InVivoMAb anti-rat CD90/mouse CD90.1 (Thy1.1)

InVivoMAb anti-rat CD90/mouse CD90.1 (Thy1.1) (CLONE: OX-7)

Liu X, Zhang J, Tang A, Xu L, Huang Y (2022). "A novel peptide ligand-coated nano-siRNA-lipoplex technology for kidney targeted gene therapy" Am J Transl Res 14(10):7362-7377. PubMed

Objectives: Small interfering RNA (siRNA) that silences specific disease-related genes holds the promise for the treatment of renal disease. However, delivery to the intended site of action remains a major obstacle. The goal of this study was to develop glomerulus-specific siRNA particles for targeted gene therapy of kidney diseases. Methods: We used a novel nanoparticle-based system comprised of siRNA in cationic liposomes (Lip) coated with non-inhibitory plasminogen activator inhibitor 1R (PAI-1R) that selectively targets glomerular cells and tested it with transforming growth factor-beta 1 (TGF-β1)-siRNA in nephritic rat model. Results: At the optimized ratio of components, three of PAI-1R, Lip and siRNA formed the compact nanostructured particles with close to neutral surface charge (+5.63 ± 1.45 mV) and relatively uniform size (68.9 ± 4.73 nm). When the fluorescence-conjugated siRNA was used, the labeled siRNA nanoparticles appeared specifically in glomeruli. Targeted delivery of siRNA specific to the TGFβ1 gene reduced elevated TGFβ1 mRNA expression and protein production in glomeruli, but had no effect on TGFβ1 mRNA levels in lung, spleen, artery or renal medulla, and in nephritic rats induced by injection of OX-7, for up to 5 days. PAI-1R-Lip-TGF-β1 siRNA administration significantly reduced increases in glomerular matrix accumulation and expression of PAI-1 and fibronectin. Conclusions: We conclude that a single dose of PAI-1R-Lip-TGF-β1 siRNA inhibited glomerular TGF-β1 gene expression thereby ameliorating glomerulosclerosis specifically and efficiently in nephritic rats without affecting most of other organs. The target silencing of genes critical for glomerular diseases may represent a promising treatment strategy for kidney disease.

Rodionova K, Veelken R, Hilgers KF, Paulus EM, Linz P, Fischer MJM, Schenker M, Reeh P, Tiegs G, Ott C, Schmieder R, Schiffer M, Amann K, Ditting T (2020). "Afferent renal innervation in anti-Thy1.1 nephritis in rats" Am J Physiol Renal Physiol 319(5):F822-F832. PubMed

Afferent renal nerves exhibit a dual function controlling central sympathetic outflow via afferent electrical activity and influencing intrarenal immunological processes by releasing peptides such as calcitonin gene-related peptide (CGRP). We tested the hypothesis that increased afferent and efferent renal nerve activity occur with augmented release of CGRP in anti-Thy1.1 nephritis, in which enhanced CGRP release exacerbates inflammation. Nephritis was induced in Sprague-Dawley rats by intravenous injection of OX-7 antibody (1.75 mg/kg), and animals were investigated neurophysiologically, electrophysiologically, and pathomorphologically 6 days later. Nephritic rats exhibited proteinuria (169.3 ± 10.2 mg/24 h) with increased efferent renal nerve activity (14.7 ± 0.9 bursts/s vs. control 11.5 ± 0.9 bursts/s, n = 11, P < 0.05). However, afferent renal nerve activity (in spikes/s) decreased in nephritis (8.0 ± 1.8 Hz vs. control 27.4 ± 4.1 Hz, n = 11, P < 0.05). In patch-clamp recordings, neurons with renal afferents from nephritic rats showed a lower frequency of high activity following electrical stimulation (43.4% vs. 66.4% in controls, P < 0.05). In vitro assays showed that renal tissue from nephritic rats exhibited increased CGRP release via spontaneous (14 ± 3 pg/mL vs. 6.8 ± 2.8 pg/ml in controls, n = 7, P < 0.05) and stimulated mechanisms. In nephritic animals, marked infiltration of macrophages in the interstitium (26 ± 4 cells/mm²) and glomeruli (3.7 ± 0.6 cells/glomerular cross-section) occurred. Pretreatment with the CGRP receptor antagonist CGRP_8-37 reduced proteinuria, infiltration, and proliferation. In nephritic rats, it can be speculated that afferent renal nerves lose their ability to properly control efferent sympathetic nerve activity while influencing renal inflammation through increased CGRP release.

Cove-Smith A, Sharpe CC, Shattock MJ, Hendry BM (2020). "Ion-Channel modulator TH1177 reduces glomerular injury and serum creatinine in chronic mesangial proliferative disease in rats" BMC Nephrol 21(1):187. PubMed

Background: T-type calcium channels (TTCC) are involved in mesangial cell proliferation. In acute thy-1 nephritis in the rat TTCC inhibition reduces glomerular damage and cell proliferation. This work is extended here by a study of the non-selective TTCC inhibitor TH1177 in a chronic model of proliferative glomerulonephritis (GN) including late treatment starting after the initial inflammation has resolved. The objective was to determine the effects of TH1177 in a model of chronic mesangioproliferative renal disease. Methods: Chronic GN was induced in WKY rats by unilateral nephrectomy (day - 7) followed by day 0 injection of Ox7 thy-1 mAb. Treatment with TH1177 (10-20 mg/Kg daily IP) was started on day 2 (early treatment) or on day 14 (late treatment) and compared to vehicle-treated controls until sacrifice at day 42. Glomerular disease was assessed with a damage score, fibrosis assay, cellular counts and renal function measured by serum creatinine. Results: Treatment with TH11777 was associated with reduced serum creatinine, less glomerular damage, reduced fibrosis and reduced glomerular cellularity. The results for early and late TH1177 treatments were essentially the same and differed significantly from vehicle. Conclusions: The ion-channel modulator TH1177 is capable of improving glomerular outcome in chronic rat GN even when treatment starts 14 days after initiation of the disease. These data are discussed in the context of the possible targets of TH1177 including TTCC, TRP family, Stim/Orai group and other cation channels. The work supports the use of genetic models to examine the roles of individual cation channels in progressive glomerulonephritis to further define the targets of TH1177.

Rodionova K, Hilgers KF, Paulus EM, Tiegs G, Ott C, Schmieder R, Schiffer M, Amann K, Veelken R, Ditting T (2020). "Neurogenic tachykinin mechanisms in experimental nephritis of rats" Pflugers Arch 472(12):1705-1717. PubMed

We demonstrated earlier that renal afferent pathways combine very likely "classical" neural signal transduction to the central nervous system and a substance P (SP)-dependent mechanism to control sympathetic activity. SP content of afferent sensory neurons is known to mediate neurogenic inflammation upon release. We tested the hypothesis that alterations in SP-dependent mechanisms of renal innervation contribute to experimental nephritis. Nephritis was induced by OX-7 antibodies in rats, 6 days later instrumented for recording of blood pressure (BP), heart rate (HR), drug administration, and intrarenal administration (IRA) of the TRPV1 agonist capsaicin to stimulate afferent renal nerve pathways containing SP and electrodes for renal sympathetic nerve activity (RSNA). The presence of the SP receptor NK-1 on renal immune cells was assessed by FACS. IRA capsaicin decreased RSNA from 62.4 ± 5.1 to 21.6 ± 1.5 mV s (*p < 0.05) in controls, a response impaired in nephritis. Suppressed RSNA transiently but completely recovered after systemic administration of a neurokinin 1 (NK1-R) blocker. NK-1 receptors occurred mainly on CD11⁺ dendritic cells (DCs). An enhanced frequency of CD11c⁺NK1R⁺ cell, NK-1 receptor⁺ macrophages, and DCs was assessed in nephritis. Administration of the NK-1R antagonist aprepitant during nephritis reduced CD11c⁺NK1R⁺ cells, macrophage infiltration, renal expression of chemokines, and markers of sclerosis. Hence, SP promoted renal inflammation by weakening sympathoinhibitory mechanisms, while at the same time, substance SP released intrarenally from afferent nerve fibers aggravated immunological processes i.e. by the recruitment of DCs.

Cove-Smith A, Mulgrew CJ, Rudyk O, Dutt N, McLatchie LM, Shattock MJ, Hendry BM (2013). "Anti-proliferative actions of T-type calcium channel inhibition in Thy1 nephritis" Am J Pathol 183(2):391-401. PubMed

Aberrant proliferation of mesangial cells (MCs) is a key finding in progressive glomerular disease. TH1177 is a small molecule that has been shown to inhibit low-voltage activated T-type Ca(2+) channels (TCCs). The current study investigates the effect of TH1177 on MC proliferation in vitro and in vivo. The effect of Ca(2+) channel inhibition on primary rat MC proliferation in vitro was studied using the microculture tetrazolium assay and by measuring bromodeoxyuridine incorporation. In vivo, rats with Thy1 nephritis were treated with TH1177 or vehicle. Glomerular injury and average glomerular cell number were determined in a blinded fashion. Immunostaining for Ki-67 and phosphorylated ERK were also performed. The expression of TCC isoforms in healthy and diseased tissue was investigated using quantitative real-time PCR. TCC blockade caused a significant reduction in rat MC proliferation in vitro, whereas L-type inhibition had no effect. Treatment of Thy1 nephritis with TH1177 significantly reduced glomerular injury (P < 0.005) and caused a 49% reduction in glomerular cell number (P < 0.005) compared to the placebo. TH1177 also reduced Ki-67-positive and pERK-positive cells per glomerulus by 52% (P < 0.01 and P < 0.005, respectively). These results demonstrate that TH1177 inhibits MC proliferation in vitro and in vivo, supporting the hypothesis that TCC inhibition may be a useful strategy for studying and modifying MC proliferative responses to injury.

Daniel C, Lüdke A, Wagner A, Todorov VT, Hohenstein B, Hugo C (2012). "Transgelin is a marker of repopulating mesangial cells after injury and promotes their proliferation and migration" Lab Invest 92(6):812-26. PubMed

Mesangial cell (MC) migration is essential during glomerular repair and kidney development. The aim of the study was to identify marker/player for glomerular progenitor/reserve cells migrating into the glomerulus after MC injury and during glomerulogenesis in the rat. Experimental mesangial proliferative nephritis was induced in Sprague Dawley rats by intravenous injection of OX-7 antibody. We investigated mRNA expression profiles in isolated glomeruli from on days 0, 1, 2, 3, and 5 after induction of anti-Thy1 nephritis using Affymetrix microarray technology. Using self-organizing maps, transgelin was identified as a new marker for repopulating glomerular cells. Expression of transgelin during anti-Thy1 nephritis was investigated by northern blot, real-time PCR, western blot, and immunohistochemistry. Migration and proliferation assays using isolated MCs after transgelin knockdown by siRNA were performed to investigate the potential role of transgelin during glomerular repopulation. Transgelin mRNA was not detected in healthy glomeruli. It was strongly upregulated during the repopulation process starting on day 1, continued to be increased until day 5 and disappeared on day 7. Transgelin was specifically expressed at the edge of the migratory front during glomerular repopulation as indicated by transgelin/OX-7 double staining. Transgelin expression was similar in migrating vs non-migrating MCs in vitro. Blocking of transgelin expression by siRNA treatment resulted in inhibition of MC migration and proliferation. Transgelin was also expressed in MCs during glomerulogenesis and in biopsies from patients with IgA nephritis. In conclusion, transgelin in the kidney is upregulated in repopulating MCs in vivo and supports their migratory and proliferative repair response after injury.

Suana AJ, Tuffin G, Frey BM, Knudsen L, Mühlfeld C, Rödder S, Marti HP (2011). "Single application of low-dose mycophenolate mofetil-OX7-immunoliposomes ameliorates experimental mesangial proliferative glomerulonephritis" J Pharmacol Exp Ther 337(2):411-22. PubMed

IgA nephropathy, one of the most frequent forms of glomerulonephritis, characterized by mesangial hypercellularity and glomerular extracellular matrix (ECM) expansion, often leads to end-stage renal disease over a prolonged period. We investigated whether antiproliferative treatment in a single low dose specifically targeted to the glomerular mesangium by immunoliposomes (ILs) results in an amelioration of mesangial proliferative glomerulonephritis in rats (anti-Thy1.1 nephritis). Mycophenolate mofetil (MMF) containing ILs was generated that targets the Thy1.1 antigen (OX-7) in rat mesangial cells. Treatment benefit of a single intravenous dose of these ILs given 2 days after disease induction was investigated by stereology, immunohistochemistry, and functional analyses (creatinine, albuminuria) until day +9 and was compared among untreated and free MMF-treated rats using six male Wistar rats per group. MMF-loaded OX7-IL prevented creatinine increase and albuminuria. Stereological analyses of MMF OX7-IL-treated animals yielded 30% reduction of mesangial cells on day +9 and a 40% reduction of glomerular ECM volume on day +5, compared with all of the other nephritic animals. Furthermore, at days +5 and +9 we observed decreased ECM content and decreased glomerular volume (day +5) in the MMF-OX7-IL-treated group compared with the nephritic group treated with free MMF. In conclusion, MMF-OX7-IL-based directed drug delivery represents a novel approach for treating mesangial cell-mediated forms of glomerulonephritis.

Tuffin G, Huwyler J, Waelti E, Hammer C, Marti HP (2008). "Drug targeting using OX7-immunoliposomes: correlation between Thy1.1 antigen expression and tissue distribution in the rat" J Drug Target 16(2):156-66. PubMed

OX7 monoclonal antibody F((ab')2) fragments directed against Thy1.1 antigen can be used for drug targeting by coupling to the surface of drug-loaded liposomes. Such OX7-conjugated immunoliposomes (OX7-IL) were used recently for drug delivery to rat glomerular mesangial cells, which are characterized by a high level of Thy1.1 antigen expression. In the present study, the relationship between OX7-IL tissue distribution and target Thy1.1 antigen localization in different organs in rat was investigated. Western blot and immunohistofluorescence analysis revealed a very high Thy1.1 expression in brain cortex and striatum, thymus and renal glomeruli. Moderate Thy1.1 levels were observed in the collecting ducts of kidney, lung tissue and spleen. Thy1.1 was not detected in liver and heart. There was a poor correlation between Thy1.1 expression levels and organ distribution of fluorescence- or (14)C-labeled OX7-IL. The highest overall organ density of OX7-IL was observed in the spleen, followed by lung, liver and kidney. Heart and brain remained negative. With respect to intra-organ distribution, a localized and distinct signal was observed in renal glomerular mesangial cells only. As a consequence, acute pharmacological (i.e. toxic) effects of doxorubicin-loaded OX7-IL were limited to renal glomeruli. The competition with unbound OX7 monoclonal antibody F((ab')2) fragments demonstrated that the observed tissue distribution and acute pharmacological effects of OX7-IL were mediated specifically by the conjugated OX7 antibody. It is concluded that both the high target antigen density and the absence of endothelial barriers are needed to allow for tissue-specific accumulation and pharmacological effects of OX7-IL. The liposomal drug delivery strategy used is therefore specific toward renal glomeruli and can be expected to reduce the risk of unwanted side effects in other tissues.

Daniel C, Albrecht H, Lüdke A, Hugo C (2008). "Nestin expression in repopulating mesangial cells promotes their proliferation" Lab Invest 88(4):387-97. PubMed

We investigated whether the intermediate filament protein and neural stem cell marker nestin characterizes the glomerular progenitor/reserve cell population immigrating the glomerulus after mesangial cell (MC) injury in the rat (anti-Thy1 nephritis). Nestin expression was investigated by immunohistochemistry and real-time PCR during anti-Thy1 nephritis. Migration and proliferation assays were used to characterize the function of nestin in isolated MCs after nestin knockdown by siRNA. After MC injury during anti-Thy1 nephritis, glomerular nestin was transiently increased during the repopulation phase. At the peak of mesangial proliferation and expansion (day 5) most OX-7-positive MCs expressed nestin largely colocalizing with the activation marker alpha-smooth muscle actin and the proliferation marker PCNA. In contrast to a healthy, non-injured mesangium in vivo, MCs in culture are considered to be in an 'activated, injured state' and express nestin in a generalized distribution with condensed localization around the nucleus as well as intensive staining of cell protrusions such as filopodia. During cell cycle, the percentage of MCs with high nestin levels was increased during S- aupnd G2-phase. Blocking of nestin using specific siRNA resulted in inhibition of cell proliferation but not cell migration. In conclusion, nestin is constitutively expressed in podocytes, but is a marker for repopulating MCs after experimental MC injury in vivo. Nestin promotes MC proliferation in vitro, suggesting a supporting role for nestin during repair reaction.

Uchimura H, Marumo T, Takase O, Kawachi H, Shimizu F, Hayashi M, Saruta T, Hishikawa K, Fujita T (2005). "Intrarenal injection of bone marrow-derived angiogenic cells reduces endothelial injury and mesangial cell activation in experimental glomerulonephritis" J Am Soc Nephrol 16(4):997-1004. PubMed

Loss of glomerular endothelial cells has been suggested to contribute to the progression of glomerular injury. Although therapeutic angiogenesis induced by administration of bone marrow-derived endothelial progenitor cells has been observed in disease models of endothelial injury, the effects on renal disease have not been clarified. Whether administration of culture-modified bone marrow mononuclear cells would mitigate the glomerular endothelial injury in anti-Thy1.1 nephritis was investigated. After cultivation under conditions that promote endothelial progenitor cell growth, bone marrow mononuclear cells were labeled with CM-DiI, a fluorescence marker, and injected into the left renal artery of Lewis rats with anti-Thy1.1 glomerulonephritis. The decrease in glomerular endothelial cells was significantly attenuated in the left kidney, as compared with the right, in nephritic rats that received the cell infusion. Glomerular injury score, the area positive for mesangial alpha-smooth muscle actin, and infiltration of macrophages were significantly decreased in the left kidney. CM-DiI-positive cells were distributed in glomeruli of the left kidney but not in those of the right kidney. Among CM-DiI-labeled cells incorporated into glomeruli, 16.5 +/- 1.2% of cells were stained with an endothelial marker, rat endothelial cell antigen-1. Culture-modified mononuclear cells secreted 281.2 +/- 85.0 pg of vascular endothelial growth factor per 10(5) cells per day. In conclusion, intra-arterial administration of culture-modified bone marrow mononuclear cells reduced endothelial injury and mesangial activation in anti-Thy1.1 glomerulonephritis. Incorporation into the glomerular endothelial lining and production of angiogenic factor(s) are likely to contribute to the protective effects of culture-modified mononuclear cells against glomerular injury.

Goldwich A, Baulmann DC, Ohlmann A, Flügel-Koch C, Schöcklmann H, Tamm ER (2005). "Myocilin is expressed in the glomerulus of the kidney and induced in mesangioproliferative glomerulonephritis" Kidney Int 67(1):140-51. PubMed

Background: Myocilin is a 55 to 57 kD secreted glycoprotein and member of the olfactomedin protein family. It is expressed in high amounts in the outflow tissues of the aqueous humor in the eye where it is supposed to contribute to outflow resistance. Myocilin is mutated in some forms of primary open angle glaucoma and affected patients show very high intraocular pressures because of an increase in resistance to aqueous humor outflow. To obtain information, if myocilin may play a comparable role in other tissues with transendothelial fluid flow, we investigated its expression in the rat kidney. Methods: The expression of myocilin in the normal rat kidney and its changes during mesangioproliferative glomerulonephritis were investigated by immunohistochemistry, one- and two-dimensional gel electrophoresis with Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR). Results: Myocilin and its mRNA were detected in isolated glomeruli. Immunohistochemistry showed specific labeling of glomerular cells, while tubular and interstitial regions were essentially negative. Double staining with the podocyte-specific markers synaptopodin and ezrin indicated that myocilin-positive cells were predominately podocytes. During mesangioproliferative glomerulonephritis, an induction of myocilin immunoreactivity was observed. Labeling for myocilin was now observed in activated mesangial cells and areas of glomerular sclerosis. In parallel cell culture experiments, mRNA for myocilin was detected in cultured murine podocytes and rat mesangial cells. Conclusion: Myocilin is expressed in podocytes of the kidney and induced in mesangial cells during experimental mesangioproliferative glomerulonephritis. The specific function of myocilin in the kidney is not clear, but in a parallel to functions of other olfactomedin proteins, it might have a role in cell-cell adhesion and/or signaling processes.

Brügger B, Graham C, Leibrecht I, Mombelli E, Jen A, Wieland F, Morris R (2004). "The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition" J Biol Chem 279(9):7530-6. PubMed

Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or "rafts" on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (</=3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained approximately 20% of prion protein. The lipid content of prion protein and Thy-1 DRMs was measured by quantitative nano-electrospray ionization tandem mass spectrometry. In four independent preparations, the lipid content was highly reproducible, with Thy-1 and prion protein DRMs differing markedly from each other and from the total DRM pool from which they were immunoprecipitated. Prion protein DRMs contained significantly more unsaturated, longer chain lipids than Thy-1 DRMs and had 5-fold higher levels of hexosylceramide. The different lipid compositions are in keeping with the different trafficking dynamics and solubility of the two proteins and show that, under the conditions used, DRMs can isolate individual membrane microenvironments. These results further identify unsaturation and glycosylation of lipids as major sources of diversity of raft structure.

Ikezumi Y, Hurst LA, Masaki T, Atkins RC, Nikolic-Paterson DJ (2003). "Adoptive transfer studies demonstrate that macrophages can induce proteinuria and mesangial cell proliferation" Kidney Int 63(1):83-95. PubMed

Background: Glomerular macrophage accumulation is a feature of proliferative human and experimental glomerulonephritis. However, our understanding of the role of macrophages in the induction of renal injury is based upon indirect evidence from depletion studies, most of which lack specificity for this cell type. Therefore, an adoptive transfer approach was used to directly assess the potential of macrophages to induce renal injury. Methods: Accelerated anti-glomerular basement membrane (anti-GBM) disease was induced in rats by immunization with sheep IgG (day -5), followed by administration of sheep anti-rat GBM serum (day 0), with animals killed on day 2. To facilitate the adoptive transfer studies, immunized animals were made leukopenic by cyclophosphamide (CyPh) given on day -2. Bone marrow-derived (BM) or NR8383 macrophages were transferred by tail vein injection 24 hours after injection of anti-GBM serum, with animals killed 3 or 24 hours after transfer. Results: Pretreatment with CyPh prevented glomerular leukocyte accumulation and completely inhibited proteinuria, glomerular cell proliferation and hypercellularity in accelerated anti-GBM disease. Adoptive transfer led to significant glomerular accumulation of BM or NR8383 macrophages within 3 hours of injection, and this was still evident 24 hours later. Adoptive transfer of BM or NR8383 macrophages induced proteinuria (63 +/- 16 BM vs. 5 +/- 2 mg/24 h CyPh control; P < 0.001), glomerular cell proliferation (5.1 +/- 1.2 BM vs. 0.5 +/- 0.1 PCNA+ cells/gcs CyPh; P < 0.001) and glomerular hypercellularity (51.2 +/- 2.0 BM vs. 41.9 +/- 0.9 nuclei/gcs CyPh; P < 0.001). The degree of renal injury correlated with the number of transferred glomerular macrophages. Two-color immunostaining demonstrated that most glomerular proliferative cell nuclear antigen+ (PCNA+) proliferating cells were OX-7+ mesangial cells. CyPh treatment did not prevent up-regulation of glomerular intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) expression or an increase in urinary monocyte chemoattractant protein-1 (MCP-1) excretion. Conclusion: This study provides the first direct evidence that macrophages can induce renal injury in terms of proteinuria and mesangial cell proliferation.

Larmonier N, Ghiringhelli F, Larmonier CB, Moutet M, Fromentin A, Baulot E, Solary E, Bonnotte B, Martin F (2003). "Freshly isolated bone marrow cells induce death of various carcinoma cell lines" Int J Cancer 107(5):747-56. PubMed

In some carcinomas such as digestive tract carcinomas, bone marrow infiltration by tumor cells is a frequent event but usually remains a micrometastatic disease and rarely induces overt bone lesions. The mechanisms responsible for the control of these metastases in the bone marrow remain poorly known. We show that freshly isolated bone marrow cells from human, murine and rat origin rapidly kill a wide range of syngeneic or xenogeneic carcinoma cell lines in culture. Further analysis of this cytotoxic process in the rat indicated that neither resident bone marrow macrophages nor NK cells were responsible for this cytotoxic effect that was restricted to a subpopulation of bone marrow cells expressing CD90 (Thy-1), a marker of hemopoietic precursors. The tumoricidal activity of these cells did not require long-term culture nor addition of exogenous cytokines or growth factors. A subset of CD90+ cells that rapidly differentiates into CD163(ED2)-expressing macrophages was observed to be responsible for tumor cell killing. These macrophages induced a non-apoptotic death of tumor cells, a process that required both a direct interaction with the tumor cell and nitric oxide (NO) production through the activation of inducible nitric oxide-synthase (iNOS). This ability of pluripotent hemopoietic stem cells to rapidly differentiate into macrophages capable of killing invasive tumor cells may account for the limited expansion of micrometastases of some carcinomas in the bone marrow.

Ishizu A, Ogawa Y, Ishikura H, Yoshiki T (2001). "Anti-Thy-1 monoclonal antibody with specific reactivity with vascular endothelial cells in rat glomeruli" Acta Histochem 103(3):279-86. PubMed

Inoculation with anti-Thy-1 antibodies (Abs) in rats induces glomerulonephritis resembling human mesangiolytic and/or mesangioproliferative diseases. Some anti-Thy-1 monoclonal Abs (mAbs) react with both mesangial and glomerular endothelial cells, whereas others react solely with mesangial cells in rat kidney. These findings suggest that the rat Thy-1 molecule possesses at least 2 variant forms, including a mesangial and a vascular endothelial isoform. However, anti-Thy-1 mAbs with specific reactivity with glomerular endothelial cells have not been available. We describe here a unique anti-rat Thy-1 mAb, TM78-8. The epitope for TM78-8 is closely related, but not identical, to that for OX-7, a commercially available anti-rat Thy-1 mAb. Immunoblotting, immunohistochemistry and immunoelectron microscopy confirm that TM78-8 reacts exclusively with Thy-1 antigens on the surface of vascular endothelial cells in rat glomeruli. TM78-8 may be a suitable marker for rat glomerular endothelial cells as well as for the vascular endothelial isoform of the rat Thy-1 molecule. Intravenous injection of TM78-8 did not induce glomerulonephritis in rats, whereas OX-7 did, indicating that TM78-8 is not nephritogenic. This finding also corresponds with the current consensus that Thy-1 antigens expressed on mesangial cells play an essential role in the development of Thy-1 nephritis.

Erben RG, Raith S, Eberle J, Stangassinger M (1998). "Ovariectomy augments B lymphopoiesis and generation of monocyte-macrophage precursors in rat bone marrow" Am J Physiol 274(3 Pt 1):E476-83. PubMed

To investigate the effects of estrogen depletion on hematopoiesis and bone turnover, female rats were either ovariectomized (OVX) or sham operated and killed at 1, 2, 3, and 4 wk postsurgery. Flow cytometric analysis of bone marrow cells (BMC) revealed that, in close temporal association with the rise in bone turnover as measured by bone histomorphometry, the number of Thy 1.1+ and KiB1R+ BMC increased two- to threefold in OVX rats relative to sham controls. The Thy 1.1+ BMC were further characterized as Thy 1.1+/KiB1R+ and Thy 1.1+/HIS24+ double-positive cells of the B cell lineage. A transient rise in ED1+ myeloid cells expressing a lysosomal antigen specific for the monocyte-macrophage and osteoclast lineage coincided with the upregulation of osteoclast numbers in OVX rats at 2 wk postsurgery, but the number of ED8+ myelomonocytic BMC remained unchanged. Administration of estradiol prevented the rise in Thy 1.1+, KiB1R+, and ED1+ BMC in OVX animals. Our study indicates that ovariectomy upregulates B lymphopoiesis in rat bone marrow and increases myeloid cell differentiation into the monocyte-macrophage and possibly also the osteoclast lineage.

Jeng CJ, McCarroll SA, Martin TF, Floor E, Adams J, Krantz D, Butz S, Edwards R, Schweitzer ES (1998). "Thy-1 is a component common to multiple populations of synaptic vesicles" J Cell Biol 140(3):685-98. PubMed

Thy-1, a glycosylphosphatidylinositol-linked integral membrane protein of the immunoglobulin superfamily, is a component of both large dense-core and small clear vesicles in PC12 cells. A majority of this protein, formerly recognized only on the plasma membrane of neurons, is localized to regulated secretory vesicles. Thy-1 is also present in synaptic vesicles in rat central nervous system. Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel. These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

Eitner F, Westerhuis R, Burg M, Weinhold B, Gröne HJ, Ostendorf T, Rüther U, Koch KM, Rees AJ, Floege J (1997). "Role of interleukin-6 in mediating mesangial cell proliferation and matrix production in vivo" Kidney Int 51(1):69-78. PubMed

Mesangial cell proliferation and matrix overproduction characterize many progressive glomerular diseases. Based on currently available data, the role of interleukin-6 (IL-6) in mediating mesangial cell proliferation and matrix production is controversial. The present study attempts to clarify this issue by showing that: (1) IL-6 knock out mice develop a normal glomerular architecture and in particular a normal mesangium. (2) Mesangioproliferative glomerulonephritis induced by Habu snake venom is equally severe in IL-6 knock out mice as in control mice. (3) A continuous seven-day intraperitoneal infusion of 50 micrograms recombinant human IL-6 into rats with a prior minimal (subnephritogenic) injury to mesangial cells does not induce glomerular cell activation, cell proliferation, matrix production, leukocyte influx, platelet influx or proteinuria. (4) A continuous seven-day IL-6 infusion into rats with mesangioproliferative nephritis (anti-Thy 1.1 nephritis) increases matrix protein transcription in the absence of detectable effects on matrix protein accumulation and otherwise has no effect on the natural course of the disease. We conclude from these findings that IL-6 is not an important mediator of mesangial cell proliferation and matrix overproduction in vivo, and that currently little rationale exists to advocate anti-IL-6 therapy in mesangioproliferative disease states.

Crook K, Hunt SV (1996). "Enrichment of early fetal-liver hemopoietic stem cells of the rat using monoclonal antibodies against the transferrin receptor, Thy-1, and MRC-OX82" Dev Immunol 4(4):235-46. PubMed

Fetal livers from inbred rat fetuses at 14 days' gestation were dispersed into a single-cell suspension by physical disruption and collagenase digestion. Pluripotent stem cells were characterized and partially purified by a combination of monoclonal antibodies. These included CD71 (anti-transferrin receptor, MRC-OX26, used for rosetting), Cdw90 (anti-Thy-1, MRC-OX7), and the newly described MRC-OX82 (reacting with myeloiid cells in peritoneal exudate), employed in FACS sorting. Enrichment was monitored by long-term reconstitution of lethally irradiated congenic rats genetically distinguishable from the donor by an allelomorphic variant of the CD45 cell-surface antigen. At intervals from 3 months to 1 year, lymph-node cells and peritoneal exudate cells were biopsied for analysis by two-color flow cytometry--one color to determine donor origin, the other to identify Th cell (CD4+), Tc cell (CD8+), B cell (sIg+ or CD45RC+), neutrophil (OX82+ or OX43-), and macrophage (OX43+) compartments. The degree of chimaerism was taken as the read out of stem-cell activity. No significant differentials between lymph-node and peritoneal exudate chimaerisms were detected in any of the recipients; therefore, the enrichment procedure revealed only pluripotent cells, not stem cells of restricted potency. All recovered stem-cell activity was in the OX26(CD71)-negative, OX7(CDw90)-positive, OX82-positive fraction. In the optimum case, an enrichment of very roughly 200-fold in cell-for-cell activity was obtained. Rat bone-marrow colony-forming units in the spleen (CFUs-12) were found to lack the surface antigens recognized by the monoclonal antibodies CD53 (MRC-OX44), MRC-OX39, MRC-OX59, and 144.2.15. These would provide a strategy for their enrichment by depletion.

Gessl A, Krugluger W, Langer K, Baumgartner I, Spittler A, Grabner G, Förster O, Boltz-Nitulescu G (1995). "Expression of MHC class II antigens on rat bone marrow cells and macrophages, and their modulation during culture with murine GM-CSF or M-CSF" Immunobiology 192(3-4):185-97. PubMed

Flow cytometric analysis employing MRC OX 6 and MRC OX17 monoclonal antibodies recognizing determinants on RT1.B or RT1.D molecules, equivalent to murine I-A and I-E, respectively, was used to detect rat MHC class II antigen (Ag) expression. Approximately 5% of freshly isolated rat bone marrow cells (BMC) expressed RT1.B and over 30% displayed RT1.D molecules. The RT1.D+ cells were W3/13+, OX 7+, OX 19- and OX 22-. After one week culture of BMC with murine recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF), regardless of concentrations, 90 to 95% of the cells were scored as bone marrow-derived macrophages (BMDM phi), and over 30% expressed both RT1.B and RT1.D Ag. GM-CSF increases the percentage of BMDM phi bearing MHC class II Ag in a concentration-dependent manner. This effect seems to be specific because antibodies to interferon-gamma, tumor necrosis factor-alpha or interleukin-4 did not reduce the number of cells expressing RT1.B and RT1.D Ag. Furthermore, GM-CSF was able to trigger expression of class II molecules on rat peritoneal macrophages (M phi) and BMDM phi resulted from cultures of BMC with mouse M phi-CSF (M-CSF), and the RT1.B and RT1.D inducing effect of GM-CSF was opposed by M-CSF, and by anti-GM-CSF antibodies. The induction of MHC class II Ag synthesis by GM-CSF on rat BMDM phi was confirmed at the mRNA level by Northern blot analysis employing cDNA probes encoding the RT1.B alpha.

Ishizu A, Ishikura H, Nakamaru Y, Takeuchi E, Kimura C, Koike T, Yoshiki T (1995). "Thy-1 induced on rat endothelium regulates vascular permeability at sites of inflammation" Int Immunol 7(12):1939-47. PubMed

We investigated the role of surface adhesion molecules on endothelial cells in regulating vascular permeability, in vitro and in vivo. Cultured rat endothelial cells (REC) express Thy-1, intercellular adhesion molecule-1 (ICAM-1), CD44 and RT1A. Permeability of albumin across the REC monolayer increased through the interaction of Thy-1 and anti-Thy-1 mAb, but not through ICAM-1 and anti-ICAM-1, CD44 and anti-CD44, and RT1A and anti-RT1A mAb. This anti-Thy-1-effect was completely inhibited when the calmodulin antagonist W-7 and the protein kinase inhibitor H-7 was combined, while the IL-6-mediated increase in REC permeability was blocked by either W-7 or H-7, independently. The anti-Thy-1-mediated permeability increase was additively augmented when IL-6 was admixed. These data suggest that intracellular signaling pathways of anti-Thy-1- and IL-6-mediated permeability regulation may be overlapping to some extent but are largely independent. As anti-Thy-1-treatment generated rearrangement of vimentin filaments within REC, alteration of the cytoskeleton distribution may possibly correlate with the regulation of permeability. Although Thy-1-expression on rat vascular endothelium in vivo was not evident, it was induced at sites of Freund's complete adjuvant-induced dermatitis. The administered anti-Thy-1 mAb exclusively located on vascular endothelial surface at the sites of inflammation. Vascular permeability in inflamed skin tissues was significantly augmented when anti-Thy-1 but not anti-ICAM-1, anti-CD44 or anti-RT1A mAb was administered i.v., without affecting populations of inflammatory cells. The collective evidence suggests that Thy-1 induced on rat endothelium is one important regulatory event in vascular permeability at sites of inflammation.

Garnett D, Barclay AN, Carmo AM, Beyers AD (1993). "The association of the protein tyrosine kinases p56lck and p60fyn with the glycosyl phosphatidylinositol-anchored proteins Thy-1 and CD48 in rat thymocytes is dependent on the state of cellular activation" Eur J Immunol 23(10):2540-4. PubMed

Cell surface glycoproteins anchored to the plasma membrane via glycosylphosphatidylinositol (GPI) structures, and hence having no cytoplasmic domains, can nevertheless transmit activation signals in lymphocytes. By immunoprecipitation from detergent lysates and in vitro immune complex kinase reactions the GPI-anchored molecules Thy-1 and CD48 are shown to be associated with multimolecular complexes of phosphoproteins including the protein tyrosine kinases p56lck and p60fyn in both rat and mouse thymocytes. Moreover, the kinase activity associated with Thy-1 on rat thymocytes is shown to be dependent on the activation state of the cells, with stimulation by the lectin, concanavalin A, producing a marked decrease in Thy-1-associated kinase activity. In such activated cells, there is an increased association of kinase activity with CD48, but this may be explained in terms of increased surface expression of CD48 and of increased total kinase activity. Additional phosphoproteins of 85, 36 and 32 kDa were consistently seen as components of the complexes.

Nakashima I, Zhang YH, Rahman SM, Yoshida T, Isobe K, Ding LN, Iwamoto T, Hamaguchi M, Ikezawa H, Taguchi R (1991). "Evidence of synergy between Thy-1 and CD3/TCR complex in signal delivery to murine thymocytes for cell death" J Immunol 147(4):1153-62. PubMed

The potential role of Thy-1 in CD3/TCR complex-mediated signal delivery to murine thymocytes was studied. Ag-mimicking cross-linked anti-CD3 mAb stimulated suspension of thymocytes from adult (6 to 8 wk old) mice for a brisk free cytoplasmic calcium ion ([Ca2+]i) rise, low level of inositol phosphate production, and marginal increase in tyrosine-specific phosphorylation of 110/120-kDa and 40-kDa cellular proteins. Weak but sustained [Ca2+]i rise, low inositol phosphate production, and weak protein tyrosine phosphorylation were also induced by the cross-linked anti-Thy-1 mAb that mimicked the putative natural ligand. The signal delivered via either of these two pathways was however insufficient for definitively promoting cell death and DNA fragmentation in the adult thymocytes. Here we demonstrated that anti-Thy-1 mAb synergized with anti-CD3 mAb for inducing a long-lasting prominent [Ca2+]i rise, definite inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakiphosphate production, and extensive tyrosine-specific phosphorylation of 110/120-, 92-, 75-, and 40-kDa proteins, which resulted in marked promotion of cell death and DNA fragmentation in the adult thymocytes. This unique anti-Thy-1 antibody activity was confirmed to be directed to glycosylphosphatidylinositol-anchored Thy-1, and was distinguished from the known anti-L3T4 activity that augmented the CD3-mediated signal transduction in a different manner. The synergistic actions of anti-CD3 and anti-Thy-1 mAb obligatorily required the cross-linking of the two mAb together. The anti-CD3 and anti-Thy-1 mAb cross-linked together acted on immature thymocytes from newborn (less than 24 h after birth) mice for rather more extensive promotion of protein tyrosine phosphorylation and cell death. In addition, they affected peripheral T lymphocytes for accelerating protein tyrosine phosphorylation but not cell death. These results suggest a novel function of glycosylphosphatidylinositol-anchored Thy-1 as a possible unique intrathymic intensifier of the CD3/TCR complex-delivered signal for negative thymocyte selection.

Mjaaland S, Fossum S (1990). "Modulation of immune responses with monoclonal antibodies. I. Effects on regional lymph node morphology and on anti-hapten responses to haptenized monoclonal antibodies" Eur J Immunol 20(7):1457-61. PubMed

Repeated injections of monoclonal antibody (mAb) culture supernatants into rat footpads increased the weights of the draining lymph nodes. Immunostained freeze sections showed that injection of MRC OX2, a mAb reacting with rat follicular dendritic cells and MRC OX7 (anti-Thy-1.1), led to gross hypertrophy primarily of the follicular areas, whereas MRC OX6 (anti-rat major histocompatibility complex class II molecules) resulted in selective stimulation of the paracortex. These findings indicate that mAb, when conjugated to certain antigens, would modulate the immune response to these antigens. Consequently, the mAb were conjugated with fluorescein isothiocyanate (FITC) and the humoral response against the hapten measured. The primary anti-FITC antibody response was tenfold stronger than after stimulation with FITC conjugated to a conventional carrier such as ovalbumin, and had some characteristics of a secondary response: a fast increase of IgG level to very high titers and a long duration without further amplification at later antigen challenges.

Dráberová L (1989). "The involvement of Thy-1 antigen in the activation of rat mast cells" Eur J Immunol 19(9):1715-20. PubMed

The Thy-1 antigen expressed on the surface of mouse T lymphocytes has been previously found to be involved in T cell activation. In the present study we have employed the anti-Thy-1.1 monoclonal antibody MRC OX7 and analyzed the expression and properties of Thy-1 antigen on the surface of peritoneal and pleural rat mast cells. Direct radioantibody binding assays, indirect immunofluorescence studies and flow cytometry analysis revealed that isolated rat mast cells express on their surface large amounts of the Thy-1 antigen. Scatchard analysis of the binding data indicated that at least one million Thy-1 molecules are expressed per cell. Immunoprecipitation studies carried out on mast cells demonstrated that MRC OX7 antibody recognizes a surface molecule of approximately 25 kDa that appears to correspond to Thy-1 antigen, and an additional molecule of 50 kDa. Incubation of the isolated mast cells with various concentrations of anti-Thy-1.1 antibody induced a rapid and concentration-dependent increase in the intracellular free calcium concentration ([Ca2+]i). The early increase in [Ca2+]i was observed in both Ca2+-supplemented and Ca2+-free media. This indicated that the initial increase in [Ca2+]i is due to a release of Ca2+ from internal stores. The [Ca2+]i increase was followed by an increase in the histamine release which was also dependent on antibody concentration. These data suggest that Thy-1 may act as an activation receptor on mast cells, analogously to its receptor function on T cells.