Cell type-specific recognition of human metapneumoviruses (HMPVs) by retinoic acid-inducible gene I (RIG-I) and TLR7 and viral interference of RIG-I ligand recognition by HMPV-B1 phosphoprotein.

Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN genes. Recently, the RNA helicase retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5' triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell type-specific manners.

IL-9 governs allergen-induced mast cell numbers in the lung and chronic remodeling of the airways.

RATIONALE: IL-9 is a pleiotropic cytokine that has multiple effects on structural as well as numerous hematopoietic cells, which are central to the pathogenesis of asthma. OBJECTIVES: The contribution of IL-9 to asthma pathogenesis has thus far been unclear, due to conflicting reports in the literature. These earlier studies focused on the role of IL-9 in acute inflammatory models; here we have investigated the effects of IL-9 blockade during chronic allergic inflammation. METHODS: Mice were exposed to either prolonged ovalbumin or house dust mite allergen challenge to induce chronic inflammation and airway remodeling. MEASUREMENTS AND MAIN RESULTS: We found that IL-9 governs allergen-induced mast cell (MC) numbers in the lung and has pronounced effects on chronic allergic inflammation. Anti-IL-9 antibody-treated mice were protected from airway remodeling with a concomitant reduction in mature MC numbers and activation, in addition to decreased expression of the profibrotic mediators transforming growth factor-ß1, vascular endothelial growth factor, and fibroblast growth factor-2 in the lung. Airway remodeling was associated with impaired lung function in the peripheral airways and this was reversed by IL-9 neutralization. In human asthmatic lung tissue, we identified MCs as the main IL-9 receptor expressing population and found them to be sources of vascular endothelial growth factor and fibroblast growth factor-2. CONCLUSIONS: Our data suggest an important role for an IL-9-MC axis in the pathology associated with chronic asthma and demonstrate that an impact on this axis could lead to a reduction in chronic inflammation and improved lung function in patients with asthma.

Patients with systemic lupus erythematosus, myositis, rheumatoid arthritis and scleroderma share activation of a common type I interferon pathway.

OBJECTIVE: To characterise activation of the type I interferon (IFN) pathway in patients with systemic lupus erythematosus (SLE), dermatomyositis (DM), polymyositis (PM), rheumatoid arthritis (RA) and systemic scleroderma (SSc) and to evaluate the potential to develop a molecular diagnostic tool from the peripheral blood that reflects this activation in disease-affected tissues. METHODS: Overexpressed transcripts were identified in the whole blood (WB) of 262 patients with SLE, 44 with DM, 33 with PM, 28 with SSc and 89 with RA and compared with 24 healthy subjects using Affymetrix microarrays. A five gene type I IFN signature was assessed in these subjects to identify subpopulations showing both activation and concordance of the type I IFN pathway in the peripheral blood and disease-affected tissues of each disease and to correlate activation of this pathway in the WB with clinical measurements. RESULTS: A common set of 36 type I IFN inducible transcripts were identified among the most overexpressed in the WB of all subjects. Significant activation of the type I IFN pathway in subgroups of each of the five diseases studied was observed. Baseline disease activity measurements correlated with a type I IFN gene signature in the WB of subjects with SLE, PM and SSc, as did various serum autoantibody levels in subjects with SLE and DM. This signature was also well correlated between disease-affected tissue and WB in subjects with SLE, DM, PM and SSc. CONCLUSIONS: The results indicate that the type I IFN pathway is activated in patient subsets of five rheumatic diseases and suggest that these subsets may benefit from anti-IFN therapy.

Safety profile, pharmacokinetics, and biologic activity of MEDI-563, an anti-IL-5 receptor alpha antibody, in a phase I study of subjects with mild asthma.

BACKGROUND: Increased eosinophil levels have been linked to airway inflammation and asthma exacerbations. IL-5 is responsible for eosinophil differentiation, proliferation, and activation; IL-5 receptors are expressed on eosinophils and their progenitors, and targeting such receptors induces eosinophil apoptosis. OBJECTIVE: To evaluate the safety profile, pharmacokinetics, and pharmacodynamics of MEDI-563, a humanized mAb targeting the IL-5 receptor alpha chain. METHODS: Single, escalating, intravenous doses (0.0003-3 mg/kg) of MEDI-563 were administered to subjects with mild atopic asthma (n = 44) over approximately 3 to 30 minutes in this open-label study. Pulmonary function, symptom scores, adverse events, MEDI-563 pharmacokinetics, and levels of C-reactive protein (CRP), IL-6, eosinophil cationic protein (ECP), and eosinophils were evaluated. RESULTS: Mean peripheral blood (PB) eosinophil levels decreased in a dose-dependent fashion (baseline +/- SD, 0.27 +/- 0.2 x 10(3)/microL; 24 hours postdose, 0.01 +/- 0.0 x 10(3)/microL); 94.0% of subjects receiving >or=0.03 mg/kg exhibited levels between 0.00 x 10(3)/microL and 0.01 x 10(3)/microL. Eosinopenia lasted at least 8 or 12 weeks with doses of 0.03 to 0.1 and 0.3 to 3 mg/kg, respectively. ECP levels were reduced from 21.4 +/- 17.2 microg/L (baseline) to 10.3 +/- 7.0 microg/L (24 hours postdose). The most frequently reported adverse events were reduced white blood cell counts (34.1%), nasopharyngitis (27.3%), and increased blood creatine phosphokinase (25.0%). Mean C-reactive protein levels increased approximately 5.5-fold at 24 hours postdose but returned to baseline by study end; mean IL-6 levels increased approximately 3.9-fold to 4.7-fold at 6 to 12 hours postdose, respectively. Pharmacokinetic activity was dose proportional at doses of 0.03 to 3 mg/kg. CONCLUSION: Single escalating doses of MEDI-563 had an acceptable safety profile and resulted in marked reduction of PB eosinophil counts within 24 hours after dosing.

MEDI-563, a humanized anti-IL-5 receptor alpha mAb with enhanced antibody-dependent cell-mediated cytotoxicity function.

BACKGROUND: Peripheral blood eosinophilia and lung mucosal eosinophil infiltration are hallmarks of bronchial asthma. IL-5 is a critical cytokine for eosinophil maturation, survival, and mobilization. Attempts to target eosinophils for the treatment of asthma by means of IL-5 neutralization have only resulted in partial removal of airway eosinophils, and this warrants the development of more effective interventions to further explore the role of eosinophils in the clinical expression of asthma. OBJECTIVE: We sought to develop a novel humanized anti-IL-5 receptor alpha (IL-5Ralpha) mAb with enhanced effector function (MEDI-563) that potently depletes circulating and tissue-resident eosinophils and basophils for the treatment of asthma. METHODS: We used surface plasmon resonance to determine the binding affinity of MEDI-563 to FcgammaRIIIa. Primary human eosinophils and basophils were used to demonstrate antibody-dependent cell-mediated cytotoxicity. The binding epitope of MEDI-563 on IL-5Ralpha was determined by using site-directed mutagenesis. The consequences of MEDI-563 administration on peripheral blood and bone marrow eosinophil depletion was investigated in nonhuman primates. RESULTS: MEDI-563 binds to an epitope on IL-5Ralpha that is in close proximity to the IL-5 binding site, and it inhibits IL-5-mediated cell proliferation. MEDI-563 potently induces antibody-dependent cell-mediated cytotoxicity of both eosinophils (half-maximal effective concentration = 0.9 pmol/L) and basophils (half-maximal effective concentration = 0.5 pmol/L) in vitro. In nonhuman primates MEDI-563 depletes blood eosinophils and eosinophil precursors in the bone marrow. CONCLUSIONS: MEDI-563 might provide a novel approach for the treatment of asthma through active antibody-dependent cell-mediated depletion of eosinophils and basophils rather than through passive removal of IL-5.

Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells.

Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.

Respiratory syncytial virus (RSV) RNA loads in peripheral blood correlates with disease severity in mice.

BACKGROUND: Respiratory Syncytial Virus (RSV) infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity. METHODS: Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood. RESULTS: RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes. CONCLUSIONS: RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.

YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages.

YKL-40 is a chitin-binding protein that is elevated in patients with various inflammatory conditions associated with ongoing remodeling. We investigated whether the levels of YKL-40 were up-regulated in the circulation and the airways of patients with chronic obstructive pulmonary disease (COPD), and whether it promoted the production of inflammatory mediators from macrophages. Serum, bronchoalveolar lavage (BAL), bronchial biopsies, lung tissue specimens, and alveolar macrophages from never-smokers (n = 15), smokers without COPD (n = 20), and smokers with COPD (n = 30) were assessed for YKL-40 levels and immunolocalization. In addition, YKL-40-induced mediator release from alveolar macrophages was examined. We found that smokers with COPD had elevated levels of YKL-40 in serum (p

CUE-101, a Novel E7-pHLA-IL2-Fc Fusion Protein, Enhances Tumor Antigen-Specific T-Cell Activation for the Treatment of HPV16-Driven Malignancies.

PURPOSE: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. EXPERIMENTAL DESIGN: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Selective targeting and potent control of tumor growth using an EphA2/CD3-Bispecific single-chain antibody construct.

The EphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in malignant cells and thus provides opportunities for selective targeting of tumor cells. We describe here the development of a novel, bispecific single-chain antibody (bscAb) referred to as bscEphA2xCD3. This molecule simultaneously targets EphA2 on tumor cells and the T-cell receptor/CD3 complex on T cells and possesses structural and functional characteristics of the recently developed BiTE technology. An EphA2-specific single-chain antibody was selected for recognition of an epitope that is preferentially exposed on malignant cells based on the concept of epitope exclusion; this was fused to a CD3-specific single-chain antibody to generate bscEphA2xCD3. The resultant bscAb redirected unstimulated human T cells to lyse EphA2-expressing tumor cells both in vitro and in vivo. In separate experiments, efficient tumor cell lysis was achieved in vitro at drug concentrations

Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract.

Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis and pneumonia in infants and children. Currently, palivizumab is the only approved monoclonal antibody (mAb) for prophylaxis of RSV. However, a small percentage of patients are not protected by palivizumab; in addition, palivizumab does not inhibit RSV replication effectively in the upper respiratory tract. We report here the development and characterization of motavizumab, an ultra-potent, affinity-matured, humanized mAb derived from palivizumab. Several palivizumab variants that enhanced the neutralization of RSV in vitro by up to 44-fold were generated; however, in vivo prophylaxis of cotton rats with these antibodies conferred only about a twofold improvement in potency over palivizumab. This unexpected small increase of in vivo potency was caused by poor serum pharmacokinetics and lung bio-availability that resulted from unexpectedly broad tissue binding. Subsequent analyses revealed that changes at three amino acids arising from the affinity maturation markedly increased the non-specific binding to various tissues. Our results suggested that k(on)-driven mutations are more likely to initiate non-specific binding events than k(off)-driven mutations. Reversion of these three residues to the original sequences greatly diminished the tissue binding. The resulting mAb, motavizumab, binds to RSV F protein 70-fold better than palivizumab, and exhibits about a 20-fold improvement in neutralization of RSV in vitro. In cotton rats, at equivalent concentrations, motavizumab reduced pulmonary RSV titers to up to 100-fold lower levels than did palivizumab and, unlike palivizumab, motavizumab very potently inhibited viral replication in the upper respiratory tract. This affinity-enhanced mAb is being investigated in pivotal clinical trials. Importantly, our engineering process offers precious insights into the improvement of other therapeutic mAbs.

Motavizumab, a neutralizing anti-Respiratory Syncytial Virus (Rsv) monoclonal antibody significantly modifies the local and systemic cytokine responses induced by Rsv in the mouse model.

Motavizumab (MEDI-524) is a monoclonal antibody with enhanced neutralizing activity against RSV. In mice, motavizumab suppressed RSV replication which resulted in significant reduction of clinical parameters of disease severity. We evaluated the effect of motavizumab on the local and systemic immune response induced by RSV in the mouse model. Balb/c mice were intranasally inoculated with 106.5 PFU RSV A2 or medium. Motavizumab was given once intraperitoneally (1.25 mg/mouse) as prophylaxis, 24 h before virus inoculation. Bronchoalveolar lavage (BAL) and serum samples were obtained at days 1, 5 (acute) and 28 (long-term) post inoculation and analyzed with a multiplex assay (Beadlyte Upstate, NY) for simultaneous quantitation of 18 cytokines: IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, KC (similar to human IL-8), IL-10, IL-12p40, IL-12p70, IL-13, IL-17, TNF-alpha, MCP-1, RANTES, IFN-gamma and GM-CSF. Overall, cytokine concentrations were lower in serum than in BAL samples. By day 28, only KC was detected in BAL specimens at low concentrations in all groups. Administration of motavizumab significantly reduced (p < 0.05) BAL concentrations of IL-1alpha, IL-12p70 and TNF-alpha on day 1, and concentrations of IFN-gamma on days 1 and 5 compared with RSV-infected untreated controls. In the systemic compartment, the concentrations of IL-10, IFN-gamma and KC were significantly reduced in the motavizumab-treated mice compared with the untreated controls. In summary, prophylactic administration of motavizumab was associated with significant reductions on RSV replication and concentrations of cytokine and chemokines, which are likely related to the improvement observed in clinical markers of disease severity.

Direct targeting of alphavbeta3 integrin on tumor cells with a monoclonal antibody, Abegrin.

The humanized monoclonal antibody Abegrin, currently in phase II trials for treatment of solid tumors, specifically recognizes the integrin alphavbeta3. Due to its high expression on mature osteoclasts, angiogenic endothelial cells, and tumor cells, integrin alphavbeta3 functions in several pathologic processes important to tumor growth and metastasis. Targeting of this integrin with Abegrin results in antitumor, antiangiogenic, and antiosteolytic activities. Here, we exploit the species specificity of Abegrin to evaluate the effects of direct targeting of tumor cells (independent of targeting of endothelia or osteoclasts). Flow cytometry analysis of human tumor cell lines shows high levels of alphavbeta3 on many solid tumors, including cancers of the prostate, skin, ovary, kidney, lung, and breast. We also show that tumor growth of alphavbeta3-expressing tumor cells is inhibited by Abegrin in a dose-dependent manner. We present a novel finding that high-dose administration can actively impair the antitumor activity of Abegrin. We also provide evidence that antibody-dependent cellular cytotoxicity contributes to in vitro and in vivo antitumor activity. Finally, it was observed that peak biological activity of Abegrin arises at serum levels that are consistent with those achieved in clinical trials. These results support a concept that Abegrin can be used to achieve selective targeting of the many tumor cells that express alphavbeta3 integrin. In combination with the well-established concept that alphavbeta3 plays a key role in cancer-associated angiogenesis and osteolytic activities, this triad of activity could provide new opportunities for therapeutic targeting of cancer.

Ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization.

We describe here the selection of ultra-potent anti-respiratory syncytial virus (RSV) antibodies for preventing RSV infection. A large number of antibody variants derived from Synagis (palivizumab), an anti-RSV monoclonal antibody that targets RSV F protein, were generated by a directed evolution approach that allowed convenient manipulation of the binding kinetics. Palivizumab variants with about 100-fold slower dissociation rates or with fivefold faster association rates were identified and tested for their ability to neutralize virus in a microneutralization assay. Our data reveal a major differential effect of the association and dissociation rates on the RSV neutralization, particularly for intact antibodies wherein the association rate plays the predominant role. Furthermore, we found that antibody binding valence also plays a critical role in mediating the viral neutralization through a mechanism that is likely unrelated to antibody size or binding avidity. We applied an iterative mutagenesis approach, and thereafter were able to identify palivizumab Fab variants with up to 1500-fold improvement and palivizumab IgG variants with up to 44-fold improvement in the ability to neutralize RSV. These anti-RSV antibodies likely will offer great clinical potential for RSV immunoprophylaxis. In addition, our findings provide insights into engineering potent antibody therapeutics for other disease targets.

Differential EphA2 epitope display on normal versus malignant cells.

The EphA2 receptor tyrosine kinase is overexpressed in many different types of human cancers where it functions as a powerful oncoprotein. Dramatic changes in the subcellular localization and function of EphA2 have also been linked with cancer, and in particular, unstable cancer cell-cell contacts prevent EphA2 from stably binding its ligand on the surface of adjoining cells. This change is important in light of evidence that ligand binding causes EphA2 to transmit signals that negatively regulate tumor cell growth and invasiveness and also induce EphA2 degradation. On the basis of these properties, we have begun to target EphA2 on tumor cells using agonistic antibodies, which mimic the consequences of ligand binding. In our present study, we show that a subset of agonistic EphA2 antibodies selectively bind epitopes on malignant cells, which are not available on nontransformed epithelial cells. We also show that such epitopes arise from differential cell-cell adhesions and that the stable intercellular junctions of nontransformed epithelial cells occlude the binding site for ligand, as well as this subset of EphA2 antibodies. Finally, we demonstrate that antibody targeting of EphA2 decreases tumor cell growth as measured using xenograft tumor models and found that the mechanism of antibody action relates to EphA2 protein degradation in vivo. Taken together, these results suggest new opportunities for therapeutic targeting of the large number of different cancers that express EphA2 in a manner that could minimize potential toxicities to normal cells.

EphA2 expression is associated with aggressive features in ovarian carcinoma.

PURPOSE: EphA2 (epithelial cell kinase) is a transmembrane receptor tyrosine kinase that has been implicated in oncogenesis. There are no published data regarding the role of EphA2 in ovarian carcinoma, which is the focus of the present study. EXPERIMENTAL DESIGN: Nontransformed (HIO-180) and ovarian cancer (EG, 222, SKOV3, and A2780-PAR) cell lines were evaluated for EphA2 by Western blot analysis. Five benign ovarian masses, 10 ovarian tumors of low malignant potential, and 79 invasive ovarian carcinomas were also evaluated for EphA2 expression by immunohistochemistry. All samples were scored in a blinded fashion. Univariate and multivariate analyses were used to determine significant associations between EphA2 expression and clinicopathological variables. RESULTS: By Western blot analysis, EG, 222, and SKOV3 cell lines overexpressed EphA2, whereas A2780-PAR and HIO-180 had low to absent EphA2 expression. All of the benign tumors had low or absent EphA2 expression. Among the invasive ovarian carcinomas examined (mean age of patients was 59.2 years), 60 (75.9%) tumors overexpressed EphA2 and the other 19 tumors had negative or minimal EphA2 expression. There was no association of EphA2 overexpression with ascites, likelihood of nodal positivity, pathological subtype, and optimum surgical cytoreduction (residual tumor <1 cm). However, EphA2 overexpression was significantly associated with higher tumor grade (P = 0.02) and advanced stage of disease (P = 0.001). The median survival for patients with tumor EphA2 overexpression was significantly shorter (median, 3.1 years; P = 0.004); the median survival for patients with low or absent EphA2 tumor expression was at least 12 years and has not yet been reached. In multivariate analysis using the Cox proportional hazards model, only volume of residual disease (P < 0.04) and EphA2 overexpression (P < 0.01) were significant and independent predictors of survival. CONCLUSIONS: EphA2 overexpression is predictive of aggressive ovarian cancer behavior and may be an important therapeutic target.

PC cell-derived growth factor expression in prostatic intraepithelial neoplasia and prostatic adenocarcinoma.

PURPOSE: PCDGF (PC cell-derived growth factor), also called progranulin, is a M(r) 88000 glycoprotein precursor of granulin. It is a novel growth factor that stimulates cell proliferation, confers epithelial tumorigenesis, and promotes tumor invasion. Here we investigate the potential of PCDGF as a therapeutic target for prostate cancer. EXPERIMENTAL DESIGN: We studied the expression of PCDGF in invasive prostate cancer, adjacent high-grade prostatic intraepithelial neoplasia (PIN), and benign prostate tissue from 99 human prostate specimens. The level of PCDGF expression was correlated with various clinicopathological characteristics. RESULTS: Normal prostate tissue did not express (53/99), or expressed low levels (46/99) of PCDGF. In the 46 normal prostate specimens that expressed PCDGF, most of them had less than 10% of cells expressing PCDGF. PCDGF expression could be detected in more than 50% of cells in all specimens of PIN and invasive prostate cancer. The expression of PCDGF in normal prostate tissue was much less intense and in a smaller fraction of cells than in PIN and invasive adenocarcinoma (P < 0.0001). There was no correlation of PCDGF expression with age, Gleason score, pathological stage, status of lymph node metastasis, extraprostatic extension, perineural invasion, surgical margins, and vascular invasion. CONCLUSIONS: Our data suggest that the induction of PCDGF expression occurs during the development of PIN. PCDGF may be a new molecular target for the treatment and prevention of prostate cancer.

Analysis of human and primate CD2 molecules by protein sequence and epitope mapping with anti-human CD2 antibodies.

A panel of anti-human CD2 monoclonal antibodies (mAb) and soluble human CD58 (LFA-3) were tested for binding to human peripheral blood mononuclear cells (PBMCs), recombinant human CD2 and mononuclear cells from Cynomolgus, Rhesus and African green monkey, Stump-tail, Pig-tail and Assamese macaque, Chimpanzee and Baboon. This analysis revealed that whilst some antibodies recognized all species, there were differential binding profiles with others. Three antibodies, MEDI-507, 6F10.3 and 4B2, recognized CD2 from human and Chimpanzee but not that from the other primates. We have cloned eight of the previously unknown primate CD2 molecules and report here their sequences for the first time. This analysis revealed that 12 amino acids formed a common set of residues in the extra cellular domain of human and Chimpanzee CD2. Using a "knock-in" mutagenesis approach starting with Baboon CD2, which does not bind MEDI-507, 6F10.3 and 4B2, we have identified three residues in the adhesion domain of human CD2 which are critical for its binding to these mAbs. These residues, N18, K55 and T59 define a region located outside of the previously described binding regions on CD2. Affinity measurements of the mutants revealed a variety of degrees of binding restoration for MEDI-507, 6F10.3 and 4B2, indicating that there are fine differences within a given epitope. Furthermore, the analysis of the competition of several of the anti-human CD2 antibodies with each other and CD58 demonstrated the existence of a continuum of overlapping epitopes on human CD2, which is in contrast to the commonly held belief that epitopes on human CD2 are clearly segregated.

Rational design and synthesis of an orally active indolopyridone as a novel conformationally constrained cannabinoid ligand possessing antiinflammatory properties.

A series of unique indazoles and pyridoindolones have been rationally designed and synthesized as novel classes of cannabinoid ligands based on a proposed bioactive amide conformation. This has led to the discovery of the novel indolopyridone 3a as a conformationally constrained cannabinoid ligand that displays high affinity for the CB2 receptor (K(i)(CB2) = 1.0 nM) and possesses antiinflammatory properties when administered orally in an in vivo murine inflammation model.

Alterations in Fas (CD 95/Apo-1) and Fas ligand (CD178) expression in acute promyelocytic leukemia during treatment with ATRA.

Over the recent years, treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) has become a widely accepted therapeutic regimen and is considered a model of differentiation therapy in malignant diseases. However, the role of ATRA treatment beyond that of the induction of differentiation of leukemic blasts is still far from being fully understood. Data from in vivo and in vitro studies have demonstrated that during ATRA treatment of APL there are significant changes not only in the expression of the apoptotic molecules Fas and Fas ligand, but also in the expression of other molecules involved both in the regulation of apoptosis and in interactions between host immune and leukemia cells. These effects may thus contribute, at least in part, to the beneficial effects of ATRA therapy in APL. In this report we review the current status of studies that contribute to our understanding of treatment with ATRA. We focus on ATRA-induced changes in apoptotic pathways, particularly as it relates to the Fas/Fas ligand system.