Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5.

There is intense interest in induction and characterization of strain-transcending neutralizing Ab against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) as a target of broadly neutralizing Abs, but there is little information regarding the functional mechanism(s) of Ab-mediated neutralization. In this study, we report that vaccine-induced polyclonal anti-PfRH5 Abs inhibit the tight attachment of merozoites to erythrocytes and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 mAbs, we provide evidence of the following: 1) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; 2) neutralizing mAbs bind spatially related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; 3) a brief exposure window of PfRH5 is likely to necessitate rapid binding of Ab to neutralize parasites; and 4) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly neutralizing anti-malaria Abs and further encourage anti-PfRH5-based malaria prevention efforts.

IL-25 blockade augments antiviral immunity during respiratory virus infection.

IL-25 is implicated in the pathogenesis of viral asthma exacerbations. However, the effect of IL-25 on antiviral immunity has yet to be elucidated. We observed abundant expression and colocalization of IL-25 and IL-25 receptor at the apical surface of uninfected airway epithelial cells and rhinovirus infection increased IL-25 expression. Analysis of immune transcriptome of rhinovirus-infected differentiated asthmatic bronchial epithelial cells (BECs) treated with an anti-IL-25 monoclonal antibody (LNR125) revealed a re-calibrated response defined by increased type I/III IFN and reduced expression of type-2 immune genes CCL26, IL1RL1 and IL-25 receptor. LNR125 treatment also increased type I/III IFN expression by coronavirus infected BECs. Exogenous IL-25 treatment increased viral load with suppressed innate immunity. In vivo LNR125 treatment reduced IL-25/type 2 cytokine expression and increased IFN-ß expression and reduced lung viral load. We define a new immune-regulatory role for IL-25 that directly inhibits virus induced airway epithelial cell innate anti-viral immunity.

GeneCalling: transcript profiling coupled to a gene database query.

We describe here the GeneCalling method for the discovery of differentially expressed genes, both known and novel, from any species and with useful sequence information to determine the potential function of novel genes captured. The method relies on transcript visualization coupled to a database query to rapidly and quantitatively identify differentially expressed transcripts. The method has been applied to a wide variety of disease models in a wide variety of species, addressing problems as diverse as identifying novel human cancer gene targets, understanding how drugs and diet affect animal models of disease, and understanding the basis of trait differences in related strains of corn.

Angioarrestin: an antiangiogenic protein with tumor-inhibiting properties.

The angiopoietins comprise a family of proteins that have pro or antiangiogenic activities. Through a proprietary technology designed to identify transcripts of all expressed genes, we isolated a cDNA encoding an angiopoietin-related protein that we designate angioarrestin. The mRNA expression profile of angioarrestin was striking in that it was down-regulated in many tumor tissues when compared with adjacent nontumor tissue, suggesting a role for this protein in tumor inhibition. To test this hypothesis, we ectopically expressed angioarrestin in HT1080 tumor cells and measured pulmonary tumor nodule formation in nude mice. HT1080 cells expressing angioarrestin showed a marked reduction in the number and size of tumor nodules. In vitro, the recombinant protein was systematically tested in a number of endothelial cell assays and found to block critical processes involved in the angiogenic cascade, such as vascular endothelial growth factor/basic fibroblast growth factor-mediated endothelial cell proliferation, migration, tubular network formation, and adhesion to extracellular matrix proteins. These findings reveal a novel function for angioarrestin as an angiogenesis inhibitor and indicate that the molecule may be a potential cancer therapeutic.

Fibroblast growth factor 7: an inhibitor of phosphate transport derived from oncogenic osteomalacia-causing tumors.

Oncogenic osteomalacia (OO), a tumor-associated phosphate-wasting syndrome, provides an opportunity to identify regulators of renal phosphate homeostasis. We established cultures from OO-associated tumors. Conditioned medium from these cultures inhibited phosphate uptake in renal tubular epithelial cells. We then compared RNA from tumor-derived cultures expressing inhibitory activity with RNA from tumor-derived cultures in which inhibitory activity was not evident and identified candidate mRNAs specifically expressed by cultures inhibiting renal phosphate transport. Testing of identified candidates revealed that one protein, fibroblast growth factor 7 (FGF7), was a potent and direct inhibitor of phosphate uptake in vitro. A neutralizing monoclonal antibody to FGF7 reversed FGF7-dependent phosphate transport inhibition and inhibitory activity in conditioned medium from tumor cell cultures. Immunoassay revealed abundant FGF7 in inhibitory conditioned medium and minimal amounts in nonconditioned medium or conditioned medium with no phosphate transport inhibitory activity. Furthermore, only small amounts of FGF23 were present in inhibitory conditioned medium, comparable to concentrations found in conditioned medium with no phosphate transport inhibitory activity. Thus, FGF7 was specifically identified when selecting for in vitro phosphate transport inhibitory activity of tumor-derived cultures and was confirmed as a potent inhibitor of phosphate transport. Finally, FGF7 message was confirmed in PCR products of mRNA extracted from fragments of each tumor. Members of the FGF family (other than FGF23) are expressed by OO-associated tumors and may play a role in mediating this syndrome.

Gene expression quantitation technology summary.

Scientists routinely talk and write about gene expression and the abundance of transcripts, but in reality they extrapolate this information from the various measurements that a variety of different technologies provide. Indeed, there are many reasons why applying different technologies to the problem of transcript abundance may give different results, owing to an incomplete understanding of the gene in question or from shortcomings in the applications of the technologies. There are nine basic considerations for making a technology choice for quantitating gene expression that will impact the overall outcome: architecture, specificity, sensitivity, sample requirement, coverage, throughput, cost, reproducibility, and data management. These considerations will be discussed in the context of available technologies.

GeneCalling: transcript profiling coupled to a gene database query.

We describe the GeneCalling method for the discovery of differentially expressed genes, both known and novel, from any species including useful sequence information to determine the potential function of novel genes captured. The method relies on transcript visualization coupled to a database query to rapidly and quantitatively identify differentially expressed transcripts. The method has been applied to a wide variety of disease models in a variety of species, addressing problems as diverse as identifying novel human cancer gene targets, understanding how drugs and diet affect animal models of disease, and understanding the basis of trait differences in related strains of corn.

Inhibition of in vitro and in vivo T cell responses by recombinant human Tim-1 extracellular domain proteins.

Members of the T cell, Ig domain and mucin domain (Tim) family of proteins have recently been implicated in the control of T cell-mediated immune responses. Tim-1 (HUGO designation HAVCR1) polymorphisms have been linked to the regulation of atopy in mice and humans, suggestive of a role in immune regulation. Tim-1 is expressed upon activation of T cells. In concert with the increased expression of Tim-1, a binding partner for the extracellular domain of Tim-1 (eTim-1) was induced on activated T cells, and mRNA expression data was consistent with the binding partner being Tim-4. We found that co-immobilized recombinant eTim-1 was able to inhibit T cell activation mediated by CD3 + CD28 mAb. eTim-1 mediated its inhibitory effects on proliferation by arresting cell cycle at G(0)/G(1) phase through regulation of cell cycle proteins. In vivo, administration of eTim-1 proteins led to a decrease in both ear (contact hypersensitivity to oxazolone) and joint (methylated BSA antigen-induced arthritis) swelling. The inhibitory activity of eTim-1 in the T(h)1-dependent models was evidence that eTim-1 is able to modulate T cell responses. Manipulation of the Tim-1 interaction with its binding partner on T cells may therefore provide a novel target for therapeutic intervention in T cell-mediated diseases.