs encoding candidate genes within the critical interval were sequenced using gene specific primers.Standard mapping methods using bulk segregant and meiotic recombination analysis of microsatellite markers  or morpholino oligonucleotides  was performed as previously described  was one of several mutant lines detected. sl19 showed a recessive Mendelian inheritance: mutants constituted 27% ± 6 of larvae in a clutch (n = 235/870 from nine clutches). By 4dpf, the sl19 line contained defects in the central projections of the epibranchial ganglia .A forward genetic screen investigating zebrafish cranial sensory circuit formation was performed using the reporter strain sl19 allele was located on Chromosome 14, and high-resolution positional cloning identified a C > T mutation located within exon 17 of adam19b  family, and is a 923 amino acid Type I transmembrane protein composed of a series of conserved domains that include: a signal sequence, a pro-domain, a metalloproteinase domain, a disintegrin domain, a cysteine-rich domain (CRD), an EGF-like domain, a transmembrane region and a C-terminus containing signaling-related motifs , we unexpectedly observed a significant increase in the number of 4 dpf larvae exhibiting the mutant phenotype: 47% (57/121) of injected embryos had a mutant phenotype compared to 25% in un-injected clutchmates . Injection of MO1 (2–4 ng) resulted in 19% of injected 4 dpf larvae exhibiting a phenotype resembling sl19: misrouting of the projections of the VIIth, IXth and Xth ganglia, but not those of the Vth . Injection of a second morpholino (MO2), designed against the e17i17 splice junction, gave similar phenotypes ; n = 0/41 or branchiomotor projections ); n = 0/32]. These results suggest that adam19b is utilized selectively by the epibranchial sensory neurons in their pathfinding to hindbrain targets.To determine if circuits , S6, we adam19b CRISPR/Cas9 targeting : the organization of the optic tectum, the cerebellum and commissural neurons appear normal.The configuration of the branchiomotor neurons and their axons was not affected at 4 dpf by argeting . This suadam19, has been reported to have a role in neural crest migration, neuromuscular junction formation and cardiac development  in the CRD of adam19b. Studies have shown that this region is critical for the post translational processing necessary for its protease and disintegrin functions  that is responsible for the defects found.We have identified a mutant,  Blobel, . Kang et Blobel, , 2004 hasl19 and not other circuits such as the trigeminal, lateral line or branchiomotor nerves. This suggests that the molecular targets of adam19b are critical for axon guidance of viscerosensory afferents, but not mechanosensory or branchiomotor axons. The identification of these substrates will require additional investigation.It is striking that only the epibranchial sensory axons are affected in zebrafish in All datasets generated for this study are included in the manuscript and/or the supplementary files.All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Saint Louis University Institutional Animal Use and Care Committee approved the procedures used (protocol #2647).Both authors contributed to the conception and design of the study, to manuscript revision, read and approved the submitted version.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.


Radiotherapy remains one of the corner stones in the treatment of various malignancies and often leads to an improvement in overall survival. Nonetheless, pre-clinical evidence indicates that radiation can entail pro-metastatic effects via multiple pathways. Via direct actions on cancer cells and indirect actions on the tumor microenvironment, radiation has the potential to enhance epithelial-to-mesenchymal transition, invasion, migration, angiogenesis and metastasis. However, the data remains ambiguous and clinical observations that unequivocally prove these findings are lacking. In this review we discuss the pre-clinical and clinical data on the local and systemic effect of irradiation on the metastatic process with an emphasis on the molecular pathways involved. Local invasion and distant metastasis are the cause of death in most malignant tumors. Tumor response, reflected by improved overall survival, and toxic side effects, deteriorating quality of life, receive major attention in clinical trials investigating the main treatment modalities used in cancer, namely, surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Yet, far less attention is paid to the effects of these treatments on tumor progression as reflected by the metastatic process.Malignant tumors consist of cancer cells and tumor-associated host cells, both participating in invasion and distant metastasis. These cells form ecosystems at the primary and at the metastatic site, mutually communicating with one another and with stem cell-generating organs such as the bone marrow. It is highly probable that therapeutic manipulation of one ecosystem affects the others, a phenomenon that needs to be analyzed in view of the increasing cellular and molecular complexity of therapy responses , its activator MMP14, tissue inhibitors of matrix metalloproteinase 2 (TIMP2), chemokine ligand 2 (CCL2), and urokinase-type plasminogen activator (uPA), the latter two being linked to the recruitment of the monocytic cells. Intravenous injection of multipotent vascular wall-resident mesenchymal stromal cells (MSCs) counteracted lung inflammation and metastasis by an as yet unknown mechanism  apoptosis and inhibited survival, proliferation, capillary tube formation on Matrigel and invasion through Matrigel  or irradiation of host sites or sites of cancer cell implantation influenced spontaneous or cancer cell-induced angiogenesis. TBI of C57/BL mice at a high dose of 15 Gy inhibited ex vivo aortic ring formation  in embryonic development and in cancer  and quantitative (e.g. the extent of stimulation) differences in their invasive response, that were not related to their organ of origin  and stimulation of invasion -positive breast cancer cells, but not negative ones, were induced to invade by ionizing radiation in a reactive oxygen species (ROS)-dependent manner  by invasive cells is mediated by lytic enzymes such as MMP2, MMP9 and membrane-bound MMP-1 (MT1-MMP). These enzymes act in balance with inhibitors such as TIMPs. Proteases not only degrade ECM but also cleave off growth factors from their precursors, e.g. proteases activate TGF-β through cleavage from its inactive precursor latency-associated peptide (LAP-β1). Both enzymatic activities are enhanced by ionizing radiation, explaining stimulation of invasion and metastasis-related cellular activities Tables . IrradiaAs stated above, activation of TGF-β through release from LAP-β1 is an extracellular process, that occurs rapidly (within 24 h), persistently (7 days) and dose-dependently (from 0.1 Gy to 5 Gy) upon irradiation of the mouse mammary gland  on cancer cells and endothelial cells  secreted by the fibroblasts and interacting with the tyrosine-protein kinase Met (c-MET) on the cancer cells by Ohuchida et al. , and was sometimes only transient.To analyze the mechanisms of metastasis, cancer cells were inoculated at various sites, orthotopic or paratopic, in immunosuppressed or syngeneic hosts Table , produciAs for irradiation of putative sites of metastasis, contradictory results have been noted. On the one hand, Travis et al. observed that irradiation of the whole lung  after the subcutaneous growth of sarcoma F tumors in CBA mice led to a decrease in lung metastases  who received either transcatheter arterial chemoembolization alone or in combination with radiotherapy (40–66 Gy in 1.8 to 2 Gy fractions). Although the study was not randomized, baseline patient characteristics and treatment variables did not differ significantly between both groups. Overall survival was similar in both groups, yet the group treated with radiotherapy quickly developed intra- and extrahepatic tumors outside the irradiation field resulting in a significantly shorter extrahepatic progression-free survival  followed 3 days later by surgery. No difference in survival was seen, yet the group treated with radiotherapy had a higher incidence of distant metastases at time of death (Strong et al., These findings bring forth the hypothesis that the beneficial local effects of radiotherapy are counteracted by its systemic pro-invasive and pro-metastatic effect and therefore does not lead to an increased overall survival. The fact that the appearance of distant metastasis has been unchanged in the field of head and neck squamous cell carcinoma over the last decades, notwithstanding new radiotherapy and chemotherapy, supports this hypothesis (Duprez et al., Furthermore, it has been shown that radiotherapy and chemotherapy can alter the metastatic pattern as compared to no treatment. De La Monte et al. found that the distribution of metastases of small cell lung carcinoma was different after radiotherapy, chemotherapy or no therapy, but with similar number of total distant metastases and similar overall survival in all groups. These cancer treatments therefore might instigate a growth advantage to certain subpopulations of the tumor, which metastasize to specific sites (de la Monte et al., Radiotherapy can also induce a local pro-invasive effect, as shown predominantly in breast cancer but also in nasopharyngeal and endometrial cancer, in which inflammatory skin recurrence, exactly demarcating the previously irradiated area, occurred (Tsujino et al., In line with this, radiotherapy of prostate cancer and rectal cancer can increase MMP activity in the tumor and the local microenvironment (Angenete et al., Evidently, radiotherapy can also yield an anti-invasive and anti-metastatic effect. Post-mastectomy radiotherapy in high-risk breast cancer patients leads to a significantly lower probability of locoregional recurrences and distant metastases (Nielsen et al., There is abundant evidence from in vivo and in vitro experiments that ionizing radiation, applied to cancer cells or host cells, to the tumor, the whole host or the putative site of tumor development may stimulate the metastatic process. Clinical observations do not directly confirm these data, leaving open the question whether or not radiotherapy enhances metastasis. The overall net outcome of radiotherapy is beneficial as it may reduce metastasis and prolong survival. This therapeutic benefit does not exclude a limitation of the benefit by negative effects of the irradiation on the cancer cells or on the host. The present review of literature suggests clinical trials to investigate correction of the pro-invasive and pro-metastatic activities of radiation by anti-inflammatory or other agents shown to be effective in preclinical settings.


Danio rerio) is a model organism for vertebrate developmental processes and, through a variety of mutant and transgenic lines, various diseases and their complications. Some of these diseases relate to proper function of the visual system. In the US, the National Eye Institute indicates >140 million people over the age of 40 have some form of visual impairment. The causes of the impairments range from refractive error to cataract, diabetic retinopathy and glaucoma, plus heritable diseases such as retinitis pigmentosa and color vision deficits. Most impairments directly affect the retina, the nervous tissue at the back of the eye. Zebrafish with long or short-wavelength color blindness, altered retinal anatomy due to hyperglycemia, high intraocular pressure, and reduced pigment epithelium are all used, and directly applicable, to study how these symptoms affect visual function. However, many published reports describe only molecular/anatomical/structural changes or behavioral deficits. Recent work in zebrafish has documented physiological responses of the different cell types to colored  light stimuli, indicating a complex level of information processing and color vision in this species. The purpose of this review article is to consolidate published morphological and physiological data from different cells to describe how zebrafish retina is capable of complex visual processing. This information is compared to findings in other vertebrates and relevance to disorders affecting color processing is discussed.Zebrafish ( GC bodies are in the most proximal layer, the GC layer (GCL).Zebrafish retina, like those of other vertebrates, contains five neural types organized into layers. Distal-most are photoreceptors (PRs) with cell bodies in the outer nuclear layer (ONL) and terminals forming synaptic contacts with the dendritic processes of bipolar (BCs) and horizontal (HCs) cells in the outer plexiform layer (OPL). The cell bodies of these second order neurons, as well as those of third order amacrine cells (ACs), reside in the inner nuclear layer (INL). BCs are presynaptic to AC and ganglion cell (GC) dendrites in the inner plexiform layer (IPL), where processes in the distal IPL, or sublamina crx), atonal (ath5) and thyroxin β2 nuclear receptor (trβ2), which are key to retinal development in mammals, also operate in zebrafish retina  responses in zebrafish larvae younger than days postfertilization  binds to an opsin molecule forming the visual pigment complex that absorbs light photons  or multiphasic  and chromaticity (C-type) HCs  introduced a 6-stratum scheme for classifying bipolar axon terminal depth within the zebrafish IPL and identified ~17 morphological types of BCs based on axonal stratification patterns , achromatic , or opponent ,” the region looking forward and upward at prey , however, patch recordings of bistratified cone BCs with axonal boutons in both sublamina a and sublamina b (Cab BCs) revealed color-opponent responses, excited by one wavelength, but inhibited by another. The Cab cells subtract signals from cones with different opsin expression, and multiple spectral patterns were observed. There was evidence that different types of BC dendritic glutamate receptors were stimulated by different spectral types of cone  responses in zebrafish AC. The dendrites of all these C-type ACs ramify deep in sublamina  Figures . In adul Figures . Biphasi Figures . Some tr Figures . Calcium Figures . The bis Figures . Taken tAs the output neuron of the retina, there have been many studies documenting light responses of retinal GCs. In fact, color-opponent GCs have been documented in a variety of species, including, but not limited to, monkey  signals are relayed directly from the principal member of the double cone to H1 HCs elements in triphasic and tetraphasic HC responses  cones contact H1, H2 and H3 HC  to AII ACs and then to ON-GCs through gap junctions with ON-BC terminals  and opponent (G-B and G-R) systems no optokinetic response a  gene, resulting in the absence of the E2 subunit of the enzyme pyruvate dehydrogenase  mutant is, therefore, a model for human PDH deficiency.One of the first mutants identified was no optokinetic response c (nrc) mutants, in addition to an absent OKR, have a diverse phenotype that includes floating ribbons in cone pedicles  gene  mutants. nof fish have a nonsense mutation in the gene coding for the α-subunit of cone transducin  allowed visualization of TαC in all zebrafish cone types, making nof mutants a model for achromatopsia , also causes cone degeneration  phenotype  is required for green cone development  genes, resulting in a loss of green cones that persists to adulthood. Interestingly, the six7 KO phenotype also includes reduced blue opsin expression , increased number of rods (larvae), and a switch in red opsin expression  expression partial optokinetic response b  and mutant lines has also identified genes specific to a given cone type. The transcription factor Allison and colleagues have developed transgenic zebrafish lines in which a single class of PR can be chemically ablated .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.


Gliomas are the most prevalent primary tumors occurring in the central nervous system (CNS), with an incidence of more than 9,000 patients per year in the United States alone. Glioblastoma (GBM) is the most frequent subtype of gliomas in adults, presenting with very aggressive and invasive nature, conferred by high cellular proliferation and migration, resulting in recurrence. Despite aggressive therapy that includes surgical resection combined with radiation and chemotherapy , the five-year survival rate of patients with GBM remains less than 10% after initial diagnosis [For the past 20 years, engineered stem cells, such as neural stem cells (NSCs) and mesenchymal stem cells (MSCs), have been used as a vehicle to carry and deliver anticancer agents to a variety of brain tumors, including gliomas . OEC disOEC is the main phagocytic cell in the olfactory epithelium (and not macrophages), and are responsible for cellular debris clearance as a result of neuronal replenishment during physiological turnover of neurons, or after olfactory nerve lesion . As suchThe use of OECs for GBM gene/cell therapy has several advantages over traditional stem cells: (1) OEC is a fully differentiated cell (and not stem cell) that can be easily obtained from the olfactory epithelium and/or bulb, a simple procedure typically performed for patients with spinal cord injury or pituitary tumor resection, allowing autologous transplantation, avoiding graft-versus-host disease; (2) OEC migration can be stimulated by cytokines associated with tumor growth, such as TNF-α and IL-6, or even by low-doses of curcumin, a known antitumor agent; (3) the intranasal pathway is a non-invasive alternative to deliver therapeutics to the brain, and is the natural route for OEC during olfactory neuron turnover; (4) No toxic or tumorigenic potential with OECs transplantation have been reported to date; Finally, given their immunomodulatory and phagocytic properties, OEC have the potential to be used not only as a vehicle to deliver therapeutic genes, but as adjuvant therapy opening new possibilities for glioma treatment that can be extended to other cancer types.Given OECs therapeutic potential for neurological pathologies and regenerative medicine, many studies are underway to optimize their collection, culturing and expansion techniques. For instance, a current clinical trial (NCT02870426) aiming at collecting OEC from brain dead or living donors undergoing elective neurosurgery and using them for patients with spinal cord injury (not autologous). As these studies evolve, they will offer a tremendous benefit in the application of OEC to GBM therapy. In all clinical applications thus far, OEC are injected directly into the area of pathology, while the proposed intranasal delivery might require higher number of cells, direct injection of OECs at the tumor site should also be explored in future studies.


Heart failure (HF) represents an important cardiovascular complication of type 2 diabetes mellitus (T2DM) associated with substantial morbidity and mortality, and is emphasized in recent cardiovascular outcome trials (CVOTs) as a critical outcome for patients with T2DM. Treatment of T2DM in patients with HF can be challenging, considering that these patients are usually elderly, frail and have extensive comorbidities, most importantly chronic kidney disease. The complexity of medical regimens, the high risk clinical characteristics of patients and the potential of HF therapies to interfere with glucose metabolism, and conversely the emerging potential of some antidiabetic agents to modulate HF outcomes, are only some of the challenges that need to be addressed in the framework of a team-based personalized approach. The presence of established HF or the high risk of developing HF in the future has influenced recent guideline recommendations and can guide therapeutic decision making. Metformin remains first-line treatment for overweight T2DM patients at moderate cardiovascular risk. Although not contraindicated, metformin is no longer considered as first-line therapy for patients with established HF or at risk for HF, since there is robust scientific evidence that treatment with other glucose-lowering agents such as sodium-glucose cotransporter 2 inhibitors (SGLT2i) should be prioritized in this population due to their strong and remarkably consistent beneficial effects on HF outcomes. Diabetes mellitus (DM) and heart failure (HF) are recognized as global epidemics, since their prevalence is increasing worldwide with alarming rates . DM and The relationship between DM and HF is bidirectional. On the one hand, DM increases more than 2-fold the risk of developing HF, both HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF), and furthermore increases the risk of progression from asymptomatic left ventricular (LV) systolic dysfunction to symptomatic HF ,15. ViceIn epidemiological terms, the association between DM and HF is stronger in women than men. This sex discrepancy, which remains still incompletely understood, was first observed in the Framingham Heart Study, where diabetic men presented with a 2-fold, while diabetic women presented with a 4-fold increased risk of HF after adjustment for age and other CV risk factors . The samThe concurrent presence of DM and HF and their shared pathophysiology underscore the need for an interdisciplinary collaborative management of both conditions . TreatmeThe multiple challenges of treating patients with DM and cardiovascular disease (CVD), especially HF, have been addressed in clinical practice guidelines and position statements issued by international scientific societies such as the European Society of Cardiology (ESC), the European Association for the Study of Diabetes (EASD), the American Heart Association (AHA) and the Heart Failure Society of America (HFSA), which aim to provide evidence-based guidance on how to optimally treat patients with DM and CV complications ,29. In t(1)Most DPP4i have neutral effects on MACEs and HF outcomes with the exception of saxagliptin, which has been shown to increase significantly the risk of HF hospitalization in high risk patients . Althoug(2)GLP1RAs have neutral impact on HF hospitalization, but some of them  reduce the risk of MACEs, including CV mortality, in diabetic patients with CVD or high CVD risk ,35,40. G(3)SGLT2i are the first antidiabetic drug class which has demonstrated the potential to reduce mortality, MACEs and HF hospitalization in diabetic patients at high CVD risk, regardless of the history of previous HF ,49,50. TOver the past 5–6 years, the landscape of T2DM treatment has been revolutionized by the introduction of novel glucose-lowering agents, which -for the first time in the history of DM pharmacotherapy- have shown the potential to improve CV outcomes in patients with T2DM. The so-called Cardiovascular Outcome Trials (CVOTs), namely randomized clinical trials originally designed to fulfil the Food and Drug Administration (FDA) requirement to assess CV safety of antidiabetic agents vs. placebo, have recently provided an overwhelming body of evidence suggesting that two specific antidiabetic drug classes exert cardioprotective effects in diabetic patients with CVD or at risk for CVD. These drug classes comprise glucagon-like peptide 1 receptor agonists (GLP1RAs) and sodium-glucose cotransporter 2 inhibitors (SGLT2i). The former have been primarily associated with a reduction in atherosclerosis-related outcomes and major adverse cardiovascular events (MACEs), whereas the latter have been linked to an improvement in HF-related endpoints. To date, several CVOTs have been published and influenced dramatically clinical guidelines and treatment algorithms: 4 for dipeptidyl peptidase 4 inhibitors (DPP4i)  ,31,32,33Interestingly, both GLP1RAs (liraglutide and semaglutide) and SGLT2i  have shown additional renoprotective effects in patients with DM. The major CVOTs of these agents have studied renal function as a secondary outcome and have shown that compared with placebo, these drugs are able to delay the progression of CKD and reduce clinically significant renal events ,41,42,43Is DCM reversible and which other mechanisms -beyond the already identified ones- may contribute to the pathophysiology of this entity?Which mechanisms could explain the divergent evidence on the association of DPP4i with HF risk showing an increased risk with saxagliptin but no increased risk with the other gliptins?Are the effects of GLP1RAs on HF outcomes neutral or potentially harmful? In case of adverse effects, are they attributable to the modest increase in heart rate induced by GLP1RAs, or are other mechanisms involved?Are SGLT2i equally safe and effective in patients with HFrEF and HFpEF?What is the role of SGLT2i as a stand-alone HF treatment in non-diabetic patients ? What are the exact mechanistic/hemodynamic effects of SGLT2i on LV remodelling and myocardial injury beyond osmotic diuresis?Which are the optimal glycemic targets for diabetic patients with different severity of HF?2.What is the optimal antidiabetic agent for patients with HF and advanced CKD? This issue is particularly relevant considering that SGLT2i are on the one hand able to confer renoprotection, but on the other hand are not indicated for use in patients with an estimated glomerular filtration rate (eGFR) below 30 mL/min/1.73 mIs the use of novel antidiabetic therapies such as SGLT2i expected to change the epidemiology of DM-associated HF?Despite the progress summarized above, a large number of unanswered questions and knowledge gaps remain still under investigation and should be thoroughly examined in the future:Future research in the field of cardiovascular diabetology should try to resolve these issues and provide definitive answers. Furthermore, it is important that future trials integrate non-invasive cardiac imaging  in order to establish novel imaging biomarkers, which would help better characterize the structural and functional abnormalities of the diabetic failing heart and facilitate monitoring of treatment effects .In conclusion, HF represents an important CV complication of DM associated with substantial morbidity and mortality, and is emphasized in recent CVOTs as a critical outcome for patients with DM. The presence of HF or the high risk for developing HF in the future has influenced recent guideline recommendations and can guide therapeutic decision making. Metformin remains first-line treatment for overweight T2DM patients without CVD and at moderate CVD risk. Although not contraindicated at any stage of HF, metformin is no longer considered as first-line therapy for diabetic patients with established HF or at risk for HF, since there is robust scientific evidence that treatment with other glucose-lowering agents such as SGLT2i should be prioritized in this population due to their strong beneficial effects on HF outcomes.


Mucosal leishmaniasis (ML) is mostly associated with Leishmania braziliensis; however, a few cases of Leishmania tropica induced mucocutaneous leishmaniasis have been reported. The standard treatment for leishmaniasis is pentavalent antimonials, but several other drugs for resistant cases have been proposed including amphotericin and miltefosine. Here we present a case of multiple treatment resistant mucocutaneous leishmaniasis with nasal involvement caused by L. tropica; cure was not achieved by multiple treatments and was eventually improved by adding thalidomide to Meglumine Antimoniate (Glucantime). To the best of our knowledge use of thalidomide in humans for leishmaniasis treatment is reported here for the first time. Here we present a case of refractory mucocutaneous leishmaniasis with nasal involvement caused by 2A 20-year-old man, accounting student, presented to cutaneous leishmaniasis clinic in Imam-Reza hospital Mashhad, Iran with an edematous mass in the right nasal nare. The patient had a history of cutaneous leishmaniasis 9 years ago (2009) on his chin and left forearm that was improved as a local outpatient treatment . However, after a short time, indurated mass on his left nasal nare developed. The new lesion showed leishmania parasite in the direct smear and was treated with one course of intramuscular meglumine antimoniate with remission of this lesion. Nevertheless, after one month, an indurated right nasal nare mass plus dyspnea and nocturnal snoring appeared. Nasal endoscopy sampling confirmed mucosal involvement by leishmaniasis. This bothersome mucocutaneous lesion  did not L. tropica. He also had anti-leishmania antibodies in his serum, but abdominal Ultrasound and bone marrow aspiration were negative and ruled out visceral leishmaniasis. Considering the location of the lesion, sinuses and nasal coronal CT scans reported no pathologies.His Immune competency laboratory test included the nitroblue tetrazolium (NBT), the dihydrorhodamine (DHR) test, blood flow cytometry T cell subtype and serum hemolytic complement (CH50) activity were within normal limit except serum Immunoglobulin E, which was more than 500. Direct smears of the lesions were repeatedly positive and the polymerase chain reaction (PCR) reported the responsible parasite to be In 2016, he underwent surgical excision of the lesion and received oral miltefosine 150 mg daily for two months but the lesion did not resolve completely and recurred after 3 month.Imam-Reza hospital with the diagnosis of multiple treatment resistant, L. tropica induced mucocutaneous leishmaniasis.At the time of admission, the patient weighed 66 kg and the presence of Leishman bodies was confirmed in the direct smear of the lesion. He was treated with combination of intramuscular glucantime (850 mg daily for 28 days) and oral thalidomide (100 mg daily for two months).Given that the first experience of this combination therapy, the minimum therapeutic dose of these drugs was used. During the first month of treatment the size of the lesion decreased significantly and during the second month the induration of the lesion disappeared and the atrophic and some retractive scar remained.No adverse effects observed during this combination therapy and the direct smears from the lesion were negative after discharge and at third and sixth months after the beginning of the treatment. No signs or symptoms of recurrence until now (February 2019) were observed during the one-year follow-up  whereas L. infantum can cause visceral leishmaniasis (VL)  . Mucosalzilliens . Howeveri Arabia , and twompletely .Mucosal leishmaniasis in new world is a devastating form of leishmaniasis that commonly affects the nasal and oral cavity and may lead to nasal deformity and even destruction of nasal septum . MoreoveL. tropica induced mucocutaneous leishmaniasis, resistant to already known treatments; meglumine antimoniate, amphotericin and miltefosine. Thalidomide as an immunomodulator was added to intramuscular meglumine antimoniate (glucantime) therapy, and resulted in complete remission. No recurrences, was observed during the one-year follow-up.Here we reported a Although production of proinflammatory cytokines, such as Interferon gamma (IFN-γ) and tumor necrosis factor-α (TNF-α), is important for Leishmania killing, an overproduction of these proinflammatory cytokines as well as a decreased ability of IL-10 and TGF-β to modulate this response may lead to severe tissue damage that have been seen in mucosal leishmaniasis patients compared to patients with classical cutaneous leishmaniasis. These abnormalities may be the basis for the pathological findings and the therapeutic challenge observed in this disease, and accordingly, the rational clinical application of immunomodulatory drugs for ML . AccordiThalidomide, once a known drug, deserted due to a series of adverse effects related to its teratogenicity. However, in recent years it has begun to attract attention especially in the field of dermatology for immunomodulatory, anti-inflammatory, and antiangiogenic properties .Levels of TNF- α are increased in serum and cultures from patients with mucosal disease that leads to an intense inflammatory reaction, so thalidomide can be useful as TNF-α inhibitor drug . Verbon About the effects of thalidomide on Interferon gamma (IFN-γ), an important cytokine in leishmaniasis pathogenesis, some studies demonstrated that an increase in IFN-γ can be observed in response to Thalidomide . AnotherIn the case of leishmaniasis, no report exists on the use of thalidomide in humans however, an animal study showed treatment potentials of thalidomide in leishmaniasis. In this study, BALB/c mice were infected with L. major and were later treated with a combination of glucantime and thalidomide. Results showed a superior efficacy of combination therapy to glucantime, thalidomide or carrier alone. They also showed that thalidomide effects were mainly due to up-regulation of IFN-γ and down-regulation of IL-10 .Considering the contradictory roles of thalidomide regarding IFN-γ and TNF-α, the results of related studies, and observations from our study, more research is needed to further understand the role of thalidomide in leishmaniasis and its effects on IFN-γ, TNF-α and immune responses of patients with leishmaniasis.Despite various applications proposed for thalidomide in dermatology, its use is limited by an infamous profile of side effects mainly its teratogenic effects that led to its withdrawal in 1962 . HoweverTo the best of our knowledge, this is the first report of the use of thalidomide in the treatment of leishmaniasis in humans. Although it showed promising results in our patient, further studies are required for establishing its efficacy in treatment of leishmaniasis.No Conflicts of interest.No financial support.