P<0.001) as compared to Non-Occl-Con pigs; however, Occl-Ex pigs exhibited partial recovery (P<0.001) intermediate to the other two groups. BK induced relaxation was reduced in LADs from Occl-Ex and Occl-Sed pigs (P<0.001), compared to Non-Occl-Con, and exercise modestly increased responses to BK (P<0.05). In addition, SNP, PE, Ang II, and ET-1 responses were not significantly different among the groups. Our results indicate that ‘simple’ occlusion of the femoral arteries induces vascular dysfunction in conduit vessels distinct from the affected hindlimbs, as evident in blunted coronary vasorelaxation responses to ADP and BK. These findings imply that PAI, even in the absence of frank atherogenic vascular disease, contributes to vascular dysfunction in the coronary arteries that could exacerbate disease outcome in patients with peripheral artery disease. Further, regular daily physical activity partially recovered the deficit observed in the coronary arteries.The present study was designed to determine whether adult swine with peripheral artery insufficiency (PAI) would exhibit vascular dysfunction in vessels distinct from the affected distal limbs, the coronary conduit arteries. Moreover, we sought to evaluate the effect of exercise training on coronary vasomotor function in PAI. Eighteen female healthy young Yucatan miniature swine were randomly assigned to either occluded exercise trained , or occluded-sedentary , or non-occluded, non-exercised control  groups. Occl-Ex pigs were progressively trained by running on a treadmill . The left descending artery (LAD) and left circumflex (LCX) coronary arteries were harvested. Vasorelaxation to adenosine diphosphate (ADP), bradykinin (BK), and sodium nitro-prusside (SNP) were assessed in LAD’s; while constrictor responses to phenylephrine (PE), angiotensin II (Ang II), and endothelin-1 (ET-1) were assessed in LCX’s. Vasorelaxation to ADP was reduced in LADs from Occl-Sed and Occl-Ex pigs ( PAD is a major cause of disability, loss of work, and lifestyle changes in the United States. It affects approximately 8 to 12 million persons in the country, and its prevalence increases with advancing age, as approximately 12 to 20% of people older than 70 years suffer from this condition.Peripheral artery disease (PAD) impedes blood flow typically to the lower extremities leading to a mismatch of oxygen delivery that becomes more evident during physical activity. The impaired perfusion observed in PAD can foster functional and structural modifications in the affected limb  which attempt to ameliorate the symptoms. This is especially true if the patients become physically active. However, PAD patients also exhibit decreased nitric oxide (NO) activity that is associated with an increased production of vasoconstrictors and sympathetic outflow,6.One half of all PAD patients older than 55 years are asymptomatic; whilst among the symptomatic, 40% experience intermittent claudication, the most common clinical manifestation of PAD, and 10% have limb ischemia at rest with increased mortality due to cardiovascular disease, especially CAD , as well as a 6.6-fold higher overall mortality than non-PAD patients. Interestingly, in patients with concomitant CAD and PAD the presence of peripheral inflammation has been postulated to contribute to coronary endothelial dysfunction, mostly due to a reduction in NO bioavailability. Indeed, many conceive that the association of PAD with CAD is simply the result of generalized atherosclerosis progressing in both arterial trees. However, an estimated 35 to 50% of all patients with PAD do not exhibit CAD or cerebral vascular disease (CVD),11. There are studies reporting a widespread incidence of normal coronary arteries among PAD patients–14. For instance, coronary angiography was found to be normal in 85% of symptomatic PAD patients . Duran et al. reported that coronary artery angiography was within normal limits in 28% of patients with PAD . Furthermore, the study by Hertzer et al. found that CAD was present in only 270 of 468 elderly patients (58%) with PAD who were living in a long-term health care facility. Nonetheless, these PAD patients in the absence of frank CAD and CVD have the same markedly increased morbidity and mortality as those PAD patients with attendant central vascular disease. It is possible that the ischemic limb muscles in PAD release substances and/or initiate neural reflex responses systemically that lead to coronary artery dysfunction and potentially greater susceptibility to CAD. It is well documented that exercise training improves walking ability and reduces cardiovascular mortality and morbidity of claudicant patients,17. Indeed, Sakamoto et al. reported that supervised exercise training reduced overall cardiovascular mortality by 52% and morbidity by 30% among PAD patients. Based on this background, the present study was designed to determine whether in an atherosclerosis free, preclinical animal model of peripheral artery insufficiency (PAI) bilateral occlusion of the femoral arteries would lead to vascular dysfunction within a vascular bed, the coronary conduit vessels, distant from the affected hindlimb vasculature. This animal model of PAI exhibits limb blood flow that is non-ischemic at rest, but is impaired during exercise,19, similar to flow patterns seen with intermittent claudication. In addition, we sought to evaluate the effect of exercise training on coronary endothelial function in this animal model of PAI. Exercise training has been shown to increase endothelial-dependent vasorelaxation of peripheral vessels in patients with intermittent claudication, and in patients with CAD. Thus, we hypothesized that a preclinical animal model of PAI would exhibit coronary dysfunction and that this would be ameliorated by exercise.PAD patients are reported to have increased incidence of coronary artery disease (CAD)–25, were used in this study. The pigs were housed in the animal care facility in rooms maintained at 20–23°C with a 12h: 12h light-dark cycle. They were fed a normal diet (Purina Laboratory 5082 Mini-Pig Breeder Chow) with ad libitum access to water. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Missouri.Healthy young female Yucatan miniature swine , a breed relatively resistant to atherogenesis.Animals were pre-anesthetized with ketamine (35 mg/ kg)-xylazine (2.25 mg/kg) intramuscular injection. A tracheal tube was inserted, and secured. Pigs were anesthetized by using 2% isoflurane gas with a closed circuit anesthesia machine and an auto-respirator (Ohmed 7000). Surgical areas were cleaned with warm soap water, sterilized with Betadine and alcohol. A 4cm incision was made on the pig’s neck to expose left carotid artery. A medical perfusion catheter  was inserted via the carotid artery and advanced into the thoracic aorta for blood pressure monitoring. Blood pressure, respiration rate, heart rate, ECG, and body temperature were monitored during the surgery procedure. A 4 cm longitudinal skin incision was made in each groin area above the femoral artery which was occluded with 2–0 surgical silk at 2 cm distal from the inguinal ligament. The femoral occlusion animal model employed in this experiment has been used extensively by our laboratoryAt the beginning of the study all pigs were familiarized with walking on a motorized treadmill for 5 days prior to surgery and then randomly assigned into exercise and sedentary groups. The occluded-exercise  were endurance trained on a treadmill at 0% grade, beginning one week post-surgery; whilst the occluded-sedentary  were limited to cage activity within a 2 x 4 m pen. Occl-Ex pigs underwent progressive daily training sessions 5 days/week for 12 weeks. The training protocol included a warm-up prolonged running and cools down stages, where the durations and intensity of exercise bouts were increased as the animals were capable. Briefly, warm-up and cool down stages lasted for 5 min each at 1.5 mph. The endurance stage was running at a constant speed of 3.1 mph, building up to a total time of 75 min as the animals were capable. This intensity could easily be achieved by the occluded animals for approximately 75 min/session within approximately 9 weeks after beginning training. Exercise stress tests were conducted for both occluded groups, once every week by running on the treadmill beginning at 1mph and increasing the speed by 1mph each min until the pig was unable to continue. Blood pressure and heart rate were monitored during the stress tests. In addition, non-occluded pigs, age-matched, served as a control group ; these pigs were not exercise neither trained nor given the stress test each week.At the end of the 12 weeks of exercise training, pigs were anesthetized with intramuscular ketamine (35 mg/ kg)-xylazine (2.25 mg/kg), and intravenous thiopenthal (25 mg/ kg) for deep anesthesia, and the heart was removed to achieve euthanasia and placed in iced Krebs solution. The left anterior descending (LAD) and left circumflex (LCX) coronary arteries were removed from the heart. The arteries were trimmed of connective tissue and fat in cold Krebs bicarbonate buffer solution; and cut into six LAD and two LCX vessel rings each 3–4 mm in length. Cut rings were photographed on an Olympus SZH video microscope which was connected to a Spot Insight camera . Then, the rings morphological characteristics  were measured using Image J software .in vitro studies, and mounted on myographs  by positioning two stainless steel wires in the lumen of each ring. Arterial rings were placed in a 20 mL bath of Krebs bicarbonate solution. The optimal point in the length-tension relationship (Lmax) was established through incremental (10% of passive outside diameter) increases in stretch in combination with potassium chloride (KCl: 50 mM) administration to the rings. The remainder of the study was performed at the experimentally determined Lmax of each ring. Contractile responses to KCl were performed prior to vasorelaxation studies. KCl (80 mM) was administered twice to all of the arterial rings until the increase in tension had reached a plateau (10 min). Following the KCl experiments, Krebs bicarbonate solution was replaced every 20 min until resting tension was achieved.Six LAD and two LCX rings were used, for −9–10−4 M) and bradykinin ; whilst the assessment of endothelium-independent vasorelaxation utilized increasing doses of sodium nitroprusside . The LAD rings were preconstricted with prostaglandin F2α  and allowed to achieve a plateau in tension development before the addition of vasodilators. For each pig’s heart ring one was left untreated (intact). Ring two was pretreated with nitro-L-arginine methyl ester  to inhibit the nitric oxide synthase (NOS) pathway. The third ring was pretreated with indomethacin  to inhibit cyclooxygenase (COX), the enzyme responsible for prostacyclin (PGI2) and prostaglandin production. Ring four was pretreated with a combination of L-NAME and Indo to assess the importance of NOS-and COX-independent mechanisms of relaxation. Ring five was pretreated with a combination of L-NAME, Indo, and potassium chloride (KCl) to block NOS, COX and the non-NOS, non-COX pathway, endothelium-derived hyperpolarizing factor (EDHF). Finally, the endothelium was removed from ring six; “denuded” by gentle rubbing of the luminal surface with fine-tipped forceps to study the endothelium- independent mechanisms, explicitly smooth muscle relaxation. Adequate ring denudation was tested by examining responses to maximal BK (30 mM). Ring six was considered to be properly denuded by the inability of BK to induce vasorelaxation (i.e. less than 5%) in PGF2α (30 μM) preconstricted rings. The order of agonists throughout the entire study was ADP, BK, and SNP. Following each agonist induced dose response; Krebs bicarbonate solution was replaced at 20min intervals until resting tension of all arterial rings was reached (roughly 60min), before the next protocol was initiated.It was designed to examine the relaxation responses. Endothelium-dependent, dose-dependent vasorelaxation was assessed in six LAD rings using cumulative addition of adenosine diphosphate , angiotensin II  and endothelin-1 . Ring one was left untreated; “intact”, whilst on ring two the endothelium was removed as described above; “denuded”.The vasoconstrictor properties were assessed in two LCX rings using cumulative addition of phenylephrine  and maintained at 37°C. Angiotensin II was purchased from Bachem Americas , and all other drugs and chemicals were purchased from Sigma Chemical .The Krebs–bicarbonate buffer solution contained (in mM) 131.5NaCl, 5.0KCl, 1.2NaH50) values, and maximal relaxation responses were determined via one-way ANOVA with a Kruskal-Wallis test using Graph Pad Prism version 5.0a. Means of the EC50 values are presented as the negative log of the molar concentration. The analysis of concentration-response curves was performed using the mixed procedure in SAS version 9. The statistical model used was a three-factor analysis of variance with one between subjects factor , and two within subject factors (treatment and dose). The Mixed procedure also allows us to model for heterogeneous variances across doses. Pairwise comparisons were made between groups for fixed treatment and dose levels using Least Squares Means. Nonparametric statistical methods were used to perform a series of tests on dose-response data in the cases where examination of residual plots from a three-factor analysis of variance model indicated that the assumption of normality of the error terms was suspect. Specifically, group differences at each dose-treatment combination were looked at with the Wilcoxon rank sum test. Treatment differences at each dose-group combination were also examined with the Wilcoxon signed-rank test on the differences. A false discovery rate adjustment was used for multiple tests in view of the large number of tests considered. Differences with false discovery rate-adjusted P-values of P<0.05 were considered significant.All values are means ± SE. Differences among groups regarding ring characteristics, half-maximal effective concentration (ECP<0.01). By the end of the training period, animals in the Occl-Ex group could run up to a higher intensity of 6–7 mph, as compared to only approximately 5mph for the Occl-Sed group during the weekly graded treadmill running test. In addition, Occl-Ex pigs showed a significantly lower peak blood pressure during the stress test than the Occl-Sed pigs (121±4 mmHg and 135±7 mmHg respectively (P<0.01). The peak heart rates were not significantly different between groups .All Occl-Ex pigs completed the training program. The body weights were Non-Occl-Con, 27.5±0.74 kg; Occl-Ex, 39.4±2.2 kg; and Occl-Sed, 42.9±4.21kg. Although we included age-matched controls, the Non-Occl-Con were significantly smaller than both, Occl-Ex and Occl-Sed, (P<0.05) of the rings from the Non-Occl-Con pigs.Structural and functional characteristics of rings harvested from LAD and LCX are presented in . There wP<0.001) in intact rings from both Occl-Ex and Occl-Sed pigs than the Non-Occl-Con pigs (P<0.05) by approximately a log-dose (P<0.001) in a dose-dependent manner, compared to the Occl-Sed group. BK-induced relaxation was partially inhibited by the addition of L-NAME in all three groups (P<0.001) (P<0.001), suggesting the presence of a prostanoid constrictor in the occluded vessels (P<0.001), indicating the presence of a COX-sensitive vasoconstrictor even in the absence of occlusion. With the combination of inhibitors, L-Name and Indo, BK-induced relaxation was reduced in all three groups (P<0.001), with a marked reduction in the Non-Occl-Con group such that the values were now below those of the Occl-Sed and Occl-Ex groups  ; while t vessels . HoweverP<0.001; . BK-indue groups . Finallye groups .P<0.001), and exhibited a greater EC50 (P<0.001) from the deficit of the Occl-Sed group. ADP-induced relaxation was in part inhibited by L-NAME in all groups (P<0.001) that of the Non-Occl-Con group with the relaxation curve of the Occl-Ex intermediate (P<0.05) in a dose-dependent manner (P<0.001), in a dose-dependent manner, when Indo was added to the bath (P<0.001) and the Occl-Ex group greater than the Occl-Sed group (P<0.05) in a dose-dependent manner. Consistently, the relative sensitivities to ADP, as evident in EC50  and increasing values in the Occl-Sed group (P<0.05) with dose-group interactions (P<0.001). It should be recognized that the ADP-induced relaxation remained rather robust for all three groups , indicating that pathways different from NOS and COX are important for vasorelaxation. In contrast, ADP-induced relaxation was markedly inhibited by L-NAME + Indo + KCl  remained. This implies that some non-NOS pathway contributes to the coronary dysfunction caused by occlusion of the femoral arteries. Further, the lack of differences in the SNP-induced responses among groups indicates that the responsiveness of the smooth muscle to NO is not an important factor underpinning the reductions in vessel relaxation observed with intact vessels from the occluded groups. Second, significant improvements in BK- & ADP-induced relaxation became evident with inhibition of the COX pathways with Indo  contribute to vasorelaxation in coronary arteries. Indeed, it has been reported that EDHF-mediated responses play an important role in conduit arteries for endothelium-dependent relaxation, especially the coronary vascular bed. The observation that BK-stimulated dilatory responses of the occluded groups were actually greater (2Y1 receptors (“purinoceptors”) located on vascular smooth muscle cells of porcine coronary arteries. However, the fact that the dilatory responses were similar in Non-Occl- Con, Occl-Sed, and Occl-Ex animals after denudation , that was evident with both BK and ADP, was eliminated by the inhibition of both the NOS and COX pathways with L-NAME and Indo, respectively. This implies that either or both NOS and COX pathways are altered by exercise training and contribute to the improved vasoresponsiveness by an enhanced NO-dependent vasorelaxation and/or a reduced COX-dependent vasoconstrictor effect. On one hand, the differences between the Occl-Ex and Occl-Sed groups  or in the presence of Indo (leading to an enhanced NOS-dependent vasodilatation). It is unclear why this was not observed with BK    were notion)  may actually be an advantage in the present experiment as it potentially eliminates confounding factors that often present as comorbidities in human PAD patients. Thus, while patients with intermittent claudication can exhibit endothelial dysfunction,6 and an increased inflammatory status with impairment of the coronary arteries it is difficult to assess the effects of PAI as an independent cause of any central vascular dysfunction. In contrast, in the present study we are able to evaluate distinct outcomes of ‘simple’ PAI in relatively young pigs that are free from atherosclerotic/inflammatory disease. Certainly, Yucatan miniature swine are highly resistant to frank atherogenesis. Minimal signs of atherogenesis are only evident after feeding them an exceptionally high-fat diet, representing 46% of their daily caloric intake, for an extended period of time (20–24 weeks)–24. Even then the extent of atherosclerosis in central and peripheral arteries is relatively modest characterized by a Stary Stage I-III classification,24,25, similar to that reported in studies of early human atherosclerosis. Thus, our experiment could expose consequences of PAI that are manifest, even in the absence of extensive comorbidities.Preclinical animal models of PAI, similar to that used in this experiment, have been well characterized and extensively used to investigate potential therapeutic approaches to treat patients with PAD. This appears to be exaggerated in PAD patients, as ischemia induced by exercise promotes oxidative stress and inflammation that has been linked to an attenuation of endothelium-dependent vascular smooth muscle relaxation. Accordingly, we could speculate that the decreased coronary vasorelaxation reported in this study might be the consequence of altered NO availability induced by excessive oxidative stress even with simple cage activity in all of the occluded pigs. However, our results indicate occlusion promulgates changes in signal pathways far more extensive than the NOS. On the other hand, it is apparent that chronic physical activity, similar to that experienced by our trained pigs, exerts an anti-inflammatory effect,38. Elevated biomarkers of chronic inflammation are reduced by exercise training in patients with chronic cardiovascular disease, including PAD,40. If this occurred in our Occl-Ex pigs, it could have contributed to the ameliorated dilatory dysfunction observed in the coronary vessels with exercise training. Second, there is the potential that PAI leads to a hypersympathetic state. Sympathetic nervous activity (SNA) is expected to increase in response to exercise  and is exacerbated during ischemic exercise. In PAD patients there is an exaggerated increase in blood pressure during walkingthat is related to walking speed and duration, as well as the severity of disease,34. This increased blood pressure has been attributed to an enhanced sympathetic output through the ischemic pressor response,42,4. Similar increases in mean arterial blood pressure, attributed to an observed increase in SNA, have been reported in studies using animal models of PAI. An increase in SNA during activity could have contributed to our results, as the associated increase in blood pressure driven by an enhanced SNA is known to be a cardiovascular risk factor leading to a reduced endothelial dependent vasodilatation. Interestingly, it has been reported that exercise training can normalize SNA, alleviate levels of vasoactive substances leading to vasoconstriction, and promote a reduction in systemic blood pressure. Consistent with these observations, our data showed that Occl-Ex pigs exhibited significantly lower peak blood pressure during the stress test than the Occl-Sed pigs .The most important finding of the present study is that coronary artery endothelial dysfunction was observed in other- wise healthy pigs following bilateral occlusion of the femoral arteries. Although the precipitating cause of this dysfunction is presently unclear, a couple of contributing factors can be proposed. First, it is now well recognized that active muscle, even in normal healthy humans, releases an abundance of potent cytokines, including factors potentially involved in the inflammatory process–14 representing approximately 35 to 50% of all patients with PAD,11. Further, exercise training partially ameliorated the dilatory deficit. The potential that muscle- derived cyotkines/factors and/or a hypersympathetic state and their amelioration by training contributed to these responses remains to be determined. To the extent that our findings apply to these human patients, this implies that individuals with PAD are at risk of coronary dysfunction, even in the absence of generalized atherosclerotic disease that affects central vessels.In conclusion, we report here that bilateral occlusion of the femoral arteries, used to establish a preclinical model of PAI, induces coronary endothelial dysfunction, evident as a reduced vasodilatory responsiveness. These observations are potentially most significant in view of the sizeable number of PAD patients that are free from frank CAD


The Sonic hedgehog (Shh) signaling pathway regulates developmental, homeostatic, and repair processes throughout the body. In the skin, touch domes develop in tandem with primary hair follicles and contain sensory Merkel cells. The developmental signaling requirements for touch dome specification are largely unknown. We found dermal Wnt signaling and subsequent epidermal Eda/Edar signaling promoted Merkel cell morphogenesis by inducing Shh expression in early follicles. Lineage-specific gene deletions revealed intraepithelial Shh signaling was necessary for Merkel cell specification. Additionally, a Shh signaling agonist was sufficient to rescue Merkel cell differentiation in Edar-deficient skin. Moreover, Merkel cells formed in Fgf20 mutant skin where primary hair formation was defective but Shh production was preserved. Although developmentally associated with hair follicles, fate mapping demonstrated Merkel cells primarily originated outside the hair follicle lineage. These findings suggest that touch dome development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and ultimately Shh signaling from primary follicles to extrafollicular Merkel cell progenitors. Shh signaling often demonstrates pleiotropic effects within a structure over time. In postnatal skin, Shh is known to regulate the self-renewal, but not the differentiation, of touch dome stem cells. Our findings relate the varied effects of Shh in the touch dome to the ligand source, with locally produced Shh acting as a morphogen essential for lineage specification during development and neural Shh regulating postnatal touch dome stem cell maintenance. Sonic hedgehog (Shh) is one of a limited set of signaling molecules that cells use to drive organ formation during development and tissue regeneration after birth. How Shh signaling achieves different biological effects in the same tissue is incompletely understood. Touch domes are unique sensory structures in the skin that contain innervated Merkel cells. Using mouse genetics, we show that touch domes develop in tandem with, but distinct from, primary hair follicles. Moreover, touch dome specification requires a cascade of cell-cell signaling that ends with Shh signaling from an adjacent primary hair follicle. It was previously shown that Shh signaling from sensory nerves regulates the maintenance of touch dome stem cells after birth. Thus, the critical role for Shh signaling in embryonic touch dome specification is dependent on locally produced Shh, whereas the renewal of touch dome stem cells requires Shh transported to the skin by sensory neurons. These observations suggest that the distinct functions of Shh in touch dome development and maintenance correspond to changes in the source of the Shh signal required for the varied effects. The Hedgehog (Hh) pathway is conserved across the Metazoa subkingdom, and is one of a small number of intercellular signaling pathways that regulate the differentiation and pattering of morphologically diverse structures during development ,2. PostnThe functional diversity of vertebrate skin depends greatly on the variety of ectodermal appendages it produces. The development of ectodermal appendages including hair follicles, teeth, sweat glands, and mammary glands is precisely regulated by networks of signaling pathways including Wnt/β-catenin, Eda/Edar, Shh, and BMP . Hair foThe touch dome (TD) is a specialized epidermal sensory structure composed of K8+ Merkel cells (MCs) arrayed among columnar basal keratinocytes that express the hair follicle keratin K17. Cells of the TD are morphologically and molecularly distinct from the adjacent interfollicular epidermis. The TD arises from the K14+ ectoderm ,10 and iTD MC development in the embryonic ectoderm is spatially and temporally associated with that of primary hair follicles. In embryonic day 14 (E14) mice, a primary wave of hair follicle placode induction takes place. These primary follicles produce guard hairs that ultimately comprise ~2% of the adult mouse coat. Secondary (E16) and tertiary E18) waves of hair follicle induction are responsible for forming the three remaining types of hair follicles  waves of. Loss ofShh or Smo in the embryonic epidermis reveled that intraepithelial Shh signaling from primary hair germs was necessary for TD MC specification. Notably, the loss of MC specification in Shh-deficient skin was observed before any disruption in hair follicle development was apparent. The importance of Shh signaling in TD MC formation was further demonstrated by using a Smo agonist to rescue MC specification in ex vivo-cultured Edar mutant skin. In contrast, Fgf20 was dispensable in TD MC development, demonstrating that the signaling cascades required for guard hair follicle development and TD formation diverge at the level of Fgf20 signaling. Distinction from the forming guard hair follicle was further demonstrated when fate mapping of the follicle lineage showed that TD MC progenitors predominantly arise in the epidermis outside the hair placode. Thus, like other ectodermal appendages, the touch dome is a distinct epidermal lineage whose specification and development requires Wnt-dependent mesenchymal-epithelial interactions and reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and subsequent Shh production by primary hair germs. The critical role for Shh signaling in embryonic TD specification is dependent on locally produced ligand, whereas the regulation of postnatal TD stem cells requires Shh transported to the skin by sensory neurons. These observations suggest that ligand source influences the differential effects of Shh signaling in the TD.To elucidate the developmental requirements for TD MC formation, we used genetically modified mice to disrupt signaling pathways known to be important in the development of other ectodermal appendages. We found that embryonic deletion of dermal β-catenin prevented TD MC formation. Similarly, Edar mutant skin failed to generate TD MCs. The mechanism of MC loss in these mice was due to failure of Shh expression by primary hair follicles, as evidenced by an absence of TD MC formation in Shh-null skin. Moreover, independent deletion of En1Cre/+; β-cateninflox/Δ mouse embryos where Cre recombination occurs in the dorsal trunk mesenchyme that forms the dermis . In. In20]. YFP Figs  and S3. Edar mutation, Shh mutation, epidermal deletion of Shh, and epidermal deletion of Smo to show that paracrine Shh signaling within the epidermis, downstream of Wnt and Eda signaling, is essential for TD MC development. However, in all of these mice, there is either a global defect in hair follicle patterning and development, a developmental defect in all hair follicles, or a defect in primary hair follicles. To separate Shh signaling from normal guard hair development, we used Fgf20-null (Fgf20LacZ/LacZ) mice [Fgf20LacZ/LacZ skin (n = 3) and found Shh levels elevated compared with those in control skin , we occasionally observed larger follicles that were comparable to guard follicles in control skin  relative to control  mice . Like ShcZ) mice . AlthougcZ) mice . We usedn n = 3, . A smalln n = 3, , suggestacZ mice . In E15.ificance , and a SVertebrate skin provides barrier, mechanical, defensive, communicative, sensory, metabolic, and homeostatic functions. This diversity of function is achieved, in part, by specialization of ectodermal appendages that form through a series of mesenchymal-epithelial interactions in the embryo. By regulating common inductive events with similar yet distinct sets of morphogens, great diversity of structure and function is achieved . AlthougEn1Cre/+; β-cateninflox/Δ skin, Edardl-J/dl-J skin, or Eda mutant Tabby skin [Shh in Eda mutant Tabby mice failed to restore primary hair follicle development [Our results support a model of TD MC development in which MC specification is tied to primary hair follicle patterning and morphogenesis up to the point of Shh production by the early follicle and can diverge from follicle development with signals such as Fgf20 that act in parallel or downstream to Shh in the hair follicle. Similarities in the developmental signaling pathways between the TD and primary hair follicles include the importance of mesenchymal Wnt signaling and ectodermal Eda/Edar signaling. Accordingly, there is no Shh expression by primary hair placodes in bby skin , and TD elopment . Downstrhairless mice, where hair follicles develop normally but undergo cystic degeneration after the first month of life [Shh from sensory neurons had no impact on TD MC formation [The coats of Fgf20 mutant mice lack guard hairs, and yet typical-appearing TDs were found at normal density and only a slightly reduced average number of MCs per TD. Interestingly, TD MCs are maintained in adult  of life , demonst of life , and embormation . It is normation . This isormation , howeverormation ,49. Shh The formation of TDs adjacent to primary hair follicles requires Shh signaling from the nascent follicles. Secondary and tertiary hair follicles also express Shh during their formation, and yet TDs do not form in association with induction of those hair follicles. Moreover, based on the broad expression of Gli1 within the developing epidermis, many embryonic epidermal cells receive Shh signaling. Thus, even though a hedgehog signaling agonist was sufficient to rescue MC differentiation in Edar mutant skin, Shh signaling alone is not sufficient to induce TD MC specification in any and all developing ectoderm. Further experimentation will be necessary to identify the factors that establish the temporal and spatial competency of developing epidermis to respond to Shh signaling with MC specification. However, Polycomb repressive complex 2 (PRC2) activity appears to be important in preventing MC specification around secondary and tertiary hair follicles . Notch signaling may also play a role in limiting MC specification, as ectopic expression of Atoh1 is able to induce MC production in some epithelial compartments of the skin, and impeding Notch signaling facilitates this process .Fate mapping showed that MCs arise predominantly from ectoderm outside the forming hair follicle. This is consistent with our observations that the first MCs appeared as Sox2+ cells in the E14.5/15.5 epidermis above and adjacent to forming primary hair germs. We confirmed that late embryonic and early postnatal guard hair follicles contain a discrete population of MCs, however these cells also tend to arise from outside the hair follicle lineage. Although the purpose of the MCs in the upper regions of primary hair follicles during development is unclear, it has been proposed that the NCAM expressed on keratinocytes around these MCs may facilitate MC innervation . CoincidGli1 is broadly expressed in the developing epidermis, it cannot be used to identify the specific K5+ Shh-responding cells required for TD MC development. Just as adult TD stem cells are Gli1+ [We have determined that intraepithelial Shh signaling from the developing hair follicle to MC precursors is a critical factor in TD MC production. However, the precise location of the target cells for Shh remain undefined. Because the Hh response gene re Gli1+ , our GliThis work elucidates the developmental signaling requirements for the sensory TD and illustrates the commonalities and contrasts that exist between TD development and development of the closely associated guard hair follicle. These results further our functional understanding of skin patterning and development, the repertoire of ectodermal appendage formation, and how specialized sensory structures form prior to interfacing with the sensory nervous system. Intriguingly, along with prior observations, these findings indicate that the distinct functions of Shh signaling in TD development and maintenance correspond to changes in the source of the Shh ligand required for the varied effects.En1Cre/+; β-cateninflox/Δ and control samples were provided by Dr. Radhika P. Atit. Some ShhGFPcre/+; R26YFP/+ samples were provided by Dr. Sunny Y. Wong. Edardl-J/+, Atoh1LacZ/+, ShhGFPcre/+, Gli1LacZ/+, Gli1CreER/+, K14-Cre, Shhflox/+, K5-tTA, TRE-cre, Smoflox/+, R26YFP/+, R26LacZ/+, and Fgf20LacZ/+ mice were described previously as cited in the text. For all embryonic and neonatal observations, littermate animals with a wildtype copy of the targeted allele and/or lacking cre recombinase activity were used as controls.Mice were housed and bred on an outcrossed Swiss Webster background in a pathogen-free facility at the National Cancer Institute (NCI), Bethesda, MD. Genotyping of mice was performed by allele-specific PCR on DNA extracted from tail tissue. All experiments were performed in accordance with institutional guidelines according to IACUC-approved protocols. K5-tTA; TRE-cre; Smoflox/flox mice were bred and maintained on a standard rodent diet without doxycycline during embryonic development. Tamoxifen (Sigma) was dissolved in corn oil (20mg/ml) and administered  to induce CreER.Skin was fixed in 4% paraformaldehyde for 15 minutes  or overnight (for immunostaining). Tissue was whole mount-stained or cryoprotected overnight in 30% sucrose, embedded in OCT, and frozen; 12-μm sections were obtained.Standard and whole mount immunostaining procedures were performed. Tissue sections on glass slides were fixed in 4% paraformaldehyde for 15 minutes before incubation in 10% serum in 0.1% PBT (0.1% Triton X-100 in PBS) for 1 hour and then in primary antibody (in 5% serum/0.1% PBT) overnight at 4°C. The primary antibodies used were: rat anti-K8 , rabbit anti-K17 , chicken anti-GFP , rabbit anti-GFP , rabbit anti-Sox2 , and rabbit anti-NCAM . Alexa Fluor-conjugated secondary antibodies  were used to detect the signals. Whole mount immunostaining followed the online protocol as described . ConcomiThe Troma-1/K8 antibody developed by Dr. Philippe Brulet and Dr. Rolf Kemler was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.2 numbers were assessed by direct visualization of immunofluorescently stained K8+ Merkel cells in whole mount dorsal trunk skin from experimental and control animals. Counting was performed by a blinded observer. In tissue sections, Merkel cells were visualized by K8 and/or Sox2 staining and were scored for co-staining with GFP. Reporter recombination in primary hair follicles was quantified by counting total GFP staining cells and dividing by total number of DAPI stained nuclei within a preselected region of each follicle.Merkel cells per touch dome and touch dome per mmTotal RNA from embryonic trunk skin was purified using RNeasy Micro Kit (Qiagen) and reverse-transcribed into cDNA following the manufacturer’s manual (Invitrogen #11752). Then quantitative RT-PCR, with actin as control, using SYBR Green was performed to detect RNA expression. Data are presented as means ± SD. The sequences of PCR primers are shown in Embryonic skin culture was performed as described . E13.5 ePopulation data sets are shown as the mean values, and error bars represent SD. For comparisons between sets, a two-tailed t-test was applied.S1 FigGli1LacZ/+ dorsal trunk skin at E17.5 and P0. (C) X-gal whole mount staining viewed from dermis side in Gli1LacZ/+ and ShhGFPcre/GFPcre; Gli1LacZ/+ skin at P0. X-gal section staining in (TIF)Click here for additional data file.S2 FigGli1CreER/+; R26YFP/+ skin at P0. Arrow, K8+ GFP+ Merkel cell. Outline, basement membrane.  Individual florescent channels for inset in B. (E) Quantification of GFP+ labeled cells in the primary follicle epithelium and Merkel cells in P0 dorsal trunk skin.(A) Schematic of the experimental design to fate map cells expressing Gli1 at E15.5. (B) GFP and K8 section staining in (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(A) Individual florescent channels for (TIF)Click here for additional data file.S5 FigAtoh1LacZ/+ skin at P0 and K8 and K17 section staining in wildtype skin at P0. (C) Confocal maximum projection oblique view of Sox2 and K8 whole mount staining in wildtype skin at P0. Arrows, touch dome in epidermis. Arrowheads, MCs in upper hair follicle. Scale bars, 50 μm.(A) En face image of Sox2 and K8 whole mount staining in wildtype P0 and P4 touch domes. (B) X-gal section staining in (TIF)Click here for additional data file.S6 FigFgf20LacZ/+) and Fgf20LacZ/LacZ skin at P50. Scale bar, 50 μm. (B) Quantification of TD density per mm2 and MC number per TD in control and Fgf20LacZ/LacZ dorsal trunk skin of adult (P50 –P103) mice.(A) K8 whole mount staining in control ((TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


In the perinatal as well as the adult CNS, the subventricular zone (SVZ) of the forebrain is the largest and most active source of neural stem cells (NSCs) that generates neurons and oligodendrocytes (OLs), the myelin forming cells of the CNS. Recent advances in the field are beginning to shed light regarding SVZ heterogeneity, with the existence of spatially segregated microdomains that are intrinsically biased to generate phenotypically distinct neuronal populations. Although most research has focused on this regionalization in the context of neurogenesis, newer findings underline that this also applies for the genesis of OLs under the control of specific patterning molecules. In this mini review, we discuss the origins as well as the mechanisms that induce and maintain SVZ regionalization. These come in the flavor of specific signaling ligands and subsequent initiation of transcriptional networks that provide a basis for subdividing the SVZ into distinct lineage-specific microdomains. We further emphasize canonical Wnts and FGF2 as essential signaling pathways for the regional genesis of OL progenitors from NSCs of the dorsal SVZ. This aspect of NSC biology, which has so far received little attention, may unveil new avenues for appropriately recruiting NSCs in demyelinating diseases. Adult CNS white matter consists largely of axons, astrocytes, NG2 glia, and OLs, that are all generated in sequential steps during late development. In the rodent forebrain, 3–4 weeks after birth oligodendrocytes (OLs) develop myelin sheaths , an embryonic form of NSCs] transforming into NSCs  generate all 3 major lineages, i.e., neuronal, astrocytic and oligodendroglial populations  promoters have collectively identified that SVZ microdomains are derived from their embryonic counterparts. For example, the medial ganglionic eminence, the lateral ganglionic eminence, and the embryonic cortex generate NSCs that populate the medial , lateral , and dorsal  aspects of the adult SVZ, respectively  during embryonic development contribute and specify oligodendrocytes progenitors (OPs), based on cells of the oligodendroglial lineage being detected in all regions of the forebrain (reviewed in Richardson et al., During postnatal life, signaling ligands are expressed by multiple sources and regulate NSC behaviors in both autocrine and paracrine manners. Expression of these ligands is observed in the various cell types forming the niche, which they also reach by the vasculature (Tavazoie et al., The initial dorsalizing trigger of the forebrain and subsequent inducer of oligodendrogenesis at birth, are derived from the choroid plexus which releases several canonical Wnt-ligands (Harrison-Uy and Pleasure, in vitro, other ligands such as IGF1 or PDGF upregulate major myelin-related genes via β-catenin activity, dependent of GSK3β signaling (Ye et al., In the postnatal forebrain, immediately following birth, relatively few specific lineage directive cues that boost the genesis of OPs from dNSCs have been identified. These, appear to ultimately converge onto activation of common TFs that are considered acting downstream of the Wnt-signaling machinery. As active Wnt canonical signaling is profusely detected in the dSVZ and absent in other SVZ microdomains Figure , few ligIn this review, the known mechanisms essential for inducing oligodendrogenesis have been discussed that altogether underline a strict spatial coding within segregated NSC populations of the postnatal dSVZ. Evidences for the existence of lineage specific microdomains in primates (Azim et al., KA: Main contributing author and wrote the article; BB and OR: Financial support, manuscript editing.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 reviewer FD and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


HTLV)‐1 causes T‐cell leukaemia and myelopathy. Together with HTLV‐2, it is endemic in some African nations. Seroprevalence data from Malawi are scarce, with no reports on associated disease incidence. HTLV seroprevalence and type were tested in 418 healthy mothers from Malawi. In addition, we tested the sera of 534 children to investigate mother‐to‐child transmission. To provide context, we conducted a systematic review and meta‐analysis of HTLV seroprevalence in African women and children.Human T‐lymphotropic virus  in mothers and 2.2% (12/534) in children. Three mothers carried HTLV‐1 alone, seven had HTLV‐2 and one was dually infected. Three children carried HTLV‐1 alone, seven had HTLV‐2 and two were dually infected. Only two corresponding mothers of the 12 HTLV‐positive children were HTLV positive. The systematic review included 66 studies of women and 13 of children conducted in 25 African countries. Seroprevalence of HTLV‐1 varied from 0 to 17% and of HTLV‐2 from 0 to 4%.HTLV‐2 was higher than that of HTLV‐1 in Malawi and one of the highest for the African region. The lack of mother–child concordance suggests alternative sources of infection among children. Our data and analyses contribute to HTLV prevalence mapping in Africa.In contrast to findings from other studies in Africa, the seroprevalence of  HTLV‐1 is the causative agent of aggressive adult T‐cell leukaemia/lymphoma (ATLL) and the progressive, chronic, disabling HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as other inflammatory conditions such as infective dermatitis and uveitis HTLV‐1, an oncogenic human RNA retrovirus, was discovered 35 years ago The African continent has a population over 1 billion and represents the largest endemic area for HTLV infection but with many data gaps. HTLV seroprevalence has been reported only once in Malawi, at 2.5%, when 159 blood donor sera were screened by ELISA, without typing or confirmatory testing This study investigated the prevalence of anti‐HTLV‐1 and HTLV‐2 antibodies in stored sera from a previous childhood cancer study. We had access to the sera of 534 children diagnosed with cancer in Blantyre, Malawi and 418 paired healthy mothers. ELISA positive tests were confirmed and typed by immunoblotting. In addition, we applied context to our results by conducting a systematic review and meta‐analysis of published HTLV seroprevalence data of African women and children.n = 534) and, where available, from their biological mothers (n = 418), were tested. Samples had been originally collected as part of a childhood malignancy and blood‐borne virus (BBV) study at the Queen Elizabeth Central Hospital in Blantyre, Malawi between 2006 and 2010 n = 174, maximum n = 276) or mothers , and data were not available for all data points. Results are, therefore, presented as % of data available.Anonymised stored serum samples from Malawian children (n = 18). DNA was isolated from frozen whole blood of serologically indeterminate samples and its integrity was confirmed by optimised PCR amplification of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) using commercially available primers. Samples were tested for the presence of HTLV‐1 DNA by PCR as previously described Replicating clinical diagnostic algorithms, the study protocol required that all ELISA reactive samples underwent immunoblot (IB) confirmatory testing. Only indeterminate IBs were tested further with polymerase chain reaction (PCR), where whole blood was available. All sera were screened in duplicate by ELISA  following the manufacturers' instructions . ELISA reactive or borderline reactive sera were tested and typed as HTLV‐1, HTLV‐2, HTLV‐1 and HTLV‐2, negative and indeterminate using an FDA approved, confirmatory, qualitative enzyme immunoblot assay (IB), (MP Diagnostics HTLV blot v2.4), which were interpreted using the HTLV European Research Network guidelines PRISMA guidelines for systematic reviews and meta‐analyses were adhered to during protocol design and analyses. The NCBI PubMed database was searched using the search terms ‘HTLV’, ‘human T‐lymphotropic virus’, ‘mother’, ‘child’, ‘transmission’ plus each respective African nation to 31 December 2014.The inclusion criteria were cross‐sectional seroprevalence studies; healthy women ; sex workers; children; living in Africa; opt‐in, opt‐out or anonymous un‐linked (blood tests/other body fluid); approved by ethics; screened for HTLV; and confirmed with a confirmatory test (CT), and studies with zero seroprevalence by ELISA were included Figure . The excApplying the above criteria at stage 1 all potential papers' titles and abstracts were screened, and at stage 2 duplicates and papers not fitting all criteria were removed  and seven HTLV‐2 (1.7%) with one (0.2%) mother being dually infected , had been born vaginally, had not been born prematurely, had been breastfed for >2 years. Only one child had received injections in the past but none had had a blood transfusion and only three had a history of malaria.At stage 1, 766 PubMed publications on HTLV in African countries were identified. At stage 2, all publications that met the outlined inclusion/exclusion criteria were collected. Specific effort was made to extract data on women and children only from the manuscripts. Data were available for 24 countries for women and eight for children and were published between 1988 and 2014 for women and between 1988 and 1999 for children. Thirty‐four studies had data for cohorts of healthy volunteers, three for blood donors, twenty‐two for antenatal care (ANC) attendees and fifteen for sex workers. Mean sample size for women was 609 (range 26–2070) and 562 for children (range 46–1323). Some manuscripts gave data on multiple subgroups of study population therefore additional data sets were created. In the first instance, analysis was conducted on 74 data sets for women  but deliberately refrained from calculating a prevalence estimate for the African continent Table . For womP = 0.3). Therefore, although they had a slightly higher risk of being positive for HTLV, this may have been due to chance variation; therefore, sex workers were not excluded from the analysis.Assuming that sex workers may have an unusually high exposure risk we compared them to other women using logistic regression with robust standard errors allowing for the clustering in studies and adjusting for region. The odds ratio of HTLV for sex workers compared to all other women was 1.34 , Nigeria 1.1% and 3.9%  To our knowledge, this is the first report on the seroprevalence and type of HTLV in Malawi combining screening with robust confirmatory testing. We had access to stored sera of children with childhood cancers and their mothers as their controls who had taken part in a previous study In this retrospective study, the mothers were of child‐bearing age with known multiple pregnancies but we can only make assumptions about their HTLV acquisition. They could have acquired these viruses through: MTCT from their own mothers; unprotected sex; contact with non‐human primates , use of unsterile instrumentation  or contaminated needles; unscreened blood products or other means not yet characterised. Bush‐meat hunting, heroin trafficking and FGM are prevalent in Malawi although not sufficiently surveyed The lack of concordance between mothers and their children remains unexplained; it might be an indication of horizontal transmission or the possibility that the child had been breastfed by someone other than their mother. The origin and route of transmission of the unusually high HTLV‐2 seroprevalence found in our study participants remains unknown and prospective study would allow verification and identification of infection source.n = 740); this region seems to have a low HTLV seroprevalence, although this should be verified with additional larger prevalence studies.Our systematic review of HTLV in women and children confirmed a distribution of HTLV in large areas of Africa . HTLV was not detected in healthy women in the three eligible studies from North Africa (et al., it is possible that HTLV‐2 was present in these blood donors.The prevalence of HTLV‐1 in many areas of the world is poorly mapped and incidence of other subtypes is even less well known. Africa is thought to be endemic for HTLV In our systematic review only two other African countries reported high rates of HTLV‐2 seroprevalence: Nigeria and Eritrea, about 2000 miles apart. HTLV‐2 was not detected in Mozambique or Zambia, neighbouring countries of Malawi (Table et al., identified an HTLV‐1 infected child, from a total of 916 children screened, whose mother had an indeterminate serological pattern and a negative HTLV PCR result et al., reported five HTLV‐1‐positive children of 476 children from a total of 460 mothers in their study; none of the mothers of the infected children had HTLV Three manuscripts in our systematic review addressed HTLV MTCT The strengths of this study were the large total and matched sample sizes, the combination of highly sensitive and specific screening and confirmation testing and the HTLV mapping of Malawi, an under surveyed country. The study has several limitations. We tested stored samples and, therefore, were unable to prospectively collect demographic and risk factor data specific to HTLV seroprevalence studies. None of the HTLV‐positive cases were HIV positive, which is interesting in itself considering that both share the same route of transmission. Samples were also a few years old which might have affected their reactivity. The cohort of children had cancer and antibody cross reactivity might have led to false positive results, although the highly specific confirmatory testing should negate this risk and HTLV infections were also found in the healthy women. Additional molecular testing of seropositive samples would have been ideal but we did not have access to the required DNA.In our systematic review, we included data only on healthy women to best match our cohort of women and reduce co‐infection/co‐morbidity bias. There were very few published reports of childhood HTLV and we excluded those who were known HIV positive. We believe data from the 1980s are valuable, although an overestimation of HTLV prevalence is possible. Furthermore, because African HTLV seroprevalence studies are limited in their number, we did not exclude any samples based on study size but calculated regional estimates rather than national estimates in an attempt to reduce dependence upon studies with small cohorts. Inherent bias from small studies and those from the 1980s was reduced by including only publications that had used serological confirmatory testing. We used appropriate study weighting based on cohort size in our analyses and regional seroprevalence estimates.In summary, our study highlights that HTLV is prevalent in Malawi. However, neither ATLL nor HAM/TSP has been reported so far. These HTLV‐1‐associated diseases are difficult to diagnose with high risk of mortality from ATLL before diagnosis has been made. They are, therefore, notoriously underreported unless specifically looked for. In addition, HTLV‐2 is not as pathogenic as HTLV‐1 and will be transmitted unknowingly from generation to generation.Prospective field studies of HTLV as well as its associated diseases are needed in Malawi to allow health policy makers to develop practical and sustainable health policies with the long‐term aim of eradicating HLTV.


An elevated human T cell lymphotropic virus 1 (HTLV)-1 proviral load (PVL) is the main risk factor for developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in HTLV-1 infected subjects, and a high cerebrospinal fluid (CSF) to peripheral blood mononuclear cell (PBMC) PVL ratio may be diagnostic of the condition. However, the standard method for quantification of HTLV-1 PVL—real-time PCR—has multiple limitations, including increased inter-assay variability in compartments with low cell numbers, such as CSF. Therefore, in this study, we evaluated a novel technique for HTVL-1 PVL quantification, digital droplet PCR (ddPCR). In ddPCR, PCR samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, and queried for fluorescent signal. Due to the high number of independent events (droplets), Poisson algorithms are used to determine absolute copy numbers independently of a standard curve, which enables highly precise quantitation. This assay has low intra-assay variability allowing for reliable PVL measurement in PBMC and CSF compartments of both asymptomatic carriers (AC) and HAM/TSP patients. It is also useful for HTLV-1-related clinical applications, such as longitudinal monitoring of PVL and identification of viral mutations within the region targeted by the primers and probe. Human T cell lymphotropic virus 1 (HTLV-1), an oncogenic human retrovirus, is the causative agent of adult-T cell leukemia (ATL) and a myelopathy termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain and Mahieux + and CD8+) and establishes a reservoir in the memory/effector and regulatory CD4+ T cells  , allows for the direct absolute quantification of a target gene in a given sample. A restriction enzyme is initially used to fragment the DNA in each sample. The PCR samples are partitioned into thousands of nanoliter-sized droplets and then subsequently amplified on a thermocycler. All droplets are then queried for fluorescent signal , droplets in the upper left quadrant are positive for HTLV-1 tax only (blue), droplets in the lower right quadrant are positive for RPP30 only (green), and droplets in the upper right quadrant are positive for both HTLV-1 tax and RPP30 (brown).Figure tax. Figure Figure Following the initial characterization of the assay as shown in Fig. p = 5.77 × 10−09; degrees of freedom = 2). When analyzed in pairs, the PBMC PVL differences between these patient populations and healthy donors were statistically significant .The median PBMC PVL for HAM/TSP patients was 13.5 % (range 3–44 %) compared to 2.3 % for AC (range 1–11 %), which is consistent with previous reports of higher PVL in HTLV-1 infected individuals with neurologic disease compared to AC , the values were normalized such that the assays could be compared.Though the goal of this study was to characterize a new assay for HTLV-1 PVL quantification, a comparison of ddPCR and quantitative real-time (qPCR) was performed, as qPCR is the most commonly used method of measuring HTLV-1 PVL. PBMC DNA from five HAM/TSP patients was run on two separate plates by both ddPCR and qPCR to directly compare the inter-assay variability of these two methods Fig. . The PVLR2 = 0.97)  Fig. , with si97) Fig. . Using t97) Fig. , suggestTo further characterize the precision and reliability of ddPCR for quantification over a wide range of HTLV-1 PVL, the intra-assay variability was evaluated. HTLV-1 PBMC PVL of eight infected individuals was measured by ddPCR in sets of ten replicates on one plate Fig. . Samplest test, p = 0.0036). This finding is in agreement with previous reports of an elevated CSF PVL to PBMC PVL ratio in HAM/TSP patients . This technique extrapolates from standard curves to quantify viral target and housekeeping genes and is limited by sensitivity and high inter-assay variability, particularly at the lower range of target input. Due to these limitations, a third-generation digital droplet PCR technology that allows for direct HTLV-1 PVL quantification was developed. In this study, we compared the precision and reproducibility of ddPCR to qPCR, demonstrated the utility of ddPCR for the detection of HTLV-1 in samples with low cell numbers (such as CSF), and identified a use for ddPCR for the identification of viral mutations in HAM/TSP patient samples.Digital droplet PCR was shown to be a highly precise and reproducible method of HTLV-1 PVL quantification. The coefficient of variation was low, less than 10 %, over a wide range of PVL. This favorably compares, if not exceeds, reliability of qPCR  compared to 25 HAM/TSP patients with characteristic fluorescence amplitudes for both viral gene targets , while the HTLV-1 gag target region . This study included 25 HAM/TSP patients, 4 HTLV-1AC, and 21 healthy volunteers  immediately after collection by lumbar puncture. The CSF supernatant was carefully removed, and the cell pellet was washed in PBS and stored at −80 °C until use. DNA was extracted from the PBMC and CSF cell pellets using DNeasy Blood and Tissue kit  according to the manufacturer’s instructions. NanoDrop 1000  was used to measure the DNA concentration and purity. The extracted DNA was then used for both ddPCR and qPCR HTLV-1 PVL quantifications.PBMC were isolated from whole blood using Ficoll-Hypaque  centrifugation . Following isolation, 3 × 10tax primers and FAM/MGB probe were designed for ddPCR using NCBI Primer Blast and Primer3Plus (Table tax (NCBI Gene ID 1491938). A temperature gradient experiment was performed to determine the optimal annealing temperature of 59 °C (data not shown). Additionally, in a duplex PCR, we amplified a cellular housekeeping gene, ribonuclease P protein subunit P30  , which was detected with a VIC/MGB probe  at one copy per cell (1 μg of TARL2 DNA contains 1.65 × 105 copies of the pX gene) and included five points with a 1:10 serial dilution . For the beta-actin standard curve, we used β-actin Detection Reagents , which included control human DNA and TaqMan primers and a FAM/TAMRA probe. The standard curve was derived from five points containing 30300, 3030, 303.3, 30.3, and 10 copies of beta-actin. The final concentrations in the qPCR reactions were 900 nM of each primer and 250 nM of the probe for HTLV-1 tax, and 100 nM of each primer and 66.6 nM of the probe for beta-actin.For HTLV-1 PVL quantification by qPCR, a different set of HTLV-1 ne Table  (Nagai ene Table . The qPCtax (or gag) and RPP30 primers and probes and Bio-Rad 2× Supermix, which was then emulsified with droplet generator oil  using a QX-100 droplet generator according to the manufacturer’s instructions. The droplets were then transferred to a 96-well reaction plate  and heat-sealed with pierceable sealing foil sheets . The duplex PCR amplification was performed in this sealed 96-well plate using a GeneAmp 9700 thermocycler  with the following cycling parameters: 10 min at 95 °C, 40 cycles consisting of a 30-s denaturation at 94 °C and a 60-s extension at 59 °C, followed by 10 min at 98 °C and a hold at 12 °C.DNA was digested with the restriction enzyme BamH1  for 30 min at 37 °C, diluted 1:5 with PCR-certified water. The digested diluted DNA was mixed with the HTLV-1 Following PCR amplification, the 96-well plate was transferred to a QX100 droplet reader . Each well was queried for fluorescence to determine the quantity of positive events (droplets), and the results were displayed as dot plots  and 2.5 μl of the primers/probe). Amplification and data acquisition were carried out in a Viia7 thermocycler . Thermal cycling conditions were as follows: hold at 50 °C for 2 min, a second hold at 95 °C for 10 min, 45 cycles of 95 °C for 15 s and 60 °C for 1 min, followed by a final hold at 25 °C for 2 min.For qPCR, the amplification of HTLV-1 For ddPCR analysis, QuantaSoft software version 1.3.2.0  was used to quantify the copies/μl of each queried target per well. Thresholds were determined manually for each experiment, according to the negative controls, which included a no template control and DNA from a healthy volunteer. Droplet positivity was determined by fluorescence intensity; only droplets above a minimum amplitude threshold were counted as positive. Negative controls were included in each experiment and resulted in less than two positive droplets. All samples were run in duplicate, unless otherwise specified, and the PVL is the average of the two measurements.tax and beta-actin. All samples were run in triplicate and the average quantity was used to calculate the normalized PVL.For qPCR analysis, Viia7 software version 1.2.1  was used to quantify HTLV-1 For both assays, the PVL was calculated as the percentage of infected cells using the following formula  was used per ROC analysis.Data normalization was accomplished by applying the log (base 2) transformation, calculating the mean and standard deviation (SD), and defining the lower (mean-2SD) and upper (mean + 2SD) values of the expected range for each assay type. These values were used to convert the log transformed values to a percentage of the expected range for each assay type, by subtracting the lower range value for the assay first then dividing by the difference of the upper and lower values of the expected range for the assay. Linear relationship of the post-normalized values was assessed using Pearson correlation. The “pROC” function supported in the statistical programming language R (


L. braziliensis or stimulated with LPS. Microbicidal activity of macrophages was determined by optic microscopy. Cytokines/chemokines from macrophage supernatants were measured by ELISA. HAM/TSP patients showed an increase frequency of intermediate monocytes, but expression of co-stimulatory molecules was similar between the groups. Macrophages from HTLV-1 infected individuals were infected with L. braziliensis at the same ratio than macrophages from HS, and all the groups had the same ability to kill Leishmania parasites. However, macrophages from HTLV-1 infected subjects produced more CXCL9 and CCL5, and less IL-10 than cells from HS. While there was no correlation between IFN-γ and cytokine/chemokine production by macrophages, there was a correlation between proviral load and TNF and CXCL10. These data showed a dissociation between the inflammatory response and microbicidal ability of macrophages from HTLV-1 infected subjects. While macrophages ability to kill an intracellular pathogen did not differ among HTLV-1 infected subjects, these cells secreted high amount of chemokines even in unstimulated cultures. Moreover the increasing inflammatory activity of macrophages was similar in HAM/TSP patients and HC and it was related to HTLV-1 proviral load rather than the high IFN-γ production observed in these subjects.The Human T lymphotropic virus type-1 (HTLV-1) infects predominantly T cells, inducing proliferation and lymphocyte activation. Additionally, HTLV-1 infected subjects are more susceptible to other infections caused by other intracellular agents. Monocytes/macrophages are important cells in the defense against intracellular pathogens. Our aims were to determine the frequency of monocytes subsets, expression of co-stimulatory molecules in these cells and to evaluate microbicidal ability and cytokine and chemokine production by macrophages from HTLV-1 infected subjects. Participants were 23 HTLV-1 carriers (HC), 22 HAM/TSP patients and 22 healthy subjects (HS) not infected with HTLV-1. The frequencies of monocyte subsets and expression of co-stimulatory molecules were determined by flow cytometry. Macrophages were infected with  Leishmania braziliensis is preserved, and these cells showed inflammatory profile, producing more CXCL9 and CCL5, and less IL-10 than macrophages from HS. It was important to determine if the exacerbated ability of macrophages to secrete cytokine was due to IFN-γ production. While there was no correlation between IFN-γ levels by PBMCs and cytokine/chemokine production by macrophages, there was a direct correlation between proviral load and TNF and CXCL10 levels. Our data indicate that despite the high production of proinflammatory mediators, macrophages from HTLV-1 infected subjects kill an intracellular pathogen in similar levels than cells from HS and pointed out for the role of viral factors inducing the inflammatory response in these cells.HTLV-1 predominantly infects T cells, inducing cell proliferation and activation. While there is a larger amount of studies regarding T cells functions in HTLV-1 infected subjects, little is known about innate immunity. We evaluated monocyte and macrophage functions in HTLV-1 infected subjects. We observed that HAM/TSP patients have an increased frequency of intermediate monocytes, but expression of co-stimulatory molecules in these cells was similar between HTLV-1 infected subjects and healthy subjects (HS). Additionally, the microbicidal ability of macrophages from HTLV-1 infected subjects to kill  Human T lymphotropic virus type 1 (HTLV-1) infects about 15 to 20 million people worldwide, with endemic foci in virtually all continents + T cells, but CD8+ T cells HTLV-1 infects predominantly CD4Mycobacterium tuberculosisburst oxidative activity), and by the decreasing in the number of neutrophils expressing CD62L and higher expression of CD66b It is well known that HTLV-1 infection increases susceptibility and severity to other infectious diseases Leishmania braziliensis. Moreover we evaluate if there are correlations between the frequency of monocytes subsets and cytokines/chemokines produced by macrophages with IFN-γ and proviral load in these individuals.These and others dysfunctions in the myeloid cell lineage may modify the immune response of HTLV-1 infected subjects to antigens. However, studies regarding the inflammatory response and microbicidal activity of monocytes and macrophages in HTLV-1 infected subjects have not been performed. The aims of the present study were to evaluate monocytes and macrophages functions in HTLV-1 infected subjects, by comparing the frequency of monocyte subsets in HTLV-1 infected subjects and the ability of monocyte-derived macrophages from HTLV-1 infected subjects to produce cytokines and chemokines and to kill the intracellular pathogen All HTLV-1 subjects have been followed at the HTLV-1 clinic of the Complexo Hospitalar Universitário Professor Edgard Santos (COM-HUPES), Federal University of Bahia, Brazil. The study was approved by the Ethics Committee from the Federal University of Bahia and all patients signed a document of informed consent.This is a cross-sectional study with the purpose of evaluating the role of myeloid lineage cells (monocytes and macrophages) from HTLV-1 infected subjects. Participants included 45 HTLV-1 infected subjects, being 23 HTLV-1 carriers (HC), 22 patients diagnosed with HAM/TSP and 22 individuals not infected with HTLV-1 constituted the healthy subjects group (HS). Pregnant woman and individuals in use of immunosupressing drugs were excluded. The diagnosis of HTLV-1 infection was established by antibody detection by ELISA  and confirmed by Western blot . Motor dysfunction and neurological involvement were determined by Osame's motor disability score (OMDS) 6 cells/mL were incubated without stimulus or stimulated with PHA (5 µg/mL) at 37°C in 5% CO2 for 72 hours and then the supernatant was frozen for later determination of IFN-γ; or c) use for the differentiation of cultured monocytes into macrophages, 5×106 cells/mL. These cells were added to 4-well plates  and incubated for 2 hours at 37°C and 5% CO2. Cells that did not adhere to the slides were removed by washing. The adherent cells (monocytes) were differentiated into macrophages after 6 days of culture at 37°C in 5% CO2, with absence of stimulus, in the presence of LPS or L. braziliensis.Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood of HTLV-1 infected subjects and healthy controls, and separated by density gradient with Ficoll-Hypaque . PBMCs from the interface were aspirated and washed with saline. After that, these cells were resuspended in RPMI 1640 culture medium with L-glutamine and 25 mM HEPES  supplemented with 10% fetal bovine serum (FBS) and 0.5% gentamicin at 10 mg/mL . PBMCs were then directed to three separate experiments: a) staining with specific antibodies for analyses of monocyte by flow cytometry; b) culture for determination of spontaneous production of IFN-γ by PBMCs. 3×10ex vivo frequency of monocyte subsets and expression of HLA-DR, CD80 and CD86 was determined using PBMCs from HC, HAM/TSP patients and HS. Cells were stained with monoclonal antibodies  for 20 minutes at 4°C. PBMCs were washed with PBS and then fixed with 2% paraformaldehyde. Cells were then analyzed on the flow cytometer . Analysis was performed using FlowJo software version 7.6 . The monocyte population was selected based on size and cell granularity and then subdivided into classical (CD14++CD16-), intermediate (CD14+CD16+) and non-classical monocytes (CD14+CD16++).The L. braziliensis isolated from a patient with cutaneous leishmaniasis from the endemic area of Corte de Pedra, Salvador, Bahia, is maintained, cryopreserved, by the Immunology Service. Parasites were initially cultivated in tubes with biphasic medium (NNN) supplemented with 10% fetal bovine serum and maintained in culture in Schneider medium  supplemented with 10% FBS and 1% penicillin streptomycin and glutamine  for expansion and proliferation of protozoa.A strain of L. braziliensis promastigotes were maintained in Schneider medium until reaching stationary (infectious) growth phase. Parasites were than centrifuged and resuspended in RPMI 1640 medium and used to infect macrophages cultured from both HTLV-1 infected subjects and HS. Unstimulated or macrophages stimulated with lipopolysaccharide (LPS) from Escherichia coli (100 ng/mL) were used as controls. Infection with L. braziliensis was performed at a ratio of 5 parasites to 1 cell for 2 hours at 37°C in 5% CO2. Following the incubation period, extracellular parasites were removed by washing, and then the cells were incubated at 37°C and 5% CO2. The percentage of macrophages infected with L. braziliensis and the number of amastigotes per 100 macrophages were evaluated by optical microscopy after 2, 48 and 72 hours of infection and staining with Giemsa. Counts were performed by two independent observers who were unaware if the slides were from an HTLV-1 infected subject or from a healthy control. The results expressed are the average of the results from both observers.L. braziliensis were not represented in the figures, because this pathogen did not induced the production of these molecules.Culture supernatants from unstimulated PBMCs or from macrophages were collected after 72 and 48 hours of incubation, respectively, and frozen at −20°C until used for determination of cytokines and chemokines. The IFN-γ levels (supernatants of PBMCs) and TNF, IL-10, CXCL9, CXCL10 and CCL5 (macrophages supernatants) were determined by ELISA, using commercial kits and following the manufacturer's instructions . Due to the limited amount of cells some experiments did not included all patients. Data about the production of cytokines and chemokines produced by macrophages after infection by 6 cells using proteinase K and salting-out method. The HTLV-1 proviral load was quantified using a real-time TaqMan PCR method 6 and expressed as the number of HTLV-1 copies/106 cells.DNA was extracted from 10L. braziliensis) compared to condition without stimulation. Spearman correlation test was used in the correlations results. Data were expressed as median and range . GraphPad Prism 5  was used to carry out the statistical evaluation and a P<0.05 was considered to indicate a significant difference.Mann-Whitney test was used to compare IFN-γ production by PBMCs and proviral load between ACs and HAM/TSP patients. Kruskal-Wallis followed by Dunn's post test was used to assess differences between the three groups studied under the same conditions. Wilcoxon test was used to evaluate the influence of stimuli , P = 0.03, and also a greater proviral load , P = 0,0004. Patients with HAM/TSP showed a median of OMDS of 6, ranging 3–9 , 23.9% of intermediate monocytes  and 5% of non-classical monocytes (P>0.05). The increasing in frequency of intermediate monocytes in HAM/TSP patients was not associated with IFN-γ production by PBMCs  in HC, HAM/TSP patients and HS were determined by flow cytometry and are shown in onocytes . There wby PBMCs .L. braziliensis at a 5∶1 ratio. The percentage of infected macrophages and the number of amastigotes/100 macrophages were evaluated by optic microscopy after 2, 48 and 72 hours of infection, as shown in L. braziliensis. Macrophages from HTLV-1 infected subjects (both HC and HAM/TSP patients) were initially infected by the parasite at the same proportion as macrophages from HS . Macrophages from HS produced 2,145 pg/mL while macrophages from HC produced 2,102 pg/mL, and macrophages from HAM/TSP produced 1,965 pg/mL, with no statistically significant differences between those three values  and HS macrophages cultured after 48 hours of incubation with or without LPS. These assays were performed by ELISA and data are shown in e values .P = 0.003) . Upon LPS stimulation, all groups showed increased IL-10 production, but macrophages from HS produced more of this cytokine (265 pg/mL) than macrophages from HC and HAM/TSP patients  .vs 6,992 pg/mL, P = 0.003). Macrophages from HC and HAM/TSP patients also produced more CXCL9 than HS's macrophages after LPS stimulus . Furthermore, stimulation with LPS did not induce the production of CXCL9 in macrophages from HS and HTLV-1 infected subjects  and HC after 48 hours of incubation, with or without LPS, as shown in subjects .P<0.03), this stimuli did not increased CXCL10 production by macrophages from HC and HAM/TSP patients  than macrophages from HS (255 pg/mL), After stimulus with LPS, we also did not observed differences between the production of this cytokine by macrophages from HS , HC  and HAM/TSP patients . However, while LPS increased the production of CXCL10 by macrophages from HS compared to the unstimulated condition . LPS was responsible to increase the production of CCL5 in all group studied (P<0.003), but we did not observe statistically significant differences between those groups   .P = 0.08), IL-10 (r = 0.09 and P = 0.74), CXCL9 (r = −0.02 and P = 0.90), CXCL10 (r = 0.41 and P = 0.09) and CCL5 (r = 0.20 and P = 0.44) in HTLV-1 infected patients.To evaluate if the spontaneous production of IFN-γ by PBMCs was associated with cytokines/chemokines produced by macrophages from HTLV-1 infected subjects, correlations were performed using Spearman correlation and r significance test. We found no correlation between spontaneous production of IFN-γ and production of TNF (r = 0.36 and P = 0.01) and CXCL10 (r = 0.63 e P = 0.05) (P = 0.26), CXCL9 (r = 0.26 and P = 0.24) and CCL5 (r = 0.35 and P = 0.21) production by macrophages from HTLV-1 infected individuals.Correlations between proviral load and cytokines/chemokines produced by macrophages were performed using Spearman correlation and r significance test. We observed a positive correlation, although weak, between proviral load and TNF (r = 0.51 and  = 0.05) . There w+ and CD8+ T cell proliferation The activity and phenotype of T cells in HTLV-1 infection have been well studied. These cells are characterized by the increased expression of proinflammatory cytokines, such as TNF and IFN-γ, and increased production of IL-2, which helps maintain CD4It is known that monocytes are a heterogeneous population of cells and can be classified, based on the expression of CD14 and CD16, as classical, intermediate or inflammatory and non-classical. Here we documented that HAM/TSP patients have a higher frequency of intermediate monocytes than HC and HS. It is known that intermediate monocytes are the main source of TNF among the three subpopulations M. tuberculosis, we sought to evaluate macrophage microbicidal function in HTLV-1 infection. To evaluate macrophage killing we used L. braziliensis, an intracellular pathogen knowing to interact with TLR2, TLR4 and TLR9 L. braziliensis was equal in the three groups. Moreover the leishmania killing, evaluated at 48 hours and 72 hours by quantifying the number of intracellular amastigotes in macrophages, was similar. This data extend our previous observations that the ability of neutrophils from HTLV-1 infected subjects to kill leishmania parasites is preserved Macrophages are capable of killing infectious agents but may also serve as habitat for intracellular pathogens. As HTLV-1 infection increases the susceptibility to infections caused by intracellular agents such as Macrophages activation has been used to indicate both, increasing ability of killing and secretion of molecules such as chemokines and cytokines. Macrophages are also a heterogeneous cells population and macrophage's subsets have been defined as classical macrophages that are associated with a type 1 immune response, and alternative macrophages that secret IL-4 and IL-10 It's known the ability of LPS to induce strong TNF production which is followed by IL-10 synthesis. The observation that HS's macrophages stimulated with LPS produced more IL-10 than cells from HTLV-1 infected subjects, both HC and HAM/TSP patients, suggests an impairment on macrophages of these individuals to secret this regulatory cytokine. Although the high production of proinflammatory mediators documented in HTLV-1 infection, especially in HAM/TSP patients, such as IFN-γ, IL-1, IL-6, could contribute to the increased production of chemokines and TNF by macrophages, we did not find a correlation between the IFN-γ production by PBMCs and TNF, IL-10, CXCL9, CXCL10 and CCL5 produced by macrophages. In contrast with the absence of correlation between IFN-γ and cytokines/chemokines levels, there was a direct correlation between proviral load and TNF and CXCL10 levels. This indicates that the HTLV-1 by itself or by inducing soluble mediators play a key role in the increased ability of macrophages to produce cytokine during HTLV-1 infection. Therefore it is important that future studies evaluate not only the role of viral proteins and other viral factors in activate innate immunity cells but also in disease expression associated to HTLV-1.It is already known that PBMCs from HAM/TSP patients produced more IFN-γ and have a higher proviral load compared to HC This is the first study to evaluate the monocyte subsets and activation, as well as the inflammatory and microbicidal activity from macrophages in HTLV-1 infection. Our data indicate that patients with HAM/TSP have an increase frequency of intermediate monocytes. We also observed that macrophages from HTLV-1 infected subjects have the same ability to phagocytize and kill an intracellular pathogen as healthy subject's macrophages, but that proinflammatory activity was enhanced in HTLV-1 infected subjects. The dissociation between microbicidal activity and production of proinflammatory cytokines and chemokines is a relevant subject and show that inflammation and killing may be independent functions. As IFN-γ is the main cytokine that activate macrophages we expected at some extension a relationship between this cytokine and the inflammatory profile observed in monocytes and macrophages. However the absence of correlation between IFN-γ and cytokine/chemokine production and a direct correlation between proviral load and TNF and CXCL10 produced by macrophages suggests innate immune cells triggered by viral factors may play an important role in the inflammatory response and in the pathogenesis of HAM/TSP.


Osteosarcoma is a rare type of cancer associated with a poor clinical outcome. Even though the pathologic characteristics of OS are well established, much remains to be understood, particularly at the molecular signaling level. The molecular mechanisms of osteosarcoma progression and metastases have not yet been fully elucidated and several evolutionary signaling pathways have been found to be linked with osteosarcoma pathogenesis, especially the hedgehog signaling (Hh) pathway. The present review will outline the importance and targeting the hedgehog signaling (Hh) pathway in osteosarcoma tumor biology. Available data also suggest that aberrant Hh signaling has pro-migratory effects and leads to the development of osteoblastic osteosarcoma. Activation of Hh signaling has been observed in osteosarcoma cell lines and also in primary human osteosarcoma specimens. Emerging data suggests that interference with Hh signal transduction by inhibitors may reduce osteosarcoma cell proliferation and tumor growth thereby preventing osteosarcomagenesis. From this perspective, we outline the current state of Hh pathway inhibitors in osteosarcoma. In summary, targeting Hh signaling by inhibitors promise to increase the efficacy of osteosarcoma treatment and improve patient outcome. Osteosarcoma (OS) is a highly malignant bone tumor which frequently leads to patient death due to pulmonary metastasis, in spite of conventional chemotherapy and surgical excision of the primary tumor. The survival rate is estimated to be around 60%–80% in patients treated with multidrug chemotherapy and local control interventions . Most coApart from treatment modalities like surgery, chemo- and radiation therapy there are no effective, alternative therapies for the treatment of malignant OS and as such, the development of novel therapeutic strategies is urgently required. Aberrant activation of Hedgehog (Hh) signaling contributes to tumor aggressiveness, affecting key tumorigenic processes such as proliferation, invasion and progression of cancer cells . Recentletc. [The Hh pathway acts as an organizer for embryonic development, patterning, growth control and mediates morphogenesis . In receetc. .The Hh sOf all the components of Hh signaling, GLI1 gene is a unique as it has no reported somatic mutations while other components of the pathway including PTCH, SMO, Suppressor of Fused SUFU, GLI2, and GLI3 have mutations ,20,21,22et al. identified SMO mutations in 50% of resistant BCCs and showed that these mutations maintain aberrant Hh signaling even in the presence of SMO inhibitors. In some of the cancers active SMO mutant proteins fail to co-localize with PTCH1 thereby allowing the activation of the pathway independently of Hh signaling [In Hh signaling, mutations and loss of heterozygosity (LOH) in PTCH1, with most of the mutations in SMO are reported . More thignaling . Severalignaling . A somatignaling . Therapeet al. reported that activation of Hh pathway varied among various OS cell lines analysed and did not correlate with the patient survival [et al. analysed Hh pathway genes in 43 human primary high-grade OS samples and determined that expression levels of genes encoding IHH, PTCH1 and GLI genes but not SMO were higher in tumour specimens [et al. analysed the transcripts of Hh genes in OS cell lines  and determined that SHh, DHh, PTCH1, SMO, GLI1 and GLI2 were overexpressed. However, only SMO, PTCH1, and GLI2 transcripts were over-expressed in human OS biopsy specimens [et al. showed that the high expression levels of GLI2 correlated with lung metastasis and has poor clinical outcome in mice [et al. demonstrated that GLI2 is involved in the migration, invasion and metastasis by regulating the cell cycle genes in vitro [et al. generated an osteoblastic OS mouse model and found that upregulated Hh signaling interacts with Yap1, the main effector of the Hippo tumor suppressor pathway and H19, a long non-coding RNA. Since YAP1 expression is also upregulated in human OS patient specimens, inhibition of Hh signaling and YAP1 together could present a significant therapeutic value [Several evolutionary signaling pathways, such as Hh, Notch, Wnt and BMP-TGF beta-activin are involved in the proper development of normal bone. It is also becoming increasingly clear that these pathways can have a crucial role in many types of cancer. Of those signaling pathways, most of the studies are now focused on Hh signaling in OS pathogenesis , renderisurvival . Howeverpecimens . Ligand-pecimens . Among t in mice  but therin vitro . The impin vitro . Treatmein vitro . Moreovein vitro . Silenciic value . More evIn recent years, Hh pathway drug discovery has been focused predominantly on targeting SMO, central transducer of the Hh signaling. SMO is widely considered as the most druggable target in the Hh signaling pathway. SMO inhibition alters transcription factors GLI1 and GLI2 to remain inactive, which prevents the expression of tumor mediating genes within the Hh pathway. A recent review on Hh inhibitors indicates the diversity of potential agents that are able to modulate the function of SMO. The chemical action of some of the small molecule Hh pathway inhibitors has been described in detail .et al. concluded that because of limited potency and remarkable side effects of cyclopamine in OS it was not suitable for clinical development [et al. showed that cyclopamine exhibited greater potent effect in inhibiting proliferation via cell cycle regulation in vitro. Cyclopamine decreased tumor growth in vivo but had side effects where mice experienced loss of body weight and dehydration and eventually died [The plant-derived compound cyclopamine first emerged as a potential drug that binds to SMO and inhibits signal transduction to the nuclear target gene GLI. Warzecha elopment . Also, tlly died . Moreovelly died . These rSaridegib is an oral, semi-synthetic derivative of the alkaloid cyclopamine and has demonstrated to exhibit potent anti-cancer activity in multiple preclinical animal models of cancer especially chondrosarcoma ,41. Theret al. showed that combination therapy of vismodegib, ATO (arsenic trioxide) and GANT61 (GLI inhibitor) at low concentrations prevented migration and metastasis of OS cells. Moreover, ATO and vismodegib significantly inhibited the metastasis of OS to the lung which suggested that the combined administration of ATO and vismodegib in xenografts might be effective for the treatment of OS metastasis [Vismodegib, a second generation cyclopamine, is a 2-arylpyridine molecule that blocks the activation of SMO. Considerable advantages over cyclopamine have been observed with better aqueous and acid stability. It is the first drug approved by the Food and Drug Administration (FDA) for the treatment of locally advanced basal cell carcinoma (BCC) that cannot be removed by either surgical resection or treated with radiation . There itastasis .Erismodegib, a SMO antagonist, induces G1 cell cycle arrest and apoptosis in many of the cancers . There iGLI transcription factors are critical mediators of the Hh signaling pathway and their aberrant activation might induce tumour cell proliferation and invasion. The Hedgehog-GLI signaling pathway is active in many cancers and is known to contribute to the growth and survival of human OS cells . The exp2O3) is an FDA-approved drug used for the treatment of patients with acute promyelocytic leukemia (APL) [et al. used ATO which was incorporated in the magnetic nanoparticles and encapsulated by poly lactic acid in vivo where MG63 OS cell lines were s.c injected into the mice. Tumour volume was significantly decreased in the nanoparticle encapsulated ATO treated group and the inhibitory effect was similar to those treated with ATO alone [et al. demonstrated that combined treatment of radiation and ATO resulted in the increased induction of autophagy and apoptosis via inhibition of the PI3K/Akt signaling pathway in OS cell lines [In vitro studies using ATO in OS cell lines showed it inhibited migration and invasion by inactivating the MAPK/ERK signaling pathway [ATO (Asia (APL) . ATO diria (APL) . Tumour ia (APL) . Li et aTO alone . Phase Ill lines . In vitr pathway . Taken tGLI activators Antagonists (GANT) which inhibits GLI-mediated transcription were discovered in a screen-based assay . Two GliIn this review, we have summarized the involvement of Hh signaling and its inhibitors for potential therapy in OS. The Hh signaling pathway might represent a valid therapeutic target in OS. Further, this pathway is activated in the progression of the disease and both ligand-dependent and independent inhibitors are effective. Hh signaling inhibitors are found to be effective in cancers like BCC and meduloblastoma where Hh signaling is constitutively activated and administration of Hh inhibitors as a single agent in those cancers are already used as first line therapy. Within the different classes of Hh inhibitors, recent drug development has focused on SMO inhibitors in controlling OS progression. Despite the fact that a lot of Hh pathway inhibitors have been developed, none of them has yet successfully reached the clinic for the treatment of OS. However, appropriate combination therapy with other signaling inhibitors might be required for clinical trials since the activated Hh pathway is not the primary driver of malignancy. Hh inhibitors have a potential to be a promising drug option, but there is still much to be learn about the biological effects of Hh pathway inhibition in order to explore the therapeutic potential of targeting the Hh signaling pathway in OS.


Mutations in hemojuvelin (HJV) are the most common cause of the juvenile-onset form of the iron overload disorder hereditary hemochromatosis. The discovery that HJV functions as a co-receptor for the bone morphogenetic protein (BMP) family of signaling molecules helped to identify this signaling pathway as a central regulator of the key iron hormone hepcidin in the control of systemic iron homeostasis. This review highlights recent work uncovering the mechanism of action of HJV and the BMP-SMAD signaling pathway in regulating hepcidin expression in the liver, as well as additional studies investigating possible extra-hepatic functions of HJV. This review also explores the interaction between HJV, the BMP-SMAD signaling pathway and other regulators of hepcidin expression in systemic iron balance. Juvenile Hemochromatosis (JH) is an autosomal recessive disorder caused by a failure to prevent excess iron entry into the bloodstream, and characterized by progressive tissue iron overload  or, more commonly, hemojuvelin  -linked membrane protein that is a member of the repulsive guidance molecule (RGM) family  signaling pathway  superfamily of ligands  on the hepcidin promoter  protein is detectable in human and rodent serum.  of endogenously produced sHJV HJV mRNA is also highly expressed in skeletal muscle and heart , that can act as feedback inhibitors of BMP-SMAD signaling and hepcidin expression   family member neogenin is also proposed to function as an HJV interacting protein that modifies BMP-SMAD signaling and iron homeostasis Figure . In partHepcidin suppression by erythropoietic drive appears to be mediated by secreted factor(s) released by proliferating red blood cell precursors in the bone marrow , epidermal growth factor (EGF), endoplasmic reticulum stress, gluconeogenic signals and the Ras/RAF and mTOR signaling pathways (Oliveira et al., Understanding the genetic basis for JH has yielded important insights into the molecular mechanisms of systemic iron homeostasis. Hepcidin and its receptor ferroportin are key regulators of body iron balance, and the BMP-SMAD pathway via the co-receptor HJV is a central regulator of hepcidin production Figure . KnowledJodie L. Babitt has ownership interest in a start-up company FerruMax Pharmaceuticals, which has licensed technology from the Massachusetts General Hospital based on the work cited here and in prior publications. All other authors declare the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HTLV-1 induces a persistent infection leading to an increased production of inflammatory cytokines. HIV, another retrovirus that causes systemic inflammation, is associated with atherosclerosis. However, few is known about HTLV-1-infection and atherosclerosis. To determine the prevalence and risk factors associated with atherosclerosis in patients infected with HTLV-1. a cross-sectional study involving 54 HTLV-1-infected patients (24 asymptomatic and 30 HAM/TSP), was carried out at the CHTLV between 2012 to 2013. Sociodemographic and cardiovascular risk factors were evaluated . Patients were submitted to Doppler echocardiography of both carotid and vertebral arteries to measure intimal-media layer. The association between intima-media thickness (IMT) and HAM/TSP diagnosis, HTLV-1 proviral load, low-density lipoprotrein cholesterol (LDL-c) or ultrasensitive C-reactive protein was determined. The mean age of patients was 57.1±12.3 years, 72.2% were women. Clinical atherosclerosis (IMT≥1.5mm) was found in nine patients (16.7%) and subclinical atherosclerosis (IMT>1 and <1.5) in 10 patients (18.5%). Atherosclerosis was more frequent in women (41%) compared with man (20%). Median age was significantly higher in patients with atherosclerosis . The mean of LDL-c level was 140.11±43.25 mg/dl (ranging from 49 to 287mg/dl). No significant differences were observed between the mean LDL-c level in individuals with subclinical (126.10±38.95mg/dl) and with atherosclerosis diagnosis (144.±43.87mg/dl), compared with individuals with normal IMT (143.21±44.65mg/dl), (p=0.4175). There was no correlation between HTLV-1 proviral load and IMT. No association was found between HTLV-1 infection and clinical or subclinical atherosclerosis as well as with the risk factors for cardiovascular diseases. A positive association between atherosclerosis and age was observed.