Independent Candidate Serum Protein Biomarkers of Response to Adalimumab and to Infliximab in Rheumatoid Arthritis: An Exploratory Study

Response to treatment of rheumatoid arthritis shows large inter-individual variability. This heterogeneity is observed with all the anti-rheumatic drugs, including the commonly used TNF inhibitors. It seems that drug-specific and target-specific factors lead individual patients to respond or not to a given drug, although this point has been challenged. The search of biomarkers distinguishing responders from non-responders has included shotgun proteomics of serum, as a previous study of response to infliximab, an anti-TNF antibody. Here, we have used the same study design and technology to search biomarkers of response to a different anti-TNF antibody, adalimumab, and we have compared the results obtained for the two anti-TNF drugs. Search of biomarkers of response to adalimumab included depletion of the most abundant serum proteins, 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) labeling, two-dimensional liquid chromatography fractionation and relative quantification with a hybrid Orbitrap mass spectrometer. With this approach, 264 proteins were identified in all the samples with at least 2 peptides and 95% confidence. Nine proteins showed differences between non-responders and responders (P < 0.05), representing putative biomarkers of response to adalimumab. These results were compared with the previous study of infliximab. Surprisingly, the non-responder/responder differences in the two studies were not correlated (r_s = 0.07; P = 0.40). This overall independence with all the proteins showed two identifiable components. On one side, the putative biomarkers of response to either adalimumab or infliximab, which were not shared and showed an inverse correlation (r_s = -0.69; P = 0.0023). On the other, eight proteins showing significant non-responder/responder differences in the analysis combining data of response to the two drugs. These results identify new putative biomarkers of response to treatment of rheumatoid arthritis and indicate that they are notably drug-specific.     

Contribution to the determination of the chemotherapeutic drug G1 in biological fluids

A sensitive gas chromatographic method for the quantitative determination of the new antibacterial and antifungal drug G1, 1-(5-bromofuran-2-yl)-2-bromo-2-nitroethene, has been optimized. The method involves a fast and single extraction step from spiked serum and urine samples. The G1 drug was quantified using an internal standard method and by means of a nitrogen-selective detector. The results are statistically significant and show that mean levels of G1 as low as 1 μg ml⁻¹ can be measured accurately.     

The Natural History of Kidney Graft Cortical Microcirculation Determined by Real-Time Contrast-Enhanced Sonography (RT-CES)

Background

Kidney transplantation is the therapy of choice for end-stage kidney disease. Graft’s life span is shorter than expected due in part to the delayed diagnosis of various complications, specifically those related to silent progression. It is recognized that serum creatinine levels and proteinuria are poor markers of mild kidney lesions, which results in delayed clinical information. There are many investigation looking for early markers of graft damage. Decreasing kidney graft cortical microcirculation has been related to poor prognosis in kidney transplantation. Cortical capillary blood flow (CCBF) can be measured by real-time contrast-enhanced sonography (RT-CES). Our aim was to describe the natural history of CCBF over time under diverse conditions of kidney transplantation, to explore the influence of donor conditions and recipient events, and to determine the capacity of CCBF for predicting renal function in medium term.

Patients and Methods

RT-CES was performed in 79 consecutive kidney transplant recipients during the first year under regular clinical practice. Cortical capillary blood flow was measured. Clinical variables were analyzed. The influence of CCBF has been determined by univariate and multivariate analysis using mixed regression models based on sequential measurements for each patient over time. We used a first-order autoregression model as the structure of the covariation between measures. The post-hoc comparisons were considered using the Bonferroni correction.

Results

The CCBF values varied significantly over the study periods and were significantly lower at 48 h and day 7. Brain-death donor age and CCBF levels showed an inverse relationship (r: -0.62, p<0.001). Living donors showed higher mean CCBF levels than brain-death donors at each point in the study. These significant differences persisted at month 12 (54.5 ± 28.2 vs 33.7 ± 30 dB/sec, living vs brain-death donor, respectively, p = 0.004) despite similar serum creatinine levels (1.5 ± 0.3 and 1.5 ± 0.5 mg/dL). A sole rejection episode was associated with lower overall CCBF values over the first year. CCBF defined better than level of serum creatinine the graft function status at medium-term.

Conclusion

RT-CES is a non-invasive tool that can quantify and iteratively estimate cortical microcirculation. We have described the natural history of cortical capillary blood flow under regular clinical conditions.     

Distribution and Characteristics of βII Tubulin-Enriched Microtubules in Interphase Cells

We have used a polyclonal antibody (Ab196) that specifically recognizes the βII tubulin isotype to examine the subcellular distribution and properties of microtubules enriched in this isotype. Antibody specificity was tested by a method that involves the analysis of its interaction with individual β isotypes. Using photoimaging analysis, we observed βII tubulin-enriched microtubules in the perinuclear region, as well as in the microtubules close to the periphery of interphase cells. The observed sorting of βII-enriched microtubules together with the reported increased levels of βII tubulin in taxol-resistant cells (M. Haberet al.,1995,J. Biol. Chem.270, 31269–31275) prompted us to study the behavior of microtubules enriched in this isotype after different depolymerizing treatments. After cold or nocodazol treatments, βII-enriched microtubules anchored at the centrosome and at the cell periphery were observed. In addition, cold-resistant microtubules were marked mainly by the specific anti-βII tubulin antibody but not by anti-acetylated α tubulin, suggesting the presence of different stable microtubule subsets enriched in particular tubulin isoforms.     

A Dinoflagellate Amylax triacantha with Plastids of the Cryptophyte Origin: Phylogeny,Feeding Mechanism,and Growth and Grazing Responses

The gonyaulacalean dinoflagellates Amylax spp. were recently found to contain plastids of the cryptophyte origin, more specifically of Teleaulax amphioxeia. However, not only how the dinoflagellates get the plastids of the cryptophyte origin is unknown but also their ecophysiology, including growth and feeding responses as functions of both light and prey concentration, remain unknown. Here, we report the establishment of Amylax triacantha in culture, its feeding mechanism, and its growth rate using the ciliate prey Mesodinium rubrum (= Myrionecta rubra) in light and dark, and growth and grazing responses to prey concentration and light intensity. The strain established in culture in this study was assigned to A. triacantha, based on morphological characteristics (particularly, a prominent apical horn and three antapical spines) and nuclear SSU and LSU rDNA sequences. Amylax triacantha grew well in laboratory culture when supplied with the marine mixotrophic ciliate M. rubrum as prey, reaching densities of over 7.5 × 10³ cells/ml. Amylax triacantha captured its prey using a tow filament, and then ingested the whole prey by direct engulfment through the sulcus. The dinoflagellate was able to grow heterotrophically in the dark, but the growth rate was approximately two times lower than in the light. Although mixotrophic growth rates of A. triacantha increased sharply with mean prey concentrations, with maximum growth rate being 0.68/d, phototrophic growth (i.e. growth in the absence of prey) was ?0.08/d. The maximum ingestion rate was 2.54 ng C/Amylax/d (5.9 cells/Amylax/d). Growth rate also increased with increasing light intensity, but the effect was evident only when prey was supplied. Increased growth with increasing light intensity was accompanied by a corresponding increase in ingestion. In mixed cultures of two predators, A. triacantha and Dinophysis acuminata, with M. rubrum as prey, A. triacantha outgrew D. acuminata due to its approximately three times higher growth rate, suggesting that it can outcompete D. acuminata. Our results would help better understand the ecophysiology of dinoflagellates retaining foreign plastids.     

Excavating Culture: Disentangling Ethnic Differences From Contextual Influences in Parenting

Historically, much of the research on parenting has not disentangled the influences of race/ethnicity, SES, and culture on family functioning and the development of children and adolescents. This special issue addresses this gap by disentangling ethnic differences in parenting behaviors from their contextual influences, thereby deepening our understanding of parenting processes in diverse families. Six members of the Parenting Section of the Study Group on Race, Culture, and Ethnicity (SGRCE) introduce and implement a novel approach toward understanding this question. The goal of this project is to study culturally related processes and the degree to which they predict parenting. An iterative process was employed to delineate the main parenting constructs (warmth, psychological and behavioral control, monitoring, communication, and self-efficacy), cultural processes, and contextual influences, and to coordinate a data analytic plan utilizing individual datasets with diverse samples to answer the research questions.     

Degradation of the antibiotics norfloxacin and ciprofloxacin by a white-rot fungus and identification of degradation products

More than 90% of the antibiotics ciprofloxacin (CIPRO) and norfloxacin (NOR) at 2 mg L⁻¹ were degraded by Trametes versicolor after 7 days of incubation in malt extract liquid medium. In in vitro assays with purified laccase (16.7 nkat mL⁻¹), an extracellular enzyme excreted constitutively by this fungus, 16% of CIPRO was removed after 20 h. The addition of the laccase mediator 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt led to 97.7% and 33.7% degradation of CIPRO and NOR, respectively. Inhibition of CIPRO and NOR degradation by the cytochrome P450 inhibitor 1-aminobenzotriazole suggests that the P450 system also plays a role in the degradation of the two antibiotics. Transformation products of CIPRO and NOR were monitored at different incubation times by triple-quadrupole and quadrupole time-of-flight mass spectrometry, and can be assigned to three different reaction pathways: (i) oxidation of the piperazinyl substituent, (ii) monohydroxylation, and (iii) formation of dimeric products.     

Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor : Effects on Full-Length and Carboxyl-Terminal Portion of Ca2+Release Channels

The ryanodine receptor (RyR)/Ca²⁺ release channel is an essential component of excitation–contraction coupling in striated muscle cells. To study the function and regulation of the Ca²⁺ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca²⁺]. Unlike the native RyR channels in muscle cells, which display localized Ca²⁺ release events (i.e., “Ca²⁺ sparks” in cardiac muscle and “local release events” in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca²⁺ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca²⁺ release events observed in muscle cells may involve gating of a group of Ca²⁺ release channels and/or interaction of RyR with muscle-specific proteins.