DA Rats Research Articles

THU0102 The Involvement of Toll-Like Receptor 9 in the Pathogenesis of Erosive Arthritis

BackgroundToll-like receptors (TLR) such as TLR7 and TLR9 that are activated by nucleic acids are known to be involved in the pathogenesis of systemic lupus erythematosus (SLE) while their role...

Combined Donor Simvastatin and Methylprednisolone Treatment Prevents Ischemia-Reperfusion Injury in Rat Cardiac Allografts Through Vasculoprotection and Immunomodulation

Ischemia-reperfusion injury (IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. Here, we investigated the effect of donor treatment with simvastatin and methylprednisolone on microvascular dysfunction and immunomodulation during IRI in rat cardiac allografts subjected to prolonged ischemia time. The DA rats received simvastatin, methylprednisolone, or both 2 hr before heart donation. The allografts were subjected to 4-hr hypothermic preservation and transplanted to the fully major histocompatibility complex-mismatched WF rat recipients. Six hours after reperfusion, donor treatment either with simvastatin alone or with high dose of methylprednisolone alone or in combination with simvastatin and methylprednisolone significantly reduced cardiac troponin T release and the number of allograft infiltrating ED1 macrophages MPO neutrophils. However, the combination donor treatment was superior in the prevention of IRI and significantly prolonged allograft survival. Donor simvastatin treatment inhibited allograft microvascular RhoA GTPase pathway activation, whereas methylprednisolone prevented activation of innate immune response and mRNA expression of hypoxia-inducible factor-1α and its multiple target genes. Our results show that donor treatment in combination with simvastatin and methylprednisolone prevents IRI and has beneficial effect on allograft survival in rat cardiac allografts. Minimizing microvascular injury and the activation of innate immunity may offer a novel therapeutic strategy to expand the donor pool and furthermore improve the function of the marginal donor organs.

Genetic variability in the rat Aplec C-type lectin gene cluster regulates lymphocyte trafficking and motor neuron survival after traumatic nerve root injury

BackgroundC-type lectin (CLEC) receptors are important for initiating and shaping immune responses; however, their role in inflammatory reactions in the central nervous system after traumatic injuries is not known. The antigen-presenting lectin-like receptor gene complex (Aplec) contains a few CLEC genes, which differ genetically among inbred rat strains. It was originally thought to be a region that regulates susceptibility to autoimmune arthritis, autoimmune neuroinflammation and infection.MethodsThe inbred rat strains DA and PVG differ substantially in degree of spinal cord motor neuron death following ventral root avulsion (VRA), which is a reproducible model of localized nerve root injury. A large F2 (DAxPVG) intercross was bred and genotyped after which global expressional profiling was performed on spinal cords from F2 rats subjected to VRA. A congenic strain, Aplec, created by transferring a small PVG segment containing only seven genes, all C-type lectins, ontoDA background, was used for further experiments together with the parental strains.ResultsGlobal expressional profiling of F2 (DAxPVG) spinal cords after VRA and genome-wide eQTL mapping identified a strong cis-regulated difference in the expression of Clec4a3 (Dcir3), a C-type lectin gene that is a part of the Aplec cluster. Second, we demonstrate significantly improved motor neuron survival and also increased T-cell infiltration into the spinal cord of congenic rats carrying Aplec from PVG on DA background compared to the parental DA strain. In vitro studies demonstrate that the Aplec genes are expressed on microglia and upregulated upon inflammatory stimuli. However, there were no differences in expression of general microglial activation markers between Aplec and parental DA rats, suggesting that the Aplec genes are involved in the signaling events rather than the primary activation of microglia occurring upon nerve root injury.ConclusionsIn summary, we demonstrate that a genetic variation in Aplec occurring among inbred strains regulates both survival of axotomized motor neurons and the degree of lymphocyte infiltration. These results demonstrate a hitherto unknown role for CLECs for intercellular communication that occurs after damage to the nervous system, which is relevant for neuronal survival.

CXCL12-γ expression is inhibited in neuroinflammation

CXCL12 plays a protective role in CNS autoimmunity. Expression of CXCL12-γ, which has distinct structural and functional properties than the other isoforms of CXCL12, was determined in spinal cords of rats immunized to develop experimental autoimmune encephalomyelitis (EAE). CNS expression of CXCL12-γ was markedly lower in EAE-prone Dark Agouti rats than in EAE-resistant Albino Oxford rats, both in spinal cord homogenates and micro-blood vessels isolated from spinal cords. Inhibition of nitric oxide (NO) synthesis in DA rats upregulated, while donation of NO in AO rats downregulated CNS expression of CXCL12-γ. NO inhibited CXCL12-γ expression in astrocytes in vitro. A splice variant of CXCL12-γ which migrates into nucleolus was not detected in spinal cord or astrocytes. Thus, CXCL12-γ is expressed in the CNS after EAE induction, but its expression is markedly suppressed in spinal cord affected with full blown inflammation. NO is an important regulator of CXCL12-γ expression in neuroinflammation.

Adoptive transfers reveal dual roles in the protection against arthritis induction in the DACP rat (P4096)

Abstract The DA rat is a commonly used inbred rat strain in inflammation research due to its susceptibility to numerous experimentally induced models of autoimmunity. We have isolated a spontaneous mutation that abrogates induction of a panel of inflammatory disorders in the DA rat and mapped it to a 2,5Mb region on chromosome 9. This new subset of the DA rat was called DA completely protected, DACP. To elucidate if the protection seen in the DACP rat is conveyed during the induction or the effector phase we utilized the Pristane-induced arthritis transfer model in a reciprocal fashion. Our results showed that cells from the DACP rat did not transfer arthritis to susceptible DA rats whereas cells from the susceptible DA rat caused a mild arthritis in DACP recipients. Looking at the cytokine profile of these cells we saw that cells from the DACP rat produced twice the amount of IL-10 compared to cells from the DA rat and that the production of RANTES was lower in the DACP rat. Differences in levels of IL-10 and RANTES could be one possible explanation for the protection seen in the DACP rat however this remains to be shown. In summary, the DACP rat was unable to develop disease upon arthritis induction but could develop a mild arthritis when receiving arthritogenic cells from the DA rat. Our findings suggest a possible dual role of the spontaneous mutation in the induction phase and the effector phase of arthritis in the DACP rat.

Strain differences influence timing and magnitude of both acute and late inflammatory reactions after intratracheal instillation of an alkylating agent in rats

The acute pulmonary responses after exposure to sulfur and nitrogen mustards are well documented whereas the late pulmonary effects are not. With a novel focus on the immune system this paper investigate whether late phase pulmonary effects developed in rats exposed to the nitrogen mustard melphalan are linked to the acute responses and whether the reactions are genetically regulated. The DA rat strain was used to establish a lung exposure model. Five other inbred rat strains (PVG, PVG.1AV1, LEW, WF and F344) were compared within the model at selected time points. All rat strains displayed a biphasic pattern of leukocyte infiltration in the lungs, dominated by neutrophils 2 days after exposure and a second peak dominated by macrophages 29 days after exposure. The number of macrophages was higher in the DA rat compared with the other strains. The infiltration of lymphocytes in the lungs varied in both time of appearance and magnitude between strains. The quantity of collagen deposition in the lungs varied between strains at day 90; LEW and WF displayed high collagen content which coincided with an increased level of cytotoxic T cells. LEW further displayed an increased number of T helper cells and natural killer (NK) T cells in the lungs. The results in this study suggest there is a link between the development of lung fibrosis and high cytotoxic cell responses and that there is a genetic influence, as there are variations in acute and late adverse reactions between rat strains in both timing and magnitude.

Collagen-induced arthritis impairs osteoclastogenesis and reactivity to sympathetic neurotransmitter stimuli in bone marrow-derived macrophages from DA rats

Osteoclasts (OC) are key players in bone destruction in rheumatoid arthritis (RA). Recent work showed that catecholaminergic nerve fibers are reduced in RA synovial tissue. Studies on sweat gland innervation revealed that catecholaminergic fibers are capable of phenotypic transition to cholinergic nerves. The sympathetic neurotransmitters norepinephrine (NE) and acetylcholine (ACh) affect osteoclastogenesis oppositely and that is why we wanted to study osteoclastogenesis at different phases of collagen-induced arthritis (CIA) in an altered neurotransmitter microenvironment. The influence of NE and ACh on differentiation and activity of bone marrow macrophage (BMM)-derived osteoclasts from arthritic and control animals were compared at various time-points after arthritis induction. The expression profile for adrenergic and ACh receptors was analyzed on mRNA level. The number of OCs was tendentially lower in arthritic animals. Stimulation with ACh yielded significantly more OCs in controls. NE decreased osteoclastogenesis via β-adrenoceptors and enhanced via α-adrenoceptor stimulation. Cells from arthritic animals were less affected. In form of a trend, osteoclasts from arthritic animals showed decreased activity in cathepsin K activity and in a pit formation assay. The receptor gene expression profile changed in the time course of arthritis. After 20 days past immunization, muscarinic ACh receptors M3 and M5 were significantly upregulated whereas after 40 days adrenoceptors α1D and α2B were significantly downregulated. We conclude that CIA suppresses OC differentiation and activity as well as reactivity to neurotransmitter stimulation but the underlying processes remain to be demonstrated.

Expression pattern of the endoplasmic reticulum stress protein gp96 in monophasic and chronic relapsing form of experimental autoimmune encephalomyelitis in rats.

Gp96 is the endoplasmic reticulum (ER)-resident molecular chaperone, which is involved in the correction of unfolded proteins, in the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, and in activation of the protein translation that modulates polypeptide traffic into the ER. Furthermore, owing to its peptide chaperone capacity and ability to interact with professional antigen-presenting cells, as well as with growth factors, integrins and Toll-like receptors, it is also endowed with crucial immunological functions acting as a "danger signal" to the innate and adaptive immunity. Considering these properties, in the present study the tissue expression of gp96 was examined during the monophasic and chronic relapsing form of experimental autoimmune encephalomyelitis (CR-EAE), induced in genetically susceptible DA rats by subcutaneous injection of myelin basic protein (MBP) or bovine brain homogenate in complete Freund's adjuvant (CFA). Immunohistochemical analyses were done in periods of attacks and remissions of EAE, and the results were compared with findings in intact rats and those treated only with CFA. The data revealed that the constitutive gp96 expression, found in several neurons and glial cells in the brain and spinal cord of intact animals, significantly diminished during the attacks of CR-EAE. On the contrary, the remission of disease was followed by high upregulation of gp96, mainly in the oligodendrocytes within the white matter, in the neurons of the hippocampal area, as well as in the motoneurons of lumbar spinal cord, suggesting that gp96 might be involved in proteostasis and immune-related pathways linked with the reparative processes in the CNS.

Accelerated Antibody-Mediated Graft Loss of Rodent Pancreatic Islets After Pretreatment With Dexamethasone-Treated Immature Donor Dendritic Cells

Allogeneic islets of Langerhans transplantation is hampered in its success as a curative treatment of type 1 diabetes by the absence of potent, specific, and nontoxic immunosuppressive drugs. Here, we assessed whether donor bone marrow-derived dexamethasone-treated dendritic cells (dexDCs) could prolong islet allograft survival in a full major histocompatibility complex mismatch rat model. Rodent allogeneic islet transplantation was performed from DA rats to Lewis rats and vice versa. Permanently immature dendritic cells were generated from the bone marrow of DA and Lewis rats by treatment with dexamethasone. Animals were either vehicle or donor dexDCs pretreated. Serum was used to monitor glucose, C-peptide, and alloreactive antibodies. The transplantation of DA islets into Lewis recipients showed direct graft failure with reduced numbers of β-cells when rats were pretreated with donor dexDCs. In the reverse model (Lewis islets into DA recipients), dexDC-treated DA recipients even showed a significantly accelerated rejection of Lewis islets. Immunohistochemical analysis of allograft tissue of dexDC-treated recipients showed a predominant natural killer cell infiltration and a presence of antibody reactivity in the absence of complement deposition. Alloreactive antibodies were solely found in dexDC-treated recipients. Our study shows that pretreatment with donor-derived dexDCs induces an antibody-mediated rejection in this islet transplantation rodent model.

Expression of Receptor Activator of Nuclear Factor-Kappa B Ligand in Leucocytes During Acute Kidney Rejection after Transplantation in Rats

Introduction: Acute cellular rejection of the transplanted kidney is an important cause of impaired graft function. One of the basic characteristics of acute cellular rejection according to the latest Banff classification of renal allograft pathology is the presence of T lymphocytes in a large number in allograft tissue. Recent studies have shown the important role of T lymphocytes and macrophages in acute cellular rejection of kidney allograft. Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B (RANK) and RANK ligand (RANKL) are three relatively novel members of the tumor necrosis factor (TNF) superfamily. They have a crucial role in physiological and pathological bone metabolism but also in immunological processes. The aim of our study was to determine the expression of RANKL and RANK by T lymphocytes and macrophages in acute cellular kidney allograft rejection in rats. Methods: The study included 15 male Wistar rats (3 months old; weight 250-300g) as recipients and 15 male DA rats (3 months old; weight 250-300g) as donors. Animals were sacrificed after 3 weeks and the transplanted kidney was extracted and processed for pathohistological analysis and immunoflorescence. The latest Banff classification of renal allograft pathology was used on all tissue samples by two experienced pathologists. In all tissue samples acute cellular rejection was proven. Acute cellular rejection kidney sections were examined by dual- labeled immunofluorescence to detect CD4, CD8 or CD68 (red) and RANK or RANKL (green) allowing clear definition of cells that co-express both (orange). Results: Cytoplasmic and on surface granular RANKL immunoreaction on fluorescence microscopy of various intensities was detected in all tissue samples. RANKL positive expression was colocalized with CD4+ and CD8+T lymphocytes in acutely rejected kidney tissue. There was no association between CD4+ and CD8+T cells with RANK expression in any obtained sample. RANK but not RANKL, was expressed by infiltrating CD68 positive macrophages in the interstitium of kidney tissue. Conclusion: RANK and RANKL are expressed by T lymphocytes and macrophages in acute cellular kidney rejection after transplantation in rats. They have a possible immunomodulatory role in acute cellular kidney rejection. Serum concentration of RANKL could be a marker for early detection of acute cellular kidney rejection.

Derivation of Germline Competent Rat Embryonic Stem Cells from DA Rats

Derivation of Germline Competent Rat Embryonic Stem Cells from DA Rats

Strain influences on inflammatory pathway activation, cell infiltration and complement cascade after traumatic brain injury in the rat

Increasing evidence suggests that genetic background affects outcome of traumatic brain injuries (TBI). Still, there is limited detailed knowledge on what pathways/processes are affected by genetic heterogeneity. The inbred rat strains DA and PVG differ in neuronal survival following TBI. We here carried out global expressional profiling to identify differentially regulated pathways governing the response to an experimental controlled brain contusion injury. One of the most differentially regulated molecular networks concerned immune cell trafficking. Subsequent characterization of the involved cells using flow cytometry demonstrated greater infiltration of neutrophils and monocytes, as well as a higher degree of microglia activation in DA compared to PVG rats. In addition, DA rats displayed a higher number of NK cells and a higher ratio of CD161bright compared to CD161dim NK cells. Local expression of complement pathway molecules such as C1 and C3 was higher in DA and both the key complement component C3 and membrane-attack complex (MAC) could be demonstrated on axons and nerve cells. A stronger activation of the complement system in DA was associated with higher cerebrospinal fluid levels of neurofilament-light, a biomarker for nerve/axonal injury. In summary, we demonstrate substantial differences between DA and PVG rats in activation of inflammatory pathways; in particular, immune cell influx and complement activation associated with neuronal/axonal injury after TBI. These findings suggest genetic influences acting on inflammatory activation to be of importance in TBI and motivate further efforts using experimental forward genetics to identify genes/pathways that affect outcome.

Naturally Occurring Variation in the Glutathione-S-Transferase 4 Gene Determines Neurodegeneration After Traumatic Brain Injury

Genetic factors are important for outcome after traumatic brain injury (TBI), although exact knowledge of relevant genes/pathways is still lacking. We here used an unbiased approach to define differentially activated pathways between the inbred DA and PVG rat strains. The results prompted us to study further if a naturally occurring genetic variation in glutathione-S-transferase alpha 4 (Gsta4) affects the outcome after TBI. Survival of neurons after experimental TBI is increased in PVG compared to the DA strain. Global expression profiling analysis shows the glutathione metabolism pathway to be the most regulated between the strains, with increased Gsta4 in PVG among top regulated transcripts. A congenic strain (R5) with a PVG genomic insert containing the Gsta4 gene on DA background displays a reversal of the strain pattern for Gsta4 expression and increased survival of neurons compared to DA. Gsta4 is known to effectively reduce 4-hydroxynonenal (4-HNE), a noxious by-product of lipid peroxidation. Immunostaining of 4-HNE was evident in both rat and human TBI. Intracerebral injection of 4-HNE resulted in neurodegeneration with increased levels of a marker for nerve injury in cerebrospinal fluid of DA compared to R5. These findings provide strong support for the notion that the inherent capability of coping with increased 4-HNE after TBI affects outcome in terms of nerve cell loss. A naturally occurring variation in Gsta4 expression in rats affects neurodegeneration after TBI. Further studies are needed to explore if genetic variability in Gsta4 can be associated to outcome also in human TBI.

Critical Role of VEGF-C/VEGFR-3 Signaling in Innate and Adaptive Immune Responses in Experimental Obliterative Bronchiolitis

Chronic inflammation, a hallmark of obliterative bronchiolitis, is known to induce lymphangiogenesis. We therefore studied the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C), its receptor VEGFR-3, and lymphangiogenesis during development of experimental obliterative bronchiolitis [ie, obliterative airway disease (OAD)] in rat tracheal allografts. The functional importance of VEGF-C was investigated by adenovirus-mediated overexpression of VEGF-C (AdVEGF-C), and by inhibition of VEGF-C activity with VEGFR-3-Ig (AdVEGFR-3-Ig). Analyses included histology, immunohistochemistry, and real-time RT-PCR 10 and 30 days after transplantation. In the course of OAD development, lymphangiogenesis was induced in the airway wall during the alloimmune response, which was reversed by cyclosporine A in a dose-dependent fashion. VEGF-C overexpression in tracheal allografts induced epithelial activation, neutrophil chemotaxis, and a shift toward a Th17 adaptive immune response, followed by enhanced lymphangiogenesis and the development of OAD. In contrast, inhibition of VEGF-C activity with VEGFR-3-Ig inhibited lymphangiogenesis and angiogenesis and reduced infiltration of CD4(+) T cells and the development of OAD. Lymphangiogenesis was linked to T-cell responses during the development of OAD, and VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts. Our results thus suggest VEGFR-3-signaling as a novel strategy to regulate T-cell responses in the development of obliterative bronchiolitis after lung transplantation.

96. Expression of TLRs in the rat intestine following chemotherapy for cancer

Gastrointestinal mucositis (GIM) is a major clinical problem caused by the cytotoxic effects of chemotherapy on the mucosa. Previous research has indicated that toll-like receptor (TLR) expression may be altered following chemotherapy and correlate with severity of GIM. As such, this study aimed to determine TLR expression in our tumor-bearing rat model of GIM. Female DA rats received irinotecan (175 mg/kg, i.p., n = 35) and were assessed over 5 days for markers of GIM (diarrhea, weight loss). Groups of rats (n = 5-8) were killed at 6, 24, 48, 72, 96 and 120 h following irinotecan. Immunohistochemistry for TLRs 2, 4, 5, and 9 was conducted on sections of jejuna and colon. All rats which received irinotecan developed GIM, with maximal diarrhea at 72 h. Similarly, peak weight loss occurred at 72 h (mean ± SD = 11.1 ± 6.6%) before recovering completly at 120 h (mean ±SD = − 0.25 ± 6.7%). Irinotecan caused a significant decrease in jejuna crypt expression of TLR4 and 5 at 96 h (p

Total body irradiation of donors can alter the course of tolerance and induce acute rejection in a spontaneous tolerance rat liver transplantation model

Liver transplantation is an established therapy for end-stage liver diseases. Graft rejection occurs unless the recipient receives immunosuppression after transplantation. This study aimed to explore the mechanism of acute rejection of liver allografts in rats pre-treated with total body irradiation to eliminate passenger lymphocytes and to define the role of CD4(+)CD25(+) regulatory T cells in the induction of immunotolerance in the recipient. Male Lewis rats were used as donors and male DA rats were recipients. Rats were randomly assigned to the following four groups: control group, homogeneity liver transplantation group, idio-immunotolerance group and acute rejection group. After transplantation, the survival time of each group, serum alanine aminotransferase, total bilirubin levels, number of Foxp3(+)CD4(+)CD25(+) regulatory T cells, expression of glucocorticoid-induced tumor necrosis factor receptor on T cell subgroups, histopathology of the hepatic graft and spleen cytotoxic T lymphocyte lytic activity were measured. In the acute rejection group, where donors were preconditioned with total body irradiation before liver transplantation, all recipients died between day 17 and day 21. On day 14, serum alanine aminotransferase increased significantly to (459.2±76.9) U L(-1), total bilirubin increased to (124.1±33.7) μmol L(-1) (P<0.05) and the ratio of Foxp3(+)CD4(+)CD25(+) regulatory T cells decreased significantly to 1.50%±0.50% (P<0.05) compared with the other groups. Analysis of the T cell subpopulations in the acute rejection group varied from the other groups. Histological analysis showed typical changes of acute rejection in the acute rejection group only. Preconditioning of the donors with total body irradiation eliminated passenger lymphocytes of the liver graft, and thus affected the course of tolerance and induced acute rejection after liver transplantation.

Overexpression of Toll‐Like Receptor 3 in Spleen is Associated with Experimental Arthritis in Rats

This study is to investigate the regulation of Toll-like receptor (TLR) expression in systemic immune reactions in different arthritis rat models, which will provide evidence to understand the mechanisms of rheumatoid arthritis further. Arthritis-susceptible DA rats were used to induce arthritis by pristane or collagen type II, and TLR2, 3, 4 and 7 expression levels in spleen were detected by real-time quantitative polymerase chain reaction. TLR3 mRNA expression in spleen of both collagen-induced arthritis and pristane-induced arthritis (PIA) rats was increased significantly at 26 and 70 days after arthritis induction. The overexpression of TLR3 was confirmed by Western blotting. Methotrexate was administrated peritoneally to PIA rats, and phytol was applied subcutaneously to PIA rats. Both methotrexate and phytol treatment could alleviate arthritis severity and block TLR3 induction. However, in arthritis-resistant E3 rats injected with pristane, TLR3 expression of spleen was unaltered. PIA in MHC congenic DA.1U rats had mild symptoms, whereas TLR3 mRNA expression in spleen of DA.1U rats showed an impaired induction at D26. So we conclude that overexpression of splenic TLR3 is strongly associated with arthritis in rats, which suggests that TLR3 should be a most vital TLR in spleen to regulate the initiation and development of experimental arthritis and may be as an intriguing therapeutic opportunity for human rheumatoid arthritis.

Erratum to: “Therapeutic effect of MBP immunodominant peptides encapsulated in nanovehicles in the development of experimental autoimmune encephalomyelitis in DA rats”

Multiple sclerosis (MS) is a severe autoimmune neurodegenerative disease. It attacks mainly young people. The development of new approaches to MS treatment is a challenge to modern immunology and pharmacology. In the present study, a high therapeutic efficacy of immunodominant peptides of myelin basic protein (MBP) incorporated into unilamellar mannosylated liposomes in the development of experimental autoimmune encephalomyelitis (EAE) is demonstrated in DA rats. MBP is a component of the oligodendrocyte membrane, which forms the axonal sheath. This protein is among the major autoantigens in MS. We have analyzed the binding pattern of anti-MBP autoantibodies from MS patients using a previously designed MBP epitope library. Utilizing the same approach, we have investigated the pool of anti-MBP antibodies from SJL/J and C57BL/6mice and DA rats with EAE. According to the autoantibody binding patterns, the rodent model most closely mimicking MS is EAE in DA rats. We have chosen three immunodominant MBP fragments encapsulated in unilamellar mannosylated liposomes for the treatment of the verified DA rodent model. MBP fragment 46–62 is the most efficient in mitigating the first EAE attack, whereas MBP 124–139 and 147–160 inhibit the development of pathology at the regression stage. Simultaneous administration of these peptides in liposomes significantly reduces the level of antibodies against MBP. The synergistic therapeutic effect of MBP fragments reduces the integral disease score by inhibiting the first EAE attack and mitigating the subsequent relapse. Thus, our findings offer new opportunities for the efficient treatment of multiple sclerosis.

Phenotypic and Gene Expression Differences between DA, BN and WOKW Rats

BackgroundBecause inbred rat strains are widely used as laboratory models, knowledge of phenotypic and genetic variations between strains will be useful to obtain insight into the relationship between different strains.Methods and ResultsWe studied phenotypic traits: of each strain – BN/K, DA/K and WOKW –10 male rats were studied for body weight and serum constituents at an age of 10 and 30 weeks. In addition, a total of 95 rats were studied for life expectancy. At an age of 30 weeks, these male rats were killed by an overdose of anesthetic (Sevofluran, Abbott), and the subcutaneous and visceral adipose tissue as well as bone tissue were removed to study the expression of 20 genes. There were significant differences in body weight, serum lipids and leptin at an age of 30 weeks between strains. Regarding life expectancy, BN rats lived longest (1072±228d). The highest gene expression was found in bone of BN rats. In adipose tissues, Nfkb1 is only expressed in subcutaneous adipocytes, and 5 genes, Col2a1, Mmp9, Tnfa, Ins1 and Cyp24a1, are not expressed in adipocytes. The ranking BN = DA>WOKW was observed in only one gene in subcutaneous (Fto) and visceral adipocytes (Col6a1). There were no significant differences in gene expression of one gene in subcutaneous adipocytes and of 3 genes in visceral adipocytes. Comparing the gene expression in visceral and subcutaneous adipocytes, only one gene showed a comparable behavior (Bmp1).ConclusionFrom these results, it can be concluded that obvious phenotypic differences are caused by genetic differences between three rat strains, BN, DA and WOKW, as supported by gene expression studies in bone and adipose tissues. Especially BN rats can be used to study the genetic basis of long life.

Liver X Receptor Regulates Rheumatoid Arthritis Fibroblast-like Synoviocyte Invasiveness, Matrix Metalloproteinase 2 Activation, Interleukin-6 and CXCL10

Fibroblast-like synoviocyte (FLS) invasiveness correlates with articular damage in rheumatoid arthritis (RA), yet little is known about its regulation. In this study we aimed to determine the role of the nuclear receptor liver X receptor (LXR) in FLS invasion. FLS were isolated from synovial tissues obtained from RA patients and from DA rats with pristane-induced arthritis. Invasion was tested on Matrigel-coated chambers in the presence of the LXR agonist T0901317, or control vehicle. FLS were cultured in the presence or absence of T0901317, and supernatants were used to quantify matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, interleukin-6 (IL-6), tumor necrosis factor-α and C-X-C motif chemokine ligand 10 (CXCL10). Nuclear factor-κB (NF-κB) (p65) and Akt activation, actin cytoskeleton, cell morphology and lamellipodia formation were also determined. The LXR agonist T0901317 significantly reduced DA FLS invasion by 99% (P ≤ 0.001), and RA FLS invasion by 96% (P ≤ 0.001), compared with control. T0901317-induced suppression of invasion was associated with reduced production of activated MMP-2, IL-6 and CXCL10 by RA FLS, and with reduction of actin filament reorganization and reduced polarized formation of lamellipodia. T0901317 also prevented both IL-1β-induced and IL-6-induced FLS invasion. NF-κB (p65) and Akt activation were not significantly affected by T0901317. This is the first description of a role for LXR in the regulation of FLS invasion and in processes and pathways implicated both in invasion as well as in inflammatory responses. These findings provide a new rationale for considering LXR agonists as therapeutic agents aimed at reducing both inflammation and FLS-mediated invasion and destruction in RA.