Maternal plasma proteomics in a rat model of pregnancy complications reveals immune and pro-coagulant gene pathway activation.

PROBLEM: The Brown Norway (BN) rat is a model of T-helper 2 immune diseases, and also a model of pregnancy disorders that include placental insufficiency, fetal loss, and pre-eclampsia-like symptoms. The aim of this study was to investigate the plasma proteomic/cytokine profile of pregnant BN rats in comparison to that of the Lewis (LEW) rat strain. METHOD OF STUDY: Plasma proteomics differences were studied at day 13 of pregnancy in pooled plasma samples by differential in-gel electrophoresis, and protein identification was performed by mass spectrometry. Key protein findings and predicted cytokine differences were validated by ELISA using plasma from rats at various pregnancy stages. Proteomics data were used for ingenuity pathway analysis (IPA). RESULTS: In-gel analysis revealed 74 proteins with differential expression between BN and LEW pregnant dams. ELISA studies confirmed increased maternal plasma levels of complement 4, prothrombin, and C-reactive protein in BN compared to LEW pregnancies. LEW pregnancies showed higher maternal plasma levels of transthyretin and haptoglobin than BN pregnancies. Ingenuity pathway analysis revealed that BN pregnancies are characterized by activation of pro-coagulant, reactive oxygen species, and immune-mediated chronic inflammation pathways, and suggested increased interleukin 6 and decreased transforming growth factor-ß1 as potential upstream events. Plasma cytokine analysis revealed that pregnant BN dams have a switch from anti- to pro-inflammatory cytokines with the opposite switch observed in pregnant LEW dams. CONCLUSION: Brown Norway rats show a maternal pro-inflammatory response to pregnancy that likely contributes to the reproductive outcomes observed in this rat strain.

Genomics studies of immune-mediated diseases using the BN-LEW rat model.

LEW and BN rats, that behave in opposite ways for their susceptibility to various immune-mediated diseases, provide a powerful model to investigate the molecular and genetic bases of immune system physiology and dysregulation. Using this model, we addressed the question of the genetic control of central nervous system autoimmunity, of xenobiotic-induced allergic diseases, and of T cell subsets that differ by their cytokine profiles. By linkage analysis and genetic dissection, using a panel of congenic rats, we identified a 120 Kb region on chromosome 9 that controls all these phenotypes, indicating that this region contains a gene or set of genes that plays an important role in the immune system homeostasis and susceptibility to immune mediated diseases. In this review, we will describe these rat genomics studies and will discuss the cellular and genetic factors that may be involved in the differences between these rat strains.

Control of Toxoplasma gondii infection by athymic LEW-Whn rnu rats.

In immunocompetent rats and humans infection with Toxoplasma gondii remains mostly without overt clinical symptoms, but can be fatal, if the T-cell response is impaired. For a better understanding of the lack of control of T. gondii infection under immunosuppressed conditions, congenitally athymic rats were used as the experimental model. Whereas athymic F344-Whn(rnu) (F344 nude) rats die from a generalized infection during the first 3 weeks after peritoneal inoculation with 10(6) tachyzoites of T. gondii strain NTE, LEW-Whn(rnu) (LEW nude) rats and euthymic LEW rats infected with a 10-fold higher number of parasites developed chronic infection. To identify underlying mechanisms of LEW rats resistance to T. gondii infection and to investigate a possible contribution of residual T-cells to LEW-Whn(rnu) rat resistance, we characterized the immune response of LEW rats by determination of cellularity and composition of lymphocyte population, antigen-specific IgG2b response as well as assays of antigen-specific proliferation and production of IL-2, IFN-gamma and TNF-alpha. As only euthymic LEW rats developed production of antigen-specific IgG and cellular in vitro responses, these results strongly suggest that the genetic background of LEW rats permits a control of the infection independent of an adaptive immune response.

The role of graft and host accommodation in a hamster-to-rat cardiac transplantation model.

BACKGROUND: We evaluated the importance and mechanism of graft and host accommodation in hamster-to-rat cardiac xenotransplantation models. METHODS: To evaluate graft accommodation, accommodated hamster grafts (Group 2) were transplanted to naïve host rats treated with FK506, and compared with naïve hamster grafts (Group 1). To evaluate host accommodation, three groups were evaluated: naive hamster hearts were transplanted to naïve hosts treated with FK506 (Group 3: 0.5 mg/kg, Group 4: 1.0 mg/kg) and splenectomy, and compared with accommodating hosts (Group 5) with FK506 0.5 mg/kg and splenectomy. We examined graft survival, histopathology, antihamster antibodies and B-1 cells in blood. RESULTS: Graft survival in Group 2 (3.4+/-0.9 days) was not significantly different from that in Group 1 (2.8+/-0.4 days). Graft survival in Groups 4 and 5 (>30 days) was significantly prolonged compared with that in Group 3 (6.0+/-0.7 days). Histopathology of Groups 1-3 showed humoral rejection, whereas Groups 4 and 5 showed normal histology and expression of protective genes. In Groups 1-3, antihamster immunoglobulin (Ig) M and B-1 cells increased significantly compared to Groups 4 and 5, where IgM and B-1 cells remained low or were reduced. CONCLUSIONS: Host accommodation was more important than graft accommodation. Accommodating grafts expressing protective genes were rejected with an elevation of both IgM and B-1 cells. In accommodated hosts, both IgM and B-1 cells decreased, suggesting that B-1 cells may be responsible for the production of antihamster antibodies. These results suggest that sufficient suppression of B-1 cells, resulting in decreased titers of antihamster antibodies, may play an important role in host accommodation.

Different characters of spleen OX-62 positive dendritic cells between Fischer and Lewis rats.

The phenotype, DNA-binding activities of NF-kappaB, cytokine production, endocytosis and stimulatory capacity of spleen OX-62-positive dendritc cells (SDCs) from Fischer rats were compared with those from Lewis rats. Results showed that the expressions of CD11b, MHC-II, CD8, CD45RA, CD54 and CD86 on SDCs were significantly higher in Fischer than those in Lewis rats. The levels of IL-2, IL-4, IL-10 and IFN-gamma in SDCs from Fischer rats were distinctly higher than those from Lewis. Both stimulatory capacity and DNA-binding activities of NF-kappaB in SDCs were all lower in Fischer than those in Lewis rats. These differences may partly contribute to rat strain-specificity in susceptibility to chronic inflammatory stimuli.

Molecular mimicry: sensitization of Lewis rats with Campylobacter jejuni lipopolysaccharides induces formation of antibody toward GD3 ganglioside.

Recently we have reported cases of demyelinating inflammatory neuropathy showing elevated titers of anti-GD3 antibodies, which occurs rarely in Guillain-Barré syndrome. To examine the correlation between the anti-GD3 antibody titer and Campylobacter jejuni infection, we sensitized female Lewis rats with lipopolysaccharides (LPSs) from serotype HS19 of C. jejuni and examined changes in nerve conduction velocity and nerve conduction block (P/D ratio). After 16 weeks of sensitization, animals revealed decreases of nerve conduction velocity and conduction block (P/D ratio) and high titer of anti-GD3 antibodies. These anti-GD3 antibodies also blocked transmission in neuromuscular junctions of spinal cord-muscle cells cocultures. The GD3 epitope was verified to be located on the Schwann cell surface and nodes of Ranvier in rat sciatic nerve. To determine the target epitope for GD3 antibodies in causing nerve dysfunction, the LPS fraction containing the GD3 epitope was purified from the total LPS by using an anti-GD3 monoclonal antibody-immobilized affinity column. Subsequently, chemical analysis of the oligosaccharide portion was performed and confirmed the presence of a GD3-like epitope as having the following tetrasaccharide structure: NeuAcalpha2-8NeuAc2-3Galbeta1-4Hep. Our data thus support the possibility of a contribution of GD3 mimicry as a potential pathogenic mechanism of peripheral nerve dysfunction.

Role of IL-27-producing dendritic [corrected] cells in Th1-immunity polarization in Lewis rats.

Lewis and Brown Norway rats are entirely different with respect to the polarization of their immune responses (Th1 and Th2, respectively). We found that naive Lewis rat splenocytes treated in vitro with heat-killed Mycobacterium tuberculosis (Mtb) upregulate the expression of both subunits of IL-27 (IL-27p28 and EBI3). Mtb treatment caused naive Lewis rat splenocytes to express 4.6-fold more IL-27p28 than Mtb-treated Brown Norway rat splenocytes 6h after the treatment. Although WSX-1, the IL-27 receptor, was not induced by Mtb treatment in splenocytes from either rat strain, Lewis rats expressed significantly higher levels of the IL-27 signal transducers T-bet and IL-12Rbeta2 than Brown Norway rats. Flow cytometric analysis of dendritic cells from bone marrow cells revealed Lewis rats had more IL-27p28-positive cells. Thus, early in the immune response, Lewis rats appear to produce higher levels of IL-27 than Brown Norway rats, resulting in polarization towards Th1-immunity.

Innate refractoriness of the Lewis rat to toxoplasmosis is a dominant trait that is intrinsic to bone marrow-derived cells.

Toxoplasmosis is a ubiquitous parasitic infection causing a wide spectrum of diseases. It is usually asymptomatic but can lead to severe ocular and neurological disorders. Among the small-animal models available to study factors that determine susceptibility to toxoplasmosis, the rat appears to be rather similar to humans, particularly in terms of resistance to acute infection. Here, we demonstrate that the Lewis (LEW) rat strain displays an unexpected refractoriness to Toxoplasma infection. Complete resistance was assessed by both negative anti-Toxoplasma serology and lack of detection of the parasite during the course of infection. In this model, sex, age, major histocompatibility complex, and inoculum size had no effect on resistance. Interestingly, progeny from F(1) hybrid crosses between Fischer (F344) or Brown Norway susceptible rats and LEW resistant rats were also fully resistant, showing a dominant effect of the gene or set of genes. Furthermore, resistance of the LEW rat was shown to be dependent on hematopoietic cells and partially abrogated by neutralization of endogenous gamma interferon. To our knowledge, this is the first observation of a rodent strain that is refractory to Toxoplasma infection. This model is therefore an attractive and powerful tool to dissect host genetic factors involved in susceptibility to toxoplasmosis.

Changes in the density and distribution of sympathetic nerves in spleens from Lewis rats with adjuvant-induced arthritis suggest that an injury and sprouting response occurs.

Previously we demonstrated reduced norepinephrine concentrations in spleens from Lewis rats with adjuvant-induced arthritis (AA), an animal model of rheumatoid arthritis. This study extends these findings, examining the anatomical localization and density of sympathetic nerves in the spleen with disease development. Noradrenergic (NA) innervation in spleens of Lewis rats was examined 28 days following adjuvant treatment to induce arthritis or vehicle for the adjuvant by using fluorescence histochemistry for catecholamines, with morphometric analysis and immunocytochemistry for tyrosine hydroxylase. In AA rats, sympathetic nerve density in the hilar regions, where NA nerves enter the spleen, was increased twofold over that observed in vehicle-treated rats. In contrast, there was a striking twofold decline in the density of NA nerves in splenic regions distal to the hilus in arthritic rats compared with nonarthritic rats. In both treatment groups, NA nerves distributed to central arterioles, white pulp regions, trabeculae, and capsule. However, NA nerve density was reduced in the white pulp but was increased in the red pulp in AA rats compared with non-AA rats. These findings indicate an injury/sprouting response with disease development whereby NA nerves die back in distal regions and undergo a compensatory sprouting response in the hilus. The redistribution of NA nerves from white pulp to red pulp suggests that these nerves signal activated immune cells localized in the red pulp in AA. Although the mechanisms of this redistribution of NA nerves into the red pulp are not known, it may be due to migration from white pulp to red pulp of target immune cells that provide trophic support for these nerves. The redistribution of NA nerves into the red pulp may be critical in modulating immune functions that contribute to the chronic inflammatory stages of arthritis.

Intraspecies variability in the dose-response relationship for Salmonella Enteritidis associated with genetic differences in cellular immune response.

To evaluate the effects of differences in host cellular immunity, we studied the dose-response relationship for infection with Salmonella enterica serovar Enteritidis (SE) in two different rat strains, skewed towards T helper 1 (Th1, Lewis rats) or T helper 2 (Th2, Brown Norway rats) immunoregulation. Rats were exposed orally to different doses of SE after overnight starvation and neutralization of gastric acid. Animals were observed for clinical signs of disease, fecal excretion and SE load in spleen and cecum, histopathology of the cecum, hematology, and cellular and humoral immune responses. Exponential dose-response models were used for binary or continuous outcomes to analyze the experimental data. Cytokine patterns, antibody isotypes, and contact hypersensitivity tests confirmed that Lewis rats are Th1 prone, whereas Brown Norway rats are Th2 prone. The probability of infection per single SE cell was approximately 100 times higher in Brown Norway rats than in Lewis rats. Cellular immune responses were more pronounced in Lewis rats but antibody responses were higher in Brown Norway rats. When infected, colonization levels and inflammation are highest in the intestinal tract of Th2 skewed rats, but systemic infection is more intense in Th1 skewed rats. Successful colonization by only one or two SE clones resulted in a marked increase of neutrophil counts by a factor of two to three in both rat strains.

Rats and mice exhibit distinct inflammatory reactions after spinal cord injury.

Spinal contusion pathology in rats and mice is distinct. Cystic cavities form at the impact site in rats while a dense connective tissue matrix occupies the injury site in mice. Because inflammatory cells coordinate mechanisms of tissue injury and repair, we evaluated whether the unique anatomical presentation in spinally injured rats and mice is associated with a species-specific inflammatory response. Immunohistochemistry was used to compare the leukocytic infiltrate between rats and mice. Microglia/macrophage reactions were similar between species; however, the onset and magnitude of lymphocyte and dendritic cell (DC) infiltration were markedly different. In rats, T-cell numbers were highest between 3 and 7 days postinjury and declined by 50% over the next 3 weeks. In mice, significant T-cell entry was not evident until 14 days postinjury, with T-cell numbers doubling between 2 and 6 weeks. Dendritic cell influx paralleled T-cell infiltration in rats but was absent in mouse spinal cord. De novo expression of major histocompatability class II molecules was increased in both species but to a greater extent in mice. Unique to mice were cells that resembled lymphocytes but did not express lymphocyte-specific markers. These cells extended from blood vessels within the fibrotic tissue matrix and expressed fibronectin, collagen I, CD11b, CD34, CD13, and CD45. This phenotype is characteristic of fibrocytes, specialized blood-borne cells involved in wound healing and immunity. Thus, species-specific neuroinflammation may contribute to the formation of distinct tissue environments at the site of spinal cord injury in mice and rats.

[Urea soluble fraction of bovine melanin associated antigen and experimental study on its uveitogenic activity].

OBJECTIVE: The purpose of this study was to investigate the uveitogenic activity of autoantigen in the bovine iris-ciliary body. METHODS: Urea soluble fraction of boveine melanin associated antigen (USF-BMAA) was isolated from the bovine iris-ciliary body biochemically and determined by SDS-PAGE as well as amino acid analysis. Lewis rats and F344 rats were immunized with USF-BMAA emulsified with equal volume complete Freud's adjuvant and Bordetella pertussis. RESULTS: A strongly stained protein band was observed in the USF-BMAA by SDS-PAGE whose molecular weight is approximate 64 000. Amino acid analysis of USF-BMAA showed that it contains 17 kinds of amino acids with high content of Glu, Leu and Asp. The experimental melanin associated antigen-induced uveitis (EMIU) was successfully incited in both eyes of the Lewis and F344 rats. The inflammation was mainly located in the anterior uvea, and spontaneously recovered. Mild focal choroiditis was present in the rats with severe lesion. However, the inflammation was not observed in the retina and pineal gland. CONCLUSIONS: USF-BMAA may be the major part of autoantigen of the uveal tract with uveitogenic activity. Unlike experimental autoimmune uveoretinitis (EAU) which is incited by the retinal soluble antigen (S-Ag) in the rats, no involvement of the retina and pineal gland is found in EMIU.

Tektin B1 demonstrates flagellar localization in human sperm.

The human flagellar protein tektin B1 (h-tekB1) in human sperm was cloned, and its sequence and subcellular location were determined. Human sperm proteins were separated by 2-dimensional electrophoresis, and a resolved protein spot of 54 kDa with an isoelectric point (pI) of 5.3 was removed from the gel, trypsinized, and microsequenced by tandem mass spectrometry. The resulting peptides did not match any protein in the (then current) protein databases. Degenerate oligonucleotides based on the microsequences were used with a polymerase chain reaction to amplify a partial cDNA clone from human testis poly(A)(+) mRNA, and subsequently a full-length 1.5-kilobase (kb) clone (GenBank AF054910) was obtained from a testis cDNA library. The open reading frame encoded a 430-amino acid protein with 47% homology to the sea urchin tektin B1. Hybridization of labeled h-tekB1 cDNA to a multiple-tissue Northern blot demonstrated a transcript of 1.7 kb in human testis, and a multiple tissue dot-blot demonstrated high levels of expression in testis, trachea, and lung, intermediate levels in fetal brain and appendix, and low levels in ovary, pituitary, and fetal kidney. Rat polyclonal serum generated against a recombinant h-tekB1 demonstrated 3 h-tekB1 isoforms of pI 5.25, 5.5, and 5.35 at 53.5 kDa on a 2-dimensional Western blot of human sperm proteins. Immunofluorescent studies localized h-tekB1 to the principal piece of human sperm, but the endpiece was unstained.

Antigen-presenting hybridoma cells expressing MHC antigens of the LEW rat.

Towards the eventual purpose of facilitating analyses of specificities and functions of LEW rat T lymphocytes involved in the induction and development of organ-specific autoimmune disorders, hybridoma cells expressing class I and class II MHC antigens of LEW rat have been developed. B cell hybridomas produced between a murine B cell tumor M12.4.5 and stimulated LEW B cells expressed high levels of LEW class II MHC antigen but the expression of LEW class I MHC antigens on these cells was rather low. The B hybridoma cells were capable of presenting soluble protein antigens to LEW CD4(+) T cells. Furthermore, The use of this hybridoma revealed antigen-specific cytolytic activity of rat CD4(+) T cells. T cell hybridomas produced between murine thymoma BW5147 and LEW T cells expressed class I MHC antigens of the LEW rat. The expression was confirmed by surface staining and specific cytolysis by rat allogeneic CTL.

Cellular and genetic factors involved in the difference between Brown Norway and Lewis rats to develop respectively type-2 and type-1 immune-mediated diseases.

The understanding of the mechanisms of immune tolerance and the unravelling of the pathophysiology of autoimmune diseases rely on animal models. In this respect, BN and LEW rats represent models of choice to study immune-mediated diseases from the cellular and genetic points of view. Indeed, BN and LEW rats are extremes with respect to their polarisation of the immune response as well as their susceptibility to autoimmune diseases. LEW rats are susceptible to Th1-mediated autoimmune diseases while BN rats are highly susceptible to Th2-mediated autoimmune disease. Comparison of the T cell compartment between LEW and BN rats revealed several important differences. 1) A MHC-dependent quantitative difference that is due to a defect in the CD8 T cell compartment in BN rats. 2) A qualitative MHC-independent difference that is related to a high frequency of CD45RClow CD4 and CD8 T cell subsets, producing IL-4, IL-13, IL-10 and TGF-beta in BN rats as compared to LEW rats. 3) Interestingly, the genetic studies showed that susceptibility to Th1-mediated experimental autoimmune encephalomyelitis, and to Th2-mediated disorders triggered by gold salts as well as the difference in the CD4SRChigh/CD45RClow ratio between LEW and BN rats are genetically determined by regions on chromosomes 9, 10 and 20.

Lewis rats are not susceptible to oral infection with Mycobacterium avium subsp. paratuberculosis.

Pathogenesis studies of Mycobacterium avium subsp. paratuberculosis infection in ruminants are hampered by the long incubation time of the disease. A laboratory animal model with a shorter incubation time would facilitate research in this field. Although small rodents are usually considered to be resistant to M.a. paratuberculosis infection, several susceptible murine strains have been found. To our knowledge, there are no detailed reports with regard to susceptibility in rats. The Lewis rat is a valuable model for inflammatory bowel disease studies as well as autoimmune diseases involving mycobacteria as inducing agents. In this study Lewis rats were used to investigate their potential as a small laboratory animal model for paratuberculosis. In total 28 female Lewis rats were orally inoculated with M.a. paratuberculosis. The rats were first inoculated at 3 weeks of age, and 12 more inoculations followed in increasing intervals during the 3 months to follow. Eight control rats received a sham inoculation. Over 9 months, two rats from each group were sacrificed at regular intervals and immunological and histopathological examinations were performed on the gastrointestinal tract, the liver and the spleen. None of the rats developed lesions which were indicative of mycobacterial infection as determined by histology with HE and Ziehl-Neelsen staining. The bacteria could not be recultured from samples taken from the gut, the liver or the spleen. The immunological tests however, showed that bacteria had entered via the intestinal tract. From this study it appears that Lewis rats are resistant to oral inoculation with M. a. paratuberculosis, and not suitable as a model to study the immunopathogenesis of paratuberculosis as it occurs in ruminants.

Leukocyte mobilization induced by hypervolemia is due to a combined alpha- and beta-adrenoceptor activation.

A phenomenon of leukocytosis induced by hypervolemic stress was discovered. Although a single injection of 350 microl of saline (equivalent to approx. 70 ml in humans, 1 ml/kg of body weight) did not have an effect on the leukocyte counts in long-term intravenously cannulated, freely behaving rats, a single injection of 750 microl of saline (equivalent to approx. 150 ml in humans, 2.1 ml/kg) induced rapid leukocytosis of 160% within 1 minute followed by a gradual increase up to 180% after 1 hour. Measurement of serum norepinephrine concentration revealed a significant increase in rats of the hypervolemic group, compared with those of the low volume group. Pretreatment with either the beta-adrenoceptor antagonist nadolol or the selective alpha2-adrenoceptor antagonist yohimbine prevented both leukocyte peaks in the high volume group, suggesting a combined receptor activation. This critical dependence of leukocyte counts on changes in blood volume should be taken into consideration in experiments with laboratory animals (the quantity of volume applications can falsify results of experiments).

Role for interferon-gamma in rat strains with different susceptibility to experimental autoimmune myasthenia gravis.

Experimental autoimmune myasthenia gravis (EAMG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular postsynaptic membrane and represents an animal model of myasthenia gravis in human. Recent studies highlighted the roles of TH1 cytokines (IFN-gamma, IL-12), rather than TH2 cytokines (IL-4), in the pathogenesis of EAMG by using homozygous (-/-) knockout mice with an EAMG-susceptible genetic background. To further evaluate a role for IFN-gamma, we injected recombinant rat IFN-gamma (rrIFN-gamma) at the time of immunization with AChR in complete Freund's adjuvant to EAMG-susceptible Lewis rats and EAMG-resistant Wistar Furth (WF) rats. RrIFN-gamma enhanced Lewis rat EAMG. The exacerbated muscular weakness was associated with higher levels of anti-AChR IgG and enhanced TNF-alpha responses. Anti-AChR IgG antibody levels were augmented to a similar extent as in Lewis rats, however, the identical immunization and IFN-gamma injection induced only mild and transient EAMG in WF rats due to the default TH3 phenotype development and inherent low TH1 responses. We conclude that IFN-gamma plays a major role in the pathogenesis of EAMG in the Lewis rat, but fails to break disease resistance in the WF rat.

Sperm mitochondria-associated cysteine-rich protein (SMCP) is an autoantigen in Lewis rats.

A common repertoire of rat sperm antigens have previously been identified by Western blotting of sperm proteins with sera obtained after vasectomy or isoimmunization with sperm. Aside from a determination of their apparent masses, however, the biochemical characteristics of these antigens have remained unknown. In this study, a rat testis cDNA expression library was screened with polyclonal antibodies obtained from rats immunized with isologous spermatozoa to identify and sequence a full-length clone encoding rat sperm mitochondria-associated cysteine-rich protein (SMCP). The open reading frame of SMCP was expressed in the pET22b vector, and recombinant SMCP (rec-SMCP) was purified. Sera from rats that had been vasectomized or hyperimmunized with isologous sperm specifically recognized rec-SMCP whereas preimmune sera from these experimental groups did not react. Rabbit antiserum produced to rec-SMCP recognized rec-SMCP on Western blots and precisely immunolocalized SMCP to the mid-piece of rat sperm. On Western blots against sperm extracts, the rabbit antibody recognized a major protein band of approximately 22-25 kDa that co-migrated with bands of identical mass that were recognized by sera from hyperimmune or vasectomized rats. These findings demonstrate that SMCP is a sperm autoantigen, recognized following vasectomy, and an isoantigen, recognized by antibodies generated through isologous immunization with sperm.