InVivoMAb anti-rat pan-CD45 (OX1)

InVivoMAb anti-rat pan-CD45 (OX1) (CLONE: OX-1)

Williams KA, Standfield SD, Smith JR, Coster DJ (2005). "Corneal graft rejection occurs despite Fas ligand expression and apoptosis of infiltrating cells" Br J Ophthalmol 89(5):632-8. PubMed

Background/aims: Constitutive expression of Fas ligand (CD95L) protects the eye against cell mediated immune responses by inducing apoptosis in infiltrating Fas bearing T cells. This study was designed to examine Fas ligand expression on acutely rejecting rat corneal grafts and to investigate the kinetics of induction of apoptosis in infiltrating leucocytes. Methods: Orthotopic penetrating corneal transplantation was performed between genetically disparate inbred rats. Fas ligand expression and the phenotype of infiltrating leucocytes were examined by immunohistochemistry. Apoptotic nuclei were visualised in sections of normal rat cornea, rejecting allografts, and time matched isografts by terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling (TUNEL) and quantified by video image analysis. Staining with Hoechst dye 33258 was used to confirm the presence of apoptotic nuclei. Results: Fas ligand was expressed on corneal endothelial and epithelial cells during acute corneal graft rejection. At all time points examined, including as early as the fifth postoperative day, the cells infiltrating both corneal isografts and allografts were TUNEL positive. By the 15th postoperative day, over 90% of all nuclei, many of which were T cells, were apoptotic. Conclusion: Expression of Fas ligand is not downregulated on the cornea during allograft rejection and infiltrating leucocytes in both isografts and allografts die rapidly in situ. Despite the death of the cells believed to be responsible for rejection, isografts survive indefinitely whereas allografts are irreparably damaged.

Foster-Cuevas M, Wright GJ, Puklavec MJ, Brown MH, Barclay AN (2004). "Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor" J Virol 78(14):7667-76. PubMed

Many viral proteins limit host immune defenses, and their genes often originate from their hosts. CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) that is implicated in locally preventing macrophage activation. Distant, but recognizable, homologues of CD200 have been identified in many herpesviruses and poxviruses. Here, we show that the product of the K14 open reading frame from human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) interacts with human CD200R and is expressed at the surfaces of infected cells solely during the lytic cycle. Despite sharing only 40% primary sequence identity, K14 and CD200 interacted with CD200R with an almost identical and low affinity (K(D) = 0.5 microM), in contrast to other characterized viral homologue interactions. Cells expressing CD200 or K14 on the cell surface were able to inhibit secretion by activated macrophages of proinflammatory cytokines such as tumor necrosis factor alpha, an effect that could be specifically relieved by addition of monoclonal antibodies and soluble monomeric CD200 protein. We conclude that CD200 delivers local down-modulatory signals to myeloid cells through direct cell-cell contact and that the K14 viral homologue closely mimics this.

Matsuda M, Shikata K, Shimizu F, Suzuki Y, Miyasaka M, Kawachi H, Kawashima H, Wada J, Sugimoto H, Shikata Y, Ogawa D, Tojo SJ, Akima K, Makino H (2002). "Therapeutic effect of sulphated hyaluronic acid, a potential selectin-blocking agent, on experimental progressive mesangial proliferative glomerulonephritis" J Pathol 198(3):407-14. PubMed

The initial event in the process of leukocyte infiltration is characterized by leukocyte rolling on the surface of the endothelium, which is mediated by selectins. P- and L-selectin bind to the sulphated sugar chains of their natural ligands, including sulphated glycolipids such as sulphatide. Recently, it has been demonstrated that sulphated glycolipids and sulphated oligosaccharides interfere with selectin binding pathways. This study synthesized sulphated hyaluronic acid (SHA), which is a potential selectin-blocking agent, and examined its therapeutic effect on the experimental progressive mesangial proliferative glomerulonephritis induced by anti-Thy-1 monoclonal antibody (1-22-3 MAb) after unilateral nephrectomy. The selectin-inhibitory effect of SHA in vitro was confirmed. SHA inhibited the binding of P- and L-selectin to sulphatide, which is a glycolipid ligand for P- and L-selectin, at a concentration of 1.5 micro g/ml and 100 micro g/ml. Immunohistochemical examination showed that P-selectin was up-regulated in the glomeruli in the 1-22-3 MAb nephritis model, while the ligands for L-selectin were not detected in the glomerular tufts. A single administration of SHA ameliorated proteinuria and glomerular leukocyte infiltration in 24 h after the injection of anti-Thy-1 MAb. Anti-P-selectin MAb, but not anti-L-selectin MAb, inhibited proteinuria and glomerular leukocyte infiltration. To examine further the therapeutic effect of SHA on chronic glomerulonephritis, SHA was administered daily from day 3 to day 14 in this model. Proteinuria and glomerular leukocyte infiltration were significantly diminished in SHA-treated rats on day 14. These results suggest that SHA ameliorated rat progressive mesangial proliferative glomerulonephritis by inhibiting P-selectin-dependent leukocyte infiltration in glomeruli. Sulphated oligosaccharides may be beneficial for the therapy of mesangial proliferative glomerulonephritis.

Morioka Y, Koike H, Ikezumi Y, Ito Y, Oyanagi A, Gejyo F, Shimizu F, Kawachi H (2001). "Podocyte injuries exacerbate mesangial proliferative glomerulonephritis" Kidney Int 60(6):2192-204. PubMed

Background: From the observations of morphology seen in early phases of the experimental models of the irreversible mesangial proliferative glomerulonephritis, we hypothesized that podocyte injury is one of the important factors in bringing upon irreversible glomerular alterations. To verify this hypothesis, we investigated whether podocyte injury induced by puromycin aminonucleoside (PAN) injection affects the mesangial alterations of anti-Thy 1.1 glomerulonephritis. Methods: Female Wistar rats were injected with 0.5 mg monoclonal antibody (mAb) 1-22-3 five days after the injection of 10 mg or 5 mg/100 g body weight (BW) of puromycin aminonucleoside (PAN), and sacrificed at 7 days or 8 weeks after the mAb 1-22-3 injection. Results: Consecutive injections of 10 mg/100 g BW of PAN and mAb 1-22-3 caused the irreversible mesangial alteration with persistent proteinuria (at week 8, proteinuria 100.3 +/- 57.8 mg/24 h, matrix score 1.13 +/- 0.52, collagen type I score 2.04 +/- 0.53, mRNA for collagen type I 227 +/- 79% to the group with a single injection of 1-22-3). Although single injection of 5 mg/100 g BW of PAN was not capable of inducing abnormal proteinuria, consecutive injections of 5 mg/100 g BW of PAN and mAb 1-22-3 also caused irreversible mesangial alteration and persistent proteinuria. Conclusions: Podocyte injury might be an important factor that exacerbates mesangial proliferation and mesangial matrix expansion. The irreversible mesangial alterations caused by consecutive injections of PAN and mAb 1-22-3 may be a novel model that could be used to analyze the mechanism of progressive mesangial alteration.

Takazoe K, Tesch GH, Hill PA, Hurst LA, Jun Z, Lan HY, Atkins RC, Nikolic-Paterson DJ (2000). "CD44-mediated neutrophil apoptosis in the rat" Kidney Int 58(5):1920-30. PubMed

Background: Apoptosis is an important mechanism by which neutrophils are removed from sites of inflammation, including the kidney. This study investigated whether ligation of the cell-surface adhesion molecule, CD44, can trigger neutrophil apoptosis. Methods: The anti-rat CD44 antibody OX-50 was used to induce apoptosis of cultured blood neutrophils, as determined by flow cytometry using annexin V staining and by transmission electron microscopy. The functional consequences of OX-50-mediated neutrophil depletion were examined in a rat model of accelerated antiglomerular basement membrane glomerulonephritis. Results: Flow cytometric analysis using the OX-50 antibody, which recognizes the common amino terminal domain of CD44, showed that rat blood neutrophils express very high levels of CD44. The addition of OX-50, but not control antibodies, rapidly induced neutrophil apoptosis in cultured rat blood leukocytes, as demonstrated by annexin V staining and by electron microscopy. Cross-linking of CD44 was essential since F(ab) fragments of the OX-50 antibody failed to induce neutrophil apoptosis. The CD44 ligand hyaluronan and an antibody to the CD44v6 isoform failed to induce neutrophil apoptosis, indicating that OX-50 antibody-mediated neutrophil apoptosis is epitope specific. This effect was specific to neutrophils since the OX-50 antibody did not induce apoptosis in other CD44-expressing cell types (lymphocytes, mesangial cells, or tubular epithelial cells). An injection of OX-50 antibody into normal rats caused a rapid and profound neutropenia, and apoptotic neutrophils could be seen in the blood by electron microscopy. Furthermore, the administration of OX-50 antibody abrogated neutrophil-dependent glomerular injury (proteinuria) on day 1 of rat antiglomerular basement membrane glomerulonephritis, whereas injury on day 10 of the disease (neutrophil independent) was largely unaffected. Conclusions: The cross-linking of specific epitopes of the CD44 molecule can rapidly induce neutrophil apoptosis in vitro and inhibit neutrophil-dependent renal injury in vivo. This finding suggests that physiological ligands of the CD44 molecule may play an important role in eliminating neutrophils from sites of inflammation, including inflammatory kidney disease.

de Water R, Noordermeer C, Houtsmuller AB, Nigg AL, Stijnen T, Schröder FH, Kok DJ (2000). "Role of macrophages in nephrolithiasis in rats: an analysis of the renal interstitium" Am J Kidney Dis 36(3):615-25. PubMed

Interstitial calcium oxalate (CaOx) crystals can be found in primary oxalosis and in secondary hyperoxaluria. In a rat model for nephrolithiasis, we investigated whether such crystals can be removed by the surrounding interstitial cells. CaOx crystals were induced by a crystal-inducing diet based on ethylene glycol (EG) and ammonium chloride (CID). Both lithogenic compounds were added to the drinking water. After 9 days, the animals received normal drinking water for 2 days. Using this CID, only the interstitial crystals are retained. Subsequently, half of the population remained on normal drinking water (normo-oxaluria), whereas the other half received a low dose of EG alone (chronic hyperoxaluria). The rats were killed at regular times thereafter. The results showed that the kidney-associated oxalate significantly declined during normo-oxaluria, but remained high during chronic hyperoxaluria. Interstitial cells positive for the leukocyte common antigen (CD45; which identifies all types of leukocytes), the ED1 antigen (which is specific for monocytes and macrophages), and the major histocompatibility class II antigen (MCHII), respectively, had increased in number, with minor differences between both rat populations. The cells around the interstitial crystals were mostly positive for ED1. Multinucleate giant cells were regularly observed. These cells were positive for CD45 and ED1 and sometimes also for MCHII. The crystals in these cells were moderately positive for acid phosphatase and carbonic anhydrase II. It is concluded that interstitial CaOx crystals can be removed under normo-oxaluric conditions and that, in all likelihood, macrophages and multinucleate giant cells are involved in that process.

de Water R, Noordermeer C, van der Kwast TH, Nizze H, Boevé ER, Kok DJ, Schröder FH (1999). "Calcium oxalate nephrolithiasis: effect of renal crystal deposition on the cellular composition of the renal interstitium" Am J Kidney Dis 33(4):761-71. PubMed

Urinary calcium oxalate (CaOx) crystals and crystal agglomerates are normally harmlessly excreted, but in nephrolithiasis they are retained by tubular epithelial cells and shifted into the renal interstitium. This crystalline material induces an inflammatory response consisting of an increase in the number of interstitial cells and an expansion of the extracellular matrix. The newly arrived cells either derive from the blood or the connective tissue or they are formed by local proliferation. Identification of the cells that surround the interstitial crystals is a first step in investigating the question of whether the interstitial cells could remove the crystalline material. Therefore, we performed an immunohistochemical study on the kidneys of rats made hyperoxaluric by ethylene glycol (EG) and ammonium chloride (AC). Attention was paid to expression of the leukocyte common antigen (LCA), which identifies all types of leukocytes, the ED1 antigen, which is specific for monocytes and macrophages, and the major histocompatibility class II antigen (MHC II), which is present on dendritic cells, B lymphocytes, and activated macrophages. The results obtained were compared with those seen in two human kidney specimens with acute and chronic oxalosis. In both rat and humans, macrophages and multinucleated giant cells are the major cells that encapsulate the interstitial crystals. This similarity in response underlines the relevance of the rat nephrolithiasis model. The rat experiments showed, furthermore, that the number of interstitial crystals and the amount of biochemically measured kidney-associated oxalate both decrease with time, if the nephrolithiatic agents EG and AC are omitted from the drinking water. Further studies must clarify whether macrophages and multinucleated giant cells are able to remove the interstitial crystals and how these cells are recruited at the inflammatory site.

Parish CR, Freeman C, Brown KJ, Francis DJ, Cowden WB (1999). "Identification of sulfated oligosaccharide-based inhibitors of tumor growth and metastasis using novel in vitro assays for angiogenesis and heparanase activity" Cancer Res 59(14):3433-41. PubMed

Inhibitors of tumor angiogenesis and metastasis are rapidly emerging as important new drug candidates for cancer therapy. To facilitate the identification of such drugs, we recently developed novel and rapid in vitro assays for human angiogenesis and for the extracellular matrix-degrading enzyme heparanase, which has been implicated in tumor metastasis. In this study, sulfated oligosaccharides, which are structural mimics of heparan sulfate, were investigated as drug candidates because these compounds may interfere with heparan sulfate recognition by many angiogenic growth factors and may inhibit cleavage of heparan sulfate by heparanase. In the preliminary screening studies, it was found that inhibitory activity in both assay systems was critically dependent on chain length and degree of sulfation, highly sulfated linear oligosaccharides of five or more monosaccharides in length being the most active. However, two sulfated oligosaccharides stood out as potential antitumor drugs, phosphomannopentaose sulfate (PI-88) and maltohexaose sulfate, both of these compounds having the important property of simultaneously being potent inhibitors of in vitro angiogenesis and heparanase activity. Due to the ease of manufacture of the starting material, phosphomannopentaose, PI-88 was studied in more detail. PI-88 was shown to inhibit the primary tumor growth of the highly invasive rat mammary adenocarcinoma 13762 MAT by approximately 50%, inhibit metastasis to the draining popliteal lymph node by approximately 40%, and reduce the vascularity of tumors by approximately 30%, all of these effects being highly significant. Acute hematogenous metastasis assays also demonstrated that PI-88 was a potent (>90%) inhibitor of blood-borne metastasis. Thus, by the use of novel in vitro screening procedures, we have identified a promising antitumor agent.

Giezeman-Smits KM, Gorter A, van Vlierberghe RL, v Eendenburg JD, Eggermont AM, Fleuren GJ, Kuppen PJ (1999). "The regulatory role of CD45 on rat NK cells in target cell lysis" J Immunol 163(1):71-6. PubMed

To investigate the role of CD45 in rat NK cell function, we developed new mAbs directed against rat CD45. mAb ANK12 binds to a high molecular isoform of CD45 and mAb ANK74 binds to the common part on all known CD45 isoforms, as has been described for the anti-rat CD45 mAb OX1. The ability of these mAbs to affect NK cell-mediated lysis was tested using the Fc receptor-positive target cell line P815. mAb ANK12 was found to significantly enhance the lysis of P815, whereas ANK74 and the anti-CD45 mAb OX1 did not. In addition, cross-linking of the CD45 isoform by ANK12 induced tyrosine phosphorylation of specific proteins in NK cells. Subsequently, the involvement of CD45 in the negative signaling after "self" MHC class I recognition by rat NK cells was investigated. The anti-CD45 mAbs were found to affect NK cell-mediated lysis of syngeneic tumor cell lines, depending upon the expression level of MHC class I on target cells. mAbs ANK74 and OX1 only inhibited lysis of the syngeneic tumor cell lines that expressed low levels of MHC class I. Furthermore, both mAbs caused an inhibition of NK cell-mediated lysis of these tumor cell lines when MHC class I molecules on the tumor cell lines were masked by an Ab. These results suggest that CD45 regulates the inhibitory signal pathway after self MHC class I recognition, supposedly by dephosphorylation of proteins.

Escudero E, Nieto M, Martín A, Molina A, Lobb RR, Sanchez-Madrid F, Mampaso F (1998). "Differential effects of antibodies to vascular cell adhesion molecule-1 and distinct epitopes of the alpha4 integrin in HgCl2-induced nephritis in Brown Norway rats" J Am Soc Nephrol 9(10):1881-91. PubMed

Four distinct epitopes (A, B1, B2, and C) have been functionally defined on the human alpha4 integrin. In this study, two cross-reactive antihuman alpha4 monoclonal antibodies (mAb) (HP2/1 and HP2/4 specific for epitopes B1 and B2, respectively) were used to functionally characterize the rat VLA-4 subunit and to define similar functional epitopes in this rodent species. It was found that B1 and B2 anti-alpha4 mAb completely block adhesion to fibronectin, but the inhibition of adhesion to vascular cell adhesion molecule-1 (VCAM-1) with HP2/1 mAb was lower than with HP2/4 mAb. It was also observed that epitope B2 HP2/4 mAb induced homotypic aggregation in rat lymphocytes, whereas epitope B1 HP2/1 mAb did not. Using the HgCl2 model of nephritis, this study shows the protective effect of both anti-alpha4 mAb against infiltration of the renal interstitium by leukocytes. Nevertheless, HP2/1 mAb, but not HP2/4 mAb, virtually abolished the anti-glomerular basement membrane antibody synthesis and glomerular deposits. These findings indicate the dual but independent role played by alpha4 integrins in both extravasation of leukocytes and in the production of antibodies. Finally, this study demonstrates that anti-rat VCAM-1 mAb showed a positive reactivity of the renal vascular endothelium and, most importantly, that administration of anti-VCAM-1 antibodies completely abrogated the interstitial cell infiltrates without affecting anti-glomerular basement membrane antibody production. These results confirm the important role played by VLA-4/VCAM-1 pathway in leukocyte infiltration, and further support the dual and independent role of alpha4 integrins in both renal infiltration and autoantibody synthesis in this model of renal disease.

Sido B, Dengler TJ, Otto G, Zimmermann R, Müller P, Meuer SC (1998). "Differential immunosuppressive activity of monoclonal CD2 antibodies on allograft rejection versus specific antibody production" Eur J Immunol 28(4):1347-57. PubMed

CD2 is a co-stimulatory receptor involved in T cell activation. Here we report on immunosuppressive effects of three mouse CD2 monoclonal antibodies (OX34, OX54, OX55) directed against non-overlapping epitopes of the rat CD2 receptor on various modes of T cell activation in vitro and in vivo. Although non-ligand-blocking OX54 and OX55, in concert, activated T cells through CD2 in vitro, they individually suppressed the mixed lymphocyte reaction (MLR) and significantly prolonged allograft survival after rat heart transplantation in vivo. Phenotype analysis revealed that OX55 significantly down-modulated CD2 in vivo, whereas OX54 depleted T cells. Graft rejection coincided with re-expression of CD2 and clearance of OX55 from serum, whereas T cell depletion by OX54 outlasted the period of graft survival. The most suppressive antibody, OX34, down-modulated CD2 and inhibited T cell activation through the TCR or CD2 and the MLR and prolonged median allograft survival time from 7 days in controls to 45 days in the absence of any additional treatment. Graft survival was clearly dose dependent and correlated with the duration of CD2 down-modulation and the presence of circulating CD2 antibody in serum. Importantly, the specific antibody production to a T cell-dependent antigen as demonstrated by immunization with keyhole limpet hemocyanin in vivo remained unaffected after treatment with OX34. These results demonstrate the pivotal role of CD2 signaling in mediating allogeneic immune reactions after vascularized organ transplantation while allowing specific humoral immune responses in vivo.

Moreira A, Takagaki Y, Brackenridge S, Wollerton M, Manley JL, Proudfoot NJ (1998). "The upstream sequence element of the C2 complement poly(A) signal activates mRNA 3' end formation by two distinct mechanisms" Genes Dev 12(16):2522-34. PubMed

The poly(A) signal of the C2 complement gene is unusual in that it possesses an upstream sequence element (USE) required for full activity in vivo. We describe here in vitro experiments demonstrating that this USE enhances both the cleavage and poly(A) addition reactions. We also show that the C2 USE can be cross-linked efficiently to a 55-kD protein that we identify as the polypyrimidine tract-binding protein (PTB), implicated previously in modulation of pre-mRNA splicing. Mutation of the PTB-binding site significantly reduces the efficiency of the C2 poly(A) site both in vivo and in vitro. Furthermore, addition of PTB to reconstituted processing reactions enhances cleavage at the C2 poly(A) site, indicating that PTB has a direct role in recognition of this signal. The C2 USE, however, also increases the affinity of general polyadenylation factors independently for the C2 poly(A) signal as detected by enhanced binding of cleavage-stimulaton factor (CstF). Strikingly, this leads to a novel CstF-dependant enhancement of the poly(A) synthesis phase of the reaction. These studies both emphasize the interconnection between splicing and polyadenylation and indicate an unexpected flexibility in the organization of mammalian poly(A) sites.

Giezeman-Smits KM, Gorter A, Nagelkerke JF, van Vlierberghe RL, van Eendenburg J, Eggermont AM, Fleuren GJ, Kuppen PJ (1997). "Characterization of three new membrane structures on rat NK cells which are involved in activation of the lytic machinery" Immunobiology 197(5):429-43. PubMed

In this study, three membrane structures on rat NK cells which activate lysis of target cells were characterized. Furthermore, the role of adhesion molecules in this activation process, in particular the CD18-associated integrins, was investigated. Three rat NK-activation structures were identified which have not been previously described. These structures are apparently unique as they differed in molecular weight from known NK-activation structures. Cross-linking of these activation structures with specific mAbs and a Fc gamma R-positive tumor cell line (P815) resulted in enhanced killing of these target cells by NK cells. If the CD18-associated integrins were masked by the anti-CD18 mAb WT.3, the redirected killing of P815 was completely blocked. This indicates that the CD18-associated integrins play a crucial role in activation of NK cells. Furthermore, our results show that rat NK cells possess multiple activation structures.

Mendola J, Corominola H, Esmatjes E, Saenz A, Fernandez-Cruz L, Gomis R (1997). "Effect of cyclosporine A treatment in vitro on pancreatic islet allograft rejection" Transplant Proc 29(5):2494-7. PubMed

Dick AD, Ford AL, Forrester JV, Sedgwick JD (1995). "Flow cytometric identification of a minority population of MHC class II positive cells in the normal rat retina distinct from CD45lowCD11b/c+CD4low parenchymal microglia" Br J Ophthalmol 79(9):834-40. PubMed

Aims: This study aimed to isolate and classify by flow cytometry, the cell surface phenotype of microglia in the normal rat retina with a view to identifying putative antigen presenting cells (APC) within the retina, which has to date not been possible by immunohistochemistry. Methods: Normal rat retinal microglia were isolated and classified using a modification of an isolation technique employing graduated Percoll density gradient cell separation and flow cytometric phenotypic criteria used for CNS microglia. Results: Retinal microglia can be defined by flow cytometry on the basis of their CD45lowCD11b/c+CD4low cell surface expression. Constitutive MHC class II expression in the normal rat retina was confined almost exclusively to a very minor population of cells expressing neither low (microglia) nor high levels of CD45. Three colour flow cytometric analysis confirmed that these MHC class II positive cells were ED2+. Conclusions: Using this sensitive isolation technique we have identified the cell surface characteristics of ramified, resident microglia, and found that they do not constitutively express MHC class II. There is, however, constitutive MHC class II expression on a phenotypically distinct population of cells (CD45low/highED2+). We propose these cells are the counterpart of the perivascular macrophages found in the CNS which present antigen to extravasating T cells, although their exact retinal location can only be confirmed by immunohistochemical analysis. The role of parenchymal microglia as APC remains undefined. Future isolation of microglia and putative perivascular cells using this technique will help identify the role these cells play in the initiation and perpetuation of immune responses within the retina.

Martín A, Molina A, Bricio T, Mampaso F (1995). "Passive dual immunization against tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta maximally ameliorates acute aminonucleoside nephrosis" Clin Exp Immunol 99(2):283-8. PubMed

Rats receiving a single dose (10 mg/100 g) of aminonucleoside of puromycin (PAN) develop heavy proteinuria and acute interstitial nephritis (AIN). Whole isolated glomeruli from rats injected with PAN secreted both TNF-alpha and IL-1 beta cytokines. TNF-alpha secretion was first and maximally detected on day 3, whereas IL-beta activity was found on day 7, when rats were heavily proteinuric and AIN developed. In vivo treatment with either anti-TNF-alpha or anti-IL-1 beta antibodies produced a drastic and simultaneous reduction in both levels of proteinuria and intensity of interstitial cell infiltrate. These effects improved when both antibodies were administered together. Our studies demonstrate the effectiveness of immunosuppressive therapy against these two cytokines in rats with PAN-induced nephrosis.

Wood MJ, Sloan DJ, Dallman MJ, Charlton HM (1992). "A monoclonal antibody to the interleukin-2 receptor enhances the survival of neural allografts: a time-course study" Neuroscience 49(2):409-18. PubMed

A time-course study of the survival and immunological characteristics of rat neural allografts was undertaken in animals treated with a murine monoclonal antibody to the alpha-chain (p55) of the rat interleukin-2 receptor. This antibody, NDS 63, was administered for ten days following grafting beginning on the day of operation. Inbred rat strains differing at both major and minor histocompatibility loci were selected as donor and host. Furthermore, the recipient strain displayed a high responder major histocompatibility complex haplotype. All grafts were placed in the lateral ventricle. Comparison was drawn between NDS 63-treated recipients and two groups of controls; an untreated group and a second group treated with the monoclonal antibody NDS 66, directed at a second epitope on the alpha-chain of the interleukin-2 receptor, which has been shown to be ineffective in competing with interleukin-2 for binding. Immunocytochemical analysis of the transplants was performed at several time-points up to 150 days following grafting. Grafts of NDS 63-treated recipients exhibited 100% survival with minimal induction of major histocompatibility complex antigens (both class I and class II) and negligible leukocyte infiltration at all time-points studied. In contrast grafts from both groups of controls showed evidence of a chronic immune response with most grafts undergoing rejection as shown by markedly elevated major histocompatibility complex antigen expression accompanied by specific immune cell infiltration. This was a protracted process with several grafts undergoing complete rejection by 60 days and a majority, but not all, by 150 days after transplantation. It is concluded that NDS 63, a monoclonal antibody to the interleukin-2 receptor, may diminish the immune response to transplanted allogeneic neural tissue and thereby enhance its prospects for long-term survival.

Beyers AD, Spruyt LL, Williams AF (1992). "Molecular associations between the T-lymphocyte antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5" Proc Natl Acad Sci U S A 89(7):2945-9. PubMed

The T-cell antigen receptor (TCR) complex is the key structure involved in signal transduction in T cells. To analyze associations between the TCR complex and other molecules, immunoprecipitations were carried out, followed by phosphorylation of molecules in vitro by tyrosine kinases associated with the precipitated molecules. This provided a sensitive assay for molecular complexes, and associations were demonstrated between the TCR complex and the surface antigens CD2, CD4, or CD8 and CD5 in normal rat T cells. The complexes were readily seen in immunoprecipitates from Brij 96 but not Nonidet P-40 detergent extracts. The multimolecular complexes are associated with the internal tyrosine kinases p56lck and p59fyn. The presence of p56lck associated with CD4 or CD8 was also examined in early thymocytes, natural killer cells, and macrophages. The kinase was present in all cases except that of normal macrophages.

Powrie F, Mason D (1990). "OX-22high CD4+ T cells induce wasting disease with multiple organ pathology: prevention by the OX-22low subset" J Exp Med 172(6):1701-8. PubMed

Congenitally athymic rats injected with CD45RBhigh CD4+ T cells from congenic euthymic donors developed a severe wasting disease with inflammatory infiltrates in liver, lung, stomach, thyroid, and pancreas. In contrast, recipients of CD45RBlow CD4+ T cells remained well and continued to gain weight. Animals given unfractionated CD4+ T cells, i.e., a mixture of approximately two-thirds CD45RBhigh and one-third CD45RBlow, were protected from the wasting disease, and the incidence of organ-specific inflammation was much reduced compared with that found in recipients of CD45RBhigh cells alone. The data suggest that this latter subset of CD4+ T cells has autoaggressive potential that is inhibited in normal animals by cells of the CD45RBlow CD4+ phenotype. The possible consequences of a breakdown in this immunoregulatory mechanism are briefly discussed.

Lazo PA, Klein-Szanto AJ, Tsichlis PN (1990). "T-cell lymphoma lines derived from rat thymomas induced by Moloney murine leukemia virus: phenotypic diversity and its implications" J Virol 64(8):3948-59. PubMed

The phenotype of 27 Moloney murine leukemia virus-induced rat thymic lymphomas and 36 cell lines derived from these tumors was determined by using 18 monoclonal antibodies directed against hematopoietic cell surface determinants. The cell lines and the primary tumors from which they were derived were clonally related as determined by the pattern of provirus integration and the pattern of rearrangement of the T-cell receptor beta and delta and Igh loci. The differentiation phenotype of the primary tumors and the cell lines derived from them were related. The differences observed between the primary tumors and the cell lines could be explained either by the selection of subpopulations of tumor cells during establishment in culture or by the phenotypic instability of the tumor cells. One cell line (LE3Sp) underwent the transition from a CD4+ CD8+ to a CD4+ CD8- phenotype following exposure to interleukin-2 in culture. Both the primary tumors and the cell lines derived from them express a wide range of phenotypes which correspond to multiple stages in T-cell development. This observation suggests that the pleiomorphism of retrovirus-induced lymphomas, which had been suggested previously from the analysis of mouse tumors, is an intrinsic property of the process of oncogenesis and is not due to the transformation of different types of cells by spontaneously arising leukemogenic variants of the inoculated virus. The wide spectrum of phenotypes expressed by these tumors suggests that Moloney murine leukemia virus may infect and transform T cells at various stages of development. Alternatively, the target cells may be immature T-cell precursors which, following transformation, continue to differentiate. A host of early findings, suggesting that the repertoire of target cells is restricted to poorly differentiated hematopoietic progenitors, and the ability of the LE3Sp cell line to differentiate in culture indicate that the latter possibility may be more likely. The data in this report address the extent and mechanism of the phenotypic variability of retrovirus-induced rodent T-cell lymphomas. In addition, they demonstrate the potential usefulness of the T-cell lymphoma lines we have established in studies of oncogenesis and T-cell differentiation.

Schluesener HJ (1986). "Inhibition of rat autoimmune T cell activation by monoclonal antibodies" J Neuroimmunol 11(4):261-70. PubMed

The essential requirement for adoptive transfer of autoimmune diseases such as experimental allergic encephalomyelitis (EAE) by T lymphoblasts from established T cell lines, is a prior activation of these cells by autoantigen or mitogen. We have investigated the possibility of modulating this activation process by using monoclonal antibodies directed against rat leukocyte differentiation antigens. We report here that antigen-driven activation of autoimmune, encephalitogenic T cells from established myelin basic protein (MBP)-specific rat T cell lines can be inhibited by some, but not all, antibodies against RT1.B Class II restriction elements. In addition, monoclonal antibodies with specificity for rat leukocyte common antigen (OX-1) and T cell differentiation antigens W3/13 and W3/25 are inhibitory, while monoclonal antibody OX-8 with specificity for T cytotoxic/suppressor cells has no effect. We also observed that concanavalin A-induced activation of the T cells is more resistant to the inhibitory effect of monoclonal antibodies, and can be blocked effectively only by antibody OX-1. This demonstration that autoimmune T cell function can be inhibited by monoclonal antibodies points the way in suggesting cellular targets for immunotherapeutic purposes.

Woollett GR, Barclay AN, Puklavec M, Williams AF (1985). "Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes" Eur J Immunol 15(2):168-73. PubMed

The molecular forms and antigenic heterogeneity of the leukocyte-common antigen (L-CA) of rat lymphocytes have been analyzed. Thymocytes show one main band at 180 kDa, T cells four bands at 180, 190, 200 and 220 kDa and B cells one broad band at about 240 kDa. T helper and T cytotoxic cell subsets show the same four bands with some differences in the proportion of each. Four mouse monoclonal antibodies (MRC OX-1, 28, 29 and 30) reacted with all molecular forms of L-CA and fell into two sets that were noncompetitive in binding to L-CA (MRC OX-1, 28, 29 vs. OX-30). The antigenic determinants seen by all these antibodies were lost when L-CA was reduced and alkylated. Three antibodies (MRC OX-22, 31 and 32) reacted selectively with B cells, T cytotoxic cells and about 2/3 of T helper cells. OX-22 and OX-31 competed for binding but were noncompetitive with OX-32. All these antibodies bound to a subfraction of the 190, 200 and 220-kDa forms of T cell L-CA but not at all to the 180-kDa form of T cells or thymocytes. One antibody bound to B cells only (MRC OX-33) and precipitated a subfraction of B cell L-CA. With all the antibodies that did not label thymocytes the antigenic determinants survived reduction and alkylation. Subsequent proteolysis with trypsin then destroyed all determinants except the one reacting with MRC OX-22 antibody. In this case tryptic peptides retained full antigenic activity which was, however, destroyed by further proteolysis with pronase.

Spickett GP, Brandon MR, Mason DW, Williams AF, Woollett GR (1983). "MRC OX-22, a monoclonal antibody that labels a new subset of T lymphocytes and reacts with the high molecular weight form of the leukocyte-common antigen" J Exp Med 158(3):795-810. PubMed

A mouse monoclonal antibody (MRC OX-22) is described that labels rat T cells which mediate graft-versus-host reactions and those responsible for the suppression of antibody synthesis in hosts undergoing these reactions. In contrast, most of the T cells that provide help for B cells are MRC OX-22 negative. These results, taken together with those published previously, demonstrate that the rat contains at least three phenotypically and functionally distinct subsets of T cells. The MRC OX-22 antibody also labels all B cells, 50% of bone marrow cells, but only 2% of thymocytes. Of these latter cells about half are found at the edge of the medulla and the remainder are randomly distributed throughout the cortex and medulla. These findings lend support to the view that mature thymocytes leave the thymus at the cortico-medullary junction, and also suggest that both cortex and medulla may be sites where thymocytes mature. Biochemical studies showed that the MRC OX-22 antibody reacts with the high molecular weight form of the leukocyte-common antigen (L-CA). Comparison with data on human L-CA suggests that the molecular and antigenic heterogeneity of this set of glycoproteins has been conserved between rat and man.

Standring R, McMaster WR, Sunderland CA, Williams AF (1978). "The predominant heavily glycosylated glycoproteins at the surface of rat lymphoid cells are differentiation antigens" Eur J Immunol 8(12):832-9. PubMed