Monoclonal Antibody 1D11 Research Articles

AB037. P008. Intratumoral regulatory T cells (Tregs) reduced by neutralization TGF-β in murine pancreatic ductal adenocarcinoma model without promising functional change

: Regulatory T cells (Treg) is a vital cell subset inducing immune tolerance in tumor microenvironment by secreting suppressive cytokines and inhibit innate immune cells. Transforming growth factor-β (TGF-β) plays an important role in this process because both the differentiation and functioning of Treg are relied on it. Elevated Tregs and TGF-β in pancreatic ductal adenocarcinoma (PDAC) microenvironment was previously explored with a protein expression array. In this study, we evaluated the quantity and quality change of Treg after neutralizing TGF-β with different doses of monoclonal antibody 1D11: the tumor volume was measured, the T cell subsets was assessed by flow cytometry in peripheral blood and spleen and was stained by immunohistochemistry in tumor, and main cytokines in tumor tissue were detected by enzyme linked immunosorbent assay in a murine PDAC model. As a result, only tumor infiltrating Tregs decreased significantly (high dose, low dose and control, 38.6±8.1, 38.6±1.8, 74.6±4.9/40× field, P=0.024) after 1D11 administration, the CD8+ T cells in the tumor microenvironment elevated in the low-dose group but remain almost the same level in the high-dose group (high dose, low dose and control, 3.1±11.9, 12.3±2.1, 5.4±0.5/40× field, P=0.016). The frequency of CD4+, CD8+ or Treg in peripheral blood and spleen showed no significant change and the typical cytokines TGF-β and inerleukin-10 secreted by Tregs and interferon-γ produced by T cells in tumor tissue also remained at the same level as untreated. In conclusion, this research indicated neutralizing TGF-β with monoclonal antibody can reduce Tregs in tumor niche, however, the functional alteration should still be evaluated in further studies.

Rescue of a cherubism bone marrow stromal culture phenotype by reducing TGFβ signaling.

We utilized a bone marrow stromal culture system to investigate changes in TGFβ signaling in a mouse model for cherubism (Sh3bp2KI/KI). Interestingly, bone marrow cultures derived from cherubism mice not only displayed impaired osteoblast differentiation, but also had spontaneous osteoclast formation. PAI1, a target gene of TGFβ signaling, was elevated 2-fold in cherubism CD11b-,CD45- cells compared to wild type cells, while the expression of BAMBI, an inhibitor of TGFβ signaling, was down-regulated. We also discovered that treatment of cherubism cultures with antagonists of the TGFβ signaling pathway could largely rescue osteoblast differentiation and markedly reduce spontaneous osteoclast formation. Treatment with the type I TGFβ receptor small molecule inhibitor SB505124 increased osteoblast reporter gene Col1a1-2.3 expression 24-fold and increased the expression of osteoblast gene markers Osterix (Sp7) 25-fold, Bone Sialoprotein (BSP) 7-fold, Osteocalcin (Bglap1) 100-fold, and Dentin Matrix Protein 1 (DMP1) 35-fold. In contrast, SB505124 treatment resulted in a significant reductions in osteoclast number and size. Gene expression analyses for RANKL, a positive regulator of osteoclast formation was 2.5-fold higher in osteoblast cultures derived from Sh3bp2KI/KI mice compared to wild type cultures, whereas OPG, an inhibitor of RANKL was 5-fold lower. However, SB505124 treatment reduced RANKL almost back down to wild type levels, while increasing OPG expression. Our studies also implicate a role for TGFβ ligands in the etiology of cherubism. Blocking of TGFβ ligands with the monoclonal antibody 1D11 increased Col1a1-2.3 reporter expression 4-fold and 13-fold in cultures derived from Sh3bp2KI/+ and Sh3bp2KI/KI mice, respectively. Serum levels of latent TGFβ1 were also 2-fold higher in SH3BP2KI/KI mice compared to wild type littermates. Taken together, these studies provide evidence that elevated levels of TGFβ signaling may contribute to the disease phenotype of cherubism and a reduction in pathway activity may be an effective therapeutic approach to treat this rare disease.

Attenuation of the DNA Damage Response by Transforming Growth Factor-Beta Inhibitors Enhances Radiation Sensitivity of Non–Small-Cell Lung Cancer Cells In Vitro and In Vivo

To determine whether transforming growth factor (TGF)-β inhibition increases the response to radiation therapy in human and mouse non-small-cell lung carcinoma (NSCLC) cells in vitro and in vivo. TGF-β-mediated growth response and pathway activation were examined in human NSCLC NCI-H1299, NCI-H292, and A549 cell lines and murine Lewis lung cancer (LLC) cells. Cells were treated in vitro with LY364947, a small-molecule inhibitor of the TGF-β type 1 receptor kinase, or with the pan-isoform TGF-β neutralizing monoclonal antibody 1D11 before radiation exposure. The DNA damage response was assessed by ataxia telangiectasia mutated (ATM) or Trp53 protein phosphorylation, γH2AX foci formation, or comet assay in irradiated cells. Radiation sensitivity was determined by clonogenic assay. Mice bearing syngeneic subcutaneous LLC tumors were treated with 5 fractions of 6 Gy and/or neutralizing or control antibody. The NCI-H1299, A549, and LLC NSCLC cell lines pretreated with LY364947 before radiation exposure exhibited compromised DNA damage response, indicated by decreased ATM and p53 phosphorylation, reduced γH2AX foci, and increased radiosensitivity. The NCI-H292 cells were unresponsive. Transforming growth factor-β signaling inhibition in irradiated LLC cells resulted in unresolved DNA damage. Subcutaneous LLC tumors in mice treated with TGF-β neutralizing antibody exhibited fewer γH2AX foci after irradiation and significantly greater tumor growth delay in combination with fractionated radiation. Inhibition of TGF-β before radiation attenuated DNA damage recognition and increased radiosensitivity in most NSCLC cells in vitro and promoted radiation-induced tumor control in vivo. These data support the rationale for concurrent TGF-β inhibition and RT to provide therapeutic benefit in NSCLC.

Effect of blocking different TGF-β isoforms on tumor immunity (TUM5P.937)

Abstract TGF-β is a pleiotropic cytokine with three isoforms that have significantly different physiological functions. This cytokine has potent immunosuppressive activity and has been shown to play critical roles in tumor development. TGF-β1 is also necessary for the development of T cell subsets as well as MDSC. We have previously reported that blockade of TGF-β by a monoclonal antibody 1D11, which neutralizes all three isoforms of TGF-β, improves immunosurveillance and vaccine efficacy in mouse tumor models. However, the role of individual isoforms of TGF-β is unknown. In this study, we examined the role of different TGF-β isoforms in tumor immunity by using anti-TGF-β with unique specificities in two tumor models, a lung metastasis of the BALB/c CT26 colon carcinoma and a subcutaneous TC1 lung epithelial cell tumor in B6. In the CT26 model, significant reduction of tumor burden was seen through the dual blockade of TGF-β1 and 2, or inhibition of all three TGF-β isoforms. In the TC1 model, the mice were treated with a peptide vaccine when the tumor size reached at least 5mm in diameter with/without anti-TGF-β. Although the vaccine alone induced significant regression of tumors, both types of anti-TGF-β facilitated the vaccine efficacy. These results suggest that blockade of only TGF-β1 and 2 is sufficient to facilitate natural immunosurveillance or enhance vaccine-induced tumor immunity. Currently we are investigating the effect of TGF-β isoform blockade on T cell subsets.

Abstract 299: Inducible Depletion of TGF-ß1 Exerts Differential Regional Effects on AngII-Induced Aortic Aneurysms

Introduction and Objectives: Infusion of AngII into normolipidemic mice leads to uniform expansion of the ascending aorta and a low incidence of abdominal aortic aneurysms. Co-administration of the monoclonal antibody 1D11, which recognizes TGFβ 1, 2, and 3, leads to greatly increased incidences of aortic rupture in both the ascending and abdominal aorta. The purpose of this study was to determine the role of TGFβ1 in AngII-induced aortic diseases. To perform this study, TGFβ1 floxed mice were bred to express an inducible form of Cre under control of the ubiquitous promoter, chicken α- actin. Methods and Results: Experimental mice were produced by breeding male Cre-ERT2 to female TGF-β1 floxed mice. At 8 weeks of age, male and female non-Cre littermates (N=16 male, 21 female) and TGF-β1 x Cre-ERT2 (N=11 male, 22 female) mice were injected with tamoxifen (25-75 mg/kg, i.p.) daily for 5 consecutive days. After 3 weeks, serum TGF-β1 was measured by ELISA. Serum concentrations of TGF-β1 decreased significantly in tamoxifen-injected Cre-ERT2 mice (P=0.001) compared to non-Cre littermates. Mice were then infused with AngII (1,000 ng/kg/min) for 28 days. AngII-induced increases in systolic blood pressures were not influenced by depletion of TGFβ1. AngII-induced expansion and rupture of the ascending aorta were not influenced by reduced TGFβ1. In contrast, there was marked enhancement of AngII-induced aortic expansion in the abdominal aorta (1.3 ± 0.3 versus 2.3 ± 0.3 mm in Cre- and Cre+ mice, respectively. P=0.019). Depletion of TGFβ1 had no effect on AngII-induced aortic pathologies in female mice. Conclusion: Depletion of TGF-β1 exerted regional-specific effects on AngII-induced aortic pathologies, with enhancement of abdominal aneurysms and no effect on ascending aortic dilation.

Interventions to improve chronic cyclosporine A nephrotoxicity through inhibiting renal cell apoptosis: a systematic review.

To reveal interventions for chronic cyclosporine A nephrotoxicity (CCN) and provide new targets for further studies, we analyzed all relevant studies about interventions in renal cell apoptosis. We collected all relevant studies about interventions for cyclosporine A (CsA)-induced renal cell apoptosis in Medline (1966 to July 2010), Embase (1980 to July 2010) and ISI (1986 to July 2010), evaluated their quality, extracted data following PICOS principles and synthesized the data. We included all relevant studies about interventions in CsA-induced renal cell apoptosis no limitation of research design and language) and excluded the duplicated articles, meeting abstracts and reviews without specific data. There were three kinds of intervention, include anti-oxidant (sulfated polysaccharides, tea polyphenols, apigenin, curcumin, spirulina, etc), biologics (recombinant human erythropoietin (rhEPO), a murine pan-specific transforming growth factor (TGF)-beta-neutralizing monoclonal antibody1D11, cartilage oligomeric matrix protein (COMP)-angiopoietin-1 and hepatocyte growth factor (HGF) gene), and other drugs (spironolactone, rosiglitazone, pirfenidone and colchicine). These interventions significantly improved the CCN, renal cell apoptosis and renal dysfunction through intervening in four apoptotic pathways in animals or protected renal cells from apoptosis induced by CsA and increased cell survival through respectively four pathways in vitro. There are three group interventions for CCN. Especially anti-oxidant drugs can significantly improve CCN, renal cell apoptosis and renal dysfunction. Many drugs can improve CCN through intervening in Fas/Fas ligand or mitochondrial pathway with sufficient evidences. Angiotensin II, nitric oxide (NO) and endoplasmic reticulum (ER) pathways will be new targets for CCN.

Immunohistochemical localization of substance P receptors in the midline glia of the developing rat medulla oblongata with special reference to the formation of raphe nuclei

Immunohistochemical localization of the substance P receptor (SPR) was examined in the developing rat medulla oblongata, with special reference to the development of substance P (SP)-immunoreactive neurons which form the medullary raphe nuclei. During development, SPR immunoreactivity was detected in cells lying lateral to the medullary midline from embryonic day 13 (E13) to postnatal day 5 (P5). The SPR-positive cell bodies were located close to the fourth ventricle, and bore long processes extending to the ventral pial surface. This SPR immunoreactivity co-localized with staining for monoclonal antibody 1D11, a specific marker of immature astrocytes. Substance P (SP)-immunoreactive neurons were first detected at E14 in the ventrolateral part of the medulla. By E16 their number had increased and they were arrayed in two rows closely parallel to the SPR-immunoreactive processes of non-neuronal cells. By P1, two separate SP-immunoreactive cell clusters could be recognized at the midline, representing dorsally the nascent raphe pallidus and ventrally the raphe obscurus. In addition, many SP-immunoreactive fibers traveled rostrocaudally in the medulla oblongata, juxtaposed to the midline sheets of SPR-immunoreactive long processes. SPR-immunoreactive processes at the midline were also immunoreactive for S-100, a glia-specific calcium-binding protein that is known to promote axonal growth of raphe neurons. These results suggest that SPR-expressing immature glial cells at the medullary midline are involved in the development of SP-immunoreactive raphe neurons, both in the formation of the medullary raphe nuclei and in axon guidance and growth.

Physical Interactions between Phospholamban and Sarco(endo)plasmic Reticulum Ca2+-ATPases Are Dissociated by Elevated Ca2+, but Not by Phospholamban Phosphorylation, Vanadate, or Thapsigargin, and Are Enhanced by ATP

Previous co-immunoprecipitation studies (Asahi, M., Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 32855-32862) revealed that physical interactions between phospholamban (PLN) and the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) were retained, even with PLN monoclonal antibody 1D11 bound to an epitope lying between PLN residues 7 and 17. Because the 1D11 antibody relieves inhibitory interaction between the two proteins, it was of interest to determine whether PLN phosphorylation or elevation of Ca(2+), which also relieves inhibitory interactions between PLN and SERCA, would disrupt physical interactions. Co-immunoprecipitation was measured in the presence of increasing concentrations of Ca(2+) or after phosphorylation of PLN by protein kinase A. Physical interactions were dissociated by elevated Ca(2+) but not by PLN phosphorylation. The addition of ATP enhanced interactions between PLN and SERCA. The further addition of vanadate and thapsigargin, both of which stabilize the E(2) conformation, did not diminish binding of PLN to SERCA. These data suggest that physical interactions between PLN and SERCA are stable when SERCA is in the Ca(2+)-free E(2) conformation but not when it is in the E(1) conformation and that phosphorylation of PLN does not dissociate physical interactions between PLN and SERCA.

Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum Ca2+ adenosinetriphosphatase.

A strong connection with nucleotide activation of Ca2+ATPase and phospholamban inhibition has been found. Phospholamban decreases the number of activatable Ca2+ATPase without affecting substrate affinity or the ability of nucleotide to serve its dual modulatory roles, i.e., catalytic and regulatory. Low concentrations of certain nucleotide mimetics, quercetin, tannin, and ellagic acid, with structural similarity to adenine can unmask phospholamban's inhibitory effect while concurrently acting as competitive inhibitors of nucleotide binding. Micromolar concentrations of tannin (EC50 approximately 0.3 microM) and ellagic acid (EC50 approximately 3 microM) stimulated Ca2+ uptake and calcium-activated ATP hydrolysis at submicromolar Ca2+ in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was followed by pronounced inhibiton at only slightly higher tannin concentrations (IC50 approximately 3 microM), whereas inhibitory effects by ellagic acid were observed at much greater concentrations (IC50 > 300 microM) than the EC50. A complex relationship between compound, SR protein, and MgATP concentration is a major determining factor in the observed effects. Stimulation was only observed under conditions of phospholamban regulation, while the inhibitory effects were observed in cardiac SR at micromolar Ca2+ and in skeletal muscle SR, which lacks phospholamban. Maximal stimulation of Ca2+ATPase was identical to that observed with the anti-phospholamban monoclonal antibody 1D11. Both compounds appear to relieve the Ca2+ATPase from phospholamban inhibition, thereby increasing the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. Tannin, even under stimulatory conditions, is a competitive inhibitor of MgATP with a linear Dixon plot. The subsequent inhibitory action of higher tannin concentrations results from competition of tannin with the nucleotide binding site of the Ca2+ATPase. In contrast, ellagic acid produced a curvilinear Dixon plot suggesting partial inhibition of nucleotide activation. The data suggest that nucleotide activation of Ca2+ATPase is functionally coupled to the phospholamban interaction site. These compounds through their interaction with the adenine binding domain of the nucleotide binding site prevent or dissociate phospholamban regulation. Clearly, this portion of Ca2+ATPase needs further study to elucidate its role in phospholamban inhibition.

Dissociation of Phospholamban Regulation of Cardiac Sarcoplasmic Reticulum Ca2+ATPase by Quercetin

Quercetin had a biphasic effect on Ca2+ uptake and calcium-stimulated ATP hydrolysis in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was observed at low quercetin concentrations (<25 microM) followed by inhibition at higher concentrations. The effects were dependent upon the SR protein concentration, the MgATP concentration, and intact phospholamban regulation of cardiac Ca2+ATPase. Only the inhibitory effects at higher quercetin concentrations were observed in skeletal muscle SR which lacks phospholamban and in cardiac SR treated to remove phospholamban regulation. Stimulation was additive with monoclonal antibody 1D11 (directed against phospholamban) at submaximal antibody concentrations; however, the maximal antibody and quercetin stimulation were identical. Quercetin increased the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. In addition, low concentrations of quercetin increased the steady-state formation of phosphoenzyme from ATP or Pi, but higher quercetin decreased phosphoenzyme levels. Quercetin, even under stimulatory conditions, was a competitive inhibitor of ATP, but appears to relieve the Ca2+ATPase from phospholamban inhibition, thereby, producing an activation. The subsequent inhibitory action of higher quercetin concentrations results from competition of quercetin with the nucleotide binding site of the Ca2+ATPase. The data suggest that quercetin interacts with the nucleotide binding site to mask phospholamban's inhibition of the SR Ca2+ATPase and suggests that phospholamban may interact at or near the nucleotide binding site.

CDNA sequence analysis and characterization of a cytokine-inducing monoclonal antibody derived from autoimmune MRL/MP-lpr/lpr mouse.

We have previously reported that a monoclonal antibody 1D11 derived from an autoimmune MRL/Mp-lpr/lpr (MRL/lpr) mouse induced synthesis or increased production of IL-3, IL-6, and TNF-alpha in the IL-3-dependent bone marrow-derived cell line FDC-P2/185-4. In this report, we analyzed a sequence of cDNA encoding the V region of 1D11, and found that VH and VL segments of 1D11 belonged to the J558 and V kappa 21 family, respectively. The nucleotide sequence of 1D11 in the VH segment was highly homologous to that of AM9, a monoclonal RF derived from MRL/lpr mouse, and the only difference was the replacement of 3 nucleotides in the framework region 1 (FR1). However, the deduced amino acid sequence of 1D11 was identical to that of AM9. In contrast with the VH segment, the sequences of the VL regions of these two antibodies were quite different from each other; 1D11 showed a 3 base deletion in the FR2 and a 24 base insertion in the FR3 compared with AM9. At present, the mechanisms of such insertions or deletions in the FRs of autoantibodies are almost unknown. It is generally accepted that the differences in specificity and affinity of these autoantibodies depend on differences of CDR sequence. However, it is possible that not only CDRs but also FRs of autoantibodies play a critical role in pathogenicity and/or specificity in autoimmune diseases.

Obstructed migration of Purkinje cells in the developing cerebellum of the reeler mutant mouse.

It has been considered that cortical malformation in the brain of the reeler mutant mouse is due to a defect in the process of neuroblast migration during development. We examined the process of Purkinje cell migration in the cerebellar primordium of the reeler mutant immunohistochemically and electron-microscopically, employing a specific marker for Purkinje cells and markers for radial glia. To facilitate the recognition of the homozygote of the reeler mutation (r1) at the embryonic stage, we introduced the chromosome carrying the autosomal semi-dominant mutation, hammer-toe (Hm), by crossbreeding and backcross into the heterozygote of the reeler mutation, which is an autosomal recessive and located on the homologous chromosome. Using this double heterozygous strain (+/rl-Hm/+), the homozygote of rl can be selected from littermates by the normal appearance of the feet. Both the heterozygous rl embryos and non-carriers harbor the Hm locus and show the Hm phenotype as a deformity of the feet that can be recognized from the 15th day of gestation. In the cerebellar primordium of control mice, Purkinje cells migrated radially from the ventricular zone towards the cortex. In contrast, most of the migratory Purkinje cells remained in the intermediate zone, and their migration towards the cortex was obstructed in the cerebellum of the reeler mutant. A disorganized arrangement of both the processes and cell bodies of the radial glia was demonstrated in the cerebellar primordium of the reeler by labeling them with the antibody against tenascin, a neuron-glial adhesion molecule, and the monoclonal antibody 1D11, a marker for immature astroglia. Electron-microscopic observations revealed apposition of the migratory cells to the radially oriented glial processes in the intermediate zone of the control cerebellum. In contrast, the apposition of leading processes of the migratory neuroblasts to disorganized processes of the radial glia was observed in the intermediate zone of the reeler cerebellum. These findings suggest that the obstructed migration and disordered cortical alignment of Purkinje cells in the reeler cerebellum is due to dysgenesis and abnormal development of radial glia, resulting in disturbance of contact guidance in the process of Purkinje cell migration.

Reversal of acute and chronic synovial inflammation by anti-transforming growth factor beta.

Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions.

Regulation of synovial cell growth. Coexpression of transforming growth factor beta and basic fibroblast growth factor by cultured synovial cells.

To demonstrate expression of transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) by cultured rheumatoid arthritis (RA) synovial cells and to investigate their role as synovial cell mitogens. Polypeptide growth factors were detected and identified by immunocytochemical staining and Western blot analysis. Messenger RNA (mRNA) transcripts encoding TGF beta and bFGF were identified by polymerase chain reaction analysis. The influence of neutralizing growth factor monoclonal antibodies (MAb) on RA synovial cell growth was investigated. TGF beta bioactivity was determined by Mv1Lu assay. Lysates of RA, as compared with normal, synovial cells contained greater amounts of TGF beta and bFGF. Western blot analysis identified a single TGF beta band (MW approximately 25 kd) in each of the cell lysates examined. Western blot analysis using MAb DE6 identified a doublet of bFGF bands (MW approximately 18.0 kd) in normal synovial cell lysates and 4 bFGF bands (MW approximately 18.0, 22.0, 22.6, and 25.2 kd) in RA synovial cell lysates. RA and normal synovial cells expressed mRNA transcripts encoding TGF beta 1 but not TGF beta 2, and FGF-2 (basic FGF). Additional mRNA transcripts encoding FGF-5 and FGF-7 were expressed by RA, but not normal, synovial cells in culture. In contrast to MAb 1D11.16, which caused a dose-dependent decrease in RA synovial cell growth, MAb DG2 (up to 100 micrograms/ml) had no effect on cell growth. RA and normal synovial cells cultured in serum-free medium express TGF beta 1 and native bFGF. However, only RA synovial cells in culture express higher molecular weight isoforms of bFGF. TGF beta 1 appears to regulate synovial cell growth in vitro through an external autocrine loop. Despite expression of high-affinity bFGF receptors on cultured synovial cells, the mechanisms by which bFGF modulates synovial cell growth are unknown.

Production and characterisation of monoclonal antibodies to the mid-region 37–67 sequence of parathyroid hormone-related protein

The production and characterisation of monoclonal antibodies (MAb) to the mid-region sequence 37–67 of human parathyroid hormone-related protein (PTHRP) is described. In spite of the poor immunogenicity of this sub-fragment of PTHRP, a high percentage of specific hybrids were produced by boosting with conjugate and free peptide prior to cell fusion. Seven of the MAbs produced cross-reacted with PTHRP37–67, PTHRP1–86 and native forms of PTHRP. Inhibition studies with peptide sub-fragments of PTHRP37–67 indicated that the majority recognised the 45–59 region. In a RIA for PTHRP1–86, detection limits ranged from 0.17 to 0.9 ng PTHRP1–86/tube, and no cross-reaction was found with PTH1–84. Two MAbs 1D11 and 4B10 were shown to be of potential use in measuring PTHRP1–86 in a two-site immunoradiometric assay in combination with either a solid phase consisting of a MAb to PTHRP1–34 or, iodinated affinity purified rabbit antibodies to PTHRP1–34, MAb 1D11 coupled to Sepharose was suitable for immunoextraction of PTHRP, and successfully localised PTHRP on immunoblots. Two additional MAbs were produced which recognised an epitope unique to PTHRP37–67 located in the 37–46 region of the peptide.

Development and migration of Purkinje cells in the mouse cerebellar primordium

The mode of Purkinje cell migration in the mouse cerebellar primordium was examined immunohistochemically, by marking Purkinje cells with anti-spot 35 antibody and labeling them with 5'-bromodeoxyuridine. The cells migrated radially from the neuroepithelium of the fourth ventricle towards the cortical surface between the 13th and 17th days (E13-E17) of gestation. Regional differences in the migratory process were evident: the final settlement of the Purkinje cells proceeded earlier in the lateral and posterior parts of the primordium, exhibiting latero-medial and posteroventral-anterodorsal diminishing sequences. To elucidate the factors involved in the migration, the arrangement of radial glial fibers, and expression of the cell adhesion molecule, tenascin, were examined immunohistochemically with the monoclonal antibody 1D11, a marker for both immature and mature astroglia, and an anti-tenascin antibody. At E14, 1D11-immunopositive fibers were seen to extend from the ventricle to the pial surface, and the cell bodies of immature glia migrated after E15 towards the cortex, shortening the radial processes whose end-feet were attached to the pia mater. Tenascin, which possesses a neuron-glial adhesiveness, was also expressed on the radial fibers during the migration of the Purkinje cells. The fibers were closely apposed to the migratory Purkinje cells, and their arrangement and orientation accorded with the migratory direction of the Purkinje cells. Further, changes in the molecular species of antigens detected by both the 1D11 and anti-tenascin antibodies were observed by immunoblotting analysis during the course of cerebellar development. These findings suggest that the arrangement of radial glia and expression of adhesion molecules may be involved in the control and guidance of Purkinje cell migration.

Autocrine regulation of rheumatoid arthritis synovial cell growth in vitro

Rheumatoid arthritis (RA), and not osteoarthritis (OA) synovial cells proliferate in serum-free medium, a finding that suggests that, in vitro, RA synovial cells may be stimulated to grow by the continuous autocrine production of at least one polypeptide growth factor. Adding monoclonal antibody 1D11.16, or rabbit polyclonal anti-tumor growth factor beta (anti-TGF-beta) antibodies (both neutralizing antibodies to TGF-beta 1 and TGF-beta 2) to RA synovial cells, in culture, caused a significant reduction in cell growth, an effect not seen when other growth factor antibodies (platelet-derived growth factor [PDGF], epidermal growth factor [EGF], or EGF receptor) were added to the culture medium. Taken together, these data are consistent with the concept that RA synovial cell growth in vitro is driven endogenous TGF-beta. Moreover, when EGF was added to the culture medium, this caused the numbers of RA, and not OA, synovial cells to increase significantly. This finding suggests that RA synovial cells are in G1 phase of the cell cycle; an effect that could be mediated by endogenous TGF-beta.

Morphological changes of astroglia in the cerebellar cortex of developing mice after neonatal administration of cytosine arabinoside

Neonatal administration of cytosine arabinoside (Ara-C) induced marked hypoplasia of the mouse cerebellum. In 17-20-day-old mice with neonatal Ara-C injections, the external granular layer was still preserved as an irregular cell laminae in the superficial part of the cerebellum, in contrast to its complete disappearance in normal animals at these stages. Immunohistochemical examination using anti-glial fibrillary acidic protein and monoclonal antibody-1D11 (Ono et al: Dev. Brain Res., 50:154-159, 1989) demonstrated malformation of a palisade-like arrangement of labeled glial fibers in the superficial part of the Ara-C treated mouse cerebellum. Furthermore, the immunoreactive astroglia were observed just beneath the pial membrane and their processes were oriented to the deeper part of the molecular layer.