Cystathionine-Gamma-Lyase-Derived Hydrogen Sulfide-Regulated Substance P Modulates Liver Sieve Fenestrations in Caecal Ligation and Puncture-Induced Sepsis.

Cystathionine-γ-lyase (CSE) isa hydrogen sulfide (H2S)-synthesizing enzyme that promotesinflammation by upregulating H2S in sepsis. Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells (liver sieve) that undergo alteration during sepsis and H2S plays a role in this process. Substance P (SP) is encoded by the preprotachykinin A (PPTA) gene, and promotes inflammation in sepsis; however, its regulation by H2S is poorly understood. Furthermore, the interaction between H2S and SP in modulating LSEC fenestrations following sepsis remains unclear. This study aimed to investigate whether CSE/H2S regulates SP and the neurokinin-1 receptor (NK-1R) andmodulates fenestrations in LSECs following caecalligation and puncture (CLP)-induced sepsis. Here we report thatthe absence of either CSE or H2S protects against liver sieve defenestration and gaps formation in LSECsin sepsis by decreased SP-NK-1R signaling. Following sepsis, there is an increased expression of liver CSE and H2S synthesis, and plasma H2S levels, which were aligned with higher SP levels in the liver, lungs and plasma and NK-1R in the liver and lungs. The genetic deletion of CSE led to decreased sepsis-induced SP and NK-1R in the liver, lungs and plasma SP suggesting H2S synthesized through CSE regulates the SP-NK-1R pathway in sepsis. Further, mice deficient in the SP-encoding gene (PPTA) preservedsepsis-induced LSEC defenestrationand gaps formation, as seen by maintenance of patent fenestrations and fewer gaps. In conclusion, CSE/H2S regulates SP-NK-1R and modulates LSEC fenestrations in sepsis.

Dietary macronutrients and the aging liver sinusoidal endothelial cell.

Fenestrations are pores within the liver sinusoidal endothelial cells (LSECs) that line the sinusoids of the highly vascularized liver. Fenestrations facilitate the transfer of substrates between blood and hepatocytes. With pseudocapillarization of the hepatic sinusoid in old age, there is a loss of fenestrations. LSECs are uniquely exposed to gut-derived dietary and microbial substrates delivered by the portal circulation to the liver. Here we studied the effect of 25 diets varying in content of macronutrients and energy on LSEC fenestrations using the Geometric Framework method in a large cohort of mice aged 15 mo. Macronutrient distribution rather than total food or energy intake was associated with changes in fenestrations. Porosity and frequency were inversely associated with dietary fat intake, while fenestration diameter was inversely associated with protein or carbohydrate intake. Fenestrations were also linked to diet-induced changes in gut microbiome, with increased fenestrations associated with higher abundance of Firmicutes and reduced abundance of Bacteroidetes Diet-induced changes in levels of several fatty acids (C16:0, C19:0, and C20:4) were also significantly inversely associated with fenestrations, suggesting a link between dietary fat and modulation of lipid rafts in the LSECs. Diet influences fenestrations and these data reflect both the key role of the LSECs in clearing gut-derived molecules from the vascular circulation and the impact these molecules have on LSEC morphology.

Scotblood 2010: key presentations of the past, present, and future of transfusion medicine to mark Scottish national blood transfusion service (SNBTS) anniversaries.

The year 2010 marked the 80th anniversary of the first volunteer blood donor panel in Scotland and the 70th anniversary of the first meeting of the Scottish National Blood Transfusion Association - the forerunner of today's SNBTS. As such the annual Scotblood meeting hosted a distinguished group of speakers to present key note and award lectures on all aspects of Transfusion Medicine including red cell antigens, solving the problems, hazards that shaped our practice, the transfusion needs of patients, donor issues, and component therapy to cellular therapy and beyond. The Iain Cook Memorial Lecture was given by Prof. Dame Marcela Contreras and was entitled "Blood Transfusion International - A Partnership with the Developing World".

Scotblood 2011 features advances in translational medicine, haemopoietic progenitor cells and milestones of blood banking systems.

Amongst the presentations featured at Scotblood 2011 were advances in diagnostic tests for antibodies to red blood cells, the establishment of an islet isolation laboratory, and the development of a clinical product for corneal stem cell transplantation. In addition, the conference comprised presentations on state-of-the-art in collection, storage, and clinical utility of haemopoietic progenitor cells. It also included a session on blood banking systems dedicated to an SNBTS colleague, the late Russell Graham. Finally, the keynote lecture was delivered by Prof. John Forsythe on behalf of the Safety of Blood, Tissues and Organs (SaBTO) members, while the Iain Cook Memorial Lecture was delivered by the recently retired SNBTS R&D director, Prof. Chris Prowse.

SCOTBLOOD 2009: the quest for understanding vCJD; Claudia's Trachea implantation; transfusion triggers; Scottish histocompatibility and immunogenetics network; and islet cell transplantation.

Scotblood 2009 consisted of a varied combination of leading edge presentations incorporating the past, present and future. Variant CJD was a major feature of the meeting comprising the quest for its understanding and the impact the disease was having on blood donation. The meeting also included the fascinating and groundbreaking story of Claudia's Trachea transplantation, along with progress in the establishment of the Scottish histocompatibility and immunogenetics network and islet transplantation. The meeting began however with a talk on facilities for the future, outlining major modernization of SNBTS in order to meet future challenges.

Scotblood 2007: Tackling local and global issues in transfusion medicine - donor recruitment, effective use of blood, stem cell plasticity, and vCJD.

This commentary briefly highlights some of the local and the global contemporary issues affecting transfusion medicine worldwide. The main areas of focus addressed this year were: donor recruitment, stem cell plasticity, the effective use of blood, and vCJD.

Scotblood 2008 celebrates 60th anniversary of the NHS through past, present, and future of transfusion medicine, encompassing Hepatitis C Scottish Odyssey, nursing, translational medicine and trauma.

On the occasion of the 60th anniversary of the UK national health service (NHS), Scotblood 2008 featured an opulent array of presentations encompassing the beginnings of the scottish national blood transfusion service (SNBTS), through featuring progress on hepatitis C, to the advancing role of nursing in transfusion medicine, translational medicine, and trauma encountered in military transfusion. This commentary comprises summaries of the presentations, based in part on the abstracts provided by the speakers.

Caloric restriction reduces age-related pseudocapillarization of the hepatic sinusoid.

Age-related changes in the hepatic sinusoid, called pseudocapillarization, may contribute to the pathogenesis of dyslipidemia. Caloric restriction (CR) is a powerful model for the study of aging because it extends lifespan. We assessed the effects of CR on the hepatic sinusoid to determine whether pseudocapillarization is preventable and hence a target for the prevention of age-related dyslipidemia. Livers from young (6 months) and old (24 months) CR and ad libitum fed (AL) F344 rats were examined using electron microscopy and immunohistochemistry. In old age, there was increased thickness of the liver sinusoidal endothelium and reduced endothelial fenestration porosity. In old CR rats, endothelial thickness was less and fenestration porosity was greater than in old AL rats. Immunohistochemistry showed that CR prevented age-related decrease in caveolin-1 expression and increase in peri-sinusoidal collagen IV staining, but did not alter the age-related increase of von Willebrand's factor. CR reduces age-related pseudocapillarization of the hepatic sinusoid and correlates with changes in caveolin-1 expression.

Age-related changes in the liver sinusoidal endothelium: a mechanism for dyslipidemia.

The liver sinusoidal endothelial cell (LSEC) influences the transfer of substrates between the sinusoidal blood and hepatocytes and has a major role in endocytosis; therefore, changes in the LSEC have significant implications for hepatic function. There are major morphological changes in the LSEC in old age called pseudocapillarization. These changes include increased LSEC thickness and reduced numbers of pores in the LSEC, which are called fenestrations. Pseudocapillarization has been found in old humans, rats, mice, and nonhuman primates. In addition, old age is associated with impaired LSEC endocytosis and increased leukocyte adhesion, which contributes to reduced hepatic perfusion. Given that fenestrations in the endothelium allow passage of some lipoproteins, including chylomicron remnants, age-related reduction in fenestrations impairs hepatic lipoprotein metabolism. In old rats, caloric restriction was associated with complete preservation of LSEC morphology and fenestrations. In conclusion, pseudocapillarization of the LSEC is a newly discovered aging change that, through its effects on lipoproteins, contributes to the association between old age, dyslipidemia, and vascular disease.

Dimethylthetin treatment causes diffuse alveolar lung damage: a pilot study in a sheep model of Continuous Ambulatory Peritoneal Dialysis (CAPD).

Total plasma homocysteine (Hcy) concentration correlates with risk of vascular disease. Over 80% of chronic renal failure patients have elevated plasma Hcy and a 10-20 times higher incidence of vascular disease. Glycine betaine lowers plasma Hcy through methylation catalysed by betaine-homocysteine methyltransferase (BHMT). Dimethylthetin (DMT), a synthetic glycine betaine analogue, is a more effective BHMT substrate. DMT is therefore a potential therapeutic agent for reducing plasma Hcy in humans and may be particularly useful in renal failure patients receiving dialysis because of chronic betaine depletion as a result of treatment. We aimed to determine whether the addition of DMT to dialysis fluid lowered plasma Hcy concentrations in a Continuous Ambulatory Peritoneal Dialysis sheep model using animals that were either in acute renal failure (n=3) or had normal renal function (n=1). Sub-acute exposure to DMT was toxic to all four animals, which died with total lung consolidation and collapse and Diffuse Alveolar Damage within 48 h of beginning treatment. Adverse side effects were observed after 4-8 doses. DMT was not detected in pre-dialysis plasma samples and the final concentration at death was 0.5-7.8 mmol/L, depending on the number of doses each animal was exposed to. Abnormalities were not observed in animals supplied standard dialysis fluid, or fluid with added glycine betaine. Toxicity associated with DMT treatment raises concerns for its use in further studies. However, sub-acute administration of DMT to sheep may provide a useful model of acute alveolar damage.

Differential Effects of Kupffer Cell Inactivation on Inflammation and The Liver Sieve Following Caecal-Ligation and Puncture-Induced Sepsis in Mice.

Sepsis remains a common clinical problem with significant mortality. Activation of the Kupffer cells during sepsis is associated with systemic inflammatory response and multiple organ failure. Kupffer cell activation also leads to structural changes in the liver sinusoidal endothelial cells (LSECs) during endotoxemia. However, these effects remain to be elucidated in caecal-ligation and puncture (CLP)-induced polymicrobial sepsis. To investigate the role of Kupffer cells on LSECs fenestrae and inflammation during CLP-induced sepsis, sepsis was induced by CLP and mice were treated with gadolinium chloride (GdCl3) before CLP-induced sepsis, to inactivate Kupffer cells. Mice were sacrificed after 8 h. Blood, liver, and lung tissues were collected and processed to measure LSECs fenestration, myeloperoxidase (MPO) activity, alanine transaminase (ALT) and aspartate aminotransferase (AST) activity, histological examination, and various cytokines/chemokines levels. LSECs fenestrae was studied using scanning electron micrographs of the LSECs. Strikingly, CLP mice treated with GdCl3 were protected against liver injury as evidenced by decreased LSECs defenestration and damage, MPO, ALT and AST activities, liver tissue damage, and inflammatory cytokines TNF-α, IL-6 and IL-1ß, and chemokines MCP-1 and MIP-2α. However, CLP mice treated with GdCl3 had no protection against increased lung MPO activity, tissue damage, inflammatory cytokines, and chemokines. Treatment with GdCl3 also had no effect on the systemic inflammatory response as shown by no change in the circulatory inflammatory cytokines and chemokines following CLP-induced sepsis. Collectively, these data suggest that inactivation of Kupffer cells by GdCl3 protects the liver but had no effect on lung injury or inflammation and systemic inflammatory response following CLP-induced sepsis.

Correction: Cystathionine-Gamma-Lyase Gene Deletion Protects Mice against Inflammation and Liver Sieve Injury following Polymicrobial Sepsis.

[This corrects the article DOI: 10.1371/journal.pone.0160521.].

Hyperlipidemia and surfactants: the liver sieve is a link.

Poloxamer 407 is a ubiquitous synthetic surfactant that causes massive hyperlipidemia and atherosclerosis in the rodent. The initial step in hepatic metabolism of lipoproteins is their transfer through 100-200 nm pores (fenestrations) in the liver sinusoidal endothelial cell, prior to receptor-mediated uptake. The 'liver sieve hypothesis' emphasizes the role of these fenestrations in the regulation of lipoprotein disposition. Here we show that P407 causes dramatic defenestration of the liver sinusoidal endothelium in vivo. By 24h after intraperitoneal administration in mice, fenestrations were reduced by approximately 80% coincident with a 10-fold increase in plasma lipids. Moreover impulse-response experiments in the perfused rat liver showed that P407 prevented the passage of small chylomicrons across the liver sinusoidal endothelium. Defenestration was also induced acutely with P407 in isolated liver sinusoidal endothelial cells, indicating this is a direct effect of P407 on fenestrations. The results establish the role of the porosity of the liver sinusoidal endothelial cell as a pivotal yet relatively unrecognised mechanism for hyperlipidemia. Furthermore, the results establish an intriguing mechanism for surfactant-induced hyperlipidemia. Thus the liver sieve is a new and untapped target for the treatment and prevention of hyperlipidemia.

What's happening - Scotblood 2006 advances in immunetolerance, information technology, microarrays and pathogen inactivation in transfusion medicine.

This commentary on Scotblood meeting aimed to provide a highlight on four areas of new development in blood transfusion medicine, based on the abstracts provided by the invited speakers. Dr. Jerard Seghatchian would like to express his sincere thanks to Prof. Robin Fraser, Chairman of the Scotblood Organising Committee, and Dr. Hagop Bessos, chairman of the Scotblood Programme Sub-Committee, for inviting him to attend this event and providing him with the essential material for this report.

Regulatory end-point assessment of the consultation competence of family practice trainees in Kuwait.

BACKGROUND: No single approach to the regulatory assessment of global consultation competence has been shown to possess the required levels of validity, reliability and feasibility. OBJECTIVE: To evaluate the approach adopted in Kuwait to the regulatory end-point assessment of the global consultation competence of family practice trainees with particular reference to validity, reliability and feasibility. METHODS: Family practice trainees in Kuwait were individually and directly observed for 3 hours in consultation with a minimum of 10 patients by a pair of examiners. Performance was judged against the explicit criteria of consultation competence as contained in the Leicester Assessment Package (LAP). RESULTS: The marks independently allocated by the pairs of examiners to 126 trainees between 1994 and 2001 were within five percentage points on 91% of occasions. A reliability coefficient of 0.82 was achieved when two examiners independently marked candidates consulting with 10 real patients; this rose to 0.95 at the critical 50% pass-fail margin. The main sources of variance contributing to the reliability of marks allocated were candidate performance (42%) and the interaction of candidate performance across cases, i.e., case specificity (30%). The clinical challenges presented by the patients were judged by both examiners to be sufficient to enable performance to be assessed across the seven LAP consultation categories as follows: behaviour and relationship with patients (100% of consultations), interviewing/history taking (100%), record keeping (99%), patient management (99%), problem solving (98%), physical examination (95%), and anticipatory care (86%). Each assessment involved a pair of examiners and lasted approximately 3.5 hours. CONCLUSION: The Kuwait clinical examination achieves high content validity and authenticity as it uses direct observation of performance, validated and explicit criteria against which performance is judged, and real patient challenges. It can discriminate between different levels of consultation performance and satisfies the recognized reliability threshold for regulatory examinations (0.82 vs 0.80). Accordingly, we recommend the use of such an approach in the regulatory end-point assessment of the global consultation competence of trainees in family practice. Such an approach is more valid, and is likely to be more feasible, than simulated surgeries or the short-case OSCE format.

Cystathionine-Gamma-Lyase Gene Deletion Protects Mice against Inflammation and Liver Sieve Injury following Polymicrobial Sepsis.

BACKGROUND: Hydrogen sulfide (H2S), produced by the activity of cystathionine-gamma-lyase (CSE), is a key mediator of inflammation in sepsis. The liver sinusoidal endothelial cells (LSECs) are important target and mediator of sepsis. The aim of this study was to investigate the role of CSE-derived H2S on inflammation and LSECs fenestrae in caecal-ligation and puncture (CLP)-induced sepsis using CSE KO mice. METHODS: Sepsis was induced by CLP, and mice (C57BL/6J, male) were sacrificed after 8 hours. Liver, lung, and blood were collected and processed to measure CSE expression, H2S synthesis, MPO activity, NF-κB p65, ERK1/2, and cytokines/chemokines levels. Diameter, frequency, porosity and gap area of the liver sieve were calculated from scanning electron micrographs of the LSECs. RESULTS: An increased CSE expression and H2S synthesizing activity in the liver and lung of wild-type mice following CLP-induced sepsis. This was associated with an increased liver and lung MPO activity, and increased liver and lung and plasma levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß, and the chemokines MCP-1 and MIP-2α. Conversely, CSE KO mice had less liver and lung injury and reduced inflammation following CLP-induced sepsis as evidenced by decreased levels of H2S synthesizing activity, MPO activity, and pro-inflammatory cytokines/chemokines production. Extracellular-regulated kinase (ERK1/2) and nuclear factor-κB p65 (NF-κB) became significantly activated after the CLP in WT mice but not in CSE KO mice. In addition, CLP-induced damage to the LSECs, as indicated by increased defenestration and gaps formation in the LSECs compared to WT sham control. CSE KO mice showed decreased defenestration and gaps formation following sepsis. CONCLUSIONS: Mice with CSE (an H2S synthesising enzyme) gene deletion are less susceptible to CLP-induced sepsis and associated inflammatory response through ERK1/2-NF-κB p65 pathway as evidenced by reduced inflammation, tissue damage, and LSECs defenestration and gaps formation.

Ultrastructure of the liver microcirculation influences hepatic and systemic insulin activity and provides a mechanism for age-related insulin resistance.

While age-related insulin resistance and hyperinsulinemia are usually considered to be secondary to changes in muscle, the liver also plays a key role in whole-body insulin handling and its role in age-related changes in insulin homeostasis is largely unknown. Here, we show that patent pores called 'fenestrations' are essential for insulin transfer across the liver sinusoidal endothelium and that age-related loss of fenestrations causes an impaired insulin clearance and hyperinsulinemia, induces hepatic insulin resistance, impairs hepatic insulin signaling, and deranges glucose homeostasis. To further define the role of fenestrations in hepatic insulin signaling without any of the long-term adaptive responses that occur with aging, we induced acute defenestration using poloxamer 407 (P407), and this replicated many of the age-related changes in hepatic glucose and insulin handling. Loss of fenestrations in the liver sinusoidal endothelium is a hallmark of aging that has previously been shown to cause deficits in hepatic drug and lipoprotein metabolism and now insulin. Liver defenestration thus provides a new mechanism that potentially contributes to age-related insulin resistance.

The hepatic sinusoid in aging and cirrhosis: effects on hepatic substrate disposition and drug clearance.

The fenestrated sinusoidal endothelium ('liver sieve') and space of Disse in the healthy liver do not impede the transfer of most substrates, including drugs and oxygen, from the sinusoidal lumen to the hepatocyte. Plasma components transfer freely in both directions through the endothelial fenestrations and into the space of Disse. The endothelium is attenuated, there is no basement membrane and there is minimum collagen in the space of Disse, thus minimising any barriers to substrate diffusion. Both cirrhosis and aging are associated with marked structural changes in the sinusoidal endothelium and space of Disse that are likely to influence bulk plasma transfer into the space of Disse, and diffusion through the endothelium and space of Disse. These changes, termed capillarisation and pseudocapillarisation in cirrhosis and aging, respectively, impede the transfer of various substrates. Capillarisation is associated with exclusion of albumin, protein-bound drugs and macromolecules from the space of Disse, and the progressive transformation of flow-limited to barrier-limited distribution of some substrates. There is evidence that the sinusoidal changes in cirrhosis and aging contribute to hepatocyte hypoxia, thus providing a mechanism for the apparent differential reduction of oxygen-dependent phase I metabolic pathways in these conditions. Structural change and subsequent dysfunction of the liver sieve warrant consideration as a significant factor in the impairment of overall substrate handling and hepatic drug metabolism in cirrhosis and aging.

Hepatic pseudocapillarization in aged mice.

Age-related changes in the hepatic sinusoid of the rat, human and baboons called pseudocapillarization have been discovered and are important because they are considered to be implicated in the pathogenesis of some age-related diseases. In this study, we investigated whether similar changes occur in the livers of old mice. Livers of young (3-4 months) and old (20-24 months) mice were perfusion-fixed and studied using electron microscopy and immunohistochemistry. The thickness of the sinusoidal endothelium was increased in old mice (154+/-4 versus 244+/-8 nm, P<0.001). There was a reduction in fenestrations within the endothelium (porosity decreased from 4.1+/-0.3 to 2.2+/-0.2%, P<0.001). There was perisinusoidal staining with Sirius red in old mice, however, expression of laminin and von Willebrands factor was similar in young and old mice. Novel perisinusoidal fat-engorged stellate cells were found extensively in the old mice. This study confirmed that pseudocapillarization is a widespread aging change in the liver, now documented in several species including the mouse. Mice are an appropriate animal model for studying aging and the hepatic sinusoid.

Practice costs of implementing guidelines for asthma and angina: findings from a randomised controlled trial.

We collected information about the costs of three interventions (dissemination of full guidelines or prioritised review criteria or criteria plus feedback) to improve care of adults with stable angina or asthma within the context of a randomised controlled trial. The cost of criteria with feedback was pound180 per practice compared to pound9.60-31.45 for the other interventions. Total mean costs borne by practices varied between pound838 and pound2127 per practice depending upon disease and intervention type.