Concentrations Of IFN-g Research Articles

Immunological aspects of the use of melatonin in experimental thermal trauma

The prevalence of thermal trauma, the high risk of infectious and non-infectious short- and long- term complications, and the limited effectiveness of the therapeutic approaches used are prerequisites for the search and pathogenetic justification of new therapies, among which the endogenous homeostasis regulator with pleiotropic properties melatonin attracts attention.The aim of the work is to investigate the immunological aspects of intraperitoneal use of melatonin (MT) in experimental thermal trauma (TT).The work was performed on 158 rats of the Wistar line, grade III TT and a relative area of 3.5% were simulated by skin immersion in water at 98-99 °C for 12 s. MT was administered intraperitoneally daily at a dose of 10 mg/kg for 5 days. The quantitative composition of blood cells was evaluated on a hematological analyzer. Plasma concentrations of IL-4, TNFa, IFNg, and CRP were determined on an automatic enzyme immunoassay using rat-specific test systems, and MT by capillary electrophoresis.With experimental TT, against the background of a progressive increase in the number of leukocytes in the blood from 5 to 20 days due to neutrophils, monocytes, basophils, the number of lymphocytes decreases. With TT, the concentration of CRP increases in serum on days 5 and 10. The content of TNFa, IL-4 increases on days 5, 10 and 20 in the absence of significant changes in the concentration of IFNg. The concentration of serum MT does not change significantly. Intraperitoneal use of MT in TT leads to a partial restoration of the number of lymphocytes in the blood on day 5. Evaluation of the cytokine profile in serum revealed a decrease in the concentration of TNFa on days 10 and 20, no significant changes in the concentration of IL-4 and IFNg were recorded, the concentration of CRP decreased on day 5. The concentration of serum MT increases by 5 days.With TT on the 5th, 10th, 20th day of the experiment, the number of neutrophils, monocytes, basophils in the blood increases, decreases – lymphocytes, the serum content of CRP, TNFa, IL-4 increases, the content of IFNg and melatonin does not change. Intraperitoneal use of MT in TT partially restores the number of lymphocytes in the blood, the concentration of CRP, TNFa. A decrease in serum concentrations of TNFa and CRP in TT under the conditions of MT use suggests a limitation of the acute phase response as a consequence of the antioxidant, anti-inflammatory effect of MT, which can accelerate healing and reduce the area of the lesion of TT.

Co-Stimulation of CD28/CD40 Signaling Molecule Potentiates CD19CAR-T Cell Functions and Stemness

Introduction: Adoptive T-cell therapy of gene-modified to express CD19 chimeric antigen receptor (CD19CAR) has achieved promising clinical outcomes in B-cell malignancies. However, the recurrences after therapies still occur due to the poor CAR-T persistence. Various approaches have been studied including signaling domain modification to improve T-cell efficacy. The nuclear factor-κB (NF-κB) which interestingly exists in both T- and B-cells is a crucial signaling pathway for T-cell persistence. Furthermore, CAR-T memory signatures drive robust T-cell proliferation. Consequently, we developed the third generation CD19CAR incorporated dual T- and B-cell costimulatory molecules; CD28 and CD40 (CD19.28.40z), to enhance CAR-T cell proliferation and tumoricidal activity. Methods: The anti-CD19CAR incorporated CD28/CD40 co-stimulatory domain; CD19scFv-CD28TM-CD28/CD40-CD3z-tEGFR, was generated. The CAR gene was subsequently cloned into retroviral packaging cells and transduced into primary CD3 cells. The CAR+-T cells were enriched using biotinylated anti-EGFR mAb and further expanded with anti-CD3/CD28 beads or gamma-irradiated EBV-LCL cells. These expanded cells were then used for downstream experiments compared with control CD19CARs including CD19.28z, CD19.BBz, and CD19.40z CAR-T cells. Results: All CD19CAR genes were successfully transduced into primary CD3cells. We first determined the downstream T-cell signaling and found the higher baseline pNF-kB and pRelB of CD19.28.40z CAR-T cells compared to others. We then investigated the ability of CD19 CAR T-cell proliferation by coculturing with gamma-irradiated CD19-expressing K562 cells. Surprisingly, the novel CD19.28.40z exhibited robust T-cell proliferation and persistence throughout 2 weeks of the culture regardless of IL-2 supplementation. The long-term co-culture assay unveiled that CD19.28.40z CAR-T cells could effectively eradicate tumor cell growth until the end of the experiment at an effector to target cell ratio of 1:16, compared to control CAR T-cells. In term of cytokine secretion assays, the similar level of IL-2, TNF-a, and IFN-g concentrations were observed among CAR constructs after being stimulated with target cells. To mimic persistent antigen stimulation in vivo, we performed three consecutive weekly antigen stimulation that exhibited the greater CD19.28.40z CAR-T cell expansion without influencing T-cell fate and exhaustion. We interrogated the intrinsic genes underlying CD19.28.40z CAR-T cell responses compared to conventional CAR-T cells following CD19 antigen exposures using RNA-sequencing. The results confirmed the significant up-regulated genes associated with T-cell proliferation and differentiation, NF-κB pathway, WNT pathway, and CD4+ phenotypes as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway (Figure 1). Regarding T-cell memory phenotypes, we observed the enrichment of genes toward T-cell stemness particularly SELL, IL-7r, TCF7, and KLF2 compared to conventional CAR-T cells. To evaluate the potential anti-tumor efficiency in vivo, we studied NALM-6- and Raji-bearing NSG mice treated with a low CD19CAR-T cell dosage to assess the functional limits of different CAR structures. The CD19.28.40z CAR-T cells demonstrated potent tumoricidal activity as well as prolonged overall survival in both NSG mice models compared to control CAR-T cells (Figure 2). Conclusions: The CD28/CD40 costimulatory molecule potentiates NF-κB signaling and memory signatures enhancing CD19CAR-T cell proliferation and persistence which translates into greater anti-tumor efficacy in both in vitro and xenograft models. The simplified structural modification of dual T-/B-cell signaling molecule could be advantageous to maximize the CAR-T cell functions and stemness. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Рівень забезпеченості вітаміном D та особливості продукції IFN-γ і IL-4 у дітей раннього віку з повторним бронхообструктивним синдромом

Актуальність. Бронхообструктивний синдром (БОС), зокрема його повторний перебіг у дітей раннього віку, є актуальною проблемою сучасної педіатрії. Обтяжений алергологічний анамнез, прояви атопії традиційно розглядаються як фактори ризику повторних епізодів БОС, які, однак, присутні не в усіх випадках. Останнім часом обговорюється можлива роль вітаміну D (VD) у формуванні схильності до повторних епізодів БОС з огляду на його протиінфекційну дію, що забезпечується шляхом активації імунних механізмів. Мета: вивчення рівня забезпеченості VD і особливостей продукції інтерферону гамма (INF-g) та інтерлейкіну-4 (IL-4) у сироватці крові в дітей раннього віку з повторними епізодами БОС. Матеріали та методи. Обстежені 120 дітей віком від 6 місяців до 3 років з клінічним діагнозом «гострий обструктивний бронхіт» (J20), які були розподілені на дві групи (I група — 60 дітей з епізодичним БОС, II група — 60 дітей із повторним БОС). Групу контролю становили 30 клінічно здорових дітей віком від 6 місяців до 3 років. Усім хворим проводили оцінку анамнестичних даних, у тому числі рівня інсоляції, ступеня тяжкості БОС за 12-бальною оціночною шкалою, загальне клінічне обстеження, пульс­оксиметрію, розраховували прогностичний індекс бронхіальної астми (API). Лабораторні дослідження включали визначення на 2–3-й день захворювання концентрації 25-гідроксивітаміну D (25(OH)D) у сироватці крові електрохемілюмінесцентним методом на аналізаторі Cobas e411 (серійний номер 1041-24, виробник Roche Diagnostics GmbH, Німеччина), сироваткових концентрацій IFN-g, IL-4 методом імуноферментного аналізу за допомогою наборів «ІФА-Бест» (виробник «Вектор-Бест», Російська Федерація) та загального кальцію (Са) за загальноприйнятою методикою. При аналізі отриманих даних використовували непараметричні статистичні критерії. Різниця між показниками, що порівнювались, вважалась вірогідною при р < 0,05. Результати. Більшість пацієнтів із повторним перебігом захворювання (56,7 %) мали 4 епізоди БОС за останні 12 місяців. Аналіз анамнестичних показників, у тому числі алергологічного анамнезу, не виявив суттєвих відмінностей між групами (p > 0,05). У 78,3 % пацієнтів II групи був негативний API. Тяжкий перебіг БОС удвічі частіше реєструвався серед дітей II групи порівняно з I групою (23,3 і 10,0 % відповідно, c2 = 4,50, р = 0,034). Середній вміст 25(OH)D у сироватці крові у дітей I групи становив 33,0 (28,19; 41,97) нг/мл; II групи — 13,68 (7,96; 19,51) нг/мл (p < 0,001). Рівень забезпеченості VD у 75 % дітей II групи відповідав критеріям дефіциту, тоді як у I групі даний показник становив 6,6 % (c2 = 57,98, p < 0,001). Виявлено обернену залежність між рівнем VD і частотою (r = –0,27, р = 0,002), та тяжкістю перебігу БОС (r = — 0,24, р = 0,008). Рівень загального Са в сироватці крові в обох групах був у межах нормальних величин. Середній рівень IFN-g у дітей І групи становив 22,69 (8,98; 41,44) пг/мл та ІІ групи — 21,08 (8,57; 37,03) пг/мл, обидва показники були вищими, ніж у групі контролю, — 12,18 (5,31; 12,92) пг/мл (p = 0,016 для обох показників). Ми виявили, що досліджувані групи відрізнялись між собою за рівнем саплементації VD, показник якого становив 60 % у I групі та 5 % — у дітей II групи (p < 0,001). Середній вміст IL-4 у сироватці крові в дітей I групи був вірогідно вищим порівняно з відповідними показниками II групи та групи контролю (р = 0,012 за U-критерієм Манна — Уїтні для обох показників). Виявлено обернений кореляційний зв’язок між частотою БОС і вмістом IFN-g у крові в дітей I групи (r = –0,45, р < 0,001) та між ступенем тяжкості БОС і сироватковою концентрацією IL-4 у дітей II групи (r = –0,28, р = 0,030). У пацієнтів із повторним БОС також був виявлений прямий кореляційний зв’язок між вмістом IL-4 та IFN-g (r = 0,41, р < 0,001). Ніяких кореляцій між VD та імунологічними показниками ми не виявили. Висновки. БОС у дітей раннього віку переважно має вірусну етіологію. Тяжкий перебіг захворювання частіше зустрічається в дітей із повторними епізодами БОС, для переважної більшості яких характерний негативний прогностичний індекс бронхіальної астми. У 3/4 пацієнтів із повторним БОС рівень забезпеченості VD знаходиться в межах дефіциту. VD обернено корелює з тяжкістю й частотою епізодів БОС. На рівень забезпеченості вітаміном D у дітей раннього віку найбільше впливає рівень його саплементації. Перебіг БОС у дітей раннього віку супроводжується підвищенням рівня IFN-g у сироватці крові в гострий період захворювання незалежно від частоти епізодів БОС в анамнезі. За вмістом IL-4 у сироватці крові досліджувані групи не відрізняються між собою, при цьому за наявності повторних епізодів даний показник прямо корелює з IFN-g. Отже, IFN-g і IL-4 суттєво не впливають на частоту та ступінь тяжкості БОС у дітей раннього віку на відміну від вітаміну D, що може розглядатися як предиктор тяжкого перебігу БОС і розвитку його повторних епізодів.

The Promotion of Peony Seed Proteolysis Product to the Immune Efficacy Induced by ptfA Gene DNA Vaccine of Avian Pasteurella multocida

Background: Avian Pasteurella multocida is one of the pathogens that affect the health of poultry. The protective efficacy of traditional attenuated vaccine is not ideal. In previous study, we prepared ptfA gene DNA vaccine of avian P. multocida. However, the protective effect of ptfA gene DNA vaccine was inferior to the attenuated vaccine. Therefore, it is necessary to improve the immune efficacy of avian P. multocida DNA vaccine, such as screening for novel adjuvant. Methods: In this study, the peony seed proteolysis product was gavaged to chickens before DNA vaccination or was added to the ptfA gene DNA vaccine as adjuvant. These vaccines were administered to chickens and the serum antibody, lymphocyte proliferation levels, IFN-g, IL-2, IL-4 and IL-6 concentrations secreted by peripheral blood lymphocytes were determined. After challenging with virulent avian P. multocida, survival time and protective efficacy was evaluated. Result: Following vaccination, no significant differences in antibody levels and concentrations of IL-4 among the DNA vaccine group, adjuvant-DNA vaccine group and gavaged group were observed. The stimulation index (SI) values, concentrations of IFN-g, IL-2 and IL-6 in adjuvant-DNA vaccine group and gavaged group were significantly higher than those in the DNA vaccine group. The protective efficacy of live attenuated vaccine group, DNA vaccine group, adjuvant-DNA vaccine group and gavaged group were 92%, 52%, 72% and 60%, respectively. This study has laid a foundation for the design and application of future DNA vaccines of P. multocida and DNA vaccine adjuvant.

Small Molecule ST2 Inhibitors Cause Reduction of Soluble ST2 and Improve Gvhd and Survival In Vivo

Activation of the membrane-bound Suppression of Tumorigenecity 2 (mST2) by Interleukin-33 (IL-33) in T cells leads to type-2 (Th2) and Foxp3+ regulatory (Tregs) immune responses. The mST2/IL-33 axis is not engaged when a ST2 splice isoform containing only the ST2 ectodomain and acting as a decoy receptor, called soluble ST2 (sST2), sequesters IL-33. Clinically, elevated plasma sST2 has been reported and used as a prognostic biomarker in cardiac allo-rejection, inflammatory bowel disease, and graft-versus-host disease (GVHD). We hypothesized that releasing IL-33 from circulating sST2 may augment the mST2/IL-33 axis activation to modulate the Th1 and Th2/Tregs responses for treating immune related diseases. This is supported by our study of a ST2 antibody in the GVHD models (Zhang et. al., Sci. Trans. Med. 2015). Advantages of small-molecule therapies over biologics include easier administration especially by oral, superior tissue penetration, modifiable pharmacokinetic properties including half-life, and lower manufacturing cost. Here, we present the proof-of-concept of this approach using three small molecule ST2 inhibitors we discovered recently. Our data show two inhibitors cause reduction of sST2, alleviate GVHD and improve survival in two in vivo GVHD models.Our high throughput screening (HTS) using the AlphaLISA assay and computational analysis led to discover three classes of small molecules inhibiting ST2 binding to IL-33 which were confirmed by the functional cell-based HEK-Blue assay. Seven compounds showing low toxicity at 66 uM to peripheral blood mononuclear cells (PBMC) were selected for the dose escalation toxicity study in mice. Six of them showed no toxicity up to 20 mg/kg in 13 days while five of them can be tolerated at 40 mg/kg. The HTS workflow is summarized in Fig. 1.Three compounds (named iST2-1-3) were evaluated in the in vivo GVHD disease models. The first model is the transplantation of human PBMC to the NOD/SCID/IL2rgnull (NSG) mice one day after the mice received 300 cGy total body irradiation (TBI). Compounds were injected via the intraperitoneal route to the mice the day before transplantation and continued daily at their respective IC50 dosages for 21 days. At Day 14, both iST2-1 and iST2-2 caused reduction of IFNg+ CD4+ T cells and increase of Foxp3+CD4+ Tregs population in the human CD45+CD4+ cells extracted from the gastrointestinal (GI), the main GVHD target organ, compared with the DMSO control (Fig. 2). More profound effect exerted by iST2-1 was observed at Day 21. Plasma concentrations of human IFNg and sST2, indicators of Th1 response, showed the highest reduction in the iST2-1 treatment group. While the hsST2 plasma concentration is maintained at < 30 pg/ml throughout 28 days in the iST2-1 treatment group, escalation of hsST2 up to 180 pg/ml was found in the DMSO control and iST2-3 treated group. Plasma hIFNg was at least 50% lower in the iST2-1 treated group at day 14 and 21. The GVHD scores and survival of NSG mouse models paralleled the changes of GI T cell populations and systemic cytokines data; iST2-1 treatment showed decreased GVHD score to 3 from 6 in the DMSO control and improvement of survival rate to 45 % on day 35 whereas all mice in the DMSO control group were dead.The second model is the minor mismatched model of allogeneic hematopoietic cell transplantation (HCT) from B6 to C3H.SW mice. We observed similar trends of IFNg+ reduction and Foxp3+ increase in CD4+ Tcells from the GI (Fig. 3). Plasma sST2 and IFNg concentrations were greatly reduced on day 14 in the iST2-1 treated group compared with DMSO control (50 versus 10 ng/ml for sST2 and 350 versus 100 pg/ml for IFNg).Finally, we have collected the Small Angle X-ray Scattering data to construct structural models between iST2-1 and sST2 to guide the inhibitor optimization. The CRISP-Cas9 technology was also used to obtain sST2 deficient B6 mice. Allo-HCT using these donor sST2 deficient T cells will delineate the specific mode of action by our ST2 inhibitors on mST2 and sST2. Our findings reveal iST2-1 is an attractive small molecule ST2 inhibitor that can be used to study the mST2/IL-33 axis in disease models and further developed as therapeutics to treat GVHD. [Display omitted] [Display omitted] [Display omitted] DisclosuresPaczesny:Viracor-IBT Laboratories: Patents & Royalties.

Prognostic value of TH1/TH2 cytokines in infants with wheezing in a three year follow-up study.

The immune system is known to play a key role in the development of viral-induced wheeze, but the pattern of cytokine response is not clear. The aim of the study was to determine whether plasma cytokine levels during the acute wheezing illness in infants are associated with the subsequent development of persistent recurrent wheezing (PRW). 43 infants admitted to Iashvili Central Children Hospital, Tbilisi, Georgia, were selected. The concentrations of IFN-g, TNF-a and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA). Total serum IgE level was also determined. IgM and IgG antibodies to RSV, Chlamydophila pneumonia and Mycoplasma pneumonia were tested by ELISA. All children were followed-up during 3 years period. There was no significant association with PRW and atopy in the infant or parental atopy, low maternal age, low birth weight, serum level of IL-6, serum level of IFN-g, serum level of total IgE and the particular pathogen. There was a tendency for the association with male sex, bottle feeding and household cigarette smoking, although the relation was not significant. There was just TNF-a which was strongly associated with the risk of PRW after three years of follow up. We have identified TNF-a, as a biomarker, which can be detected in children under 3 years of age and may be useful in the prediction of development of persistence of wheezing later. To further evaluate the usefulness of this and other markers and establish cutoff levels for prediction and use in clinical practice, a larger prospective follow-up study is needed.

Pathological mechanisms of antiphospholipid antibodies in trophoblastic cell fusion

Pathological mechanisms of antiphospholipid antibodies in trophoblastic cell fusion

Pregnancy recall in decidual NK cells

Pregnancy recall in decidual NK cells

EPA-0919 – Imbalance between glucocorticoid-induced tnfr-related protein (gitr) and il-2 in depressed copd patients modulate immunological self-tolerance mediated by treg cells and may explain amplified systemic inflammation

Mounting evidence underscores the important role of immunological and immunopathological processes in depression. Alterations in immune function secondary to increased psychological stress including enhanced secretion of proinflammatory cytokines affect the brain and elicits various symptoms of depression also in patients suffering from chronic inflammatory conditions including chronic obstructive pulmonary disease (COPD). However, mechanisms underlying increased immune response/decreased immune tolerance in people developing depression including COPD patients are largely unknown. The aim of this study was to explore the relationship between markers of systemic inflammation and depression. We hypothesized that the plausible factor contributing to amplified systemic inflammation in depressed patients is glucocorticoid-induced TNF receptor (GITR). Blood was collected from patients diagnosed with chronic obstructive pulmonary disease and comorbid depressive symptoms (COPD+DS, (N=13), individuals with either COPD (N=16) or recurrent depressive disorder (rDD) alone (N=15) and from healthy controls (N=19). Levels of IL-2, IL-6, IL-8, IFN-g, TNF-a, IL-17 and GITR were detected with ELISA. Surface phenotype expression of T regulatory and T effector cells was analysed with a flow cytometery. We observedthat COPD, depression and COPD with comorbid depression are associated with increased IL-6 levels when compared with healthy controls 42.2±1.87 pg/mL, 41.9±1.51 pg/mL, 41.7 ±1.31 and 34.7±1.36 pg/mL, respectively (p<0.05). Concentrations of IFN-g were significantly increased in COPD+DS patients when compared with controls (24.3±1.49 pg/mL and 17.8±0.70 pg/mL, respectively, p<0.05). IL-2 levels were highest in COPD+DS (3.20±0.389 pg/mL) and differed significantly when this group was compared with controls (2.30±0.176 pg/mL), p£0.05. GITR was significantly higher in COPD patients (0.332±0.04849) when compared with depressed (0.183±0.0492), when compared with COPD +depressive symptoms (0.194± 0.0357) and also when compared with controls (0.1601±0.0191), all p<0.05. We observed strong significant positive correlation between GITR and IL-2 and also between GITR and IFN-g in the study groups (p<0.001). Further, we checked the ratios of IL- 2/GITR and IFN-g /GITR. We observed significantly increased IL-2/GITR ratio in depressed groups (p<0.01). We also observed strong positive correlation between IL-2/GITR and Treg/Teffector (p<0.001). In this study we identified GITR and IL-2 as putative inflammatory agents associated with depressive symptoms in COPD patients. Furthermore our study revealed that alterations of IL-2/GITR balance modulate immunological self-tolerance mediated by Treg cells. Our observations suggest that modulation of IL-2/GITR and its effects on T cell population(s)are putative immune pathways contributing to systemic inflammation in depression.

Changes of Th1/Th2 cytokines in immunocompetent patients with pulmonary cryptococcosis

The aim of this study was to investigate the changes of Th1/Th2 cytokines in immunocompetent patients with pulmonary cryptococcosis (PC). Twenty immunocompetent patients with PC were identified by histopathological examination and were enrolled in the study along with the age- and gender-matched healthy controls. The serum concentrations of interferon-γ (IFN-γ), interleukin-4 (IL-4), and interleukin-12 (IL-12) were measured by enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMCs) in both groups were isolated and incubated with or without recombinant human IL-12 (rhIL-12) for 48 h, and the concentrations of IFN-g and IL-4 in the supernatant were measured by ELISA. Serum IFN-γ levels were greatly decreased in the patients compared with control groups (P < 0.01), whereas no significant difference was observed in serum IL-4 and IL-12 levels. The concentrations of IFN-γ and IL-4 in the supernatant of PBMCs without the stimulation of rhIL-12 showed no differences between the two groups. Treatment with rhIL-12 stimulated the release of IFN-γ, but not IL-4, into the supernatant of PBMCs in both groups, with a lower increase observed in the patients (4.3-fold) compared to that of controls (7.9-fold) (P < 0.01). Serum IFN-γ levels may be dampened in immunocompetent patients with PC with no significant changes in serum IL-4 and IL-12 levels. The deficiency in the response to IL-12 stimulation of Th1 cells may be one of the underlying mechanisms for the decline in serum IFN-γ levels.

Attenuated Acute Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation In the Absence of RORγt.

AbstractAbstract 3742CD4+ T helper (Th) cells play a critical role in the development of Graft-versus-Host Disease (GvHD). The relative contributions of particular Th subsets to GVHD pathogenesis, however, are incompletely understood. In order to clarify the contribution of the Th17 subset to GVHD induction, we made use of mice knocked out at the RORgt locus (RORgt−/−), a transcription factor crucial for Th17 polarization. Methods:Haplotype matched and complete MHC mismatched murine HSCT models were used. For the haploidentical model C57BL/6 (H-2b, B6) mice served as donors while C57BL/6 × DBA2 F1 (H-2bxd, B6D2) mice functioned as recipients. Effector T cells (Teffs) were isolated from the spleens of wild type (WT) B6 and RORgt knockout mice backcrossed 7–8 generations onto a B6 background. B6D2 mice were lethally irradiated with 900 rads on day -1 and injected intravenously with 4 × 106 Teffs from WT or RORgt−/− mice supplemented with 3 × 106 WT T cell depleted bone marrow cells (TCD BM) on day 0. For the completely MHC mismatched model, BALB/c mice (H-2d) were lethally irradiated with 800 rads on day -1 and administered 5 × 105 WT or RORgt−/− Teffs supplemented with 5 × 106 B6 TCD BM on day 0. Results:B6D2 mice that received RORgt−/− Teffs displayed significantly attenuated GvHD, recovering from weight loss by day +31 and demonstrating 100% survival on day +60. Conversely, mice that received WT Teffs showed intense disease progression with 100% mortality by day +31 (Figure A, p<0.0001 for survival comparison between WT and RORgt−/− recipients using Fisher's exact test). Similar results were seen using the completely MHC mismatched model, with superior overall survival noted in those animals receiving RORgt −/− Teffs (put in p value here). Recipients of RORgt −/− T cells demonstrated statistically significant decreased TNF in serum compared to WT recipients (Figure B, p=0.001 comparing WT and RORgt−/− recipients using student's t test). Interestingly, despite the decreased severity of GvHD, serum concentrations of IFN-g were increased in recipients transplanted with RORgt −/− T cells. Chimerism studies post-transplant revealed complete donor reconstitution in recipients of both RORgt−/− and WT Teffs. Donor Teffs isolated from recipient livers post-transplant consistently demonstrated an activated phenotype, with low L selectin and high CD25 expression. [Display omitted] Conclusions:T cell expression of the Th17 transcription factor, RORgt, is critical for the development of lethal GvHD following allogeneic stem cell transplantation in both the haploidentical and MHC complete mismatch models. GvHD attenuation in the absence of RORgt is not the result of an inability for donor T cells to undergo activation or to engraft in vivo. Interestingly, the absence of RORgt from donor T cells led to enhanced IFN-g in serum. Thus, in vivo, the Th17 pathway is critical for the induction of GvHD. Disclosures:No relevant conflicts of interest to declare.

Immune activation and neurotoxicity in patients with malignant melanoma undergoing IL-2 immunotherapy

Event Abstract Back to Event Immune activation and neurotoxicity in patients with malignant melanoma undergoing IL-2 immunotherapy Lisa Trimble1*, Caitlin Barbarita1, Erica B. Royster1, Andrew H. Miller1 and Dominique Musselman1 1 Emory University, Electrical and Electronics Engineering Department, Brain Research Laboratory, United States We sought to determine whether IL-2 stimulated increases in peripheral IFN-g plasma concentrations would be associated with increases in IL-6 plasma levels and decreases in FACT/GOG-Ntx Scores in stage IV malignant melanoma patients undergoing high-dose IL-2 therapy. IL-2 treatment is the standard of care in the United States for patients with stage IV melanoma. Although in 10-15% of cases it is effective in extending the survival of the patient it also induces many detrimental side effects including depressive, neurotoxic and even psychotic symptoms [1]. A lot of the times, these symptoms interfere with the continuation of this potentially life-saving therapy. These complications of IL-2 therapy have been posited to be caused by the activation of "secondary" cytokines through IL-2-induced stimulation of both the T-cells and B-cells of the immune system [2]. We believe that release of IFN-g, induced by IL-2 treatment, plays a significant role in the incidence of these symptoms as IFN-g activates the catabolism of the protein tryptophan. After giving informed consent, patients with Stage IV malignant melanoma were recruited. Prior to, during, and after IL-2 administration we measured plasma concentrations of IFN-g, neurobehavioral symptoms, cognitive function, and tolerance of IL-2 treatment. The Friedman test, Wilcoxon signed-rank test and Spearman's correlation were used in the analysis. IFN-g levels were undetectable until about two hours post injection. The neurotoxicity scores and IFN-g levels correlate during cycle 4 and the Ntx scores reach a low at the same time point that the IFN-g levels reach their peak. Our findings support our proposed model but further analysis of levels of TNF-a, tryptophan and quinolinic acid are still needed. Recruitment of more patients will also allow for more accurate and visible correlations.

A pilot study investigating the effects of yakult fermented milk drink (L. casei Shirota) on salivary IFN-γ, sIgA, IgA1 and IgA2 in healthy volunteers

This pilot study investigated the effects on salivary cytokine and sIgA before and after daily consumption of Lactobacillus casei Shirota (LcS) in nine healthy adult volunteers. Subjects were asked to consume two bottles each day of a fermented milk drink containing a total of 1.3 · 1010 live cells of LcS (Yakult), for 4 weeks. Volunteers were asked to avoid food and drink for at least 1 h before morning unstimulated saliva samples were collected over a 5 min period. Samples were collected at baseline, at week 1, 2 and 4 of LcS consumption, and 2 weeks after cessation of consumption. We found a significant transient increase in salivary IFN-g levels, but were unable to detect IL-12, and an increase in salivary sIgA, IgA1 and IgA2 secretion. Baseline salivary IFN-g was below the assay detection limits. However, at week 1, 4/9 subjects had detectable salivary IFN-g levels, 8/9 (P<0.01) at week 2 and 3/9 at week 4. None had detectable IFN-g levels at week 6. IFN-g concentrations ranged from 35.3 to 92.0 pg/ml. There was a significant (P<0.02) increase in salivary sIgA secretion rate at week 4 (Fig. A). At week 6, the sIgA secretion rate was greater than baseline. The IgA1 secretion rate was significantly greater than baseline at week 4 (P<0.05) and week 6 (P<0.02) (Fig. B). The IgA2 secretion was also significantly increased at week 4 (P<0.05) and week 6 (P<0.01) (Fig. B) and the IgA1:IgA2 ratio significantly greater at week 2 (P<0.01). This pilot study demonstrates that LcS can induce increased salivary IFN-g, sIgA, IgA1 and IgA2 secretion in healthy adults, which may improve mucosal immunity in the upper respiratory tract and provide health related benefits. A larger detailed study investigating cytokine networks, immunoglobulins and T cell responses in different immune system compartments in parallel with a placebo control group would define more rigorously and precisely the immunological effects of LcS (Yakult).

Open Posters: Adhesion molecules

Urease is one of the key enzymes involved in the establishment of infection by the stomach-colonizing pathogen Helicobacter pylori.The recombinant enzyme was co-administered parenterally with a range of adjuvants and their combinations. Over 6 months, we measured a number of immunological parameters to characterize both the intensity and quality of the ensuing immune responses. Of the formulations test-ed, alum þ Quil-A gave the highest peak humoral antibody titres, with chitosan and alum þ MDP reaching comparable levels. In contrast, MDP induced a lower response, however, titres were sustained throughout the duration of the study. Following splenocyte proliferation and supernatant cytokine analysis, alum þ MDP showed by far the highest proliferation index, with corresponding increased IL-2, IL-4, IL-6 and IFN-g concentrations. Alum þ Quil-A, produced the second highest proliferation index,superseding the alum þ MDP formulation only in IL-2 levels. Otherwise,IFN-g was observed only in the chitosan and alum formulations, chitosan being the only up-regulator of IL-4 alone or in combination with MDP,apart from free rURE. Surprisingly, MDP alone showed very low proliferation, with very low IL-6 and strongly down-regulated IL-4. In conclusion, the alum adjuvant combinations with MDP or Quil-A offer a significant potential in the process of eliciting CD4þ cells recently identified as essential for H. pylori clearance.

Suppression of renal disease and mortality in the female NZB x NZW F1 mouse model of systemic lupus erythematosus (SLE) by chenodeoxycholic acid.

The objective of this study was to examine the effects of ursodeoxycholic (UDCA) and chenodeoxycholic acid (CDCA) on autoimmune disease in the NZB x NZW F1 (B/W) mouse model of systemic lupus erythematosus (SLE). The development of murine lupus was assessed in female B/W mice given UDCA or CDCA. At 6 week intervals mice were examined for weight change, albuminuria, anti-DNA antibody and total IgG levels. Morbidity and mortality were assessed daily. UDCA- and CDCA-treated mice were examined at 24 weeks of age for serum cytokines, lymphocyte phenotype, and in vitro cytokine production after immunization with DNP-KLH. Liver and kidneys were examined histopathologically. The administration of UDCA and CDCA was tolerated without side effects. Weight gain in UDCA- or CDCA-treated and control mice was identical through 24 weeks of age. CDCA, but not UDCA, suppressed the development of renal disease. CDCA-treated B/W mice also had improved survival compared to UDCA-treated or control B/W mice. There were no significant effects of CDCA on anti-DNA antibodies, serum total IgG, or other immunologic parameters. CDCA-treated mice had lower serum IFN-gamma concentrations compared to control and UDCA-treated mice. The bile acid, CDCA, significantly inhibited the development of renal disease and modestly prolonged lifespan in the female B/W mouse model of SLE. Suppression of glomerulonephritis was associated with lower serum IFNgamma concentrations. Further investigation is needed to verify potential mechanisms of action, but these findings suggest that bile acids may alter the development or progression of autoimmunity.

ATTENUATION OF GVHD IN A SCID-HU MOUSE MODEL FOLLOWING THERAPY WITH A RATIONALLY DESIGNED IMMUNOMODULATORY PEPTIDE.

427 Recently, we reported on the rational design of novel immunomodulatory peptides. Administration of these peptides into mice and rats resulted in the upregulation of hemoxygenase-1 (HO-1). Upregulation of HO-1 was associated with the inhibition of several immune effector functions including lymphocyte proliferation, INFγ and TNFα production. Analysis of peptide mediated inhibition of TNFα production by RT-PCR and immunoprecipitation demonstrated that the reduction of TNFα production was due to decreased translation but not gene transcription. TNFα is a proinflammatory agent and appears to be involved in the pathology of several diseases including graft versus host disease (GVHD). The effect of one of these peptides, Allotrap 1258 was tested in a scid-hu mouse model for human graft versus host disease (GVHD). Pretreatment of scid mice with anti-asialo GM-1 antiserum and radiation allowed the successful transplantation of human peripheral blood lymphocytes into these animals. All of the Hu-PBL-scid mice died of GVHD within 5 weeks post transplantation. To test the effect of Allotrap 1258 on GVHD, mice (n=25) were treated intraperitoneally with 1 mg/kg/day Allotrap 1258 three times per week for two weeks. Peptide therapy prolonged animal survival 2.6 times (MST= 66 vs 25 days, p=0.008). Human cytokine levels in serum were determined at 3 weeks post cell infusion. Compared to control animals human TNFα and IFNg concentrations were reduced by 57% (peptide treated 8.7±3.2 units/ml vs control 20.1±4.5 units/ml; p=.001) and 47% (peptide treated 6.8±2.3 units/ml vs control 12.9±2.6 units/ml; p=.002), respectively. The human immunoglobulin levels in experimental mice were reduced by 63% compared to control animals (peptide treated 531±436 mg/l vs control 1429±329 mg/l; p=.006). Statistical analysis were done using student's t test. These results demonstrate that Allotrap 1258 not only modulates immune responses of rodent but also human lymphocytes. Peptide therapy of scid-hu mice attenuated GVHD and prolonged animal survival suggesting that Allotrap 1258 may have potential for suppressing GVHD reactions in humans.

Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages

The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL-1, rIL-2, rIL-3, rIL-5, rIL-6, rTNFa, rGM-CSF, rM-CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL-2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rIFNa and rIL-2 were ineffective in purified monocytes or macrophages. rIL-4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose-dependently inhibited by rIL-4 and this inhibition is probably due to an IL-4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.