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Biomedical subjects

N J Klein

Publications and source records attributed to N J Klein.

At least 55 records · Page 3Linked to original sources
PubMed1998

Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B.

Mannose-binding lectin (MBL) is a serum protein that has been demonstrated to activate the classical complement pathway and to function directly as an opsonin. Although MBL deficiency is associated with a common opsonic defect and a predisposition to infection, the role of the protein in bacterial infection remains unclear. We have investigated MBL binding to Neisseria meningitidis serogroup B1940 and three isogenic mutants, and the subsequent activation of the two major isoforms of C4 (C4A and C4B) by an associated serine protease, MASP. The mutants lacked expression of the capsular polysaccharide (siaD-), the lipo-oligosaccharide (LOS) outer core that prevented LOS sialylation (cpsD-), or both capsule and LOS outer core (cps-). Using flow cytometry, it was possible to detect strong MBL binding to the cps- and cpsD- mutants over a wide range of concentrations. In contrast, minimal or no MBL binding was detected on the parent organism, with binding to siaD- only at higher MBL concentrations. C4 was activated and bound by mutants that had previously bound MBL/MASP, but there was no significant difference in the amounts of C4A and C4B bound. When sialic acid residues were removed from the parent organism by neuraminidase treatment, the binding of both MBL and C4 increased significantly. Our results suggest that MBL may bind to and activate complement on these encapsulated organisms, and the major determinants of these effects are the LOS structure and sialylation.

Bacterial Proteins↗
PubMed1998

Tumor necrosis factor-alpha inhibits epithelial differentiation and morphogenesis in the mouse metanephric kidney in vitro.

Tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, has diverse actions both within and outside the immune system and has been implicated in the etiology of a wide range of pathological conditions. Evidence is accumulating that it may also have important roles in the normal development of the embryo. In this study we demonstrated that the addition of recombinant TNF-alpha to metanephric organ culture induced a dose dependent and reversible decrease in growth and development, with inhibition of ureteric bud branching and nephron formation beyond the condensate stage and despite appropriate expression of the transcription factor pax-2. TNF-alpha also increased the point prevalence of apoptosis after only 1 day of culture. We also noted that macrophages were present in renal rudiments at the inception of nephrogenesis and their numbers significantly increased during the culture period. This effect was enhanced by TNF-alpha. We have also demonstrated expression of mRNAs for TNF-alpha and its receptors in whole mouse metanephroi from the inception of renal development. TNF-alpha protein was also detected, predominantly at mesenchymal/epithelial interfaces. In addition, TNF-alpha mRNA and protein were expressed by clonal renal mesenchymal cells in vitro, suggesting that these cells are a source of TNF-alpha in vivo.

Animals↗
PubMed1997

Investigation of platelet-neutrophil interactions in whole blood by flow cytometry.

Evidence is increasing that platelets can initiate and propagate inflammatory processes by interacting with leucocytes and the vascular endothelium. Platelets have been shown to bind to neutrophils, existing as platelet/neutrophil complexes (PNC) within the circulation. We describe a simple flow cytometric method for assessing and investigating platelet interactions with neutrophils in small volumes of whole blood. Twenty-five percent (sd 6%) of circulating neutrophils from healthy adults were associated with platelets. Formation of these platelet-neutrophil complexes was CD62P (P-selectin) and divalent cation dependent. Platelet activation (with ADP or thrombin) caused a rapid and sustained rise in %PNC which differed from the pattern of free platelet activation as assessed by CD62P expression. F-met-leu-phe induced neutrophil activation but did not increase the percentage PNC. Platelet activation also caused increased neutrophil CD11b/CD18 expression which was most marked on neutrophils complexed with platelets. This straightforward technique is simple, reproducible, and allows assessment of platelet-neutrophil interactions and activation of neutrophils. It may also provide a method for estimating platelet activation in whole blood.

Adult↗
PubMed1997

Detection of fibronectin expression by human endothelial cells using a enzyme-linked immunosorbent assay (ELISA): enzymatic degradation by activated plasminogen.

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure cellular fibronectin (cFN) in association with human umbilical vein endothelial cells (HUVEC) in culture. The expression of a number of functional domains on the cFN molecule was demonstrated using three specific murine monoclonal antibodies. This system was found to be sensitive, detecting as little as 0.156 microg/ml of cFN, and required only 1.3 x 10(5) cells per well confluent cells per experimental condition. This allowed multiple experiments to be performed on one batch of endothelial cells. cFN was detected on both viable and methanol fixed endothelial cells without significant non-specific antibody binding. The utility of this experimental model was studied by exploring the effect of urokinase activated plasminogen, a potent protease, on the expression of cFN and its functional domains.

Antibodies, Monoclonal↗
PubMed1997

Induction of human endothelial tissue factor expression by Neisseria meningitidis: the influence of bacterial killing and adherence to the endothelium.

Tissue factor (TF), a small membrane bound high affinity receptor for factor VII, has an important procoagulant role in the haemostatic dysfunction associated with severe sepsis. Using an in vitro model of human endothelial TF expression, defined strains of Neisseria meningitidis were found to upregulate endothelial cell procoagulant activity (PCA) in a dose dependent manner. This TF response was detected with as little as 10(4) cfu/ml and reached similar levels to those seen with high concentrations of purified endotoxin (> 1 ng/ml). Treatment of N. meningitidis with either adult donor immune serum, penicillin or gentamicin failed to enhance this PCA. Limulus amoebocyte lysate assay of lipopolysaccharide in bacterial culture filtrates together with polymyxin B inhibition experiments suggest that endotoxin is largely responsible for endothelial TF induction by N. meningitidis. Incubation of endothelial cells with N. meningitidis B1940 and B1940 siaD- (an eight-fold more adherent unencapsulated isogenic strain), revealed a significantly greater TF response to B1940 siaD- (P < 0.01). In conclusion, bacterial adhesion to the vessel wall and therefore local levels of endotoxin may be important determinants of the endothelial procoagulant response to N. meningitidis and the consequent coagulopathy commonly associated with the disease.

Adult↗
PubMed1997

Randomised controlled trial of inhaled corticosteroids (fluticasone propionate) in cystic fibrosis.

BACKGROUND: Controlling lung inflammation may be the key to improving morbidity and mortality in cystic fibrosis. OBJECTIVE: To assess the effects of inhaled corticosteroids on lung inflammation in cystic fibrosis. DESIGN: Double blind placebo controlled randomised sequence crossover trial. Fluticasone propionate (400 micrograms/day) was given as a dry powder inhaler for six weeks with a four week washout period before crossover. OUTCOME MEASURES: Sputum inflammatory markers (interleukin-8, tumour necrosis factor-alpha (TNF-alpha) and neutrophil elastase-both free and bound to alpha 1-antiprotease), sputum interleukin-10, lung function, and symptomatology. SUBJECTS: Twenty three children from a regional cystic fibrosis centre were enrolled into the study, with mean age 10.3 years (range 7 to 17 years) and mean baseline forced expiratory volume in one second (FEV1) of 64% (range 21% to 102%) predicted for sex and height. One patient was excluded for non-compliance to the study protocol. RESULTS: No significant benefit was shown for the use of fluticasone propionate in any of the outcomes. For sputum interleukin-8 there was an estimated true treatment median difference of 142 pg/ml (95% confidence interval (CI) 8 to 2866 pg/ml) in favour of placebo; while for maximal expiratory flow at 25% (MEF25%) remaining forced vital capacity predicted for sex and height there was a 15 percentage points (pp) (95% CI 4 to 26 pp) mean treatment difference in favour of placebo. Sputum interleukin-10 was undetected in any samples and unaffected by fluticasone propionate. Neither atopic status, baseline FEV1, nor concomitant DNase therapy had any effect on response to treatment. CONCLUSIONS: Lack of benefit from fluticasone propionate was most likely due to failure of the drug to penetrate the viscid mucus lining the airways. It is suggested a large multicentre trial with higher doses given for a longer time by a different delivery system is required to assess efficacy.

Administration, Inhalation↗
PubMed1996

Congenital enterocyte heparan sulphate deficiency with massive albumin loss, secretory diarrhoea, and malnutrition.

BACKGROUND: The molecular basis of protein-losing enteropathy is unknown. However it has been shown that sulphated glycosaminoglycans may be important in regulating vascular and renal albumin loss. METHODS: We describe three baby boys who presented within the first weeks of life with massive enteric protein loss, secretory diarrhoea, and intolerance of enteral feeds. All required total parenteral nutrition and repeated albumin infusions. No cause could be found in any case despite extensive investigations, including small intestinal biopsy sampling, which were repeatedly normal. FINDINGS: By specific histochemistry, we detected gross abnormality in the distribution of small intestinal glycosaminoglycans in all three infants, with complete absence of enterocyte heparan sulphate. The distribution of vascular and lamina propria glycosaminoglycans was, however, normal. INTERPRETATION: The presentation of these infants suggests that enterocyte heparan sulphate is important in normal small intestinal function.

Albumins↗
PubMed1996

The influence of capsulation and lipooligosaccharide structure on neutrophil adhesion molecule expression and endothelial injury by Neisseria meningitidis.

Host inflammatory response to meningococcal infection is believed to be a major determinant of disease severity. Isogenic mutants of Neisseria meningitidis serogroup B1940, which differ in expression of capsular polysaccharide and lipooligosaccharide (LOS), were used to examine host responses in a whole blood model of bacteremia and a model of endothelial injury. The parent organism caused significantly less neutrophil shedding of the adhesion molecule, L-selectin, than the three mutant organisms (P < .01) and was most resistant to the bactericidal activity of whole blood. Despite marked differences in bacterial adhesion to endothelial cells (P < .05), no damage was induced by organisms alone. Endothelial injury was observed when neutrophils were incubated with adherent, capsule-deficient organisms (P < .05). The degree of endothelial damage was related to the number of neutrophils adherent to the endothelium. Thus, bacterial capsulation and LOS structure can influence neutrophil activation and endothelial injury and, as such, may be important in the pathogenesis of meningococcal sepsis.

Antigens, Bacterial↗
PubMed1996

Mucosal tumor necrosis factor-alpha production and extensive disruption of sulfated glycosaminoglycans begin within hours of birth in neonatal respiratory distress syndrome.

Many of the clinicopathologic features of neonatal respiratory distress syndrome (RDS) may be related to the inflammatory response mounted by the affected infant, although little is known about the interstitial component of this response. We have thus studied the local inflammatory response in this condition by immunohistochemical analysis of whole lung lobes, obtained at postmortem from 40 infants who died from acute RDS in the first week of life. All had demonstrated classical clinical history and histologic features. An archival subgroup from the early 1970s had never received ventilatory support. Immunohistochemical analysis demonstrated rapid temporal increase from birth in the mucosal density of CD68+ macrophages, MAC-387+ monocytes/macrophages, polymorphonuclear neutrophils, and tumor necrosis factor-alpha-immunoreactive cells, maximal in those dying at or after 72 h. Using a cationic probe specific for sulfated glycosaminoglycans (GAGs), the inflammatory infiltration was seen to be associated with striking loss of endothelial, basement membrane, and interstitial GAGs, which was almost complete by 48-72 h. GAG degradation products were found within hyaline membranes in all infants dying after 48 h. This study confirms that neonatal RDS is characterized by intense interstitial inflammation, significantly underestimated on routine staining. This begins within hours of birth and is maximal by 72 h of age. Breakdown of sulfated GAGs within the extracellular matrix follows the same time course and may explain much of the physiologic derangement characteristic of this condition.

Age of Onset↗
PubMed1996

Early production of macrophage inflammatory protein-1 alpha occurs in respiratory distress syndrome and is associated with poor outcome.

Although progression to pulmonary fibrosis in preterm infants with respiratory distress syndrome (RDS) is related to the inflammatory response, the nature of this response remains controversial. We have therefore performed sequential bronchoalveolar lavages in 30 infants with RDS (13 of whom developed bronchopulmonary dysplasia) and 7 ventilated control infants, characterizing the cells obtained by immunohistochemical analysis of lineage-specific markers and assaying macrophage-associated chemokines and cytokines in supernatant fluid. At all ages from birth, lavage supernatants demonstrated highly significant increase over controls of the beta-chemokine macrophage inflammatory protein (MIP)-1 alpha, although not of regulated upon activation, normal T cell expressed and secreted (RANTES), of the cytokines tumor necrosis factor (TNF)-alpha and IL-1 beta, and of elastase/alpha-1 antitrypsin. Significantly higher concentrations of MIP-1 alpha in particular were associated with the later development of fibrosis. Increased numbers of macrophages expressing the activation marker RM/3-1 were found at all ages in bronchopulmonary dysplasic infants, whereas neutrophil numbers were increased from d 3. Dexamethasone administered to 10 infants induced rapid decrease in inflammatory cell numbers and concentrations of MIP-1 alpha, tumor necrosis factor-alpha, IL-1 beta, and elastase/alpha-1 antitrypsin. The inflammatory response in neonatal RDS begins within the first day of life. Long-term outcome is associated with the magnitude of this early response, in particular production of MIP-1 alpha. The early introduction of specific therapy is thus likely to be beneficial.

Biomarkers↗
PubMed1995

Modulation of the endothelial procoagulant response to lipopolysaccharide and tumour necrosis factor-alpha in-vitro: the effects of dexamethasone, pentoxifylline, iloprost and a polyclonal anti-human IL-1 alpha antibody.

Endothelial expression of tissue factor (TF), a potent procoagulant molecule, is increased in response to inflammatory mediators such as lipopolysaccharide (LPS), tumour necrosis factor (TNF) and interleukin-1 (IL-1). We have examined the effects of three antiinflammatory agents and a polyclonal anti-human IL-1 alpha antibody on the human endothelial TF response to E. coli 0111:B4 LPS and recombinant TNF alpha (rTNF alpha) in vitro. In contrast to the expected inhibitory effect, dexamethasone, pentoxyfilline and iloprost failed to block TF expression when administered simultaneously or 30 minutes prior to stimulation with either LPS or rTNF alpha. Inhibition of procoagulant activity was demonstrated with the anti-IL-1 alpha antibody, suggesting that endothelial derived IL-1 alpha is partially responsible for the TF response to the agonists employed. The failure of the antiinflammatory agents to inhibit endothelial TF expression highlights the possibility that therapeutic agents that modulate the circulating monocyte response to LPS and TNF alpha may not ameliorate the endothelial dysfunction that is also induced by these inflammatory mediators.

Adjuvants, Immunologic↗
PubMed1995

Whole blood model of meningococcal bacteraemia--a method for exploring host-bacterial interactions.

An ex vivo whole blood model of meningococcal bacteraemia was developed to examine the total bactericidal activity of blood. Using a single defined donor and strains belonging to serogroups A, B and C and an unencapsulated strain, we demonstrated that the bactericidal mechanisms operating in whole blood varied with anticoagulant, serogroup and bacterial growth conditions. The choice of anticoagulant had a major effect on the survival of the serogroup A strain with 94% (SEM 7.6) survival in citrated blood compared to 19.7% (SEM 19.6) survival in heparinised blood after 60 min incubation. The serogroup C strain showed enhanced survival when grown in liquid medium compared to growth on solid medium (73.5%, SEM 7.5, and 8.2%, SEM 3.1, respectively, in citrated blood after 60 min). The pattern of survival of serogroup B and the unencapsulated strain were largely unaffected by these variables. Comparison with cell free conditions allowed the contribution of cellular components in meningococcal killing to be determined. Secreted levels of tumour necrosis factor and neutrophil elastase secreted during whole blood assays did not correlate with bacterial growth or viability indicating a lack of relationship between killing and activation of phagocytes.

Anticoagulants↗
PubMed1993

Disruption of sulphated glycosaminoglycans in intestinal inflammation.

We have studied the distribution and nature of sulphated glycosaminoglycans (GAGs) within normal and inflamed intestine. There is increasing evidence that these negatively charged polysaccharides, which both regulate the ability of albumin to leave the vasculature and inhibit thrombosis, may be affected by inflammatory cells and their products. We obtained samples of freshly resected intestinal tissue from eight controls, eleven patients with Crohn's disease, and six with ulcerative colitis. Sulphated GAGs were detected by means of a gold-conjugated poly-L-lysine probe, and the tissue density of anionic sites was assessed semiquantitatively by means of a Lennox graticule. In normal intestine there was staining in the vascular endothelium and the subepithelial basal lamina and throughout the extracellular matrix of the lamina propria and submucosa. Tissue from the patients with inflammatory bowel disease showed inflammation macroscopically and on histology. There were profound abnormalities of extracellular matrix GAGs, limited to the mucosa in ulcerative colitis and greatest in the submucosa in Crohn's disease. There was also substantial loss of GAGs from the subepithelial basal lamina in both disorders and from the vascular endothelium in submucosa in Crohn's disease. The extent of local GAG disruption was associated with the distribution of macrophages immunoreactive for tumour necrosis factor alpha and the activation marker RM 3/1. We suggest that inflammatory disruption of vascular and connective tissue GAGs may be an important pathogenetic mechanism, contributing to the leakage of protein and fluid, thrombosis, and tissue remodelling seen in inflammatory bowel disease.

Adolescent↗
PubMed1993

Detection of glycosaminoglycans on the surface of human umbilical vein endothelial cells using gold-conjugated poly-L-lysine with silver enhancement.

Endothelial glycosaminoglycans are important in a diverse range of vascular functions. In the course of a biochemical and histological study exploring the role of glycosaminoglycans in inflammation, we have investigated the use of gold-conjugated poly-L-lysine with silver enhancement to establish the nature and physical location of glycosaminoglycans on the surface of cultured human umbilical vein endothelial cells. Cationic gold was effective in locating anionic sites in both cultured endothelial cells and in paraffin-embedded renal tissue. By manipulating pH, and by using enzymes specific for degrading glycosaminoglycans, it was found that, at pH 1.2, staining was directed primarily at glycosaminoglycans. The surface of human umbilical vein endothelial cells was found to be extensively covered in heparan sulphate, the histological appearance of which was dependent upon the fixation procedure employed. Heparan sulphate was also seen to co-distribute with the extracellular matrix protein, fibronectin, when endothelial cultures were simultaneously stained with cationic gold and an antibody to cellular fibronectin.

Binding Sites↗
PubMed1993

Degradation of glycosaminoglycans and fibronectin on endotoxin-stimulated endothelium by adherent neutrophils: relationship to CD11b/CD18 and L-selectin expression.

Vascular endothelial injury observed in overwhelming sepsis may be caused by neutrophil-derived enzymes. Adherence to the endothelium, a prerequisite for this process, is mediated sequentially by the neutrophil adhesion molecules L-selectin and the beta 2 integrins including CD11b/CD18. The relationship between expression of these molecules, neutrophil adherence, endothelial activation, and consequent endothelial injury was assessed by changes in heparan sulfate and fibronectin matrices. Endothelial prestimulation with lipopolysaccharide caused both an increase in adherence and a generalized reduction in heparan sulfate; disruption of the fibronectin matrix occurred only on the further addition of FMLP. Although maximal disruption of these matrices was associated with elevation of neutrophil CD11b/CD18 and reduction in L-selectin expression, these changes did not determine either the nature or extent of endothelial damage. This model may provide further insights into the interrelationship between neutrophil activation and endothelial damage in gram-negative sepsis.

Antigens, CD↗
PubMed1993

IL-4 regulates the morphology, cytoskeleton, and proliferation of human umbilical vein endothelial cells: relationship between vimentin and CD23.

Many of the vascular endothelial changes associated with inflammation can be induced in vitro by the cytokines tumour necrosis factor, IL-1, and IFN gamma. On the other hand, although IL-4 is a powerful mediator of leucocyte function, its influence on endothelial cells has not yet been fully determined. In this study the effect of IL-4 on human umbilical vein endothelial cells has been investigated. It is shown that IL-4 stimulates DNA synthesis over the first 24 h in culture, followed by dramatic alterations in endothelial cell morphology in which the cobblestone appearance of cells grown to confluence in medium changes to a monolayer composed of islands of tightly packed polygonal cells. The morphological changes induced by IL-4 were accompanied by a reorganization of the intracellular vimentin matrix from a diffuse pattern to a perinuclear concentration observed by staining with vimentin antibodies. Similar patterns of vimentin staining were also observed with an antibody to the low affinity IgE receptor (CD23) normally associated with IL-4 activation of B lymphocytes. Our results suggest that this represents cross reactivity between CD23 and vimentin rather than the appearance of CD23 in endothelial cells. Amino acid sequence comparison between CD23 and vimentin indicated significant homology between these two molecules, including a leucine zipper-like motif. Our results suggest that IL-4 may be an important regulator of endothelial cell morphology and function in inflammation.

Amino Acid Sequence↗
PubMed1992

Reduction of the anticoagulant activity of glycosaminoglycans on the surface of the vascular endothelium by endotoxin and neutrophils: evaluation by an amidolytic assay.

The processes that underlie the coagulopathy observed in severe infection are not fully understood, but seem to be due to an imbalance in the antithrombotic, and prothrombotic properties of the vascular endothelium. Sulphated glycosaminoglycans (GAGs) present on the vessel wall represent an important component of the non-thrombogenic nature of the endothelium. We have modified an amidolytic assay to study the functional ability of GAGs on human umbilical vein endothelial cells (HUVECS), and investigate the effect of E. coli endotoxin and neutrophils on HUVEC surface anticoagulant activity (SAA). Neither endotoxin alone, nor separated neutrophils at lower concentrations (less than 10(6) neutrophils per ml), had major effects on endothelial SAA. When activated neutrophils were incubated with HUVECS pre-stimulated with endotoxin, a significant decrease in SAA was seen using either plasma (mean percentage of control 67.8% +/- sem 7.8; p < 0.02) or purified ATIII (mean percentage of control 69% +/- sem 4.6; p < 0.001). We suggest that alterations in endothelial surface GAGs may occur during sepsis and inflammation, and that this may have important consequences for vascular function. This system will allow the further study of the role of GAGs in the intravascular thrombosis of severe sepsis, and other inflammatory diseases.

Anticoagulants↗