Increases In IFN-α Research Articles

Comparative characteristics of the blood cytokine profile in gastritis in schoolchildren with a family predisposition to gastric cancer and peptic ulcer

The formation of diseases of the gastric and duodenum in the adult population begins at school age. Among the leading risk factors for their formation are family predisposition (to peptic ulcer disease (PU), gastric cancer). The basis for their formation is gastritis, the course of which is actively influenced by cytokines. Aim. To study the characteristics of cytokine levels in blood serum in schoolchildren with gastritis and a family history of gastric cancer and peptic ulcer. A total of 179 schoolchildren with gastroenterological complaints were examined. All subjects underwent an endoscopic examination with biopsies taken from the antrum of the gastric and morphological confirmation of gastritis (Sydney classification). The presence of H. pylori was also determined by morphological method. The level of cytokines in the blood (IL-2, IL-4, IL-8, IL-18, IL- 1β, IFNα, TNFα) was determined by ELISA. The study found that schoolchildren with a burden of ulcer had a decreased IL-1β in comparison with children without aggravation (p = 0.005), and with children with a burden of cancer (p = 0.030). Also, IFNα was increased in schoolchildren with a burden of ulcerative disease (p = 0.001) and gastric cancer (p = 0.038). A decrease in IL-1β was found in children with aggravated ulcer disease, both with H. pylori infection (p = 0.048) and without it (p = 0.043). IL-1β plays an important role in the immune response to H. pylori infection. In addition, it is known that during H. pylori infection the level of IL-1β increases, which is accompanied by inhibition of gastric acidity. There was a decrease in IL-1β and an increase in IFNα in children with aggravated ulcer disease, regardless of the activity of inflammation. In addition, they observed an increase in TNFα (p = 0.04) with high gastritis activity (stage 2-3). In addition, in children with ulcer burden and high gastritis activity, IL-4 is increased (p = 0.018). Thus, in children with a family history of both peptic ulcers and gastric cancer, there are features of the association of specific cytokines with gastritis, as well as its activity and H. pylori infection.

Interferons dominate damage and activity in juvenile scleroderma.

Juvenile scleroderma is a heterogeneous group of diseases associated with sclerotic skin lesions, grouped as juvenile systemic sclerosis and juvenile localized scleroderma. This study aims to measure the cytokine and chemokine levels involved in interferon (IFN) signalling in patients with juvenile scleroderma and determine their correlation with disease severity. Twenty-nine juvenile localized scleroderma, five juvenile systemic sclerosis, and nine healthy controls were included in the study. Cytokines and chemokines involved in IFN gene signalling (IL-1, IL-6, IL-8, IP-10, MCP1, TNF-α, CXCL-11, IFN-α, IFN-β, IFN-γ) and IFN-stimulated genes (ISGs), including IFI27, IFI44, ISIG15, IFIT1, OAS1, RSAD2, were measured by ELISA and RT-PCR method, respectively. A significant increase in IFN-α, IFN-β, IFN-γ, TNF-α, IL-1, IL-6 IL-8, IP-10, and MCP1 levels was observed in patients with juvenile systemic sclerosis compared with the healthy control group. Furthermore, IFN-α and IP-10 were elevated in both juvenile localized scleroderma and juvenile systemic sclerosis compared to the healthy control group. IFN-γ and IFN-α positively correlated with LoSAI and LoSDI levels, respectively. According to PGA-A analysis, IFN-β, IFN-γ, TNF-α, IL-8, IP10, MCP1, and CXCL11 were significantly higher in active disease than in the inactive state in both groups. The results suggest that IFN signalling may be impaired in patients with juvenile scleroderma. Significant changes were observed in cytokines and genes related to IFN signalling, which may have a crucial role in monitoring disease activity. In addition, we have gained important insights into the possibility of using IFN-α and IFN-γ as biomarkers for monitoring juvenile scleroderma activity and damage.

Application of metagenomic next-generation sequencing in the diagnosis of urinary tract infection in patients undergoing cutaneous ureterostomy.

Urinary tract infection (UTI) is an inflammatory response of the urothelium to bacterial invasion and is a common complication in patients with cutaneous ureterostomy (CU). For such patients, accurate and efficient identification of pathogens remains a challenge. The aim of this study included exploring utility of metagenomic next-generation sequencing (mNGS) in assisting microbiological diagnosis of UTI among patients undergoing CU, identifying promising cytokine or microorganism biomarkers, revealing microbiome diversity change and compare virulence factors (VFs) and antibiotic resistance genes (ARGs) after infection. We performed a case-control study of 50 consecutive CU patients from December 2020 to January 2021. According to the clinical diagnostic criteria, samples were divided into infected group and uninfected group and difference of urine culture, cytokines, microorganism, ARGs and VFs were compared between the two groups. Inflammatory responses were more serious in infected group, as evidenced by a significant increase in IFN-α (p=0.031), IL-1β (0.023) and IL-6 (p=0.018). Clinical culture shows that there is higher positive rate in infected group for most clinical pathogens like Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Candida auris etc. and the top three pathogens with positive frequencies were E. coli, K. pneumoniae, and Enterococcus faecalis. Benchmarking clinical culture, the total sensitivity is 91.4% and specificity is 76.3% for mNGS. As for mNGS, there was no significant difference in microbiome α- diversity between infected and uninfected group. Three species biomarkers including Citrobacter freundii, Klebsiella oxytoca, and Enterobacter cloacae are enriched in infected group based on Lefse. E. cloacae were significantly correlated with IL-6 and IL-10. K. oxytoca were significantly correlated with IL-1β. Besides, the unweighted gene number and weighted gene abundance of VFs or ARGs are significantly higher in infected group. Notablely, ARGs belonging to fluoroquinolones, betalatmas, fosfomycin, phenicol, phenolic compound abundance is significantly higher in infected group which may have bad effect on clinical treatment for patients. mNGS, along with urine culture, will provide comprehensive and efficient reference for the diagnosis of UTI in patients with CU and allow us to monitor microbial changes in urine of these patients. Moreover, cytokines (IL-6, IL-1β, and IFN-a) or microorganisms like C. freundii, K. oxytoca or E. cloacae are promising biomarkers for building effective UTI diagnostic model of patients with CU and seriously the VFs and ARGs abundance increase in infected group may play bad effect on clinical treatment.

Neutralizing interferon-α blocks inflammation-mediated vascular injury via PI3K and AMPK in systemic lupus erythematosus.

Plasmacytoid dendritic cells (pDCs) play a key role in the initiation and amplification of systemic lupus erythematosus (SLE)-associated vascular injury. In this study, we found that dsDNA induced dose- and time-dependent increase in IFN-α and Toll-like receptor 7 (TLR7), TLR9 and IRF7 expression in pDCs. Co-cultured circulating endothelial cells (ECs) with activated pDCs significantly decreased proliferation, tube formation and migration in ECs. The elevated level of cellular IFN-α increased cell adhesion, promoted cell apoptosis, induced cell senescence and arrested cells at G0/G1 phase of endothelial progenitor cells (EPCs). Additionally, the co-culture system activated MAPK and inactivated PI3K. Pristane was used to establish a in vivo SLE-like mouse model. Importantly, we showed that INF-α-neutralizing antibody (IFN-α-NA) rescued all the changes induced by IFN-α in vitro and prevented vascular injury in pristane-induced SLE model in vivo. In conclusion, we confirmed that activated pDCs promoted vascular damage and the dysfunction of ECs/EPCs via IFN-α production. IFN-α-neutralizing antibody may be a clinical implication for preventing vascular injury. PI3K signalling and AMPK signalling were associated with SLE-associated vascular functions.

High Levels of Interferon-Alpha Expressing Macrophages in Human Breast Milk During SARS-CoV-2 Infection: A Case Report

Introduction: In addition to hand washing and wearing masks, social distancing and reducing exposure time to <15 minutes are the most effective measures against the spread of COVID-19. Unfortunately, three of these guidelines are very difficult, if not impossible, for nursing babies: they cannot wear masks, stay six feet away from the lactating breasts, nor consistently finish within 15 minutes while nursing. We report a case of a nursing mother with SARS-CoV-2 infection, documenting changes of immune cells and cytokines in breast milk with and without the infection. Case Description: With Institutional Review Board (IRB) approval, we obtained expressed breast milk samples from a lactating mother before and during SARS-CoV-2 infection as documented by reverse transcription-PCR. Using flow cytometry analysis, we measured the immune cell profiles and expression of cytokines such as interferon alpha (IFNα) in milk leukocytes before and during infection. Results: There was an eightfold increase in IFNα+ milk leukocytes, from 1% before SARS-CoV-2 infection to 8% when actively infected. The milk macrophages showed the highest increase in IFNα expression. Both T and B lymphocytes showed mild increase. Innate lymphoid cells, neutrophils, and natural killer cells showed no increase in IFNα expression and the dendritic cells actually showed a reduction. Conclusion: We document the presence and high expression of IFNα in the breast milk macrophages of a lactating mother with confirmed COVID-19, compared with her milk before the infection.

Viral replication and innate immunity of feline herpesvirus-1 virulence-associated genes in feline respiratory epithelial cells

Feline herpesvirus-1 (FHV-1) infection occurs worldwide and is a leading cause of respiratory and ocular diseases in cats. Current vaccines reduce the severity of symptoms but do not prevent infection and, therefore, do not provide defense against an establishment of latency and reactivation. We hypothesize that immunomodulation of FHV-1 is the cause of lack in protection and that deletion of virulence/immune modulatory genes of FHV-1 will enhance safety and immunogenicity. Our objective was to use feline respiratory epithelial cell (FREC) cultures to define in vitro growth characteristics and immunomodulation resulting from infection of FRECs with the virulent FHV-1 strain C27 (WT) and glycoprotein C-deletion (gC-), glycoprotein E-deletion (gE-), serine/threonine protein kinase-deletion (PK-), as well as gE and thymidine kinase-double-deletion (gE-TK-) mutants generated by bacterial artificial chromosome mutagenesis.Differentiated FRECs were mock inoculated or inoculated with WT, gC-, gE-, PK-, or gE-TK- mutants. Virus titration and real-time quantitative PCR assays were performed on samples collected at 1 hpi followed by 24 h intervals between 24 and 96 hpi to determine growth kinetics. Real-time PCR was used to quantitate IFNα, TNFα, IL-1β, IL-10, and TGFβ-specific mRNA levels. Immunoassays were performed to measure the protein levels of subsets of cytokines/chemokines secreted by FRECs.Inoculation of FRECs with gE-TK- resulted in significantly lower end-point titers than inoculation with WT or gE-. Both PK- and gC- inoculated FRECs also produced significantly lower end-point titers at 96 hpi than WT. Overall, intracellular virus titers were higher than those of extracellular virus. PCR results for viral DNA paralleled the virus titration results. Further, in contrast to WT inoculation, an increase in IFNα and IL-10 mRNA expression was not observed following inoculation with gE-TK- and PK-, but inoculation with gE-TK- and PK- did result in increased TGFβ expression in FRECs compared to responses following infection with WT. Moreover, gE-TK- and PK- blocked the inhibition of IL-8 and neutrophil chemoattractant (KC), which was observed following inoculation with WT.In summary, the results obtained in FRECs may be used to predict the safety and immunogenicity characteristics of these mutants in vivo. Our study highlights the value of the FREC system for studying replication kinetics/immune modulation factors of FHV-1 and screening prospective vaccine candidates before their use in experimental cats.

Abstract # 2079 Psychological stress, immune reactivity, and attention to emotional information

There is evidence that attention to emotional stimuli can modulate the impact of stress on immunity. This study examined how three distinct attention networks – alerting, orienting, and executive control – affected salivary cytokine responses to stress. A total of 60 young adults were randomly assigned to either a high-stress or a low-stress group, with high stress being induced by (socially evaluated) Paced Auditory Serial Addition Test (PASAT). Saliva samples were collected before, 5 min after, and 45 min after completion of the stress task, and were assayed for pro- and anti-inflammatory cytokines. Attention systems were measured by the modified Emotional Attention Network Test Integration (E-ANTI). Both stressors triggered significant increases in IFN-α, IFN-γ, TNF-α, IL-2, IL-5, IL-8, IL-10, IL-12p70, and IL-13 at 45 min after stress induction (all p’s

Activation of Toll-like receptor 7 signaling in labial salivary glands of primary Sjögren's syndrome patients.

The aim of this study was to determine the expressions of Toll-like receptors (TLRs) 7-9 and type I interferon (IFN) signal in labial salivary glands (LSGs) and cultured salivary gland epithelial cells (SGECs) from primary Sjögren's syndrome (pSS) patients. We performed an immunohistochemistry analysis of LSGs from 11 patients with pSS as defined by American-European Consensus Group classification criteria and five healthy subjects. The pSS patients' SGECs were analyzed by immunofluorescence and western blotting. IFN-α expression was examined by immunosorbent assay and flow cytometry. Mononuclear cells (MNCs) from pSS patients' LSGs showed TLR-7-dominant expression. B cells, plasma cells and plasmacytoid dendritic cells (pDCs) co-expressed with TLR-7. Myeloid differentiation primary response gene 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6) and interferon regulatory factor 7 (IRF7) co-expressed with the pDC marker CD303 in LSGs. Ducts from pSS patients dominantly expressed TLR-7, and TLR-7 in the ducts co-expressed with MyD88, TRAF6 and IRF7. Type I IFNs including IFN-α and IFN-β were detected in MNCs and ducts in pSS patients' LSGs. Increased TRAF6 expression and the nuclear translocation of IRF7 in SGECs were detected by immunofluorescence following loxoribine (a TLR-7 ligand) stimulation despite IFN-β pretreatment. Western blotting showed increased TRAF6 expression in SGECs following IFN-β and loxoribine stimulation. Although no increase in IFN-α was detected in supernatant from stimulated SGECs, the IFN-α in supernatant from stimulated peripheral blood pDCs from pSS patients was significantly increased. Our findings suggest that TLR-7 is dominantly expressed in both MNCs and ducts with downstream signals for type I IFNs, indicating that TLR7-dominant innate immunity is related to the development of sialadenitis in pSS.

Attention to Emotional Information Is Associated With Cytokine Responses to Psychological Stress.

This study aimed to investigate the impact of mental stress on salivary cytokines and attention to emotional stimuli, as well as associations between stress-induced changes of immune and cognitive parameters. In a randomized order a total of 60 young adults were assigned to one of two stress conditions with varying stress intensity. High stress was induced by a socially evaluated Paced Auditory Serial Addition Test (PASAT). As a low stress task a paper-and-pencil version of PASAT was administered. Salivary cytokines were measured before, 5 min after, and 45 min after completion of the stress task, and were assayed for pro- and anti-inflammatory cytokines. Three distinct types of attention – alerting, orienting, and executive control – were measured by the modified Emotional Attention Network Test Integration (E-ANTI). IL-1β and IL-6 increased only in the high-stress group. Significant increases in IFN-α, IFN-γ, TNF-α, and IL-10 at 45 min after stress induction (all p’s < 0.05) were observed in both the high-stress and the low-stress group. Alerting attention was positively related to more pronounced increases in IFN-α and TNF-α in both groups. Further, better orienting attention after presentation of negative cues was associated with higher increases in IFN-α, TNF-α, IL-2, IL-5, and IL-10 in both groups, and higher overall levels of IFN-α, IFN-γ, and IL-12p70 in the high-stress group. There were no systematic gender differences in cytokine responses. We conclude that attention processes modulate the increases of salivary cytokines after stress exposure, and that these effects depend on stress level, particular attention network, and stimulus valence.

Type-1 interferon signaling mediates neuro-inflammatory events in models of Alzheimer's disease

A neuro-inflammatory response has been implicated in human patients and animal models of Alzheimer's disease (AD). Type-1 interferons are pleiotropic cytokines involved in the initiation and regulation of the pro-inflammatory response; however, their role in AD is unknown. This study investigated the contribution of type-1 IFN signaling in the neuro-inflammatory response to amyloid-beta (Aβ) in vitro and in the APP/PS1 transgenic mouse model of AD. Enzyme-linked immunosorbent assay confirmed a 2-fold increase in IFNα in APP/PS1 brains compared with control brains. Quantitative polymerase chain reaction also identified increased IFNα and IFNβ expression in human pre-frontal cortex from AD patients. In vitro studies in primary neurons demonstrated Aβ-induced type-1 IFN expression preceded that of other classical pro-inflammatory cytokines, IL1-β, and IL-6. Significantly, ablation of type-1 interferon-α receptor 1 expression in BE(2)M17 neuroblastoma cells and primary neurons afforded protection against Aβ-induced toxicity. This study supports a role for type-1 interferons in the pro-inflammatory response and neuronal cell death in AD and suggests that blocking type-1 interferon-α receptor 1 maybe a therapeutic target to limit the disease progression.

Cridanimod and progestin therapy in hormone-resistant endometrial cancer.

5597 Background: Cridanimod is a novel small molecule that has been shown to increase progesterone receptor (PR) expression in rat endometrium. We hypothesize that cridanimod, in combination with progestin therapy, will increase PR expression and, thus improve survival in a mouse model of advanced, high grade endometrial cancer. Methods: Hec50co cells, established in our laboratory to represent genetically and phenotypically type II endometrial cancer, were injected into the peritoneal cavity of 96 athymic mice. These mice were randomly divided into 6 groups, receiving the following therapies: control (no therapy), medroxyprogesterone acetate (MPA) alone, cridanimod 1mg IM twice a week plus MPA, cridanimod 3mg plus MPA, cridanimod 6mg plus MPA, and adenovirus-mediated PR expression plus MPA. PR expression in tumor tissue and serum interferon (IFN) levels were assayed via Western blot and ELISA, respectively. Results: Kaplan-Meier survival analysis showed that the group receiving MPA plus 6 mg cridanimod twice a week had a significantly longer survival time than the control or MPA alone (62 ± 7.0 days vs 38 ± 5 or 33 ± 3, respectively, p &lt; 0.05). The group with MPA plus 3 mg cridanimod had a significantly longer survival than the group with MPA alone (56 ± 8.0 days vs 33 ± 3, p &lt; 0.05). Western blot analysis showed that PR proteins were substantially higher in xenograft tumors from animals treated with cridanimod at doses of 3mg and 6mg when comparing to the control and MPA alone groups, ELISA data showed significant increases in IFNα and -β in cridanimod-treated mice in a dose-dependent manner. Conclusions: Combined progestin and cridanimod therapy significantly improved survival of mice with high-grade, advanced endometrial cancer. Cridanimod significantly increased PR expression, suggesting that the therapeutic benefit of combined therapy is mediated by up-regulation of PR, which is likely to be mediated through cridanimod induction of IFNα and IFNβ expression. Further investigation is warranted to determine the utility of this promising agent.

Abstract A95: Distinct roles of type I and type II interferons in radiation-induced antitumor immunity against B16 melanoma.

Abstract Endogenous type I interferons (IFN-α/β) and interferon gamma (IFN-γ) have both been implicated in immune responses against cancer. Interestingly, studies done using viral infection models have demonstrated that IFN-α/β and IFN-γ play non-redundant roles in the immune system and they act synergistically, enhancing responses that are induced by the two different signaling pathways. However, this phenomenon, known as priming, has not been well-characterized in the context of anti-tumor immunity. Our lab previously demonstrated using a B16 murine melanoma model that single local high dose (15Gy) radiation therapy induced elevated levels of intratumoral IFN-γ, which had several downstream effects like increased MHC class I expression on tumor cells that in turn enhanced recognition by CD8+ T cells. Overall, these IFN-γ;-dependent effects led to increased tumor control, evident by significant decreased rate of tumor growth following treatment. In the current study, we hypothesize that local radiation therapy (RT) increases the levels of IFN-α/β within the tumor, and this is required to enhance IFN-γ responses so that maximal therapeutic potential can be achieved. Indeed, local RT resulted in an early, transient spike in intratumoral IFN-α levels about two days after treatment. Interestingly, an increase in IFN-γ levels was observed four days post-treatment and remained elevated for at least five days. When B16 tumors were grown in mice that lack functional IFN-α/β receptors (IFNABR KO), the subsequent increase in IFN-γ following RT was completely abrogated. The ability of RT in reducing tumor growth was also less effective in IFNABR KO mice than in wild type (WT) mice. To investigate the mechanism involved, we examined the transcription of interferon-inducible genes, including the CXCR3 chemokines CXCL9 (MIG) and CXCL10 (IP-10). Following RT, intratumoral transcript levels of MIG and IP-10 were increased to similar extents in both WT and IFNγKO mice, but remained at basal levels in IFNABR KO mice. This suggests that IFN-α/β, but not IFN-γ, may be contributing to the initial chemoattraction of lymphocytes into radiation-treated tumors. Using flow cytometry analysis of immune cell populations within the tumors, we confirmed that the levels of intratumoral CXCR3 chemokines correlate strongly with tumor infiltration by T lymphocytes. Our data suggest that IFN-α/β play a critical role in the increase of CXCR3 chemokine levels within the tumor after RT. These chemokines are important for the recruitment of activated T cells into the tumor, which then produce IFN-γ, increasing IFNγ-induced immune responses locally. To investigate whether exogenous IFN-α will improve the efficacy of radiation therapy, we genetically-engineered a B16 cell line that can be induced to express IFN-α by exposing these cells, named B16-iIFN-α, to a rapamycin-analog. When inoculated in mice, B16-iIFN-α cells form established tumors similar to that of parental B16 cells. Production of IFN-α by B16-iIFN-α cells can be switched on at specific time points in tumor-bearing mice by treating the mice with the rapamycin-analog. This is a novel and invaluable tool for examining the anti-tumor effects of a local increase in IFN-α, and can be modified to study the effects of other cytokines as well. Our preliminary data demonstrated that radiation and IFN-α combination therapy resulted in promising improvement in tumor growth control compared to either of the treatments alone. We are currently studying the immunological mechanisms involved, as well as fine-tuning the treatment regimen to harness the maximal therapeutic potential of this treatment strategy. Citation Format: Joanne Y.H. Lim, Scott A. Gerber, Edith M. Lord. Distinct roles of type I and type II interferons in radiation-induced antitumor immunity against B16 melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A95.

The different expression of immune-related cytokine genes in response to velogenic and lentogenic Newcastle disease viruses infection in chicken peripheral blood

Newcastle disease virus (NDV) is an important pathogen hazardous to poultry industry, and the pathogenicity of NDV strains varies with different virulence. Peripheral blood serves as an important producer and carrier of viruses and cytokines in NDV infection. In order to explore the difference of cytokine expression in the peripheral blood between velogenic strain and lentogenic strain infection, NDV virulent strain F48E9 and vaccine strain Lasota were used to infect specific-pathogen-free (SPF) chickens separately, and peripheral blood was collected on 0, 3, 7, 10, 14, and 21days post-infection (d.p.i.). Real-time PCR was then used to detect the expression of six kinds of immune-related cytokine genes. For the F48E9 group, a sharp increase of the expression of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin-16 and IL-18 was observed on 3d.p.i. before the NDV blood peak (7d.p.i.), followed by a rapid decline to the level lower than that of control group, then the expression of IFN-α increased slowly and reached or exceeded the level of control group in the later phase of the infection, while the expression of IFN-γ, IL-16, and IL-18 fluctuated at the level of control group for the rest of study period. The increase of IL-2 expression was not obvious, and no increase of IL-15 expression was noted. For the Lasota (vaccine) group, the picture was quite different, a sharp increase of IFN-γ (but not IFN-α), IL-2 was observed on 7d.p.i. before the NDV blood peak (10d.p.i.). On the contrary, there was no dramatic increase of IL-16 and IL-18. Interestingly, in contrast to the F48E9 group, there was an increase of IL-15 on day 10d.p.i., but it remained modest. There was also an increase of IFN-α on day 21d.p.i. Our results revealed that infection with NDV strains of different virulence was associated with distinct cytokine expression patterns in peripheral blood, modulation of cytokine responses may play a key role in mediation of NDV pathogenesis.

Rational Design of Immunostimulatory siRNAs

Short-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on their sequence, can be variably sensed by the innate immune system through recruitment of toll-like receptors 7 and 8 (TLR7/8). Here, we aimed to identify sequence-based modifications allowing for the design of bifunctional siRNAs with both proinflammatory and specific silencing activities, and with potentially increased therapeutic benefits. We found that the introduction of a micro-RNA (miRNA)-like nonpairing uridine-bulge in the passenger strand robustly increased immunostimulatory activity on human immune cells. This sequence modification had no effect on the silencing efficiency of the siRNA. Increased immunostimulation with the uridine-bulge design was specific to human cells, and conserved silencing efficiency required a Dicer-substrate scaffold. The increased cytokine production with the uridine-bulge design resulted in enhanced protection against Semliki Forest virus (SFV) infection, in viral assays. Thus, we characterize a design scaffold applicable to any given siRNA sequence, that results in increased innate immune activation without affecting gene silencing. Our data suggest that this sequence modification coupled with structural modification differentially recruits human TLR8 over TLR7, and could have potential application in antiviral therapies.

419. Rational design of bifunctional siRNAs that silence genes and recruit the innate immune system to treat ectopic pregnancies

RNA interference (RNAi) is a new therapeutic approach, silencing genes to disrupt diseases. However, short interfering siRNAs (molecule used in RNAi) can have off-target effects, activating the immune system through RNA sensing toll-like receptors (TLR) 3, 7 and 8. We have previously proposed that in some diseases (cancers, ectopic pregnancies) it may be useful to enhance the immune response. A novel class of immunostimulatory siRNAs could be developed, silencing genes important to disease and recruiting the immune system to further aid disease clearance. We set out to develop a rational design strategy that enhances immunostimulatory properties to any siRNA sequence but maintains effective gene silencing. We screened a set of siRNAs targeting lamin. All were of the same sequence, except for different immunostimulatory motifs on the 3′ end of the sense strand. We also investigated a different design where we added a small micro-RNA like poly-uridine bulge (potentially immunostimulatory) on the sense strand. We used human peripheral blood mononuclear cells (PBMCs) to test for immunostimulation, and HEK 293-T-cells to test for lamin gene knockdown.Of all strategies tested, the poly-uridine bulge was best. It silenced the lamin gene as effectively as control, but caused a 2–3 fold increase of IFN-α and TNF-α. We verified this approach by adding the poly-uridine bulge onto an siRNA of low immunostimulatory potential targeting GFP. The bulge markedly enhanced immunostimulation in a dose response manner, and did not compromise gene knockdown. The addition of a poly-uridine bulge to siRNAs can increase immunostimulation without affecting gene silencing efficacy. Immunostimulatory siRNAs might be particularly efficacious to treat ectopic pregnancies where there are abundant immune cells, and functional TLR 7/8 in the trophoblast (unpublished observations). We now plan to test this immunostimulatory siRNA approach in an in vivo ectopic pregnancy model using JEG-3 cells xenographted in NOD-SCID mice.

First in Human Phase I Trial of 852A, a Novel Systemic Toll-like Receptor 7 Agonist, to Activate Innate Immune Responses in Patients with Advanced Cancer

Recent advances in the understanding of innate immunity suggest that an orchestrated sequence of events is required to elicit a productive immune response against cancer. We studied the systemic administration of the Toll-like receptor 7 agonist 852A, a small-molecule imidazoquinoline, in patients with advanced cancer. Preclinical studies showed that 852A stimulates plasmacytoid dendritic cells to produce multiple cytokines, such as IFN-alpha, interleukin-1 receptor antagonist, and IFN-inducible protein-10. Our goal was to define the tolerated dose, pharmacokinetics, pharmacodynamics, and immunologic effects of 852A in humans. Eligible adult patients with refractory solid organ tumors received i.v. 852A thrice weekly for 2 weeks. Patients who had responses or stable disease were eligible for additional cycles. Twenty-five patients (median age, 55.0 years; 72% male) were enrolled in six cohorts at dose levels of 0.15 to 2.0 mg/m(2). Serum drug levels showed dose proportionality and no evidence of drug accumulation. The maximum tolerated dose was 1.2 mg/m(2); higher doses were limited by fatigue and constitutional symptoms. Increases in IFN-alpha, interleukin-1 receptor antagonist, and IFN-inducible protein-10, immunologic activity, and clinical symptoms were observed in all patients receiving dose levels > or =0.6 mg/m(2). Significant correlations were found between pharmacodynamic biomarkers and pharmacokinetic variables, and an objective clinical response was seen. 852A was safely administered i.v. at doses up to 1.2 mg/m(2) thrice weekly for 2 weeks with transient or reversible adverse effects. This novel Toll-like receptor 7 agonist is biologically active and holds promise for stimulating innate immune responses. Future trials are warranted to assess its therapeutic role in patients with cancer.

Cognitive dysfunction in HIV encephalitic SCID mice correlates with levels of Interferon-α in the brain

Interferon alpha (IFNalpha) is an antiviral cytokine produced in response to viral infection. IFNalpha also acts as a neuromodulatory molecule in the central nervous system (CNS). Elevated IFNalpha in the CNS causes cognitive deficits. To determine if elevated levels of IFNalpha in an HIV encephalitis mouse model correlate with cognitive deficits. C57BL/6J SCID mice were inoculated intracerebrally (i.c.) with HIV infected or uninfected (control) macrophages and cognitively tested in a water escape radial arm maze. After behavioral testing was completed, immunohistochemistry and ELISA were used to examine brain pathology and IFNalpha expression. Mice injected i.c. with HIV infected macrophages exhibited significantly more working memory errors, particularly in trials with the highest memory load. Immunohistochemistry indicated increased mouse IFNalpha staining prevalent on neurons and glial cells in the brains of mice with HIV infected macrophages compared to mice with uninfected control macrophages. In addition, IFNalpha levels in the brain correlated directly with working memory errors for mice with HIV infected macrophages. These data suggest that the cognitive deficit noted for the C57BL/6J SCID mice with HIV infected macrophages is mediated by the infection induced increase in IFNalpha.

INCREASED EXPRESSION OF IFN-ALPHA AND IL-6 BY HYPERACTIVE PLASMACYTOID DENDRITIC CELLS IN BXD2 AUTOIMMUNE MICE (130.25)

Abstract BXD2 is a mouse model which exhibits several features of human SLE and arthritis. IFN-α is a known cytokine which promotes B-cell maturation and antibody CSR and SHM. ELISA results of in vitro culture of spleen cells demonstrates that IFN-α expression reaches a peak at 2 months of age (4-fold increase compared to nonautoimmune mice; p&amp;lt;0.005), suggesting that IFN-α can serve as an early trigger for B-cell autoimmunity. Immunofluorescent staining for CD11c+ antigen-presenting cells identifies an increased population (2–4 fold; p&amp;lt;0.005) of plasmacytoid dendritic cells (pDCs). Flow cytometry results demonstrate splenic pDCs are the primary cells which express IFN-α (&amp;gt;80%) and IL-6 (&amp;gt;96%) in BXD2 mice. In vivo treatment of 2 m-old BXD2 mice with DOTAP/CpG resulted in a 200-fold increase in IFN-α compared to a 4-fold increase in B6 mice. This is associated with high levels of autoantibody titers and CSR. In conclusion, BXD2 mice exhibit a significantly increased population of pDCs that express IFN-α at a young age which is associated with increased humoral response.

Chronic fatigue syndrome: Identification of distinct subgroups on the basis of allergy and psychologic variables

Background: We investigated a role for allergic inflammation and psychologic parameters in the development of chronic fatigue syndrome (CFS). Methods: The design was a comparison between subjects with CFS and age- and sex-matched control cohorts. Studies were performed on CFS subjects ( n = 18) and control cohorts consisting of normal subjects ( n = 11), allergic subjects ( n = 14), and individuals with primary depression ( n = 12). We quantified cytokines at baseline as cell-associated immunoreactive peptides and as transcripts evaluated by means of semiquantitative RNA-based polymerase chain reactions. Psychologic evaluations included administration of the Diagnostic Interview Schedule, the Structured Clinical Interview, and the Symptom Checklist 90–Revised. Results: Increases in tumor necrosis factor (TNF)-α were identified in individual subjects with CFS (50.1 ± 14.4 pg TNF-α per 10 7 peripheral blood mononuclear cells [PBMCs]; mean ± SEM) and allergic subjects (41.6 ± 7.6) in comparison with normal subjects (13.1 ± 8.8) ( P < .01 and P < .05, respectively). Similar trends were observed for interferon (IFN)-α in allergic subjects (3.0 ± 1.7 pg/10 7 PBMCs) and subjects with CFS (6.4 ± 3.4) compared with normal subjects (1.9 ± 1.4). A significant increase ( P < .05) in TNF-α transcripts was demonstrated between subjects with CFS and depressed subjects. In contrast to these proinflammatory cytokines, both subjects with CFS (2.6 ± 1.8 pg/10 7 PBMCs) and allergic subjects (3.4 ± 2.8) were associated with a statistically significant ( P < .01) decrease in IL-10 concentrations compared with normal subjects (60.2 ± 18.2). As shown in other studies, most of our subjects with CFS were allergic (15 of 18) and therefore presumably demonstrated cytokine gene activation on that basis. The seasonal exacerbation of allergy was associated with a further increase in cellular IFN-α (from 2.1 ± 1.2 to 14.2 ± 4.5 pg/10 7 PBMCs; P < .05) but no further modulation of TNF-α or IL-10. Similarly, self-reported exacerbations of CFS were associated with a further increase in IFN-α (from 2.5 ± 1.0 to 21.9 ± 7.8; P < .05) and occurred at times of seasonal exposures to allergens. This linkage does not permit making any definitive conclusions regarding a causative influence of either seasonal allergies or the increase in cellular IFN-α with the increase in CFS symptoms. The close association between atopy and CFS led us to speculate that CFS may arise from an abnormal psychologic response to the disordered expression of these proinflammatory and antiinflammatory cytokines. Psychologic variables were predictive of immune status within the CFS sample (65.9% of the variance in immune status; F (3,10) = 6.44, P < .05). Specifically, the absence of a personality disorder but greater endorsement of global psychiatric symptoms was predictive of immune activation. Conclusions: Most of our subjects with CFS were allergic, and the CFS and allergy cohorts were similar in terms of their immune status. However, the CFS subjects could be discriminated by the distinct psychologic profiles among subjects with and without immune activation. We propose that in at least a large subgroup of subjects with CFS who had allergies, the concomitant influences of immune activation brought on by allergic inflammation in an individual with the appropriate psychologic profile may interact to produce the symptoms of CFS. In a psychologically predisposed individual, symptoms associated with allergic inflammation are recognized as illness. (J Allergy Clin Immunol 1998;102:222-30.)