Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland

alpha-Melanocyte-stimulating-hormone (alpha-MSH) is an agonist at the melanocortin 3 receptor (MC3-R) and melanocortin 4 receptor (MC4-R). alpha-MSH stimulates corticosterone release from rat adrenal glomerulosa cells in vitro. Agouti-related protein (AgRP) an endogenous antagonist at the MC3-R and MC4-R, is expressed in the adrenal gland. We investigated the expression of the MC3-R and MC4-R and the role of AgRP in the adrenal gland. MC3-R and MC4-R expression was detected in rat adrenal gland using RT-PCR. The effect of AgRP on alpha-MSH-induced corticosterone release was investigated using dispersed rat adrenal glomerulosa cells. AgRP administered alone did not affect corticosterone release, but co-administration of AgRP and alpha-MSH attenuated alpha-MSH-induced corticosterone release. To investigate glucocorticoid feedback, adrenal AgRP expression was compared in rats treated with dexamethasone to controls. AgRP mRNA was increased in rats treated with dexamethasone treatment compared to controls. Our findings demonstrate that adrenal AgRP mRNA is regulated by glucocorticoids. AgRP acting via the MC3-R or MC4-R may have an inhibitory paracrine role, blocking alpha-MSH-induced corticosterone secretion.     

Xenoantisera against a murine T cell tumor product cross-react with immunoglobulin Fab determinants

Some murine monoclonal T lymphoma cells express a surface component that reacts with chicken antisera produced against the Fab fragment of normal mouse IgG. In the present study, we use a solid phase immunoadsorbent consisting of affinity-purified chicken anti-Fab coupled to Sepharose to isolate a product produced by the in vitro T cell line, WEHI-7.1. The affinity-purified T cell surface molecule (IgT) migrated on SDS-PAGE as a single band of approximately 65,000 daltons. The object of these studies was to produce xenoantisera against the purified T cell product cross-reactive with Ig determinants and to characterize the antisera. Rabbits immunized with this purified molecule produced antibodies that reacted with Fab fragments of polyclonal mouse IgG and with the myeloma proteins MOPC-104E and MOPC-41, as detected by enzyme-linked immunosorbent assay (ELISA). This binding was eliminated by adsorption of the antisera with normal polyclonal IgG; however, adsorption with fetuin did not significantly affect the reactivity of the antisera. Radioimmune precipitation assays revealed that the rabbit anti-IgT bound to normal murine spleen and thymus cells; this reactivity was abrogated by adsorption with insolubilized polyclonal IgG. Competition radioimmunoassays demonstrated that detergent extracts of the thymus and the spleen contained material that inhibited the precipitation of MOPC-41; nonlymphoid cells lacked such material. The rabbit anti-IgT serum blocked the binding of antigen by normal T cells; adsorption of the antiserum with polyclonal IgG-Sepharose abrogated this blocking capacity. A solid phase immunoadsorbent prepared from the IgG fraction of the rabbit anti-IgT isolated a single component from formic acid-solubilized mouse thymus. This molecule had an approximate mass of 65,000 to 70,000 daltons. The anti-IgT serum isolated surface IgM and IgD from lactoperoxidase-catalyzed radioiodinated B cells. The anti-IgT serum detected IgM and IgG in mouse serum with the use of immunoelectrophoresis. The anti-IgT immunoadsorbent isolated several components from normal mouse serum, that, when analyzed by SDS-PAGE under reducing conditions, revealed bands corresponding to mu-, gamma-, and light chains as well as components that migrated between mu- and gamma-chains, and another component with an approximate mass of 45,000 daltons. Our results with antibodies to a purified T cell product indicate that a surface component of normal T cells and certain monoclonal T cell tumor lines is serologically related to the Fab fragment of serum Ig and is implicated in the binding of antigen.     

Production of rabbit antisera against recombinant MHC class II beta chain and identification of immunoreactive cells in Atlantic salmon (Salmo salar)

In the present work, rabbit antisera recognising the Atlantic salmon (Salmo salar) MHC class II beta chain polypeptide were produced and used in immunoblotting, immunohistochemistry and immunogold electron microscopy. A construct encoding the beta1 and beta2 domains fused to the E. coli protein thioredoxin was used to express the recombinant MHC class II beta chain. Immunoblotting revealed a band of approximately 30kDa in total protein samples from head kidney, spleen, gills, thymus and blood leukocytes, while being absent in muscle. The distribution of MHC class II positive cells was immunohistochemically demonstrated in Atlantic salmon epithelial and haemopoietic tissues. Ultrastructural demonstration of immunoreactive organelles in mid-kidney cells was performed by immunogold electron microscopy. The results indicate expression in lymphocytes, macrophages, epithelial cells and endritic-like cells. This is the first study to address morphological MHC class II expression in a fish species.     

Activation of MAP kinase by MC4-R through PI3 kinase

The melanocortin 4 receptor (MC4-R) is a G_s-coupled receptor known to increase cAMP production following agonist stimulation. We demonstrate that the mitogen-activated protein kinases p42 (ERK2) and p44 (ERK1) are also activated by MC4-R following treatment with the MC4-R agonist NDP--MSH in stably transfected CHO-K1 cells. This time- and dose-dependent response is abolished by the MC4-R antagonist SHU-9119. p42/p44 MAPK activation is blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 but not by the protein kinase A (PKA) inhibitor Rp-cAMPS, indicating that that signal activating the p42/p44 MAPK pathway is conveyed through inositol triphosphate.     

Prolonged viral RNA detection in blood and lymphoid tissues from coxsackievirus B4 E2 orally-inoculated Swiss mice

The spreading of viral RNA within Swiss Albino mice orally inoculated with coxsackievirus B4 E2 strain (CVB4 E2) was studied by using RT-PCR and semi-nested-RT-PCR methods. Viral RNA was detected in various organs: pancreas, heart, small intestine, spleen, thymus, and blood at various postinfectious (p.i.) times ranging from 8 hr to 150 days. Our results show that (i) outbred mice can be infected with CVB4 E2 following an oral inoculation, which results in systemic spreading of viral RNA, (ii) CVB4 E2 infection can be associated with a prolonged detection of viral RNA in spleen, thymus and blood, up to 70 days p.i. and further in other organ tissues.     

Starvation-sensitive UCP 3 protein expression in thymus and spleen mitochondria

To date, UCP 3 has only been associated with skeletal muscle and brown adipose tissue (BAT). Using RT-PCR/PCR methodology, we show that human spleen and human thymus contain UCP 3. In addition, using peptide antibodies, previously demonstrated to be selective for UCP 3, we show that UCP 3 protein is present in mitochondria isolated from rat thymus and mitochondria isolated from reticulocytes, monocytes and lymphocytes of rat spleen. UCP 3 protein expression is also starvation-sensitive. UCP 3 abundance is augmented in mitochondria isolated from thymus and mitochondria isolated from lymphocytes of the spleen from fasted rats when compared to fed controls. The results are consistent with a role for UCP 3 in developing lymphocytes, thymus atrophy and fatty acid utilisation in spleen and thymus.     

Involvement of Two Types of TNF Receptor in TNF-α Induced Neutrophil Apoptosis

We previously demonstrated that tumor necrosis factor-α (TNF-α) induces rapid human neutrophil apoptosis. In this paper, we examined which of the TNF receptors, p55 kDa TNF receptor (55-R) or p75 kDa TNF receptor (75-R), or both are involved in this process using specific rabbit antisera. Antibodies to 55-R (anti55-R) or 75-R (anti75-R) alone did not induce neutrophil apoptosis. Further addition of cycloheximide and anti-rabbit immunoglobulin to anti55-R but not to anti75-R accelerated apoptosis, although anti75-R augmented the capacity of anti55-R to do so. These results suggest that 55-R is a prerequisite for TNF-α induced neutrophil apoptosis.     

Transplantation tolerance at the T-lymphocyte receptor level. I. Spontaneous release of T-cell receptors by lymphocytes from tolerant mice.

Spleen and lymph node T cells from CBA mice when cultivated in vitro almost immediately started to shed receptors for A alloantigens. In contrast, cultivated T lymphocytes from CBA mice tolerant of A alloantigens for 657 or 692 days began releasing receptors for the formerly tolerated A alloantigen after a delay of 8 h. This delay in receptor shedding coincided with that observed when nontolerant lymphocytes were treated with antisera to T-cell receptors. The results suggested a close similarity of mechanisms and indicated that transplantation tolerance may be maintained by active suppression of otherwise reactive T cells mediated by anti-receptor antibody. Lymphocytes from untreated tolerant mice and those treated with a nonspecific anti-receptor serum showed normal responses, as measured in terms of receptor release, to third-party alloantigens, as did cells from untreated normal mice. Precultivated normal lymphocytes treated with anti-receptor antibody and complement failed to release the appropriate receptor specificity but similarly treated tolerant lymphocytes were almost resistant, presumably because they expressed only few receptors at the time of treatments.     

Human leukocyte Fc (IgG) receptors: quantitation and affinity with radiolabeled affinity cross-linked rabbit IgG.

Quantitative studies have been made of Fc receptors on human leukocytes derived from peripheral blood, thymus, tonsil, and spleen. The relative affinities and average numbers of receptors per cell were determined by measuring the binding of 125I-labeled, affinity cross-linked trimers of rabbit IgG to various populations of cells. In parallel, the sizes of receptor-bearing populations were determined by fluorescence microscopy. Fc receptors could be detected on leukocytes from peripheral blood and spleen, but not from tonsil or thymus. In the peripheral blood, the highest density of receptors was found on polymorphonuclear leukocytes; a subpopulation of lymphocytes had somewhat fewer receptors per cell, and circulating monocytes had the lowest receptor density. Among splenocytes, most of the receptors were found on myeloid cells and monocytes. In all populations, the affinity of Fc receptors for the trimer was about the same. At 0 degrees C the average value for the association constant was 5 x 10(7) M-1.     

Redundancy of a functional melanocortin 1 receptor in the anti-inflammatory actions of melanocortin peptides: studies in the recessive yellow (e/e) mouse suggest an important role for melanocortin 3 receptor

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (M phi) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mphi incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro M phi activation, determined as release of the CXC chemokine KC and IL-1 beta, was inhibited by the more selective MC3-R agonist gamma(2)-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1 beta release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of gamma(2)-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal M phi leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.     

黄鳝IgM的分离纯化及兔抗黄鳝IgM抗血清的制备

目的 分离纯化黄鳝血清免疫球蛋白,制备其兔抗血清,并检测抗血清的特异性。方法 用Protein A亲和层析的方法纯化黄鳝血清免疫球蛋白,通过SDS-聚丙烯酰胺凝胶电泳检测蛋白的纯度,免疫大耳白兔制备抗血清,利用免疫双扩散检测抗血清的效价,通过western blotting检测抗血清的特异性。结果 纯化了黄鳝血清免疫球蛋白,免疫双扩散法测定兔抗黄鳝免疫球蛋白血清效价为1∶32,western blotting结果显示抗血清具有很好的特异性。结论 成功纯化了黄鳝免疫球蛋白,制备了兔抗黄鳝IgM抗血清,为建立黄鳝的血清学检测系统奠定了基础。     

Brain-associated-theta antiserum. Differential effects on lymphocyte subpopulations

Subpopulations of lymphocytes were delineated, both in vitro and in vivo, using brain associated-θ antisera. Lymphocytes were depleted from the cortical areas of the thymus and the T-dependent regions of the spleen. No depletion occurred in the medullary area of the thymus or in the deep cortical areas of the lymph node. These histological findings correlated well with the effects of brain associated-θ antisera using an in vitro cytotoxicity assay. Greater concentrations of antisera were required to cause cell death of lymphocytes from lymph node than lymphocytes from either spleen or thymus. These data would suggest that the θ antigen may be either lost or modulated during the process of lymphocyte differentiation.     

Antipeptide antibody against the human low-density-lipoprotein receptor. Characterization and cross-reactivity with bovine lymphocytes.

A dodecapeptide corresponding to the external N-terminal sequence of the human low-density-lipoprotein (LDL) receptor was synthesized. Antibodies raised in rabbits against the peptide were purified and were shown to react specifically with the peptide and with human LDL receptor of fibroblasts, HeLa cells and lymphocytes using binding studies and immunoblotting. By indirect immunogold analysis, antibodies bound to the LDL receptor of human lymphocytes could be revealed as clusters. Anti-receptor peptide immunoglobulins specifically bound to the human HeLa cell's LDL receptor with a lower affinity than LDL (Kd x 3). The anti-receptor peptide immunoglobulins and 125I-labelled-LDL competed with each other for the LDL-receptor sites. Antibodies failed to react with lymphocytes of subjects with the homozygous form of familial hypercholesterolaemia. Cross-reactivity with the dodecapeptide of the bovine LDL receptor was limited, but this cross-reactivity was confirmed by the binding of anti-receptor peptide immunoglobulins to the LDL receptor from bovine lymphocytes.     

Membrane immunoglobulin is present on thymic and splenic lymphocytes of the trout Salmo gairdneri.

Three rabbit antisera raised to trout IgM showed positive immunofluorescent reactions with lymphocytes of trout spleen and thymus. These antisera showed no detectable cross-reactivity with keyhole limpet hemocyanin, as determined by direct radioimmune precipitation and adsorption, and did not appear to react significantly with carbohydrate moieties on trout IgM, as determined by the failure of proteolytic digests of trout IgM to inhibit in radioimmunoassay against intact trout IgM. Membrane immunoglobulin determinants were detectable on the membrane of both thymocytes and splenocytes with the lactoperoxidase-catalyzed iodination reaction. Radioimmunoassay of lysates of lymphocytes confirmed the association of immunoglobulin with trout thymocytes. By radioimmunoassay, lysates of thymic and splenic lymphocytes contained similar amounts of immunoglobulin, equivalent to approximately 8 X 10(4) molecules of IgM per cell (calculated as the monomer micro 2L2).     

Prolactin receptor gene expression in rat splenocytes and thymocytes during oestrous cycle, pregnancy and lactation

Much evidence suggests that prolactin (PRL) has an immunoregulatory function. Part of this evidence is that the receptors for PRL are present on lymphocytes. Probably the effects of PRL on cells of the immune system depend on the level and specific forms of PRL-R present on the target cells. Therefore, PRL-R expression at both protein and mRNA levels was examined during oestrous cycle, pregnancy and lactation using Western blotting and PCR analysis. Antibody to the long form of PRL-R detected 84 and 42 kDa protein bands in the spleen but only 84 kDa band in the thymus. The expression pattern of these two protein bands was different in the spleen, suggesting that these two isoforms of PRL-R long form are differentially regulated by the hormones of oestrous cycle. In addition, depending on the tissue, the level of mRNA for the short and long forms of PRL-R showed a significant change at different stages of oestrous cycle. Moreover, 42 and 84 kDa PRL-R bands were detected in both spleen and thymus throughout the pregnancy and lactation; however, the expression pattern of 84 kDa protein band was different between tissues. This finding suggests that each tissue exhibits differential response to hormones which affect PRL-R content.     

Anti-receptor anti-MHC cytotoxic T lymphocytes: their role in the resistance to graft vs host reaction

Specific resistance to local graft vs host reaction (GvHR) observed in F1 hybrids pretreated s.c., i.p., or i.v. with parent strain spleen cells has been explained as being due to cytotoxic cells induced in these pretreated F1 hybrids and directed against cells bearing receptors that recognize the major histocompatibility complex (MHC) alloantigens of the opposite parent strain (anti-receptor anti-MHC cytotoxic T lymphocytes; CTL). These anti-receptor anti-MHC CTL, however, have never been detected directly in popliteal lymph nodes (PLN), which develop a specific resistance to GvHR. In this paper, we describe the detection of anti-receptor anti-D2 cytotoxic activity in both right and left PLN of B6D2F1 hybrids injected s.c. in the right footpad only with B6 spleen cells. This cytotoxic activity appears 4 days after the injection of B6 cells and diminishes by day 7. It is mediated by a Thy-1+, L3T4-, Lyt-2+ cell of B6D2F1 origin and induced by the injection of either Thy-1+L3T4+Lyt-2- or Thy-1+L3T4-Lyt-2+ B6 spleen cells. The anti-receptor anti-D2 CTL activity is not observed in PLN of B6D2F1 hybrids pretreated i.p. or i.v. with B6 spleen cells. Our results demonstrate that anti-receptor anti-MHC CTL can fully explain the specific resistance to GvHR induced by the s.c. pretreatment of F1 hybrids with parent strain spleen cells. Their role, however, in the resistance to GvHR observed in F1 hybrids i.v. or i.p. pretreated is far from being entirely proven.     

Expression of PAC1 receptor in rat thymus after irradiation

In the present work, PAC1-R (G-protein-coupled receptor specific for PACAP) was detected on cells in the normal thymus. Immunohistochemically PAC1-R was expressed strongly in stromal cells of the thymic medulla. Positive cells were also observed in the thymus of fetal and old adult rats. After 8 Gy irradiation to 9-week-old rats, PAC1-R expressions in the thymus decreased and almost recovered by day 21. The expression of PAC1-R mRNA was weak in the thymus and decreased further after irradiation. The expression almost recovered by day 28. Hip and hip/hop variants, which were not expressed in the normal thymus, were expressed in the thymus on days 3, 5 and 21 after irradiation. The expressions of IL-6 and IL-10 tended to increase initially after irradiation then decreased. Histologically, the thymic structures were destroyed on day 3 after irradiation and the thymus almost recovered by day 21. Thus PACAP is thought to be one of the important factors for cross-talk between cells involved in thymic regeneration.