Naringenin ameliorates atopic dermatitis by inhibiting inflammation and enhancing immunity through the JAK2/STAT3 pathway.

OBJECTIVE: Atopic dermatitis (AD) is an inflammatory skin disease. Naringenin (Nar) possesses an anti-inflammatory property. This paper attempts to discuss the functional mechanism of Nar in AD mice through the Janus kinase 2 (JAK2)/signal transducer and activation of transcription 3 (STAT3) pathway. METHODS: Mouse models of DNFB-induced AD were established and treated with Nar, followed by intraperitoneal injection with the JAK2/STAT3 pathway activator Coumermycin A1. Dermatitis severity was scored and the thickness of right ear was measured. The pathological changes in dorsal skin tissues were observed by HE staining. The number of infiltrated mast cells and eosinophilic granulocytes was counted by TB staining. The serum IgE level and levels of TNF-α, IL-6, IFN-γ, IL-12, and IL-5 in dorsal skin tissues were measured by ELISA. The levels of p-JAK2, JAK2, p-STAT3, and STAT3 were determined by Western blot. RESULTS: Nar decreased dermatitis scores and right ear thickness, alleviated skin lesions, and reduced the number of infiltrated mast cells and eosinophilic granulocytes in AD mice. The serum IgE level and levels of TNF-α, IL-6, IFN-γ, IL-12, and IL-5 in dorsal skin tissues of AD mice were diminished after Nar treatment in a dose-dependent manner. Nar inhibited the activation of the JAK2/STAT3 pathway. The activation of the JAK2/STAT3 pathway partially nullified the therapeutic function of Nar on AD mice. CONCLUSION: Nar protects mice from AD by inhibiting inflammation and promoting immune responses through the inhibition of the JAK2/STAT3 pathway.

Detection of IL12/23p40 via PET Visualizes Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), which includes both Crohn disease and ulcerative colitis, is a relapsing inflammatory disease of the gastrointestinal tract. Long-term chronic inflammatory conditions elevate the patient's risk of colorectal cancer (CRC). Currently, diagnosis requires endoscopy with biopsy. This procedure is invasive and requires a bowel-preparatory regimen, adding to patient burden. Interleukin 12 (IL12) and interleukin 23 (IL23) play key roles in inflammation, especially in the pathogenesis of IBD, and are established therapeutic targets. We propose that imaging of IL12/23 and its p40 subunit in IBD via immuno-PET potentially provides a new noninvasive diagnostic approach. Methods: Our aim was to investigate the potential of immuno-PET to image inflammation in a chemically induced mouse model of colitis using dextran sodium sulfate by targeting IL12/23p40 with a 89Zr-radiolabeled anti-IL12/23p40 antibody. Results: High uptake of the IL12/23p40 immuno-PET agent was exhibited by dextran sodium sulfate-administered mice, and this uptake correlated with increased IL12/23p40 present in the sera. Competitive binding studies confirmed the specificity of the radiotracer for IL12/23p40 in the gastrointestinal tract. Conclusion: These promising results demonstrate the utility of this radiotracer as an imaging biomarker of IBD. Moreover, IL12/23p40 immuno-PET can potentially guide treatment decisions for IBD management.

The protective effect of IL-12/23 neutralizing antibody in sarcopenia associated with dextran sulfate sodium-induced experimental colitis.

BACKGROUND: The improvement of colitis symptoms by treatment with IL-12/23 p40 neutralizing antibody should increase the muscle mass and the function of the sarcopenia phenotype. METHODS: An experimental colitis model was induced by oral administration of 2% dextran sulfate sodium (DSS) for 7 days. During induction of colitis, IL-12/23 p40 neutralizing antibody was injected twice on Days 3 and 5. The total body mass index was measured by dual-energy X-ray absorptiometry. The muscle function was measured by forelimb grip strength and fatigue running distance. The muscle fibre cross-sectional area (CSA) was calculated after the transverse section and haematoxylin and eosin staining, and gene expression was confirmed by RT-qPCR. Differentiated C2C12 cells were used as in vitro models and treated with recombinant IL12/23 proteins to mimic the enhanced cytokines in colitis. RESULTS: The symptoms of colitis were alleviated by injection of IL-12/23 p40 neutralizing antibody compared with phosphate-buffered saline (PBS), and the disease activity index score was significantly lower on Day 8 (0.0 ± 0.00 of cont. vs. 11.3 ± 0.9 of DSS + PBS, P < 0.0001; DSS + PBS vs. 7.7 ± 1.25 of DSS + p40Ab, P < 0.0001). The CSA of the gastrocnemius and tibialis anterior muscle fibres decreased in mice with DSS-induced colitis (gastrocnemius, 1258.2 µm2 ± 176.45 of cont. vs. 640.1 µm2 ± 59.83 of DSS + PBS, P < 0.0001; tibialis anterior, 1251.8 µm2 ± 331.48 of cont. vs. 678.9 µm2 ± 67.59 of DSS + PBS, P < 0.0001), and the treatment of IL-12/23 p40 neutralizing antibody partially restored CSA of the gastrocnemius (640.1 µm2 ± 59.83 of DSS + PBS vs. 1062.0 µm2 ± 83.41 of DSS + p40Ab, P < 0.0001) and tibialis anterior (678.9 µm2 ± 67.59 of DSS + PBS vs. 1105.3 µm2 ± 143.15 of DSS + p40Ab, P = 0.0003).vs. 640.1 µm2  ± 59.83 of DSS + PBS, P < 0.0001) and tibialis anterior (1251.8 µm2  ± 331.48 of cont. vs. 678.9 µm2  ± 67.59 of DSS + PBS, P < 0.0001), and the treatment of IL-12/23 p40 neutralizing antibody partially restored CSA of the gastrocnemius (640.1 µm2  ± 59.83 of DSS + PBS vs. 1062.0 µm2  ± 83.41 of DSS + p40Ab, P < 0.0001) and tibialis anterior (678.9 µm2  ± 67.59 of DSS + PBS vs. 1105.3 µm2  ± 143.15 of DSS + p40Ab, P = 0.0003). In the evaluation of muscle function, grip strength and fatigue distance decreased by colitis were partially restored (grip strength: 139.9 g ± 5.38 of cont. vs. 83.9 g ± 5.48 of DSS + PBS, P < 0.0001; DSS + PBS vs. 118.6 g ± 4.05 of DSS + p40Ab, P < 0.0001; fatigue distance: 872.5 m ± 104.01 of cont. vs. 58.2 m ± 107.72 of DSS + PBS, P < 0.0001; DSS + PBS vs. 328.0 m ± 109.71 of DSS + p40Ab, P = 0.0015) by injection of IL-12/23 p40 neutralizing antibody. CONCLUSIONS: Our study demonstrates that Il-12/23 acts directly on muscle to induce atrophy, and the IL-12/23 p40 neutralizing antibody is effective not only in suppressing colitis but also in maintaining muscle mass and improving muscle function in an experimental colitis model.

IL-12 Contributes to the Development of Asthma by Targeting HIF-1α/NLRP3 Pathway through Runx3.

INTRODUCTION: The aim of the study was to determine the role and mechanism of runt-related transcription factor 3 (Runx3) in the development of asthma. METHODS: An asthma mouse model was constructed and validated by hematoxylin-eosin analysis of lung tissue and noninvasive enhanced pause (Penh) evaluation of airway hyperresponsiveness. Then, the levels of Runx3 and interleukin (IL)-12 in peripheral blood and lung tissue were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. By use Runx3+/- mice, the effect of Runx3 downregulation on ovalbumin (OVA)-induced asthma was investigated. After stimulated by different doses of IL-12, the expressions of Runx3, hypoxia inducible factor-1α (HIF-1α), and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in BEAS-2B cells were tested through Western blot and immunofluorescence. Subsequently, BEAS-2B cells treated with 20 ng/mL IL-12 were divided into control, Runx3 overexpression negative control, Runx3 overexpression, HIF-1α inhibitor, and Runx3 overexpression + HIF-1α agonist groups. The Western blot, immunofluorescence, and ELISA indicators were tested repeatedly. RESULTS: The increased number of inflammatory cells and Penh value confirmed the success of the asthma mouse model. IL-12 expression was significantly increased, and Runx3 was reduced in asthma mice compared with wild-type mice. Meanwhile, the level of immunoglobulin E (IgE) in serum, cytokines in bronchoalveolar lavage fluid, and IL-12, HIF-1α, NLRP3 in the lung were significantly elevated in Runx3+/- mice. With the increase of IL-12 concentration, Runx3 protein expression decreased, while HIF-1α and NLRP3 expression increased. Further mechanistic studies suggest that Runx3 ameliorates IL-12-induced BEAS-2B injury by inhibiting HIF-1α/NLRP3 pathway. CONCLUSION: These results suggested that IL-12 contributes to the development of asthma by targeting HIF-1α/NLRP3 pathway through Runx3, thus providing a novel strategy for asthma therapy.

A Single Nucleotide Polymorphism in SH2B3/LNK Promotes Hypertension Development and Renal Damage.

BACKGROUND: SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism in SH2B3 (rs3184504) results in substitution of tryptophan (Trp) for arginine (Arg) at amino acid 262 and is a top association signal for hypertension in human genome-wide association studies. Whether this variant is causal for hypertension, and if so, the mechanism by which it impacts pathogenesis is unknown. METHODS: We used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of this SH2B3 polymorphism. Mice underwent angiotensin II (Ang II) infusion to evaluate differences in blood pressure (BP) elevation and end-organ damage including albuminuria and renal fibrosis. Cytokine production and Stat4 phosphorylation was also assessed in Arg/Arg and Trp/Trp T cells. RESULTS: Trp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8+ T cells from Ang II-infused Trp/Trp mice produce significantly more interferon gamma (IFNg) compared to Arg/Arg controls. Interleukin-12 (IL-12)-induced IFNg production is greater in Trp/Trp compared to Arg/Arg CD8+ T cells. In addition, IL-12 enhances Stat4 phosphorylation to a greater degree in Trp/Trp compared to Arg/Arg CD8+ T cells, suggesting that Trp-encoding SH2B3 exhibits less negative regulation of IL-12 signaling to promote IFNg production. Finally, we demonstrated that a multi-SNP model genetically predicting increased SH2B3 expression in lymphocytes is inversely associated with hypertension and hypertensive chronic kidney disease in humans.. CONCLUSIONS: Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

Cardiovascular Safety of Biologics Targeting Interleukin (IL)-12 and/or IL-23: What Does the Evidence Say?

There is substantial evidence regarding the association between psoriasis and the elevated risk of cardiovascular (CV) disease. Many patients with psoriasis may also be concerned that their treatments may be associated with a further increase in the risk of CV disease. In this article, we summarize the data regarding the biological role of interleukin (IL)-12/23 in atherogenesis. We performed a literature search for currently known CV safety data from trials and observational studies of treatments targeting IL-12/23 in psoriasis, i.e. the p40 inhibitors ustekinumab and briakinumab, and the p19 inhibitors guselkumab, risankizumab, and tildrakizumab. On balance, extensive evidence supports the CV safety of ustekinumab, with over 14 years of follow-up data in multiple cohort studies and randomized controlled trials (RCTs). One self-controlled study concluded ustekinumab may precipitate short-term raised CV risk, but the study had limitations hindering interpretation. The safety evidence from RCTs on the p19 inhibitors are reassuring thus far, but these studies may not detect rare CV events in real-world patients. We concluded that the overall evidence does not show that ustekinumab is associated with an increase in the risk of CV disease in patients with psoriasis, but further data are awaited to assess the CV safety of p19 inhibitors for the treatment of psoriasis.

Localized Interleukin-12 for Cancer Immunotherapy.

Interleukin-12 (IL-12) is a potent, pro-inflammatory type 1 cytokine that has long been studied as a potential immunotherapy for cancer. Unfortunately, IL-12's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. Early clinical trials in the mid-1990's showed that systemic delivery of IL-12 incurred dose-limiting toxicities. Nevertheless, IL-12's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. The development of strategies which maximize IL-12 delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. Diverse IL-12 delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. Several localized IL-12 delivery approaches have recently reached the clinical stage with several more at the precipice of translation. Taken together, localized delivery systems are supporting an IL-12 renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. This review begins with a brief historical account of cytokine monotherapies and describes how IL-12 went from promising new cure to ostracized black sheep following multiple on-study deaths. The bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for IL-12-based cancer immunotherapies. Advantages and limitations of different delivery technologies are highlighted. Finally, perspectives on how IL-12-based immunotherapies may be utilized for widespread clinical application in the very near future are offered.

Crescentic IgA nephropathy after administration of human monoclonal interleukin-12/23p40 antibody in a patient with Crohn's disease: a case report.

Ustekinumab (UST), an interleukin (IL)-12/IL-23-blocking monoclonal antibody, is a novel therapeutic option for Crohn's disease (CD). We describe a 24-year-old man with CD who showed an abrupt decline in renal function after administration of UST. Twenty-nine months previously, the patient was diagnosed with CD, and abnormal urinalysis findings in health checkup were coincidentally found at that time. Three months previously, treatment for CD was switched from infliximab to UST because of therapy-resistant severe diarrhea and bloody stools. A single dose of UST (260 mg) was initially intravenously administered, followed by single subcutaneous administration (90 mg) 2 months later. Thereafter, the patient exhibited rapid renal dysfunction with significant urinary abnormalities, although his gastrointestinal symptoms had completely disappeared. He was admitted to our hospital for further examination and treatment. Renal pathologic findings were compatible with crescentic glomerulonephritis consisting of almost fibro-cellular crescents. Immunofluorescent study showed IgA and C3 deposition in the glomerular mesangial area and IgA subclass staining revealed predominant IgA1 with concomitant mild IgA2 deposition. Furthermore, galactose-deficient IgA1 (Gd-IgA1) was also positive in the mesangial area. In addition, serum-Gd-IgA1 level was moderately increased. UST treatment was stopped and he responded to intensive steroid therapy with a parallel reduction of serum creatinine and Gd-IgA1 levels without flare of gastrointestinal symptoms. To our knowledge, this is the first case of immunoglobulin A nephropathy (IgAN) in patient with CD that might be aggravated by UST treatment. We presume that inhibition of IL-12/23 signaling with UST may cause to form crescentic IgAN by enhancing Gd-IgA1 production.

First-in-Human Phase I Trial of a Tumor-Targeted Cytokine (NHS-IL12) in Subjects with Metastatic Solid Tumors.

PURPOSE: The NHS-IL12 immunocytokine is composed of two IL12 heterodimers fused to the NHS76 antibody. Preclinical studies have shown that this antibody targets IL12 to regions of tumor necrosis by binding histones on free DNA fragments in these areas, resulting in enhanced antitumor activity. The objectives of this phase I study were to determine the maximum tolerated dose (MTD) and pharmacokinetics of NHS-IL12 in subjects with advanced solid tumors. PATIENTS AND METHODS: Subjects (n = 59) were treated subcutaneously with NHS-IL12 in a single ascending-dose cohort followed by a multiple ascending-dose cohort (n = 37 with every 4-week dosing). RESULTS: The most frequently observed treatment-related adverse events (TRAE) included decreased circulating lymphocytes, increased liver transaminases, and flu-like symptoms. Of the grade ≥3 TRAEs, all were transient and only one was symptomatic (hyperhidrosis). The MTD is 16.8 µg/kg. A time-dependent rise in IFNγ and an associated rise in IL10 were observed following NHS-IL12. Of peripheral immune cell subsets evaluated, most noticeable were increases in frequencies of activated and mature natural killer (NK) cells and NKT cells. Based on T-cell receptor sequencing analysis, increases in T-cell receptor diversity and tumor-infiltrating lymphocyte density were observed after treatment where both biopsies and peripheral blood mononuclear cells were available. Although no objective tumor responses were observed, 5 subjects had durable stable disease (range, 6-30+ months). CONCLUSIONS: NHS-IL12 was well tolerated up to a dose of 16.8 µg/kg, which is the recommended phase II dose. Early clinical immune-related activity warrants further studies, including combination with immune checkpoint inhibitors.See related commentary by Lyerly et al., p. 9.

Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent.

Interleukin-12 (IL-12) has emerged as one of the most potent agents for anti-tumor immunotherapy. However, potentially lethal toxicity associated with systemic administration of IL-12 precludes its clinical application. Here we redesign the molecule in such a way that its anti-tumor efficacy is not compromised, but toxic effects are eliminated. Deletion of the N-terminal signal peptide of IL-12 can effect such a change by preventing IL-12 secretion from cells. We use a newly designed tumor-targeted oncolytic adenovirus (Ad-TD) to deliver non-secreting (ns) IL-12 to tumor cells and examine the therapeutic and toxic effects in Syrian hamster models of pancreatic cancer (PaCa). Strikingly, intraperitoneal delivery of Ad-TD-nsIL-12 significantly enhanced survival of animals with orthotopic PaCa and cured peritoneally disseminated PaCa with no toxic side effects, in contrast to the treatment with Ad-TD expressing unmodified IL-12. These findings offer renewed hope for development of IL-12-based treatments for cancer.

α-Galactosylceramide-activated murine NK1.1(+) invariant-NKT cells in the myometrium induce miscarriages in mice.

Innate immunity, which is unable to discriminate self from allo-antigens, is thought to be important players in the induction of miscarriages. Here, we show that the administration of IL-12 to syngeneic-mated C57BL/6 mice on gestation day 7.5 (Gd 7.5), drives significant miscarriages in pregnant females. Furthermore, the administration on Gd 7.5 of α-galactosylceramide (α-GalCer), which is known to activate invariant natural killer T (iNKT) cells, induced miscarriages in both syngeneic-mated C57BL/6 mice and allogeneic-mated mice (C57BL/6 (♀) × BALB/c (♂)). Surprisingly, the percentages of both DEC-205(+) DCs and CD1d-restricted NK1.1(+) iNKT cells were higher in the myometrium of pregnant mice treated i.p. with α-GalCer than in the decidua. IL-12 secreted from α-GalCer-activated DEC-205(+) DCs stimulated the secretion of cytokines, including IL-2, IL-4, IFN-γ, TNF-α, perforin, and granzyme B, from the NK1.1(+) iNKT cells in the myometrium, leading to fetal loss in pregnant mice. Finally, the i.p. administration of IL-12 and/or α-GalCer in iNKT-deficient Jα18(-/-) (Jα18 KO) mice did not induce miscarriages. This study provides a new perspective on the importance of the myometrium, rather than the decidua, in regulating pregnancy and a mechanism of miscarriage mediated by activated DEC-205(+) DCs and NK1.1(+) iNKT cells in the myometrium of pregnant mice.

Development of a long-acting, protein-loaded, redox-active, injectable gel formed by a polyion complex for local protein therapeutics.

Although cancer immunotherapies are attracting much attention, it is difficult to develop bioactive proteins owing to the severe systemic toxicity. To overcome the issue, we designed new local protein delivery system by using a protein-loaded, redox-active, injectable gel (RIG), which is formed by a polyion complex (PIC) comprising three components, viz., cationic polyamine-poly(ethylene glycol)-polyamine triblock copolymer possessing ROS-scavenging moieties as side chains; anionic poly(acrylic acid); and a protein. The mixture formed the protein-loaded PIC flower micelles at room temperature, which immediately converted to a gel with high mechanical strength upon exposure to physiological conditions. Because the protein electrostatically interacts with the PIC gel network, RIG provided a sustained release of the protein without a significant initial burst, regardless of the types of proteins in vitro, and much longer retention of the protein at the local injection site in mice than that of the naked protein. Subcutaneous injections of IL-12@RIG in the vicinity of tumor tissue showed remarkable tumor growth inhibition in tumor-bearing mice, compared to that observed with injection of IL-12 alone, suppressing adverse events caused by IL-12-induced ROS. Our results indicate that RIG has potential as a platform technology for an injectable sustained-release carrier for proteins.

The protective effects of intranasal administration of IL-12 given before influenza virus infection and the negative effects of IL-12 treatment given after viral infection.

To investigate whether the administration of IL-12 is effective against influenza virus infection, mice were intranasally administered IL-12 for three consecutive days and then infected with a non-lethal dose of the influenza virus. The IL-12-treated mice were more resistant to the virus than control mice with respect to the remission of body weight loss, virus burden, pro-inflammatory cytokine production, and inflammatory cell infiltration in the lungs. The number of NK cells and the level of NK cell cytotoxicity significantly increased in the lungs of the mice treated with IL-12 before infection compared to that observed in control mice, leading to promptly eliminate the viral-infected cells. Unexpectedly, all of mice that received IL-12 treatment after being infected with a non-lethal dose of the virus died as a result of their high virus burden and pro-inflammatory cytokine production in the lungs. One possibility of the mechanisms was considered to be activation of myeloid-derived suppressor cell (MDSC), which has immune suppressive function, in the lungs. Thus, IL-12 treatment has opposite effects depending on whether it is administered before or after infection. These results demonstrate the potential risks of immune modulating therapies such as administration of exogenous cytokine or neutralization of cytokine. J. Med. Virol. 88:1487-1496, 2016. © 2016 Wiley Periodicals, Inc.

Phase I trial of a formulated IL-12 plasmid in combination with carboplatin and docetaxel chemotherapy in the treatment of platinum-sensitive recurrent ovarian cancer.

OBJECTIVES: The primary objective of this study was to evaluate the safety and tolerability of a formulated IL-12 plasmid administered intraperitoneally (IP) in conjunction with intravenous (IV) carboplatin/docetaxel in platinum-sensitive ovarian cancer patients. METHODS: Escalating doses of IL-12 plasmid (phIL-12) formulated with the lipopolymer PEG-PEI-Cholesterol (PPC) were administered IP every 10-11 days for a total of four treatments and the highest dose was expanded to eight treatments. Patients also received IV carboplatin (AUC 5) and docetaxel (75 mg/m(2)) every 21 days. Patients were followed for safety, biological activity and antitumor activity after phIL-12/PPC treatment. RESULTS: All 13 patients enrolled in the study received both phIL-12/PPC and chemotherapy treatment. There were 49 plasmid-associated adverse events (AEs). The most common AEs were abdominal pain, transient hypotension, low grade fever, catheter site pain, chills, dysgeusia, infusion-related reaction, and nausea. These AEs appeared to be plasmid dose related. Grade 3 AEs included manageable abdominal pain and cytokine release syndrome. There were no dose limiting toxicities and the plasmid treatment did not augment the chemotherapy-associated AEs. The best overall antitumor response (17% CR, 33% PR, 42% SD and 8% PD) was typical of the patient population enrolled for the study. Translational studies showed rise in IFN-γ and TNF-α concentrations in a dose dependent manner. CONCLUSIONS: The escalating doses and cycles of intraperitoneal phIL-12/PPC when combined with carboplatin/docetaxel chemotherapy in recurrent ovarian cancer patients were well tolerated and did not appear to exacerbate the side effects or attenuate the efficacy of the chemotherapy treatment.

Lethal NK-mediated inflammation induced by IL-12 in the absence of polymorphic and nonpolymorphic MHC class I molecules.

Interleukin-12 is a potent activator and initiator of type-1 T cell development, and can be used as an adjuvant to bias for the development of vaccine-induced Th1 immune responses. During vaccination of MHC class I deficient beta-2 microglobulin knockout mice (ß2M(-/-)) with an IL-12/αIL-4 Th1 biasing procedure, all of the mice died. None of the IL-12/αIL-4 treated wild type mice developed any noticeable complications. We hypothesized that NK cells may be activated by IL-12 treatment in these ß2M(-/-) mice, leading to necrosis and eventual death. IL-12/αIL-4 treatment of ß2M(-/-) mice resulted in increased NK cell numbers and activation status (IFN-γ(+), CD69(+)). Finally, in vivo depletion of NK cells reversed the pathology induced by IL-12/αIL-4 treatment in ß2M deficient mice. These results indicate that IL-12 combined with αIL-4 irreversibly activates NK cells leading to a disseminated inflammatory pathology and death in ß2M(-/-) mice.

Interleukin-30: a novel antiinflammatory cytokine candidate for prevention and treatment of inflammatory cytokine-induced liver injury.

UNLABELLED: The liver is the major metabolic organ and is subjected to constant attacks from chronic viral infection, uptake of therapeutic drugs, life behavior (alcoholic), and environmental contaminants, all of which result in chronic inflammation, fibrosis, and, ultimately, cancer. Therefore, there is an urgent need to discover effective therapeutic agents for the prevention and treatment of liver injury, the ideal drug being a naturally occurring biological inhibitor. Here we establish the role of IL30 as a potent antiinflammatory cytokine that can inhibit inflammation-induced liver injury. In contrast, interleukin (IL)27, which contains IL30 as a subunit, is not hepatoprotective. Interestingly, IL30 is induced by the proinflammatory signal such as IL12 through interferon-gamma (IFN-γ)/signal transducer and activator of transcription 1 signaling. In animal models, administration of IL30 by way of a gene therapy approach prevents and treats both IL12-, IFN-γ-, and concanavalin A-induced liver toxicity. Likewise, immunohistochemistry analysis of human tissue samples revealed that IL30 is highly expressed in hepatocytes, yet barely expressed in inflammation-induced tissue such as fibrous/connective tissue. CONCLUSION: These novel observations reveal a novel role of IL30 as a therapeutic cytokine that suppresses proinflammatory cytokine-associated liver toxicity.

The regulation and activity of interleukin-12.

Interleukin-12 (IL-12) is a key cytokine in the development of T helper type 1 (Th1) cell polarization, and its production of IL-12 is redundantly regulated. An important pro-inflammatory cytokine, IL-12 has been shown to have potent immunomodulatory, antitumor, and anti-infection activity in vitro and in vivo. Therefore, following a series of promising results from preclinical animal models experiments, researchers have begun to explore the clinical use of recombinant human IL-12 (rhIL-12) for treating a variety of diseases. In a series of phase I and phase II clinical trials related to cancer, viral infections, and hematopoietic stem cell transplants (HSCT), various strategies of rhIL-12 administration have been used with promising preliminary clinical results associated with tolerable toxicities.

An extension of the single threshold design for monitoring efficacy and safety in phase II clinical trials.

Tan and Machin (biStat. Med. 2002; 21:1991-2012) introduce a Bayesian two-stage design for phase II clinical trials where the binary endpoint of interest is treatment efficacy. In this paper we propose an extension of their design to incorporate safety considerations. At each stage we define the treatment successful and deserving of further study if the total number of adverse events is sufficiently small and the number of responders who simultaneously do not experience any toxicity is sufficiently large. Therefore, our criterion is based on the joint posterior probability that the true overall toxicity rate and the true efficacy-and-safety rate are, respectively, smaller and larger than conveniently pre-specified target values. The optimal two-stage sample sizes are determined specifying a minimum threshold for the above-mentioned posterior probability, computed under the assumption that favorable outcomes have occurred. Besides describing the proposed design, we suggest how to construct informative prior scenarios and we also apply the reference algorithm to derive a non-informative prior distribution. Finally, some numerical results are provided and a real data application is illustrated.

Intratumoral immunotherapy of established solid tumors with chitosan/IL-12.

IL-12 is a potent antitumor cytokine that exhibits significant clinical toxicities after systemic administration. We hypothesized that intratumoral (i.t.) administration of IL-12 coformulated with the biodegradable polysaccharide chitosan could enhance the antitumor activity of IL-12 while limiting its systemic toxicity. Noninvasive imaging studies monitored local retention of IL-12, with and without chitosan coformulation, after i.t. injection. Antitumor efficacy of IL-12 alone and IL-12 coformulated with chitosan (chitosan/IL-12) was assessed in mice bearing established colorectal (MC32a) and pancreatic (Panc02) tumors. Additional studies involving depletion of immune cell subsets, tumor rechallenge, and CTL activity were designed to elucidate mechanisms of regression and tumor-specific immunity. Coformulation with chitosan increased local IL-12 retention from 1 to 2 days to 5 to 6 days. Weekly i.t. injections of IL-12 alone eradicated ≤10% of established MC32a and Panc02 tumors, while i.t. chitosan/IL-12 immunotherapy caused complete tumor regression in 80% to 100% of mice. Depletion of CD4(+) or Gr-1(+) cells had no impact on chitosan/IL-12-mediated tumor regression. However, CD8(+) or NK cell depletion completely abrogated antitumor activity. I.t. chitosan/IL-12 immunotherapy generated systemic tumor-specific immunity, as >80% of mice cured with i.t. chitosan/IL-12 immunotherapy were at least partially protected from tumor rechallenge. Furthermore, CTLs from spleens of cured mice lysed MC32a and gp70 peptide-loaded targets. Chitosan/IL-12 immunotherapy increased local retention of IL-12 in the tumor microenvironment, eradicated established, aggressive murine tumors, and generated systemic tumor-specific protective immunity. Chitosan/IL-12 is a well-tolerated, effective immunotherapy with considerable potential for clinical translation.