Isolation of polymorphonuclear neutrophils and monocytes from a single sample of human peripheral blood.
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ABSTRACT:
SUBMITTER: Cui C
PROVIDER: S-EPMC8473578 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Polymorphonuclear neutrophils (PMNs), the main effectors of the innate immune system, have rarely been considered as an anticancer therapeutic tool. However, recent investigations using animal models and preliminary clinical studies have highlighted the potential antitumor efficacy of PMNs. In the current study, we find that PMNs from some healthy donors naturally have potent cancer-killing activity against 4 different human cancer cell lines. The killing activity appears to be cancer cell-specific since PMNs did not kill primary normal epithelial cells or an immortalized breast epithelial cell line. Transfecting the immortalized mammary cells with plasmids expressing activated forms of the rat sarcoma viral oncogene homolog (Ras) and teratocarcinoma oncogene 21 (TC21) oncogenes was sufficient to provoke aggressive attack by PMNs. However, transfection with activated Ras-related C3 botulinum toxin substrate (Rac1) was ineffective, suggesting specificity in PMN-targeting of neoplastic cells. Furthermore, PMNs from lung cancer patients were also found to exhibit relatively poor cancer-killing activity compared to the cytolytic activity of the average healthy donor. Taken together, our results suggest that PMN-based treatment regimens may represent a paradigm shift in cancer immunotherapy that may be easily introduced into the clinic to benefit a subset of patients with PMN-vulnerable tumors.
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Project description:The interaction between radioiodinated lipopolysaccharide from Escherichia coli 0111:B4 (125I-LPS) and human peripheral-blood monocytes was studied. The association of 125I-LPS with monocytes at 37 degrees C appeared to depend on binding to the cell membrane with subsequent internalization of the molecule, and was not saturable with time (up to 2 h) or 125I-LPS concentration (up to 10 micrograms/ml). There was no apparent difference in the behaviour of unlabelled LPS and 125I-LPS with respect to monocyte association. 125I-LPS association with monocytes was inhibited by LPS and O-polysaccharide from E. coli 0111:B4 and Salmonella typhi 0901, but not by lipid A or polymyxin B. We propose that the mechanism of human monocyte stimulation by LPS involves polysaccharide-dependent binding to the cell membrane followed by internalization of the LPS molecule. We were unable to demonstrate a specific LPS receptor such as that found on murine B-lymphocytes.
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Project description:BACKGROUND: CD14 is an important player in host innate immunity in that it confers lipopolysaccharide sensitivity to cell types like neutrophils, monocytes and macrophages. The study was aimed at characterizing the CD14 gene of cattle for sequence variations and to determine the effect of variations on the expression of the protein on the surfaces of monocytes and neutrophils in healthy dairy cows. RESULTS: Five SNPs were identified: two within the coding regions (g.A1908G and g.A2318G, numbering is according to GenBank No. EU148609), one in the 5' (g.C1291T) and two in the 3' (g.A2601G and g.G2621T) untranslated regions. SNP 1908 changes amino acid 175 of the protein (p.Asn175Asp, numbering is according to GenBank No. ABV68569), while SNP 2318 involves a synonymous codon change. Coding region SNPs characterized three gene alleles A (GenBank No. EU148609), A1 (GenBank No. EU148610) and B (GenBank No. EU148611) and two deduced protein variants A (ABV68569 and ABV68570) and B (ABV68571). Protein variant A is more common in the breeds analyzed. All SNPs gave rise to 3 haplotypes for the breeds. SNP genotype 1908AG was significantly (P<0.01) associated with a higher percentage of neutrophils expressing more CD14 molecules on their surfaces. The promoter region contains several transcription factor binding sites, including multiple AP-1 and SP1 sites and there is a high conservation of amino acid residues between the proteins of closely related species. CONCLUSION: The study has provided information on sequence variations within the CD14 gene and proteins of cattle. The SNP responsible for an amino acid exchange may play an important role in the expression of CD14 on the surfaces of neutrophils. Further observations involving a larger sample size are required to validate our findings. Our SNP and association analyses have provided baseline information that may be used at defining the role of CD14 in mediating bacterial infections. The computational analysis on the promoter and comparative analysis with other species has revealed regions of regulatory element motifs that may indicate important regulatory effects on the gene.
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Project description:The cytotoxic effect of Shiga-like toxin (Stx; produced by certain Escherichia coli strains) plays a central role in typical hemolytic uremic syndrome (HUS). It damages the renal endothelium by inhibiting the cellular protein synthesis. Also, the monocyte has a specific receptor for Stx but is not sensitive for the cytotoxic effect. In this work, monocytes were studied as a potential transporter for Stx to the renal endothelium. Coincubation of isolated human monocytes loaded with Stx and target cells (vero cells and human umbilical vascular endothelial cells) were performed. Transfer was determined by measuring the protein synthesis of target cells and by flow cytometry. Furthermore, the effect of a temperature shift on loaded monocytes was investigated. Stx-loaded monocytes reduced the protein synthesis of target cells. After adding an antibody against Stx, incomplete recovery occurred. Also, adding only the supernatant of coincubation was followed by protein synthesis inhibition. Stx detached from its receptor on the monocyte after a change in temperature, and no release was detected without this temperature shift. Although the monocyte plays an important role in the pathogenesis of HUS, it has no role in the transfer of Stx.
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Project description:Cerium dioxide nanoparticles (CeO(2) NPs) have diversified industrial uses, and novel therapeutic applications are actively being pursued. There is a lack of mechanistic data concerning the effects of CeO(2) NPs on primary human cells. We aimed at characterizing the cytotoxic effects of CeO(2) NPs in human peripheral blood monocytes. CeO(2) NPs and their suspensions were thoroughly characterized, including using transmission electron microscopy (TEM), dynamic light scattering, and zeta potential analysis. Blood from healthy human volunteers was drawn through phlebotomy, and CD14+ cells were isolated. Cells were exposed to CeO(2) NPs (0.5-10 ?g/mL) for 20 or 40 h, and mechanisms of cell injury were studied. TEM revealed that CeO(2) NPs are internalized by monocytes and are found either in vesicles or free in the cytoplasm. CeO(2) NP exposure leads to decrease in cell viability, and treated cells exhibit characteristic hallmarks of apoptosis (activation of Bax, loss of mitochondrial membrane potential, DNA fragmentation). CeO(2) NP toxicity is caused by mitochondrial damage and overexpression of apoptosis inducing factor, but is not due to caspase activation or reactive oxygen species production. Moreover, CeO(2) NP exposure leads to autophagy, which is further increased after pharmacological inhibition of tumor suppressor protein p53. Inhibition of autophagy partially reverses cell death by CeO(2) NPs. It is concluded that CeO(2) NPs are toxic to primary human monocytes at relatively low doses.
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Project description:Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity.
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Project description:Little is known about proteomic differences between pluripotent human peripheral blood monocytes (MN) and their terminally-differentiated pulmonary counterparts, alveolar macrophages (AM). To better characterize these cell populations, we performed a label-free shotgun proteomics assessment of matched AM and MN preparations from eight healthy volunteers. With an FDR of less than 0.45%, we identified 1754 proteins within AM and 1445 from MN. Comparison of the two proteomes revealed that 1239 of the proteins found in AM were shared with MN, whereas 206 proteins were uniquely identified in MN and 515 were unique to AM. Molecular and cellular functions, protein classes, development associations, and membership in physiological systems and canonical pathways were identified among the detected proteins. Analysis of biologic processes represented by these proteomes indicated that MN were most prominently enriched for proteins involved in cellular movement and immune cell trafficking. In contrast, AM were enriched for proteins involved in protein trafficking, molecular transport, and cellular assembly and organization. These findings provide a baseline proteomic resource for further studies aimed at better understanding of the functional differences between MN and AM in both health and disease.
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Project description:Goal was to compare if there were gene expression difference at baseline, without treatment, in the neutrophils of individuals with benign neutropenia vs individuals with normal neutrophil counts
2018-02-05 | GSE108894 | GEO