Ade Ajayi, Niyi; (2004) Glycosaminoglycans and inflammatory cells in the pathophysiology of neonatal necrotizing enterocolits. Masters thesis , UCL (University College London).

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Abstract

Fulminant neonatal necrotizing enterocolitis (NEC), is characterised by temperature instability, hypotension, disseminated intravascular coagulopathy and oliguria, all clinical manifestations of multi-system organ dysfunction. The capillary leak syndrome (CLS) is common, poses a particularly difficult management problem and is directly and indirectly responsible for many of the complications suffered by infants with this intestinal disorder. Glycosaminoglycans (GAGs) are key components of the extra-cellular matrix (ECM) and are important for structural stability, the regulation of vascular permeability and trans-endothelial inflammatory cell migration. The capillary leak phenomenon might be explained in part by matrix degradation. The primary aim of this thesis was to examine the nature and distribution of GAGs in NEC. A secondary aim was to describe the inflammatory cell infiltrate (ICI) in relation to GAGs in this disease. Histochemical experiments were performed on intestine from infants who underwent surgical resection for NEC. GAGs were detected using a cationic gold (CG) method; a 5nm gold conjugated poly-L-lysine probe at a pH of 1.5 was applied to tissue sections then developed with silver enhancer. Glycanases were employed to determine specific GAG families; sections were incubated with chondroitinase ABC or heparinase III. Control specimens from well-preserved resection margins were incubated with phosphate buffered saline. Matrix and cell surface degradation of GAGs was evident and proportionate to NEC severity. With chondroitinase ABC, GAGs in all layers of the bowel wall were digested, leaving the vascular stain intact. Whilst vascular GAGs were reduced with heparinase III, basement membrane and baso-lateral GAGs were unaffected. Inflammatory cells produce glycanases, therefore, to explore one possible mechanism of GAG degradation the cellular infiltrate in NEC was examined. The alkaline phosphatase anti-alkaline phosphatase (APAAP) method was employed. Neutrophil elastase, MAC 387, HLA-DR, CD3, CD20 and Ki67 monoclonal antibodies were used. Expression of vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and E-Selectin was also determined following high temperature citration. Neutrophils were found to be restricted to the serosa in early NEC, and did not appear in the mucosa in substantial numbers until mucosal ulceration had occurred. The pattern of activation of lymphocytes and up- regulation of adhesion molecules was consistent with an immune response to antigenic stimulation in the early stages of the disease. The hypothesis was put forward that urinary GAG levels would be elevated in NEC in proportion to disease severity. Urinary GAGs were measured in both an experimental ischaemia/reperfusion (IR) model and in human new-borns with advanced NEC. Urine from human infants was assayed using a modification of this technique. In addition, the urine from infants was subjected to two-dimensional gel electrophoresis to determine specific GAG subgroups present. In the experimental model, there was no difference between animals in the IR group and controls. Similarly in human new-borns with gangrenous bowel secondary to NEC and mechanical intestinal ischaemia, there was no significant difference in the GAG/Cr when compared with controls. However, infants with intestinal ischaemia, and in particular those with confluent intestinal gangrene had abnormal quantities of heparan sulphate (HS) relative to chondroitin sulphate (CS) detected in the urine. Our immunohistochemical results support evidence of a lymphocyte driven immunopathy as an important part of the mechanisms that underlie disease progression in NEC. Furthermore, in vivo assays of urinary GAGs demonstrated abnormal HS elevation in the presence of gangrenous bowel, suggesting that this may be a useful adjunct in detecting infants with advanced disease.

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