Golimumab, a human antibody to tumour necrosis factor {alpha} given by monthly subcutaneous injections, in active rheumatoid arthritis despite methotrexate therapy: the GO-FORWARD Study.

OBJECTIVE: The phase III GO-FORWARD study examined the efficacy and safety of golimumab in patients with active rheumatoid arthritis (RA) despite methotrexate therapy. METHODS: Patients were randomly assigned in a 3 : 3 : 2 : 2 ratio to receive placebo injections plus methotrexate capsules (group 1, n = 133), golimumab 100 mg injections plus placebo capsules (group 2, n = 133), golimumab 50 mg injections plus methotrexate capsules (group 3, n = 89), or golimumab 100 mg injections plus methotrexate capsules (group 4, n = 89). Injections were administered subcutaneously every 4 weeks. The co-primary endpoints were the proportion of patients with 20% or greater improvement in the American College of Rheumatology criteria (ACR20) at week 14 and the change from baseline in the health assessment questionnaire-disability index (HAQ-DI) score at week 24. RESULTS: The proportion of patients who achieved an ACR20 response at week 14 was 33.1% in the placebo plus methotrexate group, 44.4% (p = 0.059) in the golimumab 100 mg plus placebo group, 55.1% (p = 0.001) in the golimumab 50 mg plus methotrexate group and 56.2% (p<0.001) in the golimumab 100 mg plus methotrexate group. At week 24, median improvements from baseline in HAQ-DI scores were 0.13, 0.13 (p = 0.240), 0.38 (p<0.001) and 0.50 (p<0.001), respectively. During the placebo-controlled portion of the study (to week 16), serious adverse events occurred in 2.3%, 3.8%, 5.6% and 9.0% of patients and serious infections occurred in 0.8%, 0.8%, 2.2% and 5.6%, respectively. CONCLUSION: The addition of golimumab to methotrexate in patients with active RA despite methotrexate therapy significantly reduced the signs and symptoms of RA and improved physical function.

Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity.

ORF74 (or KSHV-vGPCR) is a highly constitutively active G protein-coupled receptor encoded by HHV8 that is regulated both positively and negatively by endogenous chemokines. When expressed in transgenic mice, this chemokine receptor induces an angioproliferative disease closely resembling Kaposi sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does not respond to agonist stimulation have a much lower incidence of angiogenic lesions and tumors. These results indicate that induction of the KS-like disease in transgenic mice by ORF74 requires not only high constitutive signaling activity but also modulation of this activity by endogenous chemokines.

Disruption of neutrophil migration in a conditional transgenic model: evidence for CXCR2 desensitization in vivo.

We developed transgenic mice conditionally expressing the neutrophil chemoattracting chemokine KC and the beta-galactosidase gene in multiple tissues. In these transgenic mice, doxycycline treatment induced a strong up-regulation in the expression of KC in several tissues, including heart, liver, kidney, skin, and skeletal muscle. Expression of KC within these tissues led to a rapid and substantial increase in the serum levels of KC (serum KC levels were higher than 200 ng/ml 24 h after treatment). Accordingly, beta-galactosidase expression was also detected after injection of doxycycline and was highest in skeletal muscle, pancreas, and liver. Surprisingly, despite expression of KC in multiple tissues, no neutrophil infiltration was observed in any of the tissues examined, including skin. Doxycycline treatment of nontransgenic mice grafted with transgenic skin caused dense neutrophilic infiltration of the grafts, but not the surrounding host skin, indicating that the KC produced in transgenic tissues was biologically active. In separate experiments, neutrophil migration toward a localized source of recombinant KC was impaired in animals overexpressing KC but was normal in response to other neutrophil chemoattractants. Analysis of transgenic neutrophils revealed that high concentrations of KC in transgenic blood had no influence on L-selectin cell surface expression but caused desensitization of the receptor for KC, CXCR2. These results confirm the neutrophil chemoattractant properties of KC and provide a mechanistic explanation for the paradoxical lack of leukocyte infiltration observed in the presence of elevated concentrations of this chemokine.

Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation, runting, infertility, and premature death.

p19, a molecule structurally related to IL-6, G-CSF, and the p35 subunit of IL-12, is a subunit of the recently discovered cytokine IL-23. Here we show that expression of p19 in multiple tissues of transgenic mice induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 mo of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and the digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-alpha and IL-1 were elevated, and the number of circulating neutrophils was increased. In addition, ubiquitous expression of p19 resulted in constitutive expression of acute phase proteins in the liver. Surprisingly, liver-specific expression of p19 failed to reproduce any of these abnormalities, suggesting specific requirements for production of biologically active p19. Bone marrow transfer experiments showed that expression of p19 by hemopoietic cells alone recapitulated the phenotype induced by its widespread expression, pointing to hemopoietic cells as the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity.

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs.

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.

Aberrant in vivo T helper type 2 cell response and impaired eosinophil recruitment in CC chemokine receptor 8 knockout mice.

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (-/-) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8(-/)- mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.

Impaired pulmonary host defense in mice lacking expression of the CXC chemokine lungkine.

Lungkine (CXCL15) is a novel CXC chemokine that is highly expressed in the adult mouse lung. To determine the biologic function of Lungkine, we generated Lungkine null mice by targeted gene disruption. These mice did not differ from wild-type mice in their hematocrits or in the relative number of cells in leukocyte populations of peripheral blood or other tissues, including lung and bone marrow. However, Lungkine null mice were more susceptible to Klebsiella pneumonia infection, with a decreased survival and increased lung bacterial burden compared with infected wild-type mice. Histologic analysis of the lung and assessment of leukocytes in the bronchioalveolar lavage revealed that neutrophil numbers were normal in the lung parenchyma, but reduced in the airspace. The production of other neutrophil chemoattractants in the Lungkine null mice did not differ from that in wild-type mice, and neutrophil migration into other tissues was normal. Taken together, these findings demonstrate that Lungkine is an important mediator of neutrophil migration from the lung parenchyma into the airspace.

Leukocytes expressing green fluorescent protein as novel reagents for adoptive cell transfer and bone marrow transplantation studies.

Transgenic mice expressing green fluorescent protein (GFP) were generated to provide a source of labeled leukocytes for cell transfer studies. The transgene comprises the GFP coding region under the transcriptional control of the chicken ss-actin promoter and human cytomegalovirus enhancer. Mice expressing this GFP transgene were generated in the B6D2 and in the 129SvEv backgrounds. Flow cytometric analysis of cells from the blood, spleen, and bone marrow of these transgenic mice revealed that most leukocytes, including dendritic cells and memory T cells, express GFP. In allogeneic cell transfers, donor GFP+ splenocytes were detected in the spleen and mesenteric lymph nodes of recipient mice within 2 hours after transfer and for at least 9 days thereafter. In syngeneic experiments using 129-derived GFP+ donor splenocytes, donor cells were detected in multiple tissues of 129 recipients from 2 hours to 3 weeks after transfer. In bone-marrow transplantation experiments using irradiated allogeneic recipients, the percent of GFP+ donor cells in recipients at 3 weeks was comparable to that seen in similar tissues of GFP+ donor mice. These data demonstrate that GFP+ transgenic mice provide a ready source of GFP-expressing primary cells that can be easily monitored after their transfer to recipient animals.

Transgenic expression of the chemokine receptor encoded by human herpesvirus 8 induces an angioproliferative disease resembling Kaposi's sarcoma.

Human herpesvirus 8 (HHV8, also known as Kaposi's sarcoma [KS]-associated herpesvirus) has been implicated as an etiologic agent for KS, an angiogenic tumor composed of endothelial, inflammatory, and spindle cells. Here, we report that transgenic mice expressing the HHV8-encoded chemokine receptor (viral G protein-coupled receptor) within hematopoietic cells develop angioproliferative lesions in multiple organs that morphologically resemble KS lesions. These lesions are characterized by a spectrum of changes ranging from erythematous maculae to vascular tumors, by the presence of spindle and inflammatory cells, and by expression of vGPCR, CD34, and vascular endothelial growth factor. We conclude that vGPCR contributes to the development of the angioproliferative lesions observed in these mice and suggest that this chemokine receptor may play a role in the pathogenesis of KS in humans.

Development of a CD28 receptor binding-based screen and identification of a biologically active inhibitor.

CD28 is a T-cell costimulatory receptor which plays a pivotal role in antigen-induced T-cell response. We have developed a cell-free and scintillation-proximity assay-based screen to search for molecules that inhibit ligand binding to CD28. The assay was shown to be versatile and adaptable to automation for high-throughput screening. Using this assay, we identified an inhibitor of CD28, NP2214. The inhibitor was shown to be active in vitro by suppressing IL-2 synthesis and proliferation of peripheral blood mononuclear cells in response to CD28 costimulation. We also demonstrated the additive effects of NP2214 and cyclosporine A which act mechanistically distinctly in inhibiting costimulation-induced IL-2 synthesis.

Characterization of potential antagonists of human interleukin 5 demonstrates their cross-reactivity with receptors for interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The ligand-binding alpha-chain of the human interleukin 5 (IL-5) receptor was expressed in its soluble form, lacking the transmembrane and cytoplasmic domains, from recombinant baculovirus. The soluble receptor was used in a scintillation proximity assay to identify two chemical compounds that inhibit binding of human IL-5 to the soluble receptor alpha chain with IC50 of 8 microM and 11 microM. These compounds also inhibited the interaction of human IL-5 with its membrane-bound receptor, composed of the ligand-binding alpha chain and signal-transducing beta chain, and prevented signaling through the receptor. Analysis by surface plasmon resonance and matrix-assisted laser-desorption/ionization mass spectrometry showed that the identified compounds bound irreversibly to the receptor at a 1:1 (mol/mol) ratio, suggesting a covalent interaction with the alpha chain of the human IL-5 receptor. Both compounds also inhibited the interaction of the receptors for interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are involved in hematopoietic differentiation and activation of immune cells, thus eliminating them as potential therapeutic agents. The inhibition of the structurally closely related receptors for IL-5, IL-3 and GM-CSF by both compounds, while binding of interleukin-4 to its receptor was not affected, suggests that a similar reactive site exists in the ligand-binding domains of the receptors for IL-5, IL-3 and GM-CSF.

Requirements for promoter activity in mouse oocytes and embryos distinguish paternal pronuclei from maternal and zygotic nuclei.

Fertilization of mouse eggs produces a 1-cell embryo containing both a paternal and maternal pronucleus. These two nuclei combine during the first mitosis to form the zygotic nuclei of 2-cell embryos. This transition is accompanied by the onset of transcription and the decline of maternal mRNA-dependent gene expression. To determine how changes in nuclear composition affect gene expression, plasmid DNA containing a promoter and an enhancer that function throughout a broad host range was injected into nuclei of oocytes and embryos. The requirements for promoter activity in paternal pronuclei of 1-cell embryos were distinct from those in maternal or zygotic nuclei: (1) Paternal pronuclei permitted high levels of promoter activity relative to maternal or zygotic nuclei. (2) Butyrate, an agent that alters chromatin structure, stimulated promoter activity in maternal or zygotic nuclei, but not in paternal pronuclei. (3) The embryo-responsive polyomavirus F101 enhancer also stimulated promoter activity, but only after formation of a 2-cell embryo. Either butyrate or the F101 enhancer stimulated promoter activity in zygotic nuclei to the level observed in paternal pronuclei. Stimulation also was observed with 2-cell embryos containing nuclei of only maternal or paternal origin, but their transcriptional capacity was more limited. These and other results support the hypothesis that the need for enhancers in 2-cell embryos results from repression by chromatin structure, and the role of enhancers is to relieve this repression.

Simian virus 40 large T antigen DNA helicase. Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements.

The ATPase of SV40 large T antigen (T antigen) which is essential for the replication of SV40 minichromosomes was recently shown to be functionally related to a newly discovered DNA helicase activity. The T antigen helicase unwinds DNA duplices of several kilobase pairs in a reaction depending on the presence of hydrolyzable ribo- or deoxyribonucleoside triphosphates. The in vitro rate of movement through duplex DNA was found to be about 100 base pairs/min at 37 degrees C. For DNA unwinding, T antigen requires a 3'-single strand extension of a partially double-stranded substrate and invades the double strand section processively, in a 3' to 5' direction. The minimum length of the single-stranded tail was determined to be less than 5 nucleotides. Unwinding studies in the presence of the Escherichia coli single strand-specific DNA-binding protein and competition experiments indicate that T antigen helicase binds preferentially at the single-stranded/double-stranded DNA junction. This DNA structure is therefore proposed to serve as an entry site for the T antigen helicase. Previously reported data suggest that T antigen is the replicative helicase of the SV40 minichromosome. The results presented here are consistent with these findings and imply that T antigen migrates actively and processively along the template for the leading strand.

Monoclonal antibodies as probes for a function of large T antigen during the elongation process of simian virus 40 DNA replication.

Various monoclonal antibodies specific for simian virus 40 large tumor antigen (T antigen) inhibit the elongation process of viral DNA replication in an in vitro system. The results provide strong evidence for a function intrinsic to T antigen during ongoing replicative-chain elongation. The antibody inhibition studies were further used to establish a correlation between the known biochemical activities of T antigen and its function during the elongation phase. The data demonstrate that, in addition to DNA binding and ATPase, a third function of T antigen is required for replicative chain elongation. This function is most probably related to the recently described DNA helicase activity of T antigen. This conclusion is based on the following results: aphidicolin treatment of actively replicating simian virus 40 minichromosomes causes a partial uncoupling of parental DNA strand separation and DNA synthesis; the strand separation reaction is blocked by the same monoclonal antibodies which strongly inhibit the elongation process. DNA helicase activity of isolated T antigen is equally well inhibited by the same set of monoclonal antibodies that affect minichromosome replication in vitro.