Vascular access thrombosis and interventions in patients missing hemodialysis sessions.

BACKGROUND: Vascular access (VA) failure is a major complication in patients with end-stage renal disease (ESRD) receiving hemodialysis (HD). Thrombosis is the most common cause of VA dysfunction, but the risk factors for VA thrombosis are not well established. While the practice of missing HD sessions (HDs) is associated with increased morbidity and mortality, its impact on VA outcomes is unknown. We evaluated the impact of missing HDs on thrombosis and intervention rates in arteriovenous (AV) accesses. METHODS: Retrospective review of prevalent HD patients using AV access was done in 2 outpatient HD centers at The Ohio State University over a one-year period. RESULTS: A total of 142 patients underwent a total of 15,692 HDs, missing 1,602 HDs. Of the 78 patients who met the inclusion criteria, 50 patients missed at least 1 HD. Those with AVF demonstrated no significant association between missing HDs and VA thrombosis. Also, the incidence rate (IR) of intervention was not significantly different for those missing and not missing HDs. However, in the AVG group, those missing HDs were more likely to experience VA thrombosis (OR 9.48, p ≈ 0.041) and had a higher IR of intervention. CONCLUSION: The practice of missing HDs was prevalent. Those missing dialysis sessions with AVG were more likely to experience VA thrombosis and needed more interventions to maintain VA patency. Our study reveals a differential impact of missing HDs on thrombosis in AVG and AVF, depicting a need to explore mechanistic explanations that may eventually help develop specific preventive strategies.

Induction of programmed cell death in a dorsal root ganglia X neuroblastoma cell line.

Growth factor-dependent neurons die when they are deprived of their specific growth factor. This "programmed" cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD.

Macrophage migration inhibitory factor interactions with glutathione and S-hexylglutathione.

Macrophage migration inhibitory factor (MIF) has been reported to interact with glutathione and S-hexylglutathione and to possess glutathione S-transferase activity. However, contrary to these reports, a recent NMR study concluded that MIF shows no affinity for glutathione. Re-examination of the glutathione-MIF interactions indicates that the reported increase in fluorescence upon addition of glutathione is because of pH-induced unfolding of the protein and not to any direct interactions. Circular dichroism shows that MIF remains folded from pH 4.5-7.5 but is 50% unfolded at pH 2.9 +/- 0.2. The reported increase in fluorescence can be achieved by acid titration. Under strongly buffered conditions, no fluorescence change is observed upon addition of glutathione. In contrast to the results with glutathione, MIF binds S-hexylglutathione with a Kd of 2.5 +/- 0.6 mM. Using NMR spectroscopy, a binding site which clusters around the N-terminal proline was identified. These data indicate that the binding site for S-hexylglutathione is the same as the catalytic site for the dopachrome tautomerase activity of MIF. Consequently, the binding of S-hexylglutathione as well as hexanethiol inhibits this catalytic activity.

Direct link between cytokine activity and a catalytic site for macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a secreted protein that activates macrophages, neutrophils and T cells, and is implicated in sepsis, adult respiratory distress syndrome and rheumatoid arthritis. The mechanism of MIF function, however, is unknown. The three-dimensional structure of MIF is unlike that of any other cytokine, but bears striking resemblance to three microbial enzymes, two of which possess an N-terminal proline that serves as a catalytic base. Human MIF also possesses an N-terminal proline (Pro-1) that is invariant among all known homologues. Multiple sequence alignment of these MIF homologues reveals additional invariant residues that span the entire polypeptide but are in close proximity to the N-terminal proline in the folded protein. We find that p-hydroxyphenylpyruvate, a catalytic substrate of MIF, binds to the N-terminal region and interacts with Pro-1. Mutation of Pro-1 to a glycine substantially reduces the catalytic and cytokine activity of MIF. We suggest that the underlying biological activity of MIF may be based on an enzymatic reaction. The identification of the active site should facilitate the development of structure-based inhibitors.

Pro-1 of macrophage migration inhibitory factor functions as a catalytic base in the phenylpyruvate tautomerase activity.

Macrophage migration inhibitory factor (MIF) is an important immunoregulatory molecule with a unique ability to suppress the anti-inflammatory effects of glucocorticoids. Although considered a cytokine, MIF possesses a three-dimensional structure and active site similar to those of 4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase. Moreover, a number of catalytic activities have been defined for MIF. To gain insight into the role of catalysis in the biological function of MIF, we have begun to characterize the catalytic activities in more detail. Here we report the crystal structure of MIF complexed with p-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF. The three binding sites for p-hydroxyphenylpyruvate in the MIF trimer lie at the interface between two subunits. The substrate interacts with Pro-1, Lys-32, and Ile-64 from one subunit and Tyr-95 and Asn-97 from an adjacent subunit. Pro-1 is positioned to function as a catalytic base. There is no functional group that polarizes the alpha-carbonyl of the substrate to weaken the adjacent C-H bond. Mutation of Pro-1 to glycine substantially reduces the catalytic activity. The insertion of an alanine between Pro-1 and Met-2 essentially abolishes activity. Structural studies of these mutants define a source of the reduced activity and provide insight into the mechanism of the catalytic reaction.

The subunit structure of human macrophage migration inhibitory factor: evidence for a trimer.

The subunit structure of human macrophage migration inhibitory factor (MIF) has been studied by preliminary X-ray analysis of wild-type and selenomethionine-MIF and dynamic light scattering. Crystal form I of MIF belongs to space group P2(1)2(1)2(1) and is grown from 2 M ammonium sulfate at pH 8.5. A native data set has been collected to 2.4 A resolution. Self-rotation studies and Van values indicate that three molecules per asymmetric unit are present. A data set to 2.8 A resolution has been collected for crystal form II, which belongs to space group P3(1)21 or P3(2)21 and grows from 2 M ammonium sulfate, 2% polyethylene glycol (average molecular mass 400) 0.1 M HEPES, pH 7.5. Three, four, five or six monomers in the asymmetric unit are consistent with Van values for this crystal form. Analysis of crystal form II containing selenomethionine-MIF indicates nine selenium sites are present per asymmetric unit. Dynamic light scattering of MIF suggests that the major form of the protein in solution is a trimer. The results of these studies are in contrast to previous reports indicating that MIF is a monomer or dimer. The subunit arrangement of MIF is similar to that of tumor necrosis factor and suggests that signal transduction might require trimerization of receptor subunits.