Peripheral Blood Monocyte Cells Research Articles

Functional interleukin-4 releasing microparticles impact THP-1 differentiated macrophage phenotype

IntroductionMacrophage cell therapies offer potential treatment in inflammatory diseases due to their ability to mobilize and stimulate their environment. However, successful treatment requires a pro-regenerative macrophage phenotype to be retained in vivo. Polymeric microparticles may provide a potential route to direct and sustain macrophage phenotype. Interleukin-4 (IL-4) is the most commonly used cytokine for in vitro modulation towards M2a macrophage phenotype. We designed IL-4 encapsulated microparticles to investigate the impact of drug release kinetics and developed a robust human peripheral blood monocyte cell (THP-1) in vitro assay to assess functional IL-4 release upon macrophage phenotype.MethodsIL-4 was encapsulated with human serum albumin (HSA) in microparticles fabricated from a blend of PLGA and a PLGA-PEG-PLGA triblock copolymer. Functional release of IL-4 and HSA over different time periods was measured using ELISAs. THP-1 differentiated macrophages were cultured either in direct contact with microparticles or indirectly through transwells. The immunomodulatory impact of microparticles on THP-1 cells were measured using ELISA and qPCR.Results and DiscussionIL-4 release kinetics fit with the first-order release kinetics model, indicating concentration dependent release. IL-4/HSA encapsulated microparticles modulated THP-1 differentiated macrophages towards pro-immunoregulatory subgroups. This strategy provides a novel approach in drug carrier development for in vitro assessments of macrophage phenotype to inform development of targeted therapies for inflammation and immune modulation.

Correlation of peripheral blood monocytic myeloid-derived suppressor cells (M-MDSC) and T-cell receptor (TCR) dynamics with clinical outcomes in patients (pts) with metastatic urothelial carcinoma (mUC) treated with nivolumab (NIVO).

4569 Background: Although PD-1 blockade is active in varied mUC treatment settings, predicting response and overcoming resistance remain unmet needs. We hypothesized that immunosuppressive M-MDSCs (CD14+Lin-/HLA-DRlow/-) and TCR dynamics in tumors and blood may correlate with outcomes from NIVO in pts with platinum-refractory mUC (NCT02553642). Methods: 69 pts with mUC were treated with NIVO 240mg intravenously every 2 weeks (wks). Pre-treatment peripheral M-MDSCs among lineage-negative CD14+ monocytes (%) were estimated by flow cytometry using an algorithm that calculates MDSC frequencies based on HLA-DR mean fluorescence intensity (Kitano et al. 2014). High throughput DNA sequencing of the CDR3 region of the TCR beta chain was performed on baseline tumors (N = 57) and pre and post-treatment (wk 2) peripheral blood mononuclear cells using Adaptive Immunosequencing (Adaptive Biotechnologies). M-MDSC levels and TCR metrics were correlated with clinical outcomes: complete or partial response (CR, PR) vs. stable or progressive disease (SD, PD) by RECIST 1.1, clinical benefit (CR + PR + SD), and progression-free and overall survival (PFS, OS). Groups were compared using Wilcoxon signed rank (paired), Wilcoxon rank sum tests (unpaired), and log-rank tests (time-to-event). Cox proportional hazards and logistic regression models were used to analyze binary and time-to-event outcomes, respectively. Results: Higher pre-treatment M-MDSCs were associated with worse PFS and OS univariably (PFS HR 1.07; 95% CI, 1.01-1.14; p = 0.025; OS HR 1.07; 95% CI, 1.00-1.14; p = 0.038), and after adjustment for liver disease and PD-L1 at baseline (PFS HR 1.12; 95% CI, 1.04-1.21, p = 0.004; OS HR 1.11; 95% CI, 1.03-1.20, p = 0.010). Baseline M-MDSCs did not significantly differ between responders and non-responders, but were significantly lower in patients with clinical benefit (median 12.8 [IQR: 10.7-15.9] vs. median 15.6 [IQR: 13.5-18.3]; p = 0.039), and remained significantly associated after adjustment for PD-L1 score in a multivariable model (OR 0.82; 95% CI, 0.67-0.97; p = 0.037). A higher number of tumor-associated clones in the blood (BTACs) at baseline was associated with response (p = 0.049). Overall, BTACs significantly increased at wk 2 (p < 0.001), and wk 2 values were associated with response (p = 0.003). PFS also significantly differed between BTAC high (high: > median 2,012 clones) and low groups at wk 2, with improved PFS in the high group (log-rank p = 0.026). OS rate for the BTAC high group was 34% vs. 16% for the low group at wk 2, but this did not achieve significance (p = 0.098). Conclusions: Peripheral M-MDSCs may promote resistance to PD-1 blockade in pts with mUC. NIVO stimulated tumor-specific TCR clones in the blood, which correlated with improved response and outcomes. Clinical trial information: NCT02553642 .

P161 The use of Novel Natural Treatments for Inflammatory Bowel Diseases

Abstract Background Background: Free radicals and reduced antioxidant levels lead to oxidative stress, which plays a role in causing Inflammatory Bowel Disease (IBD). IBD is a chronic gastrointestinal autoimmune condition that involves an abnormal immune response. This includes Crohn's disease (CD) and ulcerative colitis (UC). In the previous studies, we demonstrated the antioxidant effect of Inonotus obliquus (IO) and flavonoids such as quercetin and epicatechin on lymphocytes from IBD patients. In vitro studies have shown that Chaga extract is effective in reducing oxidative stress in lymphocytes of both healthy individuals and those with IBD. This suggests that Chaga extract may be a useful supplement for anyone looking to inhibit oxidative stress. Methods Methods: In our current study, Cord Blood Stem Cells -derived Exosomes (CBSC Exo) and their synthesised miRNAs were investigated as antioxidant/anti-inflammatory elements on lymphocytes and 3D intestinal epithelial model. There were seven miRNAs found in CBSCs Exo, and among them were two miRNAs that were identified as novel. The Peripheral Blood Monocyte Cells (PBMCs) from IBD patients and healthy individuals after challenging with H2O2 or Interleukin 6 were treated with CBSCs Exo, transfected with miRNA novels or both. The treatment method also was applied to the EpiltestinalTK 3D model from Mattek. Various techniques were utilised, including the Comet assay, Fast Microplate DNA damage assay, and CCK8. Results The findings of the study indicate that the administration of CBSC-derived Exo, Let-7c-mimic miRNA, Let7bSb miRNA, as well as Novel 1 and Novel 2 miRNAs on the 3D model intestine, significantly decreased the level of DNA damage when compared to the positive control H2O2 and IL6, as well as the untreated samples. Conclusion Conclusions: In conclusion, certain natural substances like flavonoids, Inonotus obliquus, and exosomes derived from CBSCs may have an advantage in decreasing significantly oxidative stress and DNA damage in IBD Peripheral Blood Mononuclear Cells (PBMCs) and inflamed intestinal 3D models.

Breastfeeding and Neonatal Age Influence Neutrophil-Driven Ontogeny of Blood Cell Populations in the First Week of Human Life.

The first few days of life are characterized by rapid external and internal changes that require substantial immune system adaptations. Despite growing evidence of the impact of this period on lifelong immune health, this period remains largely uncharted. To identify factors that may impact the trajectory of immune development, we conducted stringently standardized, high-throughput phenotyping of peripheral white blood cell (WBC) populations from 796 newborns across two distinct cohorts (The Gambia, West Africa; Papua New Guinea, Melanesia) in the framework of a Human Immunology Project Consortium (HIPC) study. Samples were collected twice from each newborn during the first week of life, first at Day of Life 0 (at birth) and then subsequently at Day of Life 1, 3, or 7 depending on the randomization group the newborn belongs to. The subsequent analysis was conducted at an unprecedented level of detail using flow cytometry and an unbiased automated gating algorithm. The results showed that WBC composition in peripheral blood changes along patterns highly conserved across populations and environments. Changes across days of life were most pronounced in the innate myeloid compartment. Breastfeeding, and at a smaller scale neonatal vaccination, were associated with changes in peripheral blood neutrophil and monocyte cell counts. Our results suggest a common trajectory of immune development in newborns and possible association with timing of breastfeeding initiation, which may contribute to immune-mediated protection from infection in early life. These data begin to outline a specific window of opportunity for interventions that could deliberately direct WBC composition, and with that, immune trajectory and thus ontogeny in early life. This trial is registered with NCT03246230.

Abstract A024: Systemic administration of CD3-redirected lentiviral vector efficiently targets CD19-CAR delivery to human T cells in vivo

Abstract In recent years, there has been a significant increase in interest regarding the generation of CAR-T cells directly within patients using a simple vector injection containing the CAR transgene. This promising technology has the potential to streamline the procedure and decrease its overall cost. To ensure effective in vivo delivery, these vectors must demonstrate resistance to neutralization by human serum, the ability to specifically target and transduce desired cells, and must support their subsequent expansion. Lentiviral vectors (LV) pseudotyped with VSV G-protein (G-WT) have a broad tropism, targeting various cell types through their affinity for the ubiquitously expressed LDL receptor (LDLR). G-WT LV have proven effective in generating therapeutic CAR-T cells ex vivo, however, requiring proper purification and activation of T cells beforehand. To address challenges related to their in vivo delivery, we utilized rational engineering to redesign the G protein, resulting in a modified version termed G-CD3. This redesigned G protein incorporates a CD3-specific single-chain antibody, allowing for targeted recognition of T cells while avoiding LDLR recognition. Upon testing the newly designed G-CD3 LV, we demonstrated its specificity in targeting and infecting both un-activated and activated T cells in peripheral blood monocytic cell cultures. In addition, the G-CD3 LV powerfully activates T cells, evident from their increased CD25 expression, and the infection is not hindered by the presence of human serum (25%). In further experiments, we successfully demonstrated that G-CD3-LV can infect T cells in fresh whole blood. In vivo evaluation of G-CD3-LV efficiency was performed in humanized NSG mice with more than 40 % of human CD45+ cells by intravenous (IV) or intraperitoneal (IP) LV delivery using LV encoding anti-CD19 CAR and GFP transgenes. Both IV and IP delivery of G-CD3-LV led to in vivo CAR T cell generation. However, CAR-T blood levels were higher after IP administration, reaching 13-17% of total circulating human CD45+ cells at the peak on day 21. Anti CD19-CAR-T cell generation coincided with the disappearance of endogenous CD19+ B cells, demonstrating functional in vivo CAR-T generation. Tissue analysis at day 39 after LV injection showed the presence of anti-CD19 CAR-T cells accompanied by the absence or dramatic reduction in CD19+ B cells in spleen, bone marrow and liver. In contrast, no CAR-T cell generation was detected at any time point in the mice injected with G-WT-LV, regardless of whether the virus was administered IV or IP. Taken together these data highlight the potential applicability of newly designed retargeted LV for in vivo gene delivery, offering promising prospects for future human gene therapy applications. Citation Format: Karina Krotova, Gopal Naik Nenavath, Nandakumar Packiriswamy, Rianna Vandergaast, Christopher Ziegler, Melissa Moy, Riya Narjari, Luke Schnebeck, Zachary Larson, Kyle Gromer, Chia-Hsuan Chin, Samantha Reiter, Miguel Muñoz Alía, Kah-Whye Peng, Stephen Russell. Systemic administration of CD3-redirected lentiviral vector efficiently targets CD19-CAR delivery to human T cells in vivo [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A024.

Anticancer effects of herbal medicine Emelia-M, Mshikazi and Delosma H against human leukaemia cells.

Leukaemia is one of the three major types of blood cancers that lead to the overproduction of abnormal white blood cells. Emelia M (EMB), Mshikazi and Delosma H are herbal medicines that are being used by traditional healers in KwaZulu-Natal, South Africa to treat leukaemia and other diseases. To gain insight into the safety (non-toxic effect), anti-cancer activity, mechanisms of action and phytochemical profiles of traditional herbal medicines (Emelia M (EMB), Mshikazi and Delosma H) in South Africa. The viability of human peripheral blood mononuclear cells (PBMCs), monocytic (THP-1) and T-lymphocyte (Jurkat) cell lines exposed to varying concentrations of aqueous extracts of the three herbal medicines were assessed using adenosine triphosphate (ATP) assay. All three extracts showed a dose-dependent effect on the viability of PBMCs. Cell viability decreased with increasing concentrations of extracts when compared with the untreated cells at 24 and 48 hours. The inhibitory activities (IC50) of the extract were found in the order of Mshikazi > EMB, > Delosma H. All the extracts induced apoptosis with minimal necrosis. Many bioactive compounds that have been previously reported to have anticancer effects were identified in the extracts. Mshikazi extract significantly inhibited the growth of THP-1 and Jurkat cells and induced cell death through apoptosis than the other two extracts.

Isolation and Identification of Caprine Arthritis Encephalitis Virus from Animals in the Republic of Mordovia.

This article presents the results of virological and genetic studies of an isolate of caprine arthritis encephalitis (CAE) virus from the republic of Mordovia, Russian Federation. The isolate was found during monitoring studies of goat blood samples for the viral genome, and the presence of antibodies to lentiviruses was detected. According to the recommendation of the OIE, the positive result of PCR was confirmed with nucleotide sequencing. It was found that the obtained nucleotide sequence is identical to the genome of small ruminant lentiviruses presented in the GenBank database. Phylogenetic analysis showed that the isolate "Mordovia-2018" was included in the same cluster with an isolate from the Tver region of the Russian Federation detected in 2008. The sequence of the fragment of the env-gene of the isolate from the republic of Mordovia is available in GenBank under the number MN186380.1. To isolate the virus, a fraction of peripheral blood monocyte cells from the animal's blood was added to a monolayer of lamb synovial membrane cell culture, and ten passages were carried out. The first manifestations of the cytopathic effect were observed after the third passage on the eighth day of cultivation in the form of single large cells of irregular shape with 5-7 nuclei. At the seventh passage, multiple syncytium with 7-12 nuclei were observed. At subsequent passage levels, the formation of syncytium containing more than 10-14 nuclei was observed.

Disturbance of sleep maintenance, but not sleep duration, activates nuclear factor-κB and signal transducer and activator of transcription family proteins in older adults: sex differences.

Disturbances of sleep maintenance and sleep duration are common in older adults and associated with an increased risk for age-related mortality and morbidity. Converging evidence implicates inflammation as an underlying mechanism, especially in females. However, it is unknown what specific aspects of sleep disturbance impact inflammatory mechanisms in older adults. Using data from community-dwelling older adults who participated in the Sleep Health and Aging Research (SHARE) field study (n = 262, mean age 71.9 ± 8.0 years), we conducted a secondary analysis to examine whether disturbance of sleep maintenance (i.e. greater amount of wake time after sleep onset [WASO]) and sleep duration (i.e. shorter total sleep time [TST]) assessed by sleep diary and actigraphy are associated with greater activation of nuclear factor (NF)-κB and signal transducer and activator of transcription (STAT) family proteins STAT1, STAT3, and STAT5 in peripheral blood monocytic cells. In addition, moderation effects of sex were explored. Data were available for sleep diary (n = 82), actigraphy (n = 74), and inflammatory signaling and transcriptional measures (n = 132). As assessed by sleep diary, greater amount of WASO (β = 0.39, p < 0.01), but not TST, was associated with higher levels of NF-κB. Whereas diary-assessed sleep measures were not associated with STAT family proteins, a moderation analysis revealed that greater diary-assessed WASO was associated with higher levels of STAT1 (p < 0.05), STAT3 (p < 0.05), and STAT5 (p < 0.01) in females, but not in males. Actigraphy-assessed sleep measures were not associated either with NF-κB or STAT activation. In older adults, self-reported disturbance of sleep maintenance assessed by sleep diary was uniquely associated with higher levels of NF-κB, along with higher levels of STAT family proteins in females, but not in males. Our data suggest that improvingself-reported sleep maintenance might mitigate age-related increases in inflammatory signaling and transcriptional pathways, possibly more strongly in females, with the potential to reduce mortality risk in older adults.

SPI1-mediated autophagy of peripheral blood monocyte cells as a mechanism for sepsis based on single-cell RNA sequencing.

Autophagy has been documented to participate in immune responses and inflammatory diseases, but the mechanistic actions of monocyte autophagy in sepsis remain largely unknown. This study intends to analyze the mechanism of autophagy of peripheral blood monocyte cells (PBMCs) in sepsis based on single-cell RNA sequencing (scRNA-seq). The scRNA-seq data of PBMC samples from sepsis patients were downloaded from the GEO database, followed by identification of cell marker genes, key pathways and key genes. The bioinformatics analysis showed that the PBMC samples of sepsis patients mainly contained 9 immune cell types, among which three types of monocytes showed significant changes in cell numbers in sepsis patients. Of note, the highest autophagy score was found in the intermediate monocytes. The Annexin signaling pathway was a key pathway for the communication between monocytes and other cells. More importantly, SPI1 was predicted as a key gene in the autophagy phenotype of intermediate monocytes, and SPI1 might suppress ANXA1 transcription. The high expression of SPI1 in sepsis was confirmed by RT-qPCR and Western blot analysis. Dual luciferase reporter gene assay verified that SPI1 could bind to the promoter region of ANXA1. Furthermore, it was found that SPI1 might affect monocyte autophagy in the mouse model of sepsis through regulation of ANXA1. In conclusion, we provide insight into the mechanism underlying the septic potential of SPI1, which enhances monocyte autophagy by inhibiting the transcription of ANXA1 in sepsis.

Comprehensive analysis to identify the influences of SARS-CoV-2 infections to inflammatory bowel disease.

Coronavirus disease 2019 (COVID-19) and inflammatory bowel disease (IBD) are both caused by a disordered immune response and have direct and profound impacts on health care services. In this study, we implemented transcriptomic and single-cell analysis to detect common molecular and cellular intersections between COVID-19 and IBD that help understand the linkage of COVID-19 to the IBD patients. Four RNA-sequencing datasets (GSE147507, GSE126124, GSE9686 and GSE36807) from Gene Expression Omnibus (GEO) database are extracted to detect mutual differentially expressed genes (DEGs) for IBD patients with the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to find shared pathways, candidate drugs, hub genes and regulatory networks. Two single-cell RNA sequencing (scRNA-eq) datasets (GSE150728, PRJCA003980) are used to analyze the immune characteristics of hub genes and the proportion of immune cell types, so as to find common immune responses between COVID-19 and IBD. A total of 121 common DEGs were identified among four RNA-seq datasets, and were all involved in the functional enrichment analysis related to inflammation and immune response. Transcription factors-DEGs interactions, miRNAs-DEGs coregulatory networks, and protein-drug interactions were identified based on these datasets. Protein-protein interactions (PPIs) was built and 59 hub genes were identified. Moreover, scRNA-seq of peripheral blood monocyte cells (PBMCs) from COVID-19 patients revealed a significant increase in the proportion of CD14+ monocytes, in which 38 of 59 hub genes were highly enriched. These genes, encoding inflammatory cytokines, were also highly expressed in inflammatory macrophages (IMacrophage) of intestinal tissues of IBD patients. We conclude that COVID-19 may promote the progression of IBD through cytokine storms. The candidate drugs and DEGs-regulated networks may suggest effective therapeutic methods for both COVID-19 and IBD.

Granulocytic myeloid-derived suppressor cells increase infection risk via the IDO/IL-10 pathway in patients with hepatitis B virus-related liver failure.

Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) results in high susceptibility to infection. Although granulocytic myeloid-derived suppressor cells (gMDSC) are elevated in patients with HBV-ACLF, their role in HBV-ACLF pathogenesis is unknown. To elucidate the mechanism of gMDSC expansion and susceptibility to infection in HBV-ACLF patients, we analyzed the proportion of gMDSC in the peripheral blood and organ tissues of patients with HBV-ACLF and an ACLF mouse model established by continuous injection (eight times) of Concanavalin by flow cytometry and immunohistochemistry. We found that the proportion of gMDSC increased significantly in the blood and liver of patients with HBV-ACLF. This increase was positively correlated with disease severity, prognosis, and infection. gMDSC percentages were higher in peripheral blood, liver, spleen, and bone marrow than control levels in the ACLF mouse model. Immunofluorescence revealed that the gMDSC count increased in the liver of patients with HBV-ACLF as well as in the liver and spleen of ACLF mice. We further exposed peripheral blood monocyte cells from healthy donors to plasma from HBV-ACLF patients, recombinant cytokines, or their inhibitor, and found that TNF-α led to gMDSC expansion and significant upregulation of indoleamine 2, 3-dioxygenase (IDO), while blocking TNF-α signaling decreased gMDSC. Moreover, we detected proliferation and cytokine secretion of T lymphocytes when purified gMDSC was co-cultured with Pan T cells or IDO inhibitor and found that TNF-α-induced gMDSC inhibited T cell proliferation and interferon-γ production through the IDO signaling pathway. Lastly, the ability of gMDSC to phagocytose bacteria was low in patients with HBV-ACLF. Our findings elucidate HBV-ACLF pathogenesis and provide potential therapeutic targets.

A Study on Chemical Characterization and Biological Abilities of Alstonia boonei Extracts Obtained by Different Techniques.

In the quest for novel therapeutic agents from plants, the choice of extraction solvent and technique plays a key role. In this study, the possible differences in the phytochemical profile and bioactivity (antioxidant and enzyme inhibitory activity) of the Alstonia boonei leaves and stem bark extracted using water, ethyl acetate and methanol, and different techniques, namely infusion, maceration and Soxhlet extraction, were investigated. Data collected showed that methanol extracts of both A. boonei leaves (48.34-53.08 mg gallic acid equivalent [GAE]/g dry extract) and stem bark (37.08-45.72 mg GAE/g dry extract) possessed higher phenolic content compared to the ethyl acetate extracts (leaves: 30.64-40.19 mg GAE/g; stem bark: 34.25-35.64 mg GAE/g). The methanol extracts of A. boonei leaves showed higher radical scavenging and reducing capacity, and these findings were in accordance with phenolic content results. In general, water extracts of A. boonei leaves and stem bark obtained by infusion were poor inhibitors of acetylcholinesterase, α-amylase, α-glucosidase, and tyrosinase, except for butyrylcholinesterase. The chemical profiles of the extracts were determined by UHPLC-MS and the presence of several compounds, such as phenolic acids (caffeic, chlorogenic and ferulic acids, etc.), flavonoids (rutin and isoquercetin) and flavonolignans (Cinchonain isomers). Cell viability was tested using the human peripheral blood monocytic cell line (THP-1), and the extracts were safe up to 25 μg/mL. In addition, anti-inflammatory effects were investigated with the releasing of IL-6 TNF-α and IL-1β. In particular, stem bark extracts exhibited significant anti-inflammatory effects. Data presented in this study highlight the key role of solvent choice in the extraction of bioactive secondary metabolites from plants. In addition, this study appraises the antioxidant and enzyme inhibitory action of A. boonei leaves and stem bark, which are extensively used in traditional medicine.

Potential of Lactobacillus acidophilus to modulate cytokine production by peripheral blood monocytes in patients with endometriosis.

Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. Peripheral blood monocytes cells (PBMCs) may have altered function to some extent in women with endometriosis. Lactobacillus acidophilus is a probiotic bacterium within the human body with the ability of alleviating many inflammatory diseases. Here, we examined the effect of L. acidophilus on PBMCs of endometriosis patients. In this study, peripheral blood samples were obtained from endometriosis patients (n=11) and non-endometriosis individuals (n=11). After isolation of peripheral blood mononuclear cells with Ficoll, cells were cultured in the presence and absence of phytohemagglutinin. Also, these cells were co-cultured with 1×106 CFU/ml of L. acidophilus. IL-6 and IL-1 cytokines were measured by ELISA method and the two groups were evaluated and compared. The results showed that in endometriosis patients, the production of pro-inflammatory cytokines, including IL-1 and IL-6, by PBMC was increased compared to non-endometriosis subjects, and stimuli such as PHA intensified this elevation. Also, L. acidophilus increased the levels of pro-inflammatory cytokines including IL-1 and IL-6. However, the production of these cytokines decreased due to the modulatory properties of bacterial cells after 48 h. According to the results of the current study, IL-1 and IL-6 production was significantly increased in PMBCs of endometriosis patients compared to that of the healthy controls. Also, Lactobacillus acidophilus was considered as an antigenic compound and induced IL-1 and IL-6 production. According to these results, probiotics can be further used for the treatment of endometriosis patients and more investigations are needed to confirm these results.

Early monocyte response following local ablation in hepatocellular carcinoma.

Local ablative therapies are established treatment modalities in the treatment of early- and intermediate-stage hepatocellular carcinoma (HCC). Systemic effects of local ablation on circulating immune cells may contribute to patients' response. Depending on their activation, myeloid cells are able to trigger HCC progression as well as to support anti-tumor immunity. Certain priming of monocytes may already occur while still in the circulation. By using flow cytometry, we analyzed peripheral blood monocyte cell populations from a prospective clinical trial cohort of 21 HCC patients following interstitial brachytherapy (IBT) or radiofrequency ablation (RFA) and investigated alterations in the composition of monocyte subpopulations and monocytic myeloid-derived suppressor cells (mMDSCs) as well as receptors involved in orchestrating monocyte function. We discovered that mMDSC levels increased following both IBT and RFA in virtually all patients. Furthermore, we identified varying alterations in the level of monocyte subpopulations following radiation compared to RFA. (A) Liquid biopsy liquid biopsy of circulating monocytes in the future may provide information on the inflammatory response towards local ablation as part of an orchestrated immune response.

Epithelial sodium channels in macrophage migration and polarization: role of proinflammatory cytokines TNFα and IFNγ.

Migration of monocytes-macrophages plays an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. Although epithelial Na+ channels (ENaCs) are required for normal migratory responses in other cell types, their role in macrophage migration signaling is unknown. To address this possibility, we determined whether ENaC message is present in several peripheral blood monocyte cell populations and tissue-resident macrophages in healthy humans using the Human Protein Atlas database (www.proteinatlas.org) and the mouse monocyte cell line RAW 264.7 using RT-PCR. We then determined that selective ENaC inhibition with amiloride inhibited chemotactic migration (∼50%), but not phagocytosis, of the mouse monocyte-macrophage cell line RAW 264.7. Furthermore, we generated a cell line stably expressing an NH2-terminal truncated αENaC to interrupt normal channel trafficking and found it suppressed migration. Prolonged exposure (48 h) of RAW 264.7 cells to proinflammatory cytokines interferon γ (IFNγ) and/or tumor necrosis factor α (TNFα) inhibited RAW 264.7 migration and abolished the amiloride (1 µM)-sensitive component of migration, a finding consistent with ENaC downregulation. To determine if proinflammatory cytokines regulate αENaC protein expression, cells were exposed to proinflammatory cytokines IFNγ (10 ng/mL, last 48 h) and TNFα (10 ng/mL, last 24 h). By Western blot analysis, we found whole cell αENaC protein is reduced ≥50%. Immunofluorescence demonstrated heterogeneous αENaC inhibition. Finally, we found that overnight exposure to amiloride stimulated morphological changes and increased polarization marker expression. Our findings suggest that ENaC may be a critical molecule in macrophage migration and polarization.

Antibody-dependent cell-mediated cytotoxicity using T cells with NK-like phenotype in combination with avelumab, an anti-PD-L1 antibody.

Though the PD-L1 checkpoint inhibitor avelumab has shown efficacy in the treatment of some types of cancer, improved treatment strategies are desperately needed. We evaluated whether combined treatment with avelumab and adoptively transferred T-NK cells can provide enhanced anti-cancer effects for treating PD-L1-expressing tumours. Our results demonstrate that avelumab specifically targets tumour cells with high PD-L1 expression, and that cytolytic effects are mediated by T-NK effector cells cultured from patient peripheral blood monocytic cell populations. The effects were dependent on CD16 and the perforin/granzyme pathway, supporting a role for the T-NK subpopulation. In vivo assays verified the efficacy of T-NK cells in combination with avelumab in reducing tumour growth. Furthermore, T-NK + avelumab prolonged survival in a mouse orthotopic xenograft model. Collectively, our findings provide a basis for the combined use of adoptively transferred T-NK cells with avelumab as a novel strategy for cancer treatment.

Comparative study of CNR1 and CNR2 cannabinoid receptors expression levels in COVID-19 patients with and without diabetes mellitus: Recommendations for future research targets

Background and aimsThe COVID-19 pandemic has prompted researchers to look for effective therapeutic targets. The effect of endocannabinoid system against infectious diseases is investigated for several years. In this study, we evaluated the expression level of CNR1 and CNR2 genes in patients with COVID-19 with and without diabetes to provide new insights regarding these receptors and their potential effect in COVID-19 disease. MethodsIn this study, peripheral blood monocytes cells (PBMCs) were isolated from eight different groups including COVID-19 patients, diabetic patients, and healthy individuals. RNA were extracted to evaluate the expression level of CNR1 and CNR2 genes using real-time PCR. The correlation between the expression levels of these genes in different groups were assessed. ResultsA total of 80 samples were divided into 8 groups, with each group consisting of ten samples. When comparing severe and moderate COVID-19 groups to healthy control group, the expression levels of the CNR1 and CNR2 genes were significantly higher in the severe and moderate COVID-19 groups. There were no significant differences between the mild COVID-19 group and the healthy control group. It was found that the expression levels of these genes in patients with diabetes who were infected with SARS-COV-2 did not differ across COVID-19 groups with varying severity, but they were significantly higher when compared to healthy controls. ConclusionOur study suggests the possible role of endocannabinoid system during SARS-COV-2 pathogenicity as the expression of CNR1 and CNR2 were elevated during the disease.

Multi-pin contact drawing enables production of anisotropic collagen fiber substrates for alignment of fibroblasts and monocytes

Type I collagen is the most abundant protein in the human body and is known to play important roles in numerous biological processes including tissue morphogenesis and wound healing. As such, it is one of the most frequently used substrates for cell culture, and there have been considerable efforts to develop collagen-based cell culture substrates that mimic the structural organization of collagen as it is found in native tissues, i.e., collagen fibers. However, producing collagen fibers from extracted collagen has been notoriously difficult, with existing methods providing only low throughput production of collagen fibers. In this study, we prepared collagen fibers using a highly efficient, bio-friendly, and cost-effective approach termed contact drawing, which uses an entangled polymer fluid to aid in fiber formation. Contact drawing technology has been demonstrated previously for collagen using highly concentrated dextran solutions with low concentrations of collagen. Here, we show that by replacing dextran with polyethylene oxide (PEO), high collagen content fibers may be readily formed from mixtures of soluble collagen and PEO, a polymer that readily forms fibers by contact drawing at concentrations as low as 0.5%wt. The presence of collagen and the formation of well-ordered collagen structures in the resulting fibers were characterized by attenuated total reflectance Fourier-transform infrared spectromicroscopy, Raman spectromicroscopy, and fluorescence microscopy. Corresponding to well-ordered collagen, the mechanical properties of the PEO-collagen fibers approximated those observed for native collagen fibers. Growth of cells on aligned PEO-collagen fibers attached to a polydimethyl siloxane support was examined for human dermal fibroblast (WS1) and human peripheral leukemia blood monocyte (THP-1) cell lines. WS1 and THP-1 cells readily attached, displayed alignment through migration and spreading, and proliferated on the collagen fiber substrate over the course of several days. We also demonstrated the retrieval of viable cells from the PEO-collagen fiber substrates through enzymatic digestion of the collagen substrate with collagenase IV.

PdpC, a Type Six Secretion System Substrate, is Required for Erythrocyte Invasion in Francisella tularensis LVS

Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for phagolysosomal escape and invasion of erythrocytes. An effector of the T6SS, PdpC, is required for phagosomal escape and we wanted to test if PdpC was also required for erythrocyte invasion. We constructed a pdpC-null mutant in the live vaccine strain, F. tularensis LVS. The pdpC-null strain is required for invasion of both human and sheep erythrocytes and reintroduction of a copy of pdpC, in trans, rescues this phenotype. Differential Immuno-Fluorescence Microscopy (DIFM) showed that the pdpC-null strain is affected in attachment as well as invasion. Further, a fluorescently labelled pdpC-null strain of F. tularensis LVS was unable to proliferate in THP-1 human peripheral blood monocyte cells. Finally, we constructed a fluorescent fusion of pdpC to emgfp and the resulting PdpC-EmGFP fusion protein localizes as discrete foci in a subset of broth cultured F. tularensis LVS cells. Our results confirm previous observations that PdpC is required for infection and virulence in phagocytic host cells and are the first description of an effector of the Type Six Secretion System that is required for erythrocyte invasion. The pdpC-emgfp strain will be a useful tool to further characterize the role of PdpC in both macrophage infection and red blood cell invasion. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

Immunomodulatory activity of ethanol extract of Annona reticulata L. leaf in cultured immune cells and in Swiss albino mice

BackgroundAnnona reticulata Linn, has been shown to possess antipyretic, antihelmintic, hypoglycemic, antiulcer and wound healing properties. However, its immunomodulatory role is yet to be explored. Objective(s)In the present study, we intended to investigate the effects of A. reticulata leaf ethanol extract on various components of the immune system. Material and methodsThe effects of A. reticulata leaf extract on human peripheral blood mononuclear cells, monocyte (THP1), and human macrophage (U937) cell lines were investigated. An animal study was conducted to observe the effect of the extract on humoral as well as cell mediated immunity. ResultsThe extract stimulated proliferation of human PBMC, monocytes (THP1), and macrophages (U937) significantly in a dose dependent manner; expression of transforming growth factor-beta (TGF-β) increased in western blot analysis. Additionally, the extract treated macrophages exhibited features of activation under the microscope with a significant hike in the NO production. Flow cytometry of extract treated human PBMC revealed increased proliferation of lymphocytes (CD4, CD8 & B-cells) along with enhanced intracellular expression of IL-2, IL-6. Animal study data indicate a significant rise in the antibody titer as well as a strong delayed type hypersensitivity response in the extract (150 mg/kg and 300 mg/kg) treated mice; furthermore, the expression of IL-2 and IL-6 in mice PBMC was augmented. ConclusionThe collective data evince the immunomodulatory potential of A. reticulata L. leaf.