Citrate treatment reduces endothelial death and inflammation under hyperglycaemic conditions

Hyperglycaemia and glucose degradation products (GDPs) are closely associated with oxidative stress and inflammation in diabetic patients, a condition that leads to endothelial dysfunction and cardiovascular problems. We evaluated the effect of citrate and gluconate on glucose- and GDP-induced endothelial inflammation by measuring changes in viability, inflammation and function in primary human umbilical vein endothelial cells (HUVECs). The extent of apoptosis/necrosis was measured by flow cytometry and visualised with confocal microscopy by staining with annexin V or propidium iodide, respectively. Protein kinase C-βII (PKC-βII) activation was evaluated with Western blotting. Incubation with glucose (30 mM) and GDP (50 µM) significantly increased PKC-βII expression, endothelial cell death and inflammation. The addition of citrate decreased hyperglycaemia-induced apoptosis (p = 0.021), necrosis (p = 0.04) and reduced PKC-βII expression (p = 0.021) down to background levels. Citrate improved endothelial function by reducing the inflammatory markers (p = 0.01) and by decreasing neutrophil diapedesis (p = 0.012). These results suggest that citrate may have therapeutic potential by reducing hyperglycaemia-induced endothelial inflammation and abolishing endothelial dysfunction

Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses.

Leukocyte migration into the epithelial compartment is an important feature in the active phase of mycobacterial infections. In this study, we used the Transwell model to investigate the mechanisms behind mycobacteria-induced leukocyte recruitment and investigated the role of TLR2 and TLR4 in this process. Infection of epithelial cells resulted in significantly increased secretion of the neutrophil chemotactic CXCL8 and IL-6, but no secretion of monocyte chemotactic CCL2 or TNF-α was observed. In contrast to epithelial response, mycobacteria-infected neutrophils and monocytes secreted all these cytokines. Corresponding with epithelial cytokine response, mycobacterial infection of the epithelial cells increased neutrophil diapedesis, but decreased monocyte recruitment. However, monocyte recruitment towards mycobacteria infected epithelial cells significantly increased following addition of neutrophil pre-conditioned medium. Mycobacterial infection also increases alveolar epithelial expression of TLR2, but not TLR4, as analyzed by flow cytometry, Western blotting and visualized by confocal microscopy. Blocking of TLR2 inhibited neutrophil recruitment and cytokine secretion, while blocking of TLR4 had a lesser effect. To summarize, we found that primary alveolar epithelial cells produced a selective TLR2-dependent cytokine secretion upon mycobacterial infection. Furthermore, we found that cooperation between cells of the innate immunity is required in mounting proper antimicrobial defence

Interleukin 8 Receptor Deficiency Confers Susceptibility to Acute Experimental Pyelonephritis and May Have a Human Counterpart

Neutrophils migrate to infected mucosal sites that they protect against invading pathogens. Their interaction with the epithelial barrier is controlled by CXC chemokines and by their receptors. This study examined the change in susceptibility to urinary tract infection (UTI) after deletion of the murine interleukin 8 receptor homologue (mIL-8Rh). Experimental UTIs in control mice stimulated an epithelial chemokine response and increased chemokine receptor expression. Neutrophils migrated through the tissues to the epithelial barrier that they crossed into the lumen, and the mice developed pyuria. In mIL-8Rh knockout (KO) mice, the chemokine response was intact, but the epithelial cells failed to express IL-8R, and neutrophils accumulated in the tissues. The KO mice were unable to clear bacteria from kidneys and bladders and developed bacteremia and symptoms of systemic disease, but control mice were fully resistant to infection. The experimental UTI model demonstrated that IL-8R–dependent mechanisms control the urinary tract defense, and that neutrophils are essential host effector cells. Patients prone to acute pyelonephritis also showed low CXC chemokine receptor 1 expression compared with age-matched controls, suggesting that chemokine receptor expression may also influence the susceptibility to UTIs in humans. The results provide a first molecular clue to disease susceptibility of patients prone to acute pyelonephritis

Acute pyelonephritis and renal scarring are caused by dysfunctional innate immunity in mCxcr2 heterozygous mice

The CXCR1 receptor and chemokine CXCL8 (IL-8) support neutrophil-dependent clearance of uropathogenic Escherichia coli from the urinary tract. CXCR1 is reduced in children prone to pyelonephritis, and heterozygous hCXCR1 polymorphisms are more common in this patient group than in healthy individuals, strongly suggesting a disease association. Since murine CXCR2 (mCXCR2) is functionally similar to human CXCR1, we determined effects of gene heterozygosity on the susceptibility to urinary tract infection by infecting heterozygous (mCxcr2+/−) mice with uropathogenic Escherichia coli. Clearance of infection and tissue damage were assessed as a function of innate immunity in comparison to that in knockout (mCxcr2−/−) and wild-type (mCxcr2+/+) mice. Acute sepsis-associated mortality was increased and bacterial clearance drastically impaired in heterozygous compared to wild-type mice. Chemokine and neutrophil responses were delayed along with evidence of neutrophil retention and unresolved kidney inflammation 1 month after infection. This was accompanied by epithelial proliferation and subepithelial fibrosis. The heterozygous phenotype was intermediate, between knockout and wild-type mice, but specific immune cell infiltrates that accompany chronic infection in knockout mice were not found. Hence, the known heterozygous CXCR1 polymorphisms may predispose patients to acute pyelonephritis and urosepsis

Slit2 prevents neutrophil recruitment and renal ischemia-reperfusion injury

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeriamonocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response. Copyright © 2013 by the American Society of Nephrology

Structure and ion-release mechanism of PIB-4-type ATPases.

Funder: The memorial foundation of manufacturer Vilhelm Pedersen and wife - and the Aarhus Wilson consortiumFunder: China Scholarship Council; FundRef: http://dx.doi.org/10.13039/501100004543Funder: Carl Tryggers Stiftelse för Vetenskaplig Forskning; FundRef: http://dx.doi.org/10.13039/501100002805; Grant(s): CTS 17:22Funder: Agnes og Poul Friis Fond; FundRef: http://dx.doi.org/10.13039/100009512Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here, we present structures and complementary functional analyses of an archetypal PIB-4-ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy-metal-binding domains (HMBDs), and provide fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turnover of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in for example drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens

Infusion fluids contain harmful glucose degradation products

PURPOSE: Glucose degradation products (GDPs) are precursors of advanced glycation end products (AGEs) that cause cellular damage and inflammation. We examined the content of GDPs in commercially available glucose-containing infusion fluids and investigated whether GDPs are found in patients' blood. METHODS: The content of GDPs was examined in infusion fluids by high-performance liquid chromatography (HPLC) analysis. To investigate whether GDPs also are found in patients, we included 11 patients who received glucose fluids (standard group) during and after their surgery and 11 control patients receiving buffered saline (control group). Blood samples were analyzed for GDP content and carboxymethyllysine (CML), as a measure of AGE formation. The influence of heat-sterilized fluids on cell viability and cell function upon infection was investigated. RESULTS: All investigated fluids contained high concentrations of GDPs, such as 3-deoxyglucosone (3-DG). Serum concentration of 3-DG increased rapidly by a factor of eight in patients receiving standard therapy. Serum CML levels increased significantly and showed linear correlation with the amount of infused 3-DG. There was no increase in serum 3-DG or CML concentrations in the control group. The concentration of GDPs in most of the tested fluids damaged neutrophils, reducing their cytokine secretion, and inhibited microbial killing. CONCLUSIONS: These findings indicate that normal standard fluid therapy involves unwanted infusion of GDPs. Reduction of the content of GDPs in commonly used infusion fluids may improve cell function, and possibly also organ function, in intensive-care patients

A Genetic Basis of Susceptibility to Acute Pyelonephritis

For unknown reasons, urinary tract infections (UTIs) are clustered in certain individuals. Here we propose a novel, genetically determined cause of susceptibility to acute pyelonephritis, which is the most severe form of UTI. The IL-8 receptor, CXCR1, was identified as a candidate gene when mIL-8Rh mutant mice developed acute pyelonephritis (APN) with severe tissue damage.We have obtained CXCR1 sequences from two, highly selected APN prone patient groups, and detected three unique mutations and two known polymorphisms with a genotype frequency of 23% and 25% compared to 7% in controls (p<0.001 and p<0.0001, respectively). When reflux was excluded, 54% of the patients had CXCR1 sequence variants. The UTI prone children expressed less CXCR1 protein than the pediatric controls (p<0.0001) and two sequence variants were shown to impair transcription.The results identify a genetic innate immune deficiency, with a strong link to APN and renal scarring

Pedagogisk utvecklingsplan för kurs i Patobiologi 1, termin 4, Läkarprogrammet

Jag har analyserat grundkursen i Patobiologi på termin 4 inom Läkarprogrammet som grund för pedagogisk utvecklingsplan. Kursen ges i både Lund (LÄLA42) och Malmö (LÄMA42) och har gemensam kursplan, identiska mål och kriterier för examination. Kursens syfte är att ge studenterna goda och relevanta kunskaper samt färdigheter i patobiologi, men ger även undervisning i laboratoriemedicinsk diagnostik samt farmakologisk behandling. Undervisning har PBL-fall som grund. Denna understöds av föreläsningar, laborationer, demonstrationer, obduktioner, seminarier, skriftliga uppgifter och artikelläsning. Examinationen sker med skriftlig tentamen (21 hp) samt en godkänd kursportfölj (7,5 hp). Studenterna i Lund och Malmö har samma tentamen, men undervisningen planeras av separata kursledningar i Malmö och Lund. Dessa träffas och samordnar undervisningen så att innehållet skall vara likvärdigt. Jag har analyserat Lunda-kursen och Lena Gustavsson har undersökt Malmö-kursen. Därefter jämförde vi gemensamt kurserna med avseende på upplägg och innehåll och fann dem båda väl samordnade, men med några mindre skillnader. Vi undersökte även om det går att dela upp tentamen då detta efterfrågades av flera studenter. Då vi anser att det är viktigt att säkerställa att undervisningen har samma innehåll och kvalité i Malmö och Lund har vi skrivit en gemensam utvecklingsplan

Mechanisms of Escherichia coli induced transepithelial neutrophil migration

Mucosal infections trigger an inflammatory response that includes the secretion of cytokines and the recruitment of neutrophils to the infected site. This thesis describes studies examining the molecular mechanisms of neutrophil migration to sites of mucosal bacterial infection. Escherichia coli (E. coli) infection of epithelial cell layers stimulates chemokine secretion and chemokine receptor expression. Human epithelial cells produced an array of CXC chemokines (e.g., IL-8, GRO, ENA-78, IP-10, MIG) and CC chemokines (e.g., MCP-1, MIP, RANTES) in response to bacterial infection. The chemokine repertoire was shown to be influenced by the fimbrial expression of the infecting strain. Furthermore, E. coli infection increased the expression of chemokine receptors (CXCR1 and CXCR2) on epithelial cells. IL-8 was found to be the major chemoattractant involved in neutrophil migration across infected epithelial cell layers. Transuroepithelial neutrophil migration was also shown to be depending on the level of CXCR1 expression on neutrophils and epithelial cell layers. The in vitro observations were confirmed in an experimental UTI model. Recombinant P or type 1 fimbriated E. coli stimulated chemokine responses and neutrophil recruitment more efficiently than the non-fimbriated controls. The relevance of IL-8 receptor expression was confirmed in vivo using IL-8 receptor knock-out (mIL-8Rh KO) mice. E. coli infection of mIL-8Rh KO mice caused the neutrophils to leave the blood vessels but they were unable to cross the epithelium and accumulated in the tissues. Bacterial clearance was impaired and tissues were destroyed. Subsequent human studies showed a decrease in CXCR1 expression on neutrophils from patients prone to acute pyelonephritis. These results provide a first molecular clue to the host defect in patients prone to acute pyelonephritis