Rat Interleukin Research Articles

Pericyte Microvesicles as Plasma Biomarkers Reflecting Brain Microvascular Signaling in Patients With Acute Ischemic Stroke.

Blood-based biomarkers have the potential to reflect cerebrovascular signaling after microvascular injury; yet, the detection of cell-specific signaling has proven challenging. Microvesicles retain parental cell surface antigens allowing detection of cell-specific signaling encoded in their cargo. In ischemic stroke, the progression of pathology involves changes in microvascular signaling whereby brain pericytes, perivascular cells wrapping the microcapillaries, are one of the early responders to the ischemic insult. Intercepting the pericyte signaling response peripherally by isolating pericyte-derived microvesicles may provide not only diagnostic information on microvascular injury but also enable monitoring of important pathophysiological mechanisms. Plasma samples were collected from patients with acute ischemic stroke (n=39) at 3 time points after stroke onset: 0 to 6 hours, 12 to 24 hours, and 2 to 6 days, and compared with controls (n=39). Pericyte-derived microvesicles were isolated based on cluster of differentiation 140b expression and quantified by flow cytometry. The protein content was evaluated using a proximity extension assay, and vascular signaling pathways were examined using molecular signature hallmarks and gene ontology. In this case-control study, patients with acute ischemic stroke showed significantly increased numbers of pericyte-derived microvesicles (median, stroke versus controls) at 12 to 24 hours (1554 versus 660 microvesicles/μL; P=0.0041) and 2 to 6 days after stroke (1346 versus 660 microvesicles/μL; P=0.0237). Their proteome revealed anti-inflammatory properties mediated via downregulation of Kirsten rat sarcoma virus and IL (interleukin)-6/JAK/STAT3 signaling at 0 to 6 hours, but proangiogenic as well as proinflammatory signals at 12 to 24 hours. Between 2 and 6 days, proteins were mainly associated with vascular remodeling as indicated by activation of Hedgehog signaling in addition to proangiogenic signals. We demonstrate that the plasma of patients with acute ischemic stroke reflects (1) an early and time-dependent increase of pericyte-derived microvesicles and (2) changes in the protein cargo of microvesicles over time indicating cell signaling specifically related to inflammation and vascular remodeling.

M2a macrophages regulate fibrosis and affect the outcome after stroke via PU.1/mTOR pathway in fibroblasts

The moderate formation of the fibrotic scar plays an important role in functional recovery after stroke. M2a macrophages have been identified as an important source of early fibrosis after cerebral ischemia. However, the underlying mechanisms by which macrophages interact with fibroblasts in this context remain largely unknown. Therefore, our study aimed to further investigate the potential mechanisms underlying the effects of macrophages on fibroblasts following ischemic stroke. In vitro and in vivo, recombinant rat interleukin 4 (IL4) was used to induce macrophages to polarize into M2a macrophages. In vitro, primary Sprague–Dawley newborn rat meningeal-derived fibroblasts were treated with PU.1 knockdown, the PU.1 inhibitor DB1976 or the mTOR inhibitor rapamycin, which were then co-cultured with M2a macrophage conditioned medium (MCM). In vivo, Sprague–Dawley adult rats were infected with negative control adenoviruses or PU.1-shRNA adenoviruses. Ten days after infection, an injury model of middle cerebral artery occlusion/reperfusion (MCAO/R) was constructed. Subsequently, IL4 was injected intracerebroventricularly to induce M2a macrophages polarization. In vitro, M2a MCM upregulated PU.1 expression and promoted the differentiation, proliferation, migration and extracellular matrix generation of fibroblasts, which could be reversed by treatment with the PU.1 inhibitor DB1976 or PU.1 knockdown. In vivo, PU.1 expression in fibroblasts was increased within ischemic core following MCAO/R, and this upregulation was further enhanced by exposure to IL4. Treatment with IL4 promoted fibrosis, increased angiogenesis, reduced apoptosis and infarct volume, as well as mitigated neurological deficits after MCAO/R, and these effects could be reversed by PU.1 knockdown. Furthermore, both in vivo and in vitro studies showed that IL4 treatment increased the levels of phosphorylated Akt and mTOR proteins, which were markedly decreased by PU.1 knockdown. Additionally, the use of an mTOR inhibitor rapamycin obviously suppressed the migration and differentiation of fibroblasts, and Col1 synthesis. In conclusion, our findings suggest for the first time that M2a macrophages, at least in part, regulate fibrosis and affect the outcome after cerebral ischemic stroke via the PU.1/mTOR signaling pathway in fibroblasts.

Study the effects of empagliflozin on model of chronic depression and interleukin-6 in the brain of male rats

Abstract Background: Empagliflozin (EMP) is an oral anti-diabetic drug with pleiotropic effects such as anti-inflammatory properties. Objectives: To evaluate the antidepressant effect of EMP and describe the link between stress and interleukin-6 (IL-6) level in the brains of male rats. Materials and Methods: In this experiment, 50 rats were separated into five groups G1–G5. The sucrose preference test (SPT) was used to examine the antidepressant effects of fluoxetine and EMP. Rat interleukin enzyme-linked immunosorbent assay kit was used to measure the IL-6 level in rat brain tissue. SPT was performed on each rat on days 0, 10, and 25. Chronic unpredictable stress (CUS) was performed on each rat for 24 days. Results: By the end of day 10, all rats subjected to the CUS program had a substantial (P < 0.05) reduction in sucrose intake index compared to day 0. EMP significantly increases sucrose intake compared to the stressed group. In comparison to the CUS group, fluoxetine significantly increases sucrose intake (P < 0.05). In terms of IL-6, the mean IL-6 level in G2 was considerably greater than in G1. When compared to group 2, the mean IL-6 level was considerably lower in G3 and G5. Conclusions: EMP has antidepressant-like effects and can counteract the impact of stress-increased IL-6 levels in the brains of depressed rats.

The study on the function and cell source of interleukin-6 in interstitial cystitis/bladder painful syndrome rat model.

ObjectiveThe elevated expression of interleukin‐6 (IL‐6) in patients with interstitial cystitis/bladder painful syndrome (IC/BPS) has been demonstrated, but the role of IL‐6 in IC/BPS and its source remain to be explored.MethodsIC/BPS rat model was created in female rats by using long‐term intermittent intravesical hyaluronidase (0.5 ml, 4 mg/ml). After modeling, IL‐6 stimulation group, and anti‐IL‐6R group were treated with recombinant rat IL‐6 and tocilizumab, respectively. Symptomatic changes were detected by Vonfrey pain score and urodynamics, and hematoxylin‐eosin (HE) staining, mast cell staining and Masson staining were used to evaluate the changes of inflammation in the bladder tissue of rats. Cell sources of IL‐6 was explored through enzyme linked immunosorbent assay (ELISA) test, reverse transcription polymerase chain reaction (RT‐PCR), and western‐blot test on the supernatant of coculturing rat bladder epithelial cells and rat macrophages.ResultsThe Vonfrey pain scores of the model group and IL‐6 stimulation group were significantly higher than those of the control group, while the anti‐IL‐6R group were significantly lower (p < .05). Compared with the blank control group, urodynamic results showed that the urination interval of the model group and IL‐6 stimulation group was significantly shortened, and the maximum bladder capacity was significantly reduced (p < .05), and anti‐IL‐6R treatment significantly alleviated the inflammatory response of bladder tissue. The results of HE, Mast cell staining, and Masson staining showed that the inflammatory response of bladder tissue after anti‐IL‐6R treatment was significantly reduced. Through cells coculture, the relative expression of IL‐6 from model group was found significantly higher than blank control group by RT‐PCR, ELISA, and western blot test (p < .05).ConclusionsIL‐6 played an essential role in the development of IC/BPS rat model as a proinflammation cytokine. Further evidence from coculture proved that macrophages are the cell resource of IL‐6 in IC/BPS.

To explore the therapeutic effect of Kangfuxiaomi shuan II on cervicitis in rats.

ABSTRACTPurpose:The rat cervicitis model was established with 20% phenol glue to explore the therapeutic effect of Kangfuxiaomi shuan II on rat cervicitis and its mechanism.Methods:After modeling, the rats were treated with Shuangzuotai suppository (37.84 mg/kg), Kangfuxiaoyan shuan (205.6 mg/kg) and Kangfuxiaomi shuan II (40, 80, 160 mg/kg). The histopathological changes and injury degree of cervix in rats were evaluated by vulvar inflammation score and organ index. The therapeutic effect of Kangfuxiaomi shuan II on cervicitis was evaluated by detecting the levels of copper-protein (CP), C-reactive protein (CRP), Rat interleukin 6 (IL-6), superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and epidermal growth factor (EGF), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in cervical tissue.Results:Compared with the model group, the vulvar inflammation score and cervical index of rats in other groups decreased significantly (P<0.01). Kangfuxiaomi shuan II could significantly reduce the levels of CP, CRP, and MDA in serum of rats with cervicitis, and significantly increase the activity of SOD in serum of rats with cervicitis (P<0.01). The levels of EGF and iNOS in cervical tissue of rats also increased in different degrees, while the level of COX-2 decreased significantly (P<0.01), which significantly improved the pathological degree of vulvar inflammation in rats with cervicitis.Conclusions:Kangfuxiaomi shuan II has a certain therapeutic effect on cervicitis in rats, and its mechanism may be related to the regulation of inflammatory cytokine network and immunity.

The Association of IL-1 and HRAS Gene Polymorphisms with Breast Cancer Susceptibility in a Jordanian Population of Arab Descent: A Genotype-Phenotype Study.

Breast cancer (BC) pathogenesis is poorly understood and not yet completely determined. BC susceptibility genes are responsible for 20% to 25% of breast cancer risk. The main objective of this study is to identify the genetic polymorphisms within the Harvey rat sarcoma viral oncogene homolog (HRAS1) and interleukin-1 receptor antagonist (IL1-Ra) genes in Jordanian BC female patients and to investigate the genetic association of these polymorphisms with BC. Samples were collected from 150 Jordanian BC patients and 187 healthy age-matched controls. PCR and PCR-RFLP techniques were used to identify genetic polymorphisms within these candidate genes. The single nucleotide polymorphism single nucleotide polymorphism (SNP) association web tool SNPStats (v. 3.6) was used to investigate the allelic and genotypic association with BC. Different statistical analyses were used to study the correlation between the investigated genetic variants and several prognosis factors of BC. A genetic association between BC susceptibility and Il-1β rs1143634 was found specifically at the allelic level of E1 as a risk allele (72% in the cases vs. 64.2% in the controls). Another genetic association was found in the IL-Ra gene (86-VNTR (variable number tandem repeat)), which presented one repeat allele (24.1% in cases vs. 15.59% in controls) and could be considered as a risk allele in Jordanian women. In contrast, this study found that there is no genetic association between Il-1β SNP rs16944 and BC. In addition, a significant association was found between the allelic level of the HRAS1 gene and BC susceptibility. Since this study is the first to be conducted on the genetic susceptibility of these genes to BC in the Jordanian population, more investigations on the link between BC and these variants are recommended to determine the impact of these polymorphisms on other ethnic groups.

Role of matrix metalloproteinases 2 and 9 in the development of frozen shoulder: human data and experimental analysis in a rat contracture model

Although frozen shoulder (FS) is a common shoulder disorder, its pathogenesis is not yet determined. The function of matrix metalloproteinases (MMPs) is related to extracellular matrix remodeling. The purposes of this study were to investigate the pattern of sequential expression of MMPs in a rat model of shoulder contracture and to compare the expression of MMPs in the joint capsule between patients with FS and a control group. We obtained joint capsules from rats immobilized by molding plaster (a shoulder contracture model) at baseline, 3 days, 1 week, and 3 weeks (4 rats per time point; 16 rats in total). The expression of the inflammatory cytokine interleukin 6 (IL-6), MMP-2, and MMP-9 was examined by immunohistochemistry. We also obtained joint capsules from 21 patients with FS and 13 control patients with instability to quantify the expression levels of MMP-2 and MMP-9 by immunohistochemistry. In the rat model, IL-6 and MMP-9 tended to be overexpressed in the joint capsule at 3 days and 1 week and MMP-2 at 3 days, 1 week, and 3 weeks. MMP-2 and MMP-9 were significantly overexpressed in the joint capsules of the patients with FS compared with those of control patients. The results from both human and animal studies suggest the involvement of MMP-2 and MMP-9 in the development of FS. Animal study showed that the sequential expression of IL-6 and MMPs may be associated with fibrosis of the joint capsule.

What is adverse effect of wireless local area network, using 2.45 GHz, on the reproductive system?

Aim: To investigate the inflammatory effect and testicular damage on rats exposed to low level of electromagnetic fields (EMF) at 2.45 GHz microwave radiation.Methods: Twenty two Wistar rats were divided into two groups. Group 1 was the control group and not exposed to EMF. Group 2 was exposed to low level EMF (average E-field 3.68 ± 0.36 V/m, whole body average SAR, 0.0233 W/kg, in 10 g tissue) at 2.45 GHz for 1 hour/day for 30 consecutive days. At the end of the study, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-32 (IL-32), C-reactive protein (CRP) were measured in rat serum and IL-6, IL-10, IL-32 were measured in rat testis tissue. Furthermore, testicular tissues were evaluated histopathologically in terms of spermatogenesis and coagulation necrosis.Results: Serum IL-6 and CRP levels were found to be significantly different in the study group compared to the control group (p < .05), but no significant difference was found in serum IL-10, IL-32 levels and testis tissue IL-6, IL-10, IL-32 levels compared to the control group (p > .05). On the other hand, histopathological evaluation of testicular tissue revealed a significant difference in necrosis and spermatogenesis when compared with the control group (p < .05).Conclusions: It may be concluded that low level EMF at 2.45 GHz increases inflammation and testicular damage and negative impact on male reproductive system function.

Red nucleus interleukin-6 participates in the maintenance of neuropathic pain through JAK/STAT3 and ERK signaling pathways

We previously reported that interleukin-6 (IL-6) in the red nucleus (RN) is up-regulated at 3weeks after spared nerve injury (SNI), and plays facilitated role in the later maintenance of neuropathic pain. The current study aimed to reveal the roles of different signaling pathways, including Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase/protein kinase B (PI3K/AKT), in RN IL-6-mediated pain modulation. In accord with the increase of IL-6 in the RN following SNI, the protein levels of phospho-STAT3 (p-STAT3), p-ERK and p-JNK were also up-regulated in the RN contralateral to the nerve injury side at 3weeks after SNI. The increases of p-STAT3 and p-ERK (but not p-JNK) were associated with IL-6 and could be blocked by anti-IL-6 antibody. Microinjection of JAK2 inhibitor AG490, ERK inhibitor PD98059 and also JNK inhibitor SP600125 into the RN significantly increased the paw withdrawal threshold (PWT) and alleviated SNI-induced mechanical allodynia. Further studies showed that microinjection of recombinant rat IL-6 (rrIL-6, 20ng) into the RN of normal rats significantly decreased the PWT of rats and increased the local protein levels of p-STAT3 and p-ERK, but not p-JNK. Pre-treatment with AG490 and PD98059 could prevent IL-6-induced mechanical allodynia. Whereas, p-p38 MAPK and p-AKT did not show any expression changes in the RN of rats with SNI or rats treated with rrIL-6. These results suggest that RN IL-6 participates in the later maintenance of SNI-induced neuropathic pain and plays facilitated role through activating JAK/STAT3 and ERK signaling pathways.

Interleukin-10 secreted by mesenchymal stem cells attenuates acute liver failure through inhibiting pyroptosis.

Recently, the benefit of mesenchymal stem cells (MSCs) as a cell-based therapy for acute liver failure (ALF) has gained much attention, although the mechanism of action of MSCs in the treatment of ALF remains elusive. Pyroptosis is a novel form of programmed cell death with an intense inflammatory response. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects through inhibiting pyroptosis in ALF. Mesenchymal stem cells obtained from C57BL/6 mice were isolated and cultured according to an established protocol. The MSCs were transplanted into mice with D-galactosamine (D-Gal)-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, ShIL-RNA, and MCC950 (NLRP3 inhibitor) were given to the mice to explore the therapeutic mechanism of MSCs. Statistical analyses were carried out with spss version 19.0, and all data were analyzed by independent-samples t-test. Injection of IL-10 or MSC transplantation ameliorated D-Gal-induced increase in alanine aminotransferase, aspartate aminotransferase, total bilirubin, NH3, and inflammatory cytokines. Blockage of IL-10 confirmed the therapeutic significance of this cytokine. Pyroptosis was inhibited after IL-10 infusion and inhibition of NLRP3 by MCC950 reversed liver dysfunction.

The Red Nucleus Interleukin-6 Participates in the Maintenance of Neuropathic Pain Induced by Spared Nerve Injury.

Previous studies have demonstrated that the red nucleus (RN) is involved in the regulation of neuropathic pain and plays both facilitated and inhibitory roles through different cytokines. Here, we aim to investigate the expression changes and roles of interleukin-6 (IL-6), a pleiotropic cytokine, as well as its receptor (IL-6R) in the RN of rats with neuropathic pain induced by spared nerve injury (SNI). Immunohistochemistry indicated that IL-6 and IL-6R were weakly expressed in the RN of normal rats, and were mainly co-localized with neurons and oligodendrocytes. Following SNI, the expression levels of IL-6 and IL-6R in the RN did not show obvious changes at 1week and 2weeks postinjury. However, both of them were significantly increased in the RN contralateral (but not ipsilateral) to the nerve ligation side at 3weeks postinjury, and co-localized not only with neurons and oligodendrocytes, but also with numerous astrocytes. Injection of different doses of anti-IL-6 antibody (100, 250, 500ng) into the RN contralateral to the nerve ligation side at 3weeks postinjury dose-dependently increased the paw withdrawal threshold (PWT) of rats and alleviated SNI-induced mechanical allodynia. Conversely, injection of different doses of recombinant rat IL-6 (5.0, 10, 20ng) into the unilateral RN of normal rats dose-dependently decreased the PWT of contralateral (but not ipsilateral) hind paw and evoked significant mechanical allodynia, which was similar to SNI-induced neuropathic allodynia. These results further support the conclusion that the RN is involved in the modulation of neuropathic pain, and suggest that IL-6 and IL-6R in the RN play a facilitated role in the later maintenance of SNI-induced neuropathic pain.

Interleukin-10 Contributes to Therapeutic Effect of Mesenchymal Stem Cells for Acute Liver Failure via Signal Transducer and Activator of Transcription 3 Signaling Pathway.

Background:Mesenchymal stem cells (MSCs) transplantation has been proven to have therapeutic potential for acute liver failure (ALF). However, the mechanism remains controversial. Recently, modulation of inflammation by MSCs has been regarded as a crucial mechanism. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects in ALF.Methods:MSCs isolated from Sprague-Dawley rats were identified by fluorescence-activated cell sorting analysis. Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray. MSCs and MSCs-CM were transplanted into rats with D-galactosamine-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, anti-rat IL-10 antibody, and AG490 (signal transducer and activator of transcription 3 [STAT3] signaling pathway inhibitor) were administered to explore the therapeutic mechanism of MSCs-CM. Statistical analysis was performed with SPSS version 19.0, and all data were analyzed by the independent-sample t-test.Results:There are statistical differences of the survival curve between ALF+MSCs group and ALF+Dulbecco's modified Eagle's medium (DMEM) group, as well as ALF+MSCs-CM group and ALF+DMEM group (all P < 0.05). Serum alanine aminotransferase (ALT) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (865.53±52.80 vs. 1709.75±372.12 U/L and 964.72±414.59 vs. 1709.75±372.12 U/L, respectively, all P < 0.05); meanwhile, serum aspartate aminotransferase (AST) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (2440.83±511.94 vs. 4234.35±807.30 U/L and 2739.83±587.33 vs. 4234.35±807.30 U/L, respectively, all P < 0.05). Furthermore, MSCs or MSCs-CM treatment significantly reduced serum interferon-γ (IFN-γ), IL-1β, IL-6 levels and increased serum IL-10 level compared with DMEM (all P < 0.05). Proteome profile analysis of MSCs-CM indicated the presence of anti-inflammatory factors and IL-10 was the most distinct. Blocking of IL-10 confirmed the therapeutic significance of this cytokine. Phosphorylated STAT3 was upregulated after IL-10 infusion and inhibition of STAT3 by AG490 reversed the therapeutic effect of IL-10.Conclusions:The factors released by MSCs, especially IL-10, have the potential for therapeutic recovery of ALF, and the STAT3 signaling pathway may mediate the anti-inflammatory effect of IL-10.

222 PRODUCTION OF KNOCKOUT RATS BY USING ZINC FINGER NUCLEASES AND TAL EFFECTOR NUCLEASES

The rat has been used as an important animal for understanding human diseases. Genetically engineered rat strains are used as a human disease model in various research fields. Genetically engineered rat strains are now being routinely produced, not only as transgenic animals but also using gene knockout techniques. Recently, zinc finger nucleases (ZFN) and TAL effector nucleases (TALEN) have enabled editing targeted genes without using embryonic stem cells. These techniques have been applied for production of the knockout and knockin animals. We here studied that the effects of gene targeting by ZFN and TALEN introduced into rat embryos for efficient production of knockout rats. We custom-designed ZFN and TALEN plasmids targeted rat interleukin 2 receptor gamma (Il2rg) gene. Each mRNA was transcribed in vitro from these plasmids. Final concentration of mRNA was adjusted at 10 ng μL–1 in sterilized water for microinjection. Messenger RNA was injected into rat pronuclear stage embryos. The embryos were then cultured in vitro to the 2-cell stage, and were transferred into oviducts of pseudopregnant females. The rate of development of offspring of embryos and effects of editing targeted genes were examined. Of 41 two-cell embryos introduced ZFN after embryo transfer, 9 embryos (22%) developed to offspring. Three offspring (33%) had an edited targeted gene locus. In the embryos introduced TALEN, 30% (6 offspring) of embryos developed to offspring after embryo transfer and all offspring had an edited targeted gene locus. This study demonstrated that the ZFN and TALEN mRNA was active after introduction into rat embryos. Knockout rats could be produced by introduction of ZFN and TALEN into rat embryos. ZFN and TALEN will provide a powerful new approach for targeted gene editing not only in rats but also in other animal species.

Suppressive effect of lentivirus-mediated RNA interference on interleukin-1 receptor type I gene in rat chondrocytes in vitro

Objective To construct lentiviral vectors targeting rat interleukin-1 receptor type I（IL- 1 RI） gene by RNA interference （RNAi） and to study its effects on chondrocytes in vitro. Methods Effec- tire sequence of siRNA targeting IL-1RI gene was designed and determined and 239T cells were were trans- fected with pGCL-GFP vector containing the inserted DNA sequence. The lentivirus containing the effective sequence of siRNA targeting IL-1RI gene with concentration of 1 × 107 TU/ml was obtained after supernatant of 293T cells were collected and concentrated. The recombinant lentivirus was used to infect IL-l-stimulated rat chondrocytes previously with multiple of infection （MOI） 20 and 50 seperately in vitro and the expression of IL-1RI and matrix metalloproteinase （MMP） -1 was detected by using Western blotting 3 days later. Re- suits The best MOI for lentivirus infecting chondrocytes was 50. Lentiviruses demonstrated a high （above 70% ） infection efficiency of chondrocytes. The expression of IL-1RI and MMP-1 was both repressed signifi- cantly in rat chondrocytes by RNAi. Conclusion The recombinant lentivirus shows significantly inhibitory effects on IL-1RIand MMP-1 （the downstream protein） in rat chondrocytes. Key words: Lentivirus ; Interleukin-1 ; RNA interference ; Chondrocyte ; Matrix metallo- proteinase-1

In vitro-activated tumor-bearing host T cells and the effectiveness of tumor vaccine immunotherapy

BACKGROUND AND OBJECTIVEVaccination during periods of lymphopenia may facilitate immune responses to weak self-antigens and enhance antitumor immunity. The objective of this study was to determine the effectiveness of tumor vaccine immunotherapy combined with immune reconstruction using tumor-bearing host immune cells in lymphopenia, and to investigate the role of tumor-bearing host T cells activated in vitro during immunotherapy.DESIGN AND SETTINGAnimal study conducted in the First Affiliated Hospital of Xi’an Jiaotong University from January 2009 to January 2010.PATIENTS AND METHODSLymphopenia was induced by cyclophosphamide. A reconstituted immune system with different syngeneic lymphocytes was employed, including lymphocytes from naïve rats (unsensitized group), tumor-bearing rats (tumor-bearing group), and tumor-bearing rats activated in vitro (activated group). All rats were immunized with granulocyte-macrophage colony-stimulating factor (GM-CSF)-modified NuTu-19 ovarian cancer (GM-CSF/NuTu-19) cells. Tumor vaccine-draining lymph nodes (TVDLNs) were harvested, and then stimulated to induce effector T cells (TE). TE were then adoptively transferred to rats bearing a 3-day pre-established abdominal tumor (NuTu-19), and the survival rate was calculated.RESULTSCompared with the unsensitized group, the levels of interleukin-2 (IL-2) were significantly lower in the tumor-bearing group, whereas that of IL-4 were significantly higher (P<.05). The number of CD4+ T cells secreting interferon-γ and the specific cytotoxicity of CD8+ cytotoxic T lymphocytes were significantly lower (P<.05). The survival was significantly higher in the activated group compared with the other groups.CONCLUSIONSLymphocytes from tumor-bearing rats activated in vitro can effectively reverse the immunosuppressive effects of tumor-bearing hosts.

Sinomenine reduces iNOS expression via inhibiting the T-bet IFN-γ pathway in experimental autoimmune encephalomyelitis in rats

Sinomenine is a bioactive alkaloid isolated from the Chinese medicinal plant Sinomenium acutum. It is widely used as an immunosuppressive drug for treating rheumatic and arthritic diseases. In our previous studies, we found that sinomenine reduced cellular infiltration within the spinal cord and alleviated experimental autoimmune encephalomyelitis (EAE) in rats. In this study, we further investigated the mechanisms of sinomenine treatment in EAE rats. In EAE rats, treatment with sinomenine exerted an anti-inducible NO synthase (anti-iNOS) effect, which is related to the reductions of Th1 cytokine interferon-γ (IFN-γ) and its transcription factor, T-bet, in spinal cords. Moreover, sinomenine treatment of splenocytes stimulated with anti-CD3 antibody and recombinant rat interleukin 12 reduced the expression of T-bet and IFN-γ in vitro and also reduced the capability of supernatants of splenocyte culture to induce iNOS expression by primary astrocytes. However, sinomenine had no direct inhibitory effect on iNOS produced by astrocytes cultured with IFN-γ and tumor necrosis factor αin vitro. In conclusion, the anti-iNOS effect of sinomenine on EAE is mediated via the suppression of T-bet/IFN-γ pathway.

Tumor Necrosis Factor Receptor-associated Factor-6 and Ribosomal S6 Kinase Intracellular Pathways Link the Angiotensin II AT1 Receptor to the Phosphorylation and Activation of the IκB Kinase Complex in Vascular Smooth Muscle Cells

Activation of NF-κB transcription factors by locally produced angiotensin II (Ang II) is proposed to be involved in chronic inflammatory reactions leading to atherosclerosis development. However, a clear understanding of the signaling cascades coupling the Ang II AT1 receptors to the activation of NF-κB transcription factors is still lacking. Using primary cultured aortic vascular smooth muscle cells, we show that activation of the IKK complex and NF-κB transcription factors by Ang II is regulated by phosphorylation of the catalytic subunit IKKβ on serine residues 177 and 181 in the activation T-loop. The use of pharmacological inhibitors against conventional protein kinases C (PKCs), mitogen-activated/extracellular signal-regulated kinase (MEK) 1/2, ribosomal S6 kinase (RSK), and silencing RNA technology targeting PKCα, IKKβ subunit, tumor growth factor β-activating kinase-1 (TAK1), the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor-6 (TRAF6), and RSK isoforms, demonstrates the requirement of two distinct signaling pathway for the phosphorylation of IKKβ and the activation of the IKK complex by Ang II. Rapid phosphorylation of IKKβ requires a second messenger-dependent pathway composed of PKCα-TRAF6-TAK1, whereas sustained phosphorylation and activation of IKKβ requires the MEK1/2-ERK1/2-RSK pathway. Importantly, simultaneously targeting components of these two pathways completely blunts the phosphorylation of IKKβ and the proinflammatory effect of the octapeptide. This is the first report demonstrating activation of TAK1 by the AT1R. We propose a model whereby TRAF6-TAK1 and ERK-RSK intracellular pathways independently and sequentially converge to the T-loop phosphorylation for full activation of IKKβ, which is an essential step in the proinflammatory activity of Ang II.

Anti-inflammatory effect by lentiviral-mediated overexpression of IL-10 or IL-1 receptor antagonist in rat glial cells and macrophages

Neuroinflammation, as defined by activation of local glial cells and production of various inflammatory mediators, is an important feature of many neurological disorders. Expression of pro-inflammatory mediators produced by glial cells in the central nervous system (CNS) is considered to contribute to the neuropathology observed in those diseases. To diminish the production or action of pro-inflammatory mediators, we have used lentiviral (LV) vector-mediated encoding rat interleukin-10 (rIL-10) or rat interleukin-1 receptor antagonist (rIL-1ra) to direct the local, long-term expression of these anti-inflammatory cytokines in the CNS. We have shown that cultured macrophages or astroglia transduced with LV-rIL-10 or LV-rIL-1ra produced far less tumor necrosis factor (TNF)alpha or IL-6, respectively in response to pro-inflammatory stimuli. Moreover, intracerebroventricular (i.c.v.) administration of LV-rIL-10 or LV-rIL-1ra resulted in transduction of glial cells and macrophages and, subsequently reduced TNFalpha, IL-6 and inducible nitric oxide synthase (iNOS) expression in various brain regions induced by inflammatory stimuli, whereas peripheral expression of these mediators remained unaffected. In addition, expression levels of the anti-inflammatory cytokines IL-4 and transforming growth factor-beta were not altered in either brain or pituitary gland. Furthermore, i.c.v. administration of LV-rIL-10 or LV-rIL-1ra given during the remission phase of chronic-relapsing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, improved the clinical outcome of the relapse phase. Thus, local application of LV vectors expressing anti-inflammatory cytokines could be of therapeutic interest to counteract pro-inflammatory processes in the brain without interfering with the peripheral production of inflammatory mediators.

Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases

BackgroundAlthough the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc.Methodology/Principal FindingsWe report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype.Conclusions and SignificanceThe efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

Effect of neutralization of rat interleukin 6 bioactivity on inducible nitric oxide synthase up-regulation and cerebral ischemic damage

Objectives: Our aim was to investigate whether neutralization of rat interleukin 6 (IL-6) bioactivity attenuates inducible nitric oxide synthase (iNOS) up-regulation and ameliorates cerebral ischemic damage in a model of focal central nervous system (CNS) ischemia.Methods: Seventy rats were randomly allocated to groups: Group I (n=10) consisted of normal controls; Group II (n=20) underwent surgical exposure of the middle cerebral artery but no cauterization; the remaining 40 rats were subjected to middle cerebral artery occlusion. Immediately after occlusion, each of these 40 rats was randomly assigned to either the occlusion-only group (Group III, n=20) or the occlusion plus IL-6 antibody treatment group (Group IV, n=20). Half of the rats from each of Groups II, III and IV were eternized at 24 hours and the other half at 72 hours. The samples were used for iNOS immunohistochemistry and structural analysis.Results: A single dose of the antibody had no effect on structural changes and iNOS at 24 hours after occlusion. However, administering three doses of the antibody resulted in markedly decreased quantitative and qualitative levels of iNOS-positive stained cells and milder subcellular damage compared with the findings in the occlusion-only group at 72 hours after occlusion.Discussion: Our findings prove that IL-6 bioactivity is one of the pathological events that trigger the induction of iNOS in the process of CNS ischemic injury. It appears that there may be therapeutic value in neutralization of IL-6 bioactivity to attenuate iNOS up-regulation and ameliorate cerebral ischemic damage in long-term recovery.