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1. Rapid single-molecule digital detection of protein biomarkers for continuous monitoring of systemic immune disorders

   https://doi.org/10.1182/blood.2019004399  

   Song, Yujing ; Sandford, Erin ; Tian, Yuzi ; Yin, Qingtian ; Kozminski, Andrew G. ; Su, Shiuan-Haur ; Cai, Tao ; Ye, Yuxuan ; Chung, Meng Ting ; Lindstrom, Ryan ; et al ( March 2021 , Blood)

   null (Ed.)

   Abstract Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires deployment of these assays with a rapid turnaround. Herein, we present a technology platform for ultrafast digital protein biomarker detection by using single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term “pre-equilibrium digital enzyme-linked immunosorbent assay” (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15 to 300 seconds over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell therapy cytokine release syndrome, with ∼30-minute sample-to-answer time and a sub–picograms per mL limit of detection. The rapid, sensitive, and low-input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment. 

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2. Recombinant human plasma gelsolin reverses increased permeability of the blood–brain barrier induced by the spike protein of the SARS-CoV-2 virus

   https://doi.org/10.1186/s12974-022-02642-4  

   Suprewicz, Łukasz ; Tran, Kiet A. ; Piktel, Ewelina ; Fiedoruk, Krzysztof ; Janmey, Paul A. ; Galie, Peter A. ; Bucki, Robert ( December 2022 , Journal of Neuroinflammation)

   Abstract Background Plasma gelsolin (pGSN) is an important part of the blood actin buffer that prevents negative consequences of possible F-actin deposition in the microcirculation and has various functions during host immune response. Recent reports reveal that severe COVID-19 correlates with reduced levels of pGSN. Therefore, using an in vitro system, we investigated whether pGSN could attenuate increased permeability of the blood–brain barrier (BBB) during its exposure to the portion of the SARS-CoV-2 spike protein containing the receptor binding domain (S1 subunit). Materials and methods Two- and three-dimensional models of the human BBB were constructed using the human cerebral microvascular endothelial cell line hCMEC/D3 and exposed to physiologically relevant shear stress to mimic perfusion in the central nervous system (CNS). Trans-endothelial electrical resistance (TEER) as well as immunostaining and Western blotting of tight junction (TJ) proteins assessed barrier integrity in the presence of the SARS-CoV-2 spike protein and pGSN. The IncuCyte Live Imaging system evaluated the motility of the endothelial cells. Magnetic bead-based ELISA was used to determine cytokine secretion. Additionally, quantitative real-time PCR (qRT-PCR) revealed gene expression of proteins from signaling pathways that are associated with the immune response. Results pGSN reversed S1-induced BBB permeability in both 2D and 3D BBB models in the presence of shear stress. BBB models exposed to pGSN also exhibited attenuated pro-inflammatory signaling pathways (PI3K, AKT, MAPK, NF-κB), reduced cytokine secretion (IL-6, IL-8, TNF-α), and increased expression of proteins that form intercellular TJ (ZO-1, occludin, claudin-5). Conclusion Due to its anti-inflammatory and protective effects on the brain endothelium, pGSN has the potential to be an alternative therapeutic target for patients with severe SARS-CoV-2 infection, especially those suffering neurological complications of COVID-19. 

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3. A fluorescent microbead-based microfluidic immunoassay chip for immune cell cytokine secretion quantification

   https://doi.org/10.1039/c7lc01183k  

   Cui, Xin ; Liu, Ya ; Hu, Dinglong ; Qian, Weiyi ; Tin, Chung ; Sun, Dong ; Chen, Weiqiang ; Lam, Raymond H. ( January 2018 , Lab on a Chip)

   Quantitative and dynamic analyses of immune cell secretory cytokines are essential for precise determination and characterization of the “immune phenotype” of patients for clinical diagnosis and treatment of immune-related diseases. Although multiple methods including the enzyme-linked immunosorbent assay (ELISA) have been applied for cytokine detection, such measurements remain very challenging in real-time, high-throughput, and high-sensitivity immune cell analysis. In this paper, we report a highly integrated microfluidic device that allows for on-chip isolation, culture, and stimulation, as well as sensitive and dynamic cytokine profiling of immune cells. Such a microfluidic sensing chip is integrated with cytometric fluorescent microbeads for real-time and multiplexed monitoring of immune cell cytokine secretion dynamics, consuming a relatively small extracted sample volume (160 nl) without interrupting the immune cell culture. Furthermore, it is integrated with a Taylor dispersion-based mixing unit in each detection chamber that shortens the immunoassay period down to less than 30 minutes. We demonstrate the profiling of multiple pro-inflammatory cytokine secretions ( e.g. interleukin-6, interleukin-8, and tumor necrosis factors) of human peripheral blood mononuclear cells (PBMCs) with a sensitivity of 20 pg ml −1 and a sample volume of 160 nl per detection. Further applications of this automated, rapid, and high-throughput microfluidic immunophenotyping platform can help unleash the mechanisms of systemic immune responses, and enable efficient assessments of the pathologic immune status for clinical diagnosis and immune therapy. 

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4. Baseline mucus cytokines predict 22‐item Sino‐Nasal Outcome Test results after endoscopic sinus surgery

   https://doi.org/10.1002/alr.22449  

   Chowdhury, Naweed I. ; Li, Ping ; Chandra, Rakesh K. ; Turner, Justin H. ( October 2019 , International Forum of Allergy & Rhinology)

   Mucus cytokines have been linked to baseline metrics of quality of life and olfactory function in patients with chronic rhinosinusitis (CRS). However, their potential utility in predicting postoperative outcomes has not been assessed. Therefore, in this study we evaluated the role of mucus cytokines in predicting 22‐item Sino‐Nasal Outcomes Test (SNOT‐22) scores after endoscopic sinus surgery (ESS) in a prospective cohort of CRS patients.

   One hundred forty‐seven patients with CRS electing surgical therapy were enrolled in a longitudinal cohort study. Mucus was collected intraoperatively from the middle meatus and tested for interleukin (IL)‐1β, IL‐2, ‐4, ‐5, ‐6, ‐7,‐ 8, ‐9, ‐10, ‐12, ‐13, ‐17A, and ‐21; tumor necrosis factor (TNF)‐α; interferon‐γ; eotaxin; and RANTES (regulated‐on‐activation, normal T‐cell expressed and secreted) expression using a multiplex flow‐cytometric bead assay. Sixty‐two patients were followed postoperatively (average, 10.2 months) with baseline and follow‐up SNOT‐22 surveys. Stepwise multivariate linear regression was used to model relationships between baseline cytokines, phenotype, and average postoperative SNOT‐22 total and domain scores. A machine learning approach using a random forest algorithm was also used to investigate potential nonlinear relationships.

   IL‐5 was an independent predictor of postoperative total SNOT‐22 improvement (β = −8.8,p< 0.0001), whereas IL‐2 levels predicted postoperative worsening (β = 6.97,p= 0.0015). Similar relationships were also seen for postoperative SNOT‐22 domain scores. The overall model was also noted to be significant fit for the data (adjustedR²= 0.398,p< 0.0001). The random forest model similarly identified IL‐5, TNF‐α, IL‐13, and IL‐2 as major predictors of postoperative SNOT‐22 scores.

   Mucus cytokine profiles may help identify CRS patients who are likely to obtain postoperative improvement after ESS.

    

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5. Comparative Molecular Immunological Activity of Physiological Metal Oxide Nanoparticle and its Anticancer Peptide and RNA Complexes

   https://doi.org/10.3390/nano9121670  

   DeLong, Robert K. ; Comer, Jeffrey ; Mathew, Elza Neelima ; Jaberi-Douraki, Majid ( December 2019 , Nanomaterials)

   null (Ed.)

   Currently, there is a great interest in nanoparticle-based vaccine delivery. Recent studies suggest that nanoparticles when introduced into the biological milieu are not simply passive carriers but may also contribute immunological activity themselves or of their own accord. For example there is considerable interest in the biomedical applications of one of the physiologically-based inorganic metal oxide nanoparticle, zinc oxide (ZnO). Indeed zinc oxide (ZnO) NP are now recognized as a nanoscale chemotherapeutic or anticancer nanoparticle (ANP) and several recent reports suggest ZnO NP and/or its complexes with drug and RNA induce a potent antitumor response in immuno-competent mouse models. A variety of cell culture studies have shown that ZnO NP can induce cytokines such as IFN-γ, TNF-α, IL-2, and IL-12 which are known to regulate the tumor microenvironment. Much less work has been done on magnesium oxide (MgO), cobalt oxide (Co3O4), or nickel oxide (NiO); however, despite the fact that these physiologically-based metal oxide NP are reported to functionally load and assemble RNA and protein onto their surface and may thus also be of potential interest as nanovaccine platform. Here we initially compared in vitro immunogenicity of ZnO and Co3O4 NP and their effects on cancer-associated or tolerogenic cytokines. Based on these data we moved ZnO NP forward to testing in the ex vivo splenocyte assay relative to MgO and NiO NP and these data showed significant difference for flow cytometry sorted population for ZnO-NP, relative to NiO and MgO. These data suggesting both molecular and cellular immunogenic activity, a double-stranded anticancer RNA (ACR), polyinosinic:poly cytidylic acid (poly I:C) known to bind ZnO NP; when ZnO-poly I:C was injected into B16F10-BALB/C tumor significantly induced, IL-2 and IL-12 as shown by Cohen’s d test. LL37 is an anticancer peptide (ACP) currently in clinical trials as an intratumoral immuno-therapeutic agent against metastatic melanoma. LL37 is known to bind poly I:C where it is thought to compete for receptor binding on the surface of some immune cells, metastatic melanoma and lung cells. Molecular dynamic simulations revealed association of LL37 onto ZnO NP confirmed by gel shift assay. Thus using the well-characterized model human lung cancer model cell line (BEAS-2B), poly I:C RNA, LL37 peptide, or LL37-poly I:C complexes were loaded onto ZnO NP and delivered to BEAS-2B lung cells, and the effect on the main cancer regulating cytokine, IL-6 determined by ELISA. Surprisingly ZnO-LL37, but not ZnO-poly I:C or the more novel tricomplex (ZnO-LL37-poly I:C) significantly suppressed IL-6 by >98–99%. These data support the further evaluation of physiological metal oxide compositions, so-called physiometacomposite (PMC) materials and their formulation with anticancer peptide (ACP) and/or anticancer RNA (ACR) as a potential new class of immuno-therapeutic against melanoma and potentially lung carcinoma or other cancers. 

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