Toward protein biomarkers for allergy: CD4+ T cell proteomics in allergic and nonallergic subjects sampled in and out of pollen season

Allergy is an immunological disorder of the upper airways, lung, skin, and the gut with a growing prevalence over the last decades in Western countries. Atopy, the genetic predisposition for allergy, is strongly dependent on familial inheritance and environmental factors. These observations call for predictive markers of progression from atopy to allergy, a prerequisite to any active intervention in neonates and children (prophylactic interventions/primary prevention) or in adults (immunomodulatory interventions/secondary prevention). In an attempt to identify early biomarkers of the "atopic march" using minimally invasive sampling, CD4+ T cells from 20 adult volunteers (10 healthy and 10 with respiratory allergies) were isolated and quantitatively analyzed and their proteomes were compared in and out of pollen season (± antigen exposure). The proteome study based on high-resolution 2D gel electrophoresis revealed three candidate protein markers that distinguish the CD4+ T cell proteomes of normal from allergic individuals when sampled out of pollen season, namely Talin 1, Nipsnap homologue 3A, and Glutamate-cysteine ligase regulatory protein. Three proteins were found differentially expressed between the CD4+ T cell proteomes of normal and allergic subjects when sampled during pollen season: carbonyl reductase, glutathione S-transferase ω 1, and 2,4-dienoyl-CoA reductase. The results were partly validated by Western blotting.     

Effect of drought stress at supraoptimal temperature on polyamine concentrations in transgenic soybean with increased proline levels

The effect of drought stress at supraoptimal temperature on free proline and polyamine levels was compared in wild type and transgenic soybean (Glycine max cv. Ibis) plants having increased proline levels. Since glutamate and arginine are precursors of both proline and polyamines, it was assumed that the genetic manipulation of proline levels would also affect the polyamine levels. The proline and spermine concentrations increased, while the putrescine concentration generally decreased or did not change after the treatments in both genotypes. Following drought higher proline and lower spermine levels were detected in the transgenic plants compared to the wild type ones, which could be explained by the increased use of their common precursors for proline biosynthesis in the transgenic plants.     

Local variability of the phosphoglycerate kinase-triosephosphate isomerase fusion protein from Thermotoga maritima MSB 8

The pgk-tpi gene locus of Thermotoga maritima encodes both phosphoglycerate kinase (PGK) and a bienzyme complex consisting of a fusion protein of PGK with triosephosphate isomerase (TIM). No separate tpi gene for TIM is present in T. maritima. A frame-shift at the end of the pgk gene has been previously proposed as a mechanism to regulate the expression of the two protein variants [Schurig et al., EMBO J. 14 (1995), 442-451]. Surprisingly, the complete T. maritima genome was found to contain a pgk-tpi sequence not requiring the proposed frameshift mechanism. To clarify the apparent discrepancy, a variety of DNA sequencing techniques were applied, disclosing an anomalous local variability in the pgk-tpi fusion region. The comparison of different DNA samples and the mass spectrometric analysis of the amino acid sequence of the natural fusion protein from T. maritima MSB8 confirmed the local variability of the DNA variants. Since not all peptide masses could be assigned, further variations are conceivable, suggesting an even higher heterogeneity of the T. maritima MSB8 strain.     

Loop swapping in an antisense RNA/target RNA pair changes directionality of helix progression

The binding pathway of the natural antisense RNA CopA to its target CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts followed by unidirectional helix progression into the upper stems. This involves extensive breakage of intramolecular base pairs and the subsequent formation of two intermolecular helices, B and B'. Based on the known tRNA anticodon loop structure and on results from the Sok/Hok antisense/target RNA system, it had been suggested that a U-turn (or pi-turn) in the loop of CopT might determine the directionality of helix progression. Data presented here show that the putative U-turn is one of the structural elements of antisense/target RNA pairs required to achieve fast binding kinetics. Swapping of the hypothetical U-turn structure from the target RNA to the antisense RNA retained regulatory performance in vivo and binding rates in vitro but altered the binding pathway by changing the direction in which the initiating helix was extended. In addition, our data indicate that a helical stem immediately adjacent to the target loop sequence is required to provide a scaffold for the U-turn.     

The neuroprotective antioxidant alpha-lipoic acid induces detoxication enzymes in cultured astroglial cells

-Lipoic acid (LA), an antioxidant with broad neuroprotective capacity, is thought to act by scavenging reactive oxygen species and stimulation of glutathione synthesis. LA shows structural resemblance to dithiolethiones, like anethole dithiolethione (ADT). ADT protects against oxidative damage, primarily by induction of phase II detoxication enzymes, in particular NAD(P)H:quinone oxidoreductase (NQO1) and glutathione- S -transferase (GST). Therefore, we investigated whether LA, like ADT, is capable also of inducing these protective enzymes. Our data show that LA, like ADT, induces a highly significant, time- and concentration dependent, increase in the activity of NQO1 and GST in C6 astroglial cells. The LA or ADT mediated induction of NQO1 was further confirmed by quantitative PCR and western blot analysis. This work for the first time unequivocally demonstrates LA mediated upregulation of phase II detoxication enzymes, which may highly contribute to the compounds' neuroprotective potential. Moreover, the data support the notion of a common mechanism of action of LA and ADT.     

Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: structure and stability of the recombinant enzyme and its isolated cellulose-binding domain.

The hyperthermophilic bacterium Thermotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120-kDa endo-1,4-D-xylanase exhibiting high intrinsic stability and a temperature optimum approximately 90 degrees C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C-terminal part of XynA was previously shown to be responsible for cellulose binding (Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose-binding domain within the multi-domain 120 kDa Xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol 15:431-444). In order to characterize the domain organization and the stability of XynA and its C-terminal cellulose-binding domain (CBD), the two separate proteins were expressed in Escherichia coli. CBD, because of its instability in its ligand-free form, was expressed as a glutathione S-transferase fusion protein with a specific thrombin cleavage site as linker. XynA and CBD were compared regarding their hydrodynamic and spectral properties. As taken from analytical ultracentrifugation and gel permeation chromatography, both are monomers with 116 and 22 kDa molecular masses, respectively. In the presence of glucose as a ligand, CBD shows high intrinsic stability. Denaturation/renaturation experiments with isolated CBD yield > 80% renaturation, indicating that the domain folds independently. Making use of fluorescence emission and far-UV circular dichroism in order to characterize protein stability, guanidine-induced unfolding of XynA leads to biphasic transitions, with half-concentrations c1/2 (GdmCl) approximately 4 M and > 5 M, in accordance with the extreme thermal stability. At acid pH, XynA exhibits increased stability, indicated by a shift of the second guanidine-transition from 5 to 7 M GdmCl. This can be tentatively attributed to the cellulose-binding domain. Differences in the transition profiles monitored by fluorescence emission and dichroic absorption indicate multi-state behavior of XynA. In the case of CBD, a temperature-induced increase in negative ellipticity at 217 nm is caused by alterations in the environment of aromatic residues that contribute to the far-UV CD in the native state.     

Thermodynamics of the unfolding of the cold-shock protein from Thermotoga maritima.

Proteins from (hyper-)thermophiles are known to exhibit high intrinsic stabilities. Commonly, their thermodynamic characterization is impeded by irreversible side reactions of the thermal analysis or calorimetrical problems. Small single-domain proteins are suitable candidates to overcome these obstacles. Here, the thermodynamics of the thermal denaturation of the recombinant cold-shock protein (Csp) from the hyperthermophilic bacterium Thermotoga maritima (Tm) was studied by differential scanning calorimetry. The unfolding transition can be described over a broad pH range (3.5-8.5) by a reversible two-state process. Maximum stability (DeltaG (25 degrees C)=6.5 kcal/mol) was observed at pH 5-6 where Tm Csp unfolds with a melting temperature at 95 degrees C. The heat capacity difference between the native and the denatured states is 1.1(+/-0.1) kcal/(mol K). At pH 7, thermal denaturation occurs at 82 degrees C. The corresponding free energy profile has its maximum at 30 degrees C with DeltaGN-->U=4.8(+/-0.5) kcal/mol. At the optimal growth temperature of T. maritima (80 degrees C), Tm Csp in the absence of ligands is only marginally stable, with a free energy of stabilization not far beyond the thermal energy. With the known stabilizing effect of nucleic acids in mind, this suggests a highly dynamical interaction of Tm Csp with its target molecules.     

The human anti-IL-1β monoclonal antibody ACZ885 is effective in joint inflammation models in mice and in a proof-of-concept study in patients with rheumatoid arthritis

Introduction

IL-1β is a proinflammatory cytokine driving joint inflammation as well as systemic signs of inflammation, such as fever and acute phase protein production.

Methods

ACZ885, a fully human monoclonal antibody that neutralizes the bioactivity of human IL-1β, was generated to study the potent and long-lasting neutralization of IL-1β in mechanistic animal models as well as in a proof-of-concept study in patients with rheumatoid arthritis (RA).

Results

The mouse IL-1 receptor cross-reacts with human IL-1β, and it was demonstrated that ACZ885 can completely suppress IL-1β-mediated joint inflammation and cartilage destruction in mice. This observation prompted us to study the safety, tolerability and pharmacodynamic activity of ACZ885 in RA patients in a small proof-of-concept study – the first to be conducted in humans. Patients with active RA despite treatment with stable doses of methotrexate were enrolled in this dose escalation study. The first 32 patients were split into four cohorts of eight patients each (six were randomly assigned to active treatment and two to placebo). ACZ885 doses were 0.3, 1, 3 and 10 mg/kg, administered intravenously on days 1 and 15. To explore efficacy within 6 weeks of treatment, an additional 21 patients were randomly assigned to the 10 mg/kg cohort, resulting in a total of 20 patients dosed with 10 mg/kg and 15 patients treated with placebo. There was clinical improvement (American College of Rheumatology 20% improvement criteria) at week 6 in the 10 mg/kg treatment group; however, this did not reach statistical significance (P = 0.085). A statistically significant reduction in disease activity score was observed after 4 weeks in the 10 mg/kg group. Onset of action was rapid, because most responders exhibited improvement in their symptoms within the first 3 weeks. C-reactive protein levels decreased in patients treated with ACZ885 within 1 week. ACZ885 was well tolerated. Three patients receiving ACZ885 developed infectious episodes that required treatment. No anti-ACZ885 antibodies were detected during the study.

Conclusion

ACZ885 administration to methotrexate-refractory patients resulted in clinical improvement in a subset of patients. Additional studies to characterize efficacy in RA and to determine the optimal dose regimen appear warranted.

Trial Registration

ClinicalTrials.gov identifier NCT00619905.