Adhesion receptors are differentially expressed on developing thymocytes and epithelium in human thymus.

The thymic microenvironment consists of a network of interrelated cells of epithelial, fibroblastic, endothelial, and hemopoietic origin. Within this environment, the development of specific T-lymphocyte subpopulations partially depends on the selective interaction of T-cell precursors with such cells. Human thymic epithelial cell strains, generated with a defective retroviral vector containing simian virus 40 (SV40) large T antigen and the neomycin resistance gene or by transfection with an SV40 plasmid defective in the origin of replication, provide useful tools for understanding the mechanisms contributing to the control of T-cell maturation. Because interepithelial, epithelial-macrophage, and lymphocyte-epithelial cell interactions are important for thymocyte differentiation, the distribution of integrin and nonintegrin adhesion receptors on these cells and on developing thymocytes in vivo and in vitro has been examined in detail. Our results indicate that the transformed human thymic epithelial cell strains express the common very late antigen (VLA)-beta 1 receptor and unique alpha chains VLA-2, VLA-3, and VLA-6. The cells are also positive for LFA-3 and ICAM-1 and weakly express beta 3, beta 4, and VNR alpha. They do not express the Leu-cellular adhesion molecules (CAM). This phenotypic profile on cultured thymic epithelium generally corresponds to the distribution of integrin and other receptor molecules on thymic epithelial cells in tissue sections. The majority of thymocytes also express the integrin VLA-beta 1 and -beta 2 chains as well as VLA-4, VLA-6, and LFA-1 alpha(L). Three-color flow cytometric analyses show differential levels of expression of these adhesion receptors on human thymocyte subsets. Taken together with the immunohistochemical localization of extracellular matrix molecules, these studies suggest that both the distribution of receptor-ligand pairs and the level of expression of adhesion molecules may influence T-cell development within the thymus.     

Neurotropins and their receptors are expressed in the human fetal ovary

Mammalian ovarian development is characterized by a sequential pattern of mitotic proliferation of oogonia, initiation then arrest of meiosis, and primordial follicle formation. The factors regulating these processes are poorly understood. The neurotropins are survival and differentiation factors in the nervous system, acting via high affinity receptors of the trk protooncogene family and the low affinity p75 nerve growth factor receptor, and have also been described in the rodent ovary, where changes in NT4/TrkB gene expression have been detected at the time of primordial follicle formation. There are no data on neurotropin expression in the normal human ovary. We have investigated the expression and localization of neurotropins and their receptors in the midtrimester human fetal ovary (13-21 wk gestation). Expression of mRNA for neurotropins and their receptors was detected by RT-PCR. Clusters of oogonia were found to be the predominant site of NT4 mRNA expression using in situ hybridization. However, at later gestations granulosa cells of primordial follicles showed increased expression, with lesser expression in the enclosed oocytes. NT4 protein was also localized to the granulosa cells by immunohistochemistry and at earlier developmental stages to epithelioid cells, which were mingled with clusters of oogonia not expressing NT4. TrkB receptor protein was localized by immunohistochemistry to germ cells at all gestations examined. The p75 nerve growth factor receptor protein was exclusively expressed in the ovarian stroma. These data demonstrate the expression of neurotropins and their receptors within the human fetal ovary. Developmental changes in the pattern of expression of NT4 around the time of primordial follicle formation suggest that neurotropins may be involved in signaling between somatic cells and germ cells at this crucial stage of ovarian development.     

Estrogen receptors alpha and beta are differentially expressed in developing human bone

Estrogen plays an essential role in the development and maintenance of the skeleton; its effects are mediated via interactions with two estrogen receptor (ER) subtypes, alpha and beta. The aim of this study was to establish the cellular distribution of ERalpha and ERbeta in neonatal human rib bone. ERalpha and ERbeta immunoreactivity was seen in proliferative and prehypertrophic chondrocytes in the growth plate, with lower levels of expression in the late hypertrophic zone. Different patterns of expression of the two ERs were seen in bone. In cortical bone, intense staining for ERalpha was observed in osteoblasts and osteocytes adjacent to the periosteal-forming surface and in osteoclasts on the opposing resorbing surface. In cancellous bone, ERbeta was strongly expressed in both osteoblasts and osteocytes, whereas only low expression of ERalpha was seen in these areas. Nuclear and cytoplasmic staining for ERbeta was apparent in osteoclasts. These observations demonstrate distinct patterns of expression for the two ER subtypes in developing human bone and indicate functions in both the growth plate and mineralized bone. In the latter, ERalpha is predominantly expressed in cortical bone, whereas ERbeta shows higher levels of expression in cancellous bone.     

In vitro fixation of C3d and C5b-9 on platelets by human platelet reactive antibodies

Summary Most platelet-reactive autoantibodies and alloantibodies are not able to fix complement in vitro. However, exceptions have been found. These antibodies are usually characterized by the conventional platelet complement fixation test. A recently developed competitive enzyme immunoassay for quantitation of platelet-associated immunoglobulins and a modification thereof allowed the quantitative study of fixation of C3d and the membrane attack complex (C5b-9) on platelets by HLA antibodies, human platelet autoantibodies, and drug-dependent antibodies (ddab). The highest amounts of both complement products were fixed through ddabs, whereas autoantibodies only showed moderate complement fixation. This enzyme immunoassay is a valuable tool for the characterization of the complement-fixing properties of platelet-reactive antibodies.     

Somatostatin receptors SSTR2 and SSTR5 are expressed in the human thoracic duct

Somatostatin and its analog octreotide have been used successfully to treat postoperative chylothorax, and it has been shown that octreotide binds with high affinity to somatostatin receptor (SSTR) subtypes 2 and 5. Therefore, we investigated expression of SSTR2 and SSTR5 in the human thoracic duct by immunohistochemistry. Normal rat pancreas was used as a positive control for antibodies against SSTR2 and SSTR5, and Factor VIII-related antigen, SMA, actin, elastin, or collagen type II, III, IV or V antibodies were used to identify cell types and structures within the human thoracic duct. The antibodies against SSTR2 and SSTR5 worked well and yielded positive staining in control rat islets. In the human thoracic duct, SSTR2 was present in smooth muscle cells and some scattered structures which were stained by antibodies against Factor VIII-related antigen, SMA, actin, elastin or collagen type II, III, IV or V. SSTR5 was also present in smooth muscle cells. The presence of SSTR2 and SSTR5 in the human thoracic duct sheds light on the mechanism of somatostatin and octreotide use in the successful treatment of chylothorax and offers new molecular pathways to explore for potential future therapies.     

K channels are expressed early in human T-cell development.

Mature human T lymphocytes proliferate in response to the mitogen phytohemagglutinin (PHA), but immature thymocytes lacking the T3 receptor (T3- thymocytes) do not. Because functioning K channels are required for proliferation of mature T cells, we asked whether immunoincompetent T3- thymocytes lack normal K channels. We report that T3- thymocytes have a K+ current similar to that of mature peripheral T cells--that is, similar voltage dependence, activation and inactivation kinetics, and pharmacology. Moreover, the maximal specific K+ conductance is the same for both cell types, implying a similar density of activable channels in each cell. In assessing the functional responses of the channels to PHA, we found that the K+ current of immature and mature cells responds similarly to the mitogen. Responses near the threshold voltage for activating the K+ current were variable; the K+ conductance and rate of activation were increased, decreased, or unchanged after PHA treatment. For several cells, the voltage dependence of the conductance and activation kinetics was shifted in opposite directions. At more positive voltages, PHA consistently caused a 10-20% suppression of conductance that was not due to the addition of an inward current, to changes in the time course of activation or inactivation, or to changes in the steady-state level of inactivation. The effects of PHA on the K+ current cannot be explained by a simple shift in surface potential, as has been hypothesized to be involved in its triggering of T-cell proliferation. Taken together, our findings show K channels are expressed very early in T-cell differentiation, possibly before thymic processing, differential responses of the K+ current to PHA do not account for the failure of T3- thymocytes to proliferate, and changes in surface potential are probably not a necessary early event in activation of T cells by PHA.     

N-methyl-D-aspartate receptors are transiently expressed in the developing spinal cord ventral horn.

Quantitative receptor autoradiography was used to map the distribution of N-methyl-D-aspartate (NMDA) receptors in the developing rat spinal cord. Three different specific ligands, which label partially overlapping subpopulations of NMDA receptors, were used: an agonist (L-[3H]glutamate), a noncompetitive antagonist ([3H]MK-801), and a competitive antagonist ([3H]CGP-39653). In the adult, NMDA receptors labeled with all three ligands are restricted to the substantia gelatinosa in the spinal dorsal horn. In marked distinction, at postnatal day 7 NMDA receptors labeled with L-[3H]glutamate and [3H]MK-801 are present throughout the spinal gray matter. NMDA receptors in the neonatal spinal ventral horn have a higher affinity for L-[3H]glutamate than those in the adult substantia gelatinosa. Over the second and third postnatal weeks, NMDA receptors are lost from all areas of the spinal gray matter except for the substantia gelatinosa. Neonatal NMDA receptors identified with [3H]CGP-39653 are restricted to the substantia gelatinosa. These results show that the immature ventral horn contains a subpopulation of NMDA receptors and raise the possibility that motor neurons transiently express NMDA receptors in early postnatal life. Ventral horn NMDA receptors may be a component of the mechanisms by which the mature phenotype of motor neurons is acquired through activity-dependent processes. The loss of NMDA receptors over the course of development may play a role in limiting the period of motor neuron plasticity.     

Genes encoding tumor-specific antigens are expressed in human myeloma cells.

Genes of the MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1 families encode antigenic peptides that are presented by HLA class I molecules and that are recognized on human tumors by autologous cytolytic T lymphocytes. These genes are expressed in many solid tumor types but not in normal tissues, except male germline cells. Because the latter cells are devoid of HLA molecules, the derived antigens are strictly tumor-specific and should constitute safe immunogens for cancer immunotherapy. We detected a significant expression of these genes in a high proportion of bone marrow samples from patients with advanced multiple myeloma. This observation provides a basis for clinical trials aimed at inducing a cellular immune response directed at malignant plasma cells in advanced myeloma patients.     

Subpopulations of human peripheral granulocyes and monocytes express receptors for IgA.

Receptors for the Fc portion of immunoglobin on human peripheral blood cells were enumerated by rosette formation with ox erythrocytes sensitized with rabbit IgG, IgA, and IgM. A large percentage of purified polymorphonuclear leukocytes and monocytes were found to express receptors for IgA. These receptors were also found to exist on a significantly greater percentage of lymphocytes than was previously observed. The receptors for IgA were specific, as verified by blocking studies using purified human immunogloblins. In addition, some polymorphonuclear leukocytes and monocytes were observed concomitantly to posses independent receptors for both IgG and IgA. These studies may indicate that IgA can cooperate with monocytes or polymorphonuclear leukocytes through receptors for IgA on these cells and perhaps mediate immune defense on mucosal surfaces. Initial studies on antibody-dependent cellular cytotoxicity suggested that IgA alone is ineffectual in supporting cytolysis by nonactivated human peripheral blood cells.     

CD38 expressed on human monocytes: a coaccessory molecule in the superantigen-induced proliferation

This work analyzes the hypothesis that human CD38 may cooperate with MHC Class II by acting as coreceptor in a superantigen-induced activation. The initial evidence is that CD38 ligation by specific monoclonal antibodies inhibits superantigen-induced T lymphocyte proliferation. Inhibitory effects become apparent after engagement of CD38 expressed by monocytes, whereas ligation of CD38 expressed by T lymphocytes does not apparently affect activation. The inhibition requires a cell-to-cell interaction, followed by the relevant transmembrane signaling that is reproduced by CD38 ligation. Indeed, CD38 ligation on monocytes induces tyrosine phosphorylation of several intracellular proteins including the protooncogene product c-cbl and the fgr and hck tyrosine kinases. The receptorial nature of the CD38-mediated events is confirmed by the observation of an intracellular calcium flux in monocytes secondary to CD38 ligation. These effects are additive with the similar events elicited by MHC Class II ligation, a likely indication that CD38 and MHC Class II share a common activation pathway. This conclusion is strengthened by results of comodulation experiments, indicating that CD38 and MHC Class II display lateral associations on monocytes. These results attribute to CD38 expressed by monocytes a role in the transduction of signal(s) involved in superantigen-induced activation, operating in synergy with MHC Class II.     

Orexin receptors are expressed in the adrenal medulla of the rat

Two recently discovered hypothalamic peptides, orexin-A and orexin-B, play a role as mediators in the central mechanisms that regulate feeding behavior and sleep control. These peptides bind and activate two orexins receptors that belong to the G-protein coupled receptor superfamily. Morphological studies have detected mRNA expression of orexin receptors exclusively in the rat central nervous system. In this paper we demonstrate a strong level of expression of orexin receptor 1 and 2 in the adrenal medulla of the rat by RT-PCR and immunohistochemistry. The results of the present study provide the first evidence showing that the adrenal medulla expresses orexin receptors, and thus appears to be a target tissue for orexins. This could open a new loop in which the central and autonomous nervous system may be involved in body weight homeostasis and sleep control.     

Interleukin-6 and its receptor are expressed by human megakaryocytes: in vitro effects on proliferation and endoreplication

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an important role in the megakaryocytic differentiation. Recently, we have observed that IL-6 is synthesized by several human cell lines with megakaryocytic features. In this study, we have investigated whether a similar phenomenon occurs during normal megakaryocytic differentiation. Human megakaryocytes (MK) were obtained by culturing normal marrow in liquid culture with aplastic plasma (AP). First, an IL-6 secretion in bone marrow culture enriched in MK as well as in purified MK populations was demonstrated by a biologic assay. Second, IL-6 mRNA was detected in a purified population of MK by the polymerase chain reaction and dot blot analysis. IL-6 mRNA and protein were undetectable in platelets. Third, in situ hybridization procedure demonstrated the presence of IL-6 mRNA in individual immature MK. Fourth, IL-6 protein was detected in MK at the unicellular level by an immunoalkaline phosphatase technique using a monoclonal antibody against IL-6. Furthermore, the presence of IL-6 receptor (IL-6-R) on MK was demonstrated by in situ hybridization using an IL-6-R probe and in situ autoradiography after binding with [125I]-labeled recombinant IL-6. The IL-6 endogenously produced in liquid cultures containing normal human plasma or AP was subsequently neutralized. This resulted in a 50% decrease of the MK growth with a minor shift in the ploidy distribution toward lower values. In semisolid cultures the addition of anti-IL-6 antibodies led to a 42% decrease in colony number in cultures stimulated by IL-3 but not in other conditions of culture. These results suggest that normal human megakaryocytopoiesis might be regulated in part by an IL-6 autocrine loop.     

脂联素受体在胰岛细胞表达,脂联素促进胰岛素的分泌

目的　检测脂联素受体(AR)在大鼠胰岛细胞的表达和脂联素对体外胰岛细胞分泌胰岛素的影响。方法　RT PCR和免疫细胞化学方法检测AR1、AR2的mRNA和蛋白表达;并在体外用脂联素(100μg/L)和不同浓度葡萄糖(3. 3, 5. 6, 16. 7mmol/L)处理胰岛细胞,放免法测定上清液的胰岛素浓度。结果　RT PCR扩增出胰岛AR1和AR2基因,并经直接和亚克隆测序证实;胰岛免疫细胞化学荧光染色AR1和AR2呈阳性;经脂联素处理后的胰岛细胞,在高糖(16. 7mmol/)培养 6~24h,其胰岛素分泌持续增加(均P<0. 05)。结论　胰岛细胞上存在AR1和AR2,以前者为主。在高糖情况下,一定浓度的脂联素可在体外促进胰岛细胞的胰岛素分泌和释放。