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Cranial lymphatic vessels (LVs) are involved in the transport of fluids, macromolecules and central nervous system (CNS) immune responses. Little information about spinal LVs is available, because these delicate structures are embedded within vertebral tissues and difficult to visualize using traditional histology. Here we show an extended vertebral column LV network using three-dimensional imaging of decalcified iDISCO + -clarified spine segments. Vertebral LVs connect to peripheral sensory and sympathetic ganglia and form metameric vertebral circuits connecting to lymph nodes and the thoracic duct. They drain the epidural space and the dura mater around the spinal cord and associate with leukocytes. Vertebral LVs remodel extensively after spinal cord injury and VEGF-C-induced vertebral lymphangiogenesis exacerbates the inflammatory responses, T cell infiltration and demyelination following focal spinal cord lesion. Therefore, vertebral LVs add to skull meningeal LVs as gatekeepers of CNS immunity and may be potential targets to improve the maintenance and repair of spinal tissues.

The glycolipid transfer protein, GLTP, can be found in the cytoplasm, and it has a FFAT-like motif (two phenylalanines in an acidic tract) that targets it to the endoplasmic reticulum (ER). We have previously shown that GLTP can bind to a transmembrane ER protein, vesicle-associated membrane protein-associated protein A (VAP-A), which is involved in a wide range of ER functions. We have addressed the mechanisms that might regulate the association between GLTP and the VAP proteins by studying the capacity of GLTP to recognize different N-linked acyl chain species of glucosylceramide. We used surface plasmon resonance and a lipid transfer competition assay to show that GLTP prefers shorter N-linked fully saturated acyl chain glucosylceramides, such as C8, C12, and C16, whereas long C18, C20, and C24-glucosylceramides are all bound more weakly and transported more slowly than their shorter counterparts. Changes in the intrinsic GLTP tryptophan fluorescence blueshifts, also indicate a break-point between C16- and C18-glucosylceramide in the GLTP sensing ability. It has long been postulated that GLTP would be a sensor in the sphingolipid synthesis machinery, but how this mechanistically occurs has not been addressed before. It is unclear what proteins the GLTP VAP association would influence. Here we found that if GLTP has a bound GlcCer the association with VAP-A is weaker. We have also used a formula for identifying putative FFAT-domains, and we identified several potential VAP-interactors within the ceramide and sphingolipid synthesis pathways that could be candidates for regulation by GLTP., Competing Interests: The authors have declared that no competing interests exist.

Angiocrine signals derived from endothelial cells are an important component of intercellular communication and have a key role in organ growth, regeneration and disease 1-4 . These signals have been identified and studied in multiple organs, including the liver, pancreas, lung, heart, bone, bone marrow, central nervous system, retina and some cancers 1-4 . Here we use the developing liver as a model organ to study angiocrine signals 5,6 , and show that the growth rate of the liver correlates both spatially and temporally with blood perfusion to this organ. By manipulating blood flow through the liver vasculature, we demonstrate that vessel perfusion activates β1 integrin and vascular endothelial growth factor receptor 3 (VEGFR3). Notably, both β1 integrin and VEGFR3 are strictly required for normal production of hepatocyte growth factor, survival of hepatocytes and liver growth. Ex vivo perfusion of adult mouse liver and in vitro mechanical stretching of human hepatic endothelial cells illustrate that mechanotransduction alone is sufficient to turn on angiocrine signals. When the endothelial cells are mechanically stretched, angiocrine signals trigger in vitro proliferation and survival of primary human hepatocytes. Our findings uncover a signalling pathway in vascular endothelial cells that translates blood perfusion and mechanotransduction into organ growth and maintenance.

Background: Nonoperative treatment is feasible in most patients with acute Achilles tendon rupture. Risk factors associated with failed nonoperative treatment are poorly understood. We investigated risk factors associated with rerupture after nonoperative treatment and otherwise failed nonoperative treatment of Achilles tendon rupture., Methods: All patients diagnosed with acute Achilles tendon rupture between January 2009 and June 2016 and who underwent 8 weeks of nonoperative treatment with functional rehabilitation were included in the study. Patients with rerupture or otherwise failed nonoperative treatment were identified retrospectively. Time to rerupture and association of age, sex, time from injury, diabetes, and visits to the physiotherapist for cases of reruptures and otherwise failed nonoperative treatment were investigated. A total of 210 patients were included in the study., Results: Fifteen patients sustained a rerupture. Rerupture incidence was 7.1%. Incidence of late reruptures, those occurring after return to daily activities at 12 weeks, was 1.9%. Six patients had otherwise failed nonoperative treatment. Median time to rerupture was 23 days (6 to 61) after the end of the treatment. The incidence of all-cause failure was 10.0%. Male gender was associated with reruptures ( P = .013) and failed nonoperative treatment for any reason ( P = .029)., Conclusion: It is important to highlight the increased risk of rerupture in male patients during the first month after the end of the nonoperative treatment. Age alone, even in male patients, was a poor indication for operative treatment since it did not predict early failure. Further studies will hopefully clarify the influence of activity level on the risk of rerupture., Level of Evidence: Level IV, retrospective case series.

Background: The most common cause of death of patients with idiopathic pulmonary fibrosis (IPF) has been reported to be the lung disease itself and mortality from IPF appears to be increasing. However, the causes of death in patients with IPF taking into account differences between genders and smoking histories as well as disease progression, have not been previously explored., Methods: Retrospective data from hospital register and death certificates from national database of IPF patients treated in Kuopio University Hospital (KUH) from 2002 to 2012 were collected. Mortality was also explored from the death registry database via ICD-10 code J84 revealing the numbers of deaths from pulmonary fibrosis in Finland from 1998 to 2015., Results: Out of 117 deaths, 26.5% were females and 73.5% males in KUH. The most common underlying causes of death were IPF 67.5% and ischemic heart diseases 14.8%. More males died for reasons other than IPF (39.5%) compared to females (12.9%) (p = 0.007). Pneumonia as the immediate cause of death was more common in males (27.9%) than in females (3.2%) (p = 0.004) and in ex-smokers (32.7%) compared to non-smokers (9.3%) (p = 0.007). Death register based mortality from pulmonary fibrosis is increasing in Finland., Conclusions: Even though the overall mortality was higher in males with IPF, the disease-specific mortality for IPF was higher in females i.e. in males, comorbidities were more often the underlying causes of death. Pneumonia-triggered acute exacerbations of IPF may be associated with smoking and gender since females and non-smokers were less likely to succumb to pneumonia. We conclude that disease progression at the end of life may vary depending on smoking habits and gender.

The lymphatic vessels can be selectively stimulated to grow in adult mice, rats and pigs by application of viral vectors expressing the lymphangiogenic factors VEGF-C or VEGF-D. Vice versa, lymphangiogenesis in various pathological settings can be inhibited by the blocking of the VEGF-C/VEGFR3 interaction using a ligand-binding soluble form of VEGFR3. Furthermore, the recently discovered plasticity of meningeal and lacteal lymphatic vessels provides novel opportunities for their manipulation in disease. Adenoviral and adeno-associated viral vectors (AAVs) provide suitable tools for establishing short- and long-term gene expression, respectively and adenoviral vectors have already been used in clinical trials. As an example, we describe here ways to manipulate the meningeal lymphatic vasculature in the adult mice via AAV-mediated gene delivery. The possibility of stimulation and inhibition of lymphangiogenesis in adult mice has enabled the analysis of the role and function of lymphatic vessels in mouse models of disease.

The recent discovery of meningeal lymphatic vessels (LVs) has raised interest in their possible involvement in neuropathological processes, yet little is known about their development or maintenance. We show here that meningeal LVs develop postnatally, appearing first around the foramina in the basal parts of the skull and spinal canal, sprouting along the blood vessels and cranial and spinal nerves to various parts of the meninges surrounding the central nervous system (CNS). VEGF-C, expressed mainly in vascular smooth muscle cells, and VEGFR3 in lymphatic endothelial cells were essential for their development, whereas VEGF-D deletion had no effect. Surprisingly, in adult mice, the LVs showed regression after VEGF-C or VEGFR3 deletion, administration of the tyrosine kinase inhibitor sunitinib, or expression of VEGF-C/D trap, which also compromised the lymphatic drainage function. Conversely, an excess of VEGF-C induced meningeal lymphangiogenesis. The plasticity and regenerative potential of meningeal LVs should allow manipulation of cerebrospinal fluid drainage and neuropathological processes in the CNS., (© 2017 Antila et al.)

Background: Cigarette smoking has been associated with the risk of idiopathic pulmonary fibrosis (IPF). Certain comorbidities have been associated with reduced survival although some studies have indicated that current smokers have a longer survival than ex-smokers. Comorbidities in relation to smoking history have not been previously analyzed., Methods: Retrospective data was collected and patients were categorized according to gender and smoking habits. Comorbidities and medications were collected. Predictive values for mortality were identified by COX proportional hazard analyses., Results: We examined 45 non-smokers (53.3% female), 66 ex-smokers (9.1% female) and 17 current smokers (17.6% female) with IPF. Current smokers were younger at baseline (58.1 ± 8.74 years) compared to non-smokers (71.4 ± 8.74, p < 0.001) and ex-smokers (72.5 ±7.95, p <0.001). Median survival of non-smokers and current smokers was longer (55.0 and 52.0 months, respectively) than that of ex-smokers (36.0 months) (p=0.028 and 0.034, respectively). In age and severity adjusted analyses, smoking was not related to survival. Cardiovascular diseases (CVD) (72.7 %) were the most common comorbidities, current smokers had more chronic obstructive pulmonary disease (COPD) and lung cancer compared to ex-smokers (p<0.001). CVD, COPD and use of insulin were related to poorer survival in adjusted analyses., Conclusions: Smoking seems to influence the course of disease in IPF since current smokers developed the disease at a younger age in comparison to non-smokers and ex-smokers. No significant differences in the major comorbidities were detected between IPF patients with different smoking histories. The mechanism through which smoking influences IPF progression requires further investigation.

During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE -/- mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3 + T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3 + T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development.

Background and Purposes: Permanent xerostomia as a result of radiation-induced salivary gland damage remains a common side effect of radiotherapy (RT) of the head and neck. The purpose of this study was to evaluate the usefulness of diffusion-weighted magnetic resonance imaging (DW-MRI) in assessing the post-RT salivary gland function in patients with head and neck cancer (HNC)., Materials and Methods: In this prospective study, 20 HNC patients scheduled for bilateral neck chemoradiotherapy (CRT) with weekly cisplatin went through diffusion-weighted magnetic resonance imaging (DW-MRI) and salivary gland scintigraphy (SGS) prior to and at a mean of six months after completing the treatment. The changes in apparent diffusion coefficient (ADC) before and after treatment were compared with ejection fraction (EF) measured with SGS and the radiation dose absorbed by the salivary glands., Results: As a result of gustatory stimulation with ascorbic acid, the ADC showed a biphasic response with an initial increase and subsequent decrease. This pattern was seen both before and after RT. Post-RT ADC increased as a function of RT dose absorbed by the salivary glands. A moderate statistical correlation between pre- and post-RT ADCs at rest and EF measured with SGS was found., Conclusions: DW-MRI seems a promising tool for detection of physiological and functional changes in major salivary glands after RT., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: The epidemiology of Achilles tendon ruptures and treatment strategies have undergone a major change in recent years. We investigated the incidence of acute Achilles tendon ruptures, the choice of treatment strategies and treatment implementation., Materials and Methods: The research material consisted of patients living in the catchment area of Central Finland Hospital District who had been diagnosed with an acute Achilles tendon rupture between 2010 and 2015., Results: The final sample consisted of 266 patients. Conservative treatment was started for 207 patients, and the remaining 59 were referred for surgery. During the study period, the proportion of patients undergoing surgery fell from 41% to 10%. Three patients (1.4%) were referred for surgery during conservative treatment, and 10 patients (4.7%) developed deep vein thrombosis while wearing a cast or an orthosis. Twelve patients (5.8%) sustained a re-rupture after conservative treatment. Two surgically-treated patients (3.5%) sustained a re-rupture, and one patient (1.7%) developed deep vein thrombosis., Conclusions: The strategies for treating acute Achilles tendon ruptures have clearly become more conservative in our hospital. Conservative treatment is safe and rarely fails. However, it is important to bear in mind that surgery still has a role in the treatment of acute Achilles tendon ruptures.

Vascular endothelial growth factor C (VEGF-C) is a major driver of lymphangiogenesis in embryos and adults. Vegfc gene deletion in mouse embryos results in failure of lymphangiogenesis, fluid accumulation in tissues, and lethality. The VEGF-C receptors VEGFR3 and VEGFR2 are required for embryonic blood vessel formation. The related VEGF is essential for both blood vessel formation and embryonic hematopoiesis, whereas the possible involvement of VEGF-C in hematopoiesis is unknown. Here we unveil a novel hematopoietic function of VEGF-C in fetal erythropoiesis. Deletion of Vegfc in embryonic day 7.5 (E7.5) embryos in the C57BL6 mouse genetic background led to defective fetal erythropoiesis, characterized by anemia and lack of enucleated red blood cells in blood circulation. Macrophages and erythroid cells in the fetal liver (FL) were also decreased after midgestation because of decreased cell proliferation and increased apoptosis. However, the Lin(-)Sca-1(+)c-Kit(+) stem cell compartment in E14.5 FL was not affected by Vegfc deletion. VEGF-C loss did not disrupt the generation of primitive erythroid cells or erythro-myeloid progenitors (EMPs) in the yolk sac, but it decreased the expression of α4-integrin on EMPs and compromised EMP colonization of the FL. The distribution, maturation, and enucleation of primitive erythroblasts were also impaired by Vegfc deletion. In contrast, Vegfc deletion from E10.5 onward did not compromise definitive hematopoiesis in the liver, and Vegfc deletion in adult mice did not cause anemia. These results reveal an unexpected role for VEGF-C, a major lymphangiogenic growth factor, in the transition to FL erythropoiesis., (© 2016 by The American Society of Hematology.)

Vascular endothelial growth factor C (VEGF-C) binding to its tyrosine kinase receptor VEGFR-3 drives lymphatic vessel growth during development and in pathological processes. Although the VEGF-C/VEGFR-3 pathway provides a target for treatment of cancer and lymphedema, the physiological functions of VEGF-C in adult vasculature are unknown. We show here that VEGF-C is necessary for perinatal lymphangiogenesis, but required for adult lymphatic vessel maintenance only in the intestine. Following Vegfc gene deletion in adult mice, the intestinal lymphatic vessels, including the lacteal vessels, underwent gradual atrophy, which was aggravated when also Vegfd was deleted. VEGF-C was expressed by a subset of smooth muscle cells adjacent to the lacteals in the villus and in the intestinal wall. The Vegfc-deleted mice showed defective lipid absorption and increased fecal excretion of dietary cholesterol and fatty acids. When fed a high-fat diet, the Vegfc-deficient mice were resistant to obesity and had improved glucose metabolism. Our findings indicate that the lymphangiogenic growth factors provide trophic and dynamic regulation of the intestinal lymphatic vasculature, which could be especially important in the dietary regulation of adiposity and cholesterol metabolism., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Saturated sphingolipids have high acyl chain order. Our aim was to study how palmitoylated sphingomyelin (PSM), ceramide (PCer), glucosyl (GlcPCer)-, and galactosylceramide (GalPCer) were able to order the bulk acyl chains of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in comparison with cholesterol. For this reason, we used lipid probes which had preferred phases that were either the disordered phase (1-oleoyl-2-propionyl[DPH-sn-glycero-3-phosphcholine (18:1-DPH-PC) or the ordered phase (trans parinaric acid (tPA). DPH was also used, although it has no clear phase preference. We measured steady-state anisotropy (all probes) and performed fluorescence lifetime analysis (tPA) as a function of composition and temperature. At concentrations where the saturated sphingolipids were not aggregated into ordered domains (and 23 °C), they did not increase POPC acyl chain order as determined from 18:1-DPH-PC anisotropy. As expected, cholesterol increased the POPC acyl chain order linearly as a function of concentration (0-28 mol %). Since PCer already forms ordered domains below 5 mol % (at 23 °C), we measured the acyl chain ordering effect of PCer at 50 °C (0-13 mol %) and observed that PCer ordered POPC acyl chains as efficiently as cholesterol. We conclude that the bulk acyl chain order of POPC was not markedly affected in bilayers where disordered and ordered domains coexist.

Neural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates β1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes β1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell-cell junctions. The Tie2-silenced monolayer integrity is rescued by β1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro. These results establish Ang2-mediated β1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2.

The purpose of the present study was to investigate the role of ventral pallidal opioidergic mechanisms in the control of ethanol intake by studying the effects of acute administration of morphine on the levels of GABA, glutamate, and dopamine in the ventral pallidum. The study was conducted using the alcohol-preferring Alko Alcohol (AA) and alcohol-avoiding Alko Non-Alcohol (ANA) rat lines that have well-documented differences in their voluntary ethanol intake and brain opioidergic systems. Therefore, examination of neurobiological differences between the lines is supposed to help to identify the neuronal mechanisms underlying ethanol intake, since selection pressure is assumed gradually to lead to enrichment of alleles promoting high or low ethanol intake, respectively. The effects of an acute dose of morphine (1 or 10 mg/kg s.c.) on the extracellular levels of GABA and glutamate in the ventral pallidum were monitored with in vivo microdialysis. The concentrations of GABA and glutamate in the dialyzates were determined with a high performance liquid chromatography system using fluorescent detection, while electrochemical detection was used for dopamine. The levels of glutamate in the rats injected with morphine 1 mg/kg were significantly above the levels found in the controls and in the rats receiving morphine 10 mg/kg. Morphine 10 mg/kg also increased the levels of dopamine. Morphine could not, however, modify the levels of GABA. The rat lines did not differ in any of the effects of morphine. The data suggest that the glutamatergic and dopaminergic systems in the ventral pallidum may mediate some effects of morphine. Since there were no differences between the AA and ANA lines, the basic hypothesis underlying the use of the genetic animal model suggests that the effects of morphine detected probably do not underlie the different intake of ethanol by the lines and contribute to the control of ethanol intake in these animals.

Objective: Elevated serum levels of the lymphangiogenic factors VEGF-C and -D have been observed in obese individuals but their relevance for the metabolic syndrome has remained unknown., Methods: K14-VEGFR-3-Ig (sR3) mice that constitutively express soluble-VEGFR-3-Ig in the skin, scavenging VEGF-C and -D, and wildtype (WT) mice were fed either chow or high-fat diet for 20 weeks. To assess the effect of VEGFR-3 blockage on adipose tissue growth and insulin sensitivity, we evaluated weight gain, adipocyte size and hepatic lipid accumulation. These results were complemented with insulin tolerance tests, FACS analysis of adipose tissue macrophages, in vitro 3T3-L1 differentiation assays and in vivo blocking antibody treatment experiments., Results: We show here that sR3 mice are protected from obesity-induced insulin resistance and hepatic lipid accumulation. This protection is associated with enhanced subcutaneous adipose tissue hyperplasia and an increased number of alternatively-activated (M2) macrophages in adipose tissue. We also show that VEGF-C and -D are chemotactic for murine macrophages and that this effect is mediated by VEGFR-3, which is upregulated on M1 polarized macrophages. Systemic antibody blockage of VEGFR-3 in db/db mice reduces adipose tissue macrophage infiltration and hepatic lipid accumulation, and improves insulin sensitivity., Conclusions: These results reveal an unanticipated role of the lymphangiogenic factors VEGF-C and -D in the mediation of metabolic syndrome-associated adipose tissue inflammation. Blockage of these lymphangiogenic factors might constitute a new therapeutic strategy for the prevention of obesity-associated insulin resistance.

The mammalian lymphatic vasculature is important for returning fluids from the extracellular tissue milieu back to the blood circulation. We showed previously that Prox1 dosage is important for the development of the mammalian lymphatic vasculature. The lack of Prox1 activity results in the complete absence of lymphatic endothelial cells (LECs). In Prox1 heterozygous embryos, the number of LECs is reduced because of a decrease in the progenitor pool in the cardinal vein. This reduction is caused by some progenitor cells being unable to maintain Prox1 expression. In this study, we identified Vegfr3, the cognate receptor of the lymphangiogenic growth factor Vegfc, as a dosage-dependent, direct in vivo target of Prox1. Using various mouse models, we also determined that Vegfr3 regulates Prox1 by establishing a feedback loop necessary to maintain the identity of LEC progenitors and that Vegfc-mediated activation of Vegfr3 signaling is necessary to maintain Prox1 expression in LEC progenitors. We propose that this feedback loop is the main sensing mechanism controlling the number of LEC progenitors and, as a consequence, the number of budding LECs that will form the embryonic lymphatic vasculature., (© 2014 Srinivasan et al.; Published by Cold Spring Harbor Laboratory Press.)

In glaucoma, aqueous outflow into the Schlemm's canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development., (© 2014 Zheng et al.; Published by Cold Spring Harbor Laboratory Press.)

Objective: Lymphatic vessels collect extravasated fluid and proteins from tissues to blood circulation as well as play an essential role in lipid metabolism by taking up intestinal chylomicrons. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, but clear connections between arterial pathologies and lymphatic vessels have not been described., Approach and Results: Two transgenic mouse strains with lymphatic insufficiency (soluble vascular endothelial growth factor 3 [sVEGFR3] and Chy) were crossed with atherosclerotic mice deficient of low-density lipoprotein receptor and apolipoprotein B48 (LDLR(-/-)/ApoB(100/100)) to study the effects of insufficient lymphatic vessel transport on lipoprotein metabolism and atherosclerosis. Both sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had higher plasma cholesterol levels compared with LDLR(-/-)/ApoB(100/100) control mice during both normal chow diet (16.3 and 13.7 versus 8.2 mmol/L, respectively) and Western-type high-fat diet (eg, after 2 weeks of fat diet, 45.9 and 42.6 versus 30.2 mmol/L, respectively). Cholesterol and triglyceride levels in very-low-density lipoprotein and low-density lipoprotein fractions were increased. Atherosclerotic lesions in young and intermediate cohorts of sVEGFR3×LDLR(-/-)/ApoB(100/100) mice progressed faster than in control mice (eg, intermediate cohort mice at 6 weeks, 18.3% versus 7.7% of the whole aorta, respectively). In addition, lesions in sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had much less lymphatic vessels than lesions in control mice (0.33% and 1.07% versus 7.45% of podoplanin-positive vessels, respectively)., Conclusions: We show a novel finding linking impaired lymphatic vessels to lipoprotein metabolism, increased plasma cholesterol levels, and enhanced atherogenesis., (© 2014 American Heart Association, Inc.)

Background: Arthroscopic partial meniscectomy is one of the most common orthopedic procedures, yet rigorous evidence of its efficacy is lacking., Methods: We conducted a multicenter, randomized, double-blind, sham-controlled trial in 146 patients 35 to 65 years of age who had knee symptoms consistent with a degenerative medial meniscus tear and no knee osteoarthritis. Patients were randomly assigned to arthroscopic partial meniscectomy or sham surgery. The primary outcomes were changes in the Lysholm and Western Ontario Meniscal Evaluation Tool (WOMET) scores (each ranging from 0 to 100, with lower scores indicating more severe symptoms) and in knee pain after exercise (rated on a scale from 0 to 10, with 0 denoting no pain) at 12 months after the procedure., Results: In the intention-to-treat analysis, there were no significant between-group differences in the change from baseline to 12 months in any primary outcome. The mean changes (improvements) in the primary outcome measures were as follows: Lysholm score, 21.7 points in the partial-meniscectomy group as compared with 23.3 points in the sham-surgery group (between-group difference, -1.6 points; 95% confidence interval [CI], -7.2 to 4.0); WOMET score, 24.6 and 27.1 points, respectively (between-group difference, -2.5 points; 95% CI, -9.2 to 4.1); and score for knee pain after exercise, 3.1 and 3.3 points, respectively (between-group difference, -0.1; 95% CI, -0.9 to 0.7). There were no significant differences between groups in the number of patients who required subsequent knee surgery (two in the partial-meniscectomy group and five in the sham-surgery group) or serious adverse events (one and zero, respectively)., Conclusions: In this trial involving patients without knee osteoarthritis but with symptoms of a degenerative medial meniscus tear, the outcomes after arthroscopic partial meniscectomy were no better than those after a sham surgical procedure. (Funded by the Sigrid Juselius Foundation and others; ClinicalTrials.gov number, NCT00549172.).

The skin interstitium sequesters excess Na+ and Cl- in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl- accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na+, Cl-, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure-regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3-mediated modification of cutaneous lymphatic capillary function.

Notch cell interaction mechanism governs cell fate decisions in many different cell contexts throughout the lifetime of all Metazoan species. It links the fate of one cell to that of its neighbors through cell-to-cell contacts, and binding of Notch receptors expressed on one cell to their membrane bound ligands on an adjacent cell. Environmental cues, such as growth factors and extracellular matrix molecules, superimpose a dynamic regulation on this canonical Notch signaling pathway. In this review, we will focus on Notch signaling in the vertebrate vascular and nervous systems and examine its role in angiogenesis, neurogenesis, and neurovascular interactions. We will also highlight the molecular relationships of the Notch pathway with vascular endothelial growth factors (VEGFs) and their high-affinity tyrosine kinase VEGF receptors, key regulators of both angiogenesis and neurogenesis.

Background: Little is known on racial differences in patients with diffuse large B-cell lymphoma (DLBCL). The aim of this retrospective study is to compare characteristics, prognostic factors and outcomes of Asian and Western patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP)., Methods: Patient-level data was collected from 8 centers. All patients were diagnosed with DLBCL and treated with R-CHOP. Patients were divided into Asian and Western, according to the country of report. Comparisons and univariate/multivariate survival analyses were performed., Results: 712 patients, 455 Asian and 257 Western patients were included. Westerners were more likely to present with elevated LDH (64% vs. 48%, p<0.01) and advanced stage (58% vs. 49%, p<0.01). After a median follow-up of 36 months, there was no difference in progression-free (PFS; p=0.33) or overall survival (OS; p=0.69). There were no PFS or OS differences between races when evaluating separately each age-adjusted International Prognostic Index category. In the multivariate analyses, performance status and stage were associated with PFS and OS in both races., Conclusions: There are no differences in prognostic factors, PFS and OS between Asian and Western patients with DLBCL treated with R-CHOP., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

During mammalian development, a subpopulation of endothelial cells in the cardinal vein (CV) expresses lymphatic-specific genes and subsequently develops into the first lymphatic structures, collectively termed as lymph sacs. Budding, sprouting and ballooning of lymphatic endothelial cells (LECs) have been proposed to underlie the emergence of LECs from the CV, but the exact mechanisms of lymph vessel formation remain poorly understood. Applying selective plane illumination-based ultramicroscopy to entire wholemount-immunostained mouse embryos, we visualized the complete developing vascular system with cellular resolution. Here, we report emergence of the earliest detectable LECs as strings of loosely connected cells between the CV and superficial venous plexus. Subsequent aggregation of LECs resulted in formation of two distinct, previously unidentified lymphatic structures, the dorsal peripheral longitudinal lymphatic vessel (PLLV) and the ventral primordial thoracic duct (pTD), which at later stages formed a direct contact with the CV. Providing new insights into their function, we found vascular endothelial growth factor C (VEGF-C) and the matrix component CCBE1 indispensable for LEC budding and migration. Altogether, we present a significantly more detailed view and novel model of early lymphatic development.

Background: Using positron emission tomography (PET), we compared two tracers, [11C]metomidate ([11C]MTO) and [11C]acetate ([11C]ACE), for the characterization of hepatic tumors., Methods: Thirty-three patients underwent PET with [11C]MTO and [11C]ACE and magnetic resonance imaging (MRI). Based on the histology of the tumor biopsy, 14 patients had hepatocellular carcinoma (HCC), 9 patients had focal nodular hyperplasia (FNH), and 10 patients had other types of hepatic tumors. Tumor uptake was evaluated by calculating the maximum and mean standardized uptake value and tumor-to-liver ratio., Results: Altogether, 120 hepatic lesions (59 HCC, 18 FNH, 30 metastases of different primaries, 9 adenomas, and 4 regenerating nodules of liver cirrhosis) were detected by MRI. The overall tumor detection rate was slightly higher for [11C]MTO (39%) than for [11C]ACE (33%). [11C]ACE was more sensitive for HCC detection (50% versus 43%, respectively), whereas [11C]MTO was more sensitive for FNH detection (78% versus 44%, respectively). In HCC patients, the tumor grade correlated with [11C]ACE, but not with [11C]MTO. All of the patients with liver metastases, from various primary tumors (n = 10), were negative for both tracers., Conclusions: Due to low sensitivity, [11C]MTO and [11C]ACE PET have only limited value in diagnosing hepatic tumors.

The application of different imaging techniques in the planning of and during radiation therapy enables a precise definition of the target region and healthy tissue. New three-dimensional radiotherapy techniques and advanced radiation equipment enable precise implementation of the desired dose of radiation to the treatment region. By using different imaging techniques, a higher treatment dose to the tumor region is enabled with simultaneous protection of healthy tissue structures. The likelihood of mistakes between the treatment plan and its implementation can also be reduced. Accordingly, the aim of image guided radiation therapy is to improve treatment results and at the same time reduce treatment-related side effects.

Objectives: To determine whether serum vascular endothelial growth factor (s-VEGF) levels and VEGF gene expression in tumor tissue predict survival of diffuse large B-cell lymphoma (DLBCL) patients treated with chemoimmunotherapy., Methods: VEGF levels were measured in serum samples from 102 patients <65 yrs with high-risk DLBCL using a quantitative sandwich enzyme immunoassay technique. Exon array data set of tumor tissues from 32 patients was concurrently used to determine VEGF-A exon and gene expression. All patients were treated in a Nordic phase II study with six dose-dense chemoimmunotherapy courses followed by systemic central nervous system prophylaxis., Results: After a median follow-up time of 40 months, 3-yr progression-free survival (PFS) was inferior in patients with high s-VEGF levels compared to those with low levels (59% vs. 83%, P = 0.005). The relative risk of progression or relapse was 3.1-fold (95% confidence interval 1.34-6.91, P = 0.008). The predictive capacity of s-VEGF levels on PFS was most pronounced in the DLBCLs of non-germinal center subtype. In contrast to serum data, VEGF mRNA expression in the lymphoma tissue did not predict outcome, and no correlation was found between s-VEGF levels and lymphoma VEGF expression., Conclusion: Pretreatment s-VEGF level is a predictor of PFS after chemoimmunotherapy and may help to further stratify high-risk DLBCL patients into low- and high-risk groups., (© 2012 John Wiley & Sons A/S.)

Our objective was to evaluate the non-germinal center (GC) profile as a marker for response and survival in DLBCL and to compare the characteristics of patients with GC and non-GC DLBCL treated with rituximab-containing regimens. In this patient-level meta-analysis, retrospective data from 712 newly diagnosed DLBCL patients treated with chemoimmunotherapy from 7 centers were analyzed. GC and non-GC profiles were defined according to the Hans algorithm. Although the non-GC profile showed a trend towards worse overall survival (HR 1.24, 95% CI 0.92-1.66; p=0.15) and progression-free survival (HR 1.29, 95% CI 0.96-1.73; p=0.09), it did not retain its value in the multivariate survival analysis. Additionally, the non-GC profile was independently associated with worse complete response rates (OR 0.55, 95% CI 0.37-0.83; p<0.01) in the multivariate logistic regression analysis. Interestingly, Asian patients had higher proportion of GC DLBCL (p=0.01)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background & Aims: Treatment of recurrent Clostridium difficile infection (CDI) with antibiotics leads to recurrences in up to 50% of patients. We investigated the efficacy of fecal transplantation in treatment of recurrent CDI., Methods: We reviewed records from 70 patients with recurrent CDI who had undergone fecal transplantation. Fecal transplantation was performed at colonoscopy by infusing fresh donor feces into cecum. Before transplantation, the patients had whole-bowel lavage with polyethylene glycol solution. Clinical failure was defined as persistent or recurrent symptoms and signs, and a need for new therapy., Results: During the first 12 weeks after fecal transplantation, symptoms resolved in all patients who did not have strain 027 C difficile infections. Of 36 patients with 027 C difficile infection, 32 (89%) had a favorable response; all 4 nonresponders had a pre-existing serious condition, caused by a long-lasting diarrheal disease or comorbidity and subsequently died of colitis. During the first year after transplantation, 4 patients with an initial favorable response had a relapse after receiving antibiotics for unrelated causes; 2 were treated successfully with another fecal transplantation and 2 with antibiotics for CDI. Ten patients died of unrelated illnesses within 1 year after transplantation. No immediate complications of fecal transplantation were observed., Conclusions: Fecal transplantation through colonoscopy seems to be an effective treatment for recurrent CDI and also for recurrent CDI caused by the virulent C difficile 027 strain., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.

Rationale: The selectively bred lines of alcohol-preferring alko alcohol (AA) and alcohol-avoiding alko nonalcohol (ANA) rats have been used to demonstrate differences in relevant neurotransmitters which could account for their difference in alcohol consumption. Studies have demonstrated differences in distinct components of the endogenous opioid system in various brain regions associated with the process of reinforcement between the AA and ANA lines of rats., Objectives: The goal of this current study was to investigate the hypotheses that the AA and ANA rats will show differences in the release of beta-endorphin at the level of nucleus accumbens (NAC) and in locomotor activity in response to acute systemic administration of ethanol., Materials and Methods: AA and ANA rats were unilaterally implanted with a guide cannula to aim microdialysis probes at the level of NAC. Intraperitoneal injections of 0.0, 1.5, 2.0, and 2.5 g ethanol/kg body weight were administered. Dialysate samples were collected at 30-min intervals prior to and following the injection. Radioimmunoassay specific for beta-endorphin was used to determine the dialysate beta-endorphin content., Results: The 2.5-g/kg ethanol dose induced a transient increase in extracellular beta-endorphin at the level of NAC of AA but not of ANA rats. The 2.5-g/kg ethanol dose also attenuated locomotor activity in the AA but not in the ANA rats., Conclusions: The lack of an increase in the beta-endorphin concentration in the NAC of ANA rats in response to ethanol may partially account for their lower alcohol consumption and lower alcohol-induced attenuation of locomotor activity compared to AA rats.

In this study, three single-nucleotide polymorphisms (SNPs) on the lysyl oxidase-like 1 (LOXL1) gene associated with exfoliation syndrome (XFS) and exfoliation glaucoma (XFG) were investigated in the Finnish population. A case-control study of 59 sporadic patients with XFS, 82 with XFG, 71 with primary open-angle glaucoma (POAG) and 26 individuals without these disorders from the southern Finnish population, and a family study of an extended family with 28 patients with XFS or XFG and 92 unaffected relatives from Kökar islands, Southwestern Finnish archipelago, were conducted. Anonymous blood donors (n=404) were studied as population-based controls. Three SNPs, rs1048661 (R141L), rs3825942 (G153D) and rs2165241, of the LOXL1 gene were genotyped by PCR sequencing. Association and linkage analyses were carried out. In both case-control and family materials, significant association for allele G of rs1048661 (P=2.65 x 10(-5); P=0.0007), allele G of rs3825942 (P=2.24 x 10(-8); P=0.49) and allele T of rs2165241 (P=2.62 x 10(-13); P<0.0001) was found in XFS/XFG. However, linkage was not observed for LOXL1 risk alleles. The corresponding three-locus haplotype GGT increased the risk of XFS/ XFG nearly 15-fold relative to low-risk haplotype GAC (odds ratio (OR): 14.9, P=1.6 x 10(-16)). In conclusion, the earlier reported polymorphisms of the LOXL1 gene showed significant association also in the Finnish population.

Background/aim: Liver biopsy has so far been the only method to accurately follow the progression of primary biliary cirrhosis (PBC). The stage and the severity of lymphocytic piecemeal necrosis (LPN) have been shown to be an independent factor for the development of cirrhosis. In this 3-year prospective study, we evaluated the diagnostic value of several liver function tests, surrogate markers of fibrogenesis, hyaluronic acid (HA), procollagen III N-terminal peptide (S-PIIINP), cholestanol and plant sterols in noncirrhotic PBC patients treated with ursodeoxycholic acid (UDCA) or with UDCA and budesonide to assess the stage, inflammation and fibrosis., Methods: Seventy-seven stage I-III PBC patients were included into the study, with control biopsy at 36 months. Serum liver enzymes, bile acids (BA), HA, PIIINP, immunoglobulins, lipids and cholesterol precursors and plant sterols were measured at baseline and at 36 months., Results: Aspartate aminotransferase (AST), HA, BA and PIINP were significantly different between stages I to III and differentiated mild (F0F1) from moderate (F2F3) fibrosis. The combination of these variables (PBC score) exhibited best sensitivity and specificity, compared with AST/platelet ratio, Forns' score and fibrosis index. Using a cut-off value of 66 for the PBC score, the sensitivity was 81.4% and specificity was 65.2% for classifying the stage of PBC, regarding the stage the and fibrosis in noncirrhotic PBC., Conclusions: Serum HA, BA, PIIINP and AST may serve as valuable simple tools to monitor the treatment response to UDCA in early stages of PBC. Combinations of these biomarkers into a single index further potentiate the diagnostic value of such measurements.

Objective: To examine the epidemiology of primary biliary cirrhosis (PBC) in Finland and to evaluate whether the possible increase in prevalence was attributable to the increasing incidence, better survival, or both., Material and Methods: The Hospital Discharge Register, pathology registers, and death certificates for the years 1988 99 were scrutinized, and the patients identified were followed-up for survival until 31 October 2004. The study area covered four university hospital districts: a total of 25 hospitals. The diagnosis of PBC was regarded as definite (or probable) if three (or two) of the following criteria were fulfilled: positive antimitochondrial antibodies, constantly elevated alkaline phosphatase, and compatible liver histology., Results: In the total population of the study areas, the age-standardized prevalence of PBC increased during the study period from 103 (95% CI: 97-110) to 180 (172-189) per million inhabitants. Incidence increased from 12 (10-14) to 17 (15-20) per million inhabitants per year. The annual average increase in prevalence was 5.1% (4.2-5.9%, p <0.0001) and in incidence 3.5% (0.9%-6.0%, p =0.008). In gender-specific analyses among women, the prevalence of PBC increased from 161 (151-171) to 292 (277-207) per million during the study period and the incidence from 20 (16-24) to 27 (23-32) per million per year. The death rate was 4% per year and half the deaths were from liver-related causes. Survival after diagnosis during the study period lengthened., Conclusions: The prevalence of PBC increased in Finland during 1988-99, owing to both the increased incidence and the prolonged survival.

Currently there are several compounds used as drugs or studied as new chemical entities, which have an electron withdrawing group connected to a vinylic double bond in a phenolic or catecholic core structure. These compounds share a common feature--current computational methods utilizing the Hammett type equation for the prediction of ionisation constants fail to give accurate prediction of pK(a)'s for compounds containing the vinylic moiety. The hypothesis was that the effect of electron-withdrawing substituents on the pK(a) of p-vinyl phenols is due to the delocalized electronic structure of these compounds. Thus, this effect should be additive for multiple substituents attached to the vinylic double bond and quantifiable by LFER-based methods. The aim of this study was to produce an improved equation with a reduced tendency to underestimate the effect of the double bond on the ionisation of the phenolic hydroxyl. To this end a set of 19 para-substituted vinyl phenols was used. The ionisation constants were measured potentiometrically, and a training set of 10 compounds was selected to build a regression model (r2 = 0.987 and S.E. = 0.09). The average error with an external test set of six compounds was 0.19 for our model and 1.27 for the ACD-labs 7.0. Thus, we have been able to significantly improve the existing model for prediction of the ionisation constants of substituted p-vinyl phenols.

Ursodeoxycholic acid (UDCA) is a safe medical therapy for primary biliary cirrhosis (PBC), but its effect on liver histology remains uncertain. Budesonide is a glucocorticoid with high receptor activity and high first-pass metabolism in liver. We evaluated the combination of budesonide and UDCA on liver histology and compared this with UDCA alone in a 3-year prospective, randomized, open multicenter study. Patients with PBC (n = 77), at stages I to III, were randomized into 2 treatment arms, A (n = 41): budesonide 6 mg/d and UDCA 15 mg/kg/d and B (n = 36): UDCA 15 mg/kg/d. Liver histology was assessed at the beginning and at the end of the study. Liver function tests and glucose and cortisol values were determined every 4 months. Paired liver biopsy specimens were available from 69 patients (A = 37 and B = 32). Stage improved 22% in group A but deteriorated 20% in group B (P = .009). Fibrosis decreased 25% in group A but increased 70% in group B (P = .0009). S-PIIINP decreased significantly in group A. Inflammation decreased in both groups, 34% in group A (P = .02), but only 10% in group B (P = NS). Serum liver enzymes decreased significantly in both treatment arms. Bilirubin values rose in group B but stayed stable in group A (A/B P = .002). A mild systemic glucocorticoid effect from budesonide was evident after 2 years. In conclusion, budesonide combined with UDCA improved liver histology, whereas the effect of UDCA alone was mainly on laboratory values. Studies with longer follow-up using a combination of budesonide and UDCA are warranted to confirm safety and effects.

No effective medical therapy is currently available for primary sclerosing cholangitis (PSC). Ursodeoxycholic acid (UDCA) improves liver enzymes, but its effect on liver histology is controversial. Metronidazole (MTZ) prevents PSC-like liver damage in animal models and reduces intestinal permeability. We recruited 80 patients with PSC into a randomized placebo-controlled study to evaluate the effect of UDCA and MTZ (UDCA/MTZ) compared with UDCA/placebo on the progression of PSC. Patients (41 UDCA/placebo and 39 UDCA/MTZ) were followed every third month. Assessment of liver function test, histological stage and grade, and cholangiography (via ERCP) at baseline showed no differences between the groups. After 36 months, serum aminotransferases gamma-glutamyltransferase, and alkaline phosphatase (ALP) decreased markedly in both groups, serum ALP more significantly in the UDCA/MTZ group (-337 +/- 54 U/L, P < .05) compared with the UDCA/placebo group. The New Mayo Risk Score decreased markedly only in the UDCA/MTZ group (-0.50 +/- 0.13, P < .01). The number of patients with improvement of stage (P < .05) and grade (P < .05) was higher in the combination group. ERCP findings showed no progression or improvement in 77% and 68% of patients on UDCA/MTZ and UDCA/placebo, respectively. In conclusion, combining MTZ with UDCA in PSC improved serum ALP levels and New Mayo Risk Score, but no statistically significant effect on disease progression as assessed via liver histology or ERCP was seen. Long-term studies using a higher dose of UDCA combined with MTZ in larger patient populations are indicated.

In inflammatory bowel diseases (IBD), certain chromosomal candidate loci have been repeatedly identified by independent studies in different populations. To investigate the contribution of the loci on chromosomes 1, 3, 7, 12, 14, and 16 to the susceptibility of IBD in Finnish population, where the predominant feature is the excess of ulcerative colitis (UC) families compared to Crohn's disease (CD) families, we carried out linkage analyses using 93 Finnish, multiply-affected IBD families. We observed nominal evidence for linkage to chromosome 3p21, consistent with earlier reports. The lod scores peaked at D3S2432, with a maximum two-point lod score of 1.68 (P=0.0027). In addition, we studied whether risk of IBD is associated with functional variants of two positional candidate genes; the chemokine receptor CCR5 gene on chromosome 3p21 and the interleukin-4 receptor alpha-subunit gene (IL4RA) on chromosome 16. We did not find any significant correlation between a 32-bp deletion variant of CCR5 or a single nucleotide change A1902G (Gln576Arg) of IL4RA, and IBD phenotypes, with the exception that in the UC group homozygosity for the G1902 allele of IL4RA was less frequent (0.019 vs 0.049, P=0.038). In conclusion, our study, carried out in a genetically homogenous population, suggests that chromosome 3 may contain a susceptibility gene for IBD.

Group II phospholipase A2 has been proposed to play an important role in the pathophysiology of inflammatory bowel diseases. This enzyme has also been linked to host defence mechanisms against bacteria. The current study aimed at measuring the mass concentrations of group II phospholipase A2 in serum and colonic mucosa of patients with Crohn's disease of different severity and of appropriate control patients without any inflammatory disease. The activity of the disease was determined by clinical factors (the simple index score) and endoscopic and histological scoring. The mass concentration of group II phospholipase A2 was measured by a time-resolved fluoroimmunoassay. The mass concentrations of group II phospholipase A2 in serum and colonic mucosa were significantly higher both in patients with active and inactive Crohn's disease when compared with controls. There was statistically significant difference in the mass concentration of group II phospholipase A2 in colonic mucosa but not in serum between inactive and active Crohn's disease. The current results indicate that the mass concentration of group II phospholipase A2 is increased in serum and colonic mucosa of patients with Crohn's disease and that the latter is associated with the degree of the inflammatory activity in the intestinal wall. These results support the idea that group II phospholipase A2 is involved in the local and generalised pathological processes of Crohn's disease.