Background-Previous work indicated that enzymatically remodeled LDL (E-LDL) might activate complement in atherosclerotic lesions via a C-reactive protein (CRP)-dependent and CRP-independent pathway. We sought to substantiate this contention and determine whether both pathways drive the sequence to completion. Methods and Results-E-LDL was prepared by sequential treatment of LDL with a protease and cholesteryl esterase.Trypsin, proteinase K, cathepsin H, or plasmin was used with similar results. Functional tests were used to assess total complement hemolytic activity, and immunoassays were used to demonstrate C3 cleavage and to quantify C3a, C4a, C5a, and C5b-9. E-LDL preparations activated complement to completion, independent of CRP, when present above a threshold concentration (100 to 200 g/mL in 5% serum). Below the threshold, all E-LDL preparations activated complement in dependence of CRP, but the pathway then halted before the terminal sequence. Native LDL and oxidized LDL did not activate complement under any circumstances tested. Immunohistological analyses corroborated the concept that CRP-dependent complement activation inefficiently generates C5b-9. Conclusions-Binding of CRP to E-LDL is the first trigger for complement activation in the atherosclerotic lesion, but the terminal sequence is thereby spared. This putatively protective function of CRP is overrun at higher E-LDL concentrations, so that potentially harmful C5b-9 complexes are generated.

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Atherosclerosis is widely regarded as a chronic inflammatory disease that develops as a consequence of entrapment of low density lipoprotein (LDL) in the arterial intima. Native LDL lacks inflammatory properties, so the lipoprotein must undergo biochemical alterations in order to become atherogenic. Modification is commonly regarded as being dangerous because it bestows inflammatory properties onto the lipoprotein. Most current models consider oxidation to be the decisive modifying event. Here, we submit a different concept for discussion. We propose that modification of tissue-entrapped LDL is required because it enables the lipoprotein to signal to the immune system and effect its own removal. Oxidation would be too haphazard to fulfill this function. We summarize the evidence indicating that modification occurs through the action of ubiquitous hydrolytic enzymes. Enzymatically remodeled LDL binds C-reactive protein. C-reactive protein bound to remodeled LDL not only activates complement but also regulates it by inhibiting activation of the terminal complement cascade. Simultaneously, epitopes are exposed to enable the lipoprotein to be recognized and taken up by macrophages. The high density lipoprotein-dependent reverse transport pathway concludes the sequence of events that clear tissues of cholesterol in a non-inflammatory manner very similar to what has been described for the removal of apoptotic cells. It is proposed that these physiological processes occur throughout life without harm, pathology evolving only when the machinery suffers overload. Detrimental effects are then evoked primarily by the unreigned activation of complement, macrophages, and other effectors of the immune system in the lesions.

Helicobacter pylori infects up to 50% of the human population worldwide. The infection occurs predominantly in childhood and persists for decades or a lifetime. H. pylori is believed to be transmitted from person to person. However, tremendous genetic diversity has been reported for these bacteria. In order to gain insight into the epidemiological basis of this phenomenon, we performed molecular typing of H. pylori isolates from different families. Fifty-nine H. pylori isolates from 27 members of nine families were characterized by using restriction fragment length polymorphism analysis of five PCR-amplified genes, by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA, and byvacA and cagA genotyping. The 16S rRNA gene exhibited little allelic variation, as expected for a unique bacterial species. In contrast, the vacA, flaA,ureAB, and lspA-glmM genes were highly polymorphic, with a mean genetic diversity of 0.83, which exceeds the levels recorded for all other bacterial species. In conjunction with PFGE, 59 H. pylori isolates could be differentiated into 21 clonal types. Each individual harbored only one clone, occasionally with a clonal variant. Identical strains were always found either between siblings or between a mother and her children. Statistical analysis revealed clonality of population structure in all isolates. The results of this study suggest the possible coexistence of a large array of clonal lineages that are evolving in each individual in isolation from one another. Transmission appears to occur primarily from mother to child and perhaps between siblings.

Objective-Functionally interactive proteases of the plasminogen/plasmin and the matrix metalloproteinase (MMP) system degrade and reorganize the extracellular matrix of the vessel wall in atherosclerosis. Here we investigated whether such proteases are able to confer atherogenic properties onto low density lipoprotein by nonoxidative modification. Methods and Results-Similar to the recently described enzymatically-modified low-density lipoprotein (E-LDL), native LDL exposed to plasmin or matrix MMP-2 or MMP-9 and cholesterylester-hydrolase (CEH) showed extensive deesterification, with ratios of free cholesterol to total cholesterol rising to 0.8 compared with 0.2 in native LDL. When the ratio exceeded 0.6, both plasmin/CEH-LDL and MMP/CEH-LDL fused into larger particles. In parallel, they gained C-reactive protein-dependent complement-activating capacity. E-LDL produced with any protease/CEH combination was efficiently taken up by human macrophages, whereby marked induction of MMP-2 expression by E-LDL was observed. These in vitro findings had their in vivo correlates: urokinase-type plasminogen activator, MMP-2, and MMP-9 were detectable in both early and advanced human atherosclerotic lesions in colocalization with E-LDL. Conclusions-Plasmin and MMP-2/MMP-9 may not only be involved in remodeling of the extracellular matrix in progressing plaques, but they may also be involved in lipoprotein modification during genesis and progression of atherosclerotic lesions. Key Words: atherosclerosis Ⅲ lipoproteins Ⅲ macrophages Ⅲ metalloproteinases Ⅲ plasminogen activators I t is widely held that atherogenesis is triggered by enhanced entrapment of low-density lipoprotein (LDL) in the intima, which is followed by its uptake by macrophages. Both oxidative and nonoxidative processes can generate potentially atherogenic LDL derivatives. 1 We are pursuing the concept that enzymatic remodeling of the lipoprotein is a key modification, because proteolytic cleavage of apolipoprotein B (apoB) in conjunction with hydrolysis of cholesteryl esters generates lipoprotein particles that are similar to lesionderived LDL in structure, biological properties, and composition. 2,3 Enzymatically-remodeled LDL (E-LDL) binds C-reactive protein (CRP) and activates complement. 4 E-LDL induces foam cell formation in monocytes, 5 macrophages, 3 and smooth muscle cells, 6 stimulates MCP-1 production, 7 and directly promotes adhesion and transmigration of monocytes through endothelial cell monolayers. 8 These in vitro findings have their in vivo correlates: immunohistological analyses with specific monoclonal antibodies (mAbs) have revealed extensive extracellular deposits of E-LDL at the early stages of atherosclerotic lesion formation. 9 CRP and activated complement components are also present in colocalization with E-LDL. 4,9 Like in vitro-generated E-LDL, lesioned LDL has a high content of free cholesterol; 10 -12 therefore, it is apparent that extensive deesterification of cholesteryl esters must indeed occur in the lesions.We used trypsin in c...

Genetic diversity in Helicobacter pylori strains may affect the function and antigenicity of virulence factors associated with bacterial infection and, ultimately, disease outcome. In this study, DNA diversity of H. pylori isolates was examined by analysis of vacA genotypes and by restriction fragment length polymorphism (RFLP) analysis of H. pylori-associated genes (vacA, cagA, flaA,ureAB, and ureCD). Thirty-seven H. pylori isolates from 26 patients were successfully classified into distinct vacA allelic genotypes. The signal sequence allele s1 (31 of 37) predominated over the s2 allele (6 of 37) and was significantly associated with the occurrence (past or present) of gastric ulcers. A novel midregion allele, designated as m3, has been identified in two H. pylori isolates which could not be typed with midregion allele m1- or m2-specific primers. Additionally, significant nucleotide diversity yielding different amino acid sequences was demonstrated by DNA sequencing of vacAfragments from clinical isolates of H. pylori. Furthermore, RFLP analysis of 45 H. pylori isolates (including 15 paired isolates) obtained from antrum and corpus biopsy specimens from 30 individual patients showed remarkably high interhost diversity (one patient, one H. pylori strain) and intrahost identity in gene sequences coding for VacA, CagA, flagellin, and urease. Only in a single patient was a minor genotypic variation at different anatomic sites within the stomach identified. These data warrant the detailed analysis of the effect of genetic diversity on the function and antigenicity of H. pylori-associated virulence factors.

Objective-Modification with proteases and cholesterylesterase transforms LDL to a moiety that resembles lipoproteins isolated from atherosclerotic lesions and possesses atherogenic properties.

Amoxicillin is often implemented in Helicobacter pylori treatment protocols. To date, amoxicillin-resistant H. pylori strains have rarely been detected, and only a total of 14 have been reported in the literature (1). Conspicuously, complete loss of the resistant phenotype was observed after these strains were stored at Ϫ80°C. Only one amoxicillin-resistant H. pylori strain has been isolated, in The Netherlands, in which in contrast, the amoxicillin resistance remained stable after repeated cycles of freezing and culture (5). The MIC for this strain was 8 g/ml, which is relatively low. Since 1996, we have isolated seven H. pylori strains exhibiting high-level amoxicillin resistance (MIC Ͼ 256 g/ml) (Table 1). Four of these exhibited a stable resistance phenotype (strains ACR3,-4,-5, and-7). Strains ACR1,-2, and-4 were isolated from two 11-year-old girls and one 15-year-old girl with recurrent abdominal pain. They received triple therapy in