The Role of TGF-ß in the Association Between Primary Graft Dysfunction and Bronchiolitis Obliterans Syndrome.

Primary graft dysfunction (PGD) is a possible risk factor for bronchiolitis obliterans syndrome (BOS) following lung transplantation; however, the mechanism for any such association is poorly understood. Based on the association of TGF-ß with acute and chronic inflammatory disorders, we hypothesized that it might play a role in the continuum between PGD and BOS. Thus, the association between PGD and BOS was assessed in a single-center cohort of lung transplant recipients. Bronchoalveolar lavage fluid concentrations of TGF-ß and procollagen collected within 24 h of transplantation were compared across the spectrum of PGD, and incorporated into Cox models of BOS. Immunohistochemistry localized expression of TGF-ß and its receptor in early lung biopsies posttransplant. We found an association between PGD and BOS in both bilateral and single lung recipients with a hazard ratio of 3.07 (95% CI 1.76-5.38) for the most severe form of PGD. TGF-ß and procollagen concentrations were elevated during PGD (p < 0.01), and associated with increased rates of BOS. Expression of TGF-ß and its receptor localized to allograft infiltrating mononuclear and stromal cells, and the airway epithelium. These findings validate the association between PGD and the subsequent development of BOS, and suggest that this association may be mediated by receptor/TGF-ß biology.

CXCR3 chemokine ligands during respiratory viral infections predict lung allograft dysfunction.

Community-acquired respiratory viruses (CARV) can accelerate the development of lung allograft dysfunction, but the immunologic mechanisms are poorly understood. The chemokine receptor CXCR3 and its chemokine ligands, CXCL9, CXCL10 and CXCL11 have roles in the immune response to viruses and in the pathogenesis of bronchiolitis obliterans syndrome, the predominant manifestation of chronic lung allograft rejection. We explored the impact of CARV infection on CXCR3/ligand biology and explored the use of CXCR3 chemokines as biomarkers for subsequent lung allograft dysfunction. Seventeen lung transplant recipients with CARV infection had bronchoalveolar lavage fluid (BALF) available for analysis. For comparison, we included 34 BALF specimens (2 for each CARV case) that were negative for infection and collected at a duration posttransplant similar to a CARV case. The concentration of each CXCR3 chemokine was increased during CARV infection. Among CARV infected patients, a high BALF concentration of either CXCL10 or CXCL11 was predictive of a greater decline in forced expiratory volume in 1 s, 6 months later. CXCR3 chemokine concentrations provide prognostic information and this may have important implications for the development of novel treatment strategies to modify outcomes after CARV infection.

Fungal infection in lung transplantation.

Fungal infection remains a significant cause of postoperative morbidity and mortality in lung transplant recipients. The lung recipient remains the only solid-organ allograft continuously open to the environment and to the myriad of fungal spores and pathogens. Many factors may predispose to fungal infection in these patients, including: preoperative chronic lung diseases and inherent palliative immunosuppression, intraoperative complications such as abnormalities in the bronchial anastomosis or lung injury, and postoperative complications such as enhanced immunosuppression for early rejection, graft dysfunction, concurrent viral and bacterial infections, and bronchiolitis obliterans syndrome. The risk factors and time course for fungal infection in lung transplant recipients parallel the observations in other solid-organ transplant recipients. Early fungal infections are related to surgical complications, while the period of 1-6 months reflect opportunistic, relapsed, or residual infections; fungal infections greater than 6 months and thereafter are usually associated with treatments for chronic rejection or bronchial airway mechanical abnormalities. The majority of fungal infections in lung transplant recipients involve Aspergillus species, followed by Candida, Pneumocystis, Cryptococcus, geographically-restricted agents, and newly emerging fungal pathogens. The identification of at-risk patients (preoperatively and postoperatively) is essential in implementing prophylaxis or preemptive management. Some anti-fungal strategies and future options for clinical research are discussed.

Clinical cases in transplantation.

A series of cases are presented to illustrate the complexity of the care of immunocompromised patients with established invasive fungal infections. These discussions by an expert panel serve to identify areas of controversy and for future research in the care of transplant recipients.

Safety and efficacy of Intralipid emulsions of amphotericin B.

To evaluate the clinical role of amphotericin/20% Intralipid emulsions (ILA), we conducted a Medline search of the English literature to locate the relevant case reports and clinical studies involving the use of this formulation. Due to differences in study design and definitions, we applied a set of treatment outcome definitions to determine the clinical efficacy of this treatment modality. Only 37 patients received ILA for the treatment of documented fungal infections. Using our definitions, four were considered successfully treated, one improved, two failed, and 30 were unevaluable. While infusion-related adverse events and nephrotoxicity were reportedly reduced with ILA, use of adjunctive therapies and concomitant nephrotoxic agents, and comparisons with high infusion concentrations complicate evaluation. Furthermore, incomplete and conflicting data exist regarding the physiochemical stability of ILA. The currently available data do not support recommendations for the use of this formulation for the treatment of systemic fungal infections.

Advances in medical and antibiotic management of infective endocarditis.

Infective endocarditis remains a serious medical problem despite advancements in laboratory detection, echocardiographic techniques, and newer antibiotic agents. This article summarizes the microbial agents in infective endocarditis, in addition to developments in medical and antibiotic management.

Treatment of ocular fungal infections with oral fluconazole.

PURPOSE: We treated five patients who had ocular fungal infections with oral fluconazole to determine its safety and effectiveness. METHODS: We reviewed the case histories of the five patients. One patient had coccidioidomycosis and four had endogenous Candida endophthalmitis. RESULTS: The intraocular fungal infection resolved in all patients. CONCLUSION: Fluconazole appears to be a safe and effective antifungal agent that can be administered orally and may be a useful agent for treating some intraocular fungal infections.

Binding sites for C-reactive protein on human monocytes are distinct from IgG Fc receptors.

Previous investigations have provided evidence to suggest that C-reactive protein (CRP), an acute-phase reactant, binds to human monocytes at a membrane site that is either identical to or physically associated with IgG Fc receptors. To characterize further the relationship between monocyte CRP binding sites and IgG Fc receptors, monocytes were allowed to attach to surfaces coated with IgG or CRP and binding-site redistribution was assessed. Binding was measured by using protein-coated sheep erythrocytes (E). When attached to control (gelatin or albumin) surfaces, greater than 60% and 43% of monocytes formed rosettes with E-IgG and E-CRP, respectively. Following adherence to surface immobilized CRP, the proportion of cells binding E-IgG was unchanged; however, fewer than 20% of monocytes bound E-CRP. When attached to IgG-coated surfaces, fewer than 20% of monocytes formed rosettes with either E-IgG or E-CRP. In order to determine whether the unidirectional modulation of CRP and IgG binding sites was the result of CRP binding directly to a subclass of IgG Fc receptors, fluid-phase IgG-blocking studies were performed. When monocyte monolayers were preincubated with either monomeric or heat-aggregated IgG, a dose-dependent reduction in E-IgG binding was observed. In contrast, all concentrations of fluid-phase IgG failed to inhibit monocyte binding of E-CRP. These data indicate that CRP binds to human monocytes at a site physically associated with but distinct from IgG Fc receptors.

Evidence that serum amyloid P component binds to mannose-terminated sequences of polysaccharides and glycoproteins.

Serum amyloid P component (SAP) is a normal human serum protein with pentraxin structure that has morphological and immunochemical identity to the amyloid P component found in normal tissue and amyloid deposits. In the presence of calcium, SAP binds to certain complex polysaccharides, including agarose and zymosan. While the binding of SAP to agarose involves interaction with a galactose pyruvate acetal, the ligand in zymosan has not been defined. In the present study we determined that SAP binds to ligand(s) in a soluble extract of zymosan prepared by alkaline hydrolysis, which contains the mannose oligosaccharide sequences alpha DMan1----3DMan and alpha DMan1----6DMan. SAP did not bind to the alkali-insoluble fraction of zymosan, which is predominantly a glucan polymer, and its binding to zymosan extract which had been absorbed with concanavalin A was markedly reduced, suggesting that mannose residues are involved in the binding of SAP to zymosan. We also demonstrated that SAP binds to the glycoproteins ovalbumin, thyroglobulin, beta-glucuronidase and C3bi, which contain mannose-terminated sequences, while it did not bind to native and desialized preparations of ovomucoid, alpha 1-acid glycoprotein and glycophorin, which lack terminal mannose residues. SAP did not bind to pneumococcal C polysaccharide or to N-acetylglucosamine oligosaccharides covalently linked to a protein carrier. The binding of SAP to ligand(s) in zymosan extract or ovalbumin was inhibited by the preincubation of SAP with either zymosan extract or ovalbumin glycopeptides, both of which share similar mannose oligosaccharide sequences. All of the SAP binding reactions required calcium, were maximal at approximately 1 mM calcium, and gave similar results whether purified SAP or SAP in serum was used. These findings indicate that mannose-terminated oligosaccharides of polysaccharides and glycoproteins represent a new class of ligands for SAP and suggest that SAP may function as a mannose-binding protein.

Identification of multiple forms of the P component of amyloid isolated from human serum.

We examined isolated serum amyloid P component (SAP), the circulating counterpart of the amyloid P component, and established a previously unreported heterogeneity for SAP by immunological and biochemical methods. Highly purified SAP had a gel filtration Mr of 255,000, a Stokes radius of 57 A, a calculated frictional coefficient of 1.4, and 10 subunits of Mr of approximately 25,000. We present evidence suggestive of varying affinities of SAP for agarose, to which SAP is known to adsorb in the presence of calcium, by fused rocket immunoelectrophoresis. We resolved SAP subunits by isoelectric focusing into multiple species with isoelectric points of 6.08 (two forms), 6.02, and 5.95; three of these forms were delineated by high resolution two-dimensional SDS gel electrophoresis to have an Mr = 25,500, while a fourth (pI = 6.08) had an Mr = 24,500. The observed isoelectric charge heterogeneity could not be eliminated by neuraminidase treatment event though the electrophoretic migration of native desialized SAP was impeded (alpha 1 to beta) when examined by crossed immunoelectrophoresis, being consistent with the removal of anionic charges. Further, an additional neuraminidase-generated component was detected at the beta-position which could be removed by concanavalin A affinity. These data suggest SAP may exist in microheterologous or allotypic forms, the significance of which is under investigation.

Effect of divalent metal ions and pH upon the binding reactivity of human serum amyloid P component, a C-reactive protein homologue, for zymosan. Preferential reactivity in the presence of copper and acidic pH.

The serum amyloid P component (SAP) has been found to associate in vitro with a variety of polysaccharide and proteinaceous ligands including the yeast cell wall polysaccharide preparation, zymosan, in the presence of calcium at neutral pH. In the present study, we have investigated the role of copper and zinc and other divalent cations and acidic pH on the binding of SAP to zymosan. We report that binding occurs not only in the presence of calcium, but in the presence of copper, zinc, and cadmium as well. No binding occurs in the absence of added metal, or in the presence of barium, cobalt, magnesium, manganese, or nickel. 125I-SAP binding in the presence of metals is inhibited by presaturating the zymosan surface with unlabeled SAP. Whereas calcium-mediated binding decreases by more than 50% as the pH is lowered to 5, copper-mediated binding increases substantially at the more acidic pH values while zinc-mediated binding is essentially unchanged. These data indicate that, in addition to calcium at neutral pH, copper (and zinc) at neutral and particularly acidic pH values mediates SAP binding to polysaccharide ligands. This suggests that SAP may well be considered a copper- as well as a calcium-dependent protein under certain conditions and that this reactivity is favored under those conditions of lowered pH which may result from metabolic processes occurring at local sites of inflammation.

Solubilization and electrophoretic analysis of Staphylococcus aureus membrane proteins.

The protein composition of homogeneous Staphylococcus aureus 6538P cytoplasmic membranes was examined under denaturing electrophoretic conditions. A comparative analysis on the effectiveness of a variety of membrane solubilizing agents revealed the membrane protein extracts to be qualitatively similar as determined electrophoretically but different in the quantity of protein released; Zwittergent-314, sodium dodecyl sulfate, Triton X-100, Nonidet P-40, and sodium deoxycholate all proved to be effective solubilizing agents under the conditions examined. Fifty-five to sixty protein components were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis from homogeneous late-exponential phase membranes. The profile was unaffected when phenylmethylsulfonyl fluoride was included during membrane isolation and solubilization. Analysis of the solubilized membrane proteins by two-dimensional gel electrophoresis demonstrated in excess of 100 membrane protein components in a pH gradient between 3.5 to 7.7. The profile consisted of a heterogeneous mixture of mostly acidic components with isoelectric points between pH 4 and 5 and relative molecular weights between 158,000 and 35,000. Periodic acid-Schiff staining following sodium dodecyl sulfate gel electrophoresis revealed six to ten reactive bands with two of these bands also exhibiting a reaction with concanavalin A.

Analysis of Staphylococcus aureus cytoplasmic membrane proteins by isoelectric focusing.

Cytoplasmic membranes were isolated from late-exponential phase Staphylococcus aureus 6539 P and the membrane proteins examined under non-denaturing conditions by thin-layer isoelectric focusing (TLIEF) in a pH 3.5-9.5 gradient. Isolated membrane preparations retained protein integrity as judged by the demonstration of membrane bound adenosine triphosphatase (ATPase) activity in addition to four other solubilized membrane enzyme markers. Membranes were effectively solubilized with 2.5% Triton X-100 (final concentration). Examination of Triton X-100 solubilized membrane preparations established the presence of 22 membrane proteins with isoelectric points between 3.7 and 6.0. The focused proteins displayed the following enzymatic activities and isoelectric points by zymogram methods: ATPase (EC 3.6.1.3), 4.20; malate dehydrogenase (EC 1.1.1.37), 3.90; lactate dehydrogenase (EC 1.1.1.27), 3.85; two membrane proteins exhibited multiple bands upon enzymatic staining NADH dehydrogenase (EC 1.6.99.3), 4.25, 4.35; succinate dehydrogenase (EC 1.3.99.1), 4.85, 5.10, 5.35.

Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane.

Homogeneous preparations of cytoplasmic membrane isolated from Staphylococcus aureus 6538P exhibited membrane-associated adenosine triphosphatase (ATPase) activity. Membrane ATPase activity was activated by divalent cations (4.0 mM: Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+), and ATP was hydrolyzed more readily than other nucleoside triphosphates and phosphorylated substrates. The pH optimum for the membrane ATPase was 6.5. The ATPase could not be released from the membrane by differential osmotic treatments, but detergent treatment effectively solubilized active enzyme. The nonionic detergent Triton X-100 (1%) released a protein with ATPase activity, after substrate-dependent staining in polyacrylamide gels, that differed slightly in electrophoretic migration when compared to the active enzyme solubilized with sodium dodecyl sulfate (0.1%). Membrane-associated ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (0.001 to 1 mM) and NaF (50% inhibition at 5 mM NaF). Azide and trypsin inhibited activity, whereas ouabain had a slight inhibitory effect. Diethylstilbestrol showed appreciable activation of the membrane ATPase over the range employed (0.001 to 1 mM).