Adherence of Protease-Deficient Mutants of Pseudomonas aeruginosa to a Rabbit Cornea Cell Line (SIRC) Cells

Seven protease-deficient mutants were isolated from Pseudomonas aeruginosa strain IFO 3455 which was mutagenized with nitrosoguanidine. Characterization of these mutants in vitro revealed that all mutants showed pleiotropic changes in the production of other extracellular substances. Among the mutants, two were chosen for a bacterial adherence test to Rabbit Cornea Cell Line (SIRC) cells. One mutant (IFO 3455-2) completely lost its protease activity. Another (IFO 3455-3) retained a low protease activity and was relatively similar to the parental strain with respect to extracellular products except for protease. Both mutants gave not a marked but a slight decrease of adherence as compared with the parental strain. This finding suggests that besides protease more factors are involved in the adhesion between P. aeruginosa and SIRC cells

Physical and Chemical Factors Affecting the Adherence of Pseudomonas aeruginosa to a Rabbit Cornea Cell Line (SIRC) Cells

Adherence of Pseudomonas aeruginosa to SIRC cells was examined by the use of 14C-lysine labeled organisms. Pretreatment of P. aeruginosa with heating, 3% formaldehyde, or ultraviolet caused a significant decrease in adherence to SIRC cells, whereas that with lipase, hyaluronidase, trypsin or protease did not. Treatment of SIRC cells with trypsin, protease, lipase or neuraminidase did not influence the adherence of P. aeruginosa to the cell. Treatment of P. aeruginosa with mannose or galactose inhibited the adherence, while that with fructose, lactose or glucose did not. Treatment of SIRC cells with galactosidase or mannosidase reduced the adherence of the organism. No correlation was demonstrated between the adhering ability and hydrophobicity of P. aeruginosa. The results suggest that both the viability in bacterial site and mannose and/or galactose molecules in cellular site are closely connected with the adherence of P. aeruginosa to SIRC cells

Monocyte/macrophage response to β2-microglobulin modified with advanced glycation end products

Monocyte/macrophage response to β2-microglobulin modified with advanced glycation end products. We recently found that acidic β2-microglobulin (β2m), a major isoform of β2m in amyloid fibrils of patients with dialysis-related amyloidosis (DRA), contained early Amadori products and advanced glycation end products (AGEs) formed nonenzymatically between sugar and protein. Further analysis revealed that acidic β2m induces monocyte chemotaxis and macrophage secretion of bone-resorbing cytokines, suggesting the involvement of acidic β2m in the pathogenesis of DRA. Acidic β2m, however, is a mixture of heterogeneous molecular adducts due to various types of modification. In the present study, we investigated the modification responsible for the biological activity of acidic β2m toward monocytes/macrophages. The presence of a fair amount of β2m species with deamidation was detected in acidic β2m isolated from urine of non-diabetic long-term hemodialysis patients, but deamidated β2m had no biological activity. In contrast, normal β2m acquired the activity upon incubation with glucose in vitro. Among the glycated β2m, the pigmented and fluorescent β2m that formed after a long incubation period, that is, AGE-modified β2m, exhibited biological activity, whereas β2m modified with Amadori products, major Maillard products in acidic β2m, had no such activity. These findings suggest that AGEs, although only a minor constituent of acidic β2m, are responsible for monocyte chemotaxis and macrophage secretion of cytokines, implicating the contribution of AGEs to bone and joint destruction in DRA

緑膿菌の家兎角膜培養細胞への付着性について

Adherence of Pseudomonas aeruginosa to a Rabbit Cornea Cell Line (SIRC) cells in vitro was examined. Maximal adherence occurred at pH 5.0-7.0 and when the bacterial suspension at 1.4×10^9/ml was exposed to 2×10^4/ml SIRC cells at mid-logarithmic growth phase. Adherence was not affected by temperature. In the passage of time, a temporary rise of adherence occurred within 5 min of incubation and stationary phase was maintained from 30 min through 90 min of incubation. Killing of P. aeruginosa with heating at 65°C for 30 min or 60 min resulted in a marked decrease in adherence. Similar results were obtained by pretreatment of bacteria with 3% formaldehyde. Different strains of P. aeruginosa varied in their abilities to adhere to SIRC cells. Strains which produce protease adhered more avidly to SIRC cells than those which produce no protease, while the presence of pili was not correlated with bacterial adherence

The receptor for advanced glycation end products (RAGE) is a central mediator of the interaction of AGE-beta2microglobulin with human mononuclear phagocytes via an oxidant-sensitive pathway. Implications for the pathogenesis of dialysis-related amyloidosis.

This is the published version. Copyright 1996 American Society for Clinical Investigation.An important component of amyloid fibrils in dialysis-related amyloidosis is a form of beta2microglobulin modified with advanced glycation end products (AGEs) of the Maillard reaction, known as AGE-beta2M. We demonstrate here that the interaction of AGE-beta2M with mononuclear phagocytes (MPs), cells important in the pathogenesis of the inflammatory arthropathy of dialysis-related amyloidosis, is mediated by the receptor for AGEs, or RAGE. 125I-AGE-beta2M bound to immobilized RAGE or to MPs in a specific, dose-dependent manner (Kd approximately 53.5 and approximately 81.6 nM, respectively), a process inhibited in the presence of RAGE blockade. AGE-beta2M-mediated monocyte chemotaxis was prevented by excess sRAGE or anti-RAGE IgG. Induction of tumor necrosis factor-alpha (TNF) expression by MPs exposed to AGE-beta2M resulted from engagement of RAGE, as appearances of TNF transcripts and TNF antigen release into culture supernatants were prevented by addition of sRAGE, a process mediated, at least in part, by oxidant stress. AGE-beta2M reduced cytochrome c and the elaboration of TNF by MPs was inhibited by N-acetylcysteine. Consistent with these data, immunohistochemical studies of AGE-laden amyloid deposits of a long-term hemodialysis patient revealed positive staining for RAGE in the MPs infiltrating these lesions. These data indicate that RAGE is a central binding site for AGEs formed in vivo and suggest that AGE-beta2M-MP-RAGE interaction likely contributes to the initiation of an inflammatory response in amyloid deposits of long-term hemodialysis patients, a process which may ultimately lead to bone and joint destruction

Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure

Recent studies have demonstrated a marked increase in the level of advanced glycation end products (AGEs) in the plasma, skin and amyloid fibrils of hemodialysis (HD) patients. The presence of AGEs in (beta(2)m) forming amyloid fibrils has been established in a previous immunochemical study relying on a monoclonal anti-AGE antibody. In the present study, Western blot analysis and immunohistochemistry reveal that the epitope recognized by this antibody is N-epsilon-(carboxymethyl)lysine (CML) and that CML is one of the AGE structures present in amyloid fibrils. Thus, two AGE structures, CML and pentosidine, are now recognized in dialysis-related amyloidosis. AGE accumulation in uremia is not accounted for by elevated glucose levels. Since CML and pentosidine formation are closely linked to oxidative processes, we tested the hypothesis that a high oxidative stress enhanced AGE formation in HD patients. We focused on ascorbic acid (AA) because AA is easily oxidized under oxidative stress and its oxidized form (oxiAA) is a source of CML and pentosidine. In vitro incubation of beta(2)m with AA under atmospheric oxygen resulted in: (1) the rapid appearance of characteristic physicochemical properties of AGEs (brown color, fluorescence, polymerization tendency); (2) the transformation of beta(2)m into AGE-modified beta(2)m recognized by a specific monoclonal antibody; and (3) the accelerated formation of CML in beta(2)m and beta(2)m-peptide, recognized by mass spectrometry. A similar in vitro incubation of human serum albumin disclosed a parallel production of pentosidine measured by high-performance liquid chromatographic assay. In HD patients, the degree of AA oxidation, assessed as the ratio of oxiAA to total ascorbate, was more than twice as high as that of normal subjects (0.87 +/- 0.16 vs. 0.35 +/- 0.11, P < 0.0001), suggesting the presence of an increased oxidative stress. interestingly, plasma level of oxiAA was correlated with the plasma levels of protein linked (P < 0.01, r(2) = 0.25) and free (P < 0.05, r(2) = 0.22) pentosidine. Altogether these results demonstrate that AGE, that is, CML and pentosidine, production is accelerated under oxidative stress, even in the absence of glucose. They suggest that, in uremia, CML and pentosidine production is determined both by an increased oxidative stress and the availability of precursors such as oxiAA. Finally, both CML and pentosidine contribute to the AGEs present in dialysis-related amyloid fibrils

Clearance of pentosidine, an advanced glycation end product, by different modalities of renal replacement therapy

We recently demonstrated that pentosidine, an advanced glycation end product,accumulates markedly as albumin-linked form (P-alb) and in free-form (P-free) in the plasma of patients with end-stage renal failure. The present study was undertaken to examine the clearance of P-alb and P-free by different modalities of renal replacement therapy, that is, hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD), and renal transplantation. HD cleared P-free (9.4 +/- 4.3 nmol/kg/HD) but not P-alb, by diffusion but not by membrane adsorption, whereas CAPD cleared both P-alb (4.03 +/- 2.01 nmol/kg/day) and P-free (2.43 +/- 1.24 nmol/kg/day). Plasma total pentosidine levels were significantly (P < 0.05) lower in CAPD (0.97 +/- 0.41 nmol/ml) than in HD (1.19 +/- 0.41 nmol/ml), as the result of a lower serum albumin level in the former patients. Indeed, P-alb expressed per mg albumin was virtually identical in HD and CAPD. By contrast, P-free was significantly lower in CAPD than in HD. P-alb levels were significantly correlated with plasma P-free levels in both HD and CAPD patients, but not in the CAPD dialysate. Pentosidine transport across the peritoneum occurs mainly by diffusion, both as P-alb and P-free. Interestingly, peritoneal P-alb clearance (0.17 +/- 0.07 ml/min) significantly (P < 0.00001) exceeded albumin clearance (0.11 +/- 0.05 ml/min). P-alb levels being significantly higher (P < 0.0005) in the peritoneal fluid (36.28 +/- 18.55 pmol/mg) than in the serum (27.12 +/- 11.71 pmol/mg), thus raises the possibility of a facilitated diffusion of P-alb or an active transport mechanism for protein-linked pentosidine into the peritoneal cavity. After renal transplantation, plasma P-free fell rapidly, remained barely detectable after one month, and returned to normal at six months. By contrast, P-alb fell more slowly and remained significantly above normal at six months, but returned eventually to normal levels. These findings demonstrate that: (1) both HD and CAPD remove P-free; (2) the peritoneal clearance of P-alb might contribute to the lower level of plasma pentosidine in CAPD than in HD patients; and (3) renal transplantation is the best therapeutic modality to normalize both P-alb and P-free levels

Citrobacter braakii bacteremia-induced septic shock after colonoscopy preparation with polyethylene glycol in a critically ill patient: a case report

Abstract Background Polyethylene glycol (PEG) is widely used for bowel cleaning in preparation for colonoscopy because of its safety. Septic shock after PEG preparation is an extremely rare complication. Herein, we describe a case of septic shock that occurred immediately after colonoscopy preparation with PEG. Case presentation A 75-year-old Japanese male who had previously developed diabetes after total pancreatectomy received PEG in preparation for colonoscopy. He had been admitted to the emergency intensive care unit 4 days earlier due to hematochezia presenting with shock. He ingested PEG to prepare for a colonoscopy examination, which was performed to identify the source of his bleeding over a 5-h period, but suddenly exhibited septic shock and markedly elevated procalcitonin levels. A blood culture subsequently revealed Citrobacter braakii. Immediate resuscitation and intensive care with appropriate antibiotics improved his condition. Conclusions Clinicians should be aware of the possibility of deteriorating conditions after bowel preparation with PEG among severely ill patients with recent episodes of hemorrhagic shock