Immune activation in advanced cancer patients treated with recombinant IL-21: multianalyte profiling of serum proteins

Purpose  Recombinant interleukin-21 (rIL-21) is an immune stimulating cytokine recently tested in two Phase 1 trials for immune responsive cancers. A secondary objective of these trials was to characterize pharmacodynamic responses to rIL-21 in patients. Here, we report the effects of systemic rIL-21 on serum markers of immune stimulation. Experimental design  Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 μg/kg using two distinct treatment regimens: thrice weekly (‘3/w’) for 6 weeks; or once daily for five consecutive days followed by nine dose-free days (‘5 + 9’). In the absence of dose limiting toxicity, additional cycles of dosing were initiated immediately following the nine dose-free days. An array of 70 different proteins was profiled in subject serum samples from several time points during the course of the study. Hierarchical clustering analysis was performed on a normalized subset of these data. Results  Systemic administration of rIL-21 affected the serum levels of several cytokines, chemokines, acute-phase proteins and cell adhesion proteins. The magnitude and duration of response were dose dependent for a subset of these biomarkers. The 5 + 9 dosing regimen generally produced cyclic changes that were of greater magnitude, as compared to a more chronic stimulation with the 3/w dosing regimen. Despite these differences, rIL-21 effects on many analytes were similar between regimens when averaged over the time of treatment. Based on similar temporal, between-subject and dose response changes, groups of analytes were identified that exhibited distinct components of the rIL-21-mediated immune activation. Biomarkers indicative of lymphocyte activation (increased IL-16, decreased RANTES), acute phase response (increased CRP, ferritin), myeloid activation (increased MDC, MIP-1 alpha), and leukocyte chemotaxis/trafficking (increased sCAMs, MCP-1) were strongly modulated in subjects treated with rIL-21. Conclusions  Administration of rIL-21 resulted in activation of multiple cell types and immune response pathways. The changes observed in serum proteins were consistent with coincident processes of lymphoid and myeloid cell activation and trafficking, and acute phase response. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quantification of the Nitrogen Cycle in a Prairie Stream

Nitrogen (N) was added for 35 days in the form of ¹⁵NH₄Cl to Kings Creek on Konza Prairie, Kansas. Standing stocks of N in key compartments (that is, nutrients, detritus, organisms) were quantified, and the amount of labeled N entering the compartments was analyzed. These data were used to calculate turnover and flux rates of N cycling through the food web, as well as nutrient transformation rates. Inorganic N pools turned over much more rapidly in the water column of this stream than in pelagic systems where comparable measurements have been made. As with other systems, the mass of ammonium was low but it was the key compartment mediating nutrient flux through the ecosystem, whereas dissolved organic N, the primary component of N flux through the system, is not actively cycled. Nitrification was also a significant flux of N in the stream, with rates in the water column and surface of benthos accounting for approximately 10% of the total ammonium uptake. Primary consumers assimilated 67% of the inorganic N that entered benthic algae and microbes. Predators acquired 23% of the N that consumers obtained. Invertebrate collectors, omnivorous crayfish (Orconectes spp.), and invertebrate shredders dominated the N flux associated with primary consumers. Mass balance calculations indicated that at least 23% of the 309 mg of ¹⁵N added during the 35 days of release was retained within the 210-m stream reach during the release. Overall, the rates of turnover of N in organisms and organic substrata were significantly greater when C:N was low. This ratio may be a surrogate for biological activity with regard to N flux in streams. Received 2 August 1999; accepted 18 July 2000.     

Systematic characterization of high mass accuracy influence on false discovery and probability scoring in peptide mass fingerprinting

Whereas the bearing of mass measurement error on protein identification is sometimes underestimated, uncertainty in observed peptide masses unavoidably translates to ambiguity in subsequent protein identifications. Although ongoing instrumental advances continue to make high accuracy mass spectrometry (MS) increasingly accessible, many proteomics experiments are still conducted with rather large mass error tolerances. In addition, the ranking schemes of most protein identification algorithms do not include a meaningful incorporation of mass measurement error. This article provides a critical evaluation of mass error tolerance as it pertains to false positive peptide and protein associations resulting from peptide mass fingerprint (PMF) database searching. High accuracy, high resolution PMFs of several model proteins were obtained using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Varying levels of mass accuracy were simulated by systematically modulating the mass error tolerance of the PMF query and monitoring the effect on figures of merit indicating the PMF quality. Importantly, the benefits of decreased mass error tolerance are not manifest in Mowse scores when operating at tolerances in the low parts-per-million range but become apparent with the consideration of additional metrics that are often overlooked. Furthermore, the outcomes of these experiments support the concept that false discovery is closely tied to mass measurement error in PMF analysis. Clear establishment of this relation demonstrates the need for mass error-aware protein identification routines and argues for a more prominent contribution of high accuracy mass measurement to proteomic science.     

Incorporation of xenobiotic carboxylic acids into lipids

Over thirty-six different xenobiotic carboxylic acids have been reported to form xenobiotic lipids. The majority form triacylglycerol analogs or cholesterol esters with fewer reports of polar lipids being formed. As yet there is insufficient information to deduce a relationship between the structure of the xenobiotic acid and its activity as a substrate for lipid biosynthesis, although the ability to form a CoA ester appears to be important. The action of monoacylglycerol acyltransferase, diacylglycerol acyltransferase, lecithin cholesterol acyltransferase and a carboxylesterase in synthesizing xenobiotic lipids has been demonstrated. One xenobiotic lipid has been shown to be the cause of granulomatous changes and there are some indications that others may prove to be of toxicological or pharmacological significance. Detailed investigations into several aspects of xenobiotic lipid biochemistry are still required.     

Enhanced peptide mass fingerprinting through high mass accuracy: Exclusion of non-peptide signals based on residual mass

Peptide mass fingerprinting (PMF) is among the principle methods of contemporary proteomic analysis. While PMF is routinely practiced in many laboratories, the complexity of protein tryptic digests is such that PMF based on unrefined mass spectrometric peak lists is often inconclusive. A number of data processing strategies have thus been designed to improve the quality of PMF peak lists, and the development of increasingly elaborate tools for PMF data reduction remains an active area of research. In this report, a novel and direct means of PMF peak list enhancement is suggested. Since the monoisotopic mass of a peptide must fall within a predictable range of residual values, PMF peak lists can in principle be relieved of many non-peptide signals solely on the basis of accurately determined monoisotopic mass. The calculations involved are relatively simple, making implementation of this scheme computationally facile. When this procedure for peak list processing was used, the large number of unassigned masses typical of PMF peak lists was considerably attenuated. As a result, protein identifications could be made with greater confidence and improved discrimination as compared to PMF queries submitted with raw peak lists. Importantly, this scheme for removal of non-peptide masses was found to conserve peptides bearing various post-translational and artificial modifications. All PMF experiments discussed here were performed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), which provided the high mass resolution and high mass accuracy essential for this application. Previously reported equations relating the nominal peptide mass to the permissible range of fractional peptide masses were slightly modified for this application, and these adjustments have been illustrated in detail. The role of mass accuracy in application of this scheme has also been explored.     

Haustorially expressed secreted proteins from flax rust are highly enriched for avirulence elicitors

Rust fungi, obligate biotrophs that cause disease and yield losses in crops such as cereals and soybean (Glycine max), obtain nutrients from the host through haustoria, which are specialized structures that develop within host cells. Resistance of flax (Linum usitatissimum) to flax rust (Melampsora lini) involves the induction of a hypersensitive cell death response at haustoria formation sites, governed by gene-for-gene recognition between host resistance and pathogen avirulence genes. We identified genes encoding haustorially expressed secreted proteins (HESPs) by screening a flax rust haustorium-specific cDNA library. Among 429 unigenes, 21 HESPs were identified, one corresponding to the AvrL567 gene. Three other HESPs cosegregated with the independent AvrM, AvrP4, and AvrP123 loci. Expression of these genes in flax induced resistance gene-mediated cell death with the appropriate specificity, confirming their avirulence activity. AvrP4 and AvrP123 are Cys-rich proteins, and AvrP123 contains a Kazal Ser protease inhibitor signature, whereas AvrM contains no Cys residues. AvrP4 and AvrM induce cell death when expressed intracellularly, suggesting their translocation into plant cells during infection. However, secreted AvrM and AvrP4 also induce necrotic responses, with secreted AvrP4 more active than intracellular AvrP4, possibly as a result of enhanced formation of endoplasmic reticulum-dependent disulfide bonds. Addition of an endoplasmic reticulum retention signal inhibited AvrM-induced necrosis, suggesting that both AvrM and AvrP4 can reenter the plant cell after secretion in the absence of the pathogen.