Luminex Multiplex Assay Research Articles

Altered Lipid Metabolism in the Follow Up of Cystic Fibrosis Lung Transplant Patients

<h3>Purpose</h3> This study is aimed at characterizing lipid metabolism alteration in Cystic Fibrosis (CF) patients before and after lung transplant (LuTX), in association with pharmacological therapies, dietary patterns and clinical features, at the aim of elucidating its role in LuTX rejection versus graft tolerance. In spite of reduced body weight, among CF comorbidities, plasma hypertriglyceridemia and low HDL, associated with ectopic lipid accumulation including the airways, liver steatosis and CF related diabetes are reported. An altered ability to oxidize lipids for energy requests was recently suggested in these patients and a high ratio between fat mass and lean mass correlates with clinical worsening, such as reduced FEV and recurrent infections. LuTX causes an increase in fat trunk; it is per se a cause of diabetes insurgence, partially dependent on immunosuppressive therapy. Both reduced and increased weight are considered in the prognosis of rejection and pharmacological reduction of lipid synthesis, by means of statins or activation lipid oxidation by glitazones, may implement survival in LuTX patients. Leptin and adiponectin ratio is altered in CF and in LuTX rejecting patients and CF LuTx exhibit a significantly higher risk to develop hyperlipidemia than non CF. The hypothesis of this study is that CF dyslipidemia and altered ability to use lipids for energy requirement can negatively impact on LuTX, worsening the graft implantation related comorbidities <h3>Methods</h3> The cohort includes adults CF in list for LuTX. Evaluations are performed either before and after LuTX (up to 6 months post). Lipidomic profile is obtained by untargeted LC-MS analyses from plasma, plasma derived microvesicles, explanted lungs and surveillance transbronchial biopsies. Transcriptional profile of lipid metabolism related genes is obtained in lung biopsies by RT-PCR. Lipid metabolism related hormones are evaluated by Luminex multiplex assays from patients' plasma. Clinical biochemistry, lung functional analyses, and nutritional evaluation are routinely performed. <h3>Endpoints</h3> We aim at identifying a lipid metabolism derangement in association with CF LuTX adverse prognosis. The study will provide evidence on the importance of introducing novel nutritional and pharmacological intervention in the follow up of these patients.

Blood KL-6 predicts prognosis in primary Sj\xf6gren\u2019s syndrome-associated interstitial lung disease

Interstitial lung disease associated with primary Sjögren’s syndrome (SJS-ILD) has a variable clinical course. We aimed to investigate the role of blood biomarkers in predicting prognosis for SJS-ILD. Clinical data of 46 SJS-ILD patients were retrospectively reviewed. Plasma biomarker levels, including Krebs von den Lungen-6 (KL-6), CC chemokine ligand 18 (CCL18), chitinase-3-like-1 (YKL-40), interleukin-4 receptor alpha (IL-4Ra), and matrix metalloproteinase-7 (MMP-7) were measured using the multiplex Luminex assays (R&D Systems, Minneapolis, USA). The median follow-up period was 69.0 months. The mean age of the patients was 59.4 years; 17.4% were men. The KL-6 level was significantly higher in non-survivors (n = 12; 119.6 vs. 59.5 pg/mL, P = 0.037) than survivors (n = 34), while the levels of the other biomarkers did not differ. Receiver operating characteristic analysis indicated that KL-6 shows the best performance for predicting survival (area under the curve = 0.705, P = 0.037; best cut-off value = 53.5 pg/mL). Multivariable Cox analysis that was adjusted by age and diffusing capacity for carbon monoxide suggested a high KL-6 level (> 53.5 pg/mL) as an independent prognostic factor for survival (hazard ratio = 5.939, 95% confidence interval 1.312–26.881, P = 0.021). Our results suggest that blood KL-6 might be a useful in predicting the prognosis for patients with SJS-ILD.

Different Complement Activation Pathways Underly Cognitive Impairment and Type 2 Diabetes Mellitus Combined With Cognitive Impairment.

BackgroundThe immune response and the complement system are associated with cognitive impairment and diabetes mellitus, respectively. Activation of the complement system in these diseases occurs mainly through either the classical pathway or the alternative pathway. However, the specific complement proteins involved in the development of the type 2 diabetes mellitus (T2DM) and cognitive impairment are still unclear. Here, we investigated complement proteins in serum from patients with T2DM, cognitive impairment, or both T2DM and cognitive impairment.ObjectiveTo investigate the levels of serum immune complement proteins in patients with T2DM, cognitive impairment, or T2DM combined with cognitive impairment and the associations between these complement proteins and risk factors for T2DM or cognitive impairment.MethodsClinical markers were collected from blood samples of 264 participants. Luminex multiplex assays were used to detect serum complement proteins. All statistical analyses were performed using Prism or R studio.ResultsThere was a difference in serum levels of the complement proteins C1q, C3, C3b, and FH between the three different groups. Hyperglycemia was significantly correlated with elevated C3b or reduced C3, C1q, and FH. In addition, hyperlipidemia was positively correlated with elevated levels of C3, C4, C1q, and FH proteins. There was an association between C1q, C3, C4, and FH and β-pancreas cell function, whereas only FH was associated with insulin resistance. Higher serum C1q was significantly associated with an increased risk of cognitive impairment.ConclusionSerum levels of complement proteins were closely associated with hyperglycemia and hyperlipidemia. We found that classical complement pathway activation mainly occurred in the cognitive impairment only group, whereas the alternative pathway may reflect T2DM and T2DM with cognitive impairment.

Elevated Baseline Serum PD-L1 Level May Predict Poor Outcomes from Breast Cancer in African-American and Hispanic Women.

Background: The therapeutic targeting of PD-1/PD-L1 has shown clinical efficacy in treating metastatic breast cancer. We investigated the clinical significance of measuring serum PD-L1 levels in African-American and Hispanic women with breast cancer. Methods: PD-L1 levels were measured with the ELISA method from the serum samples of 244 African-Americans and Hispanics with breast cancer and 155 women without cancers. The levels of INFα2 and TNFα were measured with a Luminex multiplex assay. The protein levels of pAkt and CD44/CD24 in tumor cells were tested with immunohistochemistry analysis. Cox regression was used to assess the predicting role of serum PD-L1 for disease-free survival (DFS). Results: PD-L1 levels were significantly elevated in breast cancer cases compared to non-cancer cases. The high PD-L1 levels were associated with HER2-positive and triple-negative breast cancer. PD-L1 level independently predicted DFS in both African-American and Hispanic women. The evaluated PD-L1 level was found to be associated with high IFNα2 and TNFα in breast cancer patients. Conclusions: PD-L1 serum levels can predict DFS in African American and Hispanic women with breast cancer. Furthermore, a high level of PD-L1 is more likely to be associated with tumor loss PTEN and the activation of Akt or with breast cancer cells expressing CD44high/CD24low. Further validation studies are needed to determine if PD-L1 could serve as a biomarker for patient selection for anti-PD-L1 therapy and assess treatment outcomes.

Comparing Concentration of Urinary Inflammatory Cytokines in Interstitial Cystitis, Overactive Bladder, Urinary Tract Infection, and Bladder Cancer

Purpose: We sought to determine if urinary cytokine concentration profiles were different between various bladder conditions. Materials and Methods: Participants at three clinical sites completed a demographics survey and provided a urine sample in a collection cup containing a room-temperature urine preservative. Participants were divided into the following categories based on physician-documented diagnosis: asymptomatic control, nonulcerative interstitial cystitis (IC), overactive bladder with incontinence (OAB wet), urinary tract infection (UTI), and bladder cancer. Urinary cytokines were measured through Luminex multiplex assay. Results: Two hundred and seventy-seven urine samples were collected from three clinical sites. Urinary pro-inflammatory cytokines had an increasing trend in bladder disease versus control, with a significant increase for chemokine (C-X-C) ligand 1 growth-regulated protein alpha CXCL1 (GRO). Further analyses demonstrated that patients with UTI had significantly higher levels of GRO and interleukin-8 (IL-8) in comparison to control, nonulcerative IC, OAB wet, and bladder cancer. Both are chemokines that stimulate chemotaxis resulting in the rapid accumulation of immune cells such as neutrophils. IL-6 levels overall were at the lower limit of assay range but were significantly increased in urine of UTI patients versus IC patients. MCP-1 (CCL2) had the least separation among the control group and the various bladder diseases. Conclusion: Urinary concentrations of GRO were higher in disease state compared to control. Specifically, levels of GRO and IL-8 were higher in urine samples from patients with UTI compared to controls and other bladder conditions. Comparing and contrasting urinary cytokines may help improve our understanding of these important bladder diseases with great unmet needs.

The survival of epidemic and sporadic MRSA on human skin mimics is determined by both host and bacterial factors.

Bacterial survival on, and interactions with, human skin may explain the epidemiological success of MRSA strains. We evaluated the bacterial counts for 27 epidemic and 31 sporadic MRSA strains on 3D epidermal models based on N/TERT cells (NEMs) after 1, 2 and 8 days. In addition, the expression of antimicrobial peptides (hBD-2, RNase 7), inflammatory cytokines (IL-1β, IL-6) and chemokine IL-8 by NEMs was assessed using immunoassays and the expression of 43 S. aureus virulence factors was determined by a multiplex competitive Luminex assay. To explore donor variation, bacterial counts for five epidemic and seven sporadic MRSA strains were determined on 3D primary keratinocyte models (LEMs) from three human donors. Bacterial survival was comparable on NEMs between the two groups, but on LEMs, sporadic strains showed significantly lower survival numbers compared to epidemic strains. Both groups triggered the expression of immune factors. Upon interaction with NEMs, only the epidemic MRSA strains expressed pore-forming toxins, including alpha-hemolysin (Hla), gamma-hemolysin (HlgB), Panton-Valentine leucocidin (LukS) and LukED. Together, these data indicate that the outcome of the interaction between MRSA and human skin mimics, depends on the unique combination of bacterial strain and host factors.

Is there a relationship between plasma, cytokine concentrations, and the subsequent risk of postpartum hemorrhage?

Postpartum hemorrhage remains the leading cause of maternal mortality. However, there is an insufficient understanding of atonic postpartum hemorrhage. Uterine atony is the most common cause of postpartum hemorrhage. Although an association between myometrium inflammatory cytokines and atonic postpartum hemorrhage has been demonstrated preliminarily, it is not clinically useful in predicting postpartum hemorrhage. Plasma is more readily available, and the assessment of its inflammatory status is more relevant to biological markers of postpartum hemorrhage and might explain the pathophysiology of atonic postpartum hemorrhage. Our objective was to examine changes in maternal plasma cytokines in women with atonic postpartum hemorrhage. This was a retrospective longitudinal case-control study of pregnant women with singleton gestations at term undergoing vaginal delivery. Cases were women with atonic postpartum hemorrhage, and 1:1 propensity-score matching was used to match the control group. Maternal plasma was collected in the first trimester, early third trimester, and late third trimester, and multiplex Luminex assay was used to determine the cytokine concentrations. Multivariate logistic regressions were used to determine the association between maternal cytokines at different stages of pregnancy and atonic postpartum hemorrhage. A total of 36 pregnant women met the clinical diagnostic criteria for atonic postpartum hemorrhage, and 36 patients without postpartum hemorrhage were matched as the control group. Concentrations were lower for most cytokines in the atonic postpartum hemorrhage group in the first and early third trimester. However, in the late third trimester, higher plasma concentrations of basic fibroblast growth factor, interleukin-1 alpha, interleukin-1 beta, interleukin-1 receptor antagonist, interleukin-2 receptor alpha, interleukin-16, interleukin-18, macrophage colony stimulating factor, macrophage inflammatory protein-1 alpha, beta-nerve growth factor, tumor necrosis factor-related apoptosis-induced ligand, and stem cell factor were significantly associated with increased risk of atonic postpartum hemorrhage. Multiple testing correction showed that basic fibroblast growth factor (P<.001; fold change [FC]=1.16), macrophage inflammatory protein-1 alpha (P<.001; FC=1.15), and stem cell factor (P=.001; FC=1.25) had the most significant difference (P<.001). The prediction model of atonic postpartum hemorrhage constructed by these significantly changed cytokines had a high predictive efficiency (area under the curve, 0.84; sensitivity, 0.78; specificity, 0.83;+likelihood ratio, 4.66;-likelihood ratio, 0.27). Higher concentrations of maternal plasma cytokines in the late third trimester are associated with high risk of subsequent atonic postpartum hemorrhage. These indicators may be potential biomarkers for predicting atonic postpartum hemorrhage.

191. High-Dose Rifampin-containing Regimens for the Treatment of TB Meningitis

191. High-Dose Rifampin-containing Regimens for the Treatment of TB Meningitis

Evaluation of the Induced Membrane for Neurotrophic Factors

Despite gaining popularity as a bridge for small and moderate nerve gaps, an acellular nerve allograft (ANA) lacks many of the neurotrophic characteristics of a nerve autograft. Pseudomembranes induced to form around temporary skeletal spacers are rich in growth factors. Induced membranes may have beneficial neurotrophic factors which could support ANA. Twenty-two male Sprague-Dawley rats underwent resection of 2 cm of the sciatic nerve. A silicone rod was inset in the defect of 11 experimental rats, and marking sutures only were placed in the nerve stumps of the remaining 11 control rats. After allowing 4 weeks for tissue maturation, tissue samples harvested from the induced membrane (experimental group) and the tissue bed (control group) were analyzed using Luminex multiplex assay to quantify differences in detectable levels of the following neurotrophic factors: nerve growth factor, glial-derived nerve factor, vascular endothelial growth factor, and transforming growth factor ß (TGF-ß) 1, 2, and 3, interleukin-1ß, and monocyte chemoattractant protein1. No difference was detected between the control and experimental groups in levels of vascular endothelial growth factor. Higher levels of TGF-ß1, TGF-ß2, TGF-ß3, glial-derived nerve factor, nerve growth factor, monocyte chemoattractant protein 1, and interleukin-1ß were detected in the experimental group. In the setting of peripheral nerve injury, an induced membrane has higher levels of several neurotrophic factors that may support nerve regeneration compared to wound bed cicatrix. This investigation provides impetus for further study examining the utility of using a staged induced membrane technique in conjunction with delayed nerve grafting in reconstruction of some peripheral nerve defects.

Apolipoprotein E4‐driven effects on inflammatory and neurotrophic factors in peripheral extracellular vesicles from cognitively impaired not demented participants converted to Alzheimer’s disease

AbstractBackgroundIn brain, extracellular vesicles (EVs) play an essential role in neuron‐glia interface and ensure the crosstalk between the brain and the periphery. Only a few studies have demonstrated the apolipoprotein E4 variant (APOE ε4)‐driven dysfunction of EVs pathway and the risk to develop Alzheimer’s disease (AD). To better understand the role of APOE ε4 in pre‐clinical AD, we determined levels of pathogenic, neurotrophic and inflammatory proteins in peripheral EVs (pEVs) and in plasma from cognitively impaired‐not demented (CIND) participants stratified upon the absence (APOE ε4‐) or the presence (APOE ε4+) of the ε4 allele of APOE.MethodLevels of 15 neurodegenerative, neurotrophic and neuroinflammatory proteins were quantified in pEVs by the multiplex Luminex assay and compared to their plasma levels from cognitively normal and CIND participantsResultFor the first time, several neurotrophic and inflammatory markers including LCN‐2, S100B, ANGPTL‐4, NPTX‐2 and α‐synuclein were evidenced in pEVs. Some proteins such as α‐Syn, NPTX‐2 and S100B were enriched in pEVs as compared to plasma. APOE ε4+ was associated with differential regulation of 7 markers and compromised the release of pEVs formed by an endosomal route. The pentraxin‐2/α‐synuclein ratio measured in pEVs was able to predict AD 5 years before the onset among APOE ε4+ CIND individuals. Discussion: Our findings suggest an alteration of the endosomal pathway in APOE ε4+ carriers and that pEVs pentraxin‐2/α‐synuclein ratio could serve as a useful early biomarker for AD susceptibility.ConclusionThe findings reported herein provide a comprehensive insight and enhance our knowledge on the emerging role of ApoE4 in abnormal pEVs cargo proteins processing and the identification of blood‐based biomarkers.

DDRE-46. REDUCED CANCER CELL SENSITIVITY TO TUMOR TREATING FIELDS (TTFields) THROUGH ACTIVATION OF THE PI3K/AKT/mTOR SIGNALING PATHWAY CAN BE MITIGATED USING PI3K INHIBITORS OR PI3K/mTOR DUAL INHIBITORS

Abstract INTRODUCTION Tumor Treating Fields (TTFields) therapy is an approved anti-cancer treatment modality applied non-invasively and loco-regionally to the tumor region. TTFields have been demonstrated to extend life, however, most patients do eventually progress. The current study aimed to identify potential molecular mechanisms involved in reduced cellular sensitivity to TTFields. METHODS Cancer cells that exhibit reduced sensitivity to TTFields were generated by continuous long duration application of TTFields (7 or 13 days, depending on the cell line). Changes in cellular signaling pathways were evaluated in ovarian A2780 and glioblastoma U-87 MG cancer cells exposed to long-term relative to short-term (3 or 7 days, depending on the cell line) treatment with TTFields using Luminex multiplex assay followed by Western blot validation. The relevance of the affected pathways was confirmed through evaluation of the response to long-term application of TTFields in combination with pharmacological pathway inhibitors by measuring cell counts, apoptosis, and clonogenicity. Relevant pathway markers in tumor sections from tumor-bearing rats treated with TTFields were examined using immunohistochemistry. RESULTS Continuous long-term application of TTFields reduced cellular sensitivity to TTFields and was accompanied by increased levels of phosphorylated AKT, mTOR and additional proteins from the PI3K/AKT/mTOR signaling pathway. Increased phosphorylation of AKT was also evident in tumor sections from rats treated with TTFields. Concomitant use of TTFields with PI3K inhibitors or PI3K/mTOR dual inhibitors sensitized A2780 cells to long-term TTFields application. CONCLUSION Our study demonstrates that decreased cancer cell sensitivity to long-term TTFields application is mediated by activation of the PI3K/AKT/mTOR signaling pathway and provides a rationale for further examining the potential benefit of combining TTFields with PI3K or PI3K/mTOR dual inhibitors.

11.6 Correlations Between Anhedonia and Adrenergic Attenuation of Immune Activation in Adolescents With Diverse Mood and Anxiety Symptoms

While the link between peripheral immune activation and mental health symptoms has been supported, the role of immune-modulating processes by the adrenergic nervous system in psychopathology remains unclear. Here, we sought to investigate the β2-agonist clenbuterol (CBL) as a promising agent to model adrenergic-driven immune attenuation following ex vivo immune activation with lipopolysaccharide (LPS). Further, we explored the relationships between CBL-modulated cytokine levels and mental health measures in our adolescent sample. Depression, anxiety, and anhedonia levels were measured with the Beck Depression Inventory (BDI), the Multidimensional Anxiety School for Children (MASC), and the Temporal Experience of Pleasure Scale (TEPS) questionnaires. Fasting whole-blood samples were acquired and stimulated with LPS (0.1 μg/mL) in the presence and absence of CBL (10-6 M) for 6 hours. Supernatants were collected and subjected to Luminex multiplex assay for 41 cytokines. Cytokine levels were compared between 3 conditions: control, LPS, and LPS+CBL. Then, an exploratory factor analysis (EFA) was utilized to dimensionally reduce cytokine data from the 3 conditions and the differences between them. Correlations between each latent factor and mental health symptoms were examined. The statistical significance of all associations was assessed after Bonferroni correction for multiple comparisons. Ten cytokines were significantly affected by CBL treatment compared to LPS alone. In each of the 6 examined conditions, EFA reduced 41 cytokines into 5 orthogonal factors. Only factor 3 of the LPS+CBL condition was found to be significantly correlated with both anticipatory anhedonia (rho = –0.39; p = 6.25 × 10-5) and consummatory anhedonia (rho = –0.36; p = 3.21 × 10-4). No significant correlation was found between any cytokine factor alone and depression or anxiety. Post hoc analysis revealed differences in intercytokine correlation structures between LPS+CBL and LPS conditions, possibly explaining CBL’s effects on the relationships between peripheral immune response and anhedonia subcomponents. Findings support our hypothesis that CBL attenuates inflammatory effects thought to underlie mental health conditions in youth. Following data-driven analyses, distinctive associations between CBL-affected immune response and dimensional anhedonia were identified.

Development of a new peptide-bead coupling method for an all peptide–based Luminex multiplexing assay for detection of Plasmodium falciparum antibody responses

Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies.Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine–lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria.The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results.Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas.

Persistent High Percentage of HLA-DR+CD38high CD8+ T Cells Associated With Immune Disorder and Disease Severity of COVID-19.

BackgroundThe global outbreak of coronavirus disease 2019 (COVID-19) has turned into a worldwide public health crisis and caused more than 100,000,000 severe cases. Progressive lymphopenia, especially in T cells, was a prominent clinical feature of severe COVID-19. Activated HLA-DR+CD38+ CD8+ T cells were enriched over a prolonged period from the lymphopenia patients who died from Ebola and influenza infection and in severe patients infected with SARS-CoV-2. However, the CD38+HLA-DR+ CD8+ T population was reported to play contradictory roles in SARS-CoV-2 infection.MethodsA total of 42 COVID-19 patients, including 32 mild or moderate and 10 severe or critical cases, who received care at Beijing Ditan Hospital were recruited into this retrospective study. Blood samples were first collected within 3 days of the hospital admission and once every 3–7 days during hospitalization. The longitudinal flow cytometric data were examined during hospitalization. Moreover, we evaluated serum levels of 45 cytokines/chemokines/growth factors and 14 soluble checkpoints using Luminex multiplex assay longitudinally.ResultsWe revealed that the HLA-DR+CD38+ CD8+ T population was heterogeneous, and could be divided into two subsets with distinct characteristics: HLA-DR+CD38dim and HLA-DR+CD38hi. We observed a persistent accumulation of HLA-DR+CD38hi CD8+ T cells in severe COVID-19 patients. These HLA-DR+CD38hi CD8+ T cells were in a state of overactivation and consequent dysregulation manifested by expression of multiple inhibitory and stimulatory checkpoints, higher apoptotic sensitivity, impaired killing potential, and more exhausted transcriptional regulation compared to HLA-DR+CD38dim CD8+ T cells. Moreover, the clinical and laboratory data supported that only HLA-DR+CD38hi CD8+ T cells were associated with systemic inflammation, tissue injury, and immune disorders of severe COVID-19 patients.ConclusionsOur findings indicated that HLA-DR+CD38hi CD8+ T cells were correlated with disease severity of COVID-19 rather than HLA-DR+CD38dim population.

The Use of Serum Matrix Metalloproteinases in Cerebral Amyloid Angiopathy-Related Intracerebral Hemorrhage and Cognitive Impairment.

Neuroimaging has played a primary role in predicting intracerebral hemorrhage (ICH) recurrence of cerebral amyloid angiopathy (CAA); however, the utilities of biomarkers in CAA-related ICH and cognitive impairment remain unexplored. To investigate the correlations of serum levels of matrix metalloproteinase-2 (MMP-2), MMP-3, and MMP-9 with CAA-related MRI markers, ICH recurrence, and cognitive status. 68 cases with first probable CAA-ICH and 69 controls were recruited. Clinical and imaging data were obtained at baseline and serum MMPs in the acute phase were measured by Luminex multiplex assays. Cognitive status was assessed with the Chinese version of Mini-Mental State Examination within 10-14 days after ICH onset. Serum MMP-2 level was significantly lower in CAA-ICH patients than controls while MMP-9 was significantly higher. In CAA-ICH patients, MMP-3 level was significantly associated with lobar cerebral microbleeds count after adjusting age, sex, and hypertension (adjusted coefficient 0.368, 95%CI 0.099-0.637, p = 0.008). During a median follow-up of 2.4 years, higher level of MMP-2 predicted lower CAA-ICH recurrence after adjusting age (adjusted HR 0.326, 95%CI 0.122-0.871, p = 0.025), ICH volume (adjusted HR 0.259, 95%CI 0.094-0.715, p = 0.009), total MRI burden of SVD score (adjusted HR 0.350, 95%CI 0.131-0.936, p = 0.037) respectively. Besides, higher level of MMP-2 was significantly associated with decreased risk of cognitive impairment independent of age and ICH volume (adjusted OR 0.054, 95%CI 0.005-0.570, p = 0.015). Serum MMP-2 in acute phase might be a promising biomarker to predict CAA-ICH recurrence and to evaluate the risk of cognitive impairment.

Circulating levels of cell adhesion molecules and risk of cardiovascular events in obstructive sleep apnea

BackgroundObstructive sleep apnea (OSA) patients are at increased risk of cardiovascular disease (CVD). Cell adhesion molecules (CAM) are increased in OSA and CAM are also implicated in the development of CVD.Research questionDo CAM (ICAM-1, VCAM-1 and E-selectin) have prognostic value in identifying risk of cardiovascular events in OSA?Study design and methodsPatients with suspected OSA referred for a polysomnogram provided a fasting blood sample. Plasma levels of ICAM-1, VCAM-1 and E-selectin were determined by multiplex Luminex Assay (Milliporesigma ON, Canada). Cardiovascular events were determined by deterministic linkage to provincial health databases.Results418 patients were included in the analysis. Mostly male (68.2%), mean age of 50.7 yrs, median AHI 16.5 events/hour, and mean BMI of 31.7 kg/m2. 36 cardiovascular events occurred in 8-yrs of follow up. Higher levels of ICAM-1 were associated with developing CVD (HR = 3.65 95% CI 1.40–9.53, 2nd and 3rd tertiles vs. 1st tertile), including in patients with OSA (HR = 3.1 95% CI 1.16–8.25). E-selectin was significantly associated with cardiovascular events in patients with moderate to severe OSA (HR = 3.31 95% CI 0.94–11.72, 2nd and 3rd tertiles vs. 1st tertile) but not in patients without moderate to severe OSA (HR = 0.67 95% CI 0.19–2.38), p-value for interaction = 0.07.InterpretationIn a suspected OSA cohort, patients with higher levels of ICAM-1 (>816 ng/ml) were significantly more likely to experience a cardiovascular event within 8 years after PSG. In moderate to severe OSA patients, a higher E-selectin (>36.4 ng/ml) was significantly associated with cardiovascular events.

QS3: CRISPR/Cas9 Editing of Autologous Dendritic Cells to Enhance Angiogenesis and Wound Healing

Purpose:Dendritic cells (DCs) are a heterogeneous cell population which critically regulates the adaptive immune response. Depending on their activation status, DCs can also promote peripheral immune tolerance, thus limiting the activation of the immune system and tissue damage. The N-myc downregulated gene 2 (Ndrg2) is highly expressed in DCs and limits the secretion of vascular endothelial growth factor (VEGF), which is critical for wound healing. Cell based therapy approaches using DCs have been approved by the FDA and clinical trials using DC immunotherapy are being performed against a variety of cancer types. However, the role of DC therapy for wound healing has not yet been investigated.Methods:Hematopoietic progenitor cells were isolated from the bone marrow of mice and differentiated into DCs over a 7-day in vitro culture period. Pharmacologic down-regulation of Ndrg2 was performed by treatment of cultures with 1,25-dihydroxyvitamin D3 (VD3) and the angiogenic potential of the treated cells was evaluated by endothelial cell (EC) tube formation assays. Cytokine secretion of DCs was measured in the conditioned media using Luminex multiplex assays. To permanently knock out Ndrg2 in DCs, a CRISPR/Cas9 gene editing approach was developed, using Cas9/sgRNA-ribonucleoproteins and electroporation. The determine the impact of genetically edited DCs on wound healing, splinted excisional wounds in C57BL6/J (wild-type) mice were treated weekly with pullulan-collagen hydrogels seeded with Ndrg2-knockout (KO) DCs, control DCs which had undergone electroporation only, or blank hydrogels. The transcriptomic impact of Ndrg2 downregulation on DC fate was evaluated by microfluidic single-cell RNA sequencing (scRNA seq) of Ndrg2-KO DCs, VD3-treated DCs and control DCs.Results:Ndrg2 down-regulation lead to a significantly stronger EC tube formation in co-cultures with VD3-treated DCs, and strongly enhanced VEGF secretion compared to untreated DCs in vitro. A CRISPR/Cas9 editing pipeline was developed for KO of Ndrg2 in DCs with a transfection rate and editing efficiency of > 90% shown by Sanger Sequencing. Excisional wounds treated with Ndrg2-KO DCs demonstrated significantly accelerated healing compared to control DCs and blank hydrogels. scRNA seq revealed that Ndrg2 downregulation strongly induced Vegfa expression and anti-oxidant transcriptomic signatures.Conclusion:Our data indicate that KO of Ndrg2 in DCs strongly enhances their secretion of VEGF, thus promoting angiogenesis and accelerating wound healing. Given the ready availability of DCs from the human blood through established leukapheresis protocols and easy multiplication in vitro, CRISPR/Cas9 editing of DCs is a promising new approach to induce wound healing and soft-tissue regeneration.

4: Prolonged Tourniquet Use Following Blast Related Lower Extremity Injuries Increase Heterotopic Ossification in a Pre-clinical Model

Purpose:Traumatic heterotopic ossification (tHO) has become a signature pathology affecting wounded military personnel who have sustained blast-associated traumatic amputations during the recent conflicts in Iraq and Afghanistan. Heterotopic ossification is characterized by the abnormal development of mature bone depositions in extra-skeletal sites such muscle, tendon, and soft tissues, leading to impaired wound healing, pain, reduced range of motion, and limited use of prostheses. While many factors influence the formation of tHO, the extended use of tourniquets to limit catastrophic hemorrhage during prolonged field care (PFC) has not been explored. Herein, we investigate the impact of tourniquet use following blast-related injury on ectopic bone formation.Methods:Utilizing an established pre-clinical model of blast-associated complex lower limb injury and traumatic amputation, we evaluated the effects of extended tourniquet use on tHO formation. Male rats (11-12-week-old) were subjected to blast overpressure exposure, femur fracture, and soft tissue crush injury. A pneumatic tourniquet (250-300mmHg) was applied to the injured limbs for either 90- and 150-minutes followed by trans-femoral amputation. Limbs were assessed for HO formation using microCT. Analysis of muscle/soft-tissue osteogenesis-related gene transcripts and multiple serum inflammatory mediators were measured by using qRT-PCR and Luminex multiplex assays, respectively.Results:At 12 weeks, volumetric analysis with microCT imaging revealed an 70% increase in total bone formation (P=0.007, n=11) near the site of injury in rats subjected to 150-minutes of tourniquet time compared to rats with no tourniquet time in the setting of blast-injuries. Rats subjected to 150-minute tourniquet usage and blast injury had increased expression of osteochondrogenic genes including Bmp2 (5.4-fold increase, P=0.01) as early as 6 hours post-injury while Hif1α (4.2-fold increase, P<0.01), Sox9 (2.8-fold increase, P<0.01), Runx2 (8.2-fold increase), and Bmp2 (7.7-fold increase, P<0.02) remained elevated for 7 days. Analysis of cytokines and chemokines in the serum demonstrated increased expression of key analytes in the tourniquet group above that induced by traumatic amputation alone in the control group in factors including IL-1 (22-44%, P<0.005) and IL-6 (13-69% p<0.03) between 6 hrs and POD7.Conclusions:These findings suggest that extended tourniquet time leads to both significant increases in key transcription factors associated with early endochondral bone formation, as well as increased systemic inflammatory mediators. Increased expression of Hif1α with prolonged tourniquet use also demonstrates the importance of tissue hypoxia and Hif1α signaling in combat applicable tHO and the potential development of targets for therapeutic inhibition. This data supports mechanisms by which extended tourniquet times under PFC conditions could result in increased local neuromuscular dysfunction and systemic inflammation, resulting in increased local tissue injury and potential further functional loss secondary to tHO in wounded military personnel.

Identification of the JNK-Active Triple-Negative Breast Cancer Cluster Associated With an Immunosuppressive Tumor Microenvironment.

Although an immunosuppressive tumor microenvironment (TME) is key for tumor progression, the molecular characteristics associated with the immunosuppressive TME remain unknown in triple-negative breast cancer (TNBC). Our previous functional proteomic study of TNBC tumors identified that C-JUN N-terminal kinase (JNK) pathway-related molecules were enriched in a cluster associated with the inflammatory pathway. However, the role of the JNK pathway in the TNBC TME is still unclear. Transcriptomic analysis was conducted using The Cancer Genome Atlas datasets. The effect of JNK-IN-8, a covalent pan-JNK inhibitor, on TNBC tumor growth, lung metastasis, and the TME was measured in TNBC syngeneic mouse models (n = 13 per group). Tumor (n = 43) or serum (n = 46) samples from TNBC patients were analyzed using multiplex immunohistochemistry or Luminex assay. All statistical tests were 2-sided. CIBERSORT analysis revealed that TNBC patients with high phosphorylated JNK level (n = 47) had more regulatory T cell (Treg) infiltration than those with a low phosphorylated JNK level (n = 47) (P = .02). Inhibition of JNK signaling statistically significantly reduced tumor growth (P < .001) and tumor-infiltrating Tregs (P = .02) while increasing the infiltration of CD8+ T cells in TNBC mouse models through the reduction of C-C motif ligand 2 (CCL2). Tumor-associated macrophages were the predominant cells secreting CCL2, and inhibition of JNK signaling reduced CCL2 secretion of human primary macrophages. Moreover, in patients with TNBC (n = 43), those with high levels of CCL2+ tumor-associated macrophages had more Treg and less CD8+ T cell infiltration (P = .04), and the serum CCL2 level was associated with poor overall survival (hazard ratio = 2.65, 95% confidence interval = 1.29 to 5.44, P = .008) in TNBC patients (n = 46). The JNK/C-JUN/CCL2 axis contributes to TNBC aggressiveness via forming an immunosuppressive TME and can offer novel therapeutic strategies for TNBC.

Urinary Cytokines Reflect Renal Inflammation in Acute Tubulointerstitial Nephritis: A Multiplex Bead-Based Assay Assessment

Background: Acute tubulointerstitial nephritis (ATIN) diagnosis lays on histological assessment through a kidney biopsy, given the absence of accurate non-invasive biomarkers. The aim of this study was to evaluate the accuracy of different urinary inflammation-related cytokines for the diagnostic of ATIN and its distinction from acute tubular necrosis (ATN). Methods: We included 33 patients (ATIN (n = 21), ATN (n = 12)), and 6 healthy controls (HC). We determined the urinary levels of 10 inflammation-related cytokines using a multiplex bead-based Luminex assay at the time of biopsy and after therapy, and registered main clinical, analytical and histological data. Results: At the time of biopsy, urinary levels of I-TAC/CXCL11, CXCL10, IL-6, TNFα and MCP-1 were significantly higher in ATIN compared to HC. A positive correlation between the extent of the tubulointerstitial cellular infiltrates in kidney biopsies and the urinary concentration of I-TAC/CXCL11, MIG/CXCL9, CXCL10, IL17, IFNα, MCP1 and EGF was observed. Notably, I-TAC/CXCL11, IL-6 and MCP-1 were significantly higher in ATIN than in ATN, with I-TAC/CXCL11 as the best discriminative classifier AUC (0.77, 95% CI 0.57–0.95, p = 0.02). A combinatory model of these three urinary cytokines increased the accuracy in the distinction of ATIN/ATN compared to the individual biomarkers. The best model resulted when combining the three cytokines with blood eosinophil and urinary leukocyte counts (LR = 9.76). Follow-up samples from 11ATIN patients showed a significant decrease in I-TAC/CXCL11, MIG/CXCL9 and CXCL10 levels. Conclusions: Urinary I-TAC/CXCL11, CXCL10, IL6 and MCP-1 levels accurately distinguish patients developing ATIN from ATN and healthy individuals and may serve as novel non-invasive biomarkers in this disease.