Presence Of Interleukin Research Articles

NF-κB oscillation profiles decode response to anti-EGFR monoclonal antibodies.

A direct connection between an inflammatory environment and cancer has been extensively proven over the years. We previously reported that the presence of interleukin 1 (IL-1) is responsible for the lack of response to monoclonal antibody targeting EGFR in colorectal cancer (CRC). Considering the driver role of NF-κB in controlling the expression of IL-1, herein, we investigate the dynamics of the oscillatory profile of the NF-κB response to monoclonal antibody, on the background of an inflammatory environment. NF-kB is a typical transcription factor that displays intrinsic oscillatory behavior, whose biological relevance in term for example of decoding response to monoclonal antibodies, remains unclear. Using live cell luciferase techniques, we recorded NF-κB activity over time in response to cetuximab (CTX) alone or in combination with IL-1 cytokines. Our results revealed an additive effect of these two agents on NF-κB activation, which was specific to CTX responsive cells. In contrast, CTX resistant cells did not display a significant change in the NF-κB profile under the IL-1 plus CTX combination. These results suggest an immediate interactive crosstalk between IL-1 and EGFR in the activation of NF-κB signaling pathways, which may lay the basis for the development of drug tolerant persistent cells (DTP), leading to CTX resistance.

Mitochondrial CLPB is a pro-survival factor at the onset of granulocytic differentiation of mouse myeloblastic cells.

Loss-of-function mutations in the CLPB gene lead to congenital neutropenia due to impaired neutrophil differentiation. CLPB, a member of the AAA+ family of proteins, resides in the intermembrane space of mitochondria. The mechanism by which a loss of CLPB elicits defects in the differentiation program of neutrophil precursor cells is not understood. Here, we used 32D clone 3 (32Dcl3) cells, an interleukin-3 (IL-3)-dependent mouse myeloblastic cell line model, to investigate the effects of CLPB knockout on myeloblast-to-neutrophil differentiation in vitro. We found that CLPB-deficient 32Dcl3 cells showed a decreased mitochondrial membrane potential and increased levels of insoluble HAX1 aggregates in mitochondria, as compared to control cells. Despite those abnormalities, CLPB loss did not affect cell proliferation rates in the presence of IL-3 but it increased apoptosis after IL-3 withdrawal and simultaneous induction of cell differentiation with granulocytic colony stimulating factor (G-CSF). CLPB-deficient cells that survived the stress associated with IL-3 withdrawal/G-CSF treatment expressed the same levels of differentiation markers as control cells. Moreover, we found that increased apoptosis of CLPB-deficient cells is linked to production of reactive oxygen species (ROS). N-acetylcysteine, exogenous free fatty acids, or exogenous citrate protected CLPB-deficient 32Dcl3 cells from apoptosis at the onset of differentiation. The protective effect of citrate was abolished by inhibition of ATP-citrate lyase (ACLY), an enzyme that converts cytosolic citrate into acetyl-CoA, a substrate for protein acetylation. We propose that citrate supplementation may help mitigate the effects of CLPB loss by facilitating ACLY-dependent ROS detoxification in granulocytic precursor cells.

Group 2 innate lymphocytes protect the balance between autophagy and apoptosis in cardiomyocytes during sepsis-induced cardiac injury

Group 2 innate lymphocytes (ILC2) have an important role in orchestrating sepsis-induced immune response. However, the impact of LC2 on sepsis-induced cardiac injury is still not fully understood. This study investigated the mechanisms governing ILC2 activation within the cardiac tissue after sepsis. In vivo experiments using wild-type and IL-33 deficient mice indicated that the presence of interleukin (IL)-33, which participates in expanding and activating ILC2 cells, was correlated with higher ILC2 levels (246 ± 34 vs. 66 ± 18, p < 0.01), reduced cardiac dysfunction, and lower markers of cardiac injury. Conversely, IL-33 deficiency led to exacerbated cardiac damage. Additionally, heart ILC2 significantly increased the expression and secretion of IL-5 (2.18 ± 0.34 ng/ml vs. 1.18 ± 0.24 ng/ml, p < 0.05) and IL-13 (10.55 ± 1.13 ng/ml vs. 7.59 ± 1.13 ng/ml, p < 0.05) following sepsis, with this response being mediated by IL-33. Moreover, IL-5 deficient mice exhibited increased cardiac dysfunction and myocardial apoptosis post-sepsis (20.7 ± 4.28% vs. 29.61 ± 4.28%, p < 0.05). Furthermore, in vitro experiments involving co-cultures of ILC2 with mice cardiomyocytes after lipopolysaccharide (LPS) administration suggested that IL-5 derived from ILC2 protects cardiomyocytes from autophagy and apoptosis. These findings imply that IL-33, released in response to sepsis, induces ILC2 activation and IL-5 secretion, orchestrating the equilibrium between autophagy and apoptosis in cardiomyocytes and offering potential therapeutic avenues for mitigating sepsis-induced cardiac injury.

Adapting the protocol for studying the functional capacity of T lymphocytes thawed from cryopreservation

Background. Immunological studies are impossible without long-term storage of cryopreserved biomaterial. There are no standard procedures for working with cryopreserved mononuclear leukocytes.The aim of the study. To optimize the protocol for culturing T lymphocytes thawed after cryopreservation by assessing their viability and proliferative capacity.Methods. Mononuclear leukocytes were isolated from the peripheral blood of relatively healthy volunteers (n = 18). Cells were subjected to controlled freezing down to –80 °C and were transferred to liquid nitrogen. First step: after thawing, the cells were stained with CFSE (carboxyfluorescein succinimidyl ester), were divided into two parts and cultured in the presence/absence of interleukin 2 (IL-2). Cell proliferation was stimulated with phytohemagglutinin (type P). Cells were incubated for 7 days. Sample analysis was performed using flow cytometry. Second stage: thawed cells were divided into three parts. Two parts were resuspended in a full growth medium with IL-2 and were placed in a thermostat (+37 °C) to “rest” for one hour or overnight. After “resting”, the cells were stained with CFSE. One third of the thawed leukocytes were stained with CFSE immediately after thawing. Cells were stimulated, cultured and analyzed the same way at both stages of the study.Results. It has been established that adding IL-2 to the culture medium contributes to a better cell survival. In the presence of IL-2, stimulated CD4+ and CD8+ T lymphocytes produced more daughter cell generations. At the end of the 7-day incubation “rested” samples had reduced leukocyte counts compared to the samples that were cultured immediately after thawing. The number of daughter cell generations formed by stimulated CD4+ and CD8+ T cells decreased when the “rest” stage was included into the study protocol.Conclusion. Adding IL-2 into culture medium can increase the viability and mitotic capacity of thawed T cells, making their state more similar to that of freshly isolated lymphocytes. Cell “rest” after thawing negatively affects the viability and proliferative activity of T lymphocytes during their weekly incubation.

Production of donor-derived cytotoxic T lymphocytes with potent anti-leukemia activity for adoptive immunotherapy in high-risk pediatric patients given haploidentical hematopoietic stem cell transplantation

Background aimsSomatic cell therapy based on the infusion of donor-derived cytotoxic T lymphocytes (CTL) able to recognize patients’ leukemia blasts (LB) is a promising approach to control leukemia relapse after allogeneic HSCT. The success of this approach strongly depends on the ex vivo generation of high-quality donor-derived anti-leukemia CTL in compliance with Good Manufacturing Practices (GMP). We previously described a procedure for generating large numbers of donor-derived anti-leukemia CTL through stimulation of CD8-enriched lymphocytes with dendritic cells (DCs) pulsed with apoptotic LB in the presence of interleukin (IL)-12, IL-7 and IL-15. Here we report that the use of IFN-DC and the addition of IFNα2b during the priming phase significantly improve the generation of an efficient anti-leukemia T cells response in vitro. MethodsUsing this approach, 20 high-risk pediatric patients given haploidentical HSCT for high-risk acute leukemia were enrolled and 51 batches of advanced therapy medical products (ATMP), anti-leukemia CTL, were produced. ResultsQuality controls demonstrated that all batches were sterile, free of mycoplasma and conformed to acceptable endotoxin levels. Genotype analysis confirmed the molecular identity of the ATMP based on the starting biological material used for their production. The majority of ATMP were CD3+/CD8+ cells, with a memory/terminal activated phenotype, including T-central memory populations. ATMP were viable after thawing, and most ATMP batches displayed efficient capacity to lyse patients’ LB and to secrete interferon-γ and tumor necrosis factor-α. ConclusionsThese results demonstrated that our protocol is highly reproducible and allows the generation of large numbers of immunologically safe and functional anti-leukemia CTL with a high level of standardization.

Bacterial antigens and asthma: a comparative study of common respiratory pathogenic bacteria

Objective: In a previous study we have shown that, in the presence of interleukin (IL)-33, repeated, per-nasal challenge of murine airways with Streptococcus pneumoniae (S. pneumoniae) organisms induces human asthma-like airways inflammation. It is not clear, however, whether this effect is unique or manifest in response to other common respiratory pathogens.Methods: To explore this, airways of BALB/c mice were repeatedly challenged per-nasally with formaldehyde-inactivated bacterial bodies in the presence or absence of murine recombinant IL-33. Serum concentrations of S.pneumoniae, Moraxella catarrhalis (M.catarrhalis) and Haemophilus influenzae (H.influenzae) lysates-specific IgE were measured in patients with asthma and control subjects.Results: We showed that in the presence of IL-33, repeated, per-nasal airways exposure to the bodies of these bacteria induced airways hyperresponsiveness (AHR) in the experimental mice. This was accompanied by cellular infiltration into bronchoalveolar lavage fluid (BALF), eosinophilic infiltration and mucous hypertrophy of the lung tissue, with elevated local expression of some type 2 cytokines and elevated, specific IgG and IgE in the serum. The precise characteristics of the inflammation evoked by exposure to each bacterial species were distinguishable.Conclusions: These results suggest that in the certain circumstances, inhaled or commensal bacterial body antigens of both Gram-positive (S. pneumoniae) and Gram-negative (M. catarrhalis and H. influenzae) respiratory tract bacteria may initiate type 2 inflammation typical of asthma in the airways. In addition, we demonstrated that human asthmatic patients manifest elevated serum concentrations of M.catarrhalis- and H.influenzae-specific IgE.

Melatonin-loaded self-healing hydrogel targets mitochondrial energy metabolism and promotes annulus fibrosus regeneration

Intervertebral disc (IVD) herniation is a major cause of chronic low back pain and disability. The current nucleus pulposus (NP) discectomy effectively relieves pain symptoms, but the annulus fibrosus (AF) defects are left unrepaired. Tissue engineering approaches show promise in treating AF injury and IVD degeneration; however, the presence of an inflammatory milieu at the injury site hinders the mitochondrial energy metabolism of AF cells, resulting in a lack of AF regeneration. In this study, we fabricated a dynamic self-healing hydrogel loaded with melatonin (an endocrine hormone well-known for its antioxidant and anti-inflammatory properties) and investigate whether melatonin-loaded hydrogel could promote AF defect repair by rescuing the matrix synthesis and energy metabolism of AF cells. The protective effects of melatonin on matrix components (e.g. type I and II collagen and aggrecan) in AF cells were observed in the presence of interleukin (IL)-1β. Additionally, melatonin was found to activate the nuclear factor erythroid 2-related factor signaling pathway, thereby safeguarding the mitochondrial function of AF cells from IL-1β, as evidenced by the increased level of adenosine triphosphate, mitochondrial membrane potential, and respiratory chain factor expression. The incorporation of melatonin into a self-healing hydrogel based on thiolated gelatin and β-cyclodextrin was proposed as a means of promoting AF regeneration. The successful implantation of melatonin-loaded hydrogel has been shown to facilitate in situ regeneration of AF tissue, thereby impeding IVD degeneration by preserving the hydration of nucleus pulposus in a rat box-cut IVD defect model. These findings offer compelling evidence that the development of a melatonin-loaded dynamic self-healing hydrogel can promote the mitochondrial functions of AF cells and represents a promising strategy for IVD regeneration.

Clonally expanded, thyrotoxic effector CD8+ T cells driven by IL-21 contribute to checkpoint inhibitor thyroiditis.

Autoimmune toxicity occurs in up to 60% of patients treated with immune checkpoint inhibitor (ICI) therapy for cancer and represents an increasing clinical challenge for expanding the use of these treatments. To date, human immunopathogenic studies of immune-related adverse events (IRAEs) have relied on sampling of circulating peripheral blood cells rather than affected tissues. Here, we directly obtained thyroid specimens from individuals with ICI-thyroiditis, one of the most common IRAEs, and compared immune infiltrates with those from individuals with spontaneous autoimmune Hashimoto's thyroiditis (HT) or no thyroid disease. Single-cell RNA sequencing revealed a dominant, clonally expanded population of thyroid-infiltrating cytotoxic CXCR6+ CD8+ T cells (effector CD8+ T cells) present in ICI-thyroiditis but not HT or healthy controls. Furthermore, we identified a crucial role for interleukin-21 (IL-21), a cytokine secreted by intrathyroidal T follicular (TFH) and T peripheral helper (TPH) cells, as a driver of these thyrotoxic effector CD8+ T cells. In the presence of IL-21, human CD8+ T cells acquired the activated effector phenotype with up-regulation of the cytotoxic molecules interferon-γ (IFN-γ) and granzyme B, increased expression of the chemokine receptor CXCR6, and thyrotoxic capacity. We validated these findings in vivo using a mouse model of IRAEs and further demonstrated that genetic deletion of IL-21 signaling protected ICI-treated mice from thyroid immune infiltration. Together, these studies reveal mechanisms and candidate therapeutic targets for individuals who develop IRAEs.

Abstract 3200: Non-engineered T cells with specific anticancer effects in KRAS-mutated colorectal cancer

Abstract Cytokine-induced killer (CIK) cell therapy is known as a useful treatment for cancer immunotherapy due to its anticancer activity and safety. However, its clinical efficacy is limited due to its lack of antigen specificity and low in vivo persistence. KRAS mutations play an important role in promoting tumorigenesis in cancer. But, its clinical application as a biomarker for cancer treatment is still difficult. Non-engineered T cell therapy approaches could be a promising strategy for treating KRAS-mutated cancer. Herein we developed non-engineered T cells empowered with specific anticancer effects in KRAS-mutated colorectal cancer (CRC), and investigated its properties and anticancer effects compared to CIK cells. A recombinant overlapping peptide was designed to overlap the KRAS protein by 15 amino acids linked by a cathepsin S cleavage site (LRMK). It contained KRASG12D, KRASG12V, KRASG13D, and KRASWT epitope peptides and other KRAS protein regions. Peripheral blood mononuclear cells (PBMCs) from CRC patients with KRAS mutations were stimulated with the recombinant overlapping peptides in vitro for two days in the presence of interleukin (IL)-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, IL-1β, and prostaglandin 2, and expanded using the 12-day CIK cell expansion method in the presence of IL-2 (200 IU/mL). To verify the enrichment of KRAS mutant-specific T cells, the expanded T cells were restimulated by pulsed monocyte-derived dendritic cells with KRAS mutant antigen for two days and the frequency of KRAS mutation-specific T cells was determined using the interferon (INF)-γ capture assay. Red fluorescent protein (RFP)-expressing HLA-matched cell lines and cell line-derived spheroids T3M10 (KRASG12D) and MCF-7 (KRASWT) were used for the Incucyte immune cell killing assay. T-cell phenotypes were determined by flow cytometry. The expanded non-engineered T cells stimulated with KRAS mutant antigen showed skewed CD4 T cell differentiation and increased central memory T cells compared to CIK cells, suggesting that they may have more long-term immunity and functionality than CIK cells. The KRASG12D-specific T cells were enriched 12-13% (9.4-fold) by in vitro stimulation compared to 1.3% in CIK cells. Anticancer activity in a 2D-culture model was significantly higher in KRASG12D-specific enriched T cells (82.5%) than in CIK cells (64.9%) co-cultured with T3M10 cells for 48 h. In a 3D spheroid-culture model, increased cytotoxicity was also detectable in the KRASG12D-specific enriched T cells (13.2% at 72 h and 14.1% at 142 h) compared to CIK cells. These results demonstrated that non-engineered T cells with KRAS mutated-specific anticancer activity are an ideal candidate for KRAS mutant cancer-targeted immunotherapy. Further studies are necessary to verify the anticancer effects in vivo. Citation Format: Sunki Lim, Yujeong Gho, Seoyoung Kim, Min Ki Kim, Jong Seob Park, Wang Jun Lee, Young-Kwan Lee, Hyoun Jong Moon, Excelsisbio.Inc, Goyang, Korea. Non-engineered T cells with specific anticancer effects in KRAS-mutated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3200.

Abstract 4056: Intratumoral delivery of autologous tumor antigen specific CD4 Th1 cells combined with dendritic cells eradicates HER2 mammary carcinoma

Abstract Human epidermal growth factor receptor 2 (HER2) overexpression accounts for 30% of invasive breast cancer (BC) and is critically associated with aggressive disease, recurrence and metastasis. Adoptive cell therapy approaches using cytotoxic CD8 T cells and natural killer cells have been shown to trigger anti-tumor immunity in BC. However, the immunosuppressive tumor microenvironment (TME) can inhibit response to these therapies and diminish the presence and function of tumor-infiltrating lymphocytes. Recently, we have shown a critical role for anti-tumor CD4 Th1 cells in dendritic cells (DC) intratumoral (i.t.) delivery in combination with anti-HER2 therapy. This combination therapy enhanced systemic and local anti-tumor immunity and eradicated tumors in HER2 positive BC with a requirement for CD4 Th1 cells. Here we investigated the efficacy of i.t. delivery of both autologous anti-tumor CD4 Th1 cells and tumor antigen pulsed type 1 polarized dendritic cells (HER2-DC1) in a HER2 mammary carcinoma model. CD4 T cells were isolated from BALB/c mice that had completely regressed (pCR) from orthotopic TUBO tumors following HER2-DC1 i.t. or combination therapy with anti-HER2 antibody. CD4 Th1 cells were then expanded by co-culturing with HER2-DC1 in the presence of interleukin (IL)-2 and IL-7 cytokines. BALB/c mice bearing orthotopic TUBO tumors were treated weekly for six weeks with anti-tumor CD4 Th1 cells i.t., non-specific CD4 Th1 cells i.t., HER2-DC1 i.t. or combination therapy. The i.t. delivery of anti-tumor CD4 Th1 cells combined with HER2-DC1 induced a strong anti-tumor response with survival benefit and complete tumor eradication in 50% of treated mice. Importantly, the i.t. delivery of anti-tumor CD4 Th1 cells were critical for providing priming signals to HER2-DC1 within the TME via CD40/CD40L engagement (licensing). This was supported by a strong anti-tumor response and complete tumor regression in 60% of orthotopic TUBO tumor bearing mice treated with CD40/CD40L licensed HER2-DC1 i.t. therapy compared to un-licensed HER2-DC1 i.t. therapy. Additionally, enhanced survival and functionality of human and mouse HER2-DC1 was observed following activation of CD40/CD40L signaling using anti-CD40 agonistic antibody in vitro. Collectively, these results suggest a new promising therapeutic strategy using DC1-anti-tumor CD4 Th1 adoptive cell therapy intratumoral delivery to induce DC1 priming and create robust anti-tumor immunity within the TME in BC. Citation Format: Ganesan Ramamoorthi, Amy Aldrich, Colin Snyder, Brian Czerniecki. Intratumoral delivery of autologous tumor antigen specific CD4 Th1 cells combined with dendritic cells eradicates HER2 mammary carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4056.

Abstract 4123: Rhus verniciflua Stokes (RVS) inhibits PD-1 expression and induces anticancer effects via enhancing T-cell function

Abstract Rhus verniciflua Stokes (RVS) has traditionally been used for centuries as a therapeutic herb and food supplement in East Asian countries. Over the last decade, the anticancer effects of RVS have been demonstrated in various preclinical and clinical studies. RVS contains major flavonoids such as fustin, fisetin, and sulfuretin, which were known to have apoptotic or anti-proliferative activities in various cancer cell lines. In clinical retrospective studies, RVS administration exclusively suggested more favorable outcomes with prolonged overall survival in solid cancer patients. However, the effect of RVS on immuno-oncology, especially the functional properties of T cells and their phenotypes remain unclear. Dried RVS grown in Wonju (Korea) was extracted with sterile distilled water after removing the allergen (urushiol). Peripheral blood mononuclear cells (PBMCs) from breast cancer patients with HLA-A*02:01 were isolated using a vacutainer tube and cultured for T cell expansion in the presence of interleukin (IL)-2 for 14 days. Cells were enriched above 90% CD3+ T cells after 14 days of expansion. Red fluorescent protein (RFP)-expressing HLA-A*02:01-matched breast cancer cell lines (MCF7 and MDA-MB-231), were used to determine the anticancer activity of T cells in the presence or absence of RVS. For in vitro killing assays, MCF7 and MDA-MB-231 cell lines expressing RFP were co-cultured with T cells with or without RVS treatment for 48 h, and cell viability was measured by the MTT assay. The phenotypic characteristics of the T cells were profiled by staining with various T cell surface markers, including CD3, CD4, CD8, PD-1, and CTLA4. RVS toxicity in the target cells (MCF7 and MDA-MB-231) was not observed at RVS concentrations up to 250 ug/mL. The anticancer activity of T cells against breast cancer cells was significantly increased by adding both 10 ug/mL and 100ug/mL of RVS. T cells co-cultured with MCF7 and MDA-MB-231 cells in the presence of 100 ug/mL of RVS showed 20.6% increases in cytotoxicity in MCF7 cells and 36.2% in MDA-MB-231 cells compared to no RVS treatment. Interestingly, relative reductions in programmed death-1 (PD-1) were found in T cells co-cultured with target cells by adding RVS, even though there was no significant difference in the other markers observed. Our findings showed that RVS could improve the function of T cells against cancer cells in the tumor microenvironment, as interpreted by reduced PD-1 expression in T cells after RVS treatment. Therefore, the components of RVS are candidates for restoring T cells exhausted against cancer. Active compounds should be identified for clinical efficacy, and further studies are necessary for combination strategies with conventional cancer treatments, such as chemotherapy and radiotherapy. Citation Format: Seoyoung Kim, Young-Kwan Lee, Hyoun Jong Moon, Sunki Lim, Yujeong Gho, Wang Jun Lee, Sanghun Lee, Excelsisbio Inc.. Rhus verniciflua Stokes (RVS) inhibits PD-1 expression and induces anticancer effects via enhancing T-cell function. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4123.

Novel antimyeloma therapeutic option with inhibition of the HDAC1-IRF4 axis and PIM kinase.

Multiple myeloma (MM) preferentially expands and acquires drug resistance in the bone marrow (BM). We herein examined the role of histone deacetylase 1 (HDAC1) in the constitutive activation of the master transcription factor IRF4 and the prosurvival mediator PIM2 kinase in MM cells. The knockdown or inhibition of HDAC1 by the class I HDAC inhibitor MS-275 reduced the basal expression of IRF4 and PIM2 in MM cells. Mechanistically, the inhibition of HDAC1 decreased IRF4 transcription through histone hyperacetylation and inhibiting the recruitment of RNA polymerase II at the IRF4 locus, thereby reducing IRF4-targeting genes, including PIM2. In addition to the transcriptional regulation of PIM2 by the HDAC1-IRF4 axis, PIM2 was markedly upregulated by external stimuli from BM stromal cells and interleukin-6 (IL-6). Upregulated PIM2 contributed to the attenuation of the cytotoxic effects of MS-275. Class I HDAC and PIM kinase inhibitors cooperatively suppressed MM cell growth in the presence of IL-6 and invivo. Therefore, the present results demonstrate the potential of the simultaneous targeting of the intrinsic HDAC1-IRF4 axis plus externally activated PIM2 as an efficient therapeutic option for MM fostered in the BM.

Correlation of Polymorphonuclear Cell Burden and Microbial Growth to the Inflammatory Cytokines in Tracheal Aspirates from Ventilated Preterm Infants.

The significance of the presence of microorganisms and polymorphonuclear cells in the tracheal aspirates (TAs) of ventilated preterm infants is not well known. Our aim was to correlate information about the presence of polymorphonuclear cells with microbial growth and the cytokine milieu in the TAs of infants who have been intubated for >7 days. TAs were collected from infants who had been intubated for 7 days or longer. Respiratory cultures were performed, and infants were stratified based on the presence and abundance of polymorphonuclear cells and microbial growth. Cytokines were measured in the TAs of each of the respective groups. In the 19 infants whose TAs were collected, the presence of at least moderate WBC with presence of microbial growth was positively associated with the presence of interleukin (IL)-10, IL-1β, IL-8, and tumor necrosis factor (TNF)-α. The presence of at least moderate WBC, with or without microbial growth, was correlated positively with the presence of IL-8 and TNF-α. There are higher levels of proinflammatory cytokines (especially, IL-10, IL-1β, and TNF-α) in TAs with higher cell counts and presence of microbial growth. The findings suggest that the presence of microbial growth correlated with inflammatory burden and warrant a larger study to see if treatment of microbial growth can ameliorate the inflammatory burden. · Concomitant evaluation of inflammatory cells, microbial growth, and cytokines in tracheal aspirates.. · Moderate TA WBC with presence of microbial growth associated with IL-10, IL-1β, IL-8, and TNF-α.. · Moderate TA WBC, with/without microbial growth, correlated with the presence of IL-8 and TNF-α.. · Higher levels of IL-10, IL-1β, and TNF-α correlated with higher TA cell counts and microbial growth..

Assessment of the activity of the immune system in patients with inflammatory bowel diseases and asymptomatic COVID-19.

Although the phenomenon of cytokine storm is well described in patients with severe COVID-19, little is known about the role of the immune system in asymptomatic patients, especially in the group with autoimmune diseases, such as inflammatory bowel disease (IBD). To assess the stimulation of the immune system expressed through the production of cytokines in IBD patients with asymptomatic COVID-19. This is a multi-centre, prospective study in which the concentration of many cytokines (IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL- 15, IL-17, IL-23, IFN-γ, TNF-α, TNF-β) was assessed in patients with IBD and asymptomatic SARS-CoV-2 infection diagnosed by serological tests. In the group of patients with a recent SARS-CoV-2 infection, defined as positive antibodies in the IgA + IgM class, a higher percentage of patients with the presence of interleukin (IL) 2 (IL-2) was found. No association with other cytokines or effects of IBD activity or treatment was found. However, the effect of the applied treatment on the concentration of some cytokines was found: a negative association of infliximab, vedolizumab, and prednisone with IL-2, a positive correlation of steroids, thiopurines with IL-10, and in the case of tumor necrosis factor-α (TNF-α), negative with infliximab, and positive with vedolizumab. The increased concentration of IL-2 may result from its regulatory role in inhibiting excessive activation of the immune system; however, considering the studies of patients with severe COVID-19, its role in the initial phase of SARS-CoV-2 infection requires further research.

Novel genetic regulators of fibrinogen synthesis identified by an in vitro experimental platform

BackgroundFibrinogen has an established, essential role in both coagulation and inflammatory pathways, and these processes are deeply intertwined in the development of thrombotic and atherosclerotic diseases. Previous studies aimed to better understand the (patho) physiological actions of fibrinogen by characterizing the genomic contribution to circulating fibrinogen levels. ObjectivesEstablish an in vitro approach to define functional roles between genes within these loci and fibrinogen synthesis. MethodsCandidate genes were selected on the basis of their proximity to genetic variants associated with fibrinogen levels and expression in hepatocytes and HepG2 cells. HepG2 cells were transfected with small interfering RNAs targeting candidate genes and cultured in the absence or presence of the proinflammatory cytokine interleukin-6. Effects on fibrinogen protein production, gene expression, and cell growth were assessed by immunoblotting, real-time polymerase chain reaction, and cell counts, respectively. ResultsHepG2 cells secreted fibrinogen, and stimulation with interleukin-6 increased fibrinogen production by 3.4 ± 1.2 fold. In the absence of interleukin-6, small interfering RNA knockdown of FGA, IL6R, or EEPD1 decreased fibrinogen production, and knockdown of LEPR, PDIA5, PLEC, SHANK3, or CPS1 increased production. In the presence of interleukin-6, knockdown of FGA, IL6R, or ATXN2L decreased fibrinogen production. Knockdown of FGA, IL6R, EEPD1, LEPR, PDIA5, PLEC, or CPS1 altered transcription of one or more fibrinogen genes. Knocking down ATXN2L suppressed inducible but not basal fibrinogen production via a post-transcriptional mechanism. ConclusionsWe established an in vitro platform to define the impact of select gene products on fibrinogen production. Genes identified in our screen may reveal cellular mechanisms that drive fibrinogen production as well as fibrin(ogen)-mediated (patho)physiological mechanisms.

Cardiac Fibroblasts Secrete Galectin‐1 After Myocardial Infarction to Communicate With Macrophages

The cardiac fibroblast is the key cell type regulating scar formation after myocardial infarction (MI). Whether the fibroblast also serves a role in the early pro‐inflammatory phase has not been evaluated. For this study we evaluated secretome from no MI day (D) 0 and MI D1 cardiac fibroblast to understand the roles of D1 fibroblast in cellular communication during the pro‐inflammatory phase of MI wound healing. Cardiac fibroblasts were isolated from day (D) 0 (n=3) left ventricle and MI D1 (n=3) infarct region, and the 24 h secretome was collected from passage 2 cells. We used mass spectrometry to catalogue and quantify the secretome. We identified galectin‐1 as a highly upregulated D1 MI secreted factor. Because galectin‐1 is known in other systems to be anti‐inflammatory, we stimulated bone marrow derived macrophages (1.5x106 cells/ well; n=8 4M/4F paired sets) with galectin‐1 (500 ng/ml) in the presence of interleukin (IL)‐1b (200 ng/ml) or IL‐10 (50 ng/ml). The conditioned media was assayed using proteome profiler mouse XL cytokine array (RnD) and immunoblotting validation. We also used a human glycosylation array 1000 (RayBio) to evaluate plasma from healthy controls (n=18) and patients 48h after MI (n=41). Galectin‐1 was 3‐fold elevated in the MI D1 secretome (p=0.007). Galectin‐1 increased IL‐10 release (p=0.02) in the macrophages treated wtih IL‐10, a finding validated by two methods. In humans, Galectin‐1 was 2.2‐fold increased at 48h after MI compared to healthy controls (p=0.013). In conclusion, the fibroblast secretes galectin‐1 to modulate the macrophage response after MI by stimulating anti‐inflammatory macrophage polarization and potentially improve MI wound healing.

Decrease of Tumor-infiltrating Regulatory T Cells Using Pentoxifylline: An Ex Vivo Analysis in Triple-negative Breast Cancer Mouse Model.

Triple-negative breast cancer (TNBC) is the most aggressive type of BC with the highest percentage of tumor-infiltrating lymphocytes (TILs). Hence, TIL therapy is considered a promising approach to target TNBC. Depletion of regulatory T cells (Tregs) in TILs can improve the antitumor function of TIL therapy. Pentoxifylline (PTXF) is a xanthine derivative that can modulate the nuclear factor kappa B (NF-κB) signaling and probably affect the Treg proportion in TILs. We aimed to evaluate the ex vivo effect of PTXF on the proportion of Treg cells in the TILs derived from a mouse model of TNBC. The 4T1 cells were inoculated subcutaneously to BALB/c mice to induce TNBC. TILs were isolated from tumor tissue by enzymatic digestion and cultured alone or with 4T1 cells for 24, 48, and 72 h in the presence of interleukin (IL)-2 and different concentrations of PTXF. The toxicity of PTXF and its effects on Tregs proportion as well as cytokine production was evaluated using MTT assay, flow cytometry, and ELISA, respectively. PTXF had no significant impact on the viability of TILs. Both 500 and 1000 mg/mL of PTXF decreased the proportion of Tregs in a dose-dependent manner. The level of interferon-g and tumor growth factor-b in TILs supernatant was increased and decreased, respectively. Our data suggest that ex vivo treatment of TILs with pentoxifylline could decrease the proportion of Tregs in the conventional IL-2-mediated TIL expansion and change the cytokine balance of TILs in favor of antitumor immune response.

The Effect of Natural-Based Formulation (NBF) on the Response of RAW264.7 Macrophages to LPS as an In Vitro Model of Inflammation.

Macrophages are some of the most important immune cells in the organism and are responsible for creating an inflammatory immune response in order to inhibit the passage of microscopic foreign bodies into the blood stream. Sometimes, their activation can be responsible for chronic inflammatory diseases such as asthma, tuberculosis, hepatitis, sinusitis, inflammatory bowel disease, and viral infections. Prolonged inflammation can damage the organs or may lead to death in serious conditions. In the present study, RAW264.7 macrophages were exposed to lipopolysaccharide (LPS; 20 ng/mL) and simultaneously treated with 20 µg/mL of natural-based formulation (NBF), mushroom–cannabidiol extract). Pro-inflammatory cytokines, chemokines, and other inflammatory markers were analyzed. The elevations in the presence of interleukin-6 (IL-6), cycloxygenase-2 (COX-2), C-C motif ligand-5 (CCL5), and nitrite response, following exposure to LPS, were completely inhibited by NBF administration. IL-1β and tumor necrosis factor alpha (TNF-α) release were inhibited by 3.9-fold and 1.5-fold, respectively. No toxic effect of NBF, as assessed by lactate dehydrogenase (LDH) release, was observed. Treatment of the cells with NBF significantly increased the mRNA levels of TLR2, and TLR4, but not NF-κB. Thus, it appears that the NBF possesses anti-inflammatory and immunomodulatory effects which can attenuate the release of pro-inflammatory markers. NBF may be a candidate for the treatment of acute and chronic inflammatory diseases and deserves further investigation.

A multifunctional micropore-forming bioink with enhanced anti-bacterial and anti-inflammatory properties

Three-dimensional (3D) bioprinting has emerged as an enabling tool for various biomedical applications, such as tissue regeneration and tissue model engineering. To this end, the development of bioinks with multiple functions plays a crucial role in the applications of 3D bioprinting technologies. In this study, we propose a new bioink based on two immiscible aqueous phases of gelatin methacryloyl (GelMA) and dextran, further endowed with anti-bacterial and anti-inflammatory properties. This micropore-forming GelMA-dextran (PGelDex) bioink exhibited excellent printability with vat-polymerization, extrusion, and handheld bioprinting methods. The porous structure was confirmed after bioprinting, which promoted the spreading of the encapsulated cells, exhibiting the exceptional cytocompatibility of this bioink formulation. To extend the applications of such a micropore-forming bioink, interleukin-4 (IL-4)-loaded silver-coated gold nanorods (AgGNRs) and human mesenchymal stem cells (MSCs) were simultaneously incorporated, to display synergistic anti-infection behavior and immunomodulatory function. The results revealed the anti-bacterial properties of the AgGNR-loaded PGelDex bioink for both Gram-negative and Gram-positive bacteria. The data also indicated that the presence of IL-4 and MSCs facilitated macrophage M2-phenotype differentiation, suggesting the potential anti-inflammatory feature of the bioink. Overall, this unique anti-bacterial and immunomodulatory micropore-forming bioink offers an effective strategy for the inhibition of bacterial-induced infections as well as the ability of immune-regulation, which is a promising candidate for broadened tissue bioprinting applications.

IL-10 contributes to gemcitabine resistance in extranodal NK/T-cell lymphoma cells via ABCC4.

BackgroundChemotherapy resistance is a main reason for treatment failure in extranodal NK/T-cell lymphoma (ENKTL). Interleukin-10 (IL-10) is closely related to the occurrence and prognosis of ENKTL. We intended to study the role and molecular mechanism of IL-10 in ENKTL resistance. MethodsFifty serum samples were collected from patients with a histologically proven diagnosis of ENKTL. Fifty healthy volunteers were enrolled as a control group. The level of serum IL-10 was detected by ELISA. The NK/T-cell lymphoma cell lines YT and NK-92 were divided into the control group (untreated), IL-10 group (treated with IL-10), IL-10 + GEM group (treated with IL-10 and gemcitabine simultaneously) and GEM group (treated with gemcitabine). A CCK8 assay and flow cytometry were employed to detect the effects of IL-10 on each group. Western blotting was applied to detect the expression of ABC membrane transporter family proteins and signaling pathway proteins in each group. ResultsSerum IL-10 levels were higher in ENKTL patients as well asin patients with ineffective treatment. The IC50 value for gemcitabine in YT and NK-92 cells increased significantly in the presence of IL-10. The effects of gemcitabine resulting in cell killing, cell cycle arrest, and apoptosis promotion were also weakened by IL-10. The expression of ABCC4, STAT1, p-STAT1, Tyk2 and p-Tyk2 was significantly increased by IL-10. ConclusionOur results indicate that IL-10 contributes to the resistance of ENKTL cells via ABCC4 and that IL-10 regulates the JAK/STAT signaling pathway in YT and NK-92 cells.