Cross-laboratory evaluation of multiplex bead assays including independent common reference standards for immunological monitoring of observational and interventional human studies

© 2018 van Meijgaarden et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Multiplex assays are increasingly applied to analyze multicomponent signatures of human immune responses, including the dynamics of cytokine and chemokine production, in observational as well as interventional studies following treatment or vaccination. However, relatively limited information is available on the performance of the different available multiplex kits, and comparative evaluations addressing this important issue are lacking. Study design To fill this knowledge gap we performed a technical comparison of multiplex bead assays from 4 manufacturers, each represented by 3 different lots, and with the assays performed by 3 different laboratories. To cross compare kits directly, spiked samples, biological samples and a newly made reference standard were included in all assays. Analyses were performed on 324 standard curves to allow for evaluation of the quality of the standard curves and the subsequent interpretation of biological specimens. Results Manufacturer was the factor which contributed most to the observed variation whereas variation in lots, laboratory or type of detection reagent contributed minimally. Inclusion of a common reference standard allowed us to overcome observed differences in cytokine and chemokine levels between manufacturers. Conclusions We strongly recommend using multiplex assays from the same manufacturer within a single study and across studies that are likely to compare results in a quantitative manner. Incorporation of common reference standards, and application of the same analysis method in assays can overcome many analytical biases and thus could bridge comparison of independent immune profiling (e.g. vaccine immunogenicity) studies. With these recommendations taken into account, the multiplex bead assays performed as described here are useful tools in capturing complex human immune-signatures in observational and interventional studies.FP7 EURIPRED (FP7-INFRA-2012 Grant Agreement No. 312661 to HMcS, HMD, THMO, MMH) and EC HORIZON2020 TBVAC2020 (Grant Agreement No. 643381EC to HMcS, HMD, THMO)

Locomotion and muscle mass measures in a murine model of collagen-induced arthritis

Background: Rheumatoid arthritis (RA) is characterized by chronic poly-arthritis, synovial hyperplasia, erosive synovitis, progressive cartilage and bone destruction accompanied by a loss of body cell mass. This loss of cell mass, known as rheumatoid cachexia, predominates in the skeletal muscle and can in part be explained by a decreased physical activity. The murine collagen induced arthritis (CIA) model has been proven to be a useful model in RA research since it shares many immunological and pathological features with human RA. The present study explored the interactions between arthritis development, locomotion and muscle mass in the CIA model. Methods: CIA was induced in male DBA/1 mice. Locomotion was registered at different time points by a camera and evaluated by a computerized tracing system. Arthritis severity was detected by the traditionally used semi-quantitative clinical scores. The muscle mass of the hind-legs was detected at the end of the study by weighing. A methotrexate (MTX) intervention group was included to study the applicability of the locomotion and muscle mass for testing effectiveness of interventions in more detail. Results: There is a strong correlation between clinical arthritis and locomotion. The correlations between muscle mass and locomotion or clinical arthritis were less pronounced. MTX intervention resulted in an improvement of disease severity accompanied by an increase in locomotion and muscle mass. Conclusion: The present data demonstrate that registration of locomotion followed by a computerized evaluation of the movements is a simple non invasive quantitative method to define disease severity and evaluate effectiveness of therapeutic agents in the CIA model.

Immunological Monitoring of Renal Transplant Recipients to Predict Acute Allograft Rejection Following the Discontinuation of Tacrolimus

Contains fulltext : 69863.pdf (publisher's version ) (Open Access)BACKGROUND: Transplant patients would benefit from reduction of immunosuppression providing that graft rejection is prevented. We have evaluated a number of immunological markers in blood of patients in whom tacrolimus was withdrawn after renal transplantation. The alloreactive precursor frequency of CD4+ and CD8+ T cells, the frequency of T cell subsets and the functional capacity of CD4+CD25+FoxP3+ regulatory T cells (Treg) were analyzed before transplantation and before tacrolimus reduction. In a case-control design, the results were compared between patients with (n = 15) and without (n = 28) acute rejection after tacrolimus withdrawal. PRINCIPAL FINDINGS: Prior to tacrolimus reduction, the ratio between memory CD8+ T cells and Treg was higher in rejectors compared to non-rejectors. Rejectors also had a higher ratio between memory CD4+ T cells and Treg, and ratios <20 were only observed in non-rejectors. Between the time of transplantation and the start of tacrolimus withdrawal, an increase in naive T cell frequencies and a reciprocal decrease of effector T cell percentages was observed in rejectors. The proportion of Treg within the CD4+ T cells decreased after transplantation, but anti-donor regulatory capacity of Treg remained unaltered in rejectors and non-rejectors. CONCLUSIONS: Immunological monitoring revealed an association between acute rejection following the withdrawal of tacrolimus and 1) the ratio of memory T cells and Treg prior to the start of tacrolimus reduction, and 2) changes in the distribution of naive, effector and memory T cells over time. Combination of these two biomarkers allowed highly specific identification of patients in whom immunosuppression could be safely reduced

Interrupting peptidoglycan deacetylation during Bdellovibrio predator-prey interaction prevents ultimate destruction of prey wall, liberating bacterial-ghosts

The peptidoglycan wall, located in the periplasm between the inner and outer membranes of the cell envelope in Gram-negative bacteria, maintains cell shape and endows osmotic robustness. Predatory Bdellovibrio bacteria invade the periplasm of other bacterial prey cells, usually crossing the peptidoglycan layer, forming transient structures called bdelloplasts within which the predators replicate. Prey peptidoglycan remains intact for several hours, but is modified and then degraded by predators escaping. Here we show predation is altered by deleting two Bdellovibrio N-acetylglucosamine (GlcNAc) deacetylases, one of which we show to have a unique two domain structure with a novel regulatory-”plug”. Deleting the deacetylases limits peptidoglycan degradation and rounded prey cell “ghosts” persist after mutant-predator exit. Mutant predators can replicate unusually in the periplasmic region between the peptidoglycan wall and the outer membrane rather than between wall and inner-membrane, yet still obtain nutrients from the prey cytoplasm. Deleting two further genes encoding DacB/PBP4 family proteins, known to decrosslink and round prey peptidoglycan, results in a quadruple mutant Bdellovibrio which leaves prey-shaped ghosts upon predation. The resultant bacterial ghosts contain cytoplasmic membrane within bacteria-shaped peptidoglycan surrounded by outer membrane material which could have promise as “bacterial skeletons” for housing artificial chromosomes

Homocysteine, vitamin B12 and folate levels in premature coronary artery disease

BACKGROUND: Hyperhomocysteinemia is known as an independent risk factor of atherosclerosis, but the probable role of hyperhomocysteinemia in premature Coronary Artery Disease (CAD) is not well studied. The aim of this study was to assess the role of hyperhomocysteinemia, folate and Vitamin B12 deficiency in the development of premature CAD. METHODS: We performed an analytical case-control study on 294 individuals under 45 years (225 males and 69 females) who were admitted for selective coronary angiography to two centers in Tehran. RESULTS: After considering the exclusion criteria, a total number of 225 individuals were enrolled of which 43.1% had CAD. The mean age of participants was 39.9 +/- 4.3 years (40.1 +/- 4.2 years in males and 39.4 +/- 4.8 years in females). Compared to the control group, the level of homocysteine measured in the plasma of the male participants was significantly high (14.9 +/- 1.2 versus 20.3 +/- 1.9 micromol/lit, P = 0.01). However there was no significant difference in homocysteine level of females with and without CAD (11.8 +/- 1.3 versus 11.5 ± 1.1 micromol/lit, P = 0.87). Mean plasma level of folic acid and vitamin B12 in the study group were 6.3 +/- 0.2 and 282.5 +/- 9.1 respectively. Based on these findings, 10.7% of the study group had folate deficiency while 26.6% had Vitamin B12 deficiency. Logistic regression analysis for evaluating independent CAD risk factors showed hyperhomocysteinemia as an independent risk factor for premature CAD in males (OR = 2.54 0.95% CI 1.23 to 5.22, P = 0.01). Study for the underlying causes of hyperhomocysteinemia showed that male gender and Vitamin B12 deficiency had significant influence on incidence of hyperhomocysteinemia. CONCLUSION: We may conclude that hyperhomocysteinemia is an independent risk factor for CAD in young patients (bellow 45 years old) – especially in men -and vitamin B12 deficiency is a preventable cause of hyperhomocysteinemia

Exploring dietitians' salient beliefs about shared decision-making behaviors

<p>Abstract</p> <p>Background</p> <p>Shared decision making (SDM), a process by which health professionals and patients go through the decision-making process together to agree on treatment, is a promising strategy for promoting diet-related decisions that are informed and value based and to which patients adhere well. The objective of the present study was to identify dietitians' salient beliefs regarding their exercise of two behaviors during the clinical encounter, both of which have been deemed essential for SDM to take place: (1) presenting patients with all dietary treatment options for a given health condition and (2) helping patients clarify their values and preferences regarding the options.</p> <p>Methods</p> <p>Twenty-one dietitians were allocated to four focus groups. Facilitators conducted the focus groups using a semistructured interview guide based on the Theory of Planned Behavior. Discussions were audiotaped, transcribed verbatim, coded, and analyzed with NVivo8 (QSR International, Cambridge, MA) software.</p> <p>Results</p> <p>Most participants stated that better patient adherence to treatment was an advantage of adopting the two SDM behaviors. Dietitians identified patients, physicians, and the multidisciplinary team as normative referents who would approve or disapprove of their adoption of the SDM behaviors. The most often reported barriers and facilitators for the behaviors concerned patients' characteristics, patients' clinical situation, and time.</p> <p>Conclusions</p> <p>The implementation of SDM in nutrition clinical practice can be guided by addressing dietitians' salient beliefs. Identifying these beliefs also provides the theoretical framework needed for developing a quantitative survey questionnaire to further study the determinants of dietitians' adoption of SDM behaviors.</p

Decrease of CD68 Synovial Macrophages in Celastrol Treated Arthritic Rats

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by cellular infiltration into the joints, hyperproliferation of synovial cells and bone damage. Available treatments for RA only induce remission in around 30% of the patients, have important adverse effects and its use is limited by their high cost. Therefore, compounds that can control arthritis, with an acceptable safety profile and low production costs are still an unmet need. We have shown, in vitro, that celastrol inhibits both IL-1β and TNF, which play an important role in RA, and, in vivo, that celastrol has significant anti-inflammatory properties. Our main goal in this work was to test the effect of celastrol in the number of sublining CD68 macrophages (a biomarker of therapeutic response for novel RA treatments) and on the overall synovial tissue cellularity and joint structure in the adjuvant-induced rat model of arthritis (AIA).FCT fellowship: (SFRH/BPD/92860/2013)

Moderate alcohol consumption increases insulin sensitivity and ADIPOQ expression in postmenopausal women: a randomised, crossover trial

Aims/hypothesis To determine whether 6 weeks of daily, moderate alcohol consumption increases expression of the gene encoding adiponectin (ADIPOQ) and plasma levels of the protein, and improves insulin sensitivity in postmenopausal women. Methods In a randomised, open-label, crossover trial conducted in the Netherlands, 36 apparently healthy postmenopausal women who were habitual alcohol consumers, received 250 ml white wine (~25 g alcohol/day) or 250 ml of white grape juice (control) daily during dinner for 6 weeks. Randomisation to treatment allocation occurred according to BMI. Insulin sensitivity and ADIPOQ mRNA and plasma adiponectin levels were measured at the end of both periods. Insulin sensitivity was estimated using the homeostasis model assessment of insulin resistance (HOMA-IR). Levels of ADIPOQ mRNA in subcutaneous adipose tissue were determined by RT-PCR. Results All subjects completed the study. Six weeks of white wine consumption reduced fasting insulin (mean¿±¿SEM 40.0¿±¿3.4 vs 46.5¿±¿3.4 pmol/l; p

The effect of clinician-patient alliance and communication on treatment adherence in mental health care: a systematic review

Background Nonadherence to mental health treatment incurs clinical and economic burdens. The clinician-patient alliance, negotiated through clinical interaction, presents a critical intervention point. Recent medical reviews of communication and adherence behaviour exclude studies with psychiatric samples. The following examines the impact of clinician-patient alliance and communication on adherence in mental health, identifying the specific mechanisms that mobilise patient engagement. Methods In December 2010, a systematic search was conducted in Pubmed, PsychInfo, Web of Science, Cochrane Library, Embase and Cinahl and yielded 6672 titles. A secondary hand search was performed in relevant journals, grey literature and reference. Results 23 studies met the inclusion criteria for the review. The methodological quality overall was moderate. 17 studies reported positive associations with adherence, only four of which employed intervention designs. 10 studies examined the association between clinician-patient alliance and adherence. Subjective ratings of clinical communication styles and messages were assessed in 12 studies. 1 study examined the association between objectively rated communication and adherence. Meta-analysis was not possible due to heterogeneity of methods. Findings were presented as a narrative synthesis. Conclusions Clinician-patient alliance and communication are associated with more favourable patient adherence. Further research of observer rated communication would better facilitate the application of findings in clinical practice. Establishing agreement on the tasks of treatment, utilising collaborative styles of communication and discussion of treatment specifics may be important for clinicians in promoting cooperation with regimens. These findings align with those in health communication. However, the benefits of shared decision making for adherence in mental health are less conclusive than in general medicine