Search

The contact system triggers the kallikrein-kinin cascade, liberating bradykinin from high-molecular-weight kininogen. Effectors of the contact system have proinflammatory and vasoactive properties. Vasculitis is a condition characterised by inflammation around vessel walls, leading to secondary tissue damage for which the underlying molecular mechanisms are poorly understood. Our aim was to investigate contact-system activation in children with vasculitis.We compared 17 children, aged 4-19 years, with vasculitis, engaging the skin, joints, intestines, or kidneys, with 21 controls, aged 2-18 years. We analysed proteolysis of high-molecular-weight kininogen by immunoblotting. Plasma bradykinin concentrations were quantified by ELISA. Kidney and skin biopsies were stained in situ for kinins. Concentrations of heparin binding protein (HBP) were quantified by ELISA.We noted extensive proteolysis of high-molecular-weight kininogen in the plasma of 13 of 17 patients, but in only one of 21 controls (p0.0001). Bradykinin concentrations were higher in the patients' plasma (median 320 ng/L, range1-19680) than in plasma from controls (11 ng/L,1-304; p=0.0004). Patients had local release of kinins at sites of inflammation in kidney and skin biopsies. HBP values were raised in patients (17.4 microg/L, 5.4-237.6) compared with controls (6 microg/L, 2.5-43.4; p=0.008).Activation of the contact system could play a part in the pathogenesis of vasculitis, and explain the inflammation, pain, vasodilatation, and oedema seen in patients.

Enterohemorrhagic Escherichia coli produce an attaching and effacing lesion upon adhering to the intestinal epithelium. Bacterial factors involved in this histopathology include the intimin adhesin and E. coli secreted proteins (Esps) A and B. In this study we investigated the serum antibody responses to recombinant E. coli O157:H7 intimin, EspA, and EspB by immunoblotting. Canadian patients with O157:H7 infection (n=10), Swedish patients with O157:H7 (n=21), non-O157 (n=18), or infection from which the serotype was not available (n=3), and asymptomatic household members (n=25) were studied and compared with Canadian (n=20) and Swedish controls (n=52). In Canadian patients, IgG antibodies to intimin, EspA, and EspB were analyzed, in Swedish patients and their household members IgA, IgG, and IgM antibodies to EspA and EspB were studied. Patients and household members mounted an antibody response to the antigens. Significantly more patients developed an acute response to EspB compared with controls (P

Iohexol clearance is an accepted, but time-consuming assay for the measurement of glomerular filtration rate (GFR). We investigated if simpler methods could predict GFR. Sixty-nine children with hematological-oncological disorders participated. A linear relationship was established by regression analysis between iohexol clearance ( n=734) and 1/s-creatinine ( r=0.45, n=727), s-cystatin C ( r=0.41, n=518), and the Schwartz ( r=0.45, n=723), Counahan-Barratt ( r=0.48, n=723), and modified Counahan-Barratt formulae ( r=0.48, n=723). These correlations improved when one GFR measurement per individual was compared with each of the five parameters. We further investigated if iohexol clearance could accurately be replaced. The degree of variation in predicting GFR was estimated by the standard deviation of the residuals (S(res)). For 1/s-creatinine and s-cystatin C, S(res) was 39 and 38 ml/min per 1.73 m(2). For the formulae of Schwartz, Counahan-Barratt, and modified Counahan-Barratt, the S(res) was 43, 40, and 40 ml/min per 1.73 m(2), respectively. The wide variations of the S(res) were not reduced when one GFR measurement per child was compared with the five parameters. Due to the large deviation in predicting GFR, we conclude that the five alternative methods studied cannot replace iohexol clearance for measurement of GFR.

Thrombocytopenia caused by platelet consumption in thrombi is a major manifestation of hemolytic uremic syndrome (HUS) associated with Shiga toxin (Stx) producing Escherichia coli. Platelets have glycosphingolipid receptors capable of binding Stx, but a direct interaction between the toxin and platelets, leading to platelet activation, has not been reported. In this study, it is shown that Stx1 and its B (binding) subunit (Stx1B), at 10 pg/mL to 10 ng/mL, bound to platelets. Toxin was internalized in platelets within 2 hours. This led to increased platelet aggregation, as demonstrated by confocal microscopy. Preincubation of Stx1B with anti-Stx1 antibody inhibited this reaction. Stx1 induced morphologic changes in platelets seen on scanning electron microscopy. In the presence of platelets and tumor necrosis factor–pretreated human umbilical vein endothelial cells (HUVEC), Stx1 and Stx1B induced the binding of platelets to the endothelial cell membrane and were present at this binding site. Incubation of Stx1 and Stx1B with whole blood increased fibrinogen binding to platelets detected by flow cytometry. Fibrinogen binding was partially inhibited by preincubation with anti-Stx1. Stx1 increased platelet retention measured in a glass bead assay. In addition, plasma from 17 patients with HUS, taken during the acute phase of the disease, increased the retention of normal platelets and normalized after recovery. Taken together, the results of this investigation show that Stx1, Stx1B, and a factor or factors in the plasma of patients with HUS activate platelets. The presence of Stx1 at the binding site of platelets to HUVEC suggests that Stx may be directly involved in the prothrombotic state seen in HUS.

Attachment to mucosal surfaces enables commensal organisms to select their ecologic niche and pathogens to select their site of infection. The tissue-specific attachment involves the coupling of microbial surface adhesins to receptor epitopes on host cell surfaces and may occur in competition with soluble receptor molecules. Gram-negative bacteria carry fimbriae with lectinlike domains that recognize receptor epitopes in cell surface glycolipids or glycoproteins. The resulting specific adherence may enhance bacterial persistence at the site of infection, but adherence also has been identified as a virulence factor that facilitates tissue attack, invasion, and mucosal inflammation. Two general mechanisms have been proposed to explain how attachment promotes tissue attack by pathogenic bacteria. In the first case, the coupling of fimbriae to their receptors activates receptor-determined signaling pathways in the host cell

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.

A fraction from human milk containing spf-multimer alpha-lactalbumin (MAL) induces apoptosis in tumor cells and immature cells but spares mature cells. The mechanism of apoptosis induction and the molecular basis for the difference in susceptibility between tumor cells and healthy cells have not been defined. In this study we examined the interaction of MAL with different cellular compartments, using confocal microscopy and subcellular fractionation. MAL was shown to accumulate in the nuclei of sensitive cells rather than in the cytosol, the vesicular fraction, or the ER-Golgi complex. Nuclear uptake occurred rapidly in cells that were susceptible to the apoptosis-inducing effect, but not in nuclei of resistant cells. Nuclear uptake was through the nuclear pore complex and was critical for the induction of DNA fragmentation, since inhibition of nuclear uptake with WGA rescued digitonin-permeabilized cells from induction of DNA fragmentation. Ca2+ was required for MAL-induced DNA fragmentation but nuclear uptake of MAL was independent of Ca2+. This way MAL differs from most previously described agents in that it crosses the plasma membrane and cytosol, and enters cell nuclei where it induces DNA fragmentation through a direct effect at the nuclear level.

SummaryThe molecular defects of the von Willebrand factor (vWF) have been studied in the patient in whom the von Willebrand disease phenotype IIC was originally described. A six nucleotide insert, AATCCC, was found in exon 11 of the vWF gene, predicting the insertion of the amino acids asparagine and proline between phenylala-nine 404 and threonine 405 of the vWF propeptide. The mutation was present in one allele. Analysis of amplification products derived from platelet vWF mRNA showed the other allele to be silent. The patient is thus a compound heterozygote for a null allele and the IIC allele, in accord with the recessive mode of inheritance of the IIC phenotype. Family studies indicated the IIC mutation to have occurred de novo, possibly as a result of a duplication event. In vitro mutagenesis and expression in COS-7 cells confirmed the detrimental effect of the mutation on vWF multimer assembly. Taken together with those of earlier studies the present findings suggest that the IIC phenotype may well be exclusively caused by mutations which result in changes of the amino acid sequence in certain regions of the vWF propeptide. Although in the recently revised classification of von Willebrand’s disease variants, the IIC type is included in the 2A category, obviously it constitutes a very distinct subtype.

Increased platelet retention in familial recurrent thrombotic thrombocytopenic purpura. We studied two brothers with recurrent thrombotic thrombocytopenic purpura (TTP). Platelet retention, measured with a modified Adeplat S glass-bead test, was found to be increased during acute episodes of TTP and during remissions. Values of platelet retention ranged between 57 to 95% (normal range 16 to 34%). The continually elevated values enabled us to investigate which fraction of the patients' blood was responsible for the increased platelet retention and to evaluate the effect of different treatments on this parameter. We found that the patients' plasma increased the retention of normal platelets and of platelets taken from a patient with von Willebrand's disease type III. This activity was located in the cryoprecipitate fraction of plasma. Unusually large von Willebrand factor (vWF) multimers were demonstrated in both children during remission and decreased during relapse. Both fresh frozen plasma (FFP) and a commercial factor VIII/vWF concentrate reduced platelet retention when tested during remission. Treatment of both siblings with FFP or factor VIII/vWF concentrate was beneficial during recurrences. We conclude that the elevated platelet retention is due to a factor in the cryoprecipitate of the childrens' plasma, and that both FFP and factor VIII/vWF concentrate are effective in decreasing platelet retention.

Context: Though typical Hemolytic Uremic Syndrome - mainly caused by Shiga toxin (Stx) produced by Escherichia coli - is a major cause of acute renal failure in children, the available treatments remain symptomatic. The aim of our experimental study was to determine whether ouabain (OB) at non-inhibitive concentrations could decrease the shigatoxin-induced apoptotic level. Methods: Experiments were performed in rat proximal tubule cells (RPTC) cultured with Stx 2-4ng/mL, and treated with OB 5-10 nM. Group comparison was measured with the Apoptotic index, DNA fragmentation, and Fluorescence activated cell sorter (FACS). Immunoprecipitation and immunostaining techniques were used to determine the pathway of the antiapototic effect of OB. Results: Stx 3-4 ng/mL significantly increased the apoptotic index in comparison to the control group. OB 5 nM significantly decreased the apoptotic index in RPTC exposed to Stx 3- 4 ng/mL (Apotptotic index %: Stx3 6.4±3.2 versus Stx3+OB 1.5±2.4, p=0.003; Stx4 8.6±8.9 vs. 2.1±1.3, p< 10-4). It also decreased the level of DNA. FACS analyses demonstrated that OB drastically protected RPTC from Stx cytotoxins. Furthermore, immunoprecipitation as well as specific immunostaining showed that OB's protective effect was mediated through the Na, K-ATPase /IP3R interaction and triggering NFkB activity. Conclusion: Our results state that ouabain may be an interesting therapeutic agent to reduce kidney damage in children with HUS.

BACKGROUND: Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)-bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF. METHODOLOGY/PRINCIPAL FINDINGS: The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF-expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery. CONCLUSIONS/SIGNIFICANCE: By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.

Recent studies have identified mutations in the complement regulatory gene factor I (CFI) that predispose to atypical hemolytic uremic syndrome (aHUS). CFI is a two-chain serine protease in which the light chain carries the catalytic domain while the heavy chain's function is unclear. It downregulates the alternative and classical complement pathways by cleaving the alpha' chains of C3b and C4b in the presence of cofactor proteins (known as cofactor activity). Many CFI mutations in aHUS result in low CFI levels with a consequent quantitative defect in complement regulation. In others, the mutant protein is present in normal amounts but the presumed functional deficiency has not yet been defined. In this report we examine the nature of the functional defect in aHUS-associated CFI mutations. The I322T, D501N and D506V mutations reside in the serine protease domain of CFI and result in secreted proteins that lack C3b and C4b cofactor activity. The delTTCAC (1446-1450) mutant leads to a protein that is not secreted. The R299W mutant lies in a region of the CFI heavy chain of no known function. Our assessments demonstrate decreased C3b and C4b cofactor activity, providing evidence that this region is important for cofactor activity. In two other heavy chain mutants and one probable polymorphic variant, no functional deficiency was identified. These defective mutant proteins will result in an inability to appropriately control the complement cascade at sites of endothelial cell injury. The excessive complement activation for a given degree of damage may result in generation of a procoagulant state and aHUS.

We investigated the phenotypic expression of factor H mutations in two patients with atypical hemolytic uremic syndrome (HUS). Factor H in serum was assayed by rocket immunoelectrophoresis, immunoblotting, and double immunodiffusion and in tissue by immunohistochemistry. Functional activity was analyzed by hemolysis of sheep erythrocytes and binding to endothelial cells. A homozygous mutation in complement control protein (CCP) domain 10 of factor H was identified in an adult man who first developed membranoproliferative glomerulonephritis and later HUS. C3 levels were very low. The patient had undetectable factor H levels in serum and a weak factor H 150 kDa band. Double immunodiffusion showed partial antigenic identity with factor H in normal serum owing to the presence of factor H-like protein 1. Strong specific labeling for factor H was detected in glomerular endothelium, mesangium and in glomerular and tubular epithelium as well as in bone marrow cells. A heterozygous mutation in CCP 20 of factor H was found in a girl with HUS. C3 levels were moderately decreased at onset. Factor H levels were normal and a normal 150 kDa band was present. Double immunodiffusion showed antigenic identity with normal factor H. Factor H labeling was minimal in the renal cortex. Factor H dysfunction was demonstrated by increased sheep erythrocyte hemolysis and decreased binding to endothelial cells. In summary, two different factor H mutations associated with HUS were examined: in one, factor H accumulated in cells, and in the other, membrane binding was reduced.