Several Strains Of Mice Research Articles

Lessons from animal models of vasculitis.

Vasculitis can occur as a primary disease or as a secondary manifestation of either another illness or a type-III hypersensitivity response to a foreign antigen. Over the past four decades, a number of animal models of vasculitis have been described. These models have served as important tools for enhancing our understanding of the basic mechanisms underlying the pathogenesis of vasculitis. In addition, animal models have made possible the preclinical testing of new therapeutic agents. Animal models of vasculitis can be broadly classified into two types--those that are experimentally induced and those that occur spontaneously. Vasculitis can be experimentally induced in animals through the stimulation of a type-III hypersensitivity response to a variety of foreign antigens, by viral or bacterial infection of vascular cells and the immune response to that infection, or by the in-vivo administration of antineutrophil cytoplasmic antibodies, estrogen, or mercuric chloride (HgCl(2)). Systemic vasculitis spontaneously develops in several strains of mice and rats. This paper reviews the current state of knowledge of several animal models of vasculitis and the lessons that have been learned from them.

Developmentally regulated gene expression of Th2 cytokines in the brain

Given the critical role of cytokines in the regulation of an inflammatory response, we investigated whether certain cytokines are expressed in the brains of normal mice during maturation that could contribute to the immune-privileged nature of the CNS or potentially influence an immune-mediated illness such as experimental allergic encephalomyelitis. The gene expression of IFNγ (Th1 cytokine) and IL-4 (Th2 cytokine) was analyzed in the brain of several strains of mice. IFNγ was not detectable. However, IL-4 was present in the brains of neonatal mice, but not adult mice. Resident CNS cells are believed to be the source of the IL-4, because mice deficient in T cells (SCID and RAG2−/−) expressed the IL-4 gene in the CNS. Further analysis indicated that the gene expression of the Th2 cytokine transcription factor, GATA-3, correlated with IL-4 and IL-10 expression in the brain. Since GATA-3-deficient mice have an abnormal CNS, brain-derived Th2 cytokines may play an important role in CNS development, as well as potentially contribute to the immune-privileged nature of the brain.

Establishment of monoclonal anti-retroviral gp70 autoantibodies from MRL/lpr lupus mice and induction of glomerular gp70 deposition and pathology by transfer into non-autoimmune mice.

Several strains of mice, including MRL/MpJ mice homozygous for the Fas mutant lpr gene (MRL/lpr mice), F(1) hybrids of New Zealand Black and New Zealand White mice, and BXSB/MpJ mice carrying a Y-linked autoimmune acceleration gene, spontaneously develop immune complex-mediated glomerulonephritis. The involvement of the envelope glycoprotein gp70 of an endogenous xenotropic virus in the formation of circulating immune complexes and their deposition in the glomerular lesions have been demonstrated, as has the pathogenicity of various antinuclear, antiphospholipid, and rheumatoid factor autoantibodies. In recent genetic linkage studies as well as in a study of cytokine-induced protection against nephritis development, the strongest association of serum levels of gp70-anti-gp70 immune complexes, rather than the levels of antinuclear autoantibodies, with the development and severity of glomerulonephritis has been demonstrated, suggesting a major pathogenic role of anti-gp70 autoantibodies in the lupus-prone mice. However, the pathogenicity of anti-gp70 autoantibodies has not yet been directly tested. To examine if anti-gp70 autoantibodies induce glomerular pathology, we established from unmanipulated MRL/lpr mice hybridoma clones that secrete monoclonal antibodies reactive with endogenous xenotropic viral env gene products. Upon transplantation, a high proportion of these anti-gp70 antibody-producing hybridoma clones induced in syngeneic non-autoimmune and severe combined immunodeficiency mice proliferative or wire loop-like glomerular lesions. Furthermore, deposition of gp70 in glomeruli and pathological changes were observed after intravenous injection of representative clones of purified anti-gp70 immunoglobulin G, demonstrating pathogenicity of at least some anti-gp70 autoantibodies.

Systemic T Helper 2 Cell Activation Is Not Sufficient for Antigen-Induced Eosinophil Recruitment into the Airways

Background: Airway eosinophilic inflammation is a characteristic feature of asthma. We have previously shown that antigen-induced eosinophil recruitment into the airways of sensitized BALB/c mice is mediated by CD4+ T cells and IL-5. However, the basis for the eosinophil recruitment into the airway are still largely unknown. Methods: To determine the regulatory mechanisms that control the magnitude of antigen-induced eosinophilic inflammation in the airways, we analyzed antigen-induced eosinophil and T cell infiltration into the trachea in several strains of mice, including BALB/c (H-2^d), BALB/b (H-2^b), C57BL/6 (H-2^b), C57BL/10 (H-2^b), and B10.D2 (H-2^d) mice. In addition, cytokine production from antigen-stimulated splenocytes and the titer of antigen-specific IgE antibody in serum were assessed. Results: Antigen-induced eosinophil recruitment into the trachea in BALB/c mice was more than 20 times as abundant as that in C57BL/6 mice, the latter of which was also mediated by CD4+ T cells. In contrast, systemic Th2 responses, which were evaluated by the titers of antigen-specific IgE antibody in serum and antigen-induced IL-4 and IL-5 production from splenocytes, were similarly observed in BALB/c mice and C57BL/6 mice. In addition, the antigen-induced eosinophil recruitment into the trachea was high in BALB/b mice, like BALB/c mice, but low in B10.D2 mice. Conclusions: Systemic Th2 responses are not sufficient for causing antigen-induced eosinophil recruitment into the airways and an undefined determinant(s) that exists in the BALB background mice rather than the H-2 haplotype is vital for the regulation of antigen-induced eosinophil recruitment into the airways.

393. Birth complications and dopamine function in animal models: implications for schizophrenia

Schizophrenia is associated with increased birth complications and altered mesocorticolimbic dopamine (DA) transmission, while stress also influences expression of psychotic symptoms. Our laboratory has used animal models to directly test if minor birth complications [Caesarean section (C-section) birth with or without a short period of apoxia] have long-term effects on DA function. At adulthood, Sprague-Dawley rats that had been born by C-section show 1) increased DA levels in nucleus accumbens (NAcc) and decreased DA in prefrontal cortex, 2) increased amphetamine-induced locomotion, 3) increased NAcc DA release in response to repeated stress and 4) increased D1 receptor levels in NAcc and infralimbio cortex, compared to vaginally born controls. Interestingly, stress at adulthood upregulated D3 receptors in vaginally born but not C-sectioned animals, and upregulated D4 receptors with C-section but not vaginal birth. Thus the individual’s previous experience of stress together with other environmental events in their history such as birth complications, are important in determining DA receptor levels. In contrast to C-section alone, rats born by C-section with added anoxia showed increased amphetamine-induced locomotion and stress-induced DA release but no change in brain DA levels or DA receptors, compared to vaginal birth. Overall, these findings highlight the sensitivity of dopaminergic systems to minor changes in birth procedure in the rat. The rat brain is somewhat less mature at birth than is the brain of the term human newborn, but guinea pig brain at birth is, in fact, more mature than the brain of the term human newborn. Similar to rats, guinea pigs born by C-section showed increased amphetamine-induced locomotion at adulthood, compared to vaginally born controls. Thus C-section birth enhances dopaminergic function in both guinea pig and rat, 2 species whose levels of CNS maturity span that of the human at birth insult. To test if genetic factors and birth complications interact to modulate dopaminergic function, we compared 3 genetically diverse strains of rats, born either vaginally or by C-section, for DA-mediated behavior. C-section birth had differing long-term effects on DA-mediated behavior depending on the animal’s genetic composition. This supports the idea that perinatal insults may have more profound effects on genetically susceptible individuals, resulting in DA dysfunction characteristics of schizophrenia. We are currently modelling similar birth complications in several strains of mice, to proceed towards quantitative trait locus analysis for genes conferring enhanced susceptibility of DAergic systems to birth insult. The epidemiology of schizophrenia provides us with leads about which factors might play a role in the etiology of the disorder. The present findings illustrate how animal models can be used to directly test if candidate etiological factors actually produce CNS changes responsible for symptoms of schizophrenia.

Biochemical Characterization of Endogenously Formed Eosinophilic Crystals in the Lungs of Mice

Crystals seldom form spontaneously within tissues of mammals, except in the urinary tract or in association with eosinophil-rich diseases in humans (Charcot-Leyden crystals). Endogenously formed eosinophilic crystals have been reported in respiratory tract and other tissues of several strains of mice, but the biochemical characterization of these crystals has not been reported. In this study, eosinophilic crystal formation was examined in homozygous C57BL/6J viable motheaten mice, lung-specific surfactant apoprotein C promoter/soluble human tumor necrosis factor p75 receptor type II fusion protein transgenic mice (C57BL/6NTac x Sv/129), and CD40L-deficient mice with spontaneous Pneumocystis carinii infection. In viable motheaten but not wild type mice, rapidly developing crystals represented a major feature of the fatal lung injury induced by macrophage dysregulation. Conversely, eosinophilic crystals did not form until 4-8 months of age in transgenic and CD40L-deficient mice and were present in 10-30% of age-matched wild type controls. Mass spectrometry analysis of proteins from bronchoalveolar lavage fluid identified the crystals as Ym1, sometimes referred to as T-lymphocyte-derived eosinophil chemotactic factor. The Ym1 sequence was homologous to chitinase, and enzymatic assays indicated a 3-5-fold increase in chitinase activity compared with control mice. Intracellular and extracellular crystals associated with epithelial damage suggested that the crystals may contribute to lung inflammation through mechanical damage and enzymatic degradation.

Identification of a Common Site of Provirus Integration in Radiation Leukemia Virus-Induced T-Cell Lymphomas in Mice

The BL/VL3 Kaplan radiation leukemia virus (RadLV-VL3) is a nondefective retrovirus that induces T cell lymphomas in several strains of mice. By using DNA probes derived from RadLV/VL3 provirus-flanking sequences cloned from the BL/VL3 cell line, we identified a DNA region rearranged in 5 of 19 tumors analysed (25%). All proviruses were integrated in the same 5′-to-3′ orientation in a small DNA region called Kis1 (Kaplan integration site 1). This region was localized on distal mouse chromosome 2 in a region not previously identified as important to lymphomagenesis. The cells rearranged at the Kis1 locus represent a clonal subpopulation of the clonal tumor masses examined, indicating a probable role of Kis1 in tumor progression.

Pathology, diagnosis and epidemiology of the rodent Helicobacter infection

Since the first isolation of Helicobacter pylori from humans in 1983, 18 Helicobacter species have been identified during the last decade in domestic and laboratory animals. Several Helicobacter species have been isolated from the gastrointestinal tracts of various mammalian species and birds. Helicobacter hepaticus, H. muridarum, H. bilis, H. rodentium and Flexispira rappini have been isolated from mice. Among these species, only H. hepaticus has been clearly recognized as a pathogen. Indeed, it displays the pathogenic potential to elicit hepatitis in several strains of mice; moreover in A/JCr mice, it is strongly associated with hepatic cancer. Among the five murine helicobacter species, apart from H. hepaticus, F. rappini has not been found associated with lesions, H. muridarum has been observed in gastric glands of mice with chronic gastritis, and H. bilis has been reported in the liver of mice with chronic hepatitis. When associated with H. rodentium, H. bilis is able to induce diarrhea in SCID mice. In no case has pathogenicity of a single species been clearly proven. In rats, H. trogontum and H. muridarum have been isolated from the intestine, without any information concerning their respective pathogenicity. H. cinaedi and H. cholecystus have been identified from the intestine and the gallbladders of hamsters, respectively. The diagnosis of Helicobacter species by polymerase chain reaction (PCR) is a rapid, specific and sensitive technique. One of the most promising diagnostic techniques of these infections seems to be the PCR detection of Helicobacter sp. from feces based on the 16S rRNA sequences, then a restriction enzyme analysis to identify the actual species. Several drug regimens have also been evaluated to eradicate H. hepaticus from mice. Helicobacter infections, particularly H. hepaticus and H. bilis, seem to be widespread in laboratory mouse colonies and have also been detected from commercial breeders.

NKT Cell Cytokine Imbalance in Murine Diabetes Mellitus

A minor subset of murine MHC class I-restricted T cells which express both the a/3 form of the T cell receptor and a NK lineage marker, termed NKT cells, is capable of secreting significant amounts of InterIeukin-4 and Interferon-γ upon activation. As such NKT cells may play a role in development of Thl and Th2 cells during T cell ontogeny or expansion of T cells expressing a dominant cytokine pattern in the effector phase. We have studied the role of NKT cells in a murine model of disease multidose streptozotocin induced diabetes mellitus (MDSDM). In MDSDM thymic and splenic NKT cells are present at normal levels but have greatly reduced capacity to secrete Interleukin-γ upon stimulation with anti-TCR antibody compared to control mice; conversely, Interferon-γ - secretion is maintained. By analysis of cytokine RNA production we found that treatment of several strains of mice with streptozotocin changes the peripheral helper T cell phenotype elicited after immunization with Keyhole Limpet Hemocyanin from a mixed Thl- and Th2-type cytokine pattern (characterized by IFN-γ and 1L-4 and IL-5 expressions, respectively) to predominately Thl-type. Furthermore, susceptibility to MDSDM is significantly enhanced when NKT cells are selectively eliminated in vivo by administration of depleting anti-CDl 22 antibody TMβ-1. In addition, antibody depletion of NKT cells from non-obese diabetic mice significantly accelerates onset of disease. Collectively these data support a model for development of murine diabetes mellitus in which NKT cell cytokine expression influences the development of Thl-type diabetogenic T cells.

Regulation of B and T cell development by anterior pituitary hormones.

Hormones produced by the anterior pituitary gland have been implicated in the regulation of primary lymphocyte development. In order to identify endocrine factors involved in that process, several strains of mice with genetic defects resulting in a selective impairment in the production of one or more anterior pituitary-derived hormones have been analysed. This study has resulted in the classification of endocrine hormones into the following four categories: (i) hormones such as prolactin with no apparent effects on primary lymphopoiesis; (ii) anabolic hormones such as growth hormone and insulin-like growth factor-I whose stimulatory effects on primary lymphopoiesis are non-lineage-specific and related to their actions as systemic mediators of growth and/or differentiation; (iii) hormones such as thyroid hormones that have an obligate role in primary B lymphopoiesis; and (iv) hormones such as oestrogens that act as negative regulators of lymphopoiesis.

Developmental studies of Brca1 and Brca2 knock-out mice.

In humans, the inheritance of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 increases the risk of developing breast and ovarian cancer. To study their biological function and to create animal models for these cancer susceptibility genes, several strains of mice mutated in the homologous genes Brca1 and Brca2 have been generated by gene targeting. Analyses of these "knock-out" mouse mutants have provided invaluable knowledge about the function of these genes. Brca1 and Brca2 null mutants are similar in phenotype: mutations in both genes result in embryonic lethality and the developing embryos show signs of a cellular proliferation defect associated with activation of the p53 pathway. The significance of this activation, as well as the role of these cancer susceptibility genes in DNA damage repair, is discussed.

Genetic background effects on dental and other craniofacial abnormalities in homozygous small eye (Pax6Sey/Pax6Sey) mice.

Small eye (Pax6Sey) is a semi-dominant mutation affecting development of the eyes, brain and nasal structures. The mutant phenotype arises from defects within the Pax6 gene and several mutant alleles have been identified. A previous study reported that Pax6Sey/Pax6Sey homozygotes, in a random-bred stock, had a median cartilaginous rod-like structure in the nasal region and 80% had supernumerary upper incisor teeth. In this study we show that supernumerary upper incisor teeth and a previously unreported nasal capsule-derived cartilaginous 'spur' occur in compound heterozygous Pax6Sey-Neu/Pax6Sey and homozygous Pax6Sey/Pax6Sey fetuses from several strains of mice. The frequencies of the abnormal phenotypes were not related to allele type but showed variable penetrance, which was dependent on genetic background. The median nasal cartilaginous rod-like structure was present in all homozygous small eye fetuses. The Pax6Sey/Pax6Sey homozygote may provide insight into the complex gene interactions involved in eye, nasal and craniofacial morphogenesis.

Genetic control of immune responses to HIV-1 env DNA vaccine.

We investigated the genetic control of immunoglobulin production and the delayed-type hypersensitivity (DTH) response produced by an HIV-specific DNA vaccine using several strains of mice. Murine antigen-specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4+8- and Ia- T cells. However, CD8+ T cells did not suppress footpad swelling. There was no difference in the induction of HIV-specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV-specific DNA vaccine is useful for immunizing various populations against HIV-1.

Myelin oligodendrocyte glycoprotein: a novel candidate autoantigen in multiple sclerosis.

Myelin oligodendrocyte glycoprotein (MOG) is a member of the immunoglobulin superfamily expressed exclusively in central nervous system (CNS) myelin. While the function of MOG is unknown, a number of studies have shown that immune responses to MOG contribute to the autoimmune-mediated demyelination seen in animals immunized with whole CNS tissue. This paper summarizes our recent studies, which unequivocally demonstrate that MOG by itself is able to generate both an encephalitogenic T cell response and an autoantibody response in Lewis rats and in several strains of mice. In Lewis rats the injection of both native MOG and MOG35-55 peptide produces a paralytic relapsing-remitting neurological disease with extensive plaque-like demyelination. The antibody response to MOG35-55 was highly restricted, as no reactivity to either other MOG peptides or myelin proteins could be detected. Fine epitope mapping showed that antibody from serum and cerebrospinal fluid of injected rats reacted strongly to MOG37-46, which is contiguous to the dominant T cell epitope contained within MOG44-55. NOD/Lt and C57BL/6 mice were also susceptible to severe neurological disease following injection with recombinant MOG or MOG35-55 peptide, indicating that this specific CNS autoantigen, or some of its determinants, can induce a pathogenic response across animal species. Severe paralysis and extensive demyelination were seen in both strains, but NOD/Lt mice experienced a chronic relapsing disease whereas C57BL/6 mice had a chronic non-remitting disease. Moreover, transfer of MOG35-55 T cells into naive NOD/Lt mice also produced severe neurological impairment as well as histological lesions. These results emphasize that a synergism between a T cell-response and anti-MOG antibodies may be important for the development of severe demyelinating disease. This, together with our demonstration that there is a predominant T cell response to MOG in patients with multiple sclerosis, clearly indicates that MOG is probably an important target autoantigen in this disease.

Characterisation of two promoters for prion protein (PrP) gene expression in neuronal cells

The neuronal membrane protein, PrP, has a key role in the development of the transmissible spongiform encephalopathies and the level of expression of the PrP gene has been shown to affect the disease profile. In order to define the sequences that are responsible for the normal expression of the PrP gene we have isolated and sequenced a 5′ region of the murine PrP gene, which includes 1.2 kb upstream from exon 1, intron 1 and exon 2. Sequencing of this region from several strains of mice identified a polymorphism linked to Sinc, the gene controlling the incubation period of scrapie in mice. We used this gene fragment and deletions of it to examine promoter mediated expression of a cat (chloramphenicol acetyl transferase) reporter gene in neuroblastoma cells (N 2a). Both promoter and suppressor elements were identified within this region. The two major areas of promoter activity were sequences adjacent to and 5′ to exons 1 and 2. The 5′ region of intron 1 was shown to contain elements that were capable of suppressing promoter activity. Transcription factor binding sites have been identified within these sequences.

Hyperproliferation of BXSB B Cells Is Linked to theYaaAllele

Systemic lupus erythematosus is characterized by polyclonal B cell activation, the production of autoantibodies, and often by renal disease. Previous studies demonstrated that unfractionated B cells from several strains of mice with lupus hyperproliferate in culture when stimulated with lipopolysaccharide (LPS) or anti-IgM. We wished to further examine proliferation of resting B cells from the BXSB mouse model of lupus and mice with theYaaallele, when activated with a number of stimuli. Our work demonstrates that: (1) resting B cells from mice containing theYaaallele hyperproliferated compared to that seen with B cells from mice lacking theYaaallele, (2) this hyperproliferation occurred whether cells were stimulated with phorbol myristate acetate/ionomycin, LPS, anti-IgM, or CD40L cross-linking, (3) this hyperproliferation is specific to B and not T cells. Taken together these data suggest that one mechanism by which theYaaallele contributes to the accelerated onset of lupus in BXSB male mice is through its influence on B cell activation.

A new Borrelia species isolated from patients with relapsing fever in Spain

Lyme disease and tick-borne relapsing fever are worldwide systemic borrelioses caused by several Borrelia species transmitted by hard ticks (family Ixodidae) and soft ticks (family Argasidae), respectively. A previous seroepidemiological study of Lyme borreliosis showed several serologically reactive patients with clinically atypical presentations, and this discovery led to the hypothesis that some of the cases of Lyme borreliosis had been caused by another borrelia organism. Blood from patients in southern Spain who had suspected Lyme disease or relapsing-fever borreliosis was cultured before treatment began. Isolates of Borrelia spp were inoculated into several strains of mice of different ages. The 16S rRNA and flagellin in genes of Borrelia spp were sequenced by PCR and assessed by phylogenetic analyses. We isolated a species of Borrelia from three patients with relapsing fever and from Ornithodorus spp ticks in southern Spain. This organism (refractory to in-vitro cultivation) caused a relapsing spirochaetaemia with multiple organ involvement in laboratory mice that recreated the human disease. Phylogenetic analysis showed that this organism is a previously unrecognised species. We have discovered a new borrelia pathogen that is closely related to the other tick-borne agents of relapsing fever in Europe and Africa, and which causes a relapsing systemic disease with serological similarities to Lyme borreliosis.

Interleukin-12 induces cytotoxic NK1+ alpha beta T cells in the lungs of euthymic and athymic mice.

We recently reported that interleukin-12 (IL-12) stimulated hepatic NK1.1 Ag+ alpha beta T cells with intermediate T-cell receptor (TCR; NK1+ TCRint cells) and enhanced their NK1 expression (NK1high TCRint), and that these cells acquire strong major histocompatibility complex (MHC) unrestricted cytotoxicity in C57BL/6 mice, both +/+ and nu/nu. In the present study, we find that although murine lung normally has few NK1+ TCRint cells, NK1high TCRint cells are induced in+/+ and nu/nu mice after systemic administration of IL-12; these cells exhibit strong MHC unrestricted cytotoxicity against NK-sensitive and -resistant targets. A small number of NK1high TCRint cells was also found in peripheral blood after increased amounts of IL-12 were administered. Cytotoxicity tests in vitro revealed that the cytotoxic activity of the lung mononuclear cells (MNC) of C57BL/6 mice induced by IL-12 was abrogated by the depletion of either NK1+ or CD3+ cells, but not of CD8+ cells, as reportedly was the case of hepatic MNC, suggesting that NK1high TCRint cells are an antimetastatic population not only in the liver but also in the lung of mice. IL-12 injection into mice markedly elevates serum interferon-gamma (IFN-gamma) levels. However, although IL-12-induced cytotoxicity of NK1high TCRint cells was significantly reduced by anti-IFN-gamma antibody injection (which decreased serum IFN-gamma to an undetectable level), the appearance of NK1high TCRint cells in the lung and liver was not so affected. These results suggest that IFN-gamma is an important mediator of the cytotoxicity of NK1high TCRint cells but is not an essential factor for induction of these cells. We also added data showing that IL-12 has a broad antimetastatic effect against various liver and lung metastatic tumours intravenously injected into several strains of mice, including NK-deficient bg/bg mice. It can be considered that, in addition to NK cells, CD8+ cytotoxic T cells and gamma delta T cells, NK1+ TCRint cells can be categorized as one of the cytotoxic effector populations. These novel type cells distinct from regular T cells may play an important role in monitoring intra- and perivascular areas.

The murine VpreB1 and VpreB2 genes both encode a protein of the surrogate light chain and are co‐expressed during B cell development

The surrogate light chain is composed of two polypeptides, VpreB and lambda 5. In the mouse there are two VpreB genes which are 99% identical within the coding regions. Extensive restriction enzyme mapping and sequencing of these two genes showed that only the coding region and immediate 5' and 3' flanking sequences exhibited such high homology. More distal sequences have diverged considerably. The region 5' of the respective gene directed transcription of a reporter gene in a pre-B cell line, indicating that it contained promoter, and perhaps enhancer function. The VpreB2 gene is functional, as it directed the production in COS cells of a 16-kDa protein that assembled with lambda 5 and was recognized by a VpreB-specific monoclonal antibody. Using transfected COS cells expressing either VpreB1 or VpreB2, a PCR assay was developed to examine the steady state level of transcripts from each gene. When this assay was applied to a number of cell lines representing early stages of B cell differentiation, co-expression of the two genes was observed in every case. VpreB1 and VpreB2 were co-expressed in the fetal liver of CB17 mice, where peak expression of each gene occurred at days 16-17 of gestation. Similarly, adult bone marrow from several strains of mice expressed both genes. In sorted bone marrow cells expression of both VpreB genes was detected in pro-B/pre-BI and large pre-BII cells, while the RNA steady state levels were at least 100-fold lower in small pre-BII and immature/mature B cells. Finally, single-cell reverse transcriptase-polymerase chain reaction on such sorted bone marrow cells detected VpreB1 and VpreB2 expression in at least 30% of all pro-B/pre-BI cells and large Ig heavy chain, surrogate light chain (pre-B receptor) expressing pre-BII cells. These results demonstrate that the control of expression of the two VpreB genes overlaps during development. They suggest that both VpreB1 and VpreB2 polypeptides can assemble with lambda 5 and mu to form pre-B cell receptor complexes.

Receptor biology of the melanocortins, a family of neuroimmunomodulatory peptides.

Melanocortins, melanocyte-stimulating hormones (MSH) and adrenocorticotropic hormone (ACTH) are homologous natural peptides derived from pro-opiomelanocortin (POMC). Recent breakthroughs in melanocortin receptor (MCR) biology are relevant to neuroimmunomodulation because melanocortins are known to modulate fever, inflammation and immunity, by acting both on peripheral targets and within the brain. During fever, endogenous melanocortins exert antipyretic effects by acting on MCR located within the brain, suggesting a protective counterregulatory role of the central melanocortin system. MCR are also found in melanocytic cells and adrenal cortical cells, the classical targets for alpha-MSH and ACTH, respectively, in myelogenous and lymphoid tissues, and in various endocrine and exocrine glands, adipocytes, and in autonomic ganglia. In the CNS, MCR are prominently distributed in close proximity to the terminal fields of melanocortinergic neurons that innervate neuroendocrine and autonomic motor nuclei as well as other subcortical brain regions important in neuroendocrine and autonomic regulation, sensory processing and various aspects of behavior. Furthermore, the presence of MCR in circumventricular organs of the brain provides direct access of systemic melanocortin hormones to central MCR. Together, these attributes provide an anatomical basis for bidirectional MCR-mediated communication between brain and periphery. A group of five G-protein-associated MCR subtypes, each of which is positively coupled to adenylate cyclase, has been identified. Among these, the adrenal ACTH receptor (MC2-R) is selectively activated by ACTH. In contrast, the other MCR subtypes (MC1-R, MC3-R, MC4-R, MC5-R) recognize a common group of ligands that includes various forms of MSH as well as ACTH; nevertheless they do exhibit important differences in ligand selectivity. MCR concentrations and MCR mRNA levels are influenced by availability of cognate ligands, by drugs, and by pathological stimuli. Two types of endogenous MCR antagonist proteins have been discovered: agouti protein and the corticostatins. Agouti protein dramatically alters coat color in mammals by antagonizing melanocytic MC1-R. Moreover, spontaneous dominant mutations of the agouti gene in several strains of mice lead to its ubiquitous overexpression and produces not only yellow coat color, but also obesity and insulin resistance, perhaps as a result of its antagonism of other MCR subtypes. The recent emergence of synthetic MCR antagonists, and the feasibility of molecular approaches for targeted inactivation of individual MCR subtypes, should facilitate elucidation of the roles and mechanisms of neuroimmunomodulation by endogenous melanocortins, and the determination of whether selective pharmacological targeting of MCR may ultimately have therapeutic utility.