Peripheral Blood Mononuclear Cells Cultures Research Articles

Mebendazole stimulates CD14+ myeloid cells to enhance T-cell activation and tumour cell killing.

Mebendazole (MBZ) was recently shown to induce a tumor suppressive M1 phenotype in THP-1 monocytes and macrophages. In the present study the immune effects of MBZ was further investigated using human peripheral blood mononuclear cells (PBMCs) co-cultured with tumour cells. The Biomap platform was used to screen for biomarkers induced from MBZ exposed co-cultures of T-cell receptor activated PBMCs, HT29 colon cancer cells and either human fibroblasts or human umbilical vein endothelial cells (HUVEC) cells. In these co-culture systems MBZ at 0.3-10 μM induced significant increases in TNFα and IFNγ indicating immune stimulation. PBMC cultures alone were subsequently tested for activation status and only in PBMCs activated by CD3/IL2 stimulation and MBZ, at a clinically achievable concentration, was able to increase PBMC clustering and release of pro-inflammatory IFNγ, TNFα, IL6 and IL1β cytokines. Moreover, when PBMC cultures were functionally tested for immune cell killing of lung cancer A549NucLightRed cells, MBZ significantly increased tumour cell apoptosis and reduced the number of surviving tumour cells. This effect was dependent on the presence of CD14 monocytes/macrophages in the co-culture. In summary, MBZ potentiated the immune stimulatory and anticancer effects of anti-CD3/IL2 activated PBMCs which could be relevant to explain the anticancer activity of MBZ observed in the clinic.

Aggregation of Recombinant Monoclonal Antibodies and Its Role in Potential Immunogenicity.

Development of new recombinant biotechnology products has greatly expanded in the field of modern pharmacy and medicine. Since biological recombinant molecules are sensitive, simple or composed proteins, their function is heavily dependent on their structure. In addition to their efficacy, biological medicinal products could show side effects such as immunogenicity. Therefore, detection and characterization of protein structural variants is essential during development and quality control of therapeutic proteins that might trigger immunogenic response in organism. This article includes proposed detection and characterization of aggregated, as well as other modified forms of monoclonal antibodies (mAb), by using selected chromatographic and spectrometric methods. Additionally, selected mAb's aggregates and modified structural variants of monoclonal antibodies were subjected to the immature monocyte-derived dendritic cells' (DC) examination experiment for monitoring of activated DC cells in order to determine potential immunogenicity of mAb structural variants. Furthermore, potential innate immunogenic response of peripheral blood mononuclear cells (PBMC) cultures to mAb aggregates was also evaluated by measuring pro-inflammatory cytokine response during early exposure of PBMCs to different mAb samples and by determining the effect of mAb aggregates on PBMC proliferation during long-term cultures. All developed and proposed analytical methods and immunological in vitro DC and PBMC assays, could be used as platform for complementary analytical characterization and determination of potential for immunogenicity for all biopharmaceutical products which contain monoclonal antibodies as active pharmaceutical ingredients.

INTERFERON GAMMA EXPRESSION CD8+-T LYMPHOCYTE WITH ESAT-6-CFP-10 FUSION ANTIGEN STIMULATION BETWEEN ACTIVE TUBERCULOSIS, LATENT TUBERCULOSIS AND HEALTHY PEOPLE

Tuberculosis (TB) is an infectious disease in the world causing a global problem. Vaccination with Purified Protein Derivative (PPD) still cannot prevent tuberculosis in Indonesia. Interferon-gamma (IFN-γ) produced by CD8+-T lymphocyte has an important role in eliminating Mycobacterium tuberculosis. The vaccine candidate antigenic test was done to observe the inducible ability of IFN-γ as a main protection cytokine. This study aim was to research the difference of IFN-γ expression CD8+-T lymphocyte percentage with ESAT-6-CFP-10 fusion antigen stimulations as a TB vaccine candidate. This research is a quasi-experimental study design in the laboratory by ESAT-6-CFP-10 fusion antigen-stimulated Peripheral Blood Mononuclear Cells (PBMC) culture in vitro in TB patients, latent TB, and healthy subjects' groups. IFN-γ expression CD8+-T lymphocyte percentage were examined by flow cytometry BDFACSCalibur with results: without antigen fusion stimulation IFN-γ expression CD8+-T lymphocyte percentage mean (TB patients 2,560, latent TB 2,173, and healthy people 2,153) and with antigen fusion (TB patients 3,039, latent TB 2,471, and healthy people 2,405). There was no significant difference in fusion antigen stimulation PBMC between TB patients, latent TB, and healthy subjects' group, and also within groups.

Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus

ObjectivesIKZF1 and IKZF3 (encoding transcription factors Ikaros and Aiolos) are susceptibility loci for systemic lupus erythematosus (SLE). The pharmacology of iberdomide (CC-220), a cereblon (CRBN) modulator targeting Ikaros and Aiolos,...

Evaluation of cytotoxic, immunomodulatory and antibacterial activities of aqueous extract from leaves of Conocarpus erectus Linnaeus (Combretaceae).

This work evaluated the antibacterial activity, cytotoxicity and immunomodulatory effect on human peripheral blood mononuclear cells (PBMCs) promoted by aqueous extract from Conocarpus erectus leaves (AELCe). The extract was characterized by thin layer chromatography and ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). Cytotoxicity of AELCe (6.25-50 μg/ml) was investigated using annexin V and propidium iodide. Cytokine and nitric oxide levels in PBMCs culture supernatants exposed or not to AELCe (12.5 μg/ml) were determined, and antibacterial activity was evaluated by disc diffusion and broth microdilution methods. AELCe contained 3',4'-OH flavonoids, phenylpropanoglycosides, saponins, polymeric proanthocyanidins and hydrolysable tannins. Moreover, 10 other compounds were identified through UPLC-MS technique. AELCe did not affect lymphocyte viability at 6.25 and 12.5 μg/ml. IL-2, IL-10, TNF-α, IFN-γ and nitric oxide was produced in higher levels by cells treated with AELCe. Proliferation and activation of CD8+ T lymphocytes were also stimulated. AELCe showed bacteriostatic activity against clinical and antibiotic-resistant strains of Staphylococcus aureus (MIC between 250 and 1000 μg/ml). AELCe showed a moderate bacteriostatic activity and promoted an immunomodulatory status through higher production of Th1 cytokines, nitric oxide release and T CD8+ lymphocytes stimulation.

P-129 - Antioxidant role of microvesicles, activated by genistein, in stem cells

Intercellular communication is an essential hallmark of multicellular organisms. Microvesicles (MVs), which are released from and taken up by most types of cells are involved in intercellular communication. The nutrients we ingest from food contact with immune cells in the bloodstream and can promote the formation of MVs. Some foods contain molecules with regulatory activity, such as genistein, a polyphenol found in soy. We aimed to study the effects of MVs released from genistein-treated immune cells on dental pulp stem cells (DPSC). We obtained peripheral blood mononuclear cells (PBMC) from 5 young women (aged 18–25). Cells were cultured and treated with 0.5 μM genistein or 0.01% DMSO as control. After 48 h, the MVs were isolated by ultracentrifugation and co-incubated with DPSC for 48 h. Then the DPSC were treated with 130 μM of H202 for 24 h. After this period, we analysed cell viability, mRNA expression, and malondialdehyde. Incubating DPSC with MVs isolated from cultures of PBMC treated with genistein protected DPSC from lysis induced by H202. This may be explained because MVs induced the expression of MnSOD and catalase. To conclude, MVs secreted by genistein-treated mononuclear cells exert a protective effect against oxidative stress in DPSC.

Different profile of cytokine production in patients with systemic sclerosis and association with clinical manifestations

Immune dysregulation is a central process in the pathogenesis of systemic sclerosis (SSc). Cytokines produced by lymphocytes and monocytes are important mediators and induce tissue damage, recruit additional inflammatory cells, and promote extracellular matrix production and fibrosis. In the present research, we aimed to study the associations between levels of cytokines in serum and culture supernatants from peripheral blood mononuclear cells (PBMCs) and clinical manifestations in SSc patients. Serum samples were obtained from 56 SSc patients and 56 unrelated age- and gender-matched healthy individuals. Resting and anti-CD3/CD28–stimulated PBMC cultures were obtained from 19 SSc patients and 8 healthy controls. IL-2, IL-4, IL-6, IL-10, IL-17A, TNF, and IFN-γ levels were measured by ELISA or CBA. Serum cytokines, except IL-17A, were below the kit detection limit in most of the patients and controls. In unstimulated PBMC, the production of TNF(p = 0.004), IL-10(p = .048), IL-2(p < 0.001), and IL-6 (p = 0.01) was higher in SSc patients than in healthy controls. After anti-CD3/CD28 stimulation, scleroderma PBMCs had lower concentrations of TNF(p = 0.009), IL-10(p = .018), and IL-2(p = .002) than HC. In unstimulated PBMC, IL-2 concentration was higher in patients with esophageal dysmotility (p = 0.04), and IL-10 levels had a positive correlation with modified Rodnan score (p = 0.03). After anti-CD3/CD28 stimulation, higher levels of IL-2 and IL-4 were observed in SSc patients with lung fibrosis (p = 0.01 and 0.006, respectively), and higher levels of IL-10 (p = 0.04) and IL-4 (p = 0.04) in patients with digital ulcers. In conclusion, SSc patients have a different profile of cytokine production and this was associated with clinical manifestations.

Increased Expression and Modulated Regulatory Activity of Coinhibitory Receptors PD-1, TIGIT, and TIM-3 in Lymphocytes From Patients With Systemic Sclerosis.

Immune dysfunction is an important component of the disease process underlying systemic sclerosis (SSc), but the mechanisms contributing to altered immune cell function in SSc remain poorly defined. This study was undertaken to measure the expression and function of the coinhibitory receptors (co-IRs) programmed cell death 1 (PD-1), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin and mucin domain 3 (TIM-3), and lymphocyte activation gene 3 (LAG-3) in lymphocyte subsets from the peripheral blood of patients with SSc. Co-IR expression levels on subsets of immune cells were analyzed using a 16-color flow cytometry panel. The functional role of co-IRs was determined by measuring cytokine production after in vitro stimulation of SSc and healthy control peripheral blood mononuclear cells (PBMCs) in the presence of co-IR-blocking antibodies. Supernatants from cultures of stimulated PBMCs were added to SSc fibroblasts, and their impact on fibroblast gene expression was measured. Mathematical modeling was used to reveal differences between co-IR functions in SSc patients and healthy controls. Levels of the co-IRs PD-1 and TIGIT were increased, and each was coexpressed, in distinct T cell subsets from SSc patients compared to healthy controls. Levels of TIM-3 were increased in SSc natural killer cells. PD-1, TIGIT, and TIM-3 antibody blockade revealed patient-specific roles of each of these co-IRs in modulating activation-induced T cell cytokine production. In contrast to healthy subjects, blockade of TIGIT and TIM-3, but not PD-1, failed to reverse inhibited cytokine production in SSc patients, indicating that enhanced T cell exhaustion is present in SSc. Finally, cytokines secreted in anti-TIM-3-treated PBMC cultures distinctly changed the gene expression profile in SSc fibroblasts. The altered expression and regulatory capacity of co-IRs in SSc lymphocytes may contribute to disease pathophysiology by modulating the cytokine-mediated cross-talk of immune cells and fibroblasts at sites of inflammation and/or fibrosis.

1-ГЕРМАТРАНОЛ-ГИДРАТ – АКТИВАТОР ТРИПТОФАНИЛ-тРНК-СИНТЕТАЗЫ

The effect of 1-germatranol hydrate, compared to trekrezan, on protein synthesis and the related systems regulating the development, differentiation, regeneration and maintenance of both cellular and tissue homeostasis was studied in vitro using the human peripheral blood mononuclear cells (PBMC) culture. The activating effect of 1-germatranol hydrate on the total activity of tryptophanyl-tRNA synthetase, one of the key enzymes in the extraribosomal stage of the tryptophanyl-tRNA synthetase protein synthesis, is shown. A 15 μg / ml dose of 1-germatranol hydrate increases the expression of tryptophanyl-tRNA synthetase by 33-45% more than a 15 μg / ml dose of trecrezan. By stimulating the biosynthesis of immunoglobulins, germatranol hydrate exhibits an actoprotective effect, thus accelerat-ing the formation of both forms of aminoacyl tRNA synthetase. Activated tryptophanyl-tRNA synthetase, suppressing pathological processes of abnormal aggressive proliferation of blood vessels, stimulates the restorative physiological development of blood vessels. The data obtained suggest that 1-germatranol hydrate can be used as a pharmaceutical exhibiting immunomodulatory and adaptogenic action.

NKG2H-Expressing T Cells Negatively Regulate Immune Responses.

The biology and function of NKG2H receptor, unlike the better characterized members of the NKG2 family NKG2A, NKG2C, and NKG2D, remains largely unclear. Here, we show that NKG2H is able to associate with the signaling adapter molecules DAP12 and DAP10 suggesting that this receptor can signal for cell activation. Using a recently described NKG2H-specific monoclonal antibody (mAb), we have characterized the expression and function of lymphocytes that express this receptor. NKG2H is expressed at the cell surface of a small percentage of peripheral blood mononuclear cell (PBMC) and is found more frequently on T cells, rather than NK cells. Moreover, although NKG2H is likely to trigger activation, co-cross-linking of this receptor with an NKG2H-specific mAb led to decreased T cell activation and proliferation in polyclonal PBMC cultures stimulated by anti-CD3 mAbs. This negative regulatory activity was seen only after cross-linking with NKG2H, but not NKG2A- or NKG2C-specific monoclonal antibodies. The mechanism underlying this negative effect is as yet unclear, but did not depend on the release of soluble factors or recognition of MHC class I molecules. These observations raise the intriguing possibility that NKG2H may be a novel marker for T cells able to negatively regulate T cell responses.

Genetic or pharmacological superoxide-hydrogen peroxide imbalances modulate the in vitro effects of lithium on glycogen synthase kinase-3β

IntroductionLithium (Li), a mood stabilizer used to treat bipolar disorder (BP) symptoms has important anti-inflammatory effects by downregulation of glycogen synthase kinase-3 beta (GSK-3β). However, sometime Li effect is not efficient in some patients suggesting genetic interference. Previous investigations described association between a genetic superoxide‑hydrogen (S-HP) imbalance caused by a superoxide dismutase manganese dependent gene polymorphism (Val16Ala-SOD2 SNP, rs4880) and differential anti-inflammatory response of some drugs and bioactive molecules. Therefore, we postulated here that S-HP imbalance could present some effect on GSK-3β modulation by Li. Methodsto test this hypothesis, a genetic and a pharmacological S-HP imbalance protocols were performed. In the two protocols, immune cells were activated by phythohemaglutin (PHA). The first one, used peripheral blood mononuclear cells (PBMCs) cultures carrying different Val16Ala-SOD2 genotypes, and the second used a commercial macrophage cell line RAW 264.7. Macrophages were exposed to paraquat to induce high S levels (VV-like cells) or porphyrin, that is a SOD2-like molecule that increase dismutation of S into HP (AA-like cells). In both protocols the Li effects on GSK-3β gene and protein modulation as evaluated in 24 h cultures. The inflammatory activation was also analyzed by cellular proliferation in 72 h cell cultures. Resultsas expected PHA exposure triggered a strong upregulation of GSK-3β gene expression (p ≤ 0.001), and Li exposure showed GSK-3β gene downregulation from 0.7 mEq/L concentrations. However, Li modulatory effects on GSk-3β gene and protein expression was directly influenced by basal S-HP balance. Presence of high S-basal levels (VV genotype and VV-like cells) induced attenuated Li anti-inflammatory effects in comparison with balanced and AA and AA-like cells (p < 0.001). Despite methodological limitations related to in vitro assays, the whole of results suggested that Li anti-inflammatory effects is influenced by S-HP basal state and is plausible that its influence could contributes to resistance of some patients to Li treatment or to increase of intensity of some side effects Li-associated.

Enhanced Expression of NLRP3 Inflammasome-Related Inflammation in Diabetic Retinopathy.

The aim of this study was to determine the association between nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) family, pyrin domain-containing 3 (NLRP3) inflammasome-induced inflammation and disease severity in diabetic retinopathy (DR). Blood samples were collected from 64 patients with diabetes (DR, 43; without DR, 21) and 25 healthy controls. The protein and mRNA expression levels of NLRP3 inflammasomes in peripheral blood mononuclear cells were determined using western blotting and quantitative real-time reverse transcription-PCR. A total of 82 vitreous samples were obtained from patients with DR (n = 60) and nondiabetic controls (n = 22). All patients were candidates for vitrectomy. Interleukin (IL)-1β and IL-18 in the peripheral blood mononuclear cell culture medium and vitreous fluid were detected by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence staining for apoptosis-associated speck-like protein with a caspase recruitment domain (ASC) and NLRP3 was performed in fibrovascular membranes from 21 proliferative DR patients and 22 controls with idiopathic epiretinal membranes. We observed increased gene and protein expression of NLRP3, ASC, and caspase-1 in peripheral blood mononuclear cells of adults with DR compared with that in normal controls. Furthermore, the elevated expressions of NLRP3 and ASC were observed in the fibrovascular membranes from 21 adults with proliferative DR when compared with the 22 controls. IL-1β and IL-18 in the peripheral blood mononuclear cells and vitreous fluid were elevated in the DR patients when compared with controls. These outcomes suggested that NLRP3 inflammasomes are upregulated in adults with DR and may play a key role in the pathogenesis and progression of DR.

Levels of inflammatory cytokines from peripheral blood mononuclear cells of children with cow's milk protein allergy.

The aim of the study was to investigate the level of cytokines in cultures of cow's milk protein- stimulated peripheral blood mononuclear cells of patients with cow's milk protein allergy. Eleven children with cow's milk protein allergy and 11 non-allergic controls were studied. Their peripheral blood mononuclear cells were cultured alone and in the presence of cow's milk α-lactalbumin; β-lactoglobulin; αS 1, αS 2, β, and κ-casein fraction mixtures; and a cow's protein mixture from whole milk. Production of cytokines, tumor necrosis factor-α, interleukin-10, and interleukin-12 were determined in culture supernatants. In cow's milk protein-stimulated peripheral blood mononuclear cell cultures of children with cow's milk protein allergy, tumor necrosis factor-α, interleukin-10, and interleukin-12 production was significantly higher than in non-allergic controls (p<0.05). No difference in cytokine production was found between cultures obtained from unstimulated peripheral blood mononuclear cell cultures of both cow's milk protein allergy and non-allergic controls. The findings of this preliminary study align with data from the literature suggesting that the investigation of tumor necrosis factor-α, interleukin-10, and interleukin-12 in cow's milk protein-stimulated peripheral blood mononuclear cell cultures of children may be taken in further consideration to explore whether they might have a predictive role for cow's milk protein allergy. Further studies are therefore needed to extensively investigate this issue.

Mechanisms of Enhanced Osteoclastogenesis in Alkaptonuria

Alkaptonuria (AKU) is a rare disorder characterized by the deficiency of the enzyme homogentisate 1,2-dioxygenase and consequent homogentisate accumulation, which leads to progressive and severe osteoarthopathy starting from the second decade of life. Thus, in AKU patients, bone involvement represents an important clinical issue, which we investigated. Serum levels of receptor activator of NF-κB ligand (RANKL), osteoprotegerin, sclerostin, Dickkopf-1, and bone remodeling markers were measured in nine AKU patients (two children and seven adults) and 22 controls, together with lumbar spine bone mineral density (LS-BMD) and femoral-BMD. In the two AKU children, the average of LS-BMD and femoral-BMD Z-scores were within the normal range, but reduced with respect to the controls. Otherwise, in the adult AKU patients, LS-BMD T-score was inside the normal range, but femoral-BMD T-score reached osteopenic levels. Consistently, in AKU adults, higher RANKL and C-terminal telopeptide of collagen type 1 and lower osteoprotegerin levels were observed than in controls. Otherwise, spontaneous osteoclastogenesis was already evident in peripheral blood mononuclear cell cultures from AKU children, together with a high percentage of circulating osteoclast precursors. Osteoclastogenesis was sustained by the high levels of tumor necrosis factor-α, RANK, RANKL, and LIGHT. In conclusion, the altered osteoclastogenesis was observed already in AKU children, despite the absence of evident injury. Thus, a preventive approach in young patients, targeting osteoclast activity, may prevent the macroscopic bone disease that appears in adult AKU.

Development and testing of species-specific ELISA assays to measure IFN-γ and TNF-α in bottlenose dolphins (Tursiops truncatus).

Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Though a number of immunological assays are readily available for humans and mice, specific assays for many veterinary species, including cetaceans such as bottlenose dolphins (Tursiops truncatus), are more limited. Herein, we describe the development of IFN-gamma (IFN-γ) and TNF-alpha (TNF-α) enzyme-linked immunosorbent assays (ELISAs) specific to bottlenose dolphins. Utilizing these assays, we monitored the immune status of bottlenose dolphins from a managed population over a period of eleven months. The ELISA assays developed for bottlenose dolphins were used to measure IFN-γ and TNF-α in serum or in culture supernatants from peripheral blood mononuclear cells (PBMCs) stimulated with varying concentrations of mitogens concanavalin A (ConA) or phytohemagglutinin (PHA). Induction of TNF-α in PBMC cultures was consistently highest with 1 μg/mL ConA, while 1 μg/mL PHA induced the highest secretion of IFN-γ. Serum levels of TNF-α and IFN-γ remained relatively constant for each animal over the time period examined. CBC and plasma chemistry variables measured concurrently in the bottlenose dolphins were then examined as independent predictors of cytokine levels. We found these clinical variables were more likely to predict linear changes in serum IFN-γ and TNF-α levels compared to concentrations of these cytokines in mitogen-stimulated PBMC culture supernatants. Cytokine assays developed will be of substantial benefit in monitoring bottlenose dolphin health as an adjunct to currently available diagnostic tests.

Dysfunction and Therapeutic Potential of Endothelial Progenitor Cells in Diabetes Mellitus.

Diabetes mellitus (DM) is a chronic, multifactorial metabolic disease whereby insulin deficiency or resistance results in hyperglycemia. Endothelial cells (ECs) form the innermost layer of the blood vessel and produce and release a variety of vasoactive substances and growth factors to regulate vascular homeostasis and angiogenesis. Hyperglycemia and insulin resistance can cause endothelial dysfunction, leading to vascular complications such as coronary artery disease, peripheral arterial disease, diabetic nephropathy, neuropathy and retinopathy. The detrimental effect exerted on ECs by hyperglycemia and insulin resistance underlines the importance of reparatory mechanisms in DM. Endothelial progenitor cells (EPCs), derived from bone marrow, have been recognized as endogenous cells involved in endothelial repair and new blood vessel formation. Initially isolated from a subset of circulating CD34+ mononuclear cells, EPCs were found to possess the ability to differentiate into ECs when cultured in vitro and incorporate into newly formed vessels upon transplantation in animal models of ischemia. Due to the low frequency of CD34+ cells in circulation, the vast majority of studies investigating EPC actions have used cells that are generated through the culture of peripheral blood mononuclear cells (PBMNCs) for 4 - 7 days in endothelial selective medium. These cells, mainly of myeloid hematopoietic cell origin, were termed “Early EPCs,” of which, few expressed stem/progenitor-cell markers. Therefore, early EPCs were also termed “myeloid angiogenic cells” (MACs). When PBMNCs are cultured for over 2 weeks, early EPCs gradually diminish while so-called late EPCs appear. Late EPCs share phenotypic features with mature ECs and are therefore also termed blood-derived ECs; they will not be addressed in this review. MAC dysfunction has been observed in a variety of disease conditions including DM. In this article we review the activities and therapeutic potential of MACs in DM. We will interchangeably use “EPCs” and “MACs” to refer to the cells procured by culture of PBMNCs in EC selective medium for approximately 7 days.

Chemical Modification of CRISPR gRNAs Eliminate type I Interferon Responses in Human Peripheral Blood Mononuclear Cells.

ObjectivesCRISPR/Cas9 is currently the primary tool used for genome editing in mammalian cells. To cleave and alter genomic DNA, both the Cas9 nuclease and a guide RNA (gRNA) must be present in the nucleus. One preferred method of introducing these reagents is direct transfection of a recombinant Cas9 protein complexed with a synthetic gRNA as a ribonucleoprotein (RNP) complex. It is well established from prior work in RNA interference that synthetic RNAs can induce a type I interferon (IFN) response that can limit the application of such methods both in vitro and in vivo. While the immunological properties of short siRNAs are well understood, little is known about the immune recognition of longer CRISPR gRNAs. The objective of our in vitro study was to investigate how the composition of the gRNA influences its recognition by human immune cells.MethodsThe study was performed in vitro in human peripheral blood mononuclear cells (PBMCs). The PBMCs from healthy donor volunteers were treated with gRNA for 24 h, and the levels of type I IFNs in culture supernatants were measured by a multiplex enzyme-linked immunosorbent chemiluminescent assay. Prior to the analysis in PBMCs, the physicochemical parameters and functionality of all nucleic acid constructs were confirmed by electrospray-ionization mass spectrometry and CRISPR/Cas9 gene editing assessment in HEK293-Cas9 cells, respectively.ResultsWe found that unmodified synthetic CRISPR gRNAs triggered a strong IFN response in PBMC cultures in vitro that could be prevented with chemical modification. Likewise, in vitro-transcribed single-guide RNAs (sgRNAs) also triggered a strong IFN response that could only be partially suppressed by phosphatase removal of the 5′-triphosphate group. However, the process by which the gRNA is prepared (i.e., chemically synthesized as a two-part crRNA:tracrRNA complex or in vitro-transcribed as an sgRNA) does not directly influence the immune response to an unmodified gRNA. When experiments were performed in the HEK293 cells, only in vitro-transcribed sgRNA containing 5′-triphosphate induced IFN secretion.ConclusionThe results of our structure-activity relationship study, therefore, suggest that chemical modifications commonly used to reduce the immunostimulation of traditional RNA therapeutics can also be used as effective tools to eliminate undesirable IFN responses to gRNAs.

AB019. 2’-fucosyllactose: a common human milk oligosaccharide with uncommon effects

: Human milk (HM) is a complex matrix containing numerous unique components compared to infant formula. One such group of components, human milk oligosaccharides (HMO), is abundant in HM and only exceeded in amount by lipid and lactose. Of the various HMOs, the most predominant HMO in HM is commonly 2’-fucosyllactose (2’FL). The increased availability of selected synthesized HMOs in recent years has allowed for the opportunity to study the preclinical and clinical effects of some of these compelling oligosaccharides. This presentation will review the roles of 2’FL as well as reviewing the emerging science as it relates to the young infant. Preclinical work supports 2’FL as a prebiotic, immune modulator, and a potential anti-nociceptive by regulating colonic contractions in mice. Additionally, more recent emerging preclinical research in rodents supports that 2’FL enhances cognition via the gut-brain axis. Learning and memory skills in both young and adult rodents that are dependent on the integrity of the vagus nerve appear to be impacted by 2’FL feeding. Recent clinical research has revealed that infants fed infant formula with 2’FL during the first 4 months of life exhibited innate immune responses closer to those of HM-fed infants than did infants fed formula without 2’FL. This included decreased levels of circulating innate inflammatory cytokines IL-1ra, IL-1α, IL-1β, IL-6, and TNF-α in HM- and 2’FL formula-fed infants. Additionally, unlike those from infants fed formula without 2’FL, ex vivo peripheral blood mononuclear cell cultures from HM- and 2’FL-fed infants had decreased concentrations of innate inflammatory cytokines IL-1ra, IL-1β, IL-6, TNF-α, and IFN-γ at 48 hours post respiratory syncytial virus infection. New preclinical and clinical work continues to reveal new functions of 2’FL. (Research funded by Abbott Nutrition, Abbott Laboratories).

The Immunomodulatory Properties of 2-Hydroxyethyl Methacrylate are Mediated by the NLRP3 Inflammasome.

The methacrylate monomer 2-hydroxyethyl methacrylate (HEMA), commonly used in dentistry, has multiple effects on the immune system. This study examined whether HEMA affects the immune system by inducing formation of the NLRP3 inflammasome. Human peripheral blood mononuclear cells (PBMCs) and the human monocyte cell line THP1 were cultured with or without 1000 μM HEMA. To block NLRP3 inflammasome activation, 130 mM KCl was also added to some of the cultures. For the in vivo studies, two different experimental setups were used. In the first experimental setup, mice were injected subcutaneously at the base of the tail with 20 μmol HEMA with or without 100 mM KCl. After 3 weeks, the animals were given an identical booster injection. Two weeks after the last injection, the mice were sacrificed and splenectomized. In the second experimental setup, HEMA (20 μmol), with or without 100 mM KCl, was injected subcutaneously into the tails of BALB/c mice. The mice were given two similar injections at 3-week intervals to allow evaluation of the local inflammation induced by HEMA. After the last inoculation, the injection site was examined daily for 4 days, after which the mice were sacrificed. Cultures of PBMCs and THP1 cells exposed to HEMA in vitro produced more IL-1ß and IL-18 than did control cells. Increased extracellular concentration of KCl inhibited the secretion of IL-1ß. HEMA exposure did not induce cytokine production in variants of the THP1 cell line unable to form the NLRP3 inflammasome. For the first experimental setup, the level of unstimulated basic splenocyte proliferation in vitro was significantly higher in cultures from mice exposed in vivo to HEMA only than in cultures from mice injected with HEMA plus KCl. In the second experimental setup of the in vivo studies, the HEMA-treated mice developed more pronounced inflammation at the site of injection compared to the group of mice given HEMA plus KCl. HEMA affects the immune system by inducing formation of the NLRP3 inflammasome.

Studies of Isolated Peripheral Blood Cells as a Model of Immune Dysfunction.

Peripheral blood mononuclear cells (PBMCs) have been used as a surrogate model of immune function in studies of multiple medical areas, such as metabolic diseases and immune dysfunction. This chapter describes a standardized technique for blood draw and preparation of PBMCs from whole blood using density gradient centrifugation, followed by cell culture. The main focus is on collection of the PBMC culture media and extraction of cellular proteins in order to provide the materials for biomarker studies.