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Biomedical subjects

D Karpman

Publications and source records attributed to D Karpman.

At least 19 recordsLinked to original sources
PubMed2006

A classification of hemolytic uremic syndrome and thrombotic thrombocytopenic purpura and related disorders.

The diagnostic terms hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are based on historical and overlapping clinical descriptions. Advances in understanding some of the causes of the syndrome now permit many patients to be classified according to etiology. The increased precision of a diagnosis based on causation is important for considering logical approaches to treatment and prognosis. It is also essential for research. We propose a classification that accommodates both a current understanding of causation (level 1) and clinical association in cases for whom cause of disease is unclear (level 2). We tested the classification in a pediatric disease registry of HUS. The revised classification is a stimulus to comprehensive investigation of all cases of HUS and TTP and is expected to increase the proportion of cases in whom a level 1 etiological diagnosis is confirmed.

Animals↗
PubMed2006

Phenotypic expression of factor H mutations in patients with atypical hemolytic uremic syndrome.

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.

Animals↗
PubMed2005

Factor H binds to washed human platelets.

BACKGROUND: Factor H regulates the alternative pathway of complement. The protein has three heparin-binding sites, is synthesized primarily in the liver and copurifies from platelets with thrombospondin-1. Factor H mutations at the C-terminus are associated with atypical hemolytic uremic syndrome, a condition in which platelets are consumed. Objectives The aim of this study was to investigate if factor H interacts with platelets. METHODS: Binding of factor H, recombinant C- or N-terminus constructs and a C-terminus mutant to washed (plasma and complement-free) platelets was analyzed by flow cytometry. Binding of factor H and constructs to thrombospondin-1 was measured by surface plasmon resonance. RESULTS: Factor H bound to platelets in a dose-dependent manner. The major binding site was localized to the C-terminus. The interaction was partially blocked by heparin. Inhibition with anti-GPIIb/IIIa, or with fibrinogen, suggested that the platelet GPIIb/IIIa receptor is involved in factor H binding. Factor H binds to thrombospondin-1. Addition of thrombospondin-1 increased factor H binding to platelets. Factor H mutated at the C-terminus also bound to platelets, albeit to a significantly lesser degree. CONCLUSIONS: This study reports a novel property of factor H, i.e. binding to platelets, either directly via the GPIIb/IIIa receptor or indirectly via thrombospondin-1, in the absence of complement. Binding to platelets was mostly mediated by the C-terminal region of factor H and factor H mutated at the C-terminus exhibited reduced binding.

Binding Sites↗
PubMed2004

Enzyme-linked immunosorbent assay for detection of Shiga toxin-producing Escherichia coli infection by antibodies to Escherichia coli secreted protein B in children with hemolytic uremic syndrome.

In order to detect immunoglobulin (Ig)A and IgG antibodies to Escherichia coli-secreted protein B in sera of children infected with Shiga toxin-producing Escherichia coli, an enzyme-linked immunosorbent assay was developed. The assay was tested using acute sera from 40 children with diarrhea-associated hemolytic uremic syndrome compared with 238 sera obtained from pediatric controls. Two cut-off values were used for children <5 (n=27) or > or =5 (n=13) years of age. Among the younger patients, 24 of 27 had IgA antibodies to Escherichia coli-secreted protein B (sensitivity, 89%; specificity, 98%) and 22 of 27 had IgG antibodies (sensitivity, 82%; specificity, 94%). Among the older patients, 13 of 13 had IgA antibodies (sensitivity, 100%; specificity, 96%) and 11 of 13 had IgG antibodies (sensitivity, 85%; specificity, 96%). This enzyme-linked immunosorbent assay detects Shiga-toxin-producing Escherichia coli independent of serogroup and could serve as a complementary assay for detection of infection.

Antibodies, Bacterial↗
PubMed2003

The host response to urinary tract infection.

The authors use the UTI model to identify basic mechanisms of disease pathogenesis, host response induction, and defense. Their studies hold the promise to provide a molecular and genetic explanation for susceptibility to UTI, and to offer more precise tools for diagnosis and therapy of these infections. There are few infections where the host response is understood in such detail and where pathologic host responses can be linked to distinct disease states. The susceptibility to UTI varies greatly in the population. The studies suggest that distinct molecular defects can cause the clinical entity of acute pyelonephritis with renal scarring, and suggest that the susceptibility to UTI in certain patient groups may have a genetic basis. In addition, the distinct signal transduction pathways explain the development of symptoms, and propose that defects in those signaling mechanisms may occur in patients with ABU. In the future, it may be useful to include these host response parameters in the diagnostic arsenal, to help in early detection of patients susceptible to recurrent UTI and renal scarring. These patients may then be offered therapies that strengthen their defense, and be offered close surveillance for recurrences and other complications.

Animals↗
PubMed2001

Platelet activation by Shiga toxin and circulatory factors as a pathogenetic mechanism in the hemolytic uremic syndrome.

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.

Adolescent↗
PubMed2001

Pathogenesis of Shiga toxin-associated hemolytic uremic syndrome.

The aim of this review is to examine recent advances in experimental and clinical research relevant to the pathogenesis of diarrhea-associated hemolytic uremic syndrome with special reference to histopathologic findings, virulence factors of Shiga toxin-producing Escherichia coli, the host response, and the prothrombotic state. Despite significant advances during the past decade, the exact mechanism by which Shiga toxin-producing E. coli leads to hemolytic uremic syndrome remains unclear. Factors such as Shiga toxin, lipopolysaccharide, the adhesins intimin and E. coli-secreted proteins A, B, and D, the 60-MD plasmid, and enterohemolysin likely contribute to the pathogenesis. Data on the inflammatory response of the host, including leukocytes and inflammatory mediators, are updated. The pathogenesis of the prothrombotic state leading to thrombocytopenia secondary to endothelial cell damage and platelet activation is also discussed. A hypothetical sequence of events from ingestion of the bacteria to the development of full-blown hemolytic uremic syndrome is proposed.

Adhesins, Bacterial↗
PubMed2001

The 'innate' host response protects and damages the infected urinary tract.

Symptoms of infection and tissue pathology are caused by the host response; not by the microbe per se. The same response is also critical for the defence and is needed to clear infection. It is therefore essential to understand how the host response is activated and to identify the critical effector mechanisms of the defence. We have studied these issues in the urinary tract infection (UTI) model. The symptoms of UTI and the host defence both rely on the so-called 'innate' immune system, making this one of the best characterized human disease models of 'innate immunity. We discuss the critical molecular events that determine whether the host response will be activated by P-fimbriated uropathogenic Escherichia coli as well as factors determining whether the patient develops acute pyelonephritis or asymptomatic bacteriuria. We will describe the glycoconjugate receptors used by the P-fimbriated bacteria adhering to host tissues, the recruitment of TLR4 co-receptors and the signalling pathways that allow progression to symptomatic disease, and discuss how these mechanisms are altered in asymptomatic carriers, presenting the possible genetic basis for unresponsiveness. We have shown that neutrophils are the critical effectors of the host defence and that neutrophil dysfunctions lead to acute pyelonephritis and renal scarring. Here we discuss the mechanisms of neutrophil-mediated, chemokine receptor (CXCR1)-dependent clearance, and the defect in interleukin-8 receptor homolog knock-out (IL-8Rh KO) mice and describe the data linking low CXCR1 expression to recurrent pyelonephritis in man, as well as the information on the genetic basis for low CXCR1 expression in affected patients. Finally, the mechanisms of renal scarring in IL8Rh KO mice will be discussed in relation to human disease. Our studies hold the promise to provide a molecular and genetic explanation for disease susceptibility in some patients with UTI and to offer more precise tools for the diagnosis and therapy of these infections.

Animals↗
PubMed2000

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.

Animals↗
PubMed1999

Multimeric alpha-lactalbumin from human milk induces apoptosis through a direct effect on cell nuclei.

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.

Animals↗
PubMed1998

Cytokine repertoire of epithelial cells lining the human urinary tract.

PURPOSE: To examine the cytokine profile of epithelial cells lining the human urinary tract with the aim of differentiating between the constitutive and disease-related cytokine production in these tissues. MATERIALS AND METHODS: Sections from the renal pelvis, ureter, bladder or urethra were obtained from 22 patients undergoing urinary tract surgery and were stained with monoclonal antibodies to interleukin(IL)-1beta, IL-4, IL-6, IL-8, interferon gamma (IFNgamma) and transforming growth factor beta (TGFbeta). Sections were classified according to the presence or absence of disease in the tissue. RESULTS: Epithelial cells lining the renal pelvis, ureter, bladder or urethra all stained for IL-8 and TGFbeta (100%) in disease-free tissues and sections with cancer or interstitial cystitis (IC). In contrast, staining for IL-1beta, IL-4, IL-6 and IFNgamma varied with the disease state of the patient. Epithelial IL-1beta staining was absent (0%) in sections from healthy bladder, but positive in tissues with IC or cancer-associated pathology (50 to 100%). IL-6 staining was detected in the epithelial layer of several patients with IC or cancer related pathology, but only in cells with non-epithelial morphology and not in disease-free tissues. IFNgamma and IL-4 staining were only observed in patients with IC and only in cells with non-epithelial morphology. CONCLUSIONS: The results show that epithelial cells from all parts of the urinary tract constitutively produce IL-8 and TGFbeta and suggest that the production of other cytokines varies with the disease of the patient. Constitutive cytokine production provides the basis for a rapid host response, in the defense against mucosal attack by microbes or toxic agents.

Adult↗
PubMed1998

Apoptosis of renal cortical cells in the hemolytic-uremic syndrome: in vivo and in vitro studies.

This study examined apoptotic cell death associated with Shiga-like toxin (Stx)-producing Escherichia coli. Renal cortices from three children with postenteropathic hemolytic-uremic syndrome (HUS) and from mice infected with E. coli O157:H7 and pediatric renal tubular epithelial cells stimulated with Stx and E. coli O157:H7 extracts were examined for apoptotic changes. Apoptotic cells were detected by terminal dUTP nick end labeling of tubuli and glomeruli from HUS patients and from mice inoculated with Stx-2-positive and Stx-negative strains. Apoptosis was more extensive and severe ultramorphological nuclear and cytoplasmic changes were seen in the Stx-2-positive group. Stx caused DNA fragmentation and ultramorphological changes indicating apoptosis in cultured pediatric tubular cells. DNA fragmentation increased when cells were pre-stimulated with tumor necrosis factor alpha. Polymyxin extracts from Stx-2-positive and Stx-negative strains induced DNA fragmentation, but only extracts from Stx-2-positive strains caused ultramorphological changes and extensive DNA fragmentation. The results indicate that HUS is accompanied by increased apoptosis of kidney cells and that bacterial factors, possibly together with host cytokines in vivo, may activate apoptotic tissue injury.

Animals↗
PubMed1998

Ins405AsnPro mutation in the von Willebrand factor propeptide in recessive type 2A (IIC) von Willebrand's disease.

The 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 phenylalanine 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.

Animals↗
PubMed1997

Bernard-Soulier syndrome Karlstad: Trp 498-->Stop mutation resulting in a truncated glycoprotein Ib alpha that contains part of the transmembranous domain.

In Bernard-Soulier syndrome, a hereditary bleeding disorder, the platelets are deficient in the glycoprotein (GP) Ib-IX-V complex, a major receptor for the von Willebrand factor. The components of the complex are encoded by separate genes. Patients with this syndrome have a variable expression level of the receptor protein on platelets depending on the specific genetic abnormality. We describe a patient with life-long bleeding symptoms, who is homozygous for a unique stop mutation. Trp 498-->Stop in the GPIb alpha gene, resulting in a truncated GPIb alpha polypeptide chain. In contrast to previously reported truncated forms of GPIb alpha, this form contains a portion of the transmembranous domain as well as the juxtamembranous cysteines engaged in a disulphide bond with GPIb beta. Flow cytometry with GPIb alpha antibodies demonstrated the presence of GPIb on the patient's platelets, although in reduced amounts compared to normal controls. GPIX was similarly detectable. Immunoblotting demonstrated that the patient synthesized a truncated GPIb alpha of the expected size of 130 K, which was, however, sensitive to proteolysis. These studies show that GPIb alpha lacking the intracytoplasmic tail can be expressed at the platelet surface provided elements of the transmembranous domain are present.

Aged↗
PubMed1997

The role of lipopolysaccharide and Shiga-like toxin in a mouse model of Escherichia coli O157:H7 infection.

The role of lipopolysaccharide (LPS) and Shiga-like toxin (SLT) in the pathogenesis of hemolytic uremic syndrome (HUS) was studied in a mouse model. Mice inoculated intragastrically with Escherichia coli O157:H7 developed gastrointestinal, neurologic, and systemic symptoms, necrotic foci in the colon, glomerular and tubular histopathology, and fragmented erythrocytes. LPS-responder (C3H/HeN) mice developed a combination of neurologic and systemic symptoms, whereas LPS-nonresponder (C3H/HeJ) mice had a biphasic course of disease, first developing systemic symptoms and later severe neurologic symptoms. Mice inoculated with SLT-II-positive strains developed severe neurotoxic symptoms and a higher frequency of systemic symptoms and glomerular pathology compared with SLT-II-negative strains. Anti-SLT-II antibodies protected against these symptoms and pathology. These results demonstrate that this model could be used to study aspects of human HUS and that both LPS and SLT are important for disease development.

Animals↗