Protrusion and retraction of lamellipodia are common features of eukaryotic cell motility. As a cell migrates through its extracellular matrix (ECM), lamellipod growth increases cell-ECM contact area and enhances engagement of integrin receptors, locally amplifying ECM input to internal signaling cascades. In contrast, contraction of lamellipodia results in reduced integrin engagement that dampens the level of ECM-induced signaling. These changes in cell shape are both influenced by, and feed back onto ECM signaling. Motivated by experimental observations on melanoma cells lines (1205Lu and SBcl2) migrating on fibronectin (FN) coated topographic substrates (anisotropic post-density arrays), we probe this interplay between intracellular and ECM signaling. Experimentally, cells exhibited one of three lamellipodial dynamics: persistently polarized, random, or oscillatory, with competing lamellipodia oscillating out of phase . Pharmacological treatments, changes in FN density, and substrate topography all affected the fraction of cells exhibiting these behaviours. We use these observations as constraints to test a sequence of hypotheses for how intracellular (GTPase) and ECM signaling jointly regulate lamellipodial dynamics. The models encoding these hypotheses are predicated on mutually antagonistic Rac-Rho signaling, Rac-mediated protrusion (via activation of Arp2/3 actin nucleation) and Rho-mediated contraction (via ROCK phosphorylation of myosin light chain), which are coupled to ECM signaling that is modulated by protrusion/contraction. By testing each model against experimental observations, we identify how the signaling layers interact to generate the diverse range of cell behaviors, and how various molecular perturbations and changes in ECM signaling modulate the fraction of cells exhibiting each. We identify several factors that play distinct but critical roles in generating the observed dynamic: (1) competition between lamellipodia for shared pools of Rac and Rho, (2) activation of RhoA by ECM signaling, and (3) feedback from lamellipodial growth or contraction to cell-ECM contact area and therefore to the ECM signaling level. Cells crawling through tissues migrate inside a complex fibrous environment called the extracellular matrix (ECM), which provides signals regulating motility. Here we investigate one such well-known pathway, involving mutually antagonistic signalling molecules  that control the protrusion and contraction of the cell edges (lamellipodia). Invasive melanoma cells were observed migrating on surfaces with topography (array of posts), coated with adhesive molecules  by Park et al., 2017. Several distinct qualitative behaviors they observed included persistent polarity, oscillation between the cell front and back, and random dynamics. To gain insight into the link between intracellular and ECM signaling, we compared experimental observations to a sequence of mathematical models encoding distinct hypotheses. The successful model required several critical factors. (1) Competition of lamellipodia for limited pools of GTPases. (2) Protrusion / contraction of lamellipodia influence ECM signaling. (3) ECM-mediated activation of Rho. A model combining these elements explains all three cellular behaviors and correctly predicts the results of experimental perturbations. This study yields new insight into how the dynamic interactions between intracellular signaling and the cell’s environment influence cell behavior. Migrating cells display polarization of many membrane and cytosolic components, and spatially inhomogeneous signaling activity. Cellular polarity can be highly dynamic, displaying random, persistent or even oscillatory patterns –3 with cAggressive cancers, such as advanced stage melanoma and glioblastoma multiforme, frequently display persistent cell migration away from the primary tumor site. In the context of melanoma, the invasive tumor spread is associated with several mutations, including the loss of functional expression of PTEN, and the corresponding increase in the activity of the PI3K-AKT signaling pathway. It is not clear how such mutations, affecting the state of the signaling and regulatory networks controlling multiple cellular functions, could influence cellular polarity dynamics and the persistence of cell migration. Migrating cells also frequently relocate to micro-environments that are distinct from those of the tissue of origin. One of the key aspects of cellular micro-environment is the organization and composition of the extracellular matrix. Alteration in the density, orientation and nano-topography of the extracellular matrix fibers and their cleaved fragments have been shown to be instrumental in onset of cellular spread and in defining the direction and persistence of cellular migration –9. RecenIn this study, we focused on testing a set of alternative models against the experimental data obtained for melanoma cell lines of different degrees of invasiveness , 14. AdvRho GTPases are central regulators that control cell polarization and migration , 16, embThe extracellular matrix (ECM) is a jungle of fibrous and adhesive material that provides a scaffold in which cells migrate, mediating adhesion and traction forces. ECM also interacts with cell-surface integrin receptors, to trigger intracellular signaling cascades. Important branches of these pathways are transduced into activating or inhibiting signals to Rho GTPases. On one hand, ECM imparts signals to regulate cell shape and cell motility. On the other hand, the deformation of a cell affects its contact area with ECM, and hence the signals it receives. The concerted effect of this chemical symphony leads to complex cell behavior that can be difficult to untangle using intuition or verbal arguments alone. This motivates our study, in which mathematical modeling of GTPases and ECM signaling, combined with experimental observations is used to gain a better understanding of cell behavior, in the context of experimental data on melanoma cells.There remains the question of how to understand the interplay between genes (cell type), environment (ECM) and signaling . We and others , 21–27 hHow does signaling and cell motility interface with external inputs to the cell? How does the change in cell shape (in protrusion/contraction) affect inputs to the signaling network and thus cell behavior?Are diverse types of cell migration  part of the same overall repertoire, or are they distinct and discrete?How do constraints such as limited GTPase availability , 29–32, Can we understand the transition to invasive cancer cells as a shift in basic parameters of the same underlying signaling system?In experiments of Park et al.  melanomaIn , cell beHere, we focus on investigating how experimental manipulations influence the fraction of cells in different phenotypes. For simplicity, we focus on the polarized and oscillatory phenotypes which can be most clearly characterized mathematically. The following experimental observations are used to test and compare our distinct models of cell signaling dynamics.(O1)Rho is known to activate Rho Kinase (ROCK), which phosphorylates myosin light chain and leads to actomyosin contraction. Inhibiting ROCK is observed to increase the fraction of polarized and decrease the fraction of oscillatory cells.(O2)More invasive melanoma cell lines (1205Lu) are enriched in PI3K and low in the antagonist PTEN. These cells exhibit a lower fraction of random cells and higher fractions of persistently polarized and oscillatory cells than the less invasive melanoma cell line SBcl2.(O3)Increasing fibronectin level on the post density array surfaces increases the fraction of oscillatory and lowers the fraction of persistently polarized cells.For a graphical summary of cell phenotypes and experimental observations, see We use experimental observations (O1–3) as indirect constraints, to distinguish hypotheses for how feedbacks between internal cellular signaling and external ECM inputs modulate cellular behaviors. Toward this end, we construct a collection of simple, predictive models for Rac-Rho-ECM signaling, and compare model predictions to observations (O1–3) to determine which hypotheses are most consistent with experimental results. The activities of the small GTPases Rac and Rho are used as surrogates for the Rac1/PI3K and RhoA/ROCK pathways that respectively promote actin-based protrusion and acto-myosin based contraction in lamellipodia. Based on prior observations , 37, 38,To compare models to data, we consider three cell states: apolar, persistently polarized, and oscillatory . We inteA schematic diagram of the signaling model we discuss is shown in It is well known that mutual inhibition  can set up bistability and hysteresis. Furthermore, bistability coupled to slow negative feedback can lead to oscillations . This id50” levels . We also scale time by GTPase inactivation times. This scaling yields a smaller number of ratios of parameters to estimate, i.e. ratios that represent scaled basal and feedback-induced activation rates. For simplicity, we assume that parameters of the Rac and Rho equations are relatively similar.As in most models of intracellular signalling, obtaining biologically accurate values of rate parameters from direct biochemical measurement is unrealistic. Hence, we use the following strategy to parametrize our models. First, we scale the state variables in terms of their inherent “ICWe discuss three model variants, each composed of (A) a subsystem endowed with bistability, and (B) a subsystem responsible for negative feedback. In short, Model 1 assumes ECM competition for (A) and feedbacks mediated by GTPases for (B). In contrast, in Model 2 we assume GTPase dynamics for (A) and ECM mediated feedbacks for (B). Model 3 resembles Model 2, but further assumes limited total pool of each GTPase (conservation), which turns out to be a critical feature. See Tables We analyze each model variant as follows: first, we determine (bi/mono)stable regimes of subsystem (A) in isolation, using standard bifurcation methods. Next, we parameterize subsystem (B) so that its slow negative feedback generates oscillations when (A) and (B) are coupled in the model as a whole. For this to work, (B) has to force (A) to transition from one monostable steady state to the other (across the bistable regime) as shown in the relaxation loop of G = R, ρ represent concentrations of active Rac and Rho, respectively, GI = RI, ρI represent inactive GTPase levels, and k = 1, 2 (e.g. Rk) indicates the level in the k’th lamellipod. We assume the inactive GTPase pool is shared by competing lamellipods, but later incorporate different hypotheses about the size of this pool and the extent to which it is depleted. In some model variants, we assume that GTPases are abundant so that RI, ρI are constant, whereas in other variants, the total GTPase is limited and conservedRT and ρT are total average concentrations of the GTPases  the Rac-Rho system will be monostable, whereas for n > 1 and appropriate parameters, the Rac-Rho system is bistable.We generally assume that mutual antagonism between Rac and Rho influences the rates of activation rosstalk , 44.) TyEk to represent both the cell-ECM contact area of the k’th lamellipod  and the “effective level of ECM signaling” in the k’th lamellipod.  Then Rac-driven cell protrusion and Rho-driven cell contraction will affect Ek on some timescale Ej, Ek) cannot expand independently. Mechanical tension or other limited shared resources mean that one lamellipod could only grow at the expense of the other. For example, Tony Y.-C. Tsai and Julie Theriot observe that neutrophil-like HL-60 cells confined to “quasi-2D” motion under an agarose pad have a constant total projected area, for which protruding and contracting lamellipodia compete . Such observations could be modelled in many different ways. Here we chose the simplest appropriate “species competition” term to capture the main idea. This suggests that a reasonable model would belc describes the strength of direct lamellipodia competition (lc = 0 indicates no competition). We consider a combination of basal and self-enhanced components of the protrusion term P, as shown in Ek interchangeably as “ECM signaling level” and “lamellipod size” in our models, with the understanding that these two cell features are intimately linked.The extracellular matrix (ECM) provides input to GTPase signaling. However, the contact area between the cell and the ECM modulates that signaling since larger or smaller lamellipodia receive different levels of stimuli from the ECM. We define the variable δρ and GTPase levels by associated Hill function “IC50 parameters” k :ϵ, δ are ratios of timescales and bR, bρ are dimensionless. BE, AE, and LE represent basal protrusion, auto-amplified protrusion, and contraction strength terms, which can depend on Rac and Rho concentrations. Rather than nondimensionalizing Ek, we retain lc, the only parameter with units of E. This allows us to easily control the strength of coupling between the two lamellipods in our simulations. To represent the fact that ECM signaling influences Rho activation 11], it iAR so that its magnitude is dependent on ECM signaling (Ek). Doing so results in a shift in the borders of regimes we have indicated in In our models, we assumed that the dominant effect of ECM signaling input is to activate Rho, rather than Rac. In reality, both GTPases are likely activated to some extent in a cell and environment-dependent manner , 42. We We have argued that conservation laws  in the cell plays an important role in the competition between lamellipods. Such conservation laws are also found to be important in a number of other settings. Fully spatial (PDE) modeling of GTPase function has shown that conservation significantly alters signaling dynamics , 31, 54.These results provide interesting insights into the biology of invasive cancer cells (in melanoma in particular), and shed light onto the mechanisms underlying the extracellular matrix-induced polarization and migration of normal cells. First, they illustrate that diverse polarity and migration patterns can be captured within the same modeling framework, laying the foundation for a better understanding of seemingly unrelated and diverse behaviors previously reported. Second, our results present a mathematical and computational platform that distills the critical aspects and molecular regulators in a complex signaling cascade; this platform could be used to identify promising single molecule and molecular network targets for possible clinical intervention.S1 Text(PDF)Click here for additional data file.


Mice were divided into three groups: FND, ANTI (anti-inflammatory therapy) and NON (control). Five, 7, 10 and 20 days after cauterization, corneas were harvested and stained with LYVE-1, CD31 to quantify (lymph)angiogenesis. The long-term survival of allografts was compared between the three groups. FND caused significant regression of both blood and lymphatic vessels compared to the control group at all time points (p < 0.05) with the most obvious effect at day 7 (p < 0.01). Graft survival was significantly prolonged when transplants were placed into the FND pretreated group (p < 0.0001). The effect of the anti-inflammatory therapy alone was less effective compared to FND (p < 0.05). This novel lymphangioregressive effect of FND can be used clinically to precondition high-risk recipients to promote graft survival.Pathological corneal hem- and lymphangiogenesis are prime risk factors for corneal graft rejection. Fine needle-diathermy (FND) is an option to regress corneal blood vessels; however, whether this treatment besides clinically visible blood vessels also affects invisible lymphatic vessels is so far unknown. Here we test the hypothesis that FND destroys not only blood but also lymphatic vessels, thereby promotes corneal high-risk graft survival. The effect of FND was studied  The avascularity of the cornea is maintained amongst others by the expression of soluble forms of the three major vascular endothelial growth factor (VEGF) receptors and anti-angiogenic and anti-lymphangiogenic factors (for review see2). This status can be destroyed by the invasion of corneal neovascularization (CoNV) from the limbal vascular arcade into the cornea, caused by a wide variety of diseases3. CoNV can be helpful in the healing process to repair and regenerate some corneal insults, however, the presence of blood and lymphatic vessels after the healing process not only interferes with corneal clarity but also disrupts the “immune privileged” status of the cornea4. Efferent blood vessels as well as afferent lymphatic vessels associated with the regional lymph node are the three structural components of the immune reflex arc which have a major role in immune responses against foreign tissue, especially in graft rejection5. Recently, Dietrich et al. showed that lymphatic vessels are more important than blood vessels in immune rejection after corneal transplantation6. Therefore, it is important to find methods to block and regress both pathological corneal angiogenesis and lymphangiogenesis.The physiologically transparent and avascular cornea plays a crucial role in good vision by providing a proper anterior refractive surface and by allowing the entry of light7. Although its effect in suppressing blood and lymphatic vessels has been so far successfully tested11, the long-term use of topical steroid leading to serious side effects  has still been a limiting aspect. An antiangiogenic antisense oligonucleotide, GS101, has been shown to significantly inhibit corneal blood vessels in phase II and III trials13. Other treatments for corneal neovascularization include argon laser, photodynamic therapy and 577 mm yellow dye laser19. However, their variable and temporary effect, their lack of availability, high cost, inaccessibility to ophthalmologists and their complications require the need for a new simple, effective and safe alternative treatment. Recently, anti-VEGF therapy has been demonstrated to be effective in attenuating corneal angiogenesis and lymphangiogenesis21. Nevertheless, isolated anti-VEGFs seem to be ineffective in regressing established, mature vessels which depend less on angiogenic growth factors22.Many different techniques have already been suggested to regress corneal blood vessels. Topical corticosteroids were first used about 60 years ago to markedly reduce corneal vascularizationet al.23. CoNV was precisely occluded by using 10-0 monofilament black nylon suture and diathermy current applied to the needle being located in the stromal vessel. This approach has shown potential also e.g. in the context of high-risk keratoplasty patients23.The long-term efficacy and safety of this therapy were documented and demonstrated by several further researches29. FND is now widely used because of its effectiveness, safety, simple procedure, and inexpensive equipment which make it accessible to any eye unit throughout the world. However, whether this treatment besides clinically visible blood vessels also affects lymphatic vessels is so far unknown. Furthermore, formal trials testing its effect on corneal graft survival in the high-risk scenario are missing. Here we test the hypothesis that fine needle diathermy destroys not only visible blood but also invisible lymphatic vessels and thereby prolongs the survival grafts of high-risk corneal transplantation.Fine needle diathermy (FND) is currently one clinical choice for managing mature pathologic corneal blood vessels. This technique was first described and clinically utilized by Pillai To identify the effect of FND on mature pathologic corneal blood and lymphatic vessels and to determine the best time point of FND treatment, corneas of high-risk eyes after 14 days of suture placement were treated and then harvested subsequently five, 7, 10 and 20 days after FND. Quantitative analysis of the neovascularised corneal area identified by immunohistochemical staining revealed that both blood and lymphatic vessels were significantly reduced in the FND group compared to the NON group at all time points (p < 0.05). The most significant effect of FND on regression of both blood and lymphatic vessels was observable at day 7 after FND treatment compared to the NON and ANTIgroups  were applied topically to reduce the additional angiogenic stimulus caused by the FND procedure itself. Indeed, the results of the corneal area covered by LYVE-1 positive macrophages at day seven showed the similarity of cell infiltration between the ANTI and FND groups (4.23 ± 1.25 and 4.67 ± 2.76 respectively) angio-regressive effect of FND, we assessed whether the preoperative regression of blood and lymphatic vessels in high-risk recipient beds by using FND could improve the graft survival after subsequent transplantation. The long-term survival of C57BL/6 donor buttons transplanted into highly vascularized BALB/C recipient beds was compared between mice receiving an intrastromal FND and anti-inflammatory eye drops (FND group) and those receiving only anti-inflammatory drops (ANTI group) or those receiving no further treatment (NON group). We could demonstrate that high-risk mice pre-treated with FND and anti-inflammatory eye drops showed a significantly improved long-term graft survival at 8 weeks after transplantation (60.9%), compared to those in eyes of ANTI  and NON group  Fine needle diathermy, besides clinically visible blood vessels, also destroys clinically invisible lymphatic vessels.(ii)Since lymphatic vessels are clinically not visible, the approach of circular perilimbal FND as used herein nearly completely regresses lymphatic vessels in pathologically prevascularized high-risk eyes.(iii)Pretreatment of pathologically prevascularized high-risk eyes with FND and anti-inflammatory eye drops significantly improves graft survival after subsequent transplantation and opens a clinically realistic treatment option for improvement of graft survival in high-risk eyes.(iv)In fact, this to our knowledge is the first evidence that preconditioning of high-risk eyes by induced regression of pathologic corneal blood and lymphatic vessels in these eyes prior to transplantation promotes subsequent graft survival.The experiments presented herein allow four important conclusions to be drawn:30. Especially the regression of already established blood and lymphatic vessels is one of the major challenges for ophthalmologists. The fine needle diathermy technique, first described by Pillai et al.23, could be proven to be effective and safe in blood vessel regression by several studies thereafter26. However, whether this treatment besides clinically visible blood vessels also affects lymphatic vessels was so far unknown.Corneal neovascularization (CoNV) threatens corneal transparency, visual acuity and greatly increases the risk of graft rejectionin vivo. Since lymphatic vessels are not visible at the slit-lamp and seem to be the primary mediator of immune reactions in vascularized high-risk eyes, imaging clinically invisible corneal lymphatics is even more challenging and important than imaging corneal blood vessels. Here a great unmet need exists in the clinic to visualize human corneal lymphatic vessels not identifiable using slit-lamp magnification. To overcome this, in fact, we just presented two novel approaches to detect these lymphatic vessels. A novel, easy to use and low priced method to image corneal lymphatic vessels using commercially available fluorescein is to inject fluorescein dye into the corneal stroma and to image lymphatics using commercially available angiography devices otherwise used for retinal angiography (e.g. HRA)31. By using this novel method, we can principally selectively destroy corneal blood and/or lymphatic vessels and thereby decrease the amount of tissue destruction caused by fine needle diathermy. Furthermore, it was recently published that high-resolution OCT imaging allows for non-contact imaging of pathologic corneal lymphatic vessels thereby also enabling a more selective and tissue-sparing destruction of corneal lymphatic vessels in patients in the future32. However, in this study, we used the suture-induced corneal neovascularization assay in the murine model which caused strong circumferential neovascularization. Therefore, the circular perilimbal approach used in this study  is a realistic option to nearly completely regress all corneal lymphatic vessels. When used alone, FND can also be disadvantageous. It may, for example, induce the release of proangiogenic factors, especially when applied to multiple vessels23. Hence, in 2015 Spiteri et al. suggested the use of corneal angiography to identify and selectively close the afferent vessels and thereby minimize tissue destruction without altering the effectiveness of FND33. Indeed, Koenig27, Elbaz28 and Hussain29 proposed combined FND and topical or subconjunctival Bevacizumab (Avastin®) to compensate for the limitation of the other. In our experiments, we combined FND with anti-inflammatory eye drops (dexamethasone) to reduce the additional angiogenic stimulus caused by the FND procedure itself. Generally, the effect of topically instilled dexamethasone on hemangiogenesis and lymphangiogenesis is well known from clinical trial and previous experimental studies . In these studies, topical dexamethasone application resulted in the regression of angiogenesis by 57–93.1% and lymphangiogenesis by 55% at day seven. In our study, blood and lymphatic vessels were significantly suppressed by corticosteroid treatment by 30% at day ten and by 47% at day twenty respectively. The difference between these studies may be because of different animals studied, different neovascularization assays and different methods of morphometry of vascularized corneal areas applied. The best effect of combined treatment of FND with dexamethasone, by contrast, in our study was observed at day seven with the reduction of hemangiogenesis by 60% (p < 0.0001) and lymphangiogenesis by approximately 80% (p < 0.0001) compared to controls. In comparison with the group treated with only dexamethasone, the combined treated group had significantly stronger inhibitory effects on hem- and lymphangiogenesis in vivo (p < 0.01). These results suggest that fine needle diathermy combined with topical dexamethasone seems to be an effective treatment to occlude not only blood vessels but also clinical invisible lymphatic vessels.Our findings demonstrate for the first time the effect of FND on regression of lymphatic vessels in the cornea 35. Graft success rate, on the contrary, falls to 10% if the corneal recipient bed is inflamed and highly vascularized. In the mouse model, corneal allograft survival is reduced from 50% after six weeks in normal-risk transplantation to nearly 0% after two weeks in high-risk recipient bed37. Our experiments showed similar results of graft survival in the control (NON) group (8.3% after 8 weeks sustained follow-up). Due to the effectiveness of FND on the regression of both corneal blood and lymphatic vessels (both arms of an immune reflex arc), the long-term high-risk corneal graft survival was significantly improved compared to controls (p < 0.0001) and isolated anti-inflammatory therapy (p < 0.05). The graft survival in ANTI group was significantly higher than that in NON group at the end of the experiment (p < 0.05). Kim et al. also indicated that corticosteroid pretreatment is effective to improve graft survival in high-risk penetrating keratoplasty8. The difference between this and our study is that in study of Kim et al. corticosteroid was injected into subconjunctival space two weeks before penetrating keratoplasty and continued for four weeks after the surgery. We applied corticosteroids topically only for one week after FND and discontinued after the corneal transplantation to avoid as much as possible the side effects of long-term usage of corticosteroids.In patients, placement of grafts into an avascular and non-inflamed bed in the so-called “normal-risk” scenario carries a 2-year survival of approximately 90%2.Taken together, our study shows that circular fine needle cautery combined with anti-inflammatory therapy can effectively destroy not only corneal blood but also clinically invisible lymphatic vessels in high-risk eyes and thereby significantly improves graft survival after subsequent corneal transplantation. This concept of lymphangioregressive pretreatment in vascularized HIGH-RISK EYES can be translated into the clinic and should next be tested in a clinical trial31. For the corneal transplantation, six-to eight-week-old female C57BL/6 mice  were used as donors and same-aged female BALB/C mice as recipients. Prior to surgery, mice were deeply anesthetized with an intraperitoneal injection of a mixture of Ketanest (8 mg/kg) and Rompun (0.1 ml/kg).All experimental procedures were approved by the local animal care and use committee  and conformed to the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmology and Vision Research. Female BALB/C mice, purchased from Charles River Laboratories, Sulzfeld, Germany (aged 6–8 weeks) were used in the mouse model of suture-induced corneal inflammation and neovascularization assay39. Three 11-0 nylon sutures  were placed intrastromally on the right eye with two stromal incursions extending over 120 degrees of corneal circumference each. The outer point of suture placement was chosen near the limbus and the inner suture point was placed near the center of cornea equidistant from the limbus to obtain standardized angiogenic responses. Sutures were left in place for 14 days and were then removed. After suture removal, study mice were divided into three groups: FND, ANTI and NON (5 mice per group) and complied with the protocols for each group mentioned below.The mouse model of suture-induced inflammatory neovascularization was performed as previously describedFND was performed after removal of the suture under topical anesthesia with Conjuncain®EDO® , by using non-alternating current from a unipolar diathermy unit also used clinically . A stainless steel 3/8 circle round-bodied, single-armed needle attached to an 11-0 nylon suture  was used with a microsurgical needle holder . The needle was inserted intrastromally close to and parallel to the corneal limbus. The power was set to 1 W and in the soft-coagulating mode, the fine angled needle  connected to the electrode handle, was brought into contact with the shaft of the surgical needle. The contact was maintained until mild blanching of the corneal stroma and/or corneal shrinkage occured  as topical drops with the same dosage and regimen compared with FND group, while the NON group did not obtain any anti-inflammatory treatment or FND after suture removal.To determine the best time point of the effect of the FND treatment on regression of both corneal blood and lymphatic vessels, mice were euthanized and corneas were harvested five, 7, 10 and 20 days post-treatment. Whole-mount double-immunohistochemistry using CD31 and LYVE-1 was performed as described below.41. First, a button of the donor cornea of C57BL/6 was excised by marking the central 2 mm of the cornea using a 2.0 mm bore and cutting with curved Vannas scissors. The recipient BALB/C mouse was anesthetized, and the graft bed was prepared on the right eye by cutting out a circular 1.8 mm central area of the cornea. Subsequently, the graft was secured to the host corneal tissue with eight equidistant interrupted 11-0 nylon sutures . Antibiotic ointment  was applied on the transplant and eyelids were sutured with an 8-0 suture  to avoid damaging the graft. Tarsorrhaphy and corneal sutures were removed after seven days, and grafts were examined once a week until week eight after transplantation. The opacity of the grafts was evaluated and scored as previously described40. Clinical scores of corneal grafts for opacity were as follows: 0, clear; +1, minimal, superficial  opacity; pupil margin and iris vessels readily visible through the cornea; +2, minimal, deep  opacity; pupil margins and iris vessels visible; +3, moderate stromal opacity; only pupil margin visible; +4, intense stromal opacity; only a portion of pupil margin visible; and +5, maximum stromal opacity; anterior chamber not visible. Grafts with opacity scores greater than +2 were considered to be rejected. The survival experiment comprised three groups with 12 mice per group. All grafted eyes with infection, abnormal graft and loss of anterior chamber were excluded from further consideration.Corneal allografting in the mouse model was performed as previously reported44. Excised corneas were rinsed two times in PBS and then fixed in acetone for 30 minutes. After washing in PBS three times, and blocking with 2% bovine serum albumin (BSA) in PBS, the corneas were stained overnight  with rabbit anti-mouse LYVE-1  for lymphatic vessels and with a FITC-conjugated rat anti-mouse CD31 antibody  for blood vessels. On the next day, LYVE-1 was detected by using a goat-anti-rabbit Cy3-conjugated secondary antibody . Double stained wholemount images were assembled automatically from nine to 12 images taken at x100 magnification with a fluorescence microscope . Afterwards, the areas covered with blood and lymphatic vessels were detected with an algorithm established in the image analyzing program Cell^F , as previously described39. Briefly, before analysis, gray value images of the whole mount images were modified successively by four software-based filters. Afterwards, the total corneal area was outlined by using the innermost vessel of the limbal arcade as the border , and the area covered by vessels defined by threshold setting was calculated (in mm2). The mean vascularized area of corneas in the NON group was defined as 100%, and the corneal vascularized areas in the other groups were then set into relation to this group. The area covered by LYVE 1 (+) cell was also quantified at day seven by using program Cell^F.The blood and lymphatic vessels in corneal wholemounts were double stained as described previouslyStatistical analyses were performed with Microsoft Excel 2010  and Prism 6 version 6.07 . Statistical significance was determined using the one-way ANOVA test. P < 0.05 was considered statistically significant. Postoperative survival of the corneal allografts was analyzed using Kaplan–Meier survival curves with Logrank test. Graphs were drawn using Prism6.The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.Supplementary figure


Human T cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia/lymphoma (ATL), HTLV-1 associated myelopathy (HAM/TSP), and of a number of inflammatory diseases with an estimated 10–20 million infected individuals worldwide. Despite a number of therapeutic approaches, a cure for ATL is still in its infancy. Conventional chemotherapy has short-term efficacy, particularly in the acute subtype. Allogeneic stem cell transplantation offers long-term disease control to around one third of transplanted patients, but few can reach to transplant. This prompted, over the past recent years, the conduction of a number of clinical trials using novel treatments. Meanwhile, new data have been accumulated on biological and molecular bases of HTLV-1 transforming and infecting activity. These data offer new rational for targeted therapies of ATL. Taking into account the double-face of ATL as an hematologic malignancy as well as a viral infectious disease, this Mini-Review seeks to provide an up-to-date overview of recent efforts in the understanding of the mechanisms involved in already used therapeutic regimens showing promising results, and in selecting novel drug targets for ATL. As a consequence, ATL patients are often refractory to intensive, conventional chemotherapy regimens. Classically used regimen are CHOP, CHOEP dose-adjusted EPOCH, and hyper-CVAD, alternating with high-dose methotrexate and cytarabine . However, an important hurdle emerged in practically all the completed studies, is the limited duration of the response. Allogeneic stem transplantation could represent an alternative, potentially curative approach (Human T cell leukemia virus type 1 (HTLV-1) is the first identified human retrovirus endemic in southwestern Japan, the Caribbean islands, inter-tropical Africa, South America, Romania and the Middle East, with an estimated 10–20 million infected individuals worldwide. HTLV-1 is known to cause adult T cell leukemia/lymphoma (ATL), HTLV-1 associated myelopathy (HAM/TSP), and a number of inflammatory diseases. Firstly detected and isolated from a cutaneous T cell lymphoma almost 40 years ago, HTLV-1 still represents a significant challenge for the scientific community engaged to disclose its oncogenic potential and to identify a focused therapy . HTLV-1 tarabine . An intetarabine . In additarabine , led to tarabine , alemtuztarabine , or dacltarabine  have beeapproach . Unfortuapproach , and thiin vivo, i.e., the mitotic and the RT-dependent ones, that might require a combined approach targeting both chronically infected host cells and direct viral replication. Actually, in the past recent years a lot of data have been accumulated on biological and molecular bases of HTLV-1 transforming as well as infecting activity. These data offer new rational for targeted therapies of ATL. Thus, taking into account the double-face of ATL as an hematologic malignancy as well as a viral infectious disease, this mini-review seeks to specifically focus on providing an up-to-date overview of recent efforts in: (i) understanding mechanisms involved in already used therapeutic regimens showing promising results, (ii) identifying and selecting novel drug targets for ATL.What makes the design of therapeutic approaches for ATL problematic is the double routes of HTLV-1 transmission Figure 1). The Tax protein has been demonstrated to trigger a number of immortalization/transformation related events in the early phase after infection, such as activation of the interaction with cAMP-responsive element-binding protein/activating transcription factor (CREB/ATF), the activation of NF-κB transcription factor, inhibition of p53, dysregulation of the cell cycle by interfering with the cellular checkpoint, impairment of cellular DNA repair mechanisms resulting in genetic instability, induction of DNA damage through production of reactive oxygen species, induction of both pro-apoptotic and anti-apoptotic activities , an orally bio-available mimetic of the Bcl-2 homology domain 3 small molecule, as compared to non-HTLV-1-associated leukemic cells  and paracrine (IL-9) loops, able to drive ex vivo spontaneous proliferation of ATL cells at an early stage (γc receptor whose expression is regulated by a family of kinase (JAK/STAT). Interestingly, JAK/STAT selective inhibitors suppressed the proliferation of smoldering/chronic ATL cells ex vivo . This could occur through direct cell-to-cell communication or paracrine stimulation through secreted Tax or various cytokines/chemokines as reported for chronic lymphocytic leukemia  showing an unexpected high response rate, particularly in previously untreated acute ATL patients , 1998. Aed cells . Neverth respond . Thus, tleukemia . Moreovehibition . Thus, ain vitro studies, AZT and tenofovir inhibit virus transmission to PBMC in short-term cultures and interfere with immortalization in the long run at a drug concentration which was poorly toxic toward uninfected cells . Tax is known to remarkably affect the host cell proliferation by directly intervening in the processes regulating DNA transcription, replication and repair. On the basis of this regulatory role of Tax in HTLV-1 transformation, a few druggable targets have been demonstrated in preclinical studies. CDK9 is a component of a transcription factor, P-TEFb, essential for transcription of most MHC class II genes and for the transcriptional elongation by phosphorylating the C-terminal domain of RNA polymerase II. The importance of CDK9 for a targeted therapy was demonstrated in advanced stage of chronic lymphocytic leukemia and multiple myeloma (ex vivo, and to decrease MCL-1 and c-Myc expression levels. In addition, BAY 1143572 decreased ATL cell migration in the liver and bone marrow in a model of ATL in vivo xenograft, in immunocompromised NOG mice (in vitro effect of deacetylase inhibitor, valproate, on ATL cells was owed to the rescue of the pre-miRNA maturation pathway (Further targets are being investigated to find new therapeutic approaches in ATL ( myeloma . An addi myeloma . In factNOG mice . Tax wasNOG mice . HelicasNOG mice . This fiNOG mice . Tax wasNOG mice . Thus, tNOG mice . The inhNOG mice , showing pathway .Theoretically, ATL cells exhibit a number of potential, different viral and cellular pharmacological targets. A number of studies suggests to take under consideration the suitability of numerous, known drugs to counteract ATL. However, it is hard to explain why, despite broad chances of potentially druggable targets, success is still limited in ATL clinical studies. Possible reasons could reside in the long latency of HTLV-1 infection, allowing the virus to escape host response, as well as the lack of suitable markers of disease progression. Hopefully, more deep knowledge of how the virus affects the regulation of host immune response and the metabolic requirements of transformed cells could represent new issues for future challenges in ATL therapy.FM-M searched for literature, wrote the paper, and performed the elaboration and graphical representation of the figures. AM, AB, and BM searched for literature and wrote the paper. SG searched for literature. OH revised the paper.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.


Deltaretrovírus that was first isolated in the 1970s, and associated with Adult T-cell Leucemia-Lymphoma (ATLL), and subsequently to Tropical Spastic Paraparesis-Myelopathy (TSP/HAM). The genetic diversity of the virus varies among geographic regions, although its mutation rate is very low (approximately 1% per thousand years) in comparison with other viruses. The present study determined the genetic diversity of HTLV-1 in the metropolitan region of Belém, in northern Brazil. Blood samples were obtained from patients at the UFPA Tropical Medicine Nucleus between January 2010 and December 2013. The DNA was extracted and the PX region of the HTLV was amplified using nested PCR. The positive samples were then digested using the Taq1 enzyme for the identification and differentiation of the HTLV-1 and HTLV-2. The 5’LTR region of the positive HTLV-1 samples were amplified by nested PCR, and then sequenced genetically. The phylogenetic analysis of the samples was based on the maximum likelihood method and the evolutionary profile was analyzed by the Bayesian approach. Overall, 78 samples tested positive for HTLV-1, and 44 were analyzed here. The aA  subtype was recorded in all the samples. The following evolutionary rates were recorded for the different subtypes–a: 2.10−3, b: 2.69. 10−2, c: 6.23. 10−2, d: 3.08. 10−2, e: 6. 10−2, f: 1.78. 10−3, g: 2.2. 10−2 mutations per site per year. The positive HTLV-1 samples tested in the present study were characterized by their low genetic diversity and high degree of stability.The Human T-cell Lymphotropic Virus (HTLV-1) is a  Deltaretrovirus that was first isolated from a blood sample taken in the 1970s from an African-American patient with a cutaneous T-cell lymphoma [The Human T-cell Lymphotropic Virus HTLV-1) is a  is a Dellymphoma .It has been estimated that, worldwide, 15 to 20 million are infected with HTLV-1 . The virIn Brazil, HTLV-1 was first detected in Japanese immigrants living in Campo Grande, in the state of Mato Grosso do Sul, in 1986 , and mosDiseases associated with HTLV-1 infection have been reported from a number of Brazilian regions . In BahiThe HTLV-1 virus replicates slowly, and studies are under way for the development of a vaccine . Few datAs this virus is associated with highly deleterious diseases and is endemic to the Amazon region, one major priority is to investigate its epidemiology and genetic variability in the metropolitan region of the city of Belém, the capital of Pará state, and the largest urban center in northern Brazil , 13, 14.The present study was transversal and retrospective.The study was based on the analysis of samples taken from seropositive patients for HTLV-1/2, as confirmed molecular procedures (PCR) for HTLV-1. These patients were treated at the Tropical Medicine Nucleus at the Federal University of Pará (UFPA), in Belém, Brazil, between January 2010 and December 2013.The study included patients of both sexes and all ages treated at the UFPA Tropical Medicine Nucleus during the study period. The study prioritized patients that agreed to participate in the research and had signed a term of informed consent.All samples considered inadequate for analysis were excluded from the study.The present study was approved by the Committee for Ethics in Research of the UFPA Tropical Medicine Nucleus, under protocol CAAE 31014114.2.0000.5172. All the norms and regulations of the UFPA were followed rigorously.Aliquots (5 mL) of blood were collected in tubes containing EDTA, with the leukocytes being separated from the plasma  and stored at -20°C until analysis. The proviral DNA was extracted from the layer of leukocyte cells following centrifugation of the total blood, using a Wizard®Genomic DNA Purification kit  according to the maker’s instructions.5’ GAAGAGCCAAGGACAGGTAC-3’) and G74 (5’-CAACTTCATCCACGTTCACC-3’) [The human β globin was amplified to evaluate the integrity of the genetic material extracted. The amplification was based on the oligonucleotides G73 (CACC-3’) .5'-TTCCCAGGGTTTGGACGAAG-3'  and HTLV_External R 5'-GGGTAAG GACCTTGAGGGTC-3' –and 1.0 μL of the DNA, for a final volume of 10 μL.Once the proviral DNA was amplified, a nested PCR was run, followed by enzymatic digestion for the confirmation of the HTLV infection and the differentiation of the virus types 1 and 2. For this, the pX region of the virus was amplified. This initial reaction was run in a solution containing 5.0 de Go Taq Green Master Mix , 2.0 μL of water, 1 μL (10 pmol) of each primer–HTLV_External F 5'CGGATACCCAGTCTACGTGTT3'  and HTLV_internal R 5'GAGCCGATAACGCGTCCATCG3' , 2.5 μL of water and 0.5 μL of the product of the first PCR, producing a fragment of 159 bps [In the second phase of the nested PCR, the same quantity of Go Taq Green Master Mix  was used, together with 1μL (10 pmol) of the primers HTLV_internal F  159 bps .Positive (a sample known to be infected) and negative controls were used for each PCR reaction. The products were electrophoresed (60 minutes at 100 V) in 2% agarose gel, stained with ethidium bromide in 1x TAE buffer , and visualized using an ultraviolet transilluminator.2O, 1.5 μL of buffer , and 0.5 μL of the TaqI 10 U/μL restriction enzyme (Promega), which was incubated for 2 hours at 65°C.To identify the HTLV type (1 or 2), the positive cases identified by PCR were processed by enzymatic digestion for the identification of RFLPs (Random Fragment Length Polymorphism). The RFLP reaction of the product of the pX gene (159 bps) was conducted in a mixture of 6.0 μL of the amplified product, 7 μL of HThe digestion of the restriction site (T/CGA) generates three fragments of 85 bps, 53 bps, and 21 bps in HTLV-2, and two (138 bps and 21 bps) in HTLV-1. The products of the digestion were electrophoresed in 3% agarose gel with 1x TAE buffer (as above) for visualization using an ultraviolet transilluminator.2, 50 mM of KCI, 10 mM Tris-HCI pH 8.3, and 0.1 U of Taq DNA polymerase. The primers used to amplify the HTLV-1 were (LTR-I.01) 5'-TGACAATGACCATGAGCCCCAA-3' and (LTR-I.02) 5'¬CGCGGAATAGGGCTAGCGCT-3', corresponding to nucleotides 1–22 and 823–842 of the ATK HTLV-1 strain [The 5'LTR region of the HTLV-1 was amplified in a total volume of 25 μL, containing 500 ng of the extracted DNA, 125 μM of each dNTP, 10 pmol/μL of each primer, 3.0 μM of MgCl1 strain .5'-GGCTTAGAGCCTCCCAGTGA-3'/ (LTR-I.04) and 5'-GCCTAGGGAATAAAGGGGCG-3', corresponding to nucleotides 30–49 and 781–800 of the HTLV-1ATK strain [In the second step of the nested PCR, 2.0 μL of the product of the first step (see above) was analyzed using primers internal to the region amplified previously. The primers were (LTR-I.03) K strain . The ampA Big Dye® terminator Cycle Sequencing kit  was used for the sequencing reaction. The reaction mixes had a final volume of 10 μL, composed of 2 μL de Big Dye, 1 μL of buffer, 2 μL of the DNA (50 ng), and 5 pmol/μL de each primer (as in the second step of the PCR of the ‘5 LTR), and 3 μL of deionized water (when necessary) in individual reactions. This mixture was processed in an automatic thermocycler , with an initial cycle of 2 minutes at 94°C, followed by 25 cycles of 45 seconds at 94°C, 30 seconds at 50°C, and 4 minutes at 60°C.et al., 1977 [Following the purification of the PCR product (5 'LTR region), the DNA was sequenced by the Sanger l., 1977  method ohttp://www.ncbi.nlm.nih.gov), using MAFFT, v. 7.The nucleotide sequences were analyzed and edited in GENEIOUS 4.8.5, and aligned with sequences of other viruses available in GenBank  of these samples were obtained from female patients, with only 23 (29.5%) from male patients. The mean age of these patients was 44 years old. It was possible to sequence the 5’LTR region of 44 of these 78 samples. All the samples presented the HTLV-1 aA  genotype see .3, b: 2.69. 10−2, c: 6.23. 10−2, d: 3.08. 10−2, e: 6. 10−2, f: 1.78. 10−3, g: 2.2. 10−2 mutations per site per year (The genetic diversity of the 44 sequences obtained in the present study (GenBank access numbers MF384328—MF384371) was compared with that of 200 HTLV-1 samples representing the seven different HTLV-1 subtypes. A total of 300 million comparative trees were generated, revealing the following rates of evolution for the different subtypes–a: 2.10−per year .Only 50 of the 78 patients that tested positive for HTLV-1 were monitored subsequently in the outpatients ward of the UFPA Tropical Medicine Nucleus. Of these patients, 16 (32%) presented symptoms of TSP/HAM, while the other 34 (68%) were asymptomatic.The HTLV-1 virus has a worldwide distribution, with considerable variation among regions in the prevalence of infection. A number of studies have highlighted the importance of historic migrations and miscegenation for the dispersal of this virus, which now infects approximately 20 million people worldwide. The present study confirmed that the aA HTLV-1 genotype is found in the metropolitan region of Belém, which is consistent with the evidence from other regions of Brazil and Latin America.The aA subtype is one of the most common HTLV-1 genotypes, especially in populations that have a long history of migration, as in northern Brazil. This may at least partly account for the high frequency of this genotype recorded in the metropolitan region of Belém, given that a similar profile of infection has been recorded in other, comparable urban environments in developing countries, in particular in Latin America , 4.In the present study, 32% of the patients were symptomatic, a relatively high rate in comparison with previous studies. This may be linked directly to the sample population, given that the outpatients department of the UFPA Tropical Medicine Nucleus attends many blood donors with positive serology for HTLV and other individuals with symptoms indicative of infection by this virus, but with unknown etiology. One other possible factor is the relatively high mean age of the infected individuals (44 years), which may contribute to the manifestation of the infection after years of latency, in addition to the immunological characteristics of the patient.All the sample that were positive for HTLV-1 were included in the subtype aA clade in the phylogenetic tree, together with samples from Colombia, Spain, Russia, Jamaica, Ecuador, Peru, Portugal, Japan, Senegal, and other countries, including Brazil. Some of the 44 samples analyzed here were tightly grouped, reflecting a high degree of conservation and similarity of the virus being disseminated through the region during the study period, which may also indicate the evolution of a new HTLV-1 a subgroup, a hypothesis that will require further, confirmatory data.The molecular investigation of HTLV-1 has permitted the identification not only of the origin of this retrovirus, but also its dispersal among the different human populations around the world. Rego et al. 2008  analyzed−7 and 6.55 x 10−7 substitutions per site per year for the env and LTR, respectively [Other studies have also shown that HTLV-1 is relatively stable, and has low genetic diversity in comparison with other retroviruses . Mutatioectively . A substectively , 25. Thiectively , althougectively , 28, 29.ectively .3 mutations per site per year for the subgroup a samples, which included all 44 analyzed in the present study. This is an extremely low rate in comparison with the evolution of the rotavirus, and the smallpox and influenza viruses. A curious detail here is that, while vaccines are available for these much more variable viruses, no vaccine has yet been developed for the HTLV-1, despite its low mutation rates.The phylogenetic tree revealed a rate of 2. 10−et al. 2005 [The phylogenetic tree was relaxed, comparing all the HTLV-1 subtypes, and it would have been expected to indicate much higher evolutionary rates, given the inclusion of highly divergent subtypes, such as c, e, f and g. However, mutation rates were low, even in these subtypes. Lemey al. 2005  applied Prevention and control are the two primary strategies for the prevention of infection by HTLV-1, and the associated vulnerability of the sufferers to highly debilitating diseases, which currently have no effective treatment. The investigation of new cases in endemic regions, such as the Amazon basin, as in the present study, will be the principal means of tracking the progress of this virus.S1 FigThe rates (estimated in mutations per site per year) were calculated by Bayesian inference. The 44 samples sequenced in this study are shown in the tree in red letters.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


OSAHS was diagnosed when AHI was ≥5. HLLV, subcutaneous and intra-abdominal fat were measured by ultrasound. After adjustment for age and BMI, both HLLV and neck circumference (NC) were independent predictors of AHI. OSAHS was found in 72% of patients; HLLV ≥ 370 cm3 was a predictor of OSAHS with a sensitivity of 66%, a specificity of 70%, a positive and negative predictive values of 85 and 44%, respectively . A multivariate logistic model was used including age, BMI, NC, and HLLV , and a cut off value for the predicted probability of OSAHS equal to 0.7 provided the best diagnostic results  in terms of sensitivity (76%), specificity (89%), negative and positive predictive values . All patients with severe OSAHS were identified by this prediction model. In conclusion, HLLV, an established index of visceral adiposity, represents an anthropometric parameter closely associated with OSAHS in severely obese women.Obstructive sleep apnea–hypopnea syndrome (OSAHS) is frequently present in patients with severe obesity, but its prevalence especially in women is not well defined. OSAHS and non-alcoholic fatty liver disease are common conditions, frequently associated in patients with central obesity and metabolic syndrome and are both the result of the accumulation of ectopic fat mass. Identifying predictors of risk of OSAHS may be useful to select the subjects requiring instrumental sleep evaluation. In this cross-sectional study, we have investigated the potential role of hepatic left lobe volume (HLLV) in predicting the presence of OSAHS. OSAHS was quantified by the apnea/hypopnea index (AHI) and oxygen desaturation index in a cardiorespiratory inpatient sleep study of 97 obese women [age: 47 ± 11 years body mass index (BMI): 50 ± 8 kg/m Obstructive sleep apnea–hypopnea syndrome (OSAHS), an emerging public health issue, is characterized by recurrent episodes of upper airway occlusion during sleep, which results in reduction or cessation of the airflow, and lead to chronic intermittent hypoxia and sleep fragmentation . The patObstructive sleep apnea–hypopnea syndrome affects a significant proportion of the adult population, mainly males, and its prevalence increases with increasing body mass index (BMI) and advancing age , 6, 7. COn the basis of animal models , it has The Cardiometabolic Think Tank convened on June 20, 2014 in Washington, DC, has tried to categorize subtypes and stages of MS in order to find an optimal care model for patients at increased cardiometabolic risk. The subtype with the excess visceral adipose tissue as main pathophysiologic mechanism is characterized by the presence of sleep disordered breathing and non-alcoholic fatty liver disease (NAFLD) .Patients with MS frequently have an increased fat (triglyceride) accumulation in the liver, called NAFLD, and hepatic insulin resistance. With the increasing worldwide prevalence of obesity, NAFLD has become the most common cause of abnormal liver function in the general adult population –23. StudThe diagnosis of NAFLD is usually established by ultrasound and can be confirmed by liver biopsy .We have shown that the ultrasound measurement of the hepatic left lobe volume (HLLV) correlated with the total volume of liver measured with MR and is an excellent indicator of visceral adiposity clustering with parameters defining MS , 26.Little is known about the prevalence of OSAHS and the impact of this medical disorder on metabolic risk factors in morbidly obese patients; especially in women in whom it frequently remains underdiagnosed .The aim of this cross-sectional study was to evaluate in a group of morbidly obese women with symptoms and signs associated with OSAHS the prevalence of OSAHS and the relationships between the severity of OSAHS and several anthropometric measurements with particular interest to HLLV.2) referred to our Obesity Center for evaluation of obesity and its comorbidities and had, therefore, been submitted to cardio-respiratory sleep study for the clinical suspicion of OSAHS . All patients signed a written consent for the treatment of their clinical data for purpose of research. The study did not require additional testing beside the protocol for evaluation of patients candidate to bariatric surgery. Exclusion criteria for this study were: history of hypothalamic–pituitary diseases, hypercortisolism, goiter, self-reported daily alcohol consumption > 20 g, current or past use of illicit drugs or hepatotoxic medications, viral hepatitis as assessed by conventional serum markers, a previous diagnosis of a disease potentially causing liver enlargement , pregnancy or breast feeding within the 12-month period before enrollment. A careful endocrinological evaluation was also performed to reveal undiagnosed dysfunctions that required specific therapy and to evaluate the presence of hormonal abnormalities associated with OSAHS. 33 subjects were taking hypoglycemic agents, 7 hypolipidemic agents, and 4 hypouricemic agents.This is a retrospective study that included 97 morbidly obese women  and intra-abdominal fat (IAF) was performed, as previously described , 26. Bri2 saturation (SpO2) was recorded during the entire length of the night. Apnea was defined by the absence of airflow for > 10 s while hypopnea was defined as any airflow reduction of >50% that lasted for >10 s and resulted in oxyhemoglobin desaturation (3% dip rate). AHI was defined as the sum of the numbers of apnea and hypopneas per hour of sleep and OSAHS was diagnosed when AHI was ≥ 5. Oxygen desaturation index (ODI) was defined as the number of desaturations per hour of sleep and OSAHS was diagnosed when ODI was ≥5.An inpatient cardiorespiratory overnight sleep study was performed by means of a polygraph . To consider a study valid, a minimum sleep duration of 5 h was required. Parameters measured included oronasal flow by nasal cannula, thoraco-abdominal movements, pulsoxymetry, snoring, and body position. The results recorded by the instrument were scored by the attending physician expert in sleep studies who was maintained blinded to the characteristics of the patient. Nocturnal OAccording to the American Sleep Disorders Association Task Force criteria , sleep-rt test and ANOVA for difference in mean values, Mann–Whitney U and Kruskal–Wallis tests for skewed variables, Pearson Chi-square test for difference in counts and frequency. The Levene’s test was used to assess the equality of variances between OSAHS groups. The Kolmogorov–Smirnov test was used to assess normality of data; logarithmic transformations were applied to skewed variables  to approximate a Gaussian distribution. Pearson (R) and Spearman (Rho) correlation coefficients were employed to quantify associations for Gaussian and skewed variables, respectively. Multiple linear regression analyses using the forward selection algorithm were carried out to identify the most significant anthropometric predictors of AHI, SpO2, and ODI.The sample size calculation was based on the number of patients needed to demonstrate an association between HLLV and AHI. A target sample size of 84 patients provided a power of 0.80 with an alpha of 0.05 to detect a modest correlation equal to 0.30. With 97 patients recruited in this study, the statistical power was equal to 0.85. Descriptive statistics were calculated for the cohort as a whole and then separately by OSAHS diagnosis. Statistical tests used to compare groups included Student’s The receiver operating characteristic (ROC) curve was calculated to identify the cutoff value of both HLLV and NC, which better discriminated between subjects with and without OSAHS in terms of highest combined sensitivity and specificity . The positive group included subjects with OSAHS (AHI ≥ 5) while the negative group those without OSAHS (AHI < 5). Sensitivity was defined as the percentage of subjects having OSAHS who were correctly classified as having this disease, while specificity was the percentage of subjects without OSAHS who were correctly classified as not having this disease.A multivariate logistic analysis including all the determinants of OSAHS as determined through univariate analysis was carried out to calculate the predicted probability of OSAHS diagnosis (AHI ≥ 5) based on the logistic formula: 1/[1 + exp(−SCORE)], where SCORE was the linear combination of logistic model coefficients multiplied for the values of the respective determinant of OSAHS. A ROC curve analysis was then performed to determine the best cutoff value of the predicted probability of OSAHS diagnosis according to the highest Youden index, as above.p-value < 0.05 was considered statistically significant. Data are presented as a mean ± SD or median with inter-quartile range (IQR), as indicated. Statistical analyses were performed in SPPS .A 2 (range: 36–81). The physical, hematological, and clinical characteristics are reported in Table 2 was lower in patients with OSAHS compared to those without (p < 0.005). Median ODI was higher in patients with OSAHS compared to those without (p < 0.001). On average, patients with OSAHS were 8-year older (p < 0.005) and had greater IAF and HLLV compared to patients without OSAHS (both p < 0.05) despite similar body weight, BMI, and SCF. In addition, higher levels of γ-GT were observed in patients with OSAHS (p < 0.05), and there was a positive association between AHI and γ-GT . No significant associations were observed between AHI and ALT (p = 0.21), AST (p = 0.22) or ALP (p = 0.49). Out of 70 patients with OSAHS, 48 (69%) and 50  patients were classified as with the MS according to ATP-III and IDF criteria, respectively.Average age of the study cohort (±SD) was 47 ± 11 years (range: 24–67) and BMI was 50 ± 8 kg/mp < 0.005) and SpO2  correlated with HLLV in a positive and negative fashion, respectively  and SpO2  were observed for NC  and with NC  in a positive fashion  and AT  were associated with AHI and SpO2 while there was no correlation with SCF . In separate multivariate models each including age and BMI, HLLV (p < 0.005) and NC (p < 0.05), but not IAF (p = 0.47), AT (p = 0.48) nor SCF (p = 0.84), were associated with AHI.Apnea/hypopnea index  and NC  were independent predictors of AHI, such that a 100-cm3 increase in HLLV and 1-cm increase in NC were independently associated with mean increases in AHI of 15 and 9%, respectively  was an independent predictor of ODI, such that a 100-cm3 increase in HLLV was independently associated with an average 13% increase in ODI  had higher AHI compared to those with an HLLV < 408 cm3 and NC < 38 cm . Similarly, NC  and HLLV  were independent predictors of SpO2 after adjustment for age and BMI  , a specificity of 70% (95% CI: 50–86%), and positive and negative predictive values (NPVs) of 85 and 44%, respectively, in identifying subjects with OSAHS. Conversely, NC did not discriminate between subjects with or without OSAHS  , but not NC, (p = 0.09) was the only independent predictor of OSAHS diagnosis after adjustment for age (p < 0.005) and BMI (p = 0.86) in the multivariate analysis. According to the results of the logistic analysis, a predicted probability of OSAHS (AHI ≥ 5) was calculated on the basis of age, BMI, NC, and HLLV applying the following formula: 1/[1 + exp(−SCORE)], where SCORE = −9.806 + age (years) × 0.075 + BMI (kg/m2) × 0.007 + NC (cm) × 0.155 + HLLV (cm3) × 0.003. The predicted probability of OSAHS achieved the highest performance in the classification of OSAHS  and a cut-off value equal to 0.70 yielded to the best diagnostic results in terms of combined sensitivity (76%), specificity (89%), NPV (59%), positive predictive value (95%), and accuracy (79%). Among subjects with a predicted probability of OSAHS ≥ 0.70, only 3 (5%) did not actually present OSAHS  while among the subjects with a predicted probability of OSAHS < 70%, 17 (41%) presented OSAHS . Results for each class of OSAHS severity are reported in Table In the whole cohort of 97 women, HLLV was a parameter significantly associated with OSAHS by ROC curve analysis  Figure A. A cutoObstructive sleep apnea–hypopnea syndrome and NAFLD are very common in patients with central obesity and MS . RegionaAge is another risk factor for OSAHS especially in women , probablFemale subjects are referred less frequently to sleep clinics probably also because of gender-related symptom differences; women with OSAHS may refer atypical symptoms such as fatigue, headaches, mood disorders , 35.In studies conducted in numerically limited populations with predominance of male subjects affected by overweight or mild and moderate obesity, higher values of BMI, waist circumference (or waist–hip ratio), neck circumference were almost always associated with the presence and severity of OSAHS , 36–39. Recognizing the risk factors and predictors of OSAHS may be of particular relevance in women. Indeed, women are more frequently addressed to bariatric surgery than men and patients with OSAHS are particularly vulnerable during anesthesia and sedation, and display an increased risk of developing respiratory and cardiopulmonary postoperative complications . IdentifFew studies have analyzed the prevalence of OSAHS in morbidly obese women; the most important risk factors identified were BMI, age, and menopausal status , 43, 45.In our sample, OSAHS was found to be present in 72% of obese women with a BMI greater than 35. Approximately half of the patients had mild OSAHS, one quarter had moderate OSAHS and one quarter of patients manifested a severe form of obstructive sleep apnea. Patients with OSAHS were older and had a greater visceral fat estimated by ultrasound compared to patients without OSAHS, despite similar body weight, BMI, NC, and SCF. γ-GT, but not transaminase, levels positively correlated with OSAHS quantified by AHI index, suggesting that OSAHS could be an independent risk factor for NAFLD. Lack of association between AHI index and serum transaminases may depend on the limited statistical power of our study sample. However, liver enzymes alone are not sensitive enough to characterize NAFLD. In this regard, it should be noted that non-invasive liver biomarkers-based scores have been developed to evaluate the extent of liver injury, which are independently associated with the severity of nocturnal hypoxia , 47.As expected, neck circumference (NC) and IAF (measured by ultrasound) correlated with AHI and ODI and with the severity of OSAHS. We also observed a positive relationship between HLLV and AHI. ODI correlated with HLLV in a positive fashion as well. Interestingly, HLLV showed a stricter correlation with parameters describing OSAHS than NC.According to the ROC curve analysis, only HLLV but not NC did discriminate between subjects with or without OSAHS. Similarly, according to the logistic regression, when dichotomizing a continuous variable (AHI) in two groups, the statistic contribution of NC was lost.These results confirm the role of liver enlargement as a marker of visceral adiposity, which may facilitate the identification of individuals who are at increased risk of OSAHS in addition to the other known risk factors of OSAHS such as age, BMI, and NC. Indeed, prediction logistic model, including age, BMI, NC, and HLLV, displayed a discriminative ability . However, the NPV was too low (59%) to propose this model in a clinical setting. While it is useful to reduce the number of false positives to avoid unnecessary instrumental sleep studies, a relevant number of false negative results may lead to a failure to treat OSAHS with potential adverse consequences. Nevertheless, among the false negative, there were no patients with severe OSAHS (AHI > 30) indicating that the model can identify all patients affected by OSAHS requiring urgent and active treatment.At present, we cannot establish whether OSHAS and NAFLD can influence each other in terms of occurrence and progression or if they are merely unrelated expressions of ectopic fat deposition. Future studies including HLLV and other non-invasive scores , 47 may In conclusion, the results of this study indicate that in severe obese women HLLV is a powerful and better anthropometric predictor of OSAHS than neck circumference, and we have identified a prediction logistic model based only on four parameters , which is capable of predicting all severe forms of OSAHS requiring active treatment.Our study is a pure retrospective analysis of data collected from a cohort of patients who gave written consent to the treatment of their clinical data for purpose of research. No additional testing was performed beside those required for the evaluation of patients’ candidate to bariatric surgery. There are no ethical issues that may require approval by our EC.GS and GQ conceived the study design, carried out data collection, and wrote the manuscript. LM performed cardiorespiratory overnight sleep study. PP performed the statistical analysis and drew the figures. SM performed ultrasound examination for measurement of hepatic left volume, subcutaneous and intra-abdominal fat. GC, PF, GS, JV, SB, and GC were involved in data interpretation and writing of the paper. AP and FS conceived the study design, contributed to data interpretation, and writing of the manuscript. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


VD). Uric acid (UA) has long been implicated as a critical cause of cardiovascular disease. Nevertheless, UA was also expected to play an important role in antioxidant and neuroprotection recently. We hypothesized that UA may have a protective role against VD. The aim of this study was to investigate the link between serum UA and cognitive dysfunction in VD.Mounting evidence suggests that oxidative stress is involved in the pathogenesis of vascular dementia  at the beginning of the trial.There were altogether 127 VD group showed lower MMSE scores and serum UA levels than nondemented controls and there was significant statistical difference between the two groups (p < .05). Demographic and clinical characteristics such as age, gender, education, body mass index (BMI), total cholesterol (TC), triglycerides (TG), high‐density lipoprotein cholesterol (HDL), low‐density lipoprotein cholesterol (LDL), blood urea nitrogen (BUN), and serum creatinine (Scr) did not differ dramatically between groups (p > .05). In VD subjects, there was a positive correlation between serum UA and MMSE scores , and this correlation was independent of demographic and clinical characteristics .The VD subjects have dramatically lower serum UA levels in comparison to nondemented controls. Lower serum UA levels are linked to cognitive dysfunction and could serve as a potential predictor for VD. UA has been known as a hazard factor of cardiovascular disease   is a most widely used scale for screening and assessing cognitive decline, which acts as one of the essential tests recommended according to the NINDS‐AIREN. The total score of MMSE is 30 points. MMSE comprises seven sections including Time orientation (5 points), Space orientation (5 points), Attention and Calculation (5 points), Retell of three words (3 points), Registration of three words (3 points), Linguistic Function (8 points), and Visual Performance (1 point)  was used for data analysis. Continuous variables  were summarized as means ± SD and categorical variables (gender) were summarized as frequency and percentages. Differences between VD patients and nondemented controls were examined using the Student's 3p = .018 vs. p < .001, respectively). Years of education of VD patients were lower than nondementia controls, though there was no distinct difference between them (p > .05). The characteristics regarding age, gender, years of education, BMI, TC, TG, HDL, LDL, BUN, and Scr were not markedly different between groups (p > .05).From January 2015 to March 2016, 127 VD patients  and 81 controls  with normal cognitive function were evaluated. A comparison of demographics and clinical characteristics at baseline are summarized in Table r = −.36, p = .034) and a significant positive correlation between MMSE scores and serum UA levels . Nevertheless, no statistically significant correlation between MMSE scores and other baseline characters including gender, years of education, BMI, TC, TG, HDL, LDL, BUN, and Scr was found (p > .05).Pearson correlation analysis between MMSE scores and baseline data in VD patients are presented in Table p = .011). And multiple linear regression analysis also showed that there was a positive correlation between MMSE scores and serum UA levels of VD subjects . However, no statistically significance between MMSE scores and other baseline characters including gender, years of education, BMI, TC, TG, HDL, LDL, BUN, and Scr was found (p > .05).The results of multiple linear regression analysis between MMSE scores and baseline data in VD patients are presented in Table 4In our study, the differences in serum UA levels and cognitive function between VD patients and nondemented controls were examined. The result suggested that VD patients have significant lower MMSE scores, in addition with reduced serum UA levels. Our study indicated that reduced serum UA levels might contribute to the pathogenesis of VD and there existed a possible link between VD and serum UA levels. We further evaluated the correlation between serum UA levels and MMSE scores. The results showed that there existed a significant positive correlation between MMSE scores and serum UA levels in patients with VD and the association was significant even after adjusting for the confounding factors. As far as we know, there are limited published papers investigating the correlation between serum UA levels and VD until now.Currently we still have poor knowledge about the pathogenesis of VD, however, there exist powerful evidence provided by numerous studies that oxidative stress is involved in the development of VD (Liu & Zhang, These findings support the theory that oxidative stress contribute to VD. It is less clear whether UA, an end oxidative product of purine metabolism accounting for 60% of the total antioxidant capacity in human plasma (Morimoto, Simao, & de Almeida, UA is considered to be one of most abundant antioxidant existing in the blood. Nevertheless, UA can serve likewise as pro‐oxidant particularly in the condition exposed to high levels of singlet oxygen and peroxynitrite (Bowman, Shannon, Frei, Kaye, & Quinn, In our study, the results suggested that there was an obvious positive correlation between MMSE scores and serum UA levels and that lower serum UA levels was an independent risk factor in VD. As far as we know, there are no published papers investigating the correlation between UA levels and MMSE scores in VD until now. Besides UA levels, age is another independent risk factor for VD (Erkinjuntti, Laaksonen, Sulkava, Syrjalainen, & Palo, Our study has some limitations. First, there were relatively few participants enrolled in our research. Second, the VD groups may comprise subjects with mixed dementia. Third, all the participants are Chinese, so whether the results in our study are appropriate for other ethnic groups is uncertain, which needs further research. Fourth, data on personal lifestyle such as the course and drug therapy history of VD, smoking status, and alcohol drinking status are not available. However, the merit of this study is that it is the first study on the relation between UA and VD in Chinese population.Taken together, our major finding of this study was that lower serum UA level is strongly associated with cognitive dysfunction and serum UA level plays a protective role against cognitive decline in VD. Nevertheless, additional prospective large clinical trials are needed to evaluate whether appropriately increasing UA level could decrease the incidences of VD. The relationship between UA and cognitive dysfunction in VD, if proven by some larger clinical trials, may have important clinical and therapeutic promise.The authors declare no conflicts of interest in this study.


TSLP) is a direct target of miR-19b. An asthma model was established via ovalbumin (OVA) sensitization and challenge in 72 female BALB/c mice. Mice were then assigned to saline, OVA-sensitized, saline+miR-19b mimics, saline+anti-TSLP, OVA-sensitized+miR-19b mimics, OVA-sensitized+mimics scramble, OVA-sensitized+anti-TSLP, and OVA-sensitized+IgG2a groups. Pathological morphology changes were detected through hematoxylin/eosin, Masson, and periodic acid-Schiff staining. miR-19b was downregulated while TSLP and Stat3 were upregulated in the OVA-sensitized group compared with the saline group. Bronchoalveolar lavage fluid samples from OVA-sensitized mice showed increased total protein, IL-4, IL-5 and IL-6 levels, numbers of inflammatory cells, eosinophils, neutrophils, mononuclear macrophages and lymphocytes, and eosinophil% compared to controls. Lung tissues from sensitized mice exhibited decreased superoxide dismutase activity and increased methane dicarboxylic aldehyde levels. The effects of OVA sensitization were reversed in the OVA-sensitized+miR-19b mimics and OVA-sensitized+anti-TSLP groups. These findings suggest miR-19b reduces airway remodeling, airway inflammation, and degree of oxidative stress by inhibiting Stat3 signaling through TSLP downregulation in a mouse asthma model.This study explored the effects of microRNA-19b (miR-19b) on airway remodeling, airway inflammation, and degree of oxidative stress in a mouse model of asthma. Bioinformatics analyses and dual luciferase reporter gene assays revealed that thymic stromal lymphopoietin ( In recen asthma . TSLP isn asthma .et al. found that Stat3 activation facilitates TSLP in airway remodeling in asthma [Signal transducers and activators of transcription (Stat) are a group of transcription factors that play key roles in cytokine signaling. Wu, n asthma . miR-19bn asthma . Decreasn asthma –16. Furtn asthma –16. It aMice in the OVA-sensitized group were initially restless, running back and forth, with increased nasal burning symptoms followed by polypnea and gasping Figure . In the microRNA.org website predicted that miR-19b targets TSLP , while TSLP-MUT 3'UTR luciferase activity was not (P>0.05). This suggests that miR-19b specifically binds the TSLP-3’-UTR region and downregulates TSLP expression at the post-transcriptional level.The P Figure , and we P Figure . CompareP<0.05; Figure P=0.036) and Stat3 , while TSLP expression was positively correlated with that of Stat3 . OVA, OVA-sensitized + mimics scramble and OVA-sensitized + IgG2a groups scored higher than the OVA-sensitized + miR-19b mimics and OVA-sensitized + anti-TSLP groups .HE staining Figure  showed cP<0.05). OVA, OVA-sensitized + mimics scramble and OVA-sensitized + IgG2a mouse N/Pi, Fib, Wat/Pi, and Wam/Pi levels were higher compared to the OVA-sensitized + miR-19b mimics and OVA-sensitized + anti-TSLP groups . No difference was observed among the saline, saline + miR-19b mimics, and saline + anti-TSLP groups . OVA, OVA-sensitized + mimics scramble, and OVA-sensitized + IgG2a PAS scores were higher compared with the OVA-sensitized + miR-19b mimics and OVA-sensitized + anti-TSLP groups , and were decreased in the OVA-sensitized + miR-19b mimics and OVA-sensitized + anti-TSLP groups compared to the OVA-sensitized group . No differences were observed among the OVA, OVA-sensitized + mimics scramble, and OVA-sensitized + IgG2a groups, or between the saline + miR-19b mimics and saline + anti-TSLP groups (P>0.05)  Table .P<0.05, Figure P<0.05). No difference was observed between the OVA-sensitized + miR-19b mimics and OVA-sensitized + anti-TSLP groups , or between the OVA, OVA-sensitized + mimics scramble, and OVA-sensitized + IgG2a groups .Saline, saline + miR-19b mimics, and saline + anti-TSLP IL-4, IL-5, and IL-6 levels were decreased in the BALF of mice compared to the OVA-sensitized groups . Increased SOD activity and decreased MDA levels were observed in the OVA-sensitized + miR-19b mimics and OVA-sensitized + anti-TSLP groups as comparison to the OVA-sensitized group . No difference was observed between the OVA-sensitized + miR-19b mimics and OVA-sensitized + anti-TSLP groups , or between the OVA, OVA-sensitized + mimics scramble, and OVA-sensitized + IgG2a groups .No difference was observed in MDA level or SOD activity among the saline, saline + miR-19b mimics, and saline + anti-TSLP groups , while no difference was observed among the OVA-sensitized + mimics scramble, OVA-sensitized + anti-TSLP, and OVA-sensitized + lgG2a groups . TSLP and Stat3 mRNA levels were downregulated in the saline + anti-TSLP and saline + miR-19b mimics groups, and upregulated in the OVA-sensitized groups as compared to the saline group . TSLP and Stat3 mRNA levels were decreased in both the OVA-sensitized + miR-19b mimics and OVA-sensitized + anti-TSLP groups as compared with the OVA-sensitized group . TSLP, Stat3, and p-Stat3 protein levels were increased in the OVA-sensitized groups and decreased in the saline + anti-TSLP and saline+miR-19b mimics groups as compared to the saline group .miR-19b mRNA was upregulated in the saline + miR-19b mimics group as compared to the saline and saline + anti-TSLP groups, and was upregulated in the OVA-sensitized group -like cytokine that influences interactions between dendritic cells, CD4+ thymocytes, and T cells in the positive selection of regulatory T cells, induction of CD4+ T cell-mediated allergic inflammation, and maintenance of peripheral CD4+ T cell homeostasis. TSLP is likely associated with the pathogenesis of asthma . He, et e models . TSLP ine models . Additioe models , 24.et al. identified epithelial Stat3 as a key regulator of allergic inflammation and airway hyper-responsiveness induced by allergens in a murine model of asthma [Stat3, critical in the regulation of asthma allergic response, is overexpressed in the mouse airway epithelium after exposure to house dustmites. Simeone-Penney, f asthma . A previf asthma . Pearsonet al. reported that the airway epithelium can produce additional inflammatory and pro-remodeling cytokines, including IL-6 and IL-8, and insufficient amounts of anti-viral interferons [et al. reported that TH2 cells and their cytokines are vital for the development of asthmatic features, such as lung eosinophilia, mucus hyper-secretion, mast cell hyperplasia, and bronchial hyper-responsiveness [et al. also reported that MDA levels and the MDA/TAC ratio are higher in asthma patients as compared with healthy subjects [In our study, HE scores, N/Pi, Fib, Wat/Pi, Wam/Pi levels, and PAS scores were increased in OVA-sensitized groups compared with the saline-treated group. BALF samples from OVA-sensitized mice showed increased total protein, numbers of inflammatory cells, EOS, neutrophils, mononuclear macrophages and lymphocytes, and EOS%. Similarly, lung tissues from sensitized mice exhibited decreased SOD activity and increased MDA levels. These effects of OVA sensitization were reversed in the OVA-sensitized + miR-19b mimics and OVA-sensitized + anti-TSLP groups. CD4+ T cells, primed by TSLP-activated DCs, differentiate into IL-4-producing TH2 cells, return to the lung, and then initiate allergic inflammation by increasing production of cytokines and chemokines . Since Ierferons . Furthersiveness . Previousiveness . Kocyigisubjects .Our results indicated that miR-19b reduces airway remodeling, airway inflammation, and degree of oxidative stress in OVA-sensitized mice by inhibiting the Stat3 signaling pathway through TSLP. Our data present a novel method for identifying molecular targets in the treatment and management of asthma, although we did not measure lung function in OVA-sensitized mice. Further investigation of the precise activities of miR-19b-targeted genes in chronic asthma-associated lung remodeling and inflammation is still needed.A total of 72, 5–7 week-old female BALB/c mice, weighing 18–25 g each, were purchased from Hunan SJA Lab Animal Ltd.  for this study. Mice received block-shaped feed, with free access to drinking water at 25–26°C. Relative humidity was maintained at 60–70%, and padding was changed twice per week. All animal experiments conformed to local principles of animal management in the Laboratory animal management and use guide of the National Institutes of Health (NIH).Construction of the asthma mouse model was performed as follows: after 7 d of adaptive feeding, mice were subcutaneously injected in the hind legs with 0.2 ml of normal saline containing 0.1 mg of ovalbumin  and 10 mg of aluminum hydroxide . The second sensitization injection was performed on d 14. For three weeks, starting on d 25, an airway challenge was carried out daily for 30 min using aerosolized 1% OVA normal saline. Control mice were injected with the same amount of normal saline only. The 72 mice were randomly distributed into: a saline group (n=12), OVA-sensitized group (n=12), saline + miR-19b mimics group (n=8), saline + anti-TSLP group (n=8), OVA-sensitized + miR-19b mimics group (n=8), OVA-sensitized + mimics scramble group (n=8), OVA-sensitized + anti-TSLP group (n=8), and OVA-sensitized + IgG2a group (n=8). Mice in the OVA-sensitized + miR-19b mimics group and the OVA-sensitized + mimics scramble group were administered with 40 ul of 20 ug miR-19b mimic (5′-UGUGCAAAUCCAUGCAAAACUGA-3′) or miR-19b scramble (5′-UUCUCCGAACGUGUCACGUTT-3′)  by nasal instillation every three days for a total of 10 times starting on the d 20. Also starting on d 20, mice in the OVA-sensitized + anti-TSLP group and the OVA-sensitized + IgG2a group were injected with 50 ul of l ug/ul TSLP antibody or IgG2a antibody . This was repeated after three days. All mice were euthanized within 24 h after their last airway obstruction. The trachea, bronchus, and lung tissues were removed, and the right mid-lung was taken out after ligation. These lung specimens were preserved in 4% paraformaldehyde and then embedded in paraffin. The remaining lung tissues were transferred to an Eppendorf (EP) tube, treated with diethyl pyrocarbonate (DEPC), and stored in liquid nitrogen.Paraffinized lung tissues were dewaxed using xylene, soaked in distilled water after an alcohol gradient, and then stained with Harris hematoxylin solution for 5 min. After rinsing, sections underwent color separation in 0.5% hydrochloric-alcohol solution for 5 s, bluing for 15 min, 70% ethyl alcohol for 5 min, 80% ethyl alcohol for 5 min, eosin solution (95% ethanol solution) for 15 s, 95% ethyl alcohol for 5min × two times, absolute ethyl alcohol 5 min × 2 times, xylene + ethyl alcohol (1:1) for 5 min, and then xylene cleaning and neutral gum sealing. Sections were then observed under a microscope and graded according to inflammatory cell infiltration degree, as follows: 0 = no inflammatory cell infiltration; 1 = a small number of infiltrated inflammatory cells; 2 = an inflammatory cell monolayer around the airway; 3 = an inflammatory cell bilayer around the airway; 4 = four layers of inflammatory cells around the airway. The experiment was repeated three times for each group.Sliced sections were dewaxed, stained with hematoxylin for 10 min, differentiated with 1% hydrochloric acid alcohol, and stained with trypan blue. After washing, sections were stained with Ponceau Staining Solution for 7 min, stained with 1% phosphomolybdate for 4 min after washing, and placed in aniline blue for 5 min for water purification. Sections were then dehydrated with 95% ethanol, dried in the oven at 60°C, and subjected to xylene transparency, fixing, and observation. After Masson staining, the Img-Pro 6.0 image analysis system  was used to determine the percentage of collagen fiber , the bronchial lumen perimeter (Pi), the bronchial wall thickness (Wat), the smooth muscle thickness (Wam), and the number of bronchial smooth muscle cells (N). These measured values were standardized as Wat/Pi and Wam/Pi for the thickness of the corresponding wall, and N/Pi for standardized smooth muscle cells and numbers. The experiment was repeated three times for each group.Fresh and dried mouse lung tissue sections were fixed with 95% alcohol for 5–10 min and dried at room temperature after rinsing. The oxidized sections were treated with 10 g/L periodic acid, dried at room temperature after rinsing, and then treated with Schiff solution for 1 h at 37°C. Sections were rinsed with ferrous sulfate solution three times, rinsed with distilled water three times, and then dried at room temperature. Finally, sections were re-dyed with 20 g/L methylgreen solution for 20 min, and observed under the microscope after rinsing and drying. Hyperplasia and mucus secretion of airway mucosa goblet cells were observed. The number of goblet cells as a percentage of total airway epithelial cells was graded as follows: <5% (0), 5–25% (1), 25–50% (2), 50–75% (3), and >75% (4). The experiment was repeated three times for each group.Twenty-four h after airway challenge, mice were anesthetized via an intraperitoneal injection of 400 mg/kg of 10% chloral hydrate. Then, the trachea was isolated and ligation was carried out on the left trachea after drawing blood. Then, 0.5 ml of phosphate buffer solution (PBS) at 4°C was infused into the trachea and collected three times. Bronchoalveolar lavage fluid (BALF) was centrifuged at 12000 rpm at 4°C for 10 min. The supernatant was stored at -80°C. The sediment was re-suspended in 1 ml of PBS, and 10 ul of the suspension was used to conduct smear staining and to count and classify inflammatory cells. Inflammatory cells in the BALF were counted via Wright staining, and eosin staining was used to count eosinophils (EOS). In total, 200 cells were observed under the microscope, and the proportion of all inflammatory cells was calculated according to morphological characteristics. The experiment was repeated three times for each group.Xanthine oxidase  was used to test superoxide dismutase (SOD) activity in mouse lung tissues. The rat total bile acide (TBA) ELISA kit (Sangon Biotech (Shanghai) Co., Ltd.) was used to assess methane dicarboxylic aldehyde (MDA) content in mouse lung tissues. A separate ELISA kit  was applied to examine IL-4, IL-5, and IL-6 levels in the BALF supernatant. Specific procedures were as follows: at room temperature, 100 ul of the diluted standard substance and specimens from each group were added to the setting plate and incubated at 37°C for 30 min. After washing, 100 ul of a relevant antibody solution (biotin anti-mouse) was added and incubated at 37°C for 30 min. After washing, 100 ul of avidin-biotin-peroxidase compound working solution was added to each well and incubated at 37°C for 30 min. After washing, 50 ul each of color developing agents A and B were mixed, added to each well, and incubated at 37°C for 15 min in the dark. Finally, 50 ul of stop buffer was added to each well, the zero setting was applied in blank wells, and optical density (OD) was assessed at a wavelength of 450 nm within 15 min of stop buffer addition. A standard curve was drawn with OD values as ordinate and the standard substance concentration as abscissa. Specimen OD values were used to identify the cytokine level of samples to be tested. The experiment was repeated three times for each group.MicroRNA.org website was used to verify whether TSLP was a direct target of miR-19b. The full length TSLP gene 3’UTR was cloned into multiple cloning sites downstream of the pmirGLO (Promega Company) expression vector luciferase gene. This 3’UTR was also used to computationally predict miR-19b binding sites. Renilla luciferase in the pRL-TK vector (TaKaRa Company) was used as an internal reference to adjust for cell number differences and assess transfection efficiency. miR-19b mimics and NC were transfected into HEK-293T cells with wild-type (WT) and mutant (MUT) luciferase reporter vectors, respectively. After 48 h of transfection, cells were collected and split, and the dual luciferase activity assay was performed according to the manufacturer's instructions .The -ΔΔCt method was used to calculate relative gene expression values. The experiment was repeated three times for each group.Trizol  was used to extract total RNA from lung tissues. RNA was dissolved with ultrapure water treated with DEPC. An ND-1000 ultraviolet/visible spectrophotometer  was used to assess RNA quality and concentration. RNA reverse transcription was performed according to the manufacturer's instructions  Co., Ltd, Beijing, China). Reaction conditions were as follows: 70°C for 10 min, ice bath for 2 min, 42°C for 60 min, and 70°C for 10 min. cDNA was preserved at -80°C. RT-qPCR  reaction conditions followed kit specifications  Co., Ltd, Beijing, China). Primer sequences are provided in Table Lung tissue total protein samples were immersed in sodium dodecyl sulfate (SDS) lysate at 100°C for 5 min. Twenty μl of the upper phase was electrophoresed in a 12% polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was rocked at room temperature for 1 h in Tris buffered saline-tween (TBS-T) containing 5% bovine serum albumin (BSA). The membrane was then incubated in TBS-T containing 5% BSA and relevant concentrations of TSLP, Stat3, p-Stat3, or β-actin antibodies  at 4°C with oscillation overnight. The next day, the membrane was rinsed with TBS-T for 3 × 10 min. After incubation with diluted secondary antibody  at 4°C for 4–6 h, the membrane was rinsed using TBS-T for 3 × 15 min. The membrane was developed using chemiluminescence reagents, and relative OD analysis was performed for all visible bands. The experiment was repeated three times for each group.t test was used to compare two groups. P<0.05 indicated a statistically significant difference.SPSS 21.0  software was utilized for data analysis. Results are shown as means ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare three or more groups and the


Xenopus, the progressive determination of the head is an extremely complex process involving the activation and localized antagonism of a number of interdependent intracellular signaling pathways including the Wingless/Int-1 (Wnt), bone morphogenetic protein (BMP), and nodal-related pathways. The sequence of events that specify the head are: dorsal-ventral polarization and head organizer specification in the blastula; gastrulation; neural induction; and patterning of the anterior-posterior and dorsal-ventral neuraxes. Wnt signaling is required for the specification of the dorsal side initially but is then inhibited within the organizer once it has formed. Similarly, Wnt signaling is required along the length of the neural tube, but must be suppressed at its rostral end for normal brain development. Nodal signaling is also necessary for induction of the mesendoderm, but is subsequently suppressed in its dorsal-anterior extreme to specify head organizer. BMP signaling is required for ventral mesoderm and non-neural ectoderm, and must also be suppressed in the head organizer region and for the differentiation of the ventral midline of the neural tube. Thus, development of the head, and indeed the body plan in general, requires precisely timed and spatially restricted activation and repression of these signaling pathways.The head represents the most dorsal and anterior extent of the body axis. In