Mechanically regulated expression of a neural glutamate transporter in bone: a role for excitatory amino acids as osteotropic agents?

Without habitual exercise, bone is lost from the skeleton. Interactions between the effects of loading of bone and other osteotropic influences are thought to regulate bone mass. In an attempt to identify potential targets for therapeutic manipulation of bone mass, we used differential RNA display to investigate early changes in osteocyte gene expression following mechanical loading of rat bone in vivo. One gene found to be down-regulated by loading had high homology to a glutamate/ aspartate transporter (GLAST) previously identified only the mammalian CNS. RT-PCR analysis using primers targeted to the coding region of the published GLAST sequence amplified identical products from bone and brain (but not a range of other tissues). The amplicons were sequenced and found to be identical to the published CNS GLAST sequence. Northern analysis confirmed expression of GLAST mRNA in bone and brain, but not other tissues. In situ hybridization localized GLAST mRNA expression in rat bone to osteoblasts and osteocytes. A GLAST antibody localized high levels of protein expression to osteoblasts, and newly incorporated osteocytes. Interestingly, older osteocytes also expressed detectable levels of GLAST. Another neural glutamate transporter, GLT-1 was immunolocalized to the pericellular region of mononuclear bone marrow cells, while a further antibody to the EAAC-1 transporter failed to bind to bone cells. Five days after loading, GLAST protein expression was undetectable in osteocytes of loaded bone but present in control nonloaded sections, confirming the downregulation detected by differential display. On quiescent periosteal surfaces, GLAST expression was almost absent, while on surfaces where loading had induced cellular proliferation and bone formation, GLAST protein expression was elevated. In the CNS, the expression of glutamate transporters on neuronal membranes is associated with reuptake of released neurotransmitters at synapses, where they have a role in the termination of transmitter action. In this study, we describe for the first time, the expression of GLAST (and GLT-1) in bone, raising the possibility that excitatory amino acids may have a role in paracrine intercellular communication in bone. Manipulation of bone cell function by moderators of glutamate action could therefore provide novel treatments for bone diseases such as osteoporosis.

Constitutive in vivo mRNA expression by osteocytes of beta-actin, osteocalcin, connexin-43, IGF-I, c-fos and c-jun, but not TNF-alpha nor tartrate-resistant acid phosphatase.

Osteocytes have been proposed to be the cells primarily responsible for sensing the effects of mechanical loading in bone. Osteocytes respond to loading in vivo, and have been shown to express osteotropic agents and their receptors, and cell/matrix adhesion molecules in vitro, but the functional significance of such findings is not clear. One obstacle to increased understanding of the role of osteocytes in the regulation of bone mass is that the cells are not easily accessible for study. In situ studies are difficult, and although it is possible to extract and culture osteocytes from neonatal bones, the responses of such cells might be very different from those in older bones in situ. We have developed a technique to investigate osteocyte gene expression in vivo, using the reverse transcriptase linked polymerase chain reaction (PCR), and have shown that they express mRNA for beta-actin (beta-ACT), osteocalcin (OC), connexin-43 (Cx43), insulin-like growth factor I (IGF-I), c-fos and c-jun, but not tumor necrosis factor alpha (TNF-alpha) or tartrate-resistant acid phosphatase (TRAP). The principle behind the method is that after removal of the periosteum, tangential cryostat sections of a tubular bone contain RNA only from osteocytes and a very small number of endothelial cells as long as the marrow cavity is not broached. Using this method, we have investigated gene expression in cells from rat ulnar cortical bone under forming and resorbing bone surfaces. In addition, we have investigated the effect on gene expression of mechanical loading which, if repeated daily, initiates new bone formation on quiescent or resorbing surfaces. Although the expression of the genes we have studied in osteocytes is different from those expressed by the periosteal surfaces overlying the cortex, we have not detected loading-related changes in osteocyte gene expression in any cortical bones. This may be because of the extreme sensitivity of the PCR technique which can only resolve large differences in expression. The use of quantitative methods in the future may allow demonstration of regulated gene expression in osteocytes.

Clinical and analytical evaluation of an immunoradiometric assay for corticotropin.

We evaluated a new IRMA developed commercially for the measurement of corticotropin (ACTH) in human plasma. The assay involves purified polyclonal goat capture antibodies specific for ACTH 26-39 and an 125I-labeled monoclonal signal antibody specific for ACTH 1-17. CVs for intraassay and total precision at ACTH concentrations between 9 and 801 ng/L ranged from 2.5% to 4.7% and from 3.3% to 9.3%, respectively, with an assay detection limit of 1.7 ng/L. The reference interval determined for adults with the new method (16-52 ng/L) differed significantly (P < 0.05) from that for an established ACTH IRMA (9-54 ng/L). Method comparison with clinical samples (n = 179) revealed a correlation coefficient of 0.970 and a best-fit equation of y (new IRMA) = (1.011 +/- 0.019)x + (4.17 +/- 3.31) with Sylx = 40.2. Both methods showed equivalent clinical sensitivity in evaluating Cushing disease, adrenal tumors, ectopic ACTH-producing tumors, hypopituitarism, steroid suppression, surgical adrenalectomy, Nelson syndrome, Addison disease, and corticotropin-releasing hormone stimulation. We conclude that the new IRMA is technically simple to perform and provides a specific and sensitive method for evaluating of adrenocortical function.

Inhibition of bone resorption and stimulation of formation by mechanical loading of the modeling rat ulna in vivo.

During normal growth of the rat ulna, bone is resorbed from the medial periosteal surface. This occurs as part of the modeling process by which the bone achieves its adult shape. By attaching strain gauges to the ulnae of rats in vivo, we measured the strains imposed on that surface of the bone during normal locomotion. We then applied mechanical loads to the ulnae of other rats in vivo for 6 consecutive days, inducing strains approximately double those we had measured. Fluorochromes were given on the 1st and 5th days. The histology of the medial ulnar periosteal surface was correlated with the amount of fluorochrome incorporation and tartrate resistant acid phosphatase (TRAP) activity in serial sections. In the nonloaded ulnae, the surfaces were lined with bone resorbing cells. Corresponding areas of the loaded bones were lined with osteoid and osteoblasts. There was insignificant label incorporation in the nonloaded bones but almost continuous label incorporation in the corresponding regions of the loaded bones, which was significantly different from the nonloaded bones. TRAP activity of the periosteal cells in the loaded bones was significantly less than in the nonloaded limbs. It is widely acknowledged that loading induces bone formation, and this implies that it also has the ability to inhibit resorption. However, to date there has been little direct evidence for the inhibition of resorption in vivo by mechanical loading. The changes we have observed are similar to the sequence of cellular events that occur during the reversal phase of bone remodeling, in which osteoclastic resorption ceases and osteoblasts are recruited and begin formation. This model may help increase understanding of that process.

Cytomegalovirus (CMV) antigenemia assay is more sensitive than shell vial cultures for rapid detection of CMV in polymorphonuclear blood leukocytes.

We compared the cytomegalovirus (CMV) antigenemia assay with shell vial cultures of polymorphonuclear leukocyte (PMNL)-enriched blood fractions for rapid diagnosis of CMV viremia. PMNL fractions of 280 blood specimens from 171 patients (170 solid-organ transplant recipients and 1 patient undergoing pretransplant evaluation) were inoculated in shell vial and conventional CMV cultures. A commercially available kit (CMV-vue kit; INCSTAR Corp.) was used for the CMV antigenemia assay, in which PMNL preparations were stained with monoclonal antibodies directed against the CMV protein pp65. Mixed-leukocyte blood fractions from the same blood specimens were inoculated in parallel shell vial and conventional cultures. CMV viremia (defined by the isolation of CMV in conventional cultures) was detected in 32 (13%) of 245 PMNL fractions included in the final analysis. Twenty-eight (87.5%) were also positive in the CMV antigenemia assay, whereas 22 (69%) were positive in shell vial cultures. Ten (4%) additional PMNL fractions positive only in the CMV antigenemia assay were from eight patients with active CMV infections (six patients), who had previous or subsequent episodes of CMV viremia (seven patients), or in whom CMV was isolated in cultures of simultaneously obtained mixed-leukocyte fractions (three patients). Overall, the CMV antigenemia assay was significantly more sensitive than shell vial cultures for detection of CMV in the PMNL fraction of blood leukocytes (P < 0.01, McNemar's test), and we recommend it as the method of choice for rapid diagnosis of CMV viremia.

Four unique monoclonal antibodies to the putative receptor binding domain of erythropoietin inhibit the biological function of the hormone.

We have produced a series of monoclonal antibodies (MoAbs) to amino acid region 99-129 of human erythropoietin (Epo) that distinguish unique structural features within this putative receptor binding domain of the hormone. The MoAbs recognize denatured Epo with widely different sensitivities on a Western blot and differentially bind to native Epo in solution. In addition, three of the four MoAbs neutralize the biological activity of Epo in a concentration-dependent fashion in vitro. Neutralization was measured both by inhibition of Epo-induced differentiation in Rauscher murine erythroleukemia cells and by inhibition of Epo-induced proliferation in normal murine splenic erythroid precursors. Characterization of the structural epitopes recognized by each of these four reagent MoAbs should provide us with important information concerning the requirements for hormone-receptor interaction.

Comparison of local and systemic immunity after intratracheal, intraperitoneal, and intravenous immunization of mice exposed to either aerosolized or ingested lead.

The humoral immunity of newborn mice exposed for 28 days to 2.5 mg/m3 aerosolized Pb(NO3)2 (Pb28-aero) or of 2-week-old mice similarly exposed for 14 days (Pb14-aero) was compared with that of both 2-week-old mice given 125 micrograms Pb(NO3)2/day by gastric intubation for 14 days (Pb14-oral) and of 4-week-old nonexposed controls. Mice from each group were immunized with 10(8) sheep red blood cells by intravenous (i.v.), intraperitoneal (ip), or intratracheal (it) routes of immunization. Immunity was assessed by both hemagglutination and the enumeration of antibody-forming cells from the spleen and thoracic lymph nodes. All treatment groups had decreased thymus/body weight and spleen/body weight ratios whereas only Pbaero groups had enlarged livers. The most significant immunosuppression occurred in the ip-immunized Pb28-aero group. A significant suppression of humoral immunity was also observed in thoracic lymph node samples from Pbaero groups immunized it or iv. There was no apparent immunosuppression in any treatment group after iv immunization. These results indicate that aerosolized lead is more immunosuppressive than equivalent amounts of ingested lead. This is most likely due to the greater absorption of inhaled lead and the subsequent cytotoxicity of lead for cells in the draining lymph nodes.

Lung localization of antibody-forming cells stimulated in distant peripheral lymph nodes.

Previous studies have shown that antibody-forming cells (AFC) produced in the lung-associated lymph nodes after lung immunization enter the blood and are subsequently extravasated into immunized lung lobes. This study evaluated AFC in blood and lung lavage fluids following simultaneous stimulation of the thoracic and popliteal lymph nodes with two antigenically distinct immunogens. Five dogs were immunized in the hind feet with rabbit red blood cells (RRBC) and in the left cardiac lung lobe with sheep red blood cells (SRBC). The number of anti-SRBC and anti-RRBC AFC in the blood and lavage fluids was periodically evaluated. The results indicated that both immunizations significantly increased the number of AFC in the blood. The number of AFC to RRBC and SRBC antigens was significantly higher in the immunized lung lobes than in the control lung lobes. A comparison of the number of RRBC and SRBC AFC in the immunized or control lung lobes, relative to the number of RRBC and SRBC AFC in the blood, indicated that AFC to both antigens entered the lung at the same rate. We conclude that AFC produced in distant lymphoid tissues enter the lung from the blood as readily as AFC produced after lung immunization.

Effects of acute nitrogen dioxide exposure on cellular immunity after lung immunization.

The effects of acute NO2 exposure on antigen-specific cell-mediated lung immunity in Fischer 344 rats were evaluated. Animals were exposed for 24 hr to either room air or 5, 10, or 26 ppm NO2 before intratracheal immunization with 10(8) sheep red blood cells (SRBC). Cellular immunity was evaluated by antigen-specific lymphocyte stimulation assays of pooled lymphoid cell suspensions from either the thoracic lymph nodes or spleens. Elevated cellular immunity was observed after exposure to NO2. The ability of the 26 ppm NO2 exposure to increase cellular immunity seemed to parallel, and in some cases even exceed, that seen in control animals immunized with SRBC mixed with 2 X 10(7) heat-killed Bacillus Calmette-Guerin. These results support the theory that lung damage, and/or alterations of regulatory populations of immune cells, induced by agents such as NO2 can be responsible for the production of abnormally elevated immune responses to antigens deposited in the damaged lung.

Local antibody production against the murine toxin of Yersinia pestis in a golf ball-induced granuloma.

The method of Garra and Baygorria, for localized antibody production, has been adapted for obtaining high-titered and monospecific antibodies against the murine toxin of Yersinia pestis. A hollow, perforated plastic golf ball surgically implanted under the skin of rabbits induced the formation of a granuloma. When the murine toxin of Y. pestis was injected directly into the granulomatous cavity, an increased amount of antibody was found in the granuloma fluid as compared with serum or with serum antibody obtained by conventional immunization. The granuloma antibody consisted mainly of immunoglobulin G, probably produced locally by lymphoid cells of the granuloma. The immune granuloma fluid and the granuloma tissue were rich in plasma cells and lymphocytes. The chemical composition of the granuloma fluid indicated that it was a transudate.

Quantitative differentiation of Yersinia pestis strains by their murine toxin and fraction I contents.

Until now the serological typing of Yersinia pestis into subgroups has not proved possible because all the antigenic components are present in each isolate. Using acrylamide disk electrophoresis it was observed that differences exist in the protein distribution patterns of aqueous extracts from various Y. pestis isolates. One component that was especially plentiful in some Javanese and South American isolates was identified as the murine toxin. The amount of murine toxin varies significantly from isolate to isolate, and so permits them to be roughly grouped. In 28 degrees C cultures the variation in the murine toxin content is independent of the variation in the F-I content. A new method of typing based on this 2-trait variation in quantities, in contrast to classical typing based on qualitative differences, might prove to be a means of differentiating Y. pestis isolates.