Recombinant Murine Interleukin Research Articles

Optimizing the synthesis of interleukin-12-loaded PLGA nanospheres (rmIL-12ns) via ultrasonication for treatment of metastatic osteosarcoma.

Clinical trials exploring bolus intravenous delivery of interleukin-12 (IL-12) for treatment of solid tumors ultimately failed due to lack of clinical response and severe dose-limiting toxicities. The present study was conducted to evaluate whether recombinant murine IL-12 (rmIL-12) could be successfully encapsulated within Poly (D, l-lactide-co-glycolide) (PLGA) nanospheres (rmIL-12ns) for safe and effective systemic delivery at pharmacologic scale. Optimal fabrication of rmIL-12ns occurs with dichloromethane as the organic solvent and emulsion formation via ultrasonication at 50% power (250 W sonicator) for 10 s (50W10s). We then determined whether utilization of synthesis modifiers including fetal bovine serum (FBS), magnesium hydroxide [Mg(OH)2 ], trehalose, or the surfactants polysorbate 80 and Span 60 alone or in combination could increase the encapsulation efficiency (EE) and/or modify the burst elution profile characteristic of the 50W10s rmIL-12ns formulation. The greatest EEs compared to the unmodified formulation were measured with modifications containing the surfactants polysorbate 80 and Span 60 (surfactant: 28.3 ± 6.10%, p = 0.29 and Surf/FBS: 85.4 ± 2.19%, p = 0.039). The Surf/FBS formulation was further modified for in vivo murine injection by substituting FBS with mouse serum albumin (MSA). The resulting Surf/MSA rmIL-12ns were then characterized before delivery at three doses (0.1, 1, and 10 mg rmIL-12ns) in our established murine model of metastatic osteosarcoma to assess efficacy. Preliminary results suggested no evidence of disease with delivery of the 0.1 mg dose in 75% of mice (3 of 4) versus a nontreated historical control (2 of 34).

Acute Interleukin‐6 Injection Influences Key Enzymes Involved in Amyloid Precursor Protein Processing in C57BL/6J Mice

Background Amyloid-beta plaques are a hallmark feature of Alzheimer's disease and are the product of aggregated amyloid-beta peptides. These peptides originate from amyloid precursor protein (APP) which is cleaved by beta secretase (BACE1), a key enzyme in the amyloidogenic cascade. Cleavage of APP by alpha secretase (ADAM10) however, does not result in amyloid-beta peptides and is a key enzyme in the non-amyloidogenic cascade. It is widely accepted that exercise reduces amyloid-beta production and plaque build-up in the brain, although it is unclear how exercise may be driving this observation. Interleukin-6 (IL-6) is an exercise induced cytokine and recent work in this area has demonstrated a role for acute exercise induced IL-6 in tissues such as muscle, adipose, and liver, however the role of acute IL-6 in the brain remains ill defined. The purpose of this study is to investigate if acute IL-6 injection will modulate key enzymes involved in APP processing, namely BACE1 and ADAM10. It is hypothesized that acute IL-6 injection will result in a shift towards the non-amyloidogenic processing of APP. Methods Male 10-week-old C57BL/6J mice were intraperitoneally injected with a weight-adjusted dose of recombinant murine IL-6 (3ng/g body mass) or an equivalent volume of saline. Brain tissue was acquired 15 minutes post-injection and the pre-frontal cortex and hippocampus regions were dissected. Results Acute IL-6 injection resulted in lower BACE1 activity in both the cortex (p=0.011) and the hippocampus (p=0.027) compared to the saline injected group. This was accompanied by lower BACE1 (p=0.007) and sAPPb(p=0.019) protein content in the hippocampus of mice injected with IL-6. No differences for BACE1 or sAPPb were observed in the cortex. ADAM10 activity was higher with IL-6 injection compared to saline in the hippocampus (p=0.018) and the cortex (p=0.055). A change in ADAM10 protein content was also observed with the ratio of pro to mature ADAM10 being higher in the cortex (p=0.033) of the IL-6 injected mice as compared to the saline injected control. Similarly, both pro (p=0.017) and mature (p=0.035) ADAM10 protein content were lower in the hippocampus of the IL-6 injected mice as compared to the saline injected control. Conclusions These results demonstrate that acute IL-6 modulates important enzymes involved in APP processing; pushing the cascade towards the non-amyloidogenic arm. This work further highlights regional brain differences in response to acute IL-6 and extends our knowledge of the function of acute IL-6 as a signalling molecule in the brain. Future work should determine if exercise induced IL-6 is in fact the main mediator of non-amyloidogenic APP processing.

CORM-3 improves emotional changes induced by hemorrhagic shock via the inhibition of pyroptosis in the amygdala

Hemorrhagic shock and resuscitation (HSR) may lead to long-term neurological dysfunction, such as depression and anxiety. Carbon monoxide (CO) has emerged as an excellent neuroprotective agent against caspase-1-associated pyroptosis, following HSR. We evaluated the effects and determined the mechanism through which CO protects against emotional changes in a model of HSR, in rats. We subjected rats to treatments with an exogenous, CO-releasing compound (CORM-3, 4 mg/kg), in vivo, after HSR. We measured sucrose preference and performed tail suspension and open field tests 7 days after HSR, assessed brain magnetic resonance imaging 12 h after HSR and evaluated pyroptosis, and neuronal and astrocyte death in the amygdala 12 h post-HSR. We also measured changes in behavior and pathology, following an injection of recombinant murine interleukin (IL)-18 into the amygdala. HSR-treated rats displayed increased depression-like and anxiety-like behaviors, increased amygdalar injury, as indicated by T2-weighted magnetic resonance imaging (MRI) and cerebral blood flow with arterial spin labeling (CBFASL), associated with both neuronal and astrocytic death and pyroptosis, and upregulated IL-18 expression was observed in astrocytes. CORM-3 administration after resuscitation, via a femoral vein injection, provided neuroprotection against HSR, and this neuroprotective effect could be partially reversed by the injection of recombinant murine IL-18 into the amygdala. Therefore, CORM-3 alleviated HSR-induced neuronal pyroptosis and emotional changes, through the downregulation of IL-18 in astrocytes.

Salidroside Attenuates Denervation-Induced Skeletal Muscle Atrophy Through Negative Regulation of Pro-inflammatory Cytokine

Skeletal muscle atrophy is associated with pro-inflammatory cytokines. Salidroside is a biologically active ingredient of Rhodiola rosea, which exhibits anti-inflammatory property. However, there is little known about the effect of salidroside on denervation-induced muscle atrophy. Therefore, the present study aimed to determine whether salidroside could protect against denervation-induced muscle atrophy and to clarify potential molecular mechanisms. Denervation caused progressive accumulation of inflammatory factors in skeletal muscle, especially interleukin 6 (IL6) and its receptor, and recombinant murine IL6 (rmIL6) local infusion could induce target muscle atrophy, suggesting that denervation induced inflammation in target muscles and the inflammation may trigger muscle wasting. Salidroside alleviated denervation-induced muscle atrophy and inhibited the production of IL6. Furthermore, the inhibition of phosphorylation of signal transducer and activator of transcription 3 (STAT3), and the decreased levels of suppressor of cytokine signaling (SOCS3), muscle RING finger protein-1 (MuRF1), atrophy F-box (atrogin-1), microtubule-associated protein light chain 3 beta (LC3B) and PTEN-induced putative kinase (PINK1) were observed in denervated muscles that were treated with salidroside. Finally, all of these responses to salidroside were replicated in neutralizing antibody against IL6. Taken together, these results suggest that salidroside alleviates denervation-induced inflammation response, thereby inhibits muscle proteolysis and muscle atrophy. Therefore, it was assumed that salidroside might be a potential therapeutic candidate to prevent muscle wasting.

IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage

Mycobacterium tuberculosis (MTB) adopts a special survival strategy to overcome the killing mechanism(s) of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3) of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ) and recombinant murine interleukin-10 (rmIL-10) on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50ng/ml) of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50ng/ml) significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.

Class A Scavenger Receptor Exacerbates Osteoclastogenesis by an Interleukin-6-Mediated Mechanism through ERK and JNK Signaling Pathways.

Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, which are important for bone health. Class A scavenger receptor (SR-A) is a multifunctional molecule that functions during differentiation of monocyte into macrophages and osteoclasts. To further characterize the role of SR-A in osteoclasts, we used the murine tooth movement model (TM) and the murine anterior cruciate ligament transection model of osteoarthritis (ACLT OA). In these two models the bones involved are of different origin and have different properties. Bone resorption was decreased in SR-A-/- mice compared to SR-A+/+ mice. Further evaluation showed that the number of multinucleated osteoclasts in SR-A-/- mice, compared to SR-A+/+ mice, was significantly decreased both in vivo and in vitro. The levels of interleukin-6 (IL-6) produced by osteoclasts were reduced in SR-A-/- mice compared to SR-A+/+ mice. In the in vitro marrow-derived osteoclast formation assay and in both mouse models, osteoclastogenesis was restored to normal in SR-A-/- mice by administration of recombinant murine IL-6. Moreover, neutralization of IL-6 reduced the number of osteoclasts formed in SR-A+/+ mice of TM model. Both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38, signaling pathways were downregulated in receptor activator of nuclear factor-κB ligand (RANKL)-stimulated SR-A-/- osteoclasts. Importantly, when treated with either ERK or JNK inhibitor, the numbers of osteoclasts generated from RANKL-induced bone marrow derived-macrophages of SR-A+/+ mice, and their IL-6 production, were significantly decreased. This suggests that SR-A activates the ERK and JNK signaling pathways, and promotes production of IL-6 by osteoclasts to further stimulate osteoclast formation.

Recombinant interleukin 6 with M cell-targeting moiety produced in Lactococcus lactis IL1403 as a potent mucosal adjuvant for peroral immunization

Development and application of safe and effective mucosal adjuvants are important to improve immunization efficiency in oral vaccine. Here, we report a novel mucosal adjuvant, IL-6-CKS9, a recombinant cytokine generated by conjugating an M cell-targeting peptide (CKS9) with c-terminus of the murine interleukin 6 (IL-6), which facilitated enhancement of mucosal immune response. Lactococcus lactis IL1403, a food-grade strain of lactic acid bacteria (LAB) which is widely used in dairy industry, was used as a host cell to express and secrete the IL-6-CKS9 for a mucosal vaccine adjuvant. The recombinant L. lactis IL1403 secreting IL-6-CKS9 was orally administered with a model antigen protein, M-BmpB (Brachyspira membrane protein B conjugated with CKS9), to BALB/c mice for mucosal immunization. ELISA analyses showed consistent enhancement tendencies in induction of anti-M-BmpB antibody levels both with mucosal (IgA) and systemic (IgG) immune responses in IL-6-CKS9-LAB treated group compared with other groups tested by conducting two separated mice immunization assays. In addition, we characterized that the oral administration of model protein antigen with live LAB producing IL-6-CKS9 could induce both Th1 and Th2 type immune responses by analysis of the specific anti-BmpB IgG1 and IgG2a isotypes in the sera and also investigated possible oral tolerance in our vaccine strategy. Collectively, our results showed successful production and secretion of recombinant murine IL-6 with M cell-targeting moiety (IL-6-CKS9) from L. lactis IL1403 and demonstrated the live recombinant LAB producing IL-6-CKS9 could have a potential to be used as an efficient adjuvant for peroral vaccination.

Enhanced Survival During Experimental Listeria monocytogenes Sepsis in Neonatal Mice Prophylactically Treated With Th1 and Macrophage Immunoregulatory Cytokines and Mediators

Impairments in T-cell and macrophage-mediated host defense lead to increased infection-related morbidity and mortality in neonates, partly because of immaturity in T helper (Th)1 function. Listeria monocytogenes (Lm) is an intracellular pathogen disproportionately causing severe disease among neonates and the immunocompromised. Intact macrophage and Th1-mediated immune responses are critical for Lm clearance. We and others have previously demonstrated downregulation of Th1 and macrophage immunoregulatory cytokines in cord blood versus adult peripheral blood. We sought to determine whether therapeutic or prophylactic single agent or combination recombinant murine interleukin (rmIL)-12, rmIL-18, recombinant murine macrophage-colony stimulating factor (rmM-CSF), recombinant murine granulocyte macrophage-colony stimulating factor (rmGM-CSF) and recombinant murine interferon (rmIFN)-γ would enhance survival during experimental neonatal Lm sepsis. A 90% lethal dose (LD90) of Lm was established in C57/BL/6 neonatal mice. rmIL-12, rmIL-18, rmM-CSF, rmGM-CSF and rmIFN-γ were administered singly or sequentially, before or after LD90 Lm inoculation; ampicillin was administered 24 hours after inoculation. Therapeutic doses of rmIL-12, rmIL-18, rmM-CSF, rmGM-CSF and rmIFN-γ as single agents and sequential therapy with rmM-CSF + (rmIL-12 and/or rmIL-18) + rmIFN-γ in addition to ampicillin were not associated with increased survival. However, prophylactic single doses of rmIL-12, rmIL-18, rmM-CSF and rmIFN-γ and prophylactic sequential doses of rmM-CSF + (rmIL-12 and/or rmIL-18) + rmIFN-γ in addition to ampicillin were associated with significantly enhanced survival compared with ampicillin alone. These data suggest prophylactic administration of macrophage and Th1 immunoregulatory cytokines can potentially overcome deficits in neonatal immunity to protect against Lm.

The antibody‐based targeted delivery of interleukin‐4 and 12 to the tumor neovasculature eradicates tumors in three mouse models of cancer

Preclinical studies with recombinant murine interleukin 4 (IL4) in models of cancer have shown potent tumor growth inhibition. However, systemic administration of human IL4 to cancer patients exhibited modest antitumor activity and considerable toxicities. To improve the therapeutic index and reduce side effects of this cytokine, we developed of a novel "immunocytokine" based on sequential fusion of murine IL4 with the antibody fragment F8 (specific to the alternatively spliced extra-domain A of fibronectin, a marker for tumor-angiogenesis) in diabody format. The resulting fusion protein, termed F8-IL4, retained full antigen-binding activity and cytokine bioactivity and was able to selectively localize on solid tumors in vivo. When used as single agent, F8-IL4 inhibited tumor growth in three different immunocompetent murine cancer models (F9 teratocarcinoma, CT26 colon carcinoma and A20 lymphoma). Furthermore, F8-IL4 showed synergistic effects when coadministered with immunocytokines based on IL2 and IL12. Indeed, combination therapy with an IL12-based immunocytokine yielded complete tumor eradication, in spite of the fact that IL4 and IL12 display opposite immunological mechanisms of action in terms of their polarization of T-cell based responses. No weight loss or any signs of toxicity were observed in treated mice, both in monotherapy and in combination, indicating a good tolerability of the immunocytokine treatment. Interestingly, mice cured from CT26 tumors acquired a durable protective antitumor immunity. Depletion experiments indicated that the antitumor activity was mediated by CD8+ T cells and by NK cells.

GW24-e1023 Advanced Glycation End Product Modified Low Density Lipoprotein Activates Mast Cells via Toll-Like 4 Receptor Pathway

ObjectivesTo investigate the effects of AGE-LDL, in murine bone marrow-derived mast cells (mBMMCs), on mast cell degranulation and their possible signal pathways.MethodsCarotid plaques were excised during Carotid Endarterectomy of diabetic...

Radioprotective effect of rmIL-12 on mice irradiated by γ-ray

The aim of this study was to investigate the radioprotective effect of recombinant murine interleukin 12 (rmIL-12) on mice irradiated by γ-ray. Fifty- six BALB/c mice were totally irradiated by 6.0 Gy of (60)Co γ-ray and randomly divided into irradiation control group,rmIL-12 treated group and recombinant murine thrombopoietin (rmTPO) treated group.The 5 and 20 µg/kg of rmIL-12 were administrated intraperitoneally at 24 h before irradiation respectively (low and high dose rmIL-12 treated group), 15 µg/kg of rmTPO was administrated subcutaneously at 30 min and 24 h following irradiation in rmTPO treated group. The general conditions of mice were observed twice a day, the changes in body weight and peripheral blood cell counts were examined once every three days, bone marrow cells were collected to perform colony cultivation at day 14 and 28 after irradiation. The results showed that the general conditions of mice in rmIL-12 treated group were better than those in irradiation control group. Compared with the irradiation control group,5 and 20 µg/kg rmIL-12 treatment significantly promoted platelet recovery, resulting in less profound nadirs (15.9% vs 8.1%,18.2% vs 8.1%,P < 0.01) and rapid recovery to normal levels (11 days vs 14 days). WBC count recovery rate in rmIL-12 treated group was faster than that in the irradiation control group. The WBC and platelet count recovery rate in 5 µg/kg rmIL-12 treated group were as fast as that in the rmTPO treated group, both of which were slower than that in 20 µg/kg rmIL-12 treated group (P > 0.05). Semi-solid bone marrow cell culture also demonstrated that rmIL-12 could stimulate bone marrow cells to form more CFU-Mix than those in the irradiation control group in vitro at day 14 and 28 after irradiation(P < 0.01).There was no significant difference between rmIL-12 and rmTPO treated groups (P > 0.05), CFU-GM counts in 5 µg/kg rmIL-12 treated group and rmTPO treated group at day 28 after irradiation were higher than those in irradiation control group(P < 0.05), but less than those in 20 µg/kg rmIL-12 treated group (P < 0.05). It is concluded that rmIL-12 has a significant radioprotective effect on mice irradiated by γ-ray.

HemaMax™, a Recombinant Human Interleukin-12, Is a Potent Mitigator of Acute Radiation Injury in Mice and Non-Human Primates

HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8–9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit–expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD50/30) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12–14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting hematopoiesis and recovery of immune functions and possibly gastrointestinal functions, likely through a network of interactions involving dendritic cells, osteoblasts, and soluble factors such as IL-12, IFN-γ, and cytoprotectant erythropoietin.

BCR-ABL but Not JAK2 V617F Inhibits Erythropoiesis through the Ras Signal by Inducing p21CIP1/WAF1

BCR-ABL is a causative tyrosine kinase (TK) of chronic myelogenous leukemia (CML). In CML patients, although myeloid cells are remarkably proliferating, erythroid cells are rather decreased and anemia is commonly observed. This phenotype is quite different from that observed in polycythemia vera (PV) caused by JAK2 V617F, whereas both oncogenic TKs activate common downstream molecules at the level of hematopoietic stem cells (HSCs). To clarify this mechanism, we investigated the effects of BCR-ABL and JAK2 V617F on erythropoiesis. Enforced expression of BCR-ABL but not of JAK2 V617F in murine LSK (Lineage(-)Sca-1(hi)CD117(hi)) cells inhibited the development of erythroid cells. Among several signaling molecules downstream of BCR-ABL, an active mutant of N-Ras (N-RasE12) but not of STAT5 or phosphatidylinositol 3-kinase (PI3-K) inhibited erythropoiesis, while N-RasE12 enhanced the development of myeloid cells. BCR-ABL activated Ras signal more intensely than JAK2 V617F, and inhibition of Ras by manumycin A, a farnesyltransferase inhibitor, ameliorated erythroid colony formation of CML cells. As for the mechanisms of Ras-induced suppression of erythropoiesis, we found that GATA-1, an erythroid-specific transcription factor, blocked Ras-mediated mitogenic signaling at the level of MEK through the direct interaction. Furthermore, enforced expression of N-RasE12 in LSK cells derived from p53-, p16(INK4a)/p19(ARF)-, and p21(CIP1/WAF1)-null/wild-type mice revealed that suppressed erythroid cell growth by N-RasE12 was restored only by p21(CIP1/WAF1) deficiency, indicating that a cyclin-dependent kinase (CDK) inhibitor, p21(CIP1/WAF1), plays crucial roles in Ras-induced suppression of erythropoiesis. These data would, at least partly, explain why respective oncogenic TKs cause different disease phenotypes.

ChemInform Abstract: Site-Directed Conjugation of Nonpeptide Groups to Peptides and Proteins via Periodate Oxidation of a 2-Amino Alcohol. Application to Modification at N-Terminal Serine.

The 2-amino alcohol structure -CH(NH2)CH(OH)- exists in proteins and peptides in N-terminal Ser or Thr and in hydroxylysine. Its very rapid oxidation by periodate at pH 7 generates an aldehyde in the peptide and is the first step in a method for site-directed labeling with biotin or a fluorescent reporter. The modifying group is a hydrazide, RCONHNH2, which reacts with the new aldehyde to form a hydrazone-peptide conjugate, RCONHN = CH-peptide. Experiments with two synthetic peptides, Ser-Ile-Gly-Ser-Leu-Ala-Lys and Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly, and with recombinant murine interleukin-1 alpha (an 18-kDa cytokine with N-terminal Ser) demonstrated this method of peptide tagging. The use of a low molar ratio of periodate to peptide minimized the potential for side reactions during the oxidation, and the desired oxidation was rapid and highly specific. The hydrazones formed were stable at pH 6-8 for at least 12 h at 22 degrees C, but were labile at more acidic pH values. Potential uses of this method include the attachment of biotin, reporter groups, metal chelating groups, imaging agents, and cytotoxic drugs to peptides.

Recombinant Murine Interleukin 4 Protein Therapy for Psoriasis in a Transgenic VEGF Mouse Model

Background: Psoriasis is a typical autoimmune disease caused by a deregulation of the Th1/Th2 balance, and immunotherapy for psoriasis has been shown to be clinically efficacious. Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis. Evidence suggests that the chronic delivery of VEGF to the skin can result in a profound inflammatory condition with many of the cellular and molecular hallmarks of human psoriasis. In this study, we investigated whether the transgenic VEGF mouse is a suitable model for antipsoriatic studies. Aim: To determine the effect of a recombinant murine interleukin 4 (rmIL-4) in the transgenic VEGF mouse model. Methods: Fifteen homozygous K14-VEGF transgenic mice were injected subcutaneously with rmIL-4 protein for 30 consecutive days with a prospective dose escalation of 0.5, 2 or 5 μg/kg. Hematoxylin-eosin staining, immunohistochemistry and real-time polymerase chain reaction analyses were performed with ear samples. Results: The rmIL-4 protein therapy was well tolerated. Tissue sections from treated skin showed improvements upon morphological and histological examinations: diminution of erythematous appearance and regression of epidermal thickness were observed, and T lymphocyte infiltration decreased significantly. The expressions of adhesion molecules, such as vascular cell adhesion molecule 1 and intracellular adhesion molecule 1, were found reduced. The level of IL-4 mRNA also increased while the level of γ-interferon mRNA decreased, resulting in a 10-fold increase in the ratio of Th1/Th2. Conclusions: Our results reveal that rmIL-4 has clinical efficacy for the treatment of K14-VEGF transgenic mice. Angiogenesis and inflammation were ameliorated by therapy with rmIL-4.

Role of interleukin 10 in murine postoperative ileus

Background and aims:Intestinal manipulation triggers an inflammatory cascade within the muscularis causing postoperative ileus (POI). The aim of this study was to investigate the recovery and therapeutic potential of interleukin...

Lipopolysaccharide-induced increases in cytokines in discrete mouse brain regions are detectable using Luminex xMAP ® technology

Methods to determine cytokine protein content in samples of interest, such as enzyme-linked immunosorbent assay (ELISA), are often labor-intensive and costly. Furthermore, because ELISA requires relatively large sample volumes and protein concentrations, it is difficult using this technique to determine protein content for multiple cytokines from individual samples. Recently, Luminex has developed an open source hardware platform combining flow cytometry- and bead-based antibody capture that is capable of detecting multiple analytes from a single sample. In the present study we employed the Luminex 200 ® platform to determine the cytokine protein content in discrete brain regions of C57BL/6J mice. In spike-and-recovery experiments, known concentrations of murine recombinant interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)α were added either singly or as a mixture of all three to whole brain homogenates containing known quantities of total protein. Spiked samples were assayed for either a single cytokine or for multiple cytokines using 1-plex or 3-plex assay kits, respectively. In whole mouse brain homogenate we recovered between 81% and 103% of the recombinant cytokines. We then injected C57BL/6J mice intraperitoneally with bacterial lipopolysaccharide (LPS) and sacrificed them 4 h later. We detected in samples taken from LPS-stimulated mice 4- to 870-fold increases in serum or spleen cytokine protein, and 1.5- to 16-fold increases in cytokine protein in discrete brain regions, relative to protein content in samples obtained from vehicle-treated animals. These results indicate that multiple cytokines may be reliably assayed from discrete regions of mouse brain using a single sample.

Culture and identification of mouse myeloid semimature dendritic cells

To investigate the methods of culturing and identifying mouse myeloid semimature dendritic cell (smDC) in vitro. Myeloid monocytes derived from 6-week-old C57 BL/6 mice were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2 ng/ml recombinant murine granulocyte macrophage-colony stimulating factor (GM-CSF), and 20 ng/ml recombinant murine interleukin (IL)-4 for 9 days. Then cells were incubated with 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) for 24 hours to obtain smDC. Meanwhile, smDC was differentiated into mature dendritic cell (mDC) or immature dendritic cell (iDC) by treatment with 1 micro/m1 lipopolysaccharide (LPS) or without LPS. The morphological features of smDC were assayed by inverted microscopy and scanning electron microscopy. Surface markers such as CD11c, CD4O, CD8O, CD86, and MHC-II were tested by flow cytometry. IL-1beta, IL-6, IL-12, and IL-10 in the supernatant were tested by ELISA. The activation of allogene lymphocyte (BALB/c mice) stimulated by C57BL/6 myeloid smDC in mixed lymphocyte reaction was examined by Cell Counting Kit-8 in vitro. The shape of smDC was round or oval-shaped, and the diameter of smDC was about 15 microm. The length of smDC dendrite was between 5 to 10 microm. smDC, iDC, and mDC all expressed high level of CD11 c. The expressions of MHC-II, CD40, CD80, and CD86 on smDC were higher than those of iDC and lower than those of mDC. IL-1beta, IL-6, and IL-12 secretion of smDC was significantly lower than that of mDC (P < 0.01), and IL-12 was significantly lower than that of iDC (P < 0.05), while no significant difference of IL-1beta and IL-6 secretion was found between smDC and iDC (P > 0.05). Furthermore, IL-10 secretion was not significantly different among these three kinds of DCs (P > 0.05). The effect of allogene lymphocytes activation on smDC was significantly lower than that of mDC and positive control (P < 0.01), but had no significant difference when compared with that of iDC and negative control (P > 0.05). smDC may be a relatively independent dendritic cell sub-population in terms of function and morphology. It is a feasible way to induce myeloid monocytes to differentiate into smDC using GM-CSF, IL-4, and TNF-alpha in vitro.

The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation.

Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1α. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10−/− B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells—perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis—identifying a strategy that appears to be conserved between at least EBV and MHV68.

Nanogel DDS enables sustained release of IL-12 for tumor immunotherapy

For a valid cytokine immunotherapy of malignancies, a suitable delivery system that ensures slow-release of cytokines is required, because short half-life in vivo of the molecules ruins therapeutic efficacy while causing severe systemic toxic effects. We previously showed that the cholesterol-bearing pullulan (CHP)-based hydrogel nanoparticles, or nanogel, encapsulates, stabilizes and releases various molecules. Here we applied this nanogel to administration in vivo of interleukin-12 (IL-12). Recombinant murine IL-12 (rmIL-12) was successfully incorporated into CHP nanogel simply by incubated with CHP at room temperature. After subcutaneously injected into mice, the CHP/rmIL-12 complex led to a prolonged elevation in IL-12 concentration in the sera. Repetitive administrations of the CHP/rmIL-12, but not rmIL-12 alone, induced drastic growth retardation of preestablished subcutaneous fibrosarcoma without causing any serious toxic event. The present study proposes a novel therapeutic intervention technology, taking advantage of slow and sustained release of bioactive cytokines from the self-assembling biocompatible nanoparticles.