Local Cytokine Concentrations Research Articles

No effect of tattoos on local sweat concentrations of select cytokines, cortisol, glucose, blood urea nitrogen, or lactate during exercise

Due to growing interest in the investigation of exercise induced sweat biomarkers to assess an individual’s health and the increasing prevalence of tattoos in the world’s population, investigators sought to determine whether local sweat concentrations and excretion rates of epidermal growth factor (EGF), interleukin (IL) -1α, IL-6, IL-8, cortisol, glucose, blood urea nitrogen (BUN), and lactate differ between tattooed and contralateral non-tattooed skin during exercise. Sixteen recreational exercisers [female (50%)] (age = 25–48 years) with ≥ 1 unilateral permanent tattoo [median tattoo age = 6 years, IQR = 5] on the arm/torso completed an outdoor group fitness session. There were no significant differences between tattooed and non-tattooed skin for sweat EGF, IL-1α, IL-8, cortisol, glucose, BUN, or lactate concentrations. There were no significant differences between tattooed and non-tattooed skin for sweat EGF, IL-1α, IL-8, cortisol, glucose, BUN, or lactate excretion rate. Findings suggest that permanent tattoos older than 1 year may not impact local sweat EGF, IL-1α, IL-8, cortisol, glucose, BUN, and lactate concentrations or excretion rates during exercise.Clinical trial identifier NCT04920266 was registered on June 9, 2021.

Hippo pathway-manipulating neutrophil-mimic hybrid nanoparticles for cardiac ischemic injury via modulation of local immunity and cardiac regeneration

The promise of regeneration therapy for restoration of damaged myocardium after cardiac ischemic injury relies on targeted delivery of proliferative molecules into cardiomyocytes whose healing benefits are still limited owing to severe immune microenvironment due to local high concentration of proinflammatory cytokines. Optimal therapeutic strategies are therefore in urgent need to both modulate local immunity and deliver proliferative molecules. Here, we addressed this unmet need by developing neutrophil-mimic nanoparticles NM@miR, fabricated by coating hybrid neutrophil membranes with artificial lipids onto mesoporous silica nanoparticles (MSNs) loaded with microRNA-10b. The hybrid membrane could endow nanoparticles with strong capacity to migrate into inflammatory sites and neutralize proinflammatory cytokines and increase the delivery efficiency of microRNA-10b into adult mammalian cardiomyocytes (CMs) by fusing with cell membranes and leading to the release of MSNs-miR into cytosol. Upon NM@miR administration, this nanoparticle could home to the injured myocardium, restore the local immunity, and efficiently deliver microRNA-10b to cardiomyocytes, which could reduce the activation of Hippo-YAP pathway mediated by excessive cytokines and exert the best proliferative effect of miR-10b. This combination therapy could finally improve cardiac function and mitigate ventricular remodeling. Consequently, this work offers a combination strategy of immunity modulation and proliferative molecule delivery to boost cardiac regeneration after injury.

Abstract LB101: Cell-generated IL12 combined with PD-1 inhibition produces local and abscopal immune activation to eradicate metastatic melanoma and pancreatic cancer

Abstract Background: Cytokines have been FDA approved for cancer immunotherapy for treatment of metastatic melanoma and renal carcinoma for over 30 years (1, 2). To overcome stability and toxicity limitations seen with high dose cytokine immunotherapy, we developed a delivery platform, called cytokine factories, composed of genetically engineered epithelial cells encapsulated in biocompatible polymers. These clinically translatable cytokine factories are able to safely deliver high local doses of pro-inflammatory cytokines, such as interleukin-12 (IL12), and allow for controlled and predictable dosing in vivo. Results: Tumor-adjacent administration of IL12-based cytokine factories in vivo created a high local cytokine concentration (IP space) without substantial leakage into the systemic circulation. In addition, administration of cytokine factories in combination with anti-PD1 checkpoint inhibitors caused reduction of tumor burden by over 60% when delivered as a monotherapeutic to mice with metastatic melanoma (3). Further, when administered in combination with local anti-PD1 checkpoint inhibitors, these cytokine factories led to reduction of intraperitoneal tumor burden by over 80% after only 10 days of treatment. Finally, we evaluated the ability of this therapy to treat pancreatic tumors which are notoriously known to be resistant to immunotherapy. We found that the median survival for control animals was 45 days. However, 8/8 of animals treated with IL12 cytokine factories survived throughout the duration of the study (110 days), demonstrating a significant extension of overall survival time. Conclusions: Our findings demonstrate efficacy of cytokine factories as single agent and combination therapeutics in preclinical animal models and provide rationale for future clinical testing for the treatment of metastatic peritoneal cancers in humans.

Abstract 4303: High-throughput 3D tumoroid models for immunotherapy and drug discovery

Abstract The tumor microenvironment (TME) is a complex 3D cellular system comprised of a diverse amalgam of cells. To characterize the TME, we developed a 3D cell culture platform which facilitates imaging-based analysis and cell viability for extended durations. We used this platform to collect in-situ spatiotemporal measurements of cytokine concentrations in the tumor vicinity, track cellular activity, and model local tumor invasion of glioblastoma (GBM) using fast-scanning confocal microscopy. The mechanism by which we achieve long-term 3D culture is perfusion, modeled as flow through a porous medium. Perfusion facilitates regulated transport of nutrients and waste within the soft microgel medium: Liquid-Like Solids (LLS). The interstitial space is tuned to mimic a capillary bed, and surface bioconjugation of the microgels promotes cellular adhesion and migration. To measure cytokine concentrations, GBM tumoroids were grown in LLS and printed into a dispersion of ELISA beads in LLS. The mechanical stability of LLS ensures the tumor and ELISA beads remain stationary without impeding cellular activity. Cytokine on and off-rates were referenced alongside measured bead fluorescence intensities and positions to fit spatiotemporal reaction-diffusion models. Fast-scanning confocal microscopy facilitated in-situ observation of the evolutionary dynamics of tumor progression. Co-culture of patient-derived tumor explants and autologous PBMCs printed into type I collagen-bioconjugated LLS enabled studies of cancer-immune interactions. In-situ cytokine measurements revealed local IL-8 concentrations reached a maximum value of 2 ng ml−1 after 10 hours. A cellular production rate was estimated at 2 molecules cell−1 s−1. Invasive behavior into the proximal space was determined to be super-diffusive; off-lattice agent-based simulations indicated this behavior is a result of the confinement of invasive fronts to the microgel interstitium. The invading glioblastoma cells used anchorage-dependent migration and were guided by geometric cues to traverse the porous bioconjugated LLS network. Cancer-immune interaction studies revealed average CD8+ speeds greater than 2.8 µm min−1 and both chemotaxis and chemokinetic behavior. CD8+ T-cell killing rates were estimated at approximately 3 cancer cells hr−1 initially, monotonically falling over 12 hours to roughly 1 cell hr−1. The development of the in-situ 3D ELISA assay and imaging-based analysis techniques have enabled the tracking of tumor-immune cell interactions, observation of dynamic tumor progression, and local cytokine concentration profiling at appreciable spatiotemporal resolutions. The combination of a physiologically relevant 3D culture platform with the capacity for in-situ qualitative and quantitative observation may lead to new, powerful, preclinical models that allow for interrogation of the TME and decrease the rate of false discovery. Citation Format: Duy Nguyen, Alexander McGhee, Diego Pedro, Alfonso Pepe, Matthew Schaller, Ryan Smolchek, Jack Famiglietti, Stephanie Warrington, W. Gregory Sawyer. High-throughput 3D tumoroid models for immunotherapy and drug discovery. [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 4303.

Local cytokine concentration in patients with non-muscle invasive bladder cancer of low malignant potential and with varying rates of recurrence

Aim. To investigate local concentrations and distribution of cytokines in tumor tissue and perifocal zone in non-muscle invasive bladder cancer of low malignant potential in patients with low and high probability of disease recurrence. Materials and methods. We have studied tumor and perifocal zone fragments of 31 patients with verified non-muscle invasive bladder cancer of low malignant potential and with different probabilities of recurrence. Fifteen (15) patients developed recurrences 6–9 months after combination treatment. The fragments of primary and recurrent tumors were echanically disaggregated and centrifuged at 1500 rpm for 10 minutes. Levels of cytokines interleukin (IL) -1β, -6, -8, -10, -18, tumor necrosis factor α (TNF-α), interferon-γ (Vektor-Best, Russia), and epithelial neutrophil activating peptide 78 (ENA-78) (CXCL-5 chemokine) (Cloud-Clone Corp., USA) were measured in the samples by ELISA. Results were statistically processed using Statistica 13 software (StatSoft Inc., USA), and presented as median and interquartile range – 25th and 75th percentile (Ме [LQ; UQ]).Results. Comparison of cytokine concentrations within the groups showed that the levels of inflammatory cytokines (TNF-α, IL-1β, IL-8, IL-6, IL-18) in tumor tissues were higher than in the perifocal zone tissues. This pattern was expected because tumor is the main site of inflammation. Comparison of these indicators between groups showed that in tumor tissues with an unfavorable course of the disease, namely disease recurrence, the levels of almost all inflammatory cytokines (TNF-α, IL-1β, IL-8, IL-6) were higher. A similar pattern was observed when comparing the levels of cytokines in the tissues of the perifocal zone. These differences were statistically significant. ENA-78 concentration was not determined in all cases.Conclusion. The data obtained during the study indicates that in patients with unfavorable disease course (recurrence), tumor growth is associated with high expression of proinflammatory cytokines, which can subsequently lead to development of disease recurrence.

Abstract 4189: Evaluation of implantable cytokine factories in combination with checkpoint inhibitors for eradication of malignant pleural mesothelioma (MPM) tumors in mice with safe and predictable dosing in non-human primates

Abstract Pro-inflammatory cytokines have been approved as a cancer immunotherapy for treatment of metastatic melanoma and renal carcinoma for over 30 years. However, widespread use of cytokine therapy in the clinic is limited by its short half-life in circulation and the associated toxicities that emerge as a result of high systemic exposure. To overcome these limitations, we developed a clinically translatable cytokine delivery platform, called cytokine factories, composed of genetically engineered epithelial cells encapsulated in biocompatible polymers. These cells are able to produce a wide range of natural cytokines (IL2, IL7, IL10, or IL12) and allow for controlled and predictable dosing in vivo. In vivo administration of cytokine factories created a high local cytokine concentration (IP space) without substantial leakage into the systemic circulation. Local, or tumor adjacent, administration of pro-inflammatory (IL-2-based) cytokine factories caused reduction of tumor burden by 70% after only 1 week of treatment when delivered as a monotherapy to mice with malignant pleural mesothelioma (MPM). Importantly, when administered in combination with local anti-PD1 injections, these cytokine factories lead to eradication of these highly aggressive tumors in 7/7 treated mice. To validate the translatability of this platform, we evaluated the safety profile in non-human primates. Significantly, this platform produced local and systemic T cell biomarker profiles that predict efficacy without renal, liver, or general toxicity in non-human primates. Our findings demonstrate the safety and efficacy of cytokine factories in preclinical animal models and provide rationale for future clinical testing for the treatment of metastatic peritoneal cancers in humans. Citation Format: Amanda Nash, Samira Aghlara-Fotovat, Andrea Hernandez, Bertha Castillo, Alexander Lu, Aarthi Pugazenthi, Peter Rios, Sofia Ghani, Ira Joshi, Douglas Isa, Chunyu Xu, Rahul Sheth, Weiyi Peng, Jose Oberholzer, Amir Jazaeri, Hee-Jin Jang, Bryan Burt, Hyun-Sung Lee, Ravi Ghanta, Omid Veiseh. Evaluation of implantable cytokine factories in combination with checkpoint inhibitors for eradication of malignant pleural mesothelioma (MPM) tumors in mice with safe and predictable dosing in non-human primates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4189.

Dynamics of cellular factors of innate immunity and antioxidant protection in the patients with chronic recurrent vulvovaginal candidosis upon exposure to cavitated physiological solution

Dysfunction of colonization resistance factors at the mucous membranes of genitourinary system during adhesion of Candida fungi is a common pathogenic situation leading to impairment of the mucous membrane regeneration, unsuccessful attempts at in vitro fertilization (IVF), increased risk of complications during gestation. The aim of present study was to substantiate the opportunity of complex therapy for chronic vulvovaginal candidiasis using cavitated saline and usage suppositories with alpha-2b human recombinant interferon a (Viferon LLC, Russia). The study involved 90 women at the age of25.45±7.16 years, with clinically and laboratory confirmed recurrencies of chronic vulvovaginal candidiasis observed, on average, 5.55±0.45 times. Group 1 consisted of 30 women with C. albicans infection of urogenital tract, at the mean age of 27.11±1.52 years, who received therapy with fluconazole (150 mg once every 7 days). Group 2 consisted of 30 women with C. albicans infection of mucous membranes, mean age 27.31±1.99 years, treated with Fluconazole (150 mg 1 time in 7 days, and 5-min. irrigation of vagina with ultrasonicated saline solution, 7 days; Low-intensity ultrasound exposure was performed using the Fotek AK100-25 hardware complex, ultrasonic vibration frequency of 25 kHz (Yekaterinburg, Russia). Group 3 consisted of 30 women whose treatment included Fluconazole (150 mg once every 6 days), irrigation of mucous membranes with ultrasonicated cavitated solution and subsequent administration of suppositories containing Viferon® 500,000 IU (1 suppository daily for 7 days). Biochemical studies included determination of isopropanol- and heptane-soluble primary, secondary end products of lipid peroxidation. Activity of the antioxidant system was studied by determination of superoxide dismutase, glutathione peroxidase, ceruloplasmin, vitamin C. Qualitative and quantitative characteristics of neutrophilic granulocytes from vaginal secretions were evaluated by testing phagocytic activity, reduction of nitroblue tetrazolium compound to diformazan. The studies of IL-8, TNFα, IL-2, and IL-10 cytokines were carried out in cell-free supernates of vaginal secretions by ELISA test systems (LLC “Vector Best”, Novosibirsk). Results: upon therapeutic exposure to low-frequency ultrasonic cavitation combined with antioxidant drug, we have observed a decreased local concentration of pro-inflammatory cytokines, the balance of lipid peroxidationantioxidant defense system was restored, and functional metabolic status of the phagocytes in vaginal secretions was normalized.

Effects of Leukocyte-Platelet-Rich Fibrin (L–PRF) on Pain, Soft Tissue Healing, Growth Factors, and Cytokines after Third Molar Extraction: A Randomized, Split-Mouth, Double-Blinded Clinical Trial

This study assessed the effects of leukocyte-platelet-rich fibrin (L–PRF) on soft tissue healing and the correlation with the local concentration of growth factors (GF) and cytokines in the dental socket of lower third molars. Forty lower-third molars (20 participants) were included in this randomized, double-blinded, split-mouth study. After extractions, randomized sides received alveolar filling with L–PRF on one side and a natural blood clot on the other side. The pain was assessed for up to seven days and soft tissue healing (Landry index) for 14 days post-extraction. Swabs were collected from the surgical sites for GF and cytokine assessment by flow luminometry. Participants reported lower postoperative pain on the sides grafted with L–PRF, which also presented increased tissue healing scores (p < 0.05). There were increased levels of all GFs and several cytokines at the L–PRF site at day one, while vascular endothelial growth factor (VEGF), IL–10, and IL1–RA remained higher throughout for seven days (p < 0.05). VEGF concentration at L–PRF sites correlated positively with the participants’ blood platelet content (ρ = 0.769). PDGF correlated negatively with pain experience on days 2 and 3, and positively with soft tissue healing scores, while FGFb presented a weak correlation with a reduction of pain on day 3. The use of L–PRF improves the soft tissue healing process and decreases postoperative pain after the third molar extractions, which correlates with an increase in the local concentration of growth factors such as PDGF and FGFb.

Novel method for effectively amplifying human peripheral blood T cells in vitro

The use of chimeric antigen receptor-modified T cells (CAR T cells) is an effective therapy for advanced cancer, especially hematological malignancies, and this method has attracted widespread attention in the last several years. The type, number and vitality of the effector cells clearly play important roles in this approach. In this study, to expand the possibility of curing cancer through adoptive cell therapy (ACT), we developed a novel method for effectively obtaining abundant T cells in vitro. The fusion proteins of three cytokines, SA-hIL-2, SA-hIL-7 and SA-hIL-21, were anchored onto biotin magnetic beads to increase the number of cytokines on the surface of the magnetic beads, which increased the local concentration of cytokines and thus promoted the binding of cytokines to T cells. Next, we examined the effects of these modified magnetic beads on the proliferation rate of T cells and CD19 CAR T cells. In this study, we report the expression and purification of the active bifunctional fusion proteins SA-hIL-2, SA-hIL-7 and SA-hIL-21, which were bound to biotin magnetic beads to develop a platform that was employed to increase the local concentration of cytokines. When the cells had been cultured for 14 days, the proliferation rate of the CD3+ T cells in the group that received cytokine-coupled biotin magnetic beads (Beads-SA-CK) was higher than that of the cells in the groups that received soluble cytokines (Soluble-SA-CK) and that of the cells in the standard group (Standard-CK). We speculate that this difference may be the result of the increased expression of Bcl-2 and the increased phosphorylation of Stat5. Moreover, our results preliminarily indicate that compared with the other two treatments, Soluble-SA-CK and Standard-CK, adding cytokine-coupled biotin magnetic beads more effectively increases the proliferation rate of CD19 CAR-T cells. As expected, the CD19 CAR-T cells stimulated by Beads-SA-CK had a stronger anticancer effect than the cells stimulated by the other two treatments. An effective method of preparing abundant T cells in vitro was developed, and it may provide a novel strategy for ACT.

Peripheral soluble epoxide hydrolase inhibition reduces hypernociception and inflammation in albumin-induced arthritis in temporomandibular joint of rats

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial tissue, joint dysfunction, and damage. Epoxyeicosatrienoic acids (EETs) are endogenous anti-inflammatory compounds, which are quickly converted by the soluble epoxide hydrolase (sEH) enzyme into a less active form with decreased biological effects. The inhibition of the sEH enzyme has been used as a strategy to lower nociception and inflammation. The goal of this study was to investigate whether the peripheral treatment with the sEH enzyme inhibitor 1- trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) could prevent the hypernociception and inflammation in the albumin-induced arthritis model in rats’ temporomandibular joint (TMJ). After the induction of experimental arthritis, animals were assessed for nociceptive behavior test, leukocyte infiltration counts and histologic analysis, ELISA to quantify several cytokines and Western blotting. The peripheral pretreatment with TPPU inhibited the arthritis-induced TMJ hypernociception and leukocyte migration. Moreover, the local concentrations of proinflammatory cytokines were diminished by TPPU, while the anti-inflammatory cytokine interleukin-10 was up-regulated in the TMJ tissue. Finally, TPPU significantly decreased protein expression of iNOS, while did not alter the expression of MRC1. This study provides evidence that the peripheral administration of TPPU reduces hypernociception and inflammation in TMJ experimental arthritis.

Natural history, dynamics, and ecology of human papillomaviruses in genital infections of young women: protocol of the PAPCLEAR cohort study

IntroductionHuman papillomaviruses (HPVs) are responsible for one-third of all cancers caused by infections. Most HPV studies focus on chronic infections and cancers, and we know little about the early stages...

Sclareol attenuates the development of atopic dermatitis induced by 2,4-dinitrochlorobenzene in mice

Context: Atopic dermatitis is a common chronic inflammatory skin disease affecting up to 20% of children and 1% of adults worldwide. Treatment of atopic dermatitis include corticosteroids and immunosuppressants, such as calcineurin inhibitors and methotrexate. However, these treatments often bring about adverse effects including skin atrophy, osteoporosis, skin cancer, and metabolic syndrome.Objective: In this study, we evaluated the therapeutic effects and mechanisms of sclareol, a natural diterpene, on atopic dermatitis (AD)-like skin lesions induced by 2,4-dinitrochlorobenzene (DNCB) in mice.Materials and methods: To evaluate the effect of sclareol in vivo model, BALB/c mice were repeatedly injected intraperitoneally with sclareol (50 and 100 mg/kg) in 2,4-dinitrochlorobenzene (DNCB)-induced AD-like murine model. Major assays were enzyme-linked immunosorbent assay, histological analysis, flow cytometry, western blot analysis.Results: Intraperitoneal administration of sclareol (50 and 100 mg/kg) significantly attenuated AD-like symptoms, such as serum IgE levels, epidermal/dermal hyperplasia, and the numbers of infiltrated mast cells. In addition, systemic sclareol treatments reduced local pro-inflammatory cytokine concentrations, including IL-6, IL-1b, TNF-a, IL-4, IFN-g, and IL-17A, on AD-like lesions. Furthermore, we demonstrated that sclareol also suppressed T cell activation and the capability of cytokine productions (IFN-g, IL-4 and IL-17A) in response to DNCB stimulation. By examining the skin homogenate, we found that sclareol inhibited the AD-like severity likely through suppressions of both NF-kB translocation and phosphorylation of the MAP kinase pathway.Discussion and conclusions: Cumulatively, our results indicate that sclareol induced anti-inflammatory effects against the atopic dermatitis elicited by DNCB. Thus, sclareol is worth of being further evaluated for its potential therapeutic benefits for the clinical treatment of AD.

Coinfection modulates inflammatory responses, clinical outcome and pathogen load of H1N1 swine influenza virus and Haemophilus parasuis infections in pigs

BackgroundRespiratory co-infections are important factor affecting the profitability of pigs production. Swine influenza virus (SIV) may predispose to secondary infection. Haemophilus parasuis (Hps) can be a primary pathogen or be associated with other pathogens such as SIV. To date, little is known about the effect of coinfection with SIV and Hps on the disease severity and inflammatory response and the role of Hps in the induction of pneumonia in the absence of other respiratory pathogens. In the study we investigated the influence of SIV and Hps coinfection on clinical course, inflammatory response, pathogens shedding and load at various time points following intranasal inoculation. The correlation between local concentration of cytokines and severity of disease as well as serum acute phase proteins (APP) concentration has been also studied.ResultsAll co-infected pigs had fever, while in single inoculated pigs fever was observed only in part of animals. Necropsy revealed lesions in the lungs all SIV-inoculated and co-inoculated pigs, while in Hps-single inoculated animals only 1 out of 11 pigs revealed gross lung lesions. The SIV shedding was the highest in co-inoculated pigs. There were no differences between Hps-single inoculated and co-inoculated groups with regard to Hps shedding. The significant increase in Hps titre in the lung has been found only in co-inoculated group. All APP increased after co-infection. In single-inoculated animals various kinetics of APP response has been observed. The lung concentrations of cytokines were induced mostly in SIV + Hps pigs in the apical and middle lobe. These results correlated well with localization of gross lung lesions.ConclusionsThe results revealed that SIV increased the severity of lung lesions and facilitated Hps (PIWetHps192/2015) replication in the porcine lung. Furthermore, Hps influenced the SIV nasal shedding. Enhanced Hps and SIV replication, together with stronger systemic and local inflammatory response contributed to a more severe clinical signs and stronger, earlier immune response in co-inoculated animals. We confirmed the previous evidence that single-Hps infection does not produce significant pneumonic lesions but it should be in mind that other strains of Hps may produce lesions different from that reported in the present study.

Far-Infrared Radiation Thermotherapy Improves Tissue Fibrosis in Chronic Extremity Lymphedema.

Fibrosis can enhance the exacerbation of lymphedema, which becomes obvious in late stage II-III lymphedema. However, whether far-infrared radiation thermotherapy (FIRT) can cure lymphedema fibrosis is still lack of research. This research was to investigate the therapeutic effect of FIRT on tissue fibrosis in the treatment of Late stage II-III lymphedema. Patients accepted only FIRT for a total of 20 sessions. The treatment session duration was 2 hours, and a stable machine temperature of 42°C was maintained throughout treatments. Clinical evaluation and laboratory evaluation were conducted before and after FIRT. Clinical outcome measures included circumference of affected extremity, skin elasticity, ultrasound, patients' subjective assessment, and quality of life (QOL). Laboratory outcome measures included serum and local lymphedema tissue fluid concentrations of fibrosis associated cytokines, tissue growth factor beta-1 (TGF-β1), interleukin (IL)-1β, IL-4, IL-18, and caspase-1. Between 2015 and 2016, clinical evaluation of 64 patients with late stage II-III lymphedema was conducted. From this group, 12 cases (18.75%) underwent simultaneous laboratory evaluation. Circumferences of affected extremities improved significantly following treatment (p < 0.001). Skin elasticity of the affected extremity improved significantly (p < 0.05). Ultrasound investigation showed reduced fiber and dense material in the affected tissue (increased gray level 6.322% ± 7.624%, p < 0.001). Patients reported a subjective improvement of their symptoms such as decreased tightness, heaviness, solidity, pain, discomfort, and numbness (p < 0.001, p < 0.001, p < 0.001, p < 0.001, p < 0.001, and p = 0.032, respectively) and improved QOL (p < 0.001). Laboratory results revealed a significant decrease in local tissue fluid concentrations of TGF-β1 (p = 0.041) and IL-18 (p = 0.049) after course completion. FIRT provides an effective treatment for lymphedema tissue fibrosis; it reduces the concentration of fibrosis cytokines in local lymphedema tissues. Consequently, this treatment can reduce the density of fibrosed tissue in the affected extremity, increase skin elasticity, significantly improve clinical symptoms, and improve QOL of patients.

Compartment-specific distribution of human intestinal innate lymphoid cells is altered in HIV patients under effective therapy.

Innate lymphocyte cells (ILCs), a novel family of innate immune cells are considered to function as key orchestrators of immune defences at mucosal surfaces and to be crucial for maintaining an intact intestinal barrier. Accordingly, first data suggest depletion of ILCs to be involved in human immunodeficiency virus (HIV)-associated damage of the intestinal mucosa and subsequent microbial translocation. However, although ILCs are preferentially localized at mucosal surfaces, only little is known regarding distribution and function of ILCs in the human gastrointestinal tract. Here, we show that in HIV(-) individuals composition and functional capacity of intestinal ILCs is compartment-specific with group 1 ILCs representing the major fraction in the upper gastrointestinal (GI) tract, whereas ILC3 are the predominant population in ileum and colon, respectively. In addition, we present first data indicating that local cytokine concentrations, especially that of IL-7, might modulate composition of gut ILCs. Distribution of intestinal ILCs was significantly altered in HIV patients, who displayed decreased frequency of total ILCs in ileum and colon owing to reduced numbers of both CD127(+)ILC1 and ILC3. Of note, frequency of colonic ILC3 was inversely correlated with serum levels of I-FABP and sCD14, surrogate markers for loss of gut barrier integrity and microbial translocation, respectively. Both expression of the IL-7 receptor CD127 on ILCs as well as mucosal IL-7 mRNA levels were decreased in HIV(+) patients, especially in those parts of the GI tract with reduced ILC frequencies, suggesting that impaired IL-7 responses of ILCs might contribute to incomplete reconstitution of ILCs under effective anti-retroviral therapy. This is the first report comparing distribution and function of ILCs along the intestinal mucosa of the entire human gastrointestinal tract in HIV(+) and HIV(-) individuals.

TNFα-induced IKKβ complex activation influences epithelial, but not stromal cell survival in endometriosis.

Can the activity of the IκB kinase (IKKβ) complex in endometriotic cells contribute to endometriotic lesion survival? There is a constitutive activity of the IKKβ catalytic complex in peritoneal and deeply infiltrating lesions that can influence epithelial, but not stromal cell viability. Endometriotic lesions exist in an inflammatory microenvironment with higher local concentrations of cytokines, such as tumour necrosis factor α (TNFα). TNFα stimulates the activation of the IKKβ complex, an important nodal point in multiple signalling pathways that influence gene transcription, proliferation and apoptosis. However, few data on the regulation of IKKβ in endometriotic tissue are currently available. A retrospective analysis of endometriotic tissue from peritoneal, ovarian and deeply infiltrating lesions from 37 women. Basal and activated (phosphorylated) IKKβ concentrations were analysed by western blotting and immunohistochemistry. The relationship between the expression and activation of these proteins and peritoneal fluid (TNFα) concentrations, measured via ELISA, was examined. A subsequent in vitro analysis of TNFα treatment on the activation of IKKβ and the effect on epithelial and stromal cell viability by its inhibition with PS1145 was also performed. Levels of the phosphorylated IKKβ complex in endometriotic lesions had a significant positive correlation with peritoneal fluid TNFα concentrations. Phosphorylated IKKβ complex was more prevalent in peritoneal and deeply infiltrating endometriosis lesions compared with ovarian lesions. IKKβ was present in both epithelial and stromal cells in all lesions but active IKKβ was limited to epithelial cells. TNFα stimulated an increased expression of phosphorylated IKKβ and the inhibition of this kinase with PS1145 significantly influenced ectopic epithelial cells viability but not eutopic epithelial cells, or endometrial stromal cells. In vitro analysis on epithelial cells was performed with immortalized cell lines and not primary cell cultures and only low sample numbers were available for the study. The regulation of aberrant signalling pathways represents a promising yet relatively unexplored area of endometriosis progression. The IKKβ complex is activated by inflammation and is critical nodal point of numerous downstream kinase-signalling pathways, includingNFκB (nuclear factor κB), mTOR (mammalian target of rapamycin) and BAD (Bcl2-antagonist of cell death). This study shows a significant relationship between peritoneal fluid TNFα and IKKβ activation in epithelial cells that will have significant consequences for the continued survival of these cells at ectopic locations through the regulation of downstream pathways. None. The study was funded by the Swiss National Science Foundation (Grant Number 320030_140774). The authors have no conflict of interest to declare.

A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

ABSTRACTIntrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI− mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

Lymph node fibroblastic reticular cell transplants show robust therapeutic efficacy in high-mortality murine sepsis.

Sepsis is an aggressive inflammatory syndrome and a global health burden estimated to kill 7.3 million people annually. Single-target molecular therapies have not addressed the multiple disease pathways triggered by septic injury. Cell therapies might offer a broader set of mechanisms of action that benefit complex, multifocal disease processes. We describe a population of immune-specialized myofibroblasts derived from lymph node tissue, termed fibroblastic reticular cells (FRCs). Because FRCs have an immunoregulatory function in lymph nodes, we hypothesized that ex vivo-expanded FRCs would control inflammation when administered therapeutically. Indeed, a single injection of ex vivo-expanded allogeneic FRCs reduced mortality in mouse models of sepsis when administered at early or late time points after septic onset. Mice treated with FRCs exhibited lower local and systemic concentrations of proinflammatory cytokines and reduced bacteremia. When administered 4 hours after induction of lipopolysaccharide endotoxemia, or cecal ligation and puncture (CLP) sepsis in mice, FRCs reduced deaths by at least 70%. When administered late in disease (16 hours after CLP), FRCs still conveyed a robust survival advantage (44% survival compared to 0% for controls). FRC therapy was dependent on the metabolic activity of nitric oxide synthase 2 (NOS2) as the primary molecular mechanism of drug action in the mice. Together, these data describe a new anti-inflammatory cell type and provide preclinical evidence for therapeutic efficacy in severe sepsis that warrants further translational study.

Local and systemic immune response in pigs during subclinical and clinical swine influenza infection

Local and systemic immune responses in pigs intranasally (IN) and intratracheally (IT) inoculated with swine influenza virus (SIV) were studied. No clinical signs were observed in IN-inoculated pigs, while IT-inoculated pigs developed typical signs of influenza. Significantly higher titres of specific antibodies and changes of haematological parameters were found only in IT-inoculated pigs. Because positive correlations between viral titre, local cytokine concentration, and lung pathology have been observed, we hypothesise that both viral load and the local secretion of cytokines play a role in the induction of lung lesions. It could be that a higher replication of SIV stimulates immune cells to secrete higher amounts of cytokines. The results of the present study indicate that pathogenesis of SIV is dependent on both, the damage caused to the lung parenchyma directly by virus, and the effects on the cells of the host's immune system.

An agent-based model of the response to angioplasty and bare-metal stent deployment in an atherosclerotic blood vessel.

PurposeWhile animal models are widely used to investigate the development of restenosis in blood vessels following an intervention, computational models offer another means for investigating this phenomenon. A computational model of the response of a treated vessel would allow investigators to assess the effects of altering certain vessel- and stent-related variables. The authors aimed to develop a novel computational model of restenosis development following an angioplasty and bare-metal stent implantation in an atherosclerotic vessel using agent-based modeling techniques. The presented model is intended to demonstrate the body’s response to the intervention and to explore how different vessel geometries or stent arrangements may affect restenosis development.MethodsThe model was created on a two-dimensional grid space. It utilizes the post-procedural vessel lumen diameter and stent information as its input parameters. The simulation starting point of the model is an atherosclerotic vessel after an angioplasty and stent implantation procedure. The model subsequently generates the final lumen diameter, percent change in lumen cross-sectional area, time to lumen diameter stabilization, and local concentrations of inflammatory cytokines upon simulation completion. Simulation results were directly compared with the results from serial imaging studies and cytokine levels studies in atherosclerotic patients from the relevant literature.ResultsThe final lumen diameter results were all within one standard deviation of the mean lumen diameters reported in the comparison studies. The overlapping-stent simulations yielded results that matched published trends. The cytokine levels remained within the range of physiological levels throughout the simulations.ConclusionWe developed a novel computational model that successfully simulated the development of restenosis in a blood vessel following an angioplasty and bare-metal stent deployment based on the characteristics of the vessel cross-section and stent. A further development of this model could ultimately be used as a predictive tool to depict patient outcomes and inform treatment options.