Hemorrhagic complications are quite common in the rare cases where thrombolysis is performed. Ischemic stroke in the aftermath of thrombolysis for a ST elevation myocardial infarction is a very rare and paradoxical complication. With these observations in mind we report two interesting cases of ischemic stroke which occurred after fibrinolytic therapy with tenecteplase for a ST elevation myocardial infarction.The first case was a 56-year-old African man who presented with an acute infero-basal ST elevation myocardial infarction 6 hours after chest pain onset. Thrombolysis with tenecteplase was performed and few minutes later an ischemic stroke occurred. The second patient was a 65-year-old African man who presented with an acute infero-basal ST elevation myocardial infarction 5 hours after chest pain onset. Thrombolysis was performed and 10 hours later an ischemic stroke occurred.Hemorrhagic stroke is not the only complication of thrombolysis, ischemic stroke can occur even if it is an extremely rare complication. The two cases on which we report shed light on the association between fibrinolytic therapy and ischemic stroke, the pathophysiology of which is not well understood. ST elevation myocardial infarction (STEMI) is due to complete occlusion of the coronary artery. The gold standard therapy for STEMI is recanalization of the infarct-related coronary artery as soon as possible by pharmacological or mechanical means. The indication for this is STEMI presenting within 12 hours of symptom onset.Primary percutaneous coronary intervention (PCI) is superior to thrombolytic therapy (TT), but there are many limitations to PCI such as the absence of a nearby angioplasty center. TT is therefore an effective treatment of choice in STEMI, as it is easy to perform anywhere and at any time .The efficacy and safety of various thrombolytic agents have been well documented in large clinical trials. The most feared complication of TT is intracranial hemorrhage which is well documented in the literature. Ischemic stroke in the aftermath of TT for STEMI worsens the patient’s prognosis; it is a paradoxical, very rare complication, the pathophysiology of which is not well understood . Hence, A 56-year-old African man presented to our emergency department 6 hours after severe chest pain onset. He was a tobacco smoker, diabetes status unknown, neither hypertensive nor dyslipidemic, and had no history of stroke. He had no personal or family medical history, including heart disease or heart rhythm disorder, and was not under any treatment prior to diagnosis. He was from a low socio-economic level. An electrocardiogram (EKG) showed a sinus rhythm with ST-segment elevation in inferior and posterior leads Fig. .Fig. 1ElAt admission he was conscious, Glasgow Coma Scale (GCS) of 15/15, without motor deficit or sensory disorder; his chest pain was constrictive irradiating to his two upper limbs without syncope. Blood pressure (BP) on admission was 140/85 mmHg symmetrical, regular rhythm at 60 beats per minute (bpm), heart sounds clearly auscultated with no heart or carotid murmur, there was no murmur of mitral insufficiency or ventricular septal defect, no signs of heart failure including no crackles, and he was without edema of his lower limbs or turgor of his jugular veins leading to a right ventricular infarction. His peripheral pulses were perceived symmetrically. The rest of the examination was strictly normal. Acute inferior and posterior STEMI was diagnosed and intravenously administered TT using tenecteplase (intravenous bolus of 30 mg considering a weight of 54 kg) was performed with adjuvant antithrombotic medication .2 body surface area (BSA) per minute; hemoglobin, 13.5 g/dl; platelets count, 500,000/mm3; plasmatic fibrinogen level, 2.49 g/l before TT and 5.89 g/l 12 hours after; white blood cells count (WBC), 15,000 mm3; and C-reactive protein (CRP), 72 mg/l. Antithrombotic medication was discontinued. His neurological condition worsened progressively and, after 1 week, he died.A few minutes later, he developed motor aphasia and right hemiplegia with altered level of consciousness: GCS of 12/15. The first cerebral computed tomography (CT) performed 1 hour later was normal and the second one performed 12 hours later showed a frontal, temporal, and parietal left ischemic stroke with a hemorrhagic infarct . Clopidogrel was stopped after 6 months.STEMI, caused by acute occlusion of the infarct-related coronary artery, is an emergency condition. The primary therapy is restoration of full antegrade flow by either PCI or TT in patients presenting within 12 hours of symptom onset. Although primary PCI is superior to TT in patients with STEMI, there are many limitations in clinical practice. TT is therefore an effective treatment of choice in STEMI as it is easy to perform anywhere and at any time.All thrombolytic agents share a common mechanism of activating plasminogen into plasmin, which in turn activates the fibrin degradation pathway. The efficacy and safety of various thrombolytic agents have been well documented in large clinical trials . TT decrBleeding after fibrinolytic treatment is due to the depletion of clotting factors and lysis of recently formed hemostatic plugs . HoweverBefore the era of TT, the incidence of stroke during a hospital stay after acute coronary syndrome (ACS) ranged from 0.7 to 2.2% with a mortality rate at 46%. Hachet and colleagues reported that typical cases occurred during the first week and 33% during the first 24 hours .Ischemic stroke after TT for STEMI is paradoxical and rare. A multivariate analysis showed a significantly low risk after TT performed within 15 minutes and a low but not significant risk after primary PCI performed within 90 minutes of first medical contact. Despite the use of fibrinolytics and primary PCI, the incidence of left ventricular thrombus formation seems to be lower but remains substantial [The Trial of ORG 10172 in Acute Stroke Treatment  studied ischemic strokes during hospital stay at the acute phase of myocardial infarction: 60% was of cardioembolic origin and 36% were of undetermined pathogenesis [The underlying pathophysiological processes of ischemic strokes after AMI are multifactorial. Ischemia itself induces a systemic procoagulant effect, facilitating cardiac thrombus formation and embolization. Furthermore, ischemia results in the release of inflammatory cytokines; this might trigger the destabilization and rupture of plaques in the cerebral circulation. Therefore, complex and unstable carotid plaques are common in patients with AMI (42% versus 8% in patients with stable angina) . These pCardioembolic strokes after AMI are mainly caused by atrial fibrillation and left ventricular thrombi . CardiacThere is also a procoagulant state of thrombolytic treatment which can explain the occurrence of a stroke after thrombolysis, hence the interest in systematically giving heparin, unfortunately that was not done with our second patient.Left ventricle thrombi formation is mainly caused by an akinetic segment of the left ventricular myocardium which can fragment and migrate to the brain after thrombolysis. It is a common complication in large anterior and apical myocardial infarction with a low LVEF (less than 40%) and the thrombotic risk increases with decrease in LVEF. The thrombi usually occur within 2 weeks after AMI; a median of 5 days after the acute event. In the prethrombolytic era, ischemic stroke after STEMI was a common complication and the incidence of thrombus formation was reported to range from 20 to 55% in patients with an anterior STEMI .et al. showed a significant risk of ischemic stroke after AMI with atrial fibrillation treated with TT [Crenshaw  with TT . Another with TT . Followi with TT .In total, several mechanisms may explain the occurrence of an ischemic stroke in the course of a thrombolysis of a STEMI. In some cases it has been explained by the procoagulant effect of thrombolytics. Embolization of microthrombi formed at the left ventricle or from carotid plaques following thrombolysis has also been reported. Some cases were explained by a paroxysmal atrial fibrillation or by thrombophilia by protein C deficiency and mutation C677T in MTHFR.In our two cases, patients were in sinus rhythm, the STEMI was inferior and posterior, LVEF was 40% or more, and no intracavitary thrombus was detected. The ischemic stroke could be explained by inflammatory reaction due to ischemia causing left ventricle microthrombi and it is presumed that TT induced lysis, fragmentation, and embolization of microthrombi in the cerebral circulation. Inflammatory reaction due to ischemia could also explain destabilization of carotid plaques. In addition, our second patient missed the first dose of heparin.Hemorrhagic stroke is not the only complication of thrombolysis, ischemic stroke can occur even if it is an extremely rare complication. Those cases represent an extremely rare clinical condition on which we report to shed light on the association between fibrinolytic therapy and ischemic stroke.Patients receiving TT for the treatment of STEMI should receive immediate antithrombotic co-therapies, have constant neurological re-evaluation, and clinicians must be prepared to handle such complications in a timely manner.Additional studies are needed to clarify the benefice of thrombolysis in high risk patients, especially the elderly or ones with a history of stroke.


In this paper, we evaluated the historical features and physical examination findings, as well as laboratory tests and ultrasound examinations, in children with testicular torsion (TT), in order to improve diagnosis and treatment in this population. A retrospective review of patients with diagnosis of TT between January 1990 and January 2010 was performed. We included 118 cases in the study, accounting for 9.01% of all cases of acute scrotum. Mean patient age was 9.3 ± 5.6 years. The left side was predominantly affected. The median duration of symptoms up to surgical exploration was 64 h. Absence of cremasteric reflex presented in 94.9% patients. All boys had an ultrasound of the scrotum; decreased or absent blood flow was observed in all orchidectomy patients. Heterogeneous echogenicity presented in all cases of orchidectomy. At surgery, viable testes were present in 46 boys (39%) and preserved; in 72 boys with nonviable testes, they were removed. The median duration of symptoms at presentation was 12 h when the testes were successfully conserved and 90 h when they were removed. Testicular salvage depends critically on early surgical intervention. Ultrasound is a useful tool for the clinical assessment of patients with TT, however, sonographic interpretation must be in conjunction with the clinical manifestations. We advocate immediate surgical exploration with suspected TT. Long-term hormonal levels are within the normal range regardless of the fate of the testis. Further follow-up is needed to confirm fertility after TT.


This concept article aims to show the rationale of targeting extracellular α-Synuclein (α-Syn) from cerebrospinal fluid (CSF) as a new strategy to remove this protein from the brain in Parkinson’s disease (PD). Misfolding and intracellular aggregation of α-synuclein into Lewy bodies are thought to be crucial in the pathogenesis of PD. Recent research has shown that small amounts of monomeric and oligomeric α-synuclein are released from neuronal cells by exocytosis and that this extracellular alpha-synuclein contributes to neurodegeneration, progressive spreading of alpha-synuclein pathology, and neuroinflammation. In PD, extracellular oligomeric-α-synuclein moves in constant equilibrium between the interstitial fluid (ISF) and the CSF. Thus, we expect that continuous depletion of oligomeric-α-synuclein in the CSF will produce a steady clearance of the protein in the ISF, preventing transmission and deposition in the brain. UnderTwo hypotheses about the structure of the protein have been proposed: An alpha-helical folded tetramer and a tetramer structure that is only adopted after membrane binding ,12,13,14The phosphorylation of α-Syn is essential in the degradation process ,17. The Recent research showed that small amounts of monomeric and oligomeric α-Syn are released from neuronal cells by unconventional exocytosis, and this extracellular α-Syn contributes to neurodegeneration, progressive spreading of α-Syn pathology, and neuroinflammation. Extracellular α-Syn is taken up by neurons through endocytosis and undergoes endocytic trafficking for degradation in lysosomes. Thus, α-Syn aggregates can be transmitted from neuron to neuron via the extracellular milieu and can propagate aggregates by a “seeding” mechanism. Moreover, extracellular α-Syn oligomers induce microglia via activation of Toll-like receptor 2 .Several studies have shown a reduction in CSF α-Syn levels in PD ,37,38 soTo control the toxicity produced by α-Syn, some methods have been found: reducing α-Syn cleavage by caspase-1 , inducinThe immunological selection of oligomeric extracellular α-Syn in animal models accelerates the clearance of this protein . The useWe have previously posed a new therapeutic hypothesis: that soluble peptides can be cleared from the brain with interventions where peptides are continuously removed from the CSF . In otheToday, we can conceive several innovative ways of removing peptides continuously from the CNS by accessing the CSF and debugging it using filtration devices. For instance, peptides can be targeted either by size—particularly the aggregated forms—or by immunological techniques, or by a combination of both. These devices can be endowed with different technologies able to capture target molecules, such as oligomeric α-Syn , from the CSF. Thus, these interventions would work as a central sink of α-Syn, reducing the levels of CSF oligomeric α-Syn, and, by means of the CSF-ISF equilibrium, would promote the efflux of oligomeric α-Syn from the ISF to the CSF .We introduce here a rationale for the “CSF sink” hypothesis and conclude that continuous depletion of oligomeric α-Syn in the CSF will produce a steady clearance of oligomeric α-Syn in the ISF. Implantable devices aimed at sequestering oligomeric α-Syn from the CSF may represent a new therapeutic strategy in PD and other α-synucleinopathies.


Cerebrospinal fluid (CSF) has been commonly accepted to drain through arachnoid projections from the subarachnoid space to the dural venous sinuses. However, a lymphatic component to CSF outflow has long been known. Here, we utilize lymphatic-reporter mice and high-resolution stereomicroscopy to characterize the anatomical routes and dynamics of outflow of CSF. After infusion into a lateral ventricle, tracers spread into the paravascular spaces of the pia mater and cortex of the brain. Tracers also rapidly reach lymph nodes using perineural routes through foramina in the skull. Using noninvasive imaging techniques that can quantify the transport of tracers to the blood and lymph nodes, we find that lymphatic vessels are the major outflow pathway for both large and small molecular tracers in mice. A significant decline in CSF lymphatic outflow is found in aged compared to young mice, suggesting that the lymphatic system may represent a target for age-associated neurological conditions. It is believed that the bulk of cerebrospinal fluid (CSF) drains through arachnoid projections from the subarachnoid space to the dural venous sinuses. Here the authors show that the major outflow pathway for CSF in mice are lymphatic vessels and that this drainage decreases as the mice age. An additional source of CSF is the interstitial fluid (ISF) from the brain tissue that is produced at the blood–brain barrier and may account for around 10% of the total volume of CSF2. Three layers of meninges surround the central nervous system (CNS), the pia mater that covers the brain and spinal cord parenchyma, and the arachnoid and dura mater layers, which line the skull and vertebral canal. The CSF flows through the SAS between the pia mater, which is semi-permeable, and the arachnoid, which forms a tight barrier to prevent flow into the dura mater4. Within the CNS itself, lymphatic vessels are believed to be absent, instead a system of paravascular spaces  is thought to provide channels for the movement of fluid and solutes from the brain interstitial space and to the CSF and vice versa5.Cerebrospinal fluid (CSF) is considered to be produced primarily by the choroid plexus and to flow through the ventricular system to reach the subarachnoid space (SAS) of the skull and spinal column7. These structures are described as a series of tubules within an outgrowth of arachnoid tissue that are continuous with the CSF of the SAS of the cranium and the spine9. They were initially believed to act as one-way valves for a pressure-driven flow from CSF to venous blood10; however, evidence from electron microscopy later indicated the presence of an intact barrier of endothelial cells11. Extensive research has not led to a consensus on the exact mechanism for flow through arachnoid villi and granulations and, despite their widespread acceptance as major outflow sites of CSF, direct in vivo physiological evidence of their function is lacking14.Based upon work in the early part of the twentieth century, the predominant outflow of CSF from the SAS is accepted to take place through arachnoid villi or granulations that project into the dural venous sinuses17. Attempts to quantify the proportion of CSF drained by the lymphatic system using cannulation of deep cervical lymphatic vessels and radiolabeled tracers have indicated that, in some species such as rabbit and sheep, lymphatic vessels were responsible for around 30–50% of total outflow, with the remainder assumed to be drained through arachnoid villi19. The outflow pathways were defined using tracers to be within sheaths around cranial nerves, with the cribriform plate route along olfactory nerves that extends to lymphatic vessels in the nasal mucosa deemed especially important21. These sheaths enclose extensions of the SAS that project extracranially with the nerves through foramina of the skull. At these locations, it was suggested that there are pathways for tracers to penetrate the arachnoid membrane to reach interstitial spaces25 or possibly directly to lymphatic vessels outside the skull27. Additional outflow pathways were found to be present around spinal nerve roots to reach lymphatic vessels in epidural tissue29. Two recent reports in mice have indicated that the dura mater of the CNS is endowed with a network of lymphatic vessels capable of draining CSF or brain ISF31. However, this potential route for drainage of CSF is controversial in light of the existence of the arachnoid barrier layer between the SAS and the dura mater4.Since an original report by Schwalbe in 1869, a large body of work in many different species has indicated a role for lymphatic vessels draining CSF in both cranial and spinal regions7. At this point, however, there is a lack of consensus on what the relative contribution for each of these outflow pathways is to total outflow of CSF. It is also not clear which pathways to reach the lymphatic system are most important. These are key questions as a functioning lymphatic vascular system draining CSF and brain ISF could be essential for many aspects of neurology including immune function and clearance of toxic proteins such as amyloid beta. As many neurological conditions are associated with the aging process, it is also critical to determine if lymphatic outflow of CSF is altered in aged conditions.Thus, the current paradigm suggests a dual-outflow system for CSF to reach the blood circulation, one directly to venous blood through arachnoid projections and one indirectly through the lymphatic system33. These bright pegylated NIR dyes avoid many of the complications of other tracers, such as direct blood vessel uptake, adherence to tissue components, or phagocytosis by macrophages, allowing quantification of lymphatic flow from the interstitial tissue of many different organs35. In this study, we first aim to use these tracers to define the major flow routes of CSF after infusion into a lateral ventricle in mice. Next, we use a recently developed tracer transport to blood assay to evaluate the dynamics of CSF outflow to the systemic circulation in comparison to visualization of outflow to collecting veins or lymphatic vessels in an attempt to shed light on the relative contribution of each pathway to total CSF outflow.We have recently developed near-infrared (NIR) tracers that in conjunction with lymphatic vessel reporter mice, allow high-resolution imaging and quantification of the anatomy and function of lymphatic vesselsWe find that the major outflow pathway from CSF for both large and small molecular tracers in mice is through perineural routes to reach the lymphatic system. Furthermore, we demonstrate significantly slower dynamics of CSF outflow in aged 18-month-old mice compared to young 2-month-old controls. Our findings implicate that the lymphatic system has a greater than previously acknowledged role in many neurological conditions.32. Using a stereotactic injector, 2.5 μL of 200 μM P40D680 was delivered to the right lateral ventricle over the course of 2.5 min in Prox1-GFP mice, a reporter line for lymphatic vasculature  glycol IRDye680 (P40D680), a pegylated NIR tracern = 5 each) to determine the pattern of spread of P40D680. As early as 10 min after the infusion, we observed that a portion of the intraventricularly infused tracer appeared to localize along the paravascular spaces of arteries within the SAS at the circle of Willis at the base of the brain with spreading within this space along the middle cerebral artery and its branches over the convexities of the cortical hemispheres  as they exit the skull  of the infused dose within the bloodstream at 60 min based on the calibration curve shown in Supplementary Fig. We infused 2.5 μL of P40D680 into the right lateral ventricle and after 5 min initiated noninvasive imaging of the saphenous vein with NIR stereomicroscopy Fig. . The ven38. As before, we infused P40D680 into the lateral ventricle and placed the Prox1-GFP mice under the stereomicroscope with a region of interest on the shaved neck skin  in the draining lymph nodes, which was significantly earlier than the increase at 25.3 ± 2.8 min representing the transit time to blood , IRDye680CW, and a 3 kDa dextran conjugated to AlexaFluor680 (3kDa-AF680). Initial experiments were performed to establish comparable doses for intraventricular infusion as well as to test the sensitivity of detection in the saphenous region. It was observed during these studies that although both IRDye680CW and 3kDa-AF680 were not retained within the blood compartment after intravenous injection, they leaked rapidly in sufficient quantities within the skin above the saphenous vein to be detected with high sensitivity  and 2-month-old (young) C57BL/6J-Tyrc-J albino mice. After 60 min, we observed less P40D680 signal in the saphenous vein of aged mice compared with the young controls  and 2-month-old (young) C57BL/6J-Tyrc-J albino mice. The infusion cannula was kept in place for 10 min to limit the backflow of tracer from the CSF and imaging was initiated at 15 min after the completion of the infusion. Once again, aged mice demonstrated a reduction in CSF outflow with lower signals apparent in the systemic blood at 60 min  through the cribriform plate to reach the nasal mucosa is the most important CSF lymphatic outflow route in several species45. Although we were able to confirm that this pathway was active, we also highlighted several other perineural routes that appear to complement the nasal route in mice. In agreement with previous literature, the perineural pathways along the optic (CN II) nerve through the orbit appeared to be important24. However, we have shown that the trigeminal nerve (CN V) also exhibited a perineural pattern and, therefore, may represent another pathway from the cranial SAS to the orbit. We also detected tracer emanating from the jugular foramina along the glossopharyngeal (CN IX), vagus (CN X), and accessory (CN XI) nerves, as well as outflow from the stylomastoid foramina along the facial nerve (CN VII), two locations that are in close proximity to the deep cervical lymph nodes. Interestingly, jugular outflow was detected in the early study in dogs by Schwalbe, however, these specific pathways through the base of the skull have otherwise been identified in only a few previous studies47. In sum, we found evidence of tracer exiting along several cranial nerves in mice after intraventricular infusion.Our study is, to our knowledge, the first to characterize in detail the lymphatic outflow pathways of CSF from the skull in mice , indicating that they are different from conventional lymphatic networks. One possible objection to a functional role for these vessels draining CSF is the presence of an arachnoid barrier layer between the CSF and the dura mater53. Similar to the barriers at the endothelium of the brain capillaries and at the epithelial lining of the choroid plexus3, there exists a barrier layer that consists of arachnoid cells containing tight junctions that serves to isolate the CSF within the SAS from the interstitial fluid of the dura mater53. As the blood vessels of the dura mater tissue are fenestrated4, it is possible that the dural lymphatic vessels exist to drain this tissue. A previous study found the presence of horseradish peroxidase in dural lymphatic vessels of the cat only under high injection pressures indicating that physical disruption of the arachnoid barrier must occur in order for CSF to reach the dura mater tissue50. Others have discussed that dural lymphatics must have a minor or accessory role to the main perineural outflow pathways54. Interestingly, anatomical studies have demonstrated lymphatic vessels in the dura around the optic and facial nerves and at the cribriform plate and jugular foramen, indicating that lymphatic uptake of CSF may occur as the cranial nerves exit the skull56. Clearly, more research is needed to determine the importance of the dural lymphatic route for CSF outflow in comparison to the more established perineural pathways to reach extracranial lymphatic vessels.The recent rediscovery of dural lymphatic vessels has attracted a great deal of attention on a role for a functional lymphatic system within the central nervous system35, with a delay in the appearance of the signal in blood followed by a steady linear increase; (2) the presence of significant levels of tracer within the lymphatic vessels and lymph nodes draining the skull before any appearance of signal in the blood; (3) the lack of any evident signal during direct imaging of the posterior facial vein which receives the blood from the transverse sinus in rodents40; and (4) the confirmation of an active pumping mechanism of collecting lymphatic vessels draining CSF that serves to propel the lymphatic fluid towards the venous system even under anesthesia. These results, indicating a lymphatic-predominant drainage of CSF, are largely in agreement with work from the group of Miles Johnston who reached a similar conclusion57. Starting in the late 1990s, a series of elegant quantitative studies in sheep and rats from this group demonstrated that radiolabeled tracers could be recovered in sufficient quantities from cannulated lymphatic vessels to account for over half of the tracers estimated within the bloodstream19. The robust outflow through lymphatic vessels under anesthesia observed in the current study is likely due to a favorable pressure gradient driving flow from the SAS within the rigid cranial cavity to the lymphatic vessels outside the skull. Measurements in other species have indicated that pressure within the SAS may be 3 or more times that of the cervical lymphatic system58. Although we have shown previously that lymphatic transport is reduced under anesthesia due to lack of uptake into initial vessels35, the pressure gradient between the skull and surrounding tissue may overcome the need for muscular movement that normally drives lymph formation. Nonetheless, the question whether CSF lymphatic outflow is increased or decreased during awake conditions remains to be answered.Our development of NIR dynamic imaging approaches has allowed real-time quantitative assessments of both lymphatic outflow and transport to blood of CSF-infused tracers that were not previously possible in animals without cannulation. The dynamic imaging results after CSF infusions of macromolecular and small molecular fluorescent tracers strongly suggest that lymphatic transport is the predominant CSF outflow pathway in mice. Supporting evidence for this conclusion is (1) the similarity of the CSF outflow pattern to patterns of lymphatic transport to blood from other tissues such as skin and peritoneal cavity59 or on ex vivo studies, which have shown in isolated arachnoid villi that particles as big as erythrocytes could pass through60. However, since a continuous lining of endothelial cells with tight junctions was found to exist on the villus11, it is still unclear how macromolecules are transported61. Previous studies have attempted to use the dynamics of accumulation of intraventricular-injected macromolecular tracers into the blood or urine as evidence of arachnoid villi transport64. In these studies there were often delays until tracer was apparent in the blood or urine compared to when the tracers were injected intravenously, similar to our findings of a delayed transport to blood. We were also unable to provide evidence of a direct route from the CSF to blood by dynamic imaging of the posterior facial vein that collects blood from the transverse sinus in mice within the first 30 min after infusion despite the presence of high levels of tracer within lymphatic vessels. These results are similar to an early cannulation study in dogs65. Studies from the group of McComb under various CSF pressure conditions were unable to detect increased tracer levels in the superior sagittal sinus compared to peripheral blood in rabbits, cats, and primates67. In rats, direct blood uptake was detected after intraventricular infusions of inulin or polystyrene beads only after reaching CSF pressures of seven to eight times normal levels68. Studies in sheep by the Johnston group determined that pressures of around threefold higher than normal levels were necessary to detect direct blood uptake13. Therefore, it appears that plenty of doubt exists regarding the role of the arachnoid projections in the drainage of fluid and macromolecules from the CSF in many species beyond mice, and that it may be time for a reexamination of this widely accepted concept. Although the present experiments cannot explicitly rule out a direct connection from CSF to blood, particularly one that is active at later time points or under high intracranial pressure, they are strongly indicative that the lymphatic outflow pathways are much more important than originally envisioned and that the lymphatic system may represent the major site between the CSF and blood compartments.These findings call into question the role of arachnoid villi or other possible direct venous connections as outflow routes of solutes from the CSF. It is perhaps not surprising, due to their location within the skull, that no direct in vivo proof of arachnoid villi uptake has ever been demonstrated. Supporting evidence for the importance of these structures draining CSF have mostly relied on examination of post-mortem tissue69. Since under homeostatic conditions, CSF inflow must equal CSF outflow, this decreased outflow in aged mice may be at least in part due to reduced CSF production by the choroid plexus or by sources from within the interstitial tissue such as the blood–brain barrier70. The choroid plexus has been shown to undergo age-related morphological alterations in its epithelial lining as well as a thickening of the basement membrane43. An additional explanation for the reduced CSF outflow observed during the current study could be previous data from humans and rats showing an increased CSF volume with aging, which would indicate that the infused tracer may have been more diluted in the CSF of aged mice71. It will be interesting to test whether a more accelerated decrease in CSF lymphatic outflow exists in mouse models of Alzheimer’s disease and whether a functional decline is associated with the development of amyloid beta plaques. If so, the lymphatic system may represent a possible new therapeutic target with the aim to enhance the clearance of toxic proteins from the CSF and the brain.To our knowledge, our study is the first to demonstrate a reduction of lymphatic outflow of CSF with aging. One previous study has shown less outflow of radiolabeled tracers to the nasal turbinates in aged rats but did not quantify outflow to the lymphatic system or the systemic blood72. Therefore, quantification of CSF outflow after intrathecal administration of NIR tracers in patients suffering from neurological disorders may be possible.In conclusion, we have identified many perineural sites for the egress of CSF from the cranium of mice. Most importantly, through NIR dynamic imaging, we have presented evidence that these outflow pathways to reach the lymphatic system appear to represent the major exit routes for both macromolecular and small molecule tracers from the CSF, rather than the commonly accepted venous route. Although demonstration of this phenomenon in humans still awaits, this study sets the stage for investigation of a potentially important role for the lymphatic system in a multitude of CNS-related pathologies. In addition, there may be potential for clinical translation of the imaging technique since noninvasive monitoring of NIR tracers in the blood already exists in the clinicTyrc-J albino mice  and Prox1-GFP mice73 on the C57BL/6J background were kept under specific pathogen-free conditions and used for experimental studies at the age of 2–3 months. For aging studies, female C57BL/6J-Tyrc-J mice were aged in-house to 18 months of age and compared with young animals from the same colony. After the completion of imaging experiments, the mice were euthanized with an overdose of anesthesia .All mouse experiments were approved by Kantonales Veterinaramt Zurich . Female C57BL/6J-32 , 0.6% Evans blue , IRDye680CW NHS ester , or 3kDa-AF680 (Thermo Fischer) at the speed of 1 µL/min was then performed with a syringe pump . The needle was left in place for 2.5 min and then slowly removed while observing if any significant backflow occurred.Mice were anesthetized  and fixed in a stereotaxic frame . The skull was thinned with a dental drill (RWD) at a location 0.95 mm lateral and 0.22 mm caudal to the bregma. A 33 G steel needle was inserted into the right lateral ventricle 2.35 mm ventral to the skull surface. Infusion of 2.5 µL of 200 μM P40D680 tracer74. After a small skin incision over the occipital bone/cervical spinal cord was performed, the three covering muscle layers were carefully dissected under a stereomicroscope using fine forceps and scissors. Subsequently, the atlanto-occipital membrane (AOM), covering the cisterna magna, was pre-punctured using a 34 G cannula. The infusion cannula was then inserted through this opening to a depth of 250 µm. A droplet of histoacryl was added to the interface of cannula and AOM to avoid reflux of CSF/tracer during the infusion. Overall, 5 µL of 100 μM P40D680 tracer was infused at the speed of 1 µL/min. After the infusion, the cannula was left in place for 10 min to avoid reflux. After withdrawal of the cannula, a droplet of histoacryl was finally added to ensure no CSF fistula.For cisterna magna infusion, mice were anesthetized as above and a surgical procedure to access this structure was performed35, fur above the saphenous vein region was shaved with a razor and depilation cream before the ventricular or cisternal infusion. After removal of the needle from the skull or the cisterna magna, the mice were quickly positioned under a Zeiss StereoLumar.V12 stereomicroscope with AxioVision software  and a Photometrics Evolve 512 camera  in a supine position on a heating pad to maintain body temperature. The autofluorescence signal on the GFP channel was used to position the saphenous blood vessels at ×25 zoom. Dynamic imaging was initiated 5 min after the completion of the ventricle infusion and 15 min after completion of the cisterna infusion by acquisition of a sequence of images (1 image every 15 s for 55 min) with a Cy5 filter set to monitor the NIR signal of the saphenous vein. Exposure time and camera gain settings were 200 ms and 200, respectively.For noninvasive imaging of tracer signals in blood32 in the shaved neck region, the ventricular infusion procedure was the same as above and image acquisition was performed with the following modifications: magnification of the imaging was at ×6 zoom on the neck region and dynamic imaging was performed for 25 min with a sequence of 1 image per 1 s.For noninvasive imaging of the lymphatic flowFor noninvasive imaging of potential tracer outflow to the posterior facial vein, the same ventricular infusion procedure was followed, but immediately afterwards, an incision to the lateral skin of the head was made to expose the junction of the internal maxillary vein with the posterior facial vein. Dynamic imaging was initiated at 10 min after injection and continued for 20 min with 1 image acquired every 15 s at ×22 zoom.t = 60 min, the linear slope of signal enhancement from t = 45 to 60 min after infusion in counts/min and the transit time in min of the arrival of tracer to the blood circulation .Using AxioVision software, a circular region of interest of radius 100 μm was placed over the saphenous vein on the acquired videos. Using the Measure Profile function, a table of fluorescence intensity in counts vs. time was exported into Microsoft Excel. As there was a loss of signal at the beginning of the scans due to photobleaching of tissue autofluorescence, baseline intensity in counts was calculated as an average signal of the lowest ten consecutive imaging frames. This baseline intensity was then subtracted from the fluorescence intensity values to plot fluorescent signal enhancement vs. time in min. For quantification of transport to blood, three assessments were made from the data of signal enhancement vs. time. These were the NIR fluorescent signal enhancement value in counts at t = 25 to 30 min after infusion and the threshold for signal enhancement to determine the transit time was set to 300 counts. The quantifications of one lymph node on each side were averaged to generate one value per mouse.For assessment of transport to mandibular lymph nodes, the procedure was the same excepting that the circular region of interest was of radius 500 μm over the mandibular lymph node. The linear slope was calculated as the signal enhancement from 33. In short, in AxioVision software, on each video of dynamic imaging of mandibular lymph nodes, a region of interest of radius 200 μm was drawn over the afferent collecting vessel on each side of the mouse. The data in mean fluorescent intensity values over time was then exported in .xml format. Contractility measures were determined for a 5 min period between 25 and 30 min after infusion using a Matlab algorithm for the frequency in contractions per min. Values from the two vessels were then averaged to obtain one value for each measure per mouse.Contractility was assessed based on our published methodsA custom-designed catheter of polyethylene (PE)—10 tubing  and a 30 G needle was placed into the tail vein of C57BL/6 J mice. An infusion pump  connected to the catheter was used for consistent infusions of tracer. For demonstration of the linear relationship between tracer dose and venous signal, either 10 μL of the P40D680 tracer (at 1 μM) or Evans blue (at 0.003%) was infused every 3 min using a stepwise infusion program. To determine the threshold amounts for detection in blood, P40D680 was diluted to 0.05 μM and Evans blue to 0.00015%, and infused in a similar manner. The number of infusions before increasing signal was detected in the blood was determined to be the threshold for detection.n = 5 mice at time points of euthanization at 10, 30, and 60 min after intraventricular infusion of P40D680. Images of P40D680 tracer spread on the surfaces of the brain, perineural exit points, and within the lymphatic vasculature were acquired with a Zeiss AxioZoom V16 microscope and a QImaging OptiMOS sCMOS camera  combined with a light-emitting diode illumination system pE-4000 . To compare signals within lymph nodes at different time points, images were acquired at identical zoom factors (×25), and exposure times (200 ms). As there were no consistent differences in signal in the lymph nodes on the injected and contralateral sides, the average value of the nodes on each side was used.Anatomical mapping of the outflow pathways in Prox1-GFP mice was performed in three groups of Mouse brains were dissected and fixed in 4% PFA at 4 °C overnight. A section of the brain between 1 mm cranial and 1 mm caudal to the needle insertion site was cut out by razor blade and imbedded with 2% low melt agarose (BIO-RAD). Cross sections were made from dorsal to ventral side at a thickness of 100 µm with Vibrating blade microtome (Leica VT1000 S). Images were acquired with a Zeiss AxioZoom V16 microscope.t-test. Means of three groups were compared with one-way ANOVA with the Tukey’s multiple comparison post hoc test. All analyses were performed using GraphPad Prism V5.0  and p < 0.05 was accepted as statistically significant.For the studies to assess paravascular spread and CSF outflow at different time points, mice were randomly allocated to different experimental groups. Owing to the nature of the aging studies, neither randomization nor blinding of investigators was possible. Group sizes were estimated based on pilot studies to determine the success rate and reproducibility of the intraventricular and cisterna magna infusions. All data are presented as mean ± SD. All groups were found to be normally distributed using the Kolmogorov–Smirnov (K–S) test. Means of two groups were compared using a two-tailed Student’s The data that support the findings of this study are available from the authors upon reasonable request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5


According to targeted treatment (TT), the whole flock is dewormed based on knowledge of the risk, or parameters that quantify the mean level of infection, whereas according to targeted selective treatment (TST), only individual animals within the grazing group are treated, based on parasitological, production and/or morbidity parameters. The aim of this study was to compare two different treatment protocols on sheep farms in Lithuania. The study was conducted from 15 April to 31 October 2014 on three sheep farms. On the TT (the whole flock) and T(S)T  farms all adult animals were treated orally with fenbendazole irrespective of EPG counts before the grazing season. The second treatment was applied with injectable ivermectin on both farms. However, on the TT farm all sheep were also treated on 2nd August regardless of their EPG counts, while on the T(S)T farm only those animals with an EPG ≥ 300 were treated on 1 July using a threshold of ≥ 300 EPG. No treatments were administered on the control farm (n = 1) during the study.3 stage larvae at the start of grazing season, while it remained significantly higher on the control farm. The positive effect of the spring treatment of ewes was reflected by increased body weight gains (BWG) in lambs in the first half of the grazing season. Following the second treatment, the weight gains in lambs on the T(S)T farm were higher compared to lambs on the TT farm, while BWG in the control lambs started to decrease. The difference was also substantiated by the body condition scores (BCS) and dag scores (DS) of lambs, which were highest on the T(S)T farm compared with those on the control and TT farms.Spring treatment of ewes significantly reduced nematode faecal egg counts (FEC) both on the TT and T(S)T farms, with the benefit of lowering pasture contamination with infective Lin refugia, helping to slow down the development of anthelmintic resistance (AR) in future.The results of this study show that both treatment strategies were useful in reducing clinical effects (BCS and DS) of gastrointestinal nematode parasitism and increasing the performance in lambs. Furthermore, on the T(S)T farm some of animals were left  Due t refugia . More sp refugia , 21. Basone time . Accordione time . Accordione time . Howeverone time –28.versus a T(S)T) and to compare these with the situation on an untreated control farm using parasitological (FEC), production (BWG) and morbidity markers  in growing lambs.The aim of this study was to compare and to evaluate two different treatment systems on two sheep farms ) in the southern Lithuania were enrolled. On the TT and T(S)T farms, different treatment regimens were applied based on FEC, while no treatments were administered on the control farm during the course of the study. The T(S)T strategy consisted of: first a TT of the whole flock (before turn-out in spring) and then a TST but only of those animals with trichostrongylid FEC ≥ 300 eggs/g faeces (EPG) in early July. The size of the flocks was 24, 57 and 28 animals, respectively. The farms were comparable in terms of grazing density , sheep breeds , location (the distance between the farms was max. 15 km) and a set stocked grazing system (i.e. no pasture rotation).3 larvae.On all three farms the lambs were grazing with ewes from 15 to 25 April until the end of October. The farms were visited every 2 weeks and individual faecal samples were collected from the rectum of all animals on the farms on each occasion. The lambs were weighed, and their BCS and DS  was also estimated. Three replicates of approximately 400 g samples of herbage were collected from each pasture to determine the contamination with L® 1%, Poland) twice a year (spring and autumn) before turn-out and at housing according to the manufactures recommendations.On the TT farm, 24 mix-breeds of Romanov and Lithuanian black-headed sheep (10 adult and 14 lambs) grazed on the 2.3-hectare pasture (339 kg/ha). In the previous 4 years the animals had been treated with injectable ivermectin (BiomectinThe T(S)T farm, had 57 (21 adult and 36 lambs) Lithuanian black-headed, Romanov and crossbreeds with Berrichon du Cher animals grazed on the 5.7-hectare pasture (371 kg/ha), which during the previous 8 years had been treated with injectable ivermectin as described above.On the control (C) farm 28 animals , mostly Romanov and crossbreeds, grazed on the 3-hectare pasture (273 kg/ha). The animals had not been treated with anthelmintics the previous 6 years.The age of lambs at the start of the study was 4–8, 2–12 and 4–8 weeks on the TT, T(S)T and C farms respectively. A total of 8 lambs both in the TT and T(S)T flocks, weighing 35–50 kg, were withdrawn from the study for slaughter from early August.Fasciola and lungworm infection on two occasions (April and September). In addition, the parasites on all farms were examined before the study for the presence of ivermectin and benzimidazole resistance using in vitro methods [On all three farms the mating season began at the end of August or October. The animals on all farms were examined for the presence of  methods , 17. It The study was performed in compliance with Lithuanian animal welfare regulations  and was approved by the Lithuanian Committee of Veterinary Medicine and Zootechnic Sciences (Protocol No. 07/2010).Moniezia infection, all the adult animals were treated orally with fenbendazole at a dose rate of 7.5 mg/kg of body weight . This was done before the grazing season (at the end of April) on both the TT and T(S)T farms and irrespective of EPG counts.Faecal samples were collected from both ewes and lambs and the average EPGs were calculated for each farm. As a part of the trichostrongylid control programme and due to ® 1%, Poland) subcutaneously behind the scapula at a dose of 0.2 mg/kg body weight on both farms. On the TT farm all ewes and lambs were treated on 2nd August regardless of their EPG counts, while on the T(S)T farm only those animals with an EPG ≥ 300 were treated on 1st July.The second treatment was applied with injectable ivermectin  and a diagnostic sensitivity of 20 EPG , 30.3) were recovered from the coprocultures using a Baermann technique [3 were identified to species or genera by microscopy according to [On each sampling occasion faecal cultures were prepared in triplicates separately for ewes and lambs on each farm. Ten grams of faeces were mixed with 10 g of vermiculite and incubated for 7 days at 27 °C meanwhile water was added to maintain an adequate moisture level. Then third-stage larvae  were recorded for lambs, as described by Campbell et al. . Dag sco® Excel 2007. The differences in FEC, weight gains in lambs, dag scores and body condition scores between farms were analysed using a repeated measures analysis of variance (ANOVA) in BMI SPSS Statistics 21 version.Descriptive statistics were calculated using MicrosoftData on monthly precipitation and average temperatures were obtained from the meteorological station situated 5–15 km from the examined farms.P < 0.05). Furthermore the number of trichostrongyle eggs in ewes has increased significantly (P < 0.01) to a level of 978 ± 1415 EPG in July on the untreated C farm compared to those on the TT and T(S)T farms. Meanwhile the FEC were very low in ewes both on the TT and T(S)T farms, and never exceeded 28 ± 31 and 63 ± 127 respectively T and C farms, respectively. Thereafter FEC decreased in the first half of May in the control ewes, reaching its lowest level of 340 ± 437 EPG in the middle of June in comparison with TT and T(S)T farms (P < 0.001) when compared to those on the TT and T(S)T farms. In contrast, there were no significant differences in FEC between the TT and T(S)T farms in ewes and lambs. Nematodirus spp. eggs were in general consistently low and did never exceed 100 EPG on the C and TT farms. The only exception was on the T(S)T farm in June, with numbers exceeding 300 EPG in five animals.At the start of the study, no nematode eggs were found in the lambs on any of the farms Fig. . EPG couTeladorsagia (54.9% vs. 46.7%), Trichostrongylus (11.6% vs. 21.4%), Cooperia (15.9% vs. 20%), Haemonchus (8.7% vs. 6.3%), Oesophagostomum (6.8% vs. 4.2%) and Chabertia (2.1% vs. 1.4%) in ewes and lambs, respectively. Teladorsagia was the dominant genus on all farms. The highest prevalence of Teladorsagia were found in May in the ewes and lambs on the C farm (76% vs. 82%), on the TT farm (52.3% vs. 46.7%) in June and on the T(S)T farm (60.7% vs. 58%) in August. Shedding of Haemonchus eggs started in June and the highest prevalence was recorded on the C farm in ewes (29.4%) and lambs (30.7%) in October.The trichostrongyle nematode population consisted mainly of 3 stage larvae was low (118–178 L3/kg of dry grass) on all farms  and T(S)T (1206 L3/kg of dry grass) farms, while pasture contamination on the C farm consistently increased during the grazing season to a peak value of 767 L3/kg of dry grass in early September. Infective larvae on pastures of the TT, T(S)T and C farms consisted of Teladorsagia , Nematodirus , Trichostrongylus , Cooperia , Haemonchus , Oesophagostomum  and Chabertia  respectively.In early May the number of infective LDuring the study rainfall was elevated, with precipitation of around 83, 84 and 116 mm when compared with an average value of 54, 63 and 74 mm in May, June and August respectively. The temperature was slightly elevated (20.9 and 18.5 °C) on all farms when compared with an average value of 17.1 and 16.2 °C in July and August respectively. There was a constant snow cover on the pastures from the end of December until the end of March before the study.P < 0.001) higher (0.27 kg/day) on the T(S)T farm. In July BWG decreased to 0.1, 0.06 and 0.06 kg per day in the TT, T(S)T and control farms respectively. From then on the BWG recovered in all groups to a level of 0.26, 0.35 and 0.24 kg per day in the TT, T(S)T and C farms respectively in the first half of August, followed by a decrease towards the end of the grazing season  and the TT farm (0.18 kg/day) during the first 2 weeks of May, whereas it was significantly (P < 0.05), while they remained comparable (0.15 kg/day vs. 0.14 kg/day) in the animals on the TT farm. Meanwhile the average BWG has decreased (0.13 kg/day vs. 0.12 kg/day) on the C farm. Accordingly, the average BWG were significantly higher both on the T(S)T (P < 0.001) and TT farms (P < 0.01) than on the C farm.Following the second treatment, the average BWG increased from 0.16 to 0.24 kg/day on the T(S)T farm (P > 0.05) from 3.4 to 3.8 ± 0.12 in lambs, while after some fluctuations BCS remained at 3.1 in the lambs on the TT farm. The average BCS in ewes followed a similar pattern to those of lambs. Following the second treatment the BC score improved gradually from 3.1 to 3.3 ± 0.25 in ewes on the TT farm, while BCS remained at a comparable level of 3.9 ± 0.55 on the T(S)T farm. The average BCS was significantly (P < 0.05) higher on the T(S)T and TT farm when compared to those on the C farm.Average BCS in lambs were 3.1 ± 0.28, 3.59 ± 0.32 and 2.89 ± 0.23 on the TT, T(S)T and C farms respectively. Following the second treatment on the T(S)T farm, the BCS has improved gradually (At the start of the study the average DS on the C farm were high both in lambs (3.2 ± 0.5) and ewes (3.4 ± 0.5), while animals on the TT and T(S)T farms were in general cleaner. Both on the TT and T(S)T farms DS remained negligible throughout the study and never exceeded 2 (lightly dagged). The only exception was on the T(S)T farm where some lambs had the highest dag score (3) starting from August until the end of grazing season. However, the average dag score in lambs on the T(S)T farm remained below 2. On the C farm at least one animal had a dag score of 3 or 4 (extensively dagged), including lambs and adult animals at every estimation point during the grazing season.in refugia and thereby slow down the selection for anthelmintic resistance [in refugia is now considered one of the most important factors in slowing down the development of AR and should be included in any potential prophylactic control programme suggested for nematode parasites [Traditionally a strategic treatment approach with up to two to three treatments per year in ewes is used on most Lithuanian sheep farms , 22. Thesistance . Maintenarasites . In the Teladorsagia survive on pasture during the winters in Lithuania. This results in an early pasture contamination with infective L3, as has previously been recorded in Lithuania on pastures grazed by sika deer [The larvae of ika deer  and goatika deer . The ini, Teladorsagia was the most prevalent genus, irrespective of the farm and age of animals. The highest concentration of Teladorsagia L3 larvae were isolated from grass sampled in May. Infective larvae of Haemonchus were only observed on the control farm , resulting in consistently low (12.1%) pasture contamination during the study.In the present studyP < 0.001) on the TT (283 ± 17 EPG) and T(S)T (378 ± 31 EPG) farms. After treatment of ewes in the spring FEC decreased and never exceeded 28 ± 31 and 63 ± 127 on the TT and T(S)T farms respectively. As anticipated before the study, the spring treatment resulted in lower pasture contamination with L3 both on the TT and T(S)T farms than on the C farm, thus preventing larval exposure resulting in high infection levels in lambs. However, grass height was significantly reduced due to low precipitation and elevated temperatures on the T(S)T and TT farms in July, resulting in a significant increase in pasture contamination for a short period in the middle of July. This could also partly be attributed to higher grazing pressure on both farms when compared to those on the control farm.At the start of the study, we observed a dramatic increase in FEC of the ewes. This rise was very pronounced especially on the C farm (3440 ± 5865 EPG), while it was significantly lower (P < 0.001) due to continuous pasture contamination. On the C farm FEC increased steeply (P < 0.001) until the end of July, reaching a peak of 3500 ± 6950 EPG. However, this was attributed to a few lambs on the C farm having extremely high EPG counts . The dominant GIN genera on the control farm in lambs during July were Teladorsagia but to a lesser extend also Haemonchus. However, due to low infection level the clinical signs (anaemia) related to Haemonchus infection in lambs was not recorded on C farm.The clinical manifestation of trichostrongylid infection can usually be observed in young kids, since a fully expressed immune response against GIN normally appears at the age of 12 months [P < 0.05) both in the ewes (845) and lambs (1109) on the C farm.On the TT farm, all animals were treated a second time on 2nd August irrespective of EPG counts. In contrast, the need for a second treatment was assessed based on FEC on the T(S)T farm and therefore only animals with ≥ 300 EPG were treated. Following the second treatment in August, the EPG on the TT farm decreased and remained below 33 for the remainder of the study. On the T(S)T farm, 14.3% of ewes and 25.0% of lambs with EPG ≥ 300 were treated in July. However, the average FEC on the TT (32.7 and 93.5) and T(S)T farms (60.5 and 79.2) were comparable at every sampling occasion, while it was significantly higher (P < 0.001), followed by those on the TT (P < 0.001) farm, when compared to those on the C farm. Due to the low grazing pressure, the pasture contamination with L3 was low but also delayed on the C farm. However the control animals still developed clinical signs of GIN infection. The spring treatment of the ewes on the TT and T(S)T farms clearly had a significant impact and reduced the shedding of nematode eggs on both farms. Thus, this had a beneficial impact and lowered the pasture contamination with infective L3 at the start of the grazing season, while it remained significantly higher on the C farm. The positive effects of the spring treatment of ewes were also reflected in increased BWG in lambs during the first half of the grazing season. Following the second treatment BWG in lambs on the T(S)T farm peaked (from 0.16 to 0.24 kg) (P < 0.05) when compared to lambs on the TT farm, while the control lambs gained weight but at a slower rate. These differences were also substantiated by the BC scores of lambs, which were highest on the T(S)T farm when compared to those on the control and TT farms (P < 0.05).Despite differences in the quality of pasture and grazing density, the average BWG at housing were highest in lambs on the T(S)T farm (in refugia. T(S)T also resulted in 77% less anthelmintics being used compared to the TT farm, which in itself may slow down the selection for AR. However, more precise studies are required involving larger flocks on comparable pastures before any final recommendations for the application of T(S)T strategies can be made.The results of this study show that targeted (selective) treatment strategy is useful in reducing the clinical effects of gastrointestinal parasitism and increased the performance especially of the lambs. On the T(S)T farm some animals were left


Overall, although this represents a significant utilization of kidneys with KDPI >85% over time (p<0.001), recent years have seen a decline in usage, probably related to regulations imposed by Centers for Medicare & Medicaid Services (CMS).There has been increasing concern in the kidney transplant community about the declining use of expanded criteria donors (ECD) despite improvement in survival and quality of life. The recent introduction of the Kidney Donor Profile Index (KDPI), which provides a more granular characterization of donor quality, was expected to increase utilization of marginal kidneys and decrease the discard rates. However, trends and practice patterns of ECD kidney utilization on a national level based on donor organ quality as per KDPI are not well known. We, therefore, performed a trend analysis of all ECD recipients in the United Network for Organ Sharing (UNOS) registry between 2002 and 2012, after calculating the corresponding KDPI, to enable understanding the trends of usage and outcomes based on the KDPI characterization. High-risk recipient characteristics  kidneys with KDPI >85%, with a hazard ratio (HR) of 0.95, a confidence interval (CI) of 0.94-0.96, and statistical significance of p<0.001. This indicates that some SCD kidneys may actually have a lower estimated quality, with a higher Kidney Donor Risk Index (KDRI), than some ECDs. The incidence of delayed graft function (DGF) in ECD recipients has significantly decreased over time from 35.2% in 2003 to 29.6% in 2011 (p=0.007), probably related to better understanding of the donor risk profile along with increased use of hypothermic machine perfusion and pretransplant biopsy to aid in optimal allograft selection.The recent decline in transplantation of KDPI >85% kidneys probably reflects risk-averse transplant center behavior. Whether discard of discordant SCD kidneys with KDPI >85% has contributed to this decline remains to be studied. The existing donor supply of kidney allografts is unable to meet the growing demand of patients awaiting transplantation. Since 2002 the number of candidates on the kidney transplant waitlist has nearly doubled from just over 50,000 to more than 96,000 by 2013. Although more than 15,000 patients on the waiting list underwent a kidney transplant in 2013 in the United States (US), more than 7,000 candidates were removed from the waiting list due to death or becoming too sick to undergo transplant -6; howevIn 2012 the Organ Procurement and Transplantation Network (OPTN) introduced KDPI, which compiles 10 donor factors (instead of four in the ECD definition) that are independently associated with all-cause allograft survival associated with the use of that particular organ. A donor KDPI >85% (DK85) is thought to be equivalent to an ECD kidney. KDPI was the basis of the new allocation policy introduced toward the end of 2014 to increase utilization of marginal kidneys and decrease the discard rates that would be expected using the older ECD classification , BMI (kg/m3. Transplant-related characteristics: Cold ischemia time (CIT) in hours, hypothermic machine perfusion  of donorThe proportion of missing data for the KDRI (60% of records), recipient cerebrovascular disease (36% of records), and recipient hypertension (35% of records) precluded the use of these parameters in the multivariable analyses.Endpoints for analysis included:-KDPI >85% utilization rate as a proportion of all cadaveric kidney transplantations.-DGF defined as the need for dialysis within the first seven days post transplantation.-All-cause graft failure defined as patient death or graft loss , whichever came first for any reason at any time during follow-up. -Graft survival calculated from the date of transplantation until the date of graft failure. Patients alive with a functioning graft were censored at the date of last known follow-up.-Overall patient survival calculated from the date of transplantation until the date of patient death. Patients alive were right censored at the date of last known follow-up.Baseline characteristics were described for the study population stratified by the year of transplantation. Normally distributed continuous data are presented as mean and standard deviation (SD), with median and interquartile range (IQR) for non-normal distributions. Categorical data are presented as counts and percentages. The Cochran-Armitage trend test was used to assess the presence of an association between binary categorical variables  and year of transplantation. Trends in continuous variables were assessed using ordinary least squares linear regression model; for categorical variables defined in more than two categories , a multinomial regression model was utilized.Multivariable binary logistic regression modeling was used to assess the simultaneous effect of year of transplantation on the risk of DGF while adjusting for donor-, recipient- and transplant-related risk factors. Odds ratio (OR) and 95% CI were calculated as measures of strength of association and precision, respectively.Graft and patient survival rates were estimated using the Kaplan-Meier method and compared between years by the log-rank test. Multivariable Cox proportional hazards modeling was used to assess the simultaneous effect of year of transplantation on the risk of graft failure, and separately for risk of patient death while adjusting for donor-, recipient-, and transplant-related risk factors. HR and 95% CI were calculated as measures of strength of association and precision, respectively. Transplantation in 1977 [A modified Bailey-Makeham model for risk of graft failure was used to describe rates of graft loss from the time of transplant. We used the graphical representation of the modified model which is a parametric model based on work published in  in 1977 . This moThe study population includes all adult recipients (age ≥ 18 years) of a deceased donor, kidney-only transplant between January 1, 2000, and December 31, 2012. All living donor transplant recipients, recipients of pediatric kidneys, multi-organ transplants, or recipients with a prior kidney transplant were excluded from the study. Study period includes three different eras: Era One, 2000-2002; Era Two, 2003-2006, which reflects changes in the allocation policy with the implementation of ECD kidneys in 2002; and Era Three, 2007-2012, which reflects implementation of the CMS program evaluation initiative in 2007. The use of ECD kidneys with KDPI >85% has significantly decreased over the years in comparison to the use of ECD kidneys with KDPI <85%, with 89.1% in Era One compared to 84.7% in Era Three. The proportion of donors with a history of diabetes and hypertension has increased between the eras (p<0.0001). Donor BMI has increased across the eras; in Era Three, 15.6% of donors had BMI >35 compared to 12.6% in Era One. Use of donor after cardiac death (DCD) kidneys significantly increased in both the KDPI groups across all three eras.Several donor characteristics were significantly associated with the eras of transplantation as seen in Table Trends in donor characteristics that defined the allografts as ECD are provided in Figure 2 increasing from 7.0 % in Era One to 10.3% in Era Three (p<0.001). Recipients of kidneys with KDPI >85% have become increasingly older over time, as seen in Table Donor kidney preservation techniques have changed over time. The use of hypothermic machine perfusion for the preservation of kidneys with KDPI > 85% increased from 56% in Era Two to 62.3% in Era Three (p<0.001). Kidneys with KDPI < 85% also saw an increased use of machine perfusion from 33.6% to 40.8%. The proportion of pretransplant biopsies increased from 83.9% in 2003 to 90.2% in 2011 (p<0.001). The mean CIT and % CIT >24 hours were not significantly different between the three eras. Median recipient wait time increased 35% from 679 days in 2003 to 915 days in 2011 (p<0.001). Table The rates of use of lower-quality kidneys were examined by era, taking into consideration both ECD and KDPI >85% designations. As seen in Figure  Figure Using time series analysis, the change in the trend of ECD utilization before and after 2008 was significant (p<0.001). To better characterize this decline, when a linear regression trend line was derived between the years of 2003 and 2008, the projected value of ECD kidney use was estimated at 23.8%. However, the actual 2011 ECD utilization rate was down to 17.7%, representing an 18% decline from the projected value of 23.8%, as seen inThe proportion of ECD transplants has increased from 18% in 2003 to a peak of 20.4% in 2008, and then declined thereafter to 17.3% in 2012.  In terms of KDPI >85%, transplants increased from 9.4% in 2003 to a peak of 12.1% in 2008 and declined to 9.7% in 2012  as shown in Figure .When graft survival is plotted with unadjusted Kaplan-Meier graft survival curves, a significant improvement in graft survival across the three eras is observed for the entire cohort (i.e. ECD and KDPI < and > 85%),as shown in Figure 8The one-, three- and five-year graft survival rates have also increased across the three eras with the use of kidneys with KDPI >85% Table .However, as expected, when comparing the hazards of graft failure by KDPI, ECD kidney with KDPI >85% has a slightly lower risk of graft failure compared to SCD kidney with KDPI >85%, with a HR of 0.95, probably related to the possibility that some SCD kidneys with a lower estimated quality (higher KDRI) have poor outcomes than some ECDs  and an inability to differentiate between kidneys of similar KDPI values . While KThe results reported here should be interpreted in the context of the limitations of the study design. This analysis was retrospective in nature; therefore, unmeasured confounders may exist. In addition, data is derived from hundreds of centers, often relying on manual data input and possibility of improper documentation or chances of missing data. Modeling techniques may not entirely account for differences in donor and recipient risk factors when comparing graft and patient survival by year of transplant. Additionally, information regarding discarded ECD kidneys is unavailable in our dataset, precluding further evaluation of causative factors underlying ECD utilization trends. The decision to use the dataset from 2002-2012 was based on its availability at the time of our prior analysis of ECD kidneys. Using the same dataset, we calculated KDPI for each ECD kidney followed by subsequent analysis. Also, using this dataset, there was enough data for observation and reporting one-, three-, and five-year survival outcomes. We do agree that analyzing an additional two years of data using kidneys with KDPI >85% may make more recent experiences available for the utilization of these high KDPI kidneys, considering changing donor age and risk profiles and increased frequency of use of machine perfusion for preservation. Additional studies may also be required to further elucidate on allograft outcomes using SCD kidneys with KDPI >85%.Transplantation outcomes using marginal kidneys have improved over time, despite worsening recipient characteristics and donor risk profiles. Despite these findings, the rate of usage of kidneys with KDPI >85% has declined in recent years. The introduction of CMS regulations that penalize centers for poor outcomes has possibly resulted in risk-averse transplant center behavior reflecting an increase in the discard of marginal kidneys with KDPI >85%. This current study analyzes the trends in utilization and outcomes of donor kidneys using KDPI categorization and may offer some understanding and guidance for use of marginal kidneys, thus preserving this valuable resource in the face of an ever-growing population of end-stage renal disease.


In this study, we investigated the impact of the single nucleotide polymorphism rs6897932 in the IL-7 receptor α-chain (IL-7Rα) which has previously been associated with several autoimmune diseases. We included 460 patients undergoing allogeneic HSCT after a myeloablative conditioning. Patients had a median age of 26.3 years (0.3–67.0 years), and 372 (80.9%) underwent HSCT for malignant diseases. Donors were matched sibling donors (n = 147), matched unrelated donors (n = 244) or mismatched unrelated donors (n = 69), and the stem cell source were either bone marrow (n = 329) or peripheral blood (n = 131). DNA from donors was genotyped for the IL-7Rα single nucleotide polymorphism (SNP) rs6897932 using an allele-specific primer extension assay . The donor T allele was associated with a higher risk of grades III–IV aGVHD  and with significantly increased risk of extensive cGVHD  after adjustment for potential risk factors. In addition, the TT genotype was associated with a higher risk of cytomegalovirus (CMV) infection post-transplant . Numbers of T cells were significantly higher on day +60 in patients receiving a rs6897932 TT graft . Donor heterozygosity for the T allele was associated with inferior overall survival  and increased treatment-related mortality , but was not associated with the risk of relapse (P = 0.35). In conclusion, the IL-7Rα rs6897932 genotype of the donor is predictive of aGVHD and cGVHD, CMV infection, and mortality following HSCT. These findings indicate that IL-7Rα SNP typing of donors may optimize donor selection and facilitate individualization of treatment in order to limit treatment-related complications.The efficacy of allogeneic hematopoietic stem cell transplantation (HSCT) is challenged by acute and chronic graft-versus-host disease (aGVHD and cGVHD) and viral infections due to long-lasting immunodeficiency. Interleukin-7 (IL-7) is a cytokine essential for  Allogeneic hematopoietic stem cell transplantation (HSCT) is a treatment of high-risk leukemia and a number of benign hematological disorders. In the treatment of leukemia, the outcome of HSCT is based on an immune-mediated cytotoxic attack on the malignant cells and persisting immune surveillance, also known as the graft-versus-leukemia effect. However, the success of HSCT is limited by long-lasting T cell dysfunction with risk of severe infections and development of graft-versus-host disease (GVHD), both contributing significantly to non-relapse mortality –3. More de novo T cell development in the thymus and homeostatic peripheral expansion of T cells (Interleukin-7 (IL-7) is a hematopoietic cytokine essential for  T cells –6. IL-7  T cells –12.Interleukin-7 receptor α-chain is expressed on lymphocyte progenitors and on naïve and memory T cells, and its expression is strictly regulated during the different developmental stages of T cells with the highest expression on naïve T cells, a lower expression on memory T cells, and downregulation of IL-7Rα upon development into effector T cells , 13. Thers6897932 SNP in the transmembrane region of the IL-7Rα increases the risk of developing multiple sclerosis, ulcerative colitis, and sarcoidosis (Single nucleotide polymorphisms (SNPs) in the exons of the IL-7Rα, which give rise to non-conservative amino-acid substitutions, have been associated with several chronic inflammatory diseases. The coidosis –20. In acoidosis –23.rs6897932 influences the rate of immune reconstitution after allogeneic HSCT with impact on infections as well as acute GVHD (aGVHD) and chronic GVHD (cGVHD).In this study, we show that the donor genotype in IL-7Rα We retrospectively studied patients undergoing allogeneic transplantation at the national HSCT center at Copenhagen University Hospital Rigshospitalet, Denmark, from 2004 to 2014. Inclusion criteria were first allogeneic HSCT, myeloablative conditioning , a matchFive-hundred twelve patients fulfilled the inclusion criteria. Deposited donor blood samples were available for 471 of these, and a donor SNP could be assigned for 460 patients (89.8%), which were all included in the study. The included patients did not differ significantly from non-participants in terms of age, diagnosis, donor, conditioning regimen, graft type, cell dose/kilogram, pre-transplant Karnofsky score, sex-mismatch, or cytomegalovirus (CMV) antibody status.The study protocol was approved by the ethics committee of the Capital Region of Denmark (#H-15006001), and written informed consent was obtained from all patients and/or their legal guardians.n = 136), acute lymphoblastic leukemia (n = 118), myelodysplastic syndrome (n = 51), chronic myeloid leukemia (n = 39), other malignancies (n = 28), or benign diseases . Donors were either fully human leukocyte antigen-A, -B, -C, -DR, and -DQ allele-matched sibling donors (n = 147), matched unrelated donors , or mismatched unrelated donors . Bone marrow (n = 329) or G-CFS mobilized peripheral blood stem cells (n = 131) were used as stem cell source, and allografts were T cell replete. Conditioning regimens consisted of total body irradiation (TBI) plus cyclophosphamide or etoposide (n = 293), cyclophosphamide plus busulphane (n = 107), or other types of chemotherapy-based conditioning (n = 60). Conditioning included anti-thymocyte globulin (ATG) in 179 patients transplanted with an unrelated donor. GVHD prophylaxis consisted of Cyclosporine A plus methotrexate for 90% of patients. All patients were monitored weekly by PCR for viral infections (reactivation or primary infection) with CMV and Epstein–Barr virus (EBV) until day +90 post-HSCT and subsequently every second week. In case of increasing viral load, pre-emptive treatment with anti-viral medication [ganciclovir for CMV infection and Rituximab for EBV infection] was commenced along with tapering of immunosuppression.The study included 153 children and 307 adults with a median age of 26.3 years (range 0.3–63.0 years). Diagnosis was acute myeloid leukemia  as described by the manufacturer.rs6897932 using a previously described multiplex bead-based assay  were measured with turbidimetry using Cobas 8000, module c502.n = 283, 62%). Peripheral blood samples were analyzed directly in a single-platform no-lyse-no-wash flow cytometry procedure. EDTA-anti-coagulated blood were incubated in Trucount tubes  for quantification of lymphocyte subsets, and a panel of monoclonal antibodies  on a FC500 flow cytometer . Lymphocytes were gated based on forward scatter and side scatter characteristics. Lymphocyte subsets were identified as CD3+ T cells, CD4+ T cells, CD8+ T cells, and CD45+ CD19+ B cells. The laboratory participates in the quality assurance program by the National External Quality Assessment Site (NEQAS).T and B cells were counted 12 months after HSCT, and in addition measured after 1, 2, 3, and 6 months in patients undergoing HSCT from 2008 to 2014  and relapse were estimated using Fine–Gray competing risk regression model. For all analyzes, transplant-related characteristics that are main risk factors for the specific transplant-related complications were included in the multivariable model for this outcome as indicated under results.9/L corresponding to one half of the minimum value of the measurements. For the multivariable analysis, all potential co-variables were included in the model and analyzed with backwards elimination. The final model included recipient age, stem cell source, ATG, and the IL-7Rα genotype. In an additional backwards elimination model, aGVHD and cGVHD were included as time-dependent variables and remained significant together with recipient age, stem cell source, ATG, and the IL-7Rα genotypes.Linear regression analyzes were used to analyze associations with cell counts and immunoglobulin levels after 1 year. Longitudinal analysis of T cell counts were performed with a linear mixed model with random slope over time since transplantation and random intercept by patient for measurements from all patients without truncation due to death, relapse, retransplantation, or donor lymphocyte infusion within the first 360 days. All cell counts were log10-transformed; and measurements equal to 0 (3.2%) were changed to 0.005 × 10To confirm these results regarding T cell counts in a model including patients, who experienced death, relapse, retransplantation, or donor lymphocyte infusion within the first 360 days, we performed a pattern mixture model including all patients and with truncation at time of the event. This model analyzed the cell counts in each stratum (defined by time of event/truncation) in turn using a standard linear mixed model. The mixed model contained a random intercept by patient and, whenever feasible due to enough data in a stratum, a random slope by time since transplantation. The IL-7Rα genotypes were compared as major allele homozygotes (CC) versus heterozygotes and minor allele homozygotes combined (CT/TT) due to limited data within each stratum in the minor allele homozygous patients. Missing and truncated were similarly distributed in the genotype groups.P-value <0.05 was considered statistically significant. All statistical analyzes were performed using R statistical software version 3.2.3 .A two-sided rs6897932 genotype in donors  corresponded to previously reported gene frequencies, and the distribution of genotypes met the criteria for Hardy–Weinberg equilibrium.The frequencies of the rs6897932 genotypes. No significant differences were found between patients in the three different groups.Table rs6897932  .251 patients (54.6%) developed aGVHD with onset at median day +24 (+6 to +106) post-HSCT; grades III–IV aGVHD were seen in 42 patients (9.1%). The risk of grades III–IV aGVHD was significantly increased in donors with one or two copies of the T allele in rs6897932 genotype with a step-wise increased risk of cGVHD for each T allele  .Extensive cGVHD occurred in 118 patients 25.7%) at median day +201 , and this was significantly associated with donor 5.7% at mrs6897932 genotype remained an independent risk factor of extensive cGVHD .To address if the effect of the donor genotype on chronic GVHD was mediated by the increased risk of aGVHD, we included aGVHD in a Cox regression model also adjusting for recipient age, donor type, stem cell source, cell dose/kilogram, TBI-based conditioning, ATG, and sex-mismatch. In this modified model, the donor 123 patients (26.7%) developed therapy-requiring CMV infection at median day +49 post-HSCT, and 34 patients (7.4%) developed therapy-requiring EBV infection at median day +57.rs6897932 TT genotype was associated with a higher occurrence of CMV infection compared with CC   for TT versus CC genotype in a multivariable Cox regression model adjusting for recipient age, donor type (matched sibling donor/matched unrelated donor/mismatched unrelated donor), stem cell source, cell dose, TBI-based conditioning, ATG and CMV mismatch between donor and recipient (D−R+ or D+R−). No association with EBV infection was observed.Donor 9 lymphocytes/L occurred in 427 patients (92.8%) within the first year post-HSCT. There was no association between donor rs6897932 genotype and time to lymphocyte recovery.Lymphocyte recovery with >10rs6897932 on recovery of T and B lymphocyte subsets within 1 year post-HSCT. Patients were excluded from the analysis in case of death, relapse, retransplantation, or donor lymphocyte infusion from the date of the event.We further investigated the impact n = 212). In an univariable model, the donor rs6897932 genotype TT was associated with significantly increased CD3+, CD4+, and CD8+ T cells at day +60 compared with the CC genotype  at day +60 compared with the CC genotype, after adjustment for age, stem cell source, and ATG .First, we studied the impact on immune reconstitution in a linear mixed model only including patients, who did not experience an event within the first 360 days n = 12. In ann = 268). At each time of follow-up, the expected change in median cell count were estimated and compared between the genotype groups. The two groups CT and TT were merged to be able to estimate all parameters, due to the limited number of donors with the TT genotype. In this model, no significant difference between cell counts for the rs6897932 genotype was found at any time point, most likely due to the limited data in each strata. Notably, missing and truncated patients were similarly distributed in the two genotype-defined groups.Next, we investigated the significance of IL-7Rα donor genotypes in a pattern mixture model with truncation due to death, relapse, retransplantation, or donor lymphocyte infusion to confirm the first results in a cohort including patients who experienced an event  patients having no events before this time point. There was no difference in cell counts of any lymphocyte subsets according to the rs6897932 TT genotype was associated with significantly lower levels of IgG and IgM compared to the CC genotype 1 year post-HSCT . However, this decrease in immunoglobulin levels was also strongly associated with occurrence of aGVHD and cGVHD, suggesting that the immunosuppressive effect of rs6897932 on late immunity might be mediated through an increased allo-response.We looked into the level of immunoglobulins to evaluate the functional interaction between T and B cells. The donor  Figures E–G, althrs6897932 donor genotype also influenced post-transplant mortality. 156 patients (33.9%) died within the follow-up time of 6.9 years (range: 1.9–12.8). Of patients transplanted for a malignant disease, 78 patients (21.0%) died of TRM and 81 patients (21.8%) relapsed.We next investigated whether the rs6897932 CT genotype was associated with inferior overall survival in an univariable Cox regression model  .Donor carriage of the rs6897932 CT genotype was associated with increased TRM in a univariable model , but not with the risk of relapse (P = 0.35) . We were not able to demonstrate a significant effect of TT homozygosity on TRM most likely reflecting the low prevalence of this genotype.In a competing risk model, donor Despite the marked improvements during the recent years, allogeneic HSCT is still challenged by severe treatment-related complications . Both aGrs6897932 TT genotype in the IL-7Rα is associated with increased risk of both aGVHD and cGVHD, CMV infection, and faster reconstitution of T cells. These results indicate that genotyping of rs6897932 could help to individualize conditioning regimens and GVHD prophylaxis, or potentially be included as a supplementary criteria for donor selection along with HLA-typing. As alloreactivity and the graft-versus-leukemia effect are often closely associated . The T allele in rs6897932 causes an amino-acid substitution in the transmembrane region of the IL-7Rα gene which reduces alternative splicing of this domain. This process results in increased expression of membrane-bound IL-7R and decreased generation of sIL-7R  and thymopoiesis directly as well as indirectly by its immunosuppressive treatment (rs6897932 genotypes.Recent studies also suggest an importance of IL-7Rα mediated signals for normal human B cell production , althougreatment . A potenrs6897932 genotype of the IL-7Rα predicts aGVHD and cGVHD, CMV infection and TRM following allogeneic HSCT, without altering the risk of relapse. The biological background for the impact of this SNP may be alterations in the levels of soluble and membrane-bound IL-7R leading to stronger IL-7 signaling. Prospective studies should address the role of IL-7R genotyping as part of the donor selection process to reduce the incidence of treatment-related complications and its application as a pre-transplant biomarker identifying patients at risk and guiding prophylactic treatment.In conclusion, we have presented solid evidence that the donor This study was carried out in accordance with the recommendations of the ethics committee of the Capital Region of Denmark. All subjects and/or their legal guardians gave written informed consent in accordance with the Declaration of Helsinki.KK, AP, LR, and KM conceived and designed the study. KK, CH, HS, and KM collected the donor samples and the clinical data. CE performed the genotyping. KK and KM analyzed the data and drafted the manuscript. All authors revised the manuscript.AP is affiliated with Merck A/S; however, the present work is unrelated to this affiliation. All other authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.


Early post-transplant hepatic fibrosis is associated with poor outcomes and may be influenced by donor/recipient genetic factors. The rs368234815 IFNL4 polymorphism is related to the previously described IL28B polymorphism, which predicts etiology-independent hepatic fibrosis. The aim of this study was to identify the impact of donor and/or recipient IFNL4 genotype on early fibrosis among patients transplanted for hepatitis C (HCV).Clinical data were collected for 302 consecutive patients transplanted for HCV. 116 patients who had available liver biopsies and donor/recipient DNA were included. 28% of these patients with stage 2 fibrosis or greater were compared to patients without significant post-transplant fibrosis with respect to clinical features as well as donor/recipient IFNL4 genotype.The IFNL4 TT/TT genotype was found in 26.0% of recipients and 38.6% of donors. Patients who developed early post-transplant fibrosis had a 3.45 adjusted odds of having donor IFNL4 TT/TT genotype (p = 0.012). Donor IFNL4 TT/TT genotype also predicted decreased overall survival compared to non-TT/TT genotypes (p = 0.016).Donor IFNL4 TT/TT genotype, a favorable predictor of spontaneous HCV clearance pre-transplant, is associated with increased early post-transplant fibrosis and decreased survival. End-stage liver disease attributable to chronic HCV infection remains a leading indication for liver transplantation . Some paSubsequent studies have identified a novel dinucleotide polymorphism  that is strongly associated with spontaneous HCV clearance and is found in strong linkage disequilibrium with rs12979860  or N (%). The prevalence of ss469425590 genotypes among donor and recipients was estimated by calculating the percentage of patients with ΔG/ΔG, TT/ΔG and TT/TT. A univariable analysis was performed to assess factors associated with developing fibrosis stage 2–4 post-transplant; Pearson’s chi-square tests were used for categorical variables and analysis of variance (ANOVA) or the non-parametric Kruskal-Wallis tests were used for continuous or ordinal factors. In addition, multivariable logistic regression was performed to assess association between genotype and presence of moderate fibrosis after adjusting for potential confounders; fibrosis 2–4 was modeled as the outcome with ss469424590 genotype and other clinical characteristics as the independent variables. Survival analysis was also performed. Follow-up time was defined as months from OLT to death and subjects who remained alive were censored on 7/1/2013. Kaplan-Meier plots were constructed and log-rank tests were used to compare the genotypes. A p<0.05 was considered statistically significant. SAS  and R  were used to perform all analyses.Data are presented as mean ± standard deviation, median  and donor TT/TT were found to be significantly associated with fibrosis. The donor or recipient rs12979860 (IL28B) polymorphism was not a significant predictor of early post-transplant fibrosis on univariable analysis. On multivariable analysis adjusting for African American race and pre-transplant HCC, donor TT/TT genotype was associated with a 3.45 odds of developing fibrosis . No othePatients who developed significant fibrosis had a higher proportion of African Americans. Therefore, we also analyzed the impact of the two polymorphisms in the African American recipient population of our cohort. In this cohort, the donor TT/TT genotype trended towards a higher odds (6.8) of developing fibrosis but the comparisons were not statistically significant due to relatively low numbers (p = 0.085).The Kaplan-Meier plots for post-transplant survival for rs368234815 genotype and rs12979860 genotype are presented in We evaluated the potential association between rs368234815 genotype and post-transplant fibrosis in patients who underwent liver transplant for HCV cirrhosis. We show that in our cohort, patients who developed early post-transplant fibrosis had a 3.45 adjusted odds of having donor IFNL4 TT/TT genotype  polymorphism was first identified as being predictive of treatment response in the non-transplant setting, with the C/C genotype being associated with a higher probability of achieving SVR to interferon-based regimens . This fiThe functional role of the IFNL4 rs368234815 allows speculation regarding the potential mechanism of increased fibrosis among patients who receive a liver with the TT/TT allele. The TT/TT allele introduces a frame-shift mutation that leads to no expression of IFNL4 . It is pThough we are in the era of near universal cure with currently available direct antiviral regimens, our data remains clinically relevant in the setting of liver transplantation. Many subjects with chronic hepatitis C and decompensated cirrhosis are not treated pre-transplant. This is because successful cure of hepatitis C may place these patients in a so called “liver purgatory” where they remain on the transplant waitlist with a decrease in MELD scores without significant clinical improvement. Pre-transplant cure of hepatitis C may also preclude them from receiving hepatitis C positive grafts. Therefore many transplant centers are waiting until after transplantation to initiate anti-viral treatment. As viremic patients continue to receive transplants and payers refuse coverage for DAA in the early post-transplant period, genetic factors that lead to rapid progression of fibrosis may help to identify patents requiring early therapy. Moreover, in subjects who receive the rs368234815 TT/TT genotype through transplantation, the increased risk of NS5A RAVs may be detrimental.We recognize several limitations in the present study. Exclusion of patients who did not have donor or recipient DNA or a liver biopsy performed between 6–18 months has the potential of selection bias. However, we separately analyzed baseline characteristics among the study population and the total population of transplanted patients and found no significant differences. Though it would be ideal to perform a genome wide association study, the sample size limited us to explore specific polymorphisms based on our hypothesis regarding specific genetic polymorphisms. The sample size may have also contributed to the lack of a significant association between IFNL3 genotype and fibrosis, a finding that has been observed in multiple larger studies ,5. In adEarly HCV-related fibrosis following liver transplantation is associated with risk of graft loss. Even in the era of direct acting anti-viral treatment, many patients with decompensated cirrhosis are now being treated post-transplant and donor genetic markers may be a useful tool to predict patients who are at risk to develop early post-transplant fibrosis. More importantly, this data offers insight into the role of novel donor genetic factors in post-transplant fibrosis in HCV.S1 Fig(TIF)Click here for additional data file.