Presence Of Interleukin Research Articles

Safety and Long-Term Outcome of Intratumoral Injection of OK432-Stimulated Dendritic Cells for Hepatocellular Carcinomas After Radiofrequency Ablation

Dendritic cell (DC)–based immunotherapies are believed to help eradicate residual tumor cells, including hepatocellular carcinoma (HCC). Here, we assessed the safety and clinical response to OK432-stimulated monocyte-derived DCs (MoDCs) in treating HCC after radiofrequency ablation (RFA). MoDCs were derived from 30 HCC patients in the presence of interleukin-4 and granulocyte-macrophage colony stimulating factor for 5 days and then cultured for 2 more days in the medium (basic protocol) or stimulated with OK432. On day 7, DCs were harvested and percutaneously injected into HCC tumors after RFA. We observed no grade 3 or 4 National Cancer Institute Common Toxicity Criteria adverse events. Kaplan-Meier analysis indicated that patients treated with RFA + OK432-stimulated DCs transfer had longer recurrence-free survival than those treated with RFA + basic-protocol DCs (median: 24.8 vs 13.0 months; P = .003). RFA with DC infusion can enhance various tumor-associated antigen (TAA)–specific T-cell responses. Additionally, the 5-year RFS rate for patients with significantly increased TAA-specific T-cell responses was much higher than for other patients (50.0% vs. 7.7%; P = .030). Our study provides useful information for development of HCC immunotherapies (trial registration: UMIN000001701).

Gamma irradiated human platelet lysate as a serum substitute for ex vivo expansion of immune cells

Background & Aim Adoptive immunotherapy is emerged as one of the leading treatments in cancer. The clinical applications require ex vivo expansion of lymphocytes to therapeutic doses in a timely manner under strict cGMP compliance. Culture medium is one important determinant for successful cell expansion. Many efficient expansion strategies incorporate serum or plasma as a supplement to provide cells with a richer milieu to grow in. However, autologous serum has supply limitations for meeting the expected commercial demand for immunotherapies. Moreover, pooled human serum or animal-derived serum bring the inherent concern about risk of infection transmission. Human platelet lysate (hPL) is well recognized as an efficient xenogenic-free alternative to FBS for manufacturing cells and cellular products. To address the risk management for pathogen transmission, we have assessed the feasibility of gamma irradiation as a pathogen reduction treatment for hPL and validated its efficacy for virus inactivation. Our gamma irradiation process demonstrated low impact on hPL potency and long-term stability. In this study, we evaluated the potential use of gamma-irradiated human platelet lysate (GI hPL) on immune cell expansion. Methods, Results & Conclusion To compare the potency of different culture supplements, peripheral blood mononuclear cells were activated and expanded in the presence of interleukin 2 (IL2). T cell expansion in GI hPL supplemented medium showed higher yields than the expansion obtained with autologous plasma. For NK cell expansion, the potency of GI hPL and plasma were comparable. The cytotoxicity of NK cells against K562 cells was high and comparable for both GI hPL and plasma. High viability was observed in all expanded lymphocytes. The results suggest that GI hPL is a viable and valuable alternative cell culture supplement for ex vivo expansion of immune cells.

A 3D iPSC-differentiation model identifies interleukin-3 as a regulator of early human hematopoietic specification.

Hematopoietic development is spatiotemporally tightly regulated by defined cell-intrinsic and extrinsic modifiers. The role of cytokines has been intensively studied in adult hematopoiesis; however, their role in embryonic hematopoietic specification remains largely unexplored. Here, we used induced pluripotent stem cell (iPSC) technology and established a 3-dimensional (3D), organoid-like differentiation system (“hemanoid”) maintaining the structural cellular integrity to evaluate the effect of cytokines on embryonic hematopoietic development. We show that defined stages of early human hematopoietic development were recapitulated within the generated hemanoids. We identified KDR+/CD34high/CD144+/CD43–/CD45– hemato-endothelial progenitors (HEP) forming organized, vasculature-like structures and giving rise to CD34low/CD144–/CD43+/CD45+ hematopoietic progenitor cells. We demonstrate that the endothelial to hematopoietic transition of HEP is dependent on the presence of interleukin 3 (IL-3). Inhibition of IL-3 signaling blocked hematopoietic differentiation and arrested the cells in the HEP stage. Thus, our data suggest an important role for IL-3 in early human hematopoiesis by supporting the endothelial to hematopoietic transition of HEP and highlight the potential of a hemanoid-based model to study human hematopoietic development.

T-cells producing multiple combinations of IFNγ, TNF and IL10 are associated with mild forms of dengue infection.

Multifunctional interleukin 10 (IL10)+ Th1 cells have been implicated in favorable evolution of many infectious diseases, promoting an efficacious immune response while limiting immunopathology. Here, we investigated the presence of multifunctional CD4+ and CD8+ T-cells that expressed interferon gamma (IFNγ), IL10 and tumor necrosis factor (TNF), or its combinations during dengue infection. Peripheral blood mononuclear cells (PBMCs) from outpatients with dengue (mild dengue forms) and hospitalized patients (or patients with dengue with warning signs and severe dengue) were cultured in the presence of envelope (ENV) or NS3 peptide libraries of DENV during critical (hospitalization period) and convalescence phases. The production of IFNγ, IL10 and TNF by CD4+ and CD8+ T-cells was assessed by flow cytometry. Our data show that patients with mild dengue, when compared with patients with dengue with warning signs and severe dengue, presented higher frequencies of multifunctional T-cells like NS3-specific IFNγ/IL10-producing CD4+ T-cells in critical phase and NS3- and ENV-specific CD8+ T-cells producing IFNγ/IL10. In addition, NS3-specific CD8+ T-cells producing high levels of IFNγ/TNF and IFNγ/TNF/IL10 were also observed in the mild dengue group. We observed that multifunctional T-cells produced higher levels of cytokines as measured by intracellular content when compared with single producer T-cells. Importantly, multifunctional CD4+ and CD8+ T-cells producing IFNγ, TNF and IL10 simultaneously displayed positive correlation with platelet levels, suggesting a protective role of this population. The presence of IL10+ Th1 and IL10+ Tc1 multifunctional cells was associated with mild dengue presentation, suggesting that these cells play a role in clinical evolution of dengue infection.

DYNAMICS OF THE LEVEL OF PRO- AND ANTI-INFLAMMATORY CYTOKINES IN THE CREVICULAR FLUID AFTER DIRECT AND INDIRECT RESTORATION

The restoration of discolored teeth is conducted by direct and indirect restoration techniques. Impairment of marginal periodontium is possible during the stages of both direct and indirect restoration in gum retraction, cofferdam application, grinding and polishing for direct restoration, removal of fixation cement residues near indirect restorations and finishing preparation of the connection boundary with the tooth. Furthermore, the restorations affect the periodontal tissues in the long term due to the formation of biofilm and dental plaque in the precervical area of the abutment teeth.
 One of the earliest ways to detect the inflammatory process in the marginal periodontium is to determine the level of pro-inflammatory and anti-inflammatory cytokines in the crevicular fluid.
 The aim of the research was to study the level of pro-inflammatory and anti-inflammatory cytokines in the crevicular fluid after direct and indirect restorations, which were performed by classical fixation and via the proposed method of fixation on the heated composite in 24 hours and 12 months.
 Materials and methods of the research. For clinical substantiation and comparison of direct and indirect ceramic restorations with classical fixation and the proposed method of fixation on the heated composite, we examined and collected the material in 24 hours and 12 months after treatment in 84 patients, aged 18-65, with dental discoloration and defects of the crown of the frontal teeth.
 All patients were divided into 7 clinical groups with 12 patients in each. Group 1 was a control, which included patients with intact dentition, without discoloration and defects of the hard tissues of the teeth of the frontal area; group 2 embraced patients who were treated by direct restoration (a day after treatment); group 3 comprised patients, treated with indirect ceramic restorations, which were fixed by conventional methods on a double-cured composite (one day after treatment); group 4 included patients, whose veneers were fixed to the heated composite (one day after treatment); group 5 embraced patients 12 months after treatment with direct restorations; group 6 included patients, treated with indirect ceramic restorations, which were fixed by conventional methods on a double-cured composite (12 months after treatment); group 7 comprised patients, whose veneers were fixed to the heated composite (12 months after treatment). The material for the study was the crevicular fluid. We determined the tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and interleukin 10 (IL-10).
 The study showed that in all three study groups in 24 hours, the significant predominance of the IL-6 content as compared to the control group was observed. The presence of IL-6 in the crevicular fluid indicates inflammation in the periodontium. The level of another pro-inflammatory cytokine, TNF-α, was higher in the groups with direct and indirect restorations, which were fixed by conventional methods to the composite of double hardening, which relates to adverse factors, since this mediator activates the processes of osteoresorption and enhances inflammatory reactions in the periodontal tissues. When comparing the concentrations of pro-inflammatory cytokines TNF-α and IL-6 in the crevicular fluid between the direct and indirect restoration group, we detected their significantly lower level in the groups where indirect restorations were performed, fixed on the composite cement and on the heated composite. The concentration of IL-10 in the groups with direct and indirect restoration, fixed on the heated composite, was the same, and in the group with indirect restoration, fixed on the composite cement, it increased significantly. After 12 months, the level of pro-inflammatory cytokines TNF-α and IL-6 was significantly higher in the direct restoration group as compared to the indirect restoration groups and with fixation on the composite cement and on the heated composite. The absence of significant differences in pro-inflammatory cytokines in this period for the groups of indirect restorations indicates the absence of inflammatory phenomena in the marginal periodontium and proves the possibility of applying the method of veneer fixation on the heated composite.

Cytotoxicity of natural killer cells on canine mammary carcinoma cells

Natural killer (NK) cells play have a crucial role in the early phase of immune responses against various pathogens. We compared characteristics of canine NK cells against two canine mammary carcinoma cell lines, REM134 and CF41.Mg. REM134 showed higher expression of progesterone receptor, proliferative cell nuclear antigen, Ki67, multiple drug resistance, Bmi-1, c-myc, E-cadherin, and human epidermal growth factor receptor type-2 than that of CF41.Mg. For specific expansion and activation of NK cells, we isolated CD5 negative cells from canine peripheral blood mononuclear cells and co-cultured K562 cells in the presence of interleukin (IL)-2, IL-15, and IL-21 for 21 days. As a result, we found that expression markers of activated NK cells such as NKp30, NKp44, NKp46, NKG2D, CD244, perforin, granzyme B, and tumor necrosis factor alpha were highly upregulated. In addition, we found there was upregulated production of interferon gamma of activated NK cells against target cells such as REM134 and CF41.Mg. Specifically, we observed that cytotoxicity of NK cells against target cells was more sensitively reacted to CF41.Mg than REM134. Based on the results of this study, we recommend the development of an experimental application of CF41Mg, which has not been reported in canine mammary carcinoma research. Keywords: canine, mammary carcinoma, natural killer cells, interferon gamma, cytotoxicity

Highly efficient resonance energy transfer in g-C3N4-Ag nanostructure: Proof-of-concept toward sensitive split-type electrochemiluminescence immunoassay

Nowadays, electrochemiluminescent resonance energy transfer (ECL-RET) as a promising signaling mechanism has been proved its promising perspective for bioassay, whereas the limitation of energy transfer efficiency remains a big challenge. Herein, we highlight a high-efficient ECL-RET mode between luminescent materials and noble metal nanoparticles, in which luminescent materials (g-C3N4 nanosheets as the energy donor) and noble metal nanoparticles (Ag nanoparticles as the energy acceptor) were innovatively combined into one nanostructure, greatly reducing the energy loss and effectively promoting the RET efficiency. To prove the concept of the RET mode in ECL bioanalysis, a split-type ECL immunosensor was developed using interleukin-6 (IL-6) as a model. In the presence of IL-6, a sandwich immunoreaction occurred in anti-IL-6 antibody-coated 96-well plate by using glucose oxidase and anti-IL-6 antibody-conjugating silicon dioxide nanoparticles (anti-IL-6-SiO2-GOx) as signal label. The carried GOx oxidized glucose to generate H2O2, which etched Ag nanoparticles in g-C3N4-Ag nanostructure, thereby destroying the ECL-RET between g-C3N4 and Ag and thus recovering the ECL intensity. The high-efficient ECL-RET in one nanostructure and GOx-induced H2O2 etching modulation in this proposed system endowed the high sensitivity of the immunoassay toward IL-6 with the detection limit of 3.2 × 10−14 g/mL and the dynamic working range of 1.0 × 10−13–1.0 × 10−8 g/mL. It’s believed that this ingenious combination of g-C3N4 and Ag into one nanostructure provides a new perspective to expand the application of ECL-RET of luminescent materials and noble metal nanoparticles.

Aptamer-modified gold nanoparticles for rapid aggregation-based detection of inflammation: an optical assay for interleukin-6

A proof-of-concept aptamer-based optical assay is described for the determination of the immuno signalling molecule interleukin-6 (IL-6), a key marker of acute inflammation. The optical assay is based on the aggregation of gold nanoparticles (AuNP) coated in two complimentary “sandwich-style” aptamers, each with different IL-6 target moieties. IL-6 will recognise the complimentary aptamer pair and bind to it, thereby causing the aggregation of the corresponding functionalised nanoparticles. The aggregation of the AuNPs after exposure to IL-6 induces a visible colour change from red to pink, with a corresponding change in the absorption maximum from 520 to 540 nm. The change in the absorption maximum can be monitored visually, or by using a spectrophotometer or a plate reader. The optimal size and functionalisation of aptamer-coated AuNPs, and the potential assay formats were investigated using UV-vis spectrophotometry, transmission electron microscopy, and dynamic light scattering. The optical assay was applied for detecting mouse IL-6 in a mixed protein solution as a representative biological sample. The assay works in the 3.3 to 125 μg·mL−1 IL-6 concentration range, and the detection limit (at S/N = 3) is 1.95 μg·mL−1. This study was performed as a proof-of-concept demonstration of this versatile assay design, with a view to developing a similar assay for use in clinical samples in future.Graphical abstractSchematic representation of the aggregation of aptamer-functionalised nanoparticles in the presence of interleukin-6 (IL-6). The presence of mouse IL-6 in a mixed protein solution leads to a visible colour change, and a change in the absorption spectrum of the nanoparticles.

Protein Translocation Inhibitors Overcome Cytokine-Induced Glucocorticoid Resistance in T-Cell Acute Lymphoblastic Leukemia

Glucocorticoids (GCs) are central to the treatment of T-cell acute lymphoblastic leukemia (T-ALL), and upfront resistance to GCs is a poor prognostic factor. We previously demonstrated that over one-third of primary patient T-ALLs are resistant to the GC dexamethasone (DEX) when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the microenvironment of leukemic blasts and that plays a well-established role in leukemogenesis. Mechanistically, we demonstrated that GCs paradoxically induce their own resistance by promoting the upregulation of IL7 receptor (IL7R) expression. In the presence of IL7, this augments signal transduction through the JAK/STAT5 axis, ultimately leading to increased STAT5 transcriptional output. This promotes the upregulation of the pro-survival protein BCL-2, which opposes DEX-induced apoptosis. Given that IL7-induced GC resistance depends on de novo synthesis of IL7R in response to DEX, and that newly synthesized IL7R reaches the cell surface via trafficking through the secretory pathway, we hypothesized that inhibiting the translocation of nascent IL7R peptide into the secretory pathway would effectively overcome IL7-induced DEX resistance. Sec61 is a protein-conducting channel in the membrane of the endoplasmic reticulum (ER) that is required for the cotranslational insertion of nascent polypeptides into the ER upon recognition of the signal sequence on secreted and cell surface proteins. To test the hypothesis that Sec61 inhibition could overcome IL7-induced DEX resistance, we utilized the human T-ALL cell line CCRF-CEM, which recapitulates the resistance phenotype observed in primary patient samples. Using a series of structurally distinct small molecule inhibitors of the Sec61 translocon, we demonstrated that Sec61 inhibition effectively overcomes the increase in cell surface IL7R expression in response to DEX. This occurs despite a persistent elevation in IL7R transcript expression following DEX exposure, confirming that Sec61 inhibitors act post-transcriptionally to attenuate cell surface IL7R expression. To determine whether the sensitivity of IL7R to Sec61 inhibitors is due specifically to the interaction between the IL7R signal sequence and Sec61 inhibitors, we generated IL7R constructs containing hydrophobic amino acid substitutions in the signal sequence, which are predicted to confer resistance to Sec61 inhibitors. Upon transient transfection of these constructs into HEK293T cells, we found that these mutations rendered IL7R resistant to the effects of Sec61 inhibition, confirming that the IL7R signal sequence confers sensitivity to these inhibitors. Using the Bliss independence model of synergy in CCRF-CEM cells, we demonstrated that Sec61 inhibitors potently synergize with DEX to overcome IL7-induced DEX resistance. Importantly, at concentrations at which synergy occurs, Sec61 inhibitors demonstrate no single-agent effect on cell survival, suggesting that these effects are not due to an overall reduction in secretory and membrane protein biogenesis. Furthermore, Sec61 inhibitors failed to sensitize CCRF-CEM cells to other chemotherapies used in T-ALL, none of which demonstrate IL7-induced resistance, thereby suggesting that these effects on DEX sensitivity are due specifically to the reduction in cell surface IL7R. To determine if Sec61 inhibitors prevent the DEX-induced increase in STAT5 transcription, we analyzed BCL-2 expression in cells exposed to DEX and IL7, and found that Sec61 inhibitors attenuate the increase in BCL-2 expression in a dose-dependent manner. We next analyzed a cohort of 34 primary patient T-ALL samples. As in CCRF-CEM cells, we found that specifically in those samples with IL7-induced DEX resistance, Sec61 inhibitors synergized with DEX to induce cell death in the presence of IL7. This effect occurred concomitantly with a reduction in cell surface IL7R expression and BCL-2 expression. Taken together, these data demonstrate the efficacy and feasibility of Sec61 inhibition as a novel and rational therapeutic strategy to overcome the IL7-induced DEX resistance phenotype that affects over one-third of newly diagnosed T-ALL patients. Disclosures Sharp: Kezar Life Sciences: Patents & Royalties. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Taunton:Global Blood Therapeutics: Equity Ownership; Principia Biopharma: Equity Ownership, Patents & Royalties; Cedilla Therapeutics: Consultancy, Equity Ownership; Pfizer: Research Funding; Kezar Life Sciences: Equity Ownership, Patents & Royalties, Research Funding.

Grape Seed Extract Eliminates Visceral Allodynia and Colonic Hyperpermeability Induced by Repeated Water Avoidance Stress in Rats.

Grape seed extract (GSE) is rich in polyphenols composed mainly of proanthocyanidins, which are known to attenuate proinflammatory cytokine production. Repeated water avoidance stress (WAS) induces visceral allodynia and colonic hyperpermeability via toll-like receptor 4 (TLR4) and proinflammatory cytokine pathways, which is a rat irritable bowel syndrome (IBS) model. Thus, we explored the effects of GSE on repeated WAS (1 h for 3 days)-induced visceral allodynia and colonic hyperpermeability in Sprague-Dawley rats. Paracellular permeability, as evaluated by transepithelial electrical resistance and flux of carboxyfluorescein, was analyzed in Caco-2 cell monolayers treated with interleukin-6 (IL-6) and IL-1β. WAS caused visceral allodynia and colonic hyperpermeability, and intragastric administration of GSE (100 mg/kg, once daily for 11 days) inhibited these changes. Furthermore, GSE also suppressed the elevated colonic levels of IL-6, TLR4, and claudin-2 caused by WAS. Paracellular permeability was increased in Caco-2 cell monolayers in the presence of IL-6 and IL-1β, which was inhibited by GSE. Additionally, GSE suppressed the claudin-2 expression elevated by cytokine stimulation. The effects of GSE on visceral changes appear to be evoked by suppressing colonic TLR4-cytokine signaling and maintaining tight junction integrity. GSE may be useful for treating IBS.

Modelling Macular Edema: The Effect of IL-6 and IL-6R Blockade on Human Blood-Retinal Barrier Integrity In Vitro.

PurposeMacular edema (ME) is a leading cause of visual loss in a range of retinal diseases and despite the use of antivascular endothelial growth factor (anti-VEGF) agents, its successful treatment remains a major clinical challenge. Based on the indirect clinical evidence that interleukin-6 (IL-6) is a key additional candidate mediator of ME, we interrogated the effect of IL-6 on blood–retinal barrier (BRB) integrity in vitro.MethodsHuman retinal pigment epithelial cell (ARPE-19) and human retinal microvascular endothelial cell (HRMEC) monolayers were used to mimic the outer and inner BRB, respectively. Their paracellular permeability was assessed by measuring the passive permeation of 40 kDa fluorescein isothiocyanate (FITC)-dextran across confluent cells in the presence of IL-6. Transendothelial/epithelial electrical resistance (TEER) then was measured and the distribution of the tight junction protein ZO-1 was assessed by immunofluorescence using confocal microscopy.ResultsTreatment with IL-6 for 48 hours significantly increased the diffusion rate of FITC-dextran, decreased TEER, and disrupted the distribution of ZO-1 in ARPE-19 cells, which constitutively express the IL-6 transmembrane receptor, and this was reversed with IL-6R blockade. In contrast, IL-6 did not affect the paracellular permeability, TEER, or ZO-1 distribution in HRMECs.ConclusionsThese in vitro data support the hypothesis that IL-6 reversibly disrupts the integrity of ARPE-19 cells, but it does not affect HRMECs.Translational RelevanceIL-6 is a candidate therapeutic target in the treatment of outer BRB driven ME.

Nicotinamide N-Methyltransferase and Its Precursor Substrate Methionine Directly and Indirectly Control Malignant Metabolism During Progression of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is one of the most common tumor diseases in adults, and new specific biomarkers are urgently needed to define diagnosis and prognosis of patients with RCC as well as monitor the outcome of therapeutic interventions. The enzyme nicotinamide N-methyltransferase (NNMT) is believed to represent such a marker molecule in RCC therapy. NNMT expression was examined by western blotting in samples from patients with RCC and in RCC cell lines. Effects of NNMT on cell growth and metabolism were assessed using the Hoechst 33342 reagent assay and Vita-Orange cell viability assay. Incubation experiments were performed to study the influence of methionine and interleukin-6 (IL6) on expression of NNMT. In patient samples, NNMT was up-regulated depending on the stage of progression. Investigations in an RCC cell culture model showed that after modulation of NNMT expression, cellular metabolism, but not cell growth was affected. This regulatory function was also dependent on the presence of the NNMT precursor substrate methionine and IL6. The metabolism-regulatory activity of NNMT depends on the precursor substrate methionine and the presence of IL6. The function of methionine appears to be dependent on the stage of progression, since in individual RCC cell lines, opposing effects on metabolism were demonstrated. This, in turn, reflects the thoroughly complex situation in the clinic.

Human Platelet Lysate Media Supplement Supports Lentiviral Transduction and Expansion of Human T Lymphocytes While Maintaining Memory Phenotype.

Immune cell therapy has emerged as a promising approach to treat malignancies that were up until recently only treated on a palliative basis. Chimeric antigen receptor- (CAR-) modified T lymphocytes (T cells) in particular have proven to be very effective for certain hematological malignancies. The production of CAR T cells usually involves viral transduction and ex vivo culture of T cells. The aim of this study was to explore the use of human platelet lysate (HPL) compared to two commonly used supplements, human AB serum (ABS) and fetal bovine serum (FBS), for modified T cell production. For studying transduction, activated T cells were transduced with lentivirus to deliver GFP transgenes with three different promoters. Transduction efficiency (percent GFP) was similar among the supplements, and a modest increase in the transgene product (mean fluorescence intensity) was observed when HPL was used as a supplement compared to ABS. To study the effect of supplements on expansion, peripheral blood mononuclear cells (PBMCs) were activated and expanded in the presence of interleukin 2 (IL2) for fourteen days. T cell expansions using HPL and ABS were comparable and slightly less than the expansion obtained with FBS. Interestingly, cells expanded in media supplemented with HPL showed a higher percentage of T cells with a central memory phenotype compared to those expanded in ABS or FBS. Protein profiling revealed that the phenotypic differences may be explained by elevated levels of several cytokines in HPL, including IL7. The results suggest that the use of HPL as a cell culture supplement during the production of modified T cells is a reasonable alternative to ABS. Furthermore, the use of HPL may enhance in vivo performance of the final product by enriching for central memory T cells that are associated with long-term persistence following adoptive transfer.

Generation of donor-derived Wilms tumor antigen 1–specific cytotoxic T lymphocytes with potent anti-leukemia activity for somatic cell therapy in children given haploidentical stem cell transplantation: a feasibility pre-clinical study

BackgroundThe Wilms tumor antigen 1 (WT1) is over-expressed in a vast majority of adult and childhood acute leukemia and myelodysplastic syndromes, being lowly or transiently expressed in normal tissues and hematopoietic stem cells (HSCs). A number of HLA-restricted WT1 epitopes are immunogenic, allowing the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs) from patients and healthy donors. AimThe aim of the study was to investigate the feasibility of producing WT1-specific CTLs suitable for somatic cell therapy to prevent or treat relapse in children with acute myeloid or lymphoblastic leukemia given haploidentical HSC transplantation (haplo-HSCT). MethodsFor WT1-specific CTL production, donor-derived either peripheral blood mononuclear cells (PBMCs) or CD8+ lymphocytes were stimulated with WT1 peptide-loaded donor dendritic cells in the presence of interleukin (IL)-7 and IL-12. Effector cells were re-stimulated once with irradiated donor PBMCs pulsed with WT1-peptides, and then expanded in an antigen-independent way. ResultsWT1-specific CTLs, displaying high-level cytotoxicity against patients’ leukemia blasts and negligible activity against patients’ non-malignant cells, were obtained from both PBMCs and CD8+ lymphocytes. WT1-specific CTLs obtained from PBMCs showed a better expansion capacity and better anti-leukemia activity than those obtained from CD8+ lymphocytes, even though the difference was not statistically significant. In CTLs derived from PBMCs, both CD8+ and CD4+ subpopulations displayed strong anti-leukemia cytotoxic activity. DiscussionResults of this pre-clinical study pave the way to a somatic cell therapy approach aimed at preventing or treating relapse in children given haplo-HSCT for WT1-positive leukemia.

The macrophage tetraspan MS4A4A enhances dectin-1-dependent NK cell–mediated resistance to metastasis

The plasma membrane tetraspan molecule MS4A4A is selectively expressed by macrophage-lineage cells, but its function is unknown. Here we report that MS4A4A was restricted to murine and human mononuclear phagocytes and was induced during monocyte-to-macrophage differentiation in the presence of interleukin 4 or dexamethasone. Human MS4A4A was co-expressed with M2/M2-like molecules in subsets of normal tissue-resident macrophages, infiltrating macrophages from inflamed synovium and tumor-associated macrophages. MS4A4A interacted and colocalized with the β-glucan receptor dectin-1 in lipid rafts. In response to dectin-1 ligands, Ms4a4a-deficient macrophages showed defective signaling and defective production of effector molecules. In experimental models of tumor progression and metastasis, Ms4a4a deficiency in macrophages had no impact on primary tumor growth, but was essential for dectin-1-mediated activation of macrophages and natural killer (NK) cell-mediated metastasis control. Thus, MS4A4A is a tetraspan molecule selectively expressed in macrophages during differentiation and polarization, essential for dectin-1-dependent activation of NK cell-mediated resistance to metastasis.

Long-term prognostic significance of interleukin-17-producing T cells in patients with non-small cell lung cancer.

The presence of interleukin (IL)‐17‐producing T cells has recently been reported in non‐small cell lung cancer (NSCLC) patients. However, the long‐term prognostic significance of these populations in NSCLC patients remains unknown. In the present study, we collected peripheral blood from 82 NSCLC patients and 22 normal healthy donors (NC). Percentages of IL‐17‐producing CD4+T (Th17), CD8+T (Tc17) and γδT cells (γδT17) were measured to determine their association with clinical outcomes and overall survival (OS) in NSCLC. All NSCLC patients were followed up until July 2018. Median follow‐up time was 13.5 months (range 1‐87 months). The 3‐ and 5‐year survival rate was 27% and 19.6%, respectively. We found that Th17 cells and γδT17 cells were significantly increased, whereas Tc17 cells were markedly decreased in patients with NSCLC compared with those in NC. In addition, Th17 cells were significantly positively associated with T helper type 1 cells (Th1), whereas γδT17 cells were significantly negatively associated with γδT + interferon (IFN)‐γ+ cells. High percentages of peripheral Tc17 cells were significantly associated with favorable 5‐year OS (P = .025), especially in patients with early TNM stage (P = .016). Furthermore, high percentages of peripheral Th17 cells were positively associated with favorable 5‐year OS in patients with late TNM stage (P = .002). However, no significant association was observed between γδT17 cells and OS, regardless of the TNM stage. In conclusion, our findings suggest that enhanced Th17 and reduced Tc17 cells in the peripheral blood could be a significant predictor of a favorable prognosis for NSCLC patients.

IL-21 Expands HIV-1-Specific CD8+ T Memory Stem Cells to Suppress HIV-1 Replication In Vitro.

Due to the existence of viral reservoirs, the rebound of human immunodeficiency virus type 1 (HIV-1) viremia can occur within weeks after discontinuing combined antiretroviral therapy. Immunotherapy could potentially be applied to eradicate reactivated HIV-1 in latently infected CD4+ T lymphocytes. Although the existence of HIV-1-specific CD8+ T memory stem cells (TSCMs) is well established, there are currently no reports regarding methods using CD8+ TSCMs to treat HIV-1 infection. In this study, we quantified peripheral blood antigen-specific CD8+ TSCMs and then expanded HIV-1-specific TSCMs that targeted optimal antigen epitopes (SL9, IL9, and TL9) in the presence of interleukin- (IL-) 21 or IL-15. The suppressive capacity of the expanded CD8+ TSCMs on HIV-1 production was measured by assessing cell-associated viral RNA and performing viral outgrowth assays. We found that the number of unmutated TL9-specific CD8+ TSCMs positively correlated with the abundance of CD4+ T cells and that the expression of IFN-γ was higher in TL9-specific CD8+ TSCMs than that in non-TL9-specific CD8+ TSCMs. Moreover, the antiviral capacities of IL-21-stimulated CD8+ TSCMs exceeded those of conventional CD8+ memory T cells and IL-15-stimulated CD8+ TSCMs. Thus, we demonstrated that IL-21 could efficiently expand HIV-1-specific CD8+ TSCMs to suppress HIV-1 replication. Our study provides new insight into the function of IL-21 in the in vitro suppression of HIV-1 replication.

Metabolic disturbances in synovial fluid are involved in the onset of synovitis in heifers with acute ruminal acidosis

Acute ruminal acidosis (ARA) is the result of increased intake of highly fermentable carbohydrates, which frequently occurs in dairy cattle and is associated with aseptic polysynovitis. To characterise the metabolic changes in the joints of animals with ARA, we performed an untargeted gas chromatography–mass spectrometry (GC-MS)-based metabolomic analysis of synovial fluid. Seven heifers were challenged with an intraruminal oligofructose overload (13 g/kg of body weight [BW]) dissolved in water. Synovial fluid samples were collected at 0, 9 and 24 h post-overload. Metabolome analysis revealed the presence of 67 metabolites. At 9 h post-overload, glyceric acid, cellobiose, fructose and lactic acid were all increased, whereas at 24 h, sorbitol, lactic acid and fructose levels were all increased >10-fold. At 24 h, citric acid and threonine levels were significantly reduced. We detected increased L- and D-lactate, and the presence of interleukin-6 (IL-6) in synovial fluid. Furthermore, using bovine fibroblast-like synoviocytes, we observed that D-lactate induces IL-6 synthesis. Our results suggest that ARA produces severe metabolomic changes in synovial fluid, including disturbances in starch and sucrose metabolism, and increased lactate levels. These changes were observed prior to the appearance of synovitis, suggesting a potential role in the onset of polysynovitis.

Inhibitory Effects of Three Dimensional Adipose Tissue-Derived Mesenchymal Stem Cell Conditioned Medium on Immune Response and Efficacy Evaluation of its Cream

Purpose: This thesis aims to confirm the skin-smoothing effects of 3D adiposetissue derived mesenchymal stem cell conditioned medium (3D-ADMSC-CM) and study how 3D-ADMSC-CM facilitates skin stimulation recovery. Methods: Since confirming that 3D-ADMSC-CM inhibits cell growth and immune reactions, we have continued to evaluate the effects of cream containing 3D-ADMSC-CM on human skin. Results: 3D-ADMSC-CM showed superior self-replication and cell growth rate (at 1, 3 and 5days), and the presence of interleukin 1 alpha (IL-1α) was reduced by 1.4 times, compared to LPS treated. We then applied 3D-ADMSC-CM to human skin. The skin irritation improvement and sedative effects by external stimuli (chemical stimulation) showed a decrease of 25.88% in the applied area and 22.20% in the non-dermal area after 3 weeks of use compared with before the test substance use (after SLS stimulation). Skin improvement due to the external stimulation (chemical) and soothing has been reduced after 3 weeks after use up to 25.88% of applied part and 22.20% of non-applied part compared to test material before use. The skin irritation sedative effect by physical stimulation (friction) immediately after one use showed 12.53% reduction of applied area and 8.71% reduction of non-applied part. Skin roughness was reduced after 3 weeks after use up to 15.91% of applied part and 3.16% of non-applied part compared to test material before use. The final results suggested improved keratin excitation and exfoliation has been reduced after 3 weeks after use up to 78.28% of applied part and 12.26% of non-applied part compared to test material before use. Conclusion: 3D-ADMSC-CM is effective and provides adequate skin stimulation. Cream contained 3D-ADMSC-CM soothed skin irritation improved the overall tone and texture of skin, and recovery of exfoliation by skin stimulation (chemical & physical stimulation).

Culturing the Human Natural Killer Cell Line NK-92 in Interleukin-2 and Interleukin-15 - Implications for Clinical Trials.

The human natural killer cell line NK-92 is increasingly being used in adoptive cell immunotherapies, either in vitro or in animal models transduced with different chimeric antigen receptor (CAR) constructs. Herein, NK-92 cells were analyzed with respect to their proliferation and cytotoxicity, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15). A time-resolved fluorometric assay (TDA-labeled K562 target cells) was used for measuring the cytotoxic activity of NK-92 cells treated with IL-2, IL-4, IL-7 and/or IL-15. Their proliferation, in the presence of these common cytokine receptor γ chain (γc)-dependent cytokines, was measured by traditional tritiated thymidine ([3H]-TdR) incorporation. IL-2 and IL-15, but not IL-4 or IL-7, were able to induce a dose-dependent proliferation of NK-92 cells. IL-15 was, depending on the dose and culture time, up to 10 times more potent compared to corresponding concentrations of IL-2, whereas their combination could potentiate the NK-activity almost equally well. No synergistic effects could be noticed with respect to the cytotoxicity and the proliferation of these cells. Data presented here indicate that of the common gamma chain receptor-dependent cytokines tested here, IL-15 alone is able to cultivate and trigger NK-92 cells to such an extent so that they can be used for immune-based cancer therapies. Implications with respect to CAR-transduced NK-92 cells are also discussed.