Leukocyte Proliferation Research Articles

Anticancer Effectivity of Nanocrystals Derived from Mangosteen (Garcinia mangostana) Peel Extract on Leukemia HL-60 Cells

Leukemia, characterized by abnormal leukocyte proliferation, ranks ninth in Indonesia as the most common cancer. While treatments such as chemotherapy and radiation effectively target cancer cells, they also risk damaging healthy blood cells. This has spurred interest in exploring low-toxicity herbal compounds as potential therapies, with mangosteen peel emerging as a widely researched option. Nanotechnology, which has the potential to enhance the bioavailability of herbal compounds, is also a focus of extensive research. This study objective was to assess the impact of Mangosteen Peel Nanocrystal (MPN) on HL-60 leukemia cells by analyzing various parameters, including cytotoxicity, reactive oxygen species (ROS) levels, senescence, and gene expression changes. MPN was prepared with high-speed milling and characterized using particle size analyzers, microscopy, and stability assessments. HL-60 cells were cultured and subjected to MPN treatment. Cytotoxicity was evaluated using WST-8 assays, ROS levels were assessed using flow cytometry, and senescence analyses using Senescence-Associated b-Galactosidase Staining. AKT and FLT-1 gene expression were determined via qRT-PCR. MPN has been successfully characterized as a nanoparticle based on size, stability, and morphology. MPN has an impact on leukemia cells by increasing cytotoxicity, decreasing ROS levels, inducing senescence, and modulating AKT and FLT-1 gene expressions. The findings suggest potential implications for MPN in targeting leukemia cells. The study sheds light on the promising effects of MPN in leukemia cell models, indicating its potential applications in targeting cancer cells, inducing senescence, decreasing ROS levels, and modulating gene expressions related to cell survival and proliferation.

Identifying Genes Associated With Proliferation, Immunity and Thrombosis in Paroxysmal Nocturnal Haemoglobinuria.

PIGA mutation cannot fully explain the proliferative advantage of abnormal clones and thrombosis tendency in paroxysmal nocturnal haemoglobinuria (PNH), and additional genes may play a role, justifying further investigation. CD59+ and CD59- peripheral blood mononuclear cells from six PNH patients were sorted and subjected to whole-exon sequencing (WES) and whole-transcriptome sequencing respectively. Six age- and sex-matched healthy volunteers were enrolled as controls. Genes related to proliferation, immunity and thrombosis were selected by gene ontology (GO) analysis. The selected gene mutant alleles were then identified in the WES results for 40 patients and verified by the Sanger method in another 40 PNH patients. CD59+ and CD59- peripheral blood mononuclear cells from seven patients were sorted, and the RNA and protein expression levels of target genes were assessed via quantitative real-time PCR (RT-qPCR), flow cytometry and western blotting. The final selected genes were then related to clinical features to analyse. T-cell activation-related genes were upregulated, whereas platelet degranulation, coagulation, haemostasis, leukocyte proliferation and platelet activation-related genes were downregulated in both CD59+ and CD59- cells. The mRNA or protein expression levels of SELP, FLT1, NRP1 and vWF were either different from those in healthy controls or different between CD59+ and CD59- cells. Moreover, platelet aggregation was greater in patients with mutations in these genes than in patients without such mutations. Except for PIGA, other genes may be involved in the proliferation and coagulopathy that occur in PNH patients.

Study of Retinal and Peripapillary Vascular Changes in Newly Diagnosed Acute Leukemic Patients Using Optical Coherence Tomography Angiography

Abstract Background Leukemia is a neoplastic blood disorder characterized by an abnormal proliferation of leukocytes. Knowledge of ocular involvement in leukemia is important because the eye is the only site where the leukemic involvement of nerves and blood vessels can be directly observed, because the eye symptoms may be the initial mode of presentation of the systemic illness. Almost all ocular structures have been found to be involved in patients with various types of leukemia. Objective To detect retinal and peripapillary vascular changes in newly diagnosed acute leukemic patients prior to chemotherapy treatment using optical coherence tomography angiography. Patients and Methods This comparative cross sectional study was carried out on 52 eyes of 26 patients and 52 eyes of 26 healthy subjects. Optical coherence tomography angiography was performed for all subjects. Central macular thickness. Retinal nerve fiber layer thickness, ganglion cell complex thickness, superficial and deep vessel density, foveal avascular zone area and radial peripapillary capillaries density were measured and analyzed. Results Significant decrease in the superficial capillary plexus and the radial peripapillary capillary density in the cases group. The FAZ area is larger in the cases group. The central macular thickness is lower in the cases group. But the RNFL thickness, and C/D parameters were higher in the case group. However, there was no significant difference between the two groups regarding the GCC and the DCP density. Conclusion Acute leukemia causes decrease in superficial capillary plexus density and radial peripapillary capillaries density and decreased macular thickness with no affection in deep capillary vessel density.

Multiplexed immunohistochemical evaluation of small bowel inflammatory and epithelial parameters in environmental enteric dysfunction

BackgroundEnvironmental enteric dysfunction (EED) is characterized by reduced absorptive capacity and barrier function of the small intestine, leading to poor ponderal and linear childhood growth. ObjectivesTo further define gene expression patterns that are associated with EED to uncover new pathophysiology of this disorder. MethodsDuodenal biopsies from cohorts of children with EED from Bangladesh, Pakistan and Zambia were analyzed by immunohistochemistry (IHC) to interrogate gene products that distinguished differentiation and various biochemical pathways in immune and epithelial cells, some identified by prior bulk RNA sequence analyses. Immunohistochemical staining was digitally quantified from scanned images and compared to cohorts of North American children with celiac disease (gluten-sensitive enteropathy) or with no known enteric disease and no pathologic abnormality (NPA) detected in their clinical biopsies. ResultsAfter multivariable statistical analysis, we identified statistically significant (P < 0.05, 2-tailed t-test) elevated signals representing cluster of differentiation 45 (80%; 95% confidence interval [CI]: 24%, 127%), lipocalin 2 (659%; 95% CI: 198%, 1838%), and regenerating family 1 beta (221%; 95% CI: 47%, 600%) and lower signals corresponding to granzyme B (−74%; 95% CI: −82%, −62%), and sucrase isomaltase (−58%; 95% CI: −75%, −29%) in EED biopsies compared with NPA biopsies. Computerized algorithms also detected statistically significant elevation in intraepithelial lymphocytes (49%; 95% CI: 9%, 105%) and proliferation of leukocytes (267%; 95% CI: 92%, 601%) in EED biopsies compared with NPA biopsies. ConclusionsOur results support a model of chronic epithelial stress that decreases epithelial differentiation and absorptive function. The close association of several IHC parameters with manual histologic scoring suggests that automated digital quantification of IHC panels complements traditional histomorphologic assessment in EED.

Immunological properties of silica nanoparticles: a structure–activity relationship study

Silica nanoparticles are increasingly considered for drug delivery applications. These applications require an understanding of their biocompatibility, including their interactions with the immune system. However, systematic studies for silica nanoparticle immunological safety profiles are lacking. To fill this gap, we conducted an in vitro study investigating various aspects of silica nanoparticles’ interactions with blood and immune cells. Four types of silica nanoparticles with variations in size and porosity were studied. These included nonporous Stöber silica nanoparticles with average diameters of approximately 50 and 100 nm (SNP50 and SNP100), mesoporous silica nanoparticles of approximately 100 nm (Meso100), and hollow mesoporous silica nanoparticles of approximately 100 nm (HMSNP100) in diameter, respectively. The hematological compatibility was assessed using hemolysis, complement activation, platelet aggregation, and plasma coagulation assays. The effects of nanoparticles on immune cell function were studied using in vitro phagocytosis, chemotaxis, natural killer cell cytotoxicity, leukocyte proliferation, human lymphocyte activation, colony-forming unit granulocyte-macrophage, and leukocyte procoagulant activity assays. The in vitro findings suggest that at high concentrations, corresponding to the in vivo human dose of 40 mg/kg, silica nanoparticles demonstrated an array of immunotoxic effects that depended on their physicochemical properties. However, all types of silica nanoparticles studied were not immunotoxic at concentrations corresponding to lower doses (≤ 8 mg/kg) comparable to that of nanocarriers in other nanomedicines currently used in the clinic. These findings are promising for using silica nanoparticles for the systemic delivery of bioactive and imaging agents.

Effects of injectable trace minerals on the inflammatory cytokines’ response to vaccination in dairy calves

Trace mineral supplementation concurrent with the bovine respiratory diseases complex (BRDC) vaccine boosts antibody production and leukocyte proliferation. However, the mecha­nism underlying this effect is not well understood. On the other hand, cytokines response post-vaccination is an important driver of both humoral and cellular immunity. Therefore, we hypothesized that trace mineral supplementation may improve BRD vaccine immunogenicity and effectiveness by modulating inflammatory cytokines in dairy calves. Our objective was to characterize the effects of injectable trace minerals (ITM) on the cytokines response to the BRD modified-live virus (MLV) vaccine in post-weaning dairy calves.

16S rRNA seq-identified Corynebacterium promotes pyroptosis to aggravate diabetic foot ulcer

BackgroundDiabetic foot ulcer (DFU) is one of the main chronic complications caused by diabetes, leading to amputation in severe cases. Bacterial infection affects the wound healing in DFU.MethodsDFU patients who met the criteria were selected, and the clinical data were recorded in detail. The pus exudate from the patient’s foot wound and venous blood were collected for biochemical analysis. The distribution of bacterial flora in pus exudates of patients was analyzed by 16S rRNA sequencing, and the correlation between DFU and pathogenic variables, pyroptosis and immunity was analyzed by statistical analysis. Then, the effects of key bacteria on the inflammation, proliferation, apoptosis, and pyroptosis of polymorphonuclear leukocytes were investigated by ELISA, CCK-8, flow cytometry, RT-qPCR and western blot.ResultsClinical data analysis showed that Wagner score was positively correlated with the level of inflammatory factors, and there was high CD3+, CD4+, and low CD8+ levels in DFU patients with high Wagner score. Through alpha, beta diversity analysis and species composition analysis, Corynebacterium accounted for a large proportion in DFU. Logistics regression model and Person correlation analysis demonstrated that mixed bacterial infections could aggravate foot ulcer, and the number of bacteria was closely related to inflammatory factors PCT, PRT, immune cells CD8+, and pyroptosis-related proteins GSDMD and NLRP3. Through in vitro experiments, Corynebacterium inhibited cell proliferation, promoted inflammation (TNF-α, PCT, CRP), apoptosis and pyroptosis (IL-1β, LDH, IL-18, GSDMD, NLRP3, and caspase-3).ConclusionMixed bacterial infections exacerbate DFU progression with a high predominance of Corynebacterium, and Corynebacterium promotes inflammation, apoptosis and pyroptosis to inhibit DFU healing.

An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

Abstract 3867: Identification of novel biomarkers of immune toxicity from CAR T-cell therapy using ultrasensitive NULISA™ proteome technology

Abstract Introduction: The efficacy of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in patients with relapsed/refractory large B-cell lymphoma (LBCL) is limited by acute toxic events, most notably cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Identification of biomarkers of toxicities can allow for selection of high-risk patients and elucidate targetable mechanisms for mitigation. Previous biomarker studies have been constrained by relatively narrow panels of potential mediators and limited time points. Here, we employed NULISA, a novel ultrasensitive assay capable of simultaneously quantifying 204 inflammatory proteins from a single sample, to identify temporal proteome associations with acute toxicities in anti-CD19 CAR T-cell treated patients. Methods: Baseline and post-treatment peripheral blood samples from 80 patients with LBCL who underwent anti-CD19 CAR-T cell therapy were collected within specific time intervals. Plasma samples (n=480) were analyzed with NULISA. CRS and ICANS were graded according to ASTCT consensus criteria. Patients were grouped according to their maximum CRS and ICANS scores. Differential protein abundance across the toxicity groups was assessed using linear mixed effects models fit for each protein, including a time by toxicity group interaction. Proteins showing significant associations were subjected to pathway enrichment and network analysis. Results: Severe CRS and ICANS patient groups showed proteome upregulation beginning on day 1-2 and peaking at day 6-9, followed by extensive downregulation on day 11-16. The strongest upregulated pathway associations of severe CRS and ICANS were inflammatory response, IL-17 signaling, non-genomic action of vitamin D3, regulation of leukocyte proliferation, and cellular extravasation. Downregulated pathways included anti-microbial humoral response and TNFs binding to receptors. In addition to the previously identified inflammatory proteins IL-6, IFNγ, sIL-2Rα, CXCL8, CCL2, our analysis revealed significant association of several novel immune regulators and mediators. These included upregulated Th2 cytokines, IL-17A, IL-22, GZMB, CTLA4, IFNA1;IFNA13, and downregulated S100A12, IL-12B, CCL22, BDNF and TNFSFs, revealing exquisite temporal orchestration of lymphoid and myeloid activities during CRS and ICANS development. Conclusions: This study represents the most comprehensive characterization of immune response to CAR T-cell therapy to date. Using the novel NULISA technology, we identified new proteins and functional pathways associated with CAR T-cell-induced toxicity, implicating them as potential biomarkers. These previously unidentified factors also provide a platform to further investigate the causative immune mechanisms of acute toxicities in CAR-T cell therapy. Citation Format: Riley Kirkpatrick, Joanne Beer, Manishkumar S. Patel, Akansha Jalota, Agrima Mian, Ishara S. Ariyapala, Qinyu Hao, Wei Feng, Xiao-Jun Ma, Yuling Luo, Brian T. Hill, Neetu Gupta. Identification of novel biomarkers of immune toxicity from CAR T-cell therapy using ultrasensitive NULISA™ proteome technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3867.

Pan-cancer analysis of DDIT4 identifying its prognostic value and function in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a hematological malignancy derived from the accumulation of abnormal proliferation of infantile leukocytes in the hematopoietic system. DNA-damage-inducible transcript 4 (DDIT4) acting as a negative regulator of rapamycin inhibitor is involved in various cellular functions. Many studies have suggested that DDIT4 plays a key role in tumorigenesis. However, the role of DDIT4 in AML has been poorly studied.MethodIn this study, we analyzed the expression of DDIT4 in AML patients using The Cancer Genome Atlas and real-time polymerase chain reaction. The Chi-square test was used to assess the correlation between DDIT4 and clinical characters in AML patients. Loss-of-function experiments were implemented to investigate the role of DDIT4 in AML carcinogenesis. The R package was applied to evaluate the correlation between DDIT4 expression and immune cells.ResultsResults showed that the expression of DDIT4 was associated with Age, Cytogenetic risk, Cytogenetics and OS event. Moreover, high expression of DDIT4 led to a terrible prognosis. KEGG analysis showed that differently expressed genes (DEGs) were involved in the PI3-Akt signaling pathway. GSEA enrichment analysis displayed DEGs were correlated with apoptosis. Functional experiments presented that knocking down DDIT4 suppressed cell cycle transition/proliferation and facilitated apoptosis. In addition, DDIT4 is associated with immune infiltration.ConclusionOur research verified that DDIT4 can be used as a prognostic marker and a potential therapeutic target for AML.

Low expression of KLRB1 predicts poor survival outcomes and is associated with immune infiltration in breast cancer.

KLRB1 is downregulated in various cancer types. Nevertheless, the specific involvement of KLRB1 in the context of breast cancer (BRCA) has not been fully elucidated. This research aimed to explore its clinical value in BRCA. A dataset comprising 1,109 BRCA samples and 113 healthy samples was retrieved from The Cancer Genome Atlas (TCGA) database to establish the association between KLRB1 expression and pan-cancer. Subsequently, an analysis was executed to explore the link between KLRB1 and BRCA. T-tests and Chi-squared tests were conducted to assess the expression of KLRB1 and its clinical implications in BRCA. The prognosis-predictive value of KLRB1 in BRCA was assessed using the Kaplan-Meier method and Cox regression analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses screened biological pathways to analyze the association between the immune infiltration level and KLRB1 expression in BRCA. Lastly, the conclusion was validated through quantitative polymerase chain reaction (qPCR), immunohistochemistry (IHC), and Cell Counting Kit-8 (CCK8) assays. KLRB1 exhibited low expression in patients with BRCA. Furthermore, KLRB1 demonstrated strong diagnostic potential, as indicated by an area under curve (AUC) of 0.712. Kaplan-Meier survival and Cox regression analyses indicated that attenuated expression of KLRB1 was independently linked to unfavorable clinical outcomes. GO and KEGG enrichment analyses were performed on the top 10 genes that exhibited positive and negative correlations with KLRB1. Analysis of genes positively correlated with KLRB1 revealed associations with signaling receptor activator activity, lymphocyte proliferation, mononuclear cell proliferation, leukocyte proliferation, receptor-ligand activity, immunoglobulin binding, and hematopoietic cell lineage signaling pathway. KLRB1 expression exhibited significant correlations with all immune cells. Furthermore, qPCR and IHC outcomes demonstrated that KLRB1 was significantly downregulated in BRCA tissues. CCK8 findings showed a decrease in the proliferation of BRCA MCF7 cells upon knockout of KLRB1. This research investigated the mechanism and potential therapeutic target of the KLRB1 gene in BRCA. By analyzing the expression and function of the KLRB1 gene, the study aims to find its significant role in the onset and progression of BRCA. This research endeavors to offer novel strategies and approaches for treating BRCA.

Investigation of sex-based differences in the immunotoxicity of silver nanoparticles

The growing application of silver nanoparticles (AgNPs) in consumer, healthcare, and industrial products has raised concern over potential health implications due to increasing exposure. The evaluation of the immune response to nanomaterials is one of the key criteria to assess their biocompatibility. There are well-recognized sex-based differences in innate and adaptive immune responses. However, there is limited information available using human models. The aim was to investigate the potential sex-based differences in immune functions after exposure to AgNPs using human peripheral blood mononuclear cells (PBMCs) and plasma from healthy donors. These functions include inflammasome activation, cytokine expression, leukocyte proliferation, chemotaxis, plasma coagulation, and complement activation. AgNPs were characterized by dynamic light scattering and transmission electron microscopy. Inflammasome activation by AgNPs was measured after 6- and 24-hours incubations. AgNPs-induced inflammasome activation was significantly higher in the females, especially for the 6-hour exposure. No sex-based differences were observed for Ag ions controls. Younger donors exhibited significantly more inflammasome activation than older donors after 24-hours exposure. IL-10 was significantly suppressed in males and females after exposure. AgNPs suppressed leukocyte proliferation similarly in males and females. No chemoattractant effects, no alterations in plasma coagulation, or activation of the complement were observed after AgNPs exposure. In conclusion, the results highlight that there are distinct sex-based differences in inflammasome activation after exposure to AgNPs in human PBMCs. The results highlight the importance of considering sex-based differences in inflammasome activation induced by exposure to AgNPs in any future biocompatibility assessment for products containing AgNPs.

Uterine Immunoprivileged Cells Restore Cardiac Function of Male Recipients After Myocardial Infarction.

It has been documented that the uterus plays a key cardio-protective role in pre-menopausal women, which is supported by uterine cell therapy, to preserve cardiac functioning post-myocardial infarction, being effective among females. However, whether such therapies would also be beneficial among males is still largely unknown. In this study, we aimed to fill in this gap in knowledge by examining the effects of transplanted uterine cells on infarcted male hearts. We identified, based on major histocompatibility complex class I (MHC-I) expression levels, 3 uterine reparative cell populations: MHC-I(neg), MHC-I(mix), and MHC-I(pos). In vitro, MHC-I(neg) cells showed higher levels of pro-angiogenic, pro-survival, and anti-inflammatory factors, compared to MHC-I(mix) and MHC-I(pos). Furthermore, when cocultured with allogeneic mixed leukocytes, MHC-I(neg) had lower cytotoxicity and leukocyte proliferation. In particular, CD8+ cytotoxic T cells significantly decreased, while CD4+CD25+ Tregs and CD4-CD8- double-negative T cells significantly increased when cocultured with MHC-I(neg), compared to MHC-I(mix) and MHC-I(pos) cocultures. In vivo, MHC-I(neg) as well as MHC-I(mix) were found under both syngeneic and allogeneic transplantation in infarcted male hearts, to significantly improve cardiac function and reduce the scar size, via promoting angiogenesis in the infarcted area. All of these findings thus support the view that males could also benefit from the cardio-protective effects observed among females, via cell therapy approaches involving the transplantation of immuno-privileged uterine reparative cells in infarcted hearts.

Immune-Related Genes Associated with Interstitial Lung Disease in Dermatomyositis.

Interstitial lung disease (ILD) is one of the significant complications of dermatomyositis (DM), but the mechanisms by which it occurs remain incompletely elucidated. This study aimed to explore further the possible genetic mechanisms by which this complication occurs. Gene expression profiles for DM (GSE39454, GSE46239, GSE143323) and ILD (GSE32537, GSE110147, GSE150910) were downloaded from the Gene Expression Omnibus (GEO) database. After identifying common differentially expressed genes (DEGs) to DM and ILD using the "limma" R package and the "VennDiagram" R package, functional annotation, relationship to immune cell infiltration, identification of transcription factors (TFs), we also collected clinical cases of DM-associated ILD (DM-ILD), including 3 cases of rapidly progressive ILD (RP-ILD) and 3 cases of none-RP-ILD, and explored whether there were differences in serum lymphocyte subpopulations. A total of 4 common DEGs (SLAMF7, SPP1, TDO2, and VCAM1) were screened and Gene Ontology (GO) enrichment analysis showed that these genes were mainly enriched in T cell activation, regulation of lymphocyte activation, lymphocyte differentiation, leukocyte proliferation and regulation of T cell activation. In terms of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, the three significantly enriched pathways were the PI3K-Akt signaling pathway, MAPK signaling pathway, and Cytokine-cytokine receptor interaction. In lung and muscle tissues, 21 and 3 TFs may regulate the expression of these genes, respectively. Finally, by analysing the serum lymphocyte subpopulations, we also found a decrease in the absolute number of CD8+ T cells and an increase in the CD4+ /CD8+ T cell ratio in DM combined with RP-ILD. These common pathways and key genes may provide new ideas for further research into DM-ILD.

Rab27a via Its effector JFC1 Localizes to Anaplasma Inclusions and Promotes Anaplasma Proliferation in Leukocytes

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes tick-borne zoonosis called human granulocytic anaplasmosis. Mechanisms by which Anaplasma replicates inside of the membrane-bound compartment called “inclusion” in neutrophils is incompletely understood. A small GTPase Rab27a is found in the secretory granules and multivesicular endosomes. In this study we found Rab27a-containing granules were localized to Anaplasma inclusions in guanine nucleotide-dependent manner, and constitutively active Rab27a enhanced Anaplasma infection and dominant-negative Rab27a inhibited Anaplasma infection. Rab27a effector, JFC1 is known to mediate docking/fusion of Rab27a-bearing granules for exocytosis in leukocytes. shRNA stable knockdown of Rab27a or JFC1 inhibited Anaplasma infection in HL-60 cells. Similar to Rab27a, JFC1 were localized to Anaplasma inclusions by immunostaining, and fluorescent probe transfection in live cells. The JFC1 C2A domain that binds 3'-phosphoinositides, was sufficient and required for JFC1 and Rab27a localization to Anaplasma inclusions which were enriched with phosphatidylinositol 3-phosphate. Nexinhib20, the small molecule inhibitor specific to Rab27a and JFC1 binding, inhibited Anaplasma infection. Taken together, these results imply elevated phosphatidylinositol 3-phosphate in the inclusion membrane recruits JFC1 to mediate Rab27a-bearing granules/vesicles to dock/fuse with Anaplasma inclusions, the lumen of which is topologically equivalent to the exterior of the cell to benefit Anaplasma proliferation.

IL-34 and two types of CSF-1R from snakehead (Channa argus) participate in its immune response to two bacterial challenges, promote leukocyte proliferation, and activate NF-κB and AP-1 signaling

Interleukin-34 (IL-34), a relatively newly identified cytokine, binds to colony stimulating factor 1 receptor (CSF-1R) to activate several signaling pathways. It thus performs essential roles in immune surveillance, the inflammatory response, cell proliferation, and cell migration. In this study, the homologues of IL-34 (designated ‘CaIL-34′) and two types of CSF-1R (designated ‘CaCSF-1R1′ and ‘CaCSF-1R2′) are identified and described in the snakehead, Channa argus. Analysis of the amino acid sequence revealed that CaIL-34 has relatively conservative structural characteristics. CaCSF-1R1 and CaCSF-1R2 both contain four immunoglobulin-like domains and a tyrosine kinase domain. Their transcriptions were detected in all the tissues examined in healthy individuals, with relatively high transcription levels in immune-related tissues. The two major bacterial pathogens, Nocardia seriolae and Aeromonas schubertii, both induced their transcription in the head kidney and spleen tissues of snakeheads in vivo. The three classical pathogen-associated molecular patterns, lipopolysaccharide (LPS), lipoteichoic acid (LTA), and polyinosinic–polycytidylic acid (poly[I:C]), and six recombinant cytokines of the snakehead, recombinant tumor necrosis factor-α1 (TNF-α1), TNF-α2, IL-1β, IL-18, IL-20, and IL-21, all increased their transcription in the head kidney leukocytes (HKLs) in vitro, albeit at different levels. Recombinant CaIL-34 (rCaIL-34) protein was expressed in a prokaryotic system. After stimulation with rCaIL-34, the transcription of the IL-1β, TNF-α1, TNF-α2, and interferon-γ (IFN-γ) genes, and endogenous CaIL-34, CaCSF-1R1, and CaCSF-1R2, was enhanced in HKLs, and HKL proliferation was promoted. A subcellular localization analysis showed that CaIL-34 was exclusively located in the cytoplasm of HEK293T cells. The overexpression of CaIL-34 activated the nuclear translocation of nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) signaling, albeit to different levels. In summary, CaIL-34 and two types of CSF-1R of C. argus participate strongly in its antibacterial responses, leukocyte proliferation, and the activation of NF-κB and AP-1 signaling.

Evaluation of [18F]RoSMA-18-d6 as a CB2 PET Radioligand in Nonhuman Primates.

The cannabinoid type 2 receptor (CB2) has been implicated in a variety of central and peripheral inflammatory diseases, prompting significant interest in the development of CB2-targeted diagnostic and therapeutic agents. A validated positron emission tomography (PET) radioligand for imaging CB2 in the living human brain as well as in peripheral tissues is currently lacking. As part of our research program, we have recently identified the trisubstituted pyridine, [18F]RoSMA-18-d6, which proved to be highly suitable for in vitro and in vivo mapping of CB2 in rodents. The aim of this study was to assess the performance characteristics of [18F]RoSMA-18-d6 in nonhuman primates (NHPs) to pave the way for clinical translation. [18F]RoSMA-18-d6 was synthesized from the respective tosylate precursor according to previously reported procedures. In vitro autoradiograms with NHP spleen tissue sections revealed a high binding of [18F]RoSMA-18-d6 to the CB2-rich NHP spleen, which was significantly blocked by coincubation with the commercially available CB2 ligand, GW405833 (10 μM). In contrast, no specific binding was observed by in vitro autoradiography with NHP brain sections, which was in agreement with the notion of a CB2-deficient healthy mammalian brain. In vitro findings were corroborated by PET imaging experiments in NHPs, where [18F]RoSMA-18-d6 uptake in the spleen was dose-dependently attenuated with 1 and 5 mg/kg GW405833, while no specific brain signal was observed. Remarkably, we observed tracer uptake and retention in the NHP spinal cord, which was reduced by GW405833 blockade, pointing toward a potential utility of [18F]RoSMA-18-d6 in probing CB2-expressing cells in the bone marrow. If these observations are substantiated in NHP models of enhanced leukocyte proliferation in the bone marrow, [18F]RoSMA-18-d6 may serve as a valuable marker for hematopoietic activity in various pathologies. In conclusion, [18F]RoSMA-18-d6 proved to be a suitable PET radioligand for imaging CB2 in NHPs, supporting its translation to humans.

Molecular and functional characterization of two granulocyte colony stimulating factors in goldfish (Carassius auratus L.).

Granulocyte colony-stimulating factor (GCSF) is a member of the hematopoietic growth factor family that acts primarily on neutrophils and neutrophilic precursors to promote cell proliferation and differentiation. Although multiple GCSF genes have been found in teleosts, knowledge of their functions during fish hematopoietic development is still limited. Here, we report for the first time the molecular and functional characterization of two goldfish GCSFs (gfGCSF-a and gfGCSF-b). The open reading frame (ORF) of the gfGCSF-a and gfGCSF-b cDNA transcript consisted respectively of 624bp and 678bp with its ORF encoding 207 and 225 amino acids (aa), with a 17 aa signal peptide for each gene and a conserved domain of the IL-6 superfamily. Treatment of goldfish head kidney leukocytes (HKLs) with LPS increased gfGCSF-a and gfGCSF-b mRNA expression levels, also exposure of HKLs to either heat-killed or live A. hydrophila, induced transcriptional upregulation of gfGCSF-a and gfGCSF-b levels. Recombinant gfGCSF-a and gfGCSF-b protein (rgGCSF-a and rgGCSF-b) induced a dose-dependent production of TNFα and IL-1β from goldfish neutrophils. In vitro experiments showed rgGCSF-a and rgGCSF-b differentially promoted the proliferation and differentiation of leukocytes in goldfish. Furthermore, treatment of HKLs with rgGCSF-a showed significant upregulation of mRNA levels of the hematopoietic transcription factor GATA2, Runx1, MafB, and cMyb, while gfGCSF-b induces not only all four transcriptional factors mentioned above but also CEBPα. Our results indicate that goldfish GCSF-a and GCSF-b are important regulators of neutrophil proliferation and differentiation, which could stimulate different stages and lineages of hematopoiesis.

Inactivation of TRPM7 Kinase Targets AKT Signaling and Cyclooxygenase-2 Expression in Human CML Cells.

Cyclooxygenase-2 (COX-2) is a key regulator of inflammation. High constitutive COX-2 expression enhances survival and proliferation of cancer cells, and adversely impacts antitumor immunity. The expression of COX-2 is modulated by various signaling pathways. Recently, we identified the melastatin-like transient-receptor-potential-7 (TRPM7) channel-kinase as modulator of immune homeostasis. TRPM7 protein is essential for leukocyte proliferation and differentiation, and upregulated in several cancers. It comprises of a cation channel and an atypical α-kinase, linked to inflammatory cell signals and associated with hallmarks of tumor progression. A role in leukemia has not been established, and signaling pathways are yet to be deciphered. We show that inhibiting TRPM7 channel-kinase in chronic myeloid leukemia (CML) cells results in reduced constitutive COX-2 expression. By utilizing a CML-derived cell line, HAP1, harboring CRISPR/Cas9-mediated TRPM7 knockout, or a point mutation inactivating TRPM7 kinase, we could link this to reduced activation of AKT serine/threonine kinase and mothers against decapentaplegic homolog 2 (SMAD2). We identified AKT as a direct in vitro substrate of TRPM7 kinase. Pharmacologic blockade of TRPM7 in wildtype HAP1 cells confirmed the effect on COX-2 via altered AKT signaling. Addition of an AKT activator on TRPM7 kinase-dead cells reconstituted the wildtype phenotype. Inhibition of TRPM7 resulted in reduced phosphorylation of AKT and diminished COX-2 expression in peripheral blood mononuclear cells derived from CML patients, and reduced proliferation in patient-derived CD34+ cells. These results highlight a role of TRPM7 kinase in AKT-driven COX-2 expression and suggest a beneficial potential of TRPM7 blockade in COX-2-related inflammation and malignancy.