Cytokine Profiling in Plasma from Patients with Brain Tumors Versus Healthy Individuals using 2 Different Multiplex Immunoassay Platforms

We compared the performance of two 96-well multiplex immunoassay platforms in assessing plasma cytokine concentrations in patients with glioblastoma (GBM; n = 27), individuals with melanoma, breast or lung cancer metastases to the brain (n = 17), and healthy volunteers (n = 11). Assays included a bead-based fluorescence MILLIPLEX® assay/Luminex (LMX) platform and 4 planar electrochemiluminescence kits from Meso Scale Discovery (MSD). The LMX kit evaluated 21 cytokines and the 3 MSD kits evaluated 20 cytokines in total, with 19 overlapping human cytokines between platforms (GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-21, IL-23, MIP-1α, MIP-1β, MIP-3α, TNFα). The MSD platform had lower LLoQs (lower limits of quantification) than LMX for 17/19 cytokines, and higher LLoQs for IFN-γ and IL-21. The ULoQs were higher in LMX versus MSD assays for 17/19 shared analytes, but lower than MSD for IL-17A and IL-21. With LMX, all 19 shared analytes were quantifiable in each of 55 samples. Although MSD recombinant protein standard curves indicated lower LLoQs than LMX for most cytokines, MSD detected 7/19 (37%) native analytes in <75% of samples, including 0% detection for IL-21 and 8% for IL-23. The LMX platform categorized identical samples at greater concentrations than the MSD system for most analytes (MIP-1β the sole exception), sometimes by orders of magnitude. This mismatched quantification paradigm was supported by Bland-Altman analysis. LMX identified significantly elevated levels of 10 of 19 circulating cytokines in GBM: GM-CSF, IFN-γ, IL-1β, IL-5, IL-10, IL-17A, IL-21, IL-23, MIP-1α, and MIP-3α, consistent with prior findings and confirming the utility of applying appropriate multiplex immunoassay technologies toward developing a cytokine signature profile for GBM.

Molecular markers of T2DM in obesity: expression profile of inflammatory response genes in peripheral blood mononuclear cells

Сахарный диабет 2 типа (СД2) - метаболическое многофакторное, генетически обусловленное, опасное для жизни заболевание. Развитие осложнений связано с хроническим иммуновоспалительным процессом и с образованием иммунных комплексов. Целью исследования явился анализ профиля экспрессии комплекса функционально взаимосвязанных генов (цитокинов, хемокинов и другие ключевые молекулы воспалительного ответа) в мононуклеарах периферической крови у пациентов с СД2 (N=10) и в контрольной группе (N=10). Пациенты и контроль были подобраны по полу, возрасту, принадлежали к этнической группе татар. Профиль экспрессии 84 генов иммунного ответа исследовали методом OT-ПЦР на приборе BioRad CFX96TM набором RT2 Profiler PCR Arrays «Human Cytokines & Chemokines PCR Array» (Qiagen). Значимые изменения экспрессии (p<0,05) в общей группе больных СД2 по сравнению с контролем были установлены для генов FASLG, IL16, OSM, CSF1, CXCL16, BMP6. При этом профиль экспрессии этих генов у больных СД2 характеризуется повышением уровня от 2,1 до 6,7 раз по сравнению со здоровыми индивидами. Профиль экспрессии генов CXCL8, CXCL1, CXCL2, CXCL10, CCL2, IL1RN характеризуется снижением уровня от 2,1 до 8,78 раз по сравнению со здоровыми индивидами. Значимые статистические различия при снижении экспрессии были получены для генов IL1RN (fold-change (FCh)=-3,31, p=0,013), CXCL1 (FCh=-2,74, p=0,027), CXCL2 (FCh=-2,66, p=0,038). Наиболее значимое повышение транскрипционной активности было показано для генов IL16 (FCh=6,72, p=0,037) и FASLG (FCh=2,83 p=0,034) среди больных СД2 по сравнению с контрольной группой. Type 2 diabetes mellitus (T2DM) is a metabolic multifactorial, genetically determined, life-threatening disease. The development of complications is associated with a chronic immuno-inflammatory process and with the formation of immune complexes. The aim of the study was to analyze the expression profile of a complex of functionally interconnected genes (cytokines, chemokines, and other key molecules of the inflammatory response) in peripheral blood mononuclear cells in patients with T2DM (N = 10) and in the control group (N = 10). Patients and controls were matched by gender, age, and belonged to the Tatars ethnic group. The expression profile of 84 immune response genes was studied by OT-PCR on a BioRad CFX96TM instrument using the Human Cytokines & Chemokines PCR Array RT2 Profiler PCR Arrays kit (Qiagen). Significant changes in expression (p <0.05) in the general group of patients with T2DM compared with the control were found for the FASLG, IL16, OSM, CSF1, CXCL16, BMP6 genes. Moreover, the expression profile of these genes in patients with type 2 diabetes is characterized by an increase in the level from 2.1 to 6.7 times in comparison with healthy individuals. The gene expression profile of CXCL8, CXCL1, CXCL2, CXCL10, CCL2, IL1RN is characterized by a decrease in the level from 2.1 to 8.78 times in comparison with healthy individuals. Significant statistical differences with a decrease in expression were obtained for the genes IL1RN (fold-change (FCh) = - 3.31, p = 0.013), CXCL1 (FCh = -2.74, p = 0.027), CXCL2 (FCh = -2.66, p = 0.038) . The most significant increase in transcriptional activity was shown for the genes IL16 (FCh = 6.72, p = 0.037) and FASLG (FCh = 2.83 p = 0.034) among patients with type 2 diabetes compared with the control group.

Recent Advancements and Applications of Human Immune System Mice in Preclinical Immuno-Oncology

Significant advances in immunotherapies have resulted in the increasing need of predictive preclinical models to improve immunotherapeutic drug development, treatment combination, and to prevent or minimize toxicity in clinical trials. Immunodeficient mice reconstituted with human immune system (HIS), termed humanized mice or HIS mice, permit detailed analysis of human immune biology, development, and function. Although this model constitutes a great translational model, some aspects need to be improved as the incomplete engraftment of immune cells, graft versus host disease and the lack of human cytokines and growth factors. In this review, we discuss current HIS platforms, their pathology, and recent advances in their development to improve the quality of human immune cell reconstitution. We also highlight new technologies that can be used to better understand these models and how improved characterization is needed for their application in immuno-oncology safety, efficacy, and new modalities therapy development.

Engraftment of chronic myelomonocytic leukemia cells in immunocompromised mice supports disease dependency on cytokines

Key Points Transgenic mice expressing 3 human cytokines enable expansion of CMML cells with limited stem cell engraftment. The mutational profile of CMML cells that expand in mice mirrors that of patient monocytes.