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This study addresses the question whether susceptibility versus resistance to experimental autoimmune uveoretinitis (EAU) is connected to a Th1-type (interferon-gamma high, interleukin-4 low), versus a Th2-type (IFN-gamma low, IL-4 high) response. Primed lymph node cells of susceptible Lewis rats produced IFN-gamma in response to antigen in culture and transferred EAU to syngeneic recipients, whereas lymph node cells of resistant F344 rats made no IFN-gamma and did not transfer disease. Reversal of the disease pattern, by treatment of F344 rats with B. pertussis toxin and immunisation of Lewis rats with antigen in incomplete Freund's adjuvant, resulted in a parallel reversal of these response patterns. Neither strain produced significant IL-4 responses. A study of the response patterns in mice confirmed that high Th1 responders were susceptible, whereas low Th1 responders and Th2 responders were resistant. We conclude that susceptibility to EAU is connected with a Th1-dominant response, but resistance can involve either a 'null', F344-like response (Th1-low/Th2-low) or a Th2-dominant response.

Osteopetrosis, a skeletal disorder of inadequate bone resorption with an abnormal increase in skeletal mass, results from a variety of independent single gene mutations that affect osteoclast differentiation and/or function. The osteopetrotic defect, op, is one of four spontaneous, nonallelic mutations in rats that result in osteopetrosis. In intercross progeny of (BN/SsN x LEW/SsN. +/op) F1 carriers, we mapped this locus by linkage analysis with microsatellite markers to rat chromosome 10. The linkage group contained, as well as op, 15 anonymous DNA loci and 9 DNA loci associated with genes (interleukin-3, myosin heavy chain [skeletal, embryonic], asialoglycoprotein receptor [hepatic lectin]-1, vesicle-associated membrane protein [synaptobrevin-2], sex hormone binding globulin, aldolase C, nitric oxide synthase [inducible], erythroblastic leukemia avian viral oncogene homolog-2, and proline-rich protein). The markers for these loci include nine not previously reported. The op locus mapped to the end of the chromosome 10 linkage group, within 1 cM of the anonymous DNA locus, D10Mit6. Based on its location, the op gene is likely to be distinct from seven described mutations in mice as well as three other mutations in rats. These results may permit a positional cloning strategy to be undertaken to identify the gene and mutation underlying the op defect.

A genomic DNA fragment containing the rat epimorphin gene sequence was cloned from a rat DNA cosmid library using a mouse epimorphin cDNA probe. Within the cosmid insert, nine epimorphin exons were identified and sequenced. The predicted amino acid sequence of the rat epimorphin protein exhibited 96 and 86% identity with the mouse and human epimorphin proteins, respectively. Consistent with the developmentally related expression pattern of the mouse epimorphin gene, transcripts of the rat epimorphin gene were detected in 17-day postfertilization rat embryos. The gene, designated Epim, was assigned to rat chromosome 12 by somatic cell hybrid analysis and localized to 12q16 by fluorescence in situ hybridization. The mouse and human homologs of this gene were localized on mouse chromosome 5 and human chromosome 7 by linkage analysis and chromosomal in situ hybridization, respectively.

We describe an adaptation of competitive RT-PCR to quantitate rat IFN-gamma mRNA expression. An IFN-gamma DNA mimic that shared the same primers and had an identical sequence to the target mRNA except for deletion of 66 nucleotides was created by a simple PCR amplification from target cDNA. To reduce variations of initial RNA concentrations, beta-actin cDNAs from each target RNA sample were normalized using the densitometric data. A known amount of pretitrated DNA competitor was then used to analyze the relative levels of target cDNA in different samples by PCR co-amplification. The amplification efficiency for both target and competitor remained constant throughout the PCR reaction, and the ratio of target to competitor PCR product remained proportional to the initial ratio of target to competitor. Relative mRNA levels among samples determined by this method were comparable to levels determined by northern blot analysis. They were also comparable to levels of IFN-gamma protein estimated by ELISA. We conclude that this method can be used to estimate the relative abundance of the target mRNA. This method is adaptable to quantitation of other cytokines and is particularly valuable if there are numerous samples or if the amount of initial mRNA is limited.

Interleukin-12 (IL-12) is a key inducer of differentiation of uncommitted T helper (TH) cells toward the TH1 phenotype, which regulates cellular immunity, whereas IL-10 inhibits TH1 functions and potentiates TH2-regulated responses (i.e., humoral immunity). To examine the potential effects of stress on TH1/TH2 balance, we studied the ability of three prototype stress hormones-dexamethasone (a synthetic glucocorticoid) and the catecholamines norepinephrine and epinephrine-to alter the production of IL-12 (p70) and IL-10 induced by bacterial lipopolysaccharide (LPS) in human whole blood. Dexamethasone inhibited LPS-induced bioactive IL-12 production in a dose-dependent fashion and at physiologically relevant concentrations; it had no effect on IL-10 secretion. The glucocorticoid-induced reduction of IL-12 production was antagonized by RU 486, a glucocorticoid-receptor antagonist, suggesting that it was mediated by the glucocorticoid receptor. Norepinephrine and epinephrine also suppressed IL-12 production in a dose-dependent fashion and at physiological concentrations; both catecholamines, however, dose-dependently increased the production of IL-10. The effects of either catecholamine on IL-12 or IL-10 secretion were blocked completely by propranolol, a beta-adrenoreceptor antagonist, indicating that they were mediated by the beta-adrenergic receptor. These findings suggest that the central nervous system may regulate IL-12 and IL-10 secretion and, hence, TH1/TH2 balance via the peripheral end-effectors of the stress system. Thus, stress may cause a selective suppression of TH1 functions and a shift toward a TH2 cytokine pattern rather than generalized TH suppression. The TH1-to-TH2 shift may be responsible for the stress-induced susceptibility of the organism to certain infections. Through the same or a reciprocal mechanism, states associated with chronic hyperactivity or hypoactivity of the stress system might influence the susceptibility of an individual to certain autoimmune, allergic, infectious, or neoplastic diseases.

Identification of specific genetic loci that contribute to susceptibility to rheumatoid arthritis (RA) in humans has been hampered by several factors, including: i) multiple interacting genetic loci contributing to susceptibility; ii) complex interactions of environmental and genetic factors; iii) genetic heterogeneity; and iv) low penetrance. We have, therefore, mapped quantitative trait loci (QTLs) that control inflammatory arthritis susceptibility and/or severity in progeny of two inbred rat strains with significantly different susceptibilities to collagen-induced arthritis (CIA), an animal model for RA. Not surprisingly, we identified a major susceptibility factor, Cia1, on chromosome 20 in the vicinity of the rat major histocompatibility complex (MHC). However, by limiting the analysis to animals with arthritis-susceptible MHC genotypes and using genome-wide QTL analytic techniques, we also found four non-MHC QTLs-Cia2, 3, 4 and 5-on chromosomes 1, 4, 7 and 10, that contributed to disease severity. In addition, a QTL on chromosome 8 was suggestive for linkage. Characterization of the genes underlying these QTLs will facilitate the identification of key biochemical pathways regulating experimental autoimmune arthritis in rats and may provide insights into RA and other human autoimmune diseases. These genes may also represent novel targets for therapy.

Genetic linkage maps of loci defined by polymorphic DNA markers on rat chromosome 1 were constructed by genotyping F2 progeny of F344/N x LEW/N, BN/SsN x LEW/N, and DA/Bkl x F344/Hsd inbred rat strains. In total, 43 markers were mapped, of which 3 were restriction fragment length polymorphisms and the others were simple sequence length polymorphisms. Nineteen of these markers were associated with genes. Six markers for five genes, gamma-aminobutyric acid receptor beta3 (Gabrb3), syntaxin 2 (Stx2), adrenergic receptor beta1 (Adrb1), carcinoembryonic antigen gene family member 1 (Cgm1), and lipogenic protein S14 (Lpgp), and 20 anonymous loci were not previously reported. Thirteen gene loci (Myl2, Aldoa, Tnt, Igf2, Prkcg, Cgm4, Calm3, Cgm3, Psbp1, Sa, Hbb, Ins1, and Tcp1) were previously mapped. Comparative mapping analysis indicated that a large portion of rat chromosome 1 is homologous to mouse chromosome 7, although the homologs of two rat genes are located on mouse chromosomes 17 and 19. Homologs of the rat chromosome 1 genes that we mapped are located on human chromosomes 6, 10, 11, 12, 15, 16, and 19.

Objective: Treatment with high-dose (400 mg/kg/day) intravenous immunoglobulin (IVIg) shows benefit in many autoimmune diseases but is very expensive. Low-dose IVIg has also been shown to be effective in inhibiting adjuvant arthritis in the rat. This pilot, randomized, double-blind, placebo-controlled trial was conducted to assess the use of low-dose IVIg in patients with treatment-refractory rheumatoid arthritis (RA)., Methods: Twenty patients with active RA were recruited. Ten patients received IVIg and 10 received albumin. Study subjects were given 6 courses of either IVIg (5 mg/kg) or albumin (5 mg/kg), once every 3 weeks. Baseline medications were continued and not changed throughout the study., Results: There were no complications. Five patients dropped out before the 18-week followup visit. No significant differences between treatment groups were noted during the 18-week trial in terms of global activity indices (patient or physician assessment), joint swelling, joint pain or tenderness, erythrocyte sedimentation rate, C-reactive protein level, or rheumatoid factor. The protocol was terminated prematurely because of reported contamination of IVIg by hepatitis C virus. None of the patients showed evidence of hepatitis C infection by serologic analysis or by polymerase chain reaction., Conclusion: Low-dose IVIg, as administered in this trial, does not show a therapeutic effect in patients with refractory RA.

Hormones, particularly those involved in the hypothalamic-pituitary-gonadal and -adrenal axes (HPG and HPA), play important roles in various animal models of autoimmunity such as systemic lupus erythematosus in mice and collagen-induced arthritis (CIA) in mice and rats, and the streptococcal cell wall, adjuvant and avridine arthritis models in rats. Intimately linked to the subject of hormones and autoimmunity are gender, sex chromosomes and age. The importance of these factors in the various animal models is emphasized in this chapter. Several major themes are apparent. First, oestrogens promote B-cell dependent immune-complex mediated disease (e.g. lupus nephritis) but suppress T-cell dependent pathology (CIA in mice and rats), and vice versa. Second, testosterone's effects are complicated and depend on species and disease model. In rats, testosterone suppresses both T-cell and B-cell immunity. In mice, the effects are complex and difficult to interpret, e.g. they tend to enhance CIA arthritis and suppress lupus. Sex chromosome/sex hormone interactions are clearly involved in generating these complicated effects. Third, studies in Lewis and Fischer F344 rats exemplify the importance of corticosteroids, corticotrophin releasing hormone and the HPA axis in the regulation of inflammation and the predisposition to autoimmune diseases. Fourth, the HPA axis is intimately linked to the HPG axis and is sexually dimorphic. Oestrogens stimulate higher corticosteroid responses in females. The animal model data have major implications for understanding autoimmunity in humans. In particular, adrenal and gonadal hormone deficiency is likely to facilitate T-cell dependent diseases like rheumatoid arthritis, while high oestrogen levels or effects, relative to testosterone, are likely to promote B-cell dependent immune-complex-mediated diseases such as lupus nephritis.

Arginine vasopressin (AVP) acts synergistically with corticotropin-releasing hormone (CRH) to stimulate ACTH release from the anterior pituitary. In a previous study of bilateral simultaneous inferior petrosal sinus (IPS) sampling in healthy human subjects, we observed lateralized ACTH secretion, suggesting lateralized secretion of an ACTH-regulating hypothalamic factor. To investigate this possibility, we measured ACTH, CRH, AVP, and oxytocin (OT) levels in the IPS and the peripheral circulation in nine normal volunteers, before and after 1 microgram/kg i.v. bolus ovine CRH (oCRH). At baseline, ACTH, AVP, and OT exhibited a significant (P < 0.05) two to threefold intersinus gradient (ISG), indicating the existence of a dominant petrosal sinus. Endogenous CRH was undetectable in all samples. Despite similar exogenous oCRH levels in both petrosal sinuses, oCRH caused a significant increase (P < 0.001) in the ACTH ISG (15.8 +/- 5.6, mean +/- SEM), suggesting increased responsiveness of one dominant side of the anterior pituitary. This was associated with an ipsilateral CRH-induced AVP release and a significant increase (P < 0.01) in the AVP ISG (8.6 +/- 2.3), suggesting lateralized AVP secretion by the hypothalamus. Furthermore, the increased AVP ISG after oCRH correlated strongly with the ACTH ISG (r = 0.92, P < 0.01). oCRH administration did not affect OT. These findings suggest that there is a dominant petrosal sinus in healthy volunteers that appears to reflect a dominant side of the adenohypophysis, characterized by increased functional activity and/or responsiveness of the pituitary corticotrophs. This may reflect lateralized hypothalamic and/or suprahypothalamic function resulting in CRH-responsive lateralized secretion of AVP from parvocellular and/or magnocellular axons in the median eminence and the posterior pituitary. Although the functional and teleologic significance of these findings remains to be investigated, our data suggest a novel mechanism for CRH-mediated ACTH release, namely CRH-induced release of AVP which then enhances CRH action on the corticotrophs. Furthermore, our data represent the first direct evidence for the concept of brain lateralization with respect to neuroendocrine secretion.

Rheumatoid arthritis (RA) is a multifactorial disease in which both environmental and genetic factors play a role. Data also suggest that neuroendocrine factors are involved. I briefly summarize observations that support this hypothesis. RA is characterized by striking age-sex disparities. The incidence of disease in women increases steadily from the age of menarche to its maximal incidence around menopause. The disease is uncommon in men under age 45, but its incidence increases rapidly in older men and approaches the incidence in women. These observations strongly suggest that androgens play a major suppressive role, and, in fact, testosterone levels are depressed in most men with RA. Mechanistically, many data indicate that testosterone suppresses both cellular and humoral immune responses. Dehydroepiandrosterone (DHEA), an adrenal product, is the major androgen in women. Its production is strikingly dependent upon age. Peak production is in the 2nd and 3rd decades, but levels decline precipitously thereafter. DHEA levels are low in both men and women with RA, and recent data show that levels of this hormone may be depressed before the onset of disease. The role of DHEA in immune diseases, however, is controversial. The menopausal peak of RA onset suggests estrogen and/or progesterone deficiency play a role in the disease, and many data indicate that estrogens suppress cellular immunity but stimulate humoral immunity, i.e., deficiency promotes cellular (Th1-type) immunity. Recent data also indicate that progesterone stimulates a switch for Th1 to Th2-type immune responses. RA often develops or flares in the postpartum period, particularly if the mother breastfeeds. This is again consistent with gonadal steroid deficiency playing a role in the onset of disease. Breastfeeding is associated with blunted hypothalamic-pituitary-adrenal function and elevated prolactin synthesis. Gonadal and adrenal steroid hormone deficiency, plus elevated prolactin, probably greatly facilitates the expression of Th1-type immunity, which is widely believed to be critical in the pathogenesis of RA. By contrast, RA typically remits during pregnancy, in parallel with the increasing levels of corticosteroids, estrogens, and progesterone. Pregnancy is characterized by a shift in immune function from Th1-type to Th2-type. Oral contraceptives, which generate a condition of pseudopregnancy, also decrease the risk of RA. These data argue that adrenal and gonadal steroid hormones suppress the development of RA. Several studies indicate that corticosteroid production is inappropriately low in patients with RA, and are reminiscent of observations in Lewis rat models of chronic erosive arthritis. In summary, a growing body of data indicate that RA develops as a consequence of a deficiency in both adrenal and gonadal steroid hormone production. This hypothesis clearly has potential clinical implications.

Linkage maps of rat chromosomes 15, 16, 17, 19, and X were constructed by multipoint genetic linkage analysis of 22 polymorphic markers in 40 F2 progeny of Fischer (F344/N) and Lewis (LEW/N) inbred rat strains. These markers are associated with eight genes (angiotensin receptor A, M3 muscarinic acetylcholine receptor, heme oxygenase, endothelin receptor A, haptoglobin, tyrosine aminotransferase, phosphoribosylpyrophosphate synthetase subunit II, and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) and 14 anonymous loci. Linkage analysis placed the markers into five linkage groups covering 11.7,7.9,11.6,42.5, and 5.1cM. These linkage groups were assigned to rat chromosomes 15, 16, 17, 19, and X, respectively, either by mouse x rat somatic cell hybrid analysis or based on previously identified locations of severalloci. In polymorphism analysis, these markers exhibited two to nine different alleles in 16 inbred rat strains.

Skin involvement is common in patients with SLE, and in some cases is related to the menstrual period. We describe the clinical course of 3 patients with menstrual related rashes who experienced a significant improvement from their skin disease after the initiation of oral contraceptives. The potential role of hormones in the manifestations of SLE is discussed.

Corticotropin releasing hormone (CRH) is a key hormone in integrated response to stress, acting as the major regulator of the hypothalamic-pituitary-adrenal axis. Recently, local production of CRH has been detected in normal human colonic enterochromaffin cells. CRH is locally secreted in granulomatous and arthritic tissues in rats and humans, where it seems to act as a local proinflammatory agent. To find out if CRH is present in colonic tissues of patients with ulcerative colitis, this study examined the expression of this peptide in the large bowel of patients with ulcerative colitis. Colonic tissues of patients with ulcerative colitis obtained by endoscopic biopsy were immunostained with anti-CRH antibody. CRH messenger (m) RNA was also examined in biopsy specimens of ulcerative colitis by the reverse transcribed polymerase chain reaction method and by in situ hybridisation. Considerably enhanced expression of immunoreactive CRH was found in mucosal inflammatory cells. Intense staining with anti-CRH antibody was also shown in mucosal macrophages. CRH mRNA was expressed in mucosal epithelial cells. The expression of immunoreactive CRH in colonic mucosal epithelial cells of ulcerative colitis slightly increased, but not significantly, compared with normal colonic mucosal epithelial cells. These results suggest that CRH may play a part in the modulation of intestinal immune and inflammatory system, and as a modulator in the pathogenesis of ulcerative colitis.

Inflammatory mediators such as prostaglandin E2 (PGE2) and interleukin-1 (IL-1) induce angiogenesis by yet undefined mechanisms. We demonstrate that PGE2 and IL-1 induces the expression of vascular endothelial growth factor (VEGF), a selective angiogenic factor by rheumatoid synovial fibroblast cells. Transcripts for the EP1 and EP2 subtypes of PGE receptors are expressed in synovial fibroblasts. Activators of protein kinase A pathway stimulated the expression of VEGF whereas down-regulation of protein kinase C did not influence the PGE effect, suggesting that signalling from the EP2 receptor via the protein kinase A pathway is important. The induction of VEGF expression by PGE2 and interleukin-1 alpha may be an important mechanism in inflammatory angiogenesis.

Glucocorticoids are potent antiinflammatory agents. They inhibit leukocyte chemotaxis and vascular permeability and generally suppress the expression of many inflammatory mediators. Recent reports suggested that somatostatin (Sms) had significant immunomodulatory properties in vitro and in vivo. In this study we examined the effects of glucocorticoids on immunoreactive somatostatin expression in aseptic inflammatory sites of Sprague-Dawley rats given carrageenin sc. The progress of the inflammatory reaction was studied over a 7-h period with respect to the volume and cellularity of the exudate and the levels of the inflammatory mediators expressed in the inflammatory site, including immunoreactive substance P (sP), corticotropin-releasing hormone (CRH), and tumor necrosis factor-alpha (TNF alpha). Dexamethasone significantly reduced the volume and cellularity of the inflammatory exudates; in parallel, the levels of immunoreactive sP, CRH, and TNF alpha were significantly suppressed by this glucocorticoid. In contrast, immunoreactive Sms was stimulated by dexamethasone in a time-dependent fashion. These findings suggest another mechanism for suppression of the inflammatory reaction by glucocorticoids via stimulation of local Sms expression, which occurs in parallel to the inhibition of the local inflammatory mediators sP, CRH, and TNF alpha.

Several studies indicate that nonsteroidal anti-inflammatory drugs including indomethacin, aspirin, sulindac, and piroxicam reduce the risk of colon cancer. Furthermore, nonsteroidal anti-inflammatory drugs that inhibit the cyclooxygenase (COX) enzyme were shown to inhibit the development of colon cancer in animal models of carcinogenesis. Non-steroidal anti-inflammatory drugs inhibit the enzymatic activity of both the constitutive (COX-1) and inducible (COX-2) isoforms of COX enzyme. We have investigated the expression of COX-1 and COX-2 polypeptides in human colon cancer tissues using immunohistochemistry. Enhanced COX-2 expression was observed in colon cancer tissues from 15 subjects with clinically diagnosed colorectal cancer. Marked COX-2 expression was observed in cancer cells, inflammatory cells, vascular endothelium, and fibroblasts of the lesional tissues compared with the nonlesional and normal colon tissues. The extent and intensity of the immunoreactive COX-2 in cancer cells was much greater than that of the other cell types. In contrast, the expression of COX-1 polypeptide was weak in both normal and cancerous specimens. These data suggest that the enhanced expression of the COX-2 gene in colon cancer tissues may contribute to the enhanced synthesis of prostaglandin E2 by the colon cancer tissues. Enhanced expression of COX-2 may play a role in the pathogenesis of colon cancer. Furthermore, selective inhibition of COX-2 may prove to be more efficacious in the retardation of colon cancer development.

Sixteen polymorphic markers, including markers for eight new loci, forming three linkage groups, were assigned to rat Chromosome (Chr) 4 by linkage analysis of the progeny of an F2 intercross of Fischer (F344/N) and Lewis (LEW/N) inbred rats. One gene, Igk, was mapped by restriction fragment length polymorphism (RFLP) analysis. One marker for Tcrb was identified by the polymorphic insertion of a repetitive LINE element. The remaining 14 markers contained polymorphic simple sequence repeats (SSRs). Ten were identified in genes (Tgfa, Npy, Prss1, Prss2, Aldr1, Iapp, Prp, Eno2, Cacnl1a1, and Il6), one was identified in a sequence related to a gene (Egr4l1), and three were identified in anonymous DNA segments. The SSR markers were highly polymorphic in 16 inbred rat strains. These markers expand the genetic map of the rat and should be useful in future genetic studies of inbred rats.

Lewis (LEW/N) and Fischer (F344) rats are inbred strains that respond antithetically to administration of several inflammatory stimuli. Thus, in response to streptococcal cell wall-derived peptidoglycan/polysaccharide, 6-week-old female Lewis rats develop acute and chronic polyarthritis, whereas age- and sex-matched Fischer rats are arthritis-resistant. The susceptibility of Lewis rats to development of chronic severe inflammatory disease has been attributed to their inability to appropriately activate their hypothalamic-pituitary-adrenal axis in response to inflammatory stimuli, leading to a functional glucocorticoid deficiency. To investigate whether the acute neurogenic inflammatory response was also different in the two strains, we studied the air-pouch model of carrageenin-induced neurogenic inflammation in adult male Lewis and Fischer rats. Both the volume and the leukocyte concentration of the inflammatory exudate were significantly higher in Lewis than in Fischer rats, suggesting that the known differences in the handling of chronic inflammation between the two strains pertain to the acute neurogenic type of inflammation as well. To confirm that glucocorticoids play a major role in the differential response of the two strains to this inflammatory stimulus, we administered graded doses of the glucocorticoid agonist dexamethasone or antagonist RU 486 to both strains and examined their responses to concomitantly administered carrageenin. RU 486 increased, whereas dexamethasone decreased, the inflammatory response of Fischer and Lewis rats, respectively, to approach the magnitude of each other's natural response, suggesting that glucocorticoids are involved in this phenomenon as well. To rule out any differences in end-organ sensitivity to glucocorticoids between the two strains, we evaluated dose-response relations of whole body, thymus, spleen, and adrenal weights after 1 week daily administration of graded doses of dexamethasone. We found similar ED50 for both Lewis and Fischer rats. We conclude that the differences in the susceptibility to acute, carrageenin-induced, neurogenic inflammation between the LEW/N and F344 rat strains are similar to those of chronic inflammatory responses in these strains and likewise glucocorticoid-dependent. No apparent major differences exist in the sensitivity of target tissues to exogenous glucocorticoids between Lewis and Fischer rats.

Nine microsatellite loci were mapped to rat Chromosome (Chr) 7 by genetic linkage and somatic cell hybrid analysis. These loci include the gene encoding a member of the IID sub-family of cytochrome P450 (Cyp2d), a gene with repetitive sequences expressed during myotube formation (D7Arb1e), four anonymous loci, D7Arb81, D7Arb208, D7Arb569, D7Arb609a, and three DNA loci defined by MapPair markers R245, R513, and R1071. The nine loci were all identified by PCR-based microsatellite polymorphism analysis and were characterized in 40 F2 intercross progeny of Fischer (F344/N) and Lewis (LEW/N) rats for segregation analysis. These markers formed a single linkage group spanning 76.8 cM with the following order and distances: D7Arb569-11.4 cM-D7Arb81-9.7 cM-R513-2.6 cM-Cyp2d-0.0 cM-R245-1.3 cM-D7Arb1e-10.4 cM-R1071-15.9 cM-D7Arb609a-15.4 cM-D7Arb208. Physical mapping of Cyp2d by somatic cell hybrid analysis allowed us to assign this linkage group to rat Chr 7. For each marker, two to six alleles were detected in a panel of 16 inbred rat strains (ACI/N, BN/SsN, BUF/N, DA/Bkl, F344/N, LER/N, LEW/N, LOU/MN, MNR/N, MR/N, SHR/N, SR/Jr, SS/Jr, WBB1/N, WBB2/N, WKY/N).

Because rheumatoid arthritis rarely remits, refractory disease is common. Attention should be paid to aggravating comorbid conditions. Pharmacologic options currently available include cyclosporin, high-dose methotrexate, combination second-line agents, and creative use of corticosteroids. The available literature is reviewed in this article and the potential risks and benefits of the various options are discussed.

Five genes and three anonymous DNA loci were mapped to rat chromosome 6 by genetic linkage and somatic cell hybrid analyses. The eight loci were all identified by PCR-based microsatellite polymorphism analysis and were characterized in 40 F2 intercross progeny of Fischer (F344/N) and Lewis (LEW/N) inbred rats for segregation analysis. These markers formed two linkage groups spanning, respectively, 58.1 cM and 4.0 cM. The first linkage group is comprised of two anonymous DNA loci and four genes with the following map order and distances: D6Cep8 (previously D3)-17.9 cM-D6Arb309-2.5 cM-Vsnl1 (neural visinin-like protein)-20.4 cM-Prkar2b (type IIb regulatory subunit of cAMP-dependent protein kinase)-8.8 cM-Fkhl1 (forkhead-like transcription factor BF-1)-8.5 cM-Rnu1c (18-3A U1 RNA). The second linkage group is comprised of one gene, Ckb (creatine kinase, brain) and one anonymous DNA locus, D6Arb54, separated by 4.0 cM. For each marker, two to eight alleles were detected in a panel of 16 inbred rat strains (ACI/N, BN/SsN, BUF/N, DA/Bk1, F344/N, LER/N, LEW/N, LOU/MN, MNR/N, MR/N, SHR/N, SR/Jr, SS/Jr, WBB1/N, WBB2/N, and WKY/N). Comparative mapping information indicated that rat chromosome 6 exhibits syntenic conservation with mouse chromosome 12. Homologs of the rat chromosome 6 loci have been identified on human chromosomes 2, 7, and 14.

The concept of an integrated bidirectionally regulated neuroendocrine-immune adaptive response to stress has strong experimental support. The quality and intensity of this coordinated response to stress varies depending upon age, gender, reproductive status, and other genetically determined factors as well as the types and magnitudes of environmental challenges. These factors and dysfunctional communication between the nervous, endocrine, and immune systems appear to contribute to the development of autoimmune diseases in the Lewis and BB rats, the OS chicken, and the NOD, MRL, NZB, NZW, and NZB/NZW F1 mice. Neuroendocrine-immune dysfunction also contributes to the pathogenesis of human autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, thyroid diseases, and others. This review highlights these concepts. It includes discussions on various aspects of the stress response, the hypothalamic-pituitary-adrenal and -gonadal axes, corticotropin releasing hormone, luteinizing hormone releasing hormone, interleukin-1 and -6, corticosteroids, estrogens, testosterone, dehydroepiandrosterone, growth hormone, prolactin, and thyroid hormone. The role of the nervous and endocrine systems in regulating thymopoiesis and T cell development is also emphasized.

Objective: To examine basal and stimulated hypothalamic-pituitary-adrenal (HPA) axis and related hormone levels, including adrenocorticotropin (ACTH), cortisol, arginine vasopressin (AVP), and neuropeptide Y (NPY), in patients with fibromyalgia (FM)., Methods: Basal and ovine corticotropin-releasing hormone (oCRH)-stimulated HPA axis function were assessed in 12 patients with FM and in age- and sex-matched normal subjects. Basal plasma AVP levels and AVP release after postural change were assessed, and plasma NPY levels were measured in the same samples., Results: Patients with FM had low 24-hour urinary free cortisol, but normal peak and elevated trough plasma cortisol levels, compared with normal subjects. The net integrated ACTH response to oCRH in FM was not significantly different from that in normal subjects, but tended toward an exaggerated response. There was a significant decrease in net integrated cortisol response to oCRH in FM patients, indicating adrenal hyporesponsiveness. AVP levels were not significantly different between FM patients and control subjects, but variability was greater among the FM patients. Plasma NPY levels were significantly lower in FM patients than in normal subjects., Conclusion: These data support the view that HPA axis function is perturbed in patients with FM. Further study is required to ascertain the cause of HPA axis perturbations and their relationship to symptoms in patients with FM.

Eleven polymorphic DNA markers were mapped to rat Chromosome (Chr) 3 by linkage analysis of F2 progeny of F344/N and LEW/N rat strains. The markers, including seven genes and four anonymous loci, formed a single linkage group covering approximately 112 cM with the following order: Ptgs1 (prostaglandin G/H synthase I)-D3Arb178-Scn2a (sodium channel, type II, alpha-polypeptide)-D3Arb1-Cat (catalase)-Bdnf (brain-derived neurotropic factor)-D3Arb219-D3Arb2-Sus2 (seminal vesicle secretion II protein)-Sdc4 (ryudocan/syndecan4)-Stn1 (statin-like protein). Eight of these markers were analyzed for polymorphisms in 14 additional inbred rat strains. Three to five alleles were detected for each marker, suggesting that they are highly polymorphic and useful for genetic mapping studies with inbred rat strains. Chromosomal syntenic conservation among rats, mice and humans is also discussed.

Over the past year, convincing direct evidence that rheumatoid arthritis is caused by a retrovirus has not been presented. Strong support for this hypothesis, however, comes from an impressive body of recent work demonstrating that human T-cell lymphotropic virus type I causes a destructive arthropathy that has many of the same features as rheumatoid arthritis. Additional strong support for the hypothesis is provided by the resemblance of the arthropathies induced by caprine arthritis encephalitis and the ovine maedi-visna viruses to rheumatoid arthritis. These retroviral infections emphasize the potential complexity of detecting retroviral involvement and proving its importance in the causation of rheumatoid arthritis. Retroviruses clearly possess diverse pathways for involvement in autoimmune diseases.

Background/aims: Corticotropin-releasing hormone (CRH) is a regulator of the hypothalamic-pituitary-adrenal axis and a coordinator of the gastrointestinal response to stress. In addition to its central effects, CRH has peripheral effects on the immune system. CRH is present in several human tissues, such as the brain, spinal cord, adrenal medulla, lung, liver, peripheral blood leukocytes, as well as the gastrointestinal tract. The current study examined the local production of CRH in the normal human colon., Methods: Normal human colonic tissues obtained by endoscopic biopsy were immunostained with anti-CRH and anti-5-hydroxytryptamine antibody and analyzed for CRH messenger (m)RNA by a reverse-transcribed polymerase chain reaction method and by in situ hybridization., Results: Immunoreactive CRH and CRH mRNA were detected in the colonic mucosal cells in the neighborhood of the base of the crypts. The mucosal cells that expressed CRH mRNA also immunostained with anti-5-hydroxytryptamine antibody., Conclusions: Normal human colonic mucosal enterochromaffin cells produce CRH. CRH in the colonic mucosa may play a role in the modulation of the intestinal immune system and/or other gastrointestinal functions basally during stressful conditions.

High levels of immunoreactive cyclooxygenase (Cox; prostaglandin H synthase) are present in synovia from patients with rheumatoid arthritis (RA). We now show that the recently identified inducible isoform of Cox, Cox-2, is expressed in synovia from patients with RA. To further explore modulation of the Cox isoforms in RA synovial tissues, we examined the expression and modulation of Cox-1 and -2 in rheumatoid synovial explant cultures and cultured rheumatoid synovial fibroblast-like cells (synoviocytes). Immunoprecipitation of in vitro labeled proteins and Western blot analysis demonstrated the presence of both Cox-1 and -2 under basal conditions in freshly explanted rheumatoid synovial tissues. De novo synthesis of Cox-2 polypeptide was enhanced by IL-1 beta or PMA, and dramatically suppressed by dexamethasone (dex). Cox-1 expression, under the same conditions, showed only minor variation. Since mRNA for Cox-2 is highly unstable, we examined the regulation of Cox-2 transcripts in cultured rheumatoid synoviocytes. Under basal conditions both Cox-1 and -2 mRNAs were present at low levels, but Cox-2 mRNA was markedly increased by treatment with IL-1 beta or PMA. dex markedly suppressed the induction of Cox-2 mRNA. In sharp contrast, Cox-1 transcripts were not modulated by IL-1 beta or dex. These data suggest that modulation of Cox-2 expression by IL-1 beta and corticosteroids may be an important component of the inflammatory process in synovial tissues from patients with RA.

Two genes and two anonymous DNA loci were mapped to rat chromosome 11 using F2 intercross progeny of Fischer (F344/N) and Lewis (LEW/N) inbred rats. These four loci formed a single linkage group covering 21.5 cM with the following map order: somatostatin (SST)-D11N161-D11N18-cell surface protein (MOX2). These four loci were typed by PCR-based simple sequence repeat (SSR) length polymorphism detection. For each marker four to seven different alleles were detected using a panel of 13 inbred rat strains (F344/N, LEW/N, BN/SsN, BUF/N, LER/N, MR/N, MNR/N, LOU/MN, ACI/N, WBB1/N, WBB2/N, SHR/N, WKY/N). Comparative gene mapping analysis suggests syntenic conservation between rat chromosome 11 and mouse Chromosome 16.

Five genes and four anonymous polymorphic markers, forming two linkage groups, were mapped in F2 intercross progeny of F344/N x LEW/N rats using polymerase chain reaction (PCR) techniques. Both linkage groups were assigned to rat chromosome 8 because they contained genetic loci previously mapped to this chromosome. The first group was comprised of markers for three anonymous loci and two gene loci, thymus cell antigen-1 (Thy1) and tropoelastin (Eln). The second group was comprised of markers for one anonymous locus and three gene loci, cellular retinol binding protein II (Rbp2), matrin F/G (Matr1), and acyl-peptide hydrolase (Apeh). Seven markers (identified by simple sequence repeat associated length polymorphisms) were characterized in an additional 13 inbred rat strains (ACI/N, BN/SsN, BUF/N, LER/N, LOU/MN, MNR/N, MR/N, SHR/N, SR/Jr, SS/Jr, WBB1/N, WBB2/N, and WKY/N). Two to six alleles were detected for each marker. The reported markers should facilitate genetic mapping and monitoring of inbred rat strains.

Glucocorticoids are pleiotropic hormones that at pharmacologic doses prevent or suppress inflammation and other immunologically mediated processes. At the molecular level, glucocorticoids form complexes with specific receptors that migrate to the nucleus where they interact with selective regulatory sites within DNA; this results in positive and negative modulation of several genes involved in inflammatory and immune responses. At the cellular level, glucocorticoids inhibit the access of leukocytes to inflammatory sites; interfere with the functions of leukocytes, endothelial cells, and fibroblasts; and suppress the production and the effects of humoral factors involved in the inflammatory response. Clinically, several modes of glucocorticoid administration are used, depending on the disease process, the organ involved, and the extent of involvement. High doses of daily glucocorticoids are usually required in patients with severe diseases involving major organs, whereas alternate-day regimens may be used in patients with less aggressive diseases. Intravenous glucocorticoids (pulse therapy) are frequently used to initiate therapy in patients with rapidly progressive, immunologically mediated diseases. The benefits of glucocorticoid therapy can easily be offset by severe side effects; even with the greatest care, side effects may occur. Moreover, for certain complications (for example, infection diathesis, peptic ulcer, osteoporosis, avascular necrosis, and atherosclerosis), other drug toxicities and pathogenic factors overlap with glucocorticoid effects. Minimizing the incidence and severity of glucocorticoid-related side effects requires carefully decreasing the dose; using adjunctive disease-modifying immunosuppressive and anti-inflammatory agents; and taking general preventive measures.

IL-4 inhibits production of certain proinflammatory cytokines, including IL-1 beta, TNF-alpha, and IL-6, by activated monocytes. Although monocytes are a major source of IL-6, other cell types such as fibroblasts and endothelial cells can also express this cytokine. To determine whether IL-4 inhibits IL-6 expression in non-hemopoietic cells, we investigated the effects of IL-4 on IL-6 production in both primary human fibroblasts and fibroblast lines. Rheumatoid synovial fibroblasts were evaluated in these studies because, like monocytes, they produce high levels of IL-6 when stimulated with IL-1. Although peripheral blood monocytes did not constitutively express IL-6 mRNA or protein, stimulation with IL-1 or LPS induced de novo IL-6 expression in these cells. In contrast, synovial fibroblasts displayed a significant basal level of IL-6 production, which was markedly increased after stimulation with IL-1. IL-4 suppressed IL-6 expression in monocytes, but did not inhibit IL-6 production in synovial fibroblasts. The inability of IL-4 to suppress IL-6 synthesis in rheumatoid synovial fibroblasts was not caused by a lack of IL-4R and was not unique to these cells because IL-4 also failed to inhibit IL-6 production in normal fibroblast lines derived from other tissues. Inhibition of IL-6 production by IL-4 in monocytes was associated with decreased nuclear NF-kappa B levels. However, IL-4 does not globally suppress the activity of all DNA-binding proteins because IL-4 treatment did not reduce the levels of NF-IL-6 or NF-IL-1 beta B in the same cells. Because NF-kappa B activation is required for transcription of many cytokine genes, including IL-6, the ability of IL-4 to suppress NF-kappa B activity in monocytes suggests a potential mechanism by which this molecule may inhibit the expression of multiple cytokines.

Seven polymorphic markers comprising a single linkage group were assigned to rat Chromosome (Chr) 5 by linkage analysis of the progeny of an F2 intercross of Fischer (F344/N) and Lewis (LEW/N) inbred rats. Three genes, alpha-L-fucosidase 1 (FUCA1), mitochondrial superoxide dismutase (SOD2), and glucose transporter (GLUT1), were mapped by restriction fragment length polymorphism (RFLP) analysis. Two genes, glucose transporter (GTG3) and elastase II (ELAII), one pseudogene for alpha tubulin (TUBAPS), and one sequence related to the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFBP1-related sequence) were mapped by simple sequence repeat (SSR) polymorphism analysis. The loci are in the following order: SOD2, GTG3/GLUT1, FUCA1, ELAII/PFKFBP1-related sequence, and TUBAPS. This linkage group covered 68.3 cM of rat Chr 5. The SSR markers were highly polymorphic in 13 inbred rat strains (SHR/N, WKY/N, MNR/N, MR/N, LOU/MN, BN/SsN, BUF/N, WBB1/N, WBB2/N, ACI/N, LER/N, F344/N, and LEW/N). These markers, located on rat Chr 5, will be useful in genetic studies of inbred rats.

A genetic map of nine loci defined by polymorphic DNA markers was created using a single cross of F344/N and LEW/N rats. The markers contained polymorphic simple sequence repeats identified in five genes, renin (Ren), cardiac troponin T (Tnnt3), synaptotagmin (Syt2), Na+,K(+)-ATPase catalytic subunit (Atp1a2), and the Asp-, Gly-, Glu-, and Leu-tRNA gene cluster (Trnegl), as well as four anonymous DNA segments. Analysis of the segregation of the alleles of these markers in F2 intercross progeny of F344/N and LEW/N rats indicated the following locus order and distances between pairs of loci: D13N1-5 cM-Ren-1 cM-Tntt3-0 cM-Syt2-12 cM-D13N2-25 cM-Atp1a2-0 cM-Trnegl-7 cM-D13N3-4 cM-D13N4. Three of the loci, Ren, Trnegl, and Atp1a2, have previously been assigned to rat chromosome 13. Except for Ren, none of the loci have previously been mapped by linkage analysis. The markers for these loci were characterized in a total of 13 inbred rat strains (F344/N, LEW/N, LOU/MN, WBB1/N, WBB2/N, MR/N, MNR/N, ACI/N, SHR/N, WKY/N, BN/SsN, BUF/N, and LER/N) and were found to be highly polymorphic, with two to eight alleles detected for each marker. These markers expand the genetic map of the rat and should be valuable tools for future genetic studies. An examination of human and mouse comparative map information for all loci assigned to rat chromosome 13 shows significant synteny conservation with the q arm of human chromosome 1 and the distal portion of mouse chromosome 1.

Four PCR-typable polymorphic markers were mapped to rat chromosome 12 by linkage analysis of F2 intercross progeny of Fischer (F344/N) and Lewis (LEW/N) rat strains. The markers formed a single linkage group, covering 27.7 cM, with the following order and distance between markers: plasminogen activator inhibitor (Planh)--0.0 cM--phosphoenolpyruvate carboxykinase-related sequence 2 (Pepckr2)--15.4 cM--anonymous marker (D12N155)--12.3 cM--serine dehydratase (Sdh). All markers were identified and genotyped by PCR analysis of simple sequence repeats. The gene encoding Planh was previously assigned to rat chromosome 12, which allowed us to assign the entire linkage group to this chromosome. These markers were highly polymorphic in 13 additional inbred rat strains (BUF/N, BN/SsN, WKY/N, MNR/N, LER/N, WBB1/N, WBB2/N, MR/N, LOU/MN, SHR/N, ACI/N, SR/Jr, and SS/Jr). These markers should be useful tools for further genetic studies in rats.

Experimental autoimmune uveoretinitis (EAU) is a prototypic T cell-mediated autoimmune disease, whose target tissue is the neural retina, that is used as a model for a number of human blinding ocular diseases of a presumed autoimmune nature. EAU in rats can be induced by adoptive transfer of small numbers of retinal antigen-specific CD4+ T cell lines. Although recruitment mechanisms were assumed to play a role in the immunopathogenesis of uveitis, there is no direct evidence that would permit assessment of the importance of recruited non-antigen-specific T cells in retinal autoimmunity. In the present study, we addressed this question by using congenitally athymic Lewis rats (LEW.rnu/rnu), that are deficient in functional endogenous T cells, but are otherwise syngeneic with the euthymic Lewis rats that develop characteristically severe EAU. The uveitogenic stimulus was delivered in the form of phenotypically and functionally homogeneous pathogenic T cell lines, specific to the major pathogenic epitope of either the intracellular photoreceptor protein, S-Ag, or the extracellular photoreceptor matrix protein, IRBP. Depending on the T cell line used, EAU in athymic rats was either drastically reduced in severity (IRBP), or essentially absent (S-Ag). Susceptibility was restored when the athymic animals were reconstituted with immunocompetent T cells from syngeneic euthymic donors. While the intraocular infiltrate in euthymic rats was predominantly lymphocytic, with smaller numbers of monocyte/macrophages and even fewer neutrophils, the sparse infiltrate in athymics was largely monocytic, and with a relatively high proportion of neutrophils and eosinophils. Reconstituted animals had an intermediate histological picture with respect to the infiltrating cell types and disease severity. Our data are consistent with the interpretation that recruitment of naive T cells constitutes an amplification mechanism that is central to the expression and pathogenesis of uveitis. The extent of dependence on this phenomenon appears to be influenced by the antigenic specificity of the T cell line, and could be connected to the 'accessibility' of the target antigen in vivo.

Inflammation normally results in enhanced synthesis and secretion of hypothalamic corticotropin-releasing hormone (CRH) which, in turn, exerts antiinflammatory effects by virtue of increased adrenal glucocorticoid production. CRH and CRH binding sites are also expressed in the peripheral nervous and immune systems. Our groups have recently shown that CRH is secreted locally in acute carrageenin-induced inflammation in rats and has predominantly proinflammatory effects. We have also shown that CRH is expressed in the joints of Lewis rats with experimental arthritis. To determine if CRH is present in human inflammatory arthritis, we examined synovial fluids and tissues from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) and normal individuals. We found markedly enhanced expression of immunoreactive CRH in situ in synovium from patients, which was significantly greater in RA than in OA (p < 0.01). CRH concentrations were also significantly higher in RA (140 +/- 33 pg/ml, mean +/- SEM; n = 10) than OA (25 +/- 4 pg/ml; n = 6) synovial fluids (p < 0.005). HPLC showed immunoreactive CRH extracted from RA and OA synovial tissues and fluids coeluted with CRH 1-41. CRH mRNA was present in low levels in synovial tissue from patients with RA and, to a lesser extent, OA. In summary, immunoreactive CRH is locally secreted in the synovium of patients with RA and, at lower levels, OA. These data support the view that CRH functions as an autocrine and/or paracrine mediator of inflammation in humans.

To analyze the structure and formally ascertain the B-1a cellular origin of IgM rheumatoid factor (RF) autoantibodies, we generated 4 IgM RF mAb-producing cell lines using sorted (surface CD5+) B-1a cells from a patient with active rheumatoid arthritis. The RF mAb111, mAb112, mAb113, and mAb114 were monoreactive and displayed a relatively high affinity for human IgG Fc fragment (Kd, 3.1 x 10(-7) to 6.8 x 10(-7) M). The B-1a origin of the lymphocytes that gave rise to the IgM RF was confirmed by the expression of surface CD5 and specific CD5 mRNA by all mAb-producing cell lines. Analysis of the genes encoding the RF mAb VH and VL regions revealed that members of the VHI and VHIII families were utilized in conjunction with V kappa IIIa, V kappa IIIb, or V lambda I genes. JH3 and JH4 genes were each utilized twice. The H chain CDR3 sequences were divergent and variable in length. The RF mAb VH genes were identical or closely related to those expressed in the "restricted" fetal B cell repertoire and/or were JH-proximal. For instance, mAb111 VH gene likely constituted a mutated variant of the expressed fetal 20P3 which is the second most JH-proximal gene (125 kb from JH). In addition, the expressed VH and VL genes were among those that have been found to encode other RF, different autoantibodies, high affinity antibodies induced by exogenous Ag, and natural autoantibodies in the adult and neonatal B cell repertoires. When compared with those of known germline genes, the expressed V gene sequences displayed a number of differences. By cloning and sequencing DNA from PMN of the same patient whose B lymphocytes were used for the mAb generation, we showed that such differences resulted from somatic hypermutation in the RF mAb112 VH gene. The germline gene (112GL) that presumably gave rise to the RF mAb112 VH segment was identical to the expressed fetal 51P1 gene. The distribution and the high replacement to silent mutation ratio of the nucleotide mutations in RF mAb112 VH segment were highly consistent with their selection by Ag. RF mAb113 was clonally related to RF mAb112, as shown by the utilization of the same sets of VHI-D-JH4 and V kappa IIIb-J kappa 4 genes, displaying identical junctional sequences, and the presence of two identical replacement and one silent mutations.(ABSTRACT TRUNCATED AT 400 WORDS)

Lewis (LEW/N) and Fisher (F344/N) rats are histocompatible inbred strains characterized respectively by susceptibility and resistance to inflammatory disease. LEW/N rats have deficient corticotropin-releasing hormone (CRH), ACTH and corticosterone responses to inflammation, and increased circulating and hypothalamic concentrations of arginine vasopressin (AVP). CRH is produced locally at inflammatory sites, where it acts as a proinflammatory agent, while AVP has been reported to exert immunopotentiating effects in vivo and in vitro. In order to further investigate the mechanism of increased AVP secretion in LEW/N rats, we measured AVP and CRH mRNA in several hypothalamic nuclei in LEW/N and F344/N rats, using in situ hybridization histochemistry. LEW/N rats had increased AVP mRNA concentrations in the supraoptic (SON) and paraventricular (PVN), but not in the suprachiasmatic (SCN) nuclei. CRH mRNA, on the other hand, was decreased in the PVN of LEW/N rats. To examine the potential role of AVP and CRH in the exaggerated inflammatory responses of LEW/N rats, we pretreated young female Lewis and Fischer rats with AVP- and/or CRH-neutralizing rabbit antisera and elicited subsequently an inflammatory response by a nuchal subcutaneous injection of carrageenin. We demonstrated that both antisera decreased significantly the leukocyte concentration in the inflammatory exudate in LEW/N rats, but found no synergistic or addictive effects between them. We conclude that previously observed differences in hypothalamic AVP and CRH contents between LEW/N and F344/N rats correspond to differences in the steady state mRNA levels of the two neuropeptides and that both AVP and CRH participate in the excessive inflammatory response of Lewis rats as locally active proinflammatory agents.

Twelve polymorphic markers identified by restriction fragment length polymorphism (RFLP) analysis or simple sequence repeat (SSR) polymorphism analysis were assigned to rat chromosome 1 by linkage analysis of F2 intercross progeny of F344/N and LEW/N inbred rat strains. One linkage group, covering 46.3 cM, consisted of eight markers including five genes, TNT (fast skeletal troponin T), IGF2 (insulin-like growth factor 2), MYL2 (MLC2 gene for muscle myosin light chain 2), ALDOA (aldolase A), and HBB (hemoglobin beta-chain); one anonymous locus, D1N64; one marker related to the carboxypeptidase B gene, CARB07-related sequence; and one marker related to the parathyroid hormone gene, PTH-related sequence. A second linkage group, covering 45.0 cM, consisted of three markers including two anonymous loci, 2B1 and D1N40, and one gene, TCP1 (T-complex 1). INS1 (insulin 1), which has been previously assigned to rat chromosome 1, was not linked to these markers. The SSR markers were highly polymorphic in 13 inbred rat strains (SHR/N, WKY/N, MNR/N, MR/N, LOU/MN, BN/SsN, BUF/N, WBB1/N, WBB2/N, ACl/N, LER/N, F344/N, and LEW/N). These markers, located on chromosome 1, will be useful in genetic studies in rats.

Fibroblast growth factor (FGF)-1 and PDGF-B-like factors have been implicated in the pathobiology of RA and animal models of this disease. Since the receptors for FGF-1 and PDGF are tyrosine kinases, we examined the expression of tyrosine phosphorylated proteins (phosphotyrosine, P-Tyr) in synovial tissues from patients with RA and osteoarthritis (OA), and rats with streptococcal cell wall (SCW) and adjuvant arthritis (AA). Synovia from patients with RA and LEW/N rats with SCW and AA arthritis, in contrast to controls, stained intensely with anti-P-Tyr antibody. The staining colocalized with PDGF-B and FGF-1 staining. Comparative immunoblot analysis showed markedly enhanced expression of a 45-kD P-Tyr protein in the inflamed synovia. Treatment with physiological concentrations of dexamethasone suppressed both arthritis and P-Tyr expression in AA. P-Tyr was only transiently expressed in athymic nude Lewis rats and was not detected in relatively arthritis-resistant F344/N rats. These data suggest that (a) FGF-1 and PDGF-B-like factors are upregulated and may induce tyrosine phosphorylation of proteins in vivo in inflammatory joint diseases, (b) persistent high level P-Tyr expression is T lymphocyte dependent, correlates with disease severity, and is strain dependent in rats, (c) corticosteroids, in physiological concentrations, downregulate P-Tyr expression in these lesions.

The nude trait in the rat is transmitted in an autosomal recessive manner and is associated with thymic aplasia, T-cell deficiency, and hairlessness. Congenic rats homozygous for the RNU (Rowett nude) locus are important models in the study of inflammatory disease, tumor growth, and transplant rejection. The RNU locus has not been previously mapped, and the nature of the gene product is unknown. To determine the map location of this gene, a single F344.rnu/rnu (athymic nude congenic Fischer rat) male congenic rat was bred with 3 LEW/N (NIH stock Lewis rat) female rats to produce F1 progeny. Twelve F1 brother-sister breeding pairs were established. Forty-nine phenotypically nude F2 offspring (198 total) were obtained. Linkage analysis done on F2 DNA revealed highly significant cosegregation between the nude phenotype and eight polymorphic markers located on Chromosome (Chr) 10. The tightest linkages were with: MYH3 (embryonic, skeletal myosin heavy chain) and SHBG (sex hormone-binding globulin), giving 2 point lod scores of 20.2, and 20.0, respectively. The map order and map distances, determined by multipoint linkage calculations, were: RR24-(16.1 cM)-MYH3-(3.5 cM)-SHBG-(4.7 cM)-RNU-(11.9 cM)-F16F2-(24.1 cM)-CLATP (citrate lyase ATPase)-(2.4 cM)-ACE (angiotensin converting enzyme)/PPY (pancreatic polypeptide)-(14.1 cM)-RR1023. The position of the RNU locus in the rat corresponds closely with that of the recently reported nu locus in the mouse. This finding suggests that the nude phenotype in the rat and the mouse arise from defects in homologous genes.

Seven polymorphic markers identified by polymerase chain reaction (PCR) amplification, including markers for six genes--DRD1L (dopamine receptor, D1-like-2), GLUKA (glucokinase), PF4 (platelet factor 4), ALB (albumin), AFP (alpha-fetoprotein), and BSP (bone sialoprotein)--and one anonymous locus (D14N52), were mapped to a single 67-cM linkage group with F2 intercross progeny of F344/N and LEW/N inbred rat strains. Two of these markers, ALB and AFP, have previously been assigned to rat Chromosome (Chr) 14, allowing assignment of this entire linkage group. Five of the markers--DRD1L, PF4, ALB, AFP, and BSP--have been physically mapped to a large region of human Chr 4 encompassing the p arm and the q arm to band q28. Homologs of two of the markers, ALB and AFP, have been mapped to Chr 5 in the mouse. Comparison of human Chr 4 with the homologous regions on Chr 14 of the rat and Chr 5 of the mouse indicated that linkage conservation with human Chr 4 extends over a greater region in the rat than in the mouse. The markers described here were found to be highly polymorphic in twelve inbred strains (F344/N, LEW/N, ACI/N, BUF/N, BN/SsN, LOU/MN, MNR/N, MR/N, SHR/N, WBB1/N, WBB2/N, and WKY/N). These polymorphic markers should be useful in genetic linkage studies of important phenotypes in rats.

A genetic linkage map of seven polymorphic markers was created with F2 intercross progeny of F344/N and LEW/N rats and assigned to rat Chromosome (Chr) 18. Five of the markers described were defined by simple sequence length polymorphisms (SSLPs) associated with five genes: transthyretin (TTR), trypsin inhibitor-like protein (TILP), beta 2 adrenergic receptor (ADRB2), olfactory neuron-specific G protein (OLF), and gap junction protein (GJA1). One marker was defined by a restriction fragment length polymorphism (RFLP) detected with a probe for the human colony stimulating factor 1 receptor (CSF1R) gene. The D18N1R locus was defined by an anonymous DNA fragment amplified by the randomly amplified polymorphic DNA (RAPD) technique with a single short primer. These seven DNA loci formed a single genetic linkage group 30.4 cM in length with the following order: TTR-6.8 cM-D18N1R-9.1 cM-TILP-4.3 cM-CSF1R-0 cM-ADRB2-10.2 cM-OLF-0 cM-GJA1. The five SSLP markers were highly polymorphic. In a total of 13 inbred rat strains analyzed (F344/N, LEW/N, LOU/MN, WBB1/N, WBB2/N, MR/N, MNR/N, ACI/N, SHR/N, WKY/N, BN/SsN, BUF/N, and LER/N), three to six alleles were detected for each marker. Remarkable linkage conservation was detected between the region of rat Chr 18 mapped and a region of mouse Chr 18. However, genes associated with these markers have been mapped to three different human chromosomes (Chrs 5, 6, and 18). The markers described here should be useful for genetic mapping studies and genetic monitoring of inbred rat strains.

Hypothalamic secretion of corticotropin releasing hormone (CRH) triggers the secretion of pituitary ACTH and antiinflammatory adrenal corticosteroids. CRH is also secreted in local inflammatory sites such as the arthritic joint, but its action appears to be proinflammatory. This review notes the complex interrelationships of the nervous, endocrine, immune and inflammatory systems and suggests that abnormalities in these interactions may play a role in susceptibility to rheumatoid arthritis and various animal models of autoimmune diseases.