Higher Cytokine Concentrations Research Articles

Cytokine Storm in COVID-19: The Current Evidence and Treatment Strategies.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is the pathogen that causes coronavirus disease 2019 (COVID-19). As of 25 May 2020, the outbreak of COVID-19 has caused 347,192 deaths around the world. The current evidence showed that severely ill patients tend to have a high concentration of pro-inflammatory cytokines, such as interleukin (IL)-6, compared to those who are moderately ill. The high level of cytokines also indicates a poor prognosis in COVID-19. Besides, excessive infiltration of pro-inflammatory cells, mainly involving macrophages and T-helper 17 cells, has been found in lung tissues of patients with COVID-19 by postmortem examination. Recently, increasing studies indicate that the “cytokine storm” may contribute to the mortality of COVID-19. Here, we summarize the clinical and pathologic features of the cytokine storm in COVID-19. Our review shows that SARS-Cov-2 selectively induces a high level of IL-6 and results in the exhaustion of lymphocytes. The current evidence indicates that tocilizumab, an IL-6 inhibitor, is relatively effective and safe. Besides, corticosteroids, programmed cell death protein (PD)-1/PD-L1 checkpoint inhibition, cytokine-adsorption devices, intravenous immunoglobulin, and antimalarial agents could be potentially useful and reliable approaches to counteract cytokine storm in COVID-19 patients.

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

Early results of negative pressure wound therapy in treatment of chronic wounds

Abstract Background Chronic wounds may affect only the epidermis and dermis, or they may affect tissues all the way to the fascia. Chronic wounds seem to be detained in one or more of the phases of wound healing. For example, chronic wounds often remain in the inflammatory stage for too long. Aim of the work to assess the effect of negative pressure wound therapy in management of chronic wound. Patients and Methods This is prospective study was conducted on 30 Egyptian volunteers participated; they were 20 males and 10 females, between the age of 18 and 70 years. This study was conducted from July 2017 to July 2018 at Ain shams university specialized hospitals and Alexandria armed forces hospital. Results VAC therapy, together with periodical surgical wound debridement and specific antibiotic therapy, could be helpful to promote and accelerate wound healing of foot lesions after restoration of an adequate distal blood flow through surgical revascularization or angioplasty. Conclusion Negative-pressure wound therapy (NPWT) devices are designed to apply controlled suction to a wound bed at continuous or intermittent pressure settings to stimulate wound closure. Negative pressure can result in numerous alterations in the wound environment, including removal of excess exudate containing high concentrations of proteases and inflammatory cytokines, stimulation of senescent cells, mobilization of macrophages, and stimulation of angiogenesis.

Cytokine concentrations throughout pregnancy and risk for psychosis in adult offspring: a longitudinal case-control study

Schizophrenia has been associated with pregnancy and birth complications and fetal exposure to inflammation is thought to be a common underlying mechanism. However, whether the risk is specific to particular phases of pregnancy is unclear. The aim of this study was to characterise and compare longitudinal patterns of maternal serum concentrations of cytokines across pregnancy between offspring who were later ascertained to have a psychotic disorder, non-psychotic siblings of these cases, and unrelated, non-psychotic individuals who served as controls. The National Collaborative Perinatal Project was a large-scale prospective longitudinal study that assessed the effects of perinatal factors on infant and child development. At sites across the USA, over 50 000 pregnant women were enrolled during prenatal clinical visits between 1959 and 1965. The present study draws from the Philadelphia cohort, which includes 9236 surviving offspring of 6753 pregnant women. Psychotic disorder diagnoses in adulthood were assessed with review of medical records and were confirmed with a validation study. Concentrations of TNFα, IL-1β, IL-5, IL-6, IL-8, IL-10, and IL-17a were assessed using a multiplex bead assay in archived maternal serum samples collected across prenatal visits and birth. We characterized cytokine patterns with linear mixed models. Our final sample comprised 90 cases, 79 siblings (of 40 cases), and 273 matched controls. Concentrations of proinflammatory cytokines TNFα, IL-1β, and IL-6 were significantly higher in maternal serum of offspring who later developed psychosis compared with maternal serum of matched controls. These differences were greatest in the first half of pregnancy (7-20 weeks), with no difference observed during the second half of pregnancy. Our results suggest that exposure to high maternal proinflammatory cytokine concentrations in early pregnancy might play a part in psychosis. These findings place the timing of risk associated with maternal inflammation much earlier in prenatal development than previously documented in humans and provide insight into a potential developmental pathway to the disorder. National Institute of Mental Health (P50) Silvio O Conte Center at Johns Hopkins, Stanley Foundation, March of Dimes, Yale University, National Science Foundation, and National Institute of Child Health and Human Development/Division of Intramural Population Health Research.

Vascular Leak and Hypercytokinemia Associated with Severe Fever with Thrombocytopenia Syndrome Virus Infection in Mice.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral hemorrhagic fever (VHF) endemic to China, South Korea, Japan, and Vietnam. Here we characterize the pathogenesis and natural history of disease in IFNAR-/- mice challenged with the HB29 strain of SFTS virus (SFTSV) and demonstrate hallmark features of VHF such as vascular leak and high concentrations of proinflammatory cytokines in blood and tissues. Treatment with FX06, a natural plasmin digest product of fibrin in clinical development as a treatment for vascular leak, reduced vascular permeability associated with SFTSV infection but did not significantly improve survival outcome. Further studies are needed to assess the role of vascular compromise in the SFTS disease process modeled in IFNAR-/- mice.

Is Platelet Rich Plasma (PRP) an Effective Treatment for Pressure Ulcers?

Background: Platelet rich plasma (PRP) has been used as an effective treatment over the years indifferent medical and surgical fields. It has a high concentration of cytokines and growth factors that play a significant role in the wound healing process. This literature review is looking at whether PRP is effective in treating pressure ulcers as they are a major problem causing a significant increase in health care costs, have a huge impact on patients’ quality of life and cause great morbidity and mortality. Study design and methods: A comprehensive review of the literature was performed. Pubmed, Ovid Medline, OvidEMBASE and CINHAL were used to conduct the literature search using the following MESH headings: PRP, Platelet rich plasma, Chronic wound*, pressure ulcer*, pressure sore*. Cochrane library was searched for systemic reviews and reference lists of relevant publications were used to identify further studies. Results: The literature search identified studies that looked at PRP’s effect on different chronic wounds including pressure ulcers in terms of healing. Some studies looked at pressure ulcers separately. The included studies in this literature review are: one randomised double blind controlled trial, three randomised open label controlled trial, one observation study, five case series and two case reports. Conclusion: PRP promotes the healing of chronic wounds. Individual case series and case reports showed that PRP aids the healing of pressure ulcers improving their vascularity and decreasing ulcer area and volume. Also, some reported significant reduction in healing time. However, further research and randomised controlled trials focusing on the effect of PRP on pressure ulcers need to be conducted in order to support the hypothesis.

Anthropometry before Day 46 and Growth Velocity before 6 Months of Guatemalan Breastfed Infants Are Associated with Subclinical Mastitis and Milk Cytokines, Minerals, and Trace Elements

Subclinical mastitis (SCM) is an inflammatory condition of the mammary gland, but its association with human milk composition and infant growth is not well described. We investigated whether SCM, milk cytokines, and/or estimated intakes of milk minerals and trace elements were associated with infant anthropometry or growth velocity before 6 mo. Breast milk was collected from Mam-Mayan mothers (n=114) at both early (2-46 d) and established (4-6 mo) lactation. Concentrations of 9 elements (sodium, potassium, calcium, magnesium, copper, iron, manganese, selenium, zinc) analyzed by inductively-coupled plasma mass spectrometry were used to estimate infants' daily intakes. Concentrations of 4 cytokines were measured. Milk concentrations and infants' estimated elemental intakes and anthropometric measurements during early and established lactation were compared by SCM status. Multiple regression was used to identify factors associated with infant growth attainment (<46 d) for infant weight- (WAZ), length- (LAZ), and head circumference-for-age (HCAZ) and weight-for-length (WLZ) z scores and with growth velocity (expressed as Δ/d) from early to established lactation. SCM prevalence was higher in early (30%) than established (10%) lactation. Breast milk of SCM mothers had higher cytokine concentrations and higher magnesium in early and higher selenium concentrations in both early and established lactation (Padj<0.0121). At day 46, regression models showed inverse associations of SCM with WLZ and IL-1β with LAZ (Padj<0.0150). In contrast, linear growth velocity was positively associated with IL-1β measured in early lactation (Padj<0.0124), whereas cranial growth velocity was positively associated with IL-8 measured during established lactation ( Padj<0.0124). Despite a high prevalence of inadequate intakes, only infants' intake of milk magnesium during early lactation was associated with linear growth velocity in breastfed infants <6 mo. Evidence shows that SCM, breast-milk cytokines, and infants' estimated intakes of select elements are independently associated with growth attainment and growth velocity during lactation.

ENVIRONMENTAL OPTIMIZATION ENABLES MAINTENANCE OF QUIESCENT HEMATOPOIETIC STEM CELLS EX VIVO

Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our novel culture system provides a means to evaluate properties of steady state HSCs and test effects of defined factors in vitro under near-physiological conditions.

Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo

Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate invitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our culture system provides a means to evaluate properties of steady-state HSCs and test effects of defined factors invitro under near-physiological conditions.

Assessment of Cytokines, Biochemical Markers of Malnutrition and Frailty Syndrome Patients Considered for Lung Transplantation

BackgroundThe aim of the study was to investigate the serum concentration of cytokines and biochemical markers of malnutrition in correlation with frailty syndrome in patients qualified for lung transplantation (LTx). MethodsThe study population comprised 31 potential lung recipients, including 18 patients with idiopathic lung disease, 12 patients with chronic obstructive pulmonary disease, and 1 patient with bronchiectasis who qualified for a LTx. Cytokine serum levels were assessed using commercially available enzyme-linked immunosorbent assay kits and the Luminex 200 platform (ProcartaPlex Hu Cytokine/Chemokine Panel 1A 34plex, Invitrogen, Carlsbad, Calif., United States). The patients were also asked to complete a questionnaire, the Clinical Frailty Scale. ResultsAll patients were found to have higher cytokine concentrations (IL6, IL 2,IL18, IL23, IL 12p70, IL 10, IL 7). No statistically significant differences in the analyzed cytokines were noted when the men's results were compared to those of the women. There were no significant differences between patients who scored 6 vs 7 points on the Canadian Study of Health and Aging Function Scale. In comparing chronic obstructive pulmonary disease to idiopathic lung disease patients, no significant differences were observed in the analyzed cytokine values. Significant correlations were observed between the analyzed cytokines and age of the patients, C-reactive protein, triglycerides, transferrin, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. ConclusionsOur findings indicate that cytokines may not have a statistically significant effect on the parameters of the frailty syndrome. The results require further investigations on larger study groups. The findings suggest that the analyzed cytokines may play a proinflammatory role in the end stages of lung diseases, but further studies are needed to evaluate whether these cytokines could be used as biomarkers in this group of patients.

Repeated Measures of Cervicovaginal Cytokines during Healthy Pregnancy: Understanding "Normal" Inflammation to Inform Future Screening.

This study aimed to describe characteristics of cervicovaginal cytokines obtained during pregnancy from women who subsequently delivered at term. We used repeated measures of 20 cervicovaginal cytokines, collected on average on a monthly basis, from the second to the ninth month of gestation among 181 term pregnancies in the Mexico City Pregnancy Research on Inflammation, Nutrition, & City Environment: Systematic Analyses cohort (2009-2014). Cytokines were quantified using multiplex assay. Cytokine distributions differed more between than within cytokines. Across trimesters, cytokines interleukin (IL)-1Ra, IL-1α, and IL-8 consistently had high concentrations compared with other measured cytokines. Cytokine intraclass correlation coefficients ranged from 0.41 to 0.82. Spearman's correlation coefficients among cytokine pairs varied but correlation directions were stable; 95.3% of the 190 correlation pairs remained either negative or positive across trimesters. Mean longitudinal patterns of log-transformed cytokines from Tobit regression varied across but less within cytokines. Although mean concentrations of cervicovaginal cytokines among term pregnancies were high, they were largely stable over time. The high cytokine concentrations corroborate that pregnancy is associated with an active inflammatory state. These characterizations may serve as a baseline for comparison to other obstetric outcomes, which may be helpful in understanding deviations from normal gestational inflammation.

Biological Characteristics of Subsilicone Oil Fluid and Differences With Other Ocular Humors.

PurposeSubsilicone oil fluid (SOF) in eyes with silicone oil (SO) endotamponade possibly has a role in complications (e.g., vision loss); thus, we aimed to examine inflammatory cytokine and electrolyte levels and retinal glial cell viability in SOF.MethodsWe measured major inflammatory cytokine levels and electrolytes in SOF and compared them with those in vitreous fluid (VF) and anterior chamber fluid (ACF). We analyzed the correlation between inflammatory cytokines and retinal thickness in SO-filled eyes. Further, we measured the MIO-M1 cell viability in medium with SOF and compared it with that containing VF.ResultsWe collected and examined 57 SOF, 22 ACF, and 21 VF samples from eyes with PVR, PDR, RD, and MH. Interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 levels in SOF were significantly higher than those in ACF. There was no significant difference for all cytokines between SOF and VF. Retinal thickness changes during SO endotamponade were not correlated with the presence of any inflammatory cytokines. Levels of ferrous iron, but not of potassium, showed a significant decrease in SOF compared with VF. The WST-1 assay showed that SOF-added medium induced higher MIO-M1 cell viability than VF-added medium.ConclusionsWe found no significant correlation between the change in the retinal thickness and cytokine levels, but SOF contains higher concentrations of cytokines and lower concentrations of ferrous iron and can be biologically distinguished from ACF and VF.Translational RelevanceNovel knowledge of inflammatory cytokine levels and electrolytes in SOF provides better understanding of pathology of SO-filled eyes.

Robustness and Information Transfer within IL-6-induced JAK/STAT Signalling

Cellular communication via intracellular signalling pathways is crucial. Expression and activation of signalling proteins is heterogenous between isogenic cells of the same cell-type. However, mechanisms evolved to enable sufficient communication and to ensure cellular functions. We use information theory to clarify mechanisms facilitating IL-6-induced JAK/STAT signalling despite cell-to-cell variability. We show that different mechanisms enabling robustness against variability complement each other. Early STAT3 activation is robust as long as cytokine concentrations are low. Robustness at high cytokine concentrations is ensured by high STAT3 expression or serine phosphorylation. Later the feedback-inhibitor SOCS3 increases robustness. Channel Capacity of JAK/STAT signalling is limited by cell-to-cell variability in STAT3 expression and is affected by the same mechanisms governing robustness. Increasing STAT3 amount increases Channel Capacity and robustness, whereas increasing STAT3 tyrosine phosphorylation reduces robustness but increases Channel Capacity. In summary, we elucidate mechanisms preventing dysregulated signalling by enabling reliable JAK/STAT signalling despite cell-to-cell heterogeneity.

Twice-weekly exercise training reduces oxidative stress and proinflammatory cytokine levels in elder women

Abstract Aim: Reactive oxygen species and high concentrations of proinflammatory cytokines are related to diseases that are often triggered during the aging process. This study aimed to investigate the effect of moderate-intensity physical exercise, twice a week, on oxidative stress and inflammation biomarkers. Methods: Participants were older women aged 60-70 years, engaged in a moderate-intensity exercise program carried out in 60 minutes sessions, twice a week, for 24 weeks. Exercise sessions consisted of step, resistance and stretch exercises. Thiobarbituric acid reactive substances (TBARS), serum iron and cytokines IL-6, IL-1β and IL-1ra were determined using specific kits. Physical fitness was assessed using tests provided by AAPHERD. A paired t test was performed. Results: Moderate-intensity exercise program provided a reduction in lipid peroxidation (27%) and in the serum release of prooxidant iron ions (40%). Cytokine levels were reduced by 37% for IL-6, 16% for IL-1β and 32% for IL-1ra. Improvements in cardiorespiratory fitness (13%), muscular endurance (11%) and flexibility (12%) were also observed. Conclusion: Diminished redox state and inflammation were obtained using a twice-weekly exercise program. These results have important implications for older adults who are unable or unwilling to attend exercise programs more than twice a week. Moreover, these results could re-establish the minimum exercise activity necessary for obtaining health benefits in the elderly population.

THE CONTENT OF INTERLEUKINS IN ORAL FLUID OF PATIENTS WITH NON-REMOVABLE METAL CERAMIC PROSTHESES

The dental bridges are often used to replace missing teeth. Currently, the most common are the metal-ceramic structures, which have the high mechanical properties and are aesthetic and functional, because they restore chewing efficiency by 85-100% and provide a high level of adaptation to them. However, in the presence of metallic and combined prostheses in the oral cavity, the pathological changes may occur, taking into account that in the oral cavity, the non-removable dentures are foriegn bodies affecting the tissues and the environment of the oral cavity. The effect of almost all artificial bridges on the tissues of the marginal periodontal disease depends on the level of the location of the bridge margin and the depth of the immersion under the gum. In the presence of such processes, the main factor is the specific antimicrobial protection, which is carried out by immunoglobulins; in the oral cavity, there are present only IgA, IgG, IgM of six classes. Thus, in the prosthetic dentistry one of the important problems is the connection of prosthetic constructions and the state of the oral cavity – homeostasis. The dental prostheses activate the lipid peroxidation and reduce the antioxidant defense factors. On the 7th day after bridge fixation, there is a significant decrease in the level of antioxidant enzymes and immunoglobulins and high concentrations of anti-inflammatory cytokines and interleukins. It is found that on the first stage of dental prosthesis the activity of lysozyme is sharply reduced and then restored very slowly. There is reduced not only non-specific reactivity (lysozyme), but also specific (lgA, IgG, and IgE) affected by dental prostheses, in particular, from acrylic plastics.&#x0D; The clinical data of 46 patients, age between 29 – 73 years old (56% of women, 44% of men), which had the non-removable metal ceramic crowns and bridge prostheses, and the indicators of immune markers in the oral fluid were used. In a determination of IL-1β concentration in the saliva of the tested patients, there was found a significant increase by a factor of 12 in the control parameters, it indicates on the high activity of the monocyte-macrophage lineage cells. In the saliva, the increased content of IL-1β confirms its role in the local inflammatory process and indicates the activation of endothelial cells and connective tissue. In the group of patients, IL-6 salivary content exceeds the level in healthy people by 3.66 times, indicating a protective mechanism that is necessary for the initial phase of inflammation. The IL-6 high level is a prognostic adverse factor, which can indicate the progression of the inflammatory process and the increase of membrane-destructive disorders in the cells.&#x0D; Monocytes and macrophages, activated by periodontopathogenic microbes, produce all cascade of proinflammatory interleukins, causing an imbalance between them. The damage of the periodontal tissue and resorption of the alveolar bone occur. In periodontal disease, the most damaging effect is characteristic for IL-1β and TNF-α.&#x0D; In the saliva of patients, there was detected an increased TNF-α level by 2.89 times compared to control, that has a negative inotropic effect, stimulates apoptosis, and increases vascular wall permeability. In prolonged prosthesis in the saliva of patients, there was found the higher level of IL-4 than in healthy individuals by 1.24 times. IL-4 is an anti-inflammatory cytokine, an activator and chemokinetic factor for phagocytes.&#x0D; The cytokines ensure the consistency and completeness of the immune response. Most of them induce inflammatory reaction and acute phase response of the organism and can exhibit the immunopathological effect on the tissues. At the systemic level, cytokines modulate the key protective reactions of the organism.

Inflammatory Cytokine Profiles of Semen Influence Cytokine Responses of Cervicovaginal Epithelial Cells.

Genital inflammatory cytokine responses increase HIV risk. Since male partner semen is a complex mixture of immune-modulatory prostaglandins and cytokines, we hypothesized that exposure to semen may influence genital inflammation in women. Here, we investigated cytokine response kinetics of cervical cells following stimulation with seminal plasma from HIV-negative and HIV-positive men characterized as having low or high concentrations of inflammatory cytokines. Irrespective of the HIV status or semen cytokine profile, in vitro stimulation of cervical cells with seminal plasma resulted in significantly elevated concentrations of secreted IL-6, IL-8, TNF-β, MCP-1, GM-CSF, and VEGF within 8 h of stimulation, which tended to decline by 24 h, although this was only significant for TNF-β. Consistent with this, cervical cells responded to seminal plasma with increases in IL-8 and IL-1β mRNA expression of 10-fold. These findings suggest that the impact of semen on local female genital cytokines is likely transient. Although these findings suggest that the impact of semen on local female genital cytokines may not be sustained long-term, this heightened genital inflammation may have implications for HIV risk in women.

Pretreatment Cerebrospinal Fluid Bacterial Load Correlates With Inflammatory Response and Predicts Neurological Events During Tuberculous Meningitis Treatment.

The Mycobacterium tuberculosis load in the brain of individuals with tuberculous meningitis (TBM) may reflect the host's ability to control the pathogen, determine disease severity, and determine treatment outcomes. We used the GeneXpert assay to measure the pretreatment M. tuberculosis load in cerebrospinal fluid (CSF) specimens from 692 adults with TBM. We sought to understand the relationship between CSF bacterial load and inflammation, and their respective impact on disease severity and treatment outcomes. A 10-fold higher M. tuberculosis load was associated with increased disease severity (odds ratio, 1.59; P = .001 for the comparison between grade 1 and grade 3 severity), CSF neutrophil count (r = 0.364 and P < .0001), and cytokine concentrations (r = 0.438 and P < .0001). A high M. tuberculosis load predicted new neurological events after starting treatment (P = .005, by multinomial logistic regression) but not death. Patients who died had an attenuated inflammatory response at the start of treatment, with reduced cytokine concentrations as compared to survivors. In contrast, patients with high pretreatment CSF bacterial loads, cytokine concentrations, and neutrophil counts were more likely to subsequently experience neurological events. The pretreatment GeneXpert-determined M. tuberculosis load may be a useful predictor of neurological complications occurring during TBM treatment. Given the evidence for the divergent pathogenesis of TBM-associated neurological complications and deaths, therapeutic strategies to reduce them may need reassessment.

Robust erythroid differentiation system for rhesus hematopoietic progenitor cells allowing preclinical screening of genetic treatment strategies for the hemoglobinopathies

γ-globin expression can be induced by various gene modification strategies, which could be beneficial for hemoglobin (Hb) disorders. To translate promising ideas into clinics, large animal models have proven valuable to evaluate safety and efficacy of the approaches; however, in vitro erythroid differentiation methods have not been established to determine whether they can be modeled in nonhuman primates. We optimized erythroid differentiation culture to produce high-level adult Hb from rhesus hematopoietic progenitor cells by using low (LC) or high cytokine concentration (HC) protocols with or without feeder cells. In addition, we established rhesus globin protein analysis using reverse-phase high performance liquid chromatography and mass spectrometry. Robust adult Hb production at protein levels was observed in the LC protocol when feeder cells were used, whereas the HC protocol resulted in higher baseline fetal Hb levels (P < 0.01). We then compared lentiviral transduction of rhesus cells between serum-containing LC media and serum-free StemSpan-based differentiation media, revealing 100-fold more efficient transduction in serum-free differentiation media (P < 0.01). Finally, rhesus CD34+ cells were transduced with lentiviral vectors encoding artificial zinc finger proteins (ZF-Ldb1), which can reactivate γ-globin expression via tethering the transcriptional co-regulator Ldb1 to γ-globin promoters, and were differentiated in the optimized erythroid differentiation method. This resulted in marked increases of γ-globin levels compared with control groups (P < 0.01). In conclusion, we developed an efficient rhesus erythroid differentiation protocol from hematopoietic progenitor cells with low fetal and high adult Hb production. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells.

The functional potency of natural killer cells in response to IL-2/IL-15/IL-21 stimulation is limited by a concurrent upregulation of Tim-3 in bladder cancer

For reasons not completely clear, natural killer (NK) cells from tumor patients displayed multiple exhaustion features and could not be completely restored even when the inhibitory signals from the intratumoral environment had ceased to exist. Here, we found that the circulating NK cells from bladder cancer patients presented significantly reduced cytotoxicity than the circulating NK cells from healthy volunteers. This impairment in cytotoxicity resulted in part from an overrepresentation of Tim-3+ NK cells in bladder cancer patients. Interestingly, patients with higher frequency of Tim-3+ NK cells tended to present higher frequency of Gal-9+ cells in tumor. Exogenous Gal-9 significantly reduced the cytotoxicity of Tim-3+, but not Tim-3-, NK cells. Patients with better prognosis presented lower levels of Tim-3+ NK cells and Gal-9+ tumor cells. We then attempted to improve the cytotoxicity of NK cells using a combination of exogenous cytokines. IL-2 + IL-15 and IL-2 + IL-21 significantly enhanced, but could not completely restore, the cytotoxicity of NK cells in bladder cancer patients. Notably, when the cytokine concentration increased from intermediate levels to high levels, the cytotoxicity of NK cells from healthy volunteers significantly increased with a strong upward trend, whereas the cytotoxicity of NK cells from bladder cancer patients plateaued at intermediate levels. Further examination revealed that high cytokine concentration significantly increased the Tim-3 expression in NK cells from bladder cancer patients. Blocking Tim-3 not only improved the cytotoxicity of NK cells from bladder cancer patients, but also eliminated the plateauing effect when the NK cells were stimulated with high concentrations of cytokines. Together, these data suggested that proinflammatory cytokines could moderately improve NK cell cytotoxicity in bladder cancer patients. However, the effect was limited due to a concurrent upregulation of Tim-3.

Abstract IA13: Spatio-temporal quirks of IFN-γ regulation of immune responses in tumors

Abstract Recent preclinical and clinical studies have highlighted how downregulation of IFN-γ responsiveness allow tumors to resist T cell-driven immunotherapy. Given the significance of IFN-γ in upregulating antigen presentation and T cell responses, we investigated the spatio-temporal characteristics of inflammation and the interplay between tumors and immune infiltrates. Cytokine-mediated communication allows immune cells to achieve a context-appropriate response, but the distance and the timing over which this communication happens in immune tissues and tumors is not known. We show that a simple diffusion-consumption mechanism quantitatively describes the spatial spread of cytokines (IL2, IFNg, IL-4) both in model systems in vitro and in vivo; this mechanism explains how localized niches of high cytokine concentrations self-assemble. The niche sizes are determined by the competition between the cytokine diffusion and the consumption rate of the cells around the cytokine source. In turn, the variability of niche sizes accounts for the cell-to-cell heterogeneity of cytokine responses in dense tissues. We also uncover a surprising mechanism to enforce tissue-level memory of inflammation: cancer and immune cells are shown to rely on their surface phosphatidylserine to mediate cytokine catch and release and convert transient exposure to Th1 cytokines into long-term inflammation. To conclude, we will discuss how quantitative approaches can help address fundamental issues in systems immunology and predict their significance for cancer immunotherapies. Citation Format: Altan-Bonnet Grégoire. Spatio-temporal quirks of IFN-γ regulation of immune responses in tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA13.