The error-prone DNA polymerase ι provides quantitative resistance to lung tumorigenesis and mutagenesis in mice.

Opposite undamaged nucleotide T, DNA polymerase ι (Polι) preferentially incorporates G rather than A, violating the Watson-Crick rule. Although the actual biological role of Polι remains enigmatic, we have identified its coding gene as a candidate for pulmonary adenoma resistance 2 (Par2), a mouse quantitative trait locus modulating chemically induced lung tumor susceptibility. Notably, the most tumor-sensitive Par2 allele possessed by the 129X1/SvJ mouse is associated with a loss-of-function mutation in Polι. To determine whether the nonfunctional Polι is responsible for the 129X1/SvJ-specific Par2 phenotype, we knocked out Polι in a C57BL/6J mouse carrying a less tumor-sensitive Par2 allele. Disruption of the C57BL/6J Polι conferred 129X1/SvJ-like sensitivity on the C57BL/6J Par2 locus and increased the in vivo mutation frequency in the lung, providing definitive proof that Polι causes the Par2 effect and inhibits tumorigenesis and mutagenesis, despite its extreme replication infidelity.

Unique contribution of IRF-5-Ikaros axis to the B-cell IgG2a response.

IRF-5 is a transcription factor activated by toll like receptor (TLR)7 and TLR9 during innate immune responses. IRF-5 activates not only Type I IFN, but also inflammatory cytokines. Most importantly, a genetic variation in the IRF-5 gene shows a strong association with autoimmune diseases such as Lupus. Here, we report that IRF5-deficient mice have attenuated IgG2a/c responses to T-cell-dependent and -independent antigens and to polyoma virus infection. This defect is due to the intrinsic deletion of IRF-5 in B cells, as SCID mice reconstituted with Irf5-/- B cells show a decrease in IgG2a/c expression after viral infection compared with mice that received wild-type B cells. Irf5-/-B cells in vitro have diminished TLR and cytokine-induced class switching to IgG2a/c. Addressing the molecular mechanism, we show that IRF-5 regulates IgG2a/c expression by decreasing Ikaros expression; reconstitution of IRF-5 in Irf5-/- B cells downregulates Ikaros levels and increases switching to IgG2a/c. The IRF site in ikzf1 promoter binds IRF-5, IRF-4 and IRF-8. We show that IRF-8 but not IRF-4 activates the ikzf1 promoter, and IRF-5 inhibits the transcriptional activity of IRF-8. Collectively, these results identify the IRF-5-Ikaros axis as a critical modulator of IgG2a/c class switching.

Preclinical development of CAT-354, an IL-13 neutralizing antibody, for the treatment of severe uncontrolled asthma.

BACKGROUND AND PURPOSE: IL-13 is a pleiotropic Th2 cytokine considered likely to play a pivotal role in asthma. Here we describe the preclinical in vitro and in vivo characterization of CAT-354, an IL-13-neutralizing IgG4 monoclonal antibody (mAb), currently in clinical development. EXPERIMENTAL APPROACH: In vitro the potency, specificity and species selectivity of CAT-354 was assayed in TF-1 cells, human umbilical vein endothelial cells and HDLM-2 cells. The ability of CAT-354 to modulate disease-relevant mechanisms was tested in human cells measuring bronchial smooth muscle calcium flux induced by histamine, eotaxin generation by normal lung fibroblasts, CD23 upregulation in peripheral blood mononuclear cells and IgE production by B cells. In vivo CAT-354 was tested on human IL-13-induced air pouch inflammation in mice, ovalbumin-sensitization and challenge in IL-13 humanized mice and antigen challenge in cynomolgus monkeys. KEY RESULTS: CAT-354 has a 165 pM affinity for human IL-13 and functionally neutralized human, human variant associated with asthma and atopy (R130Q) and cynomolgus monkey, but not mouse, IL-13. CAT-354 did not neutralize human IL-4. In vitro CAT-354 functionally inhibited IL-13-induced eotaxin production, an analogue of smooth muscle airways hyperresponsiveness, CD23 upregulation and IgE production. In vivo in humanized mouse and cynomolgus monkey antigen challenge models CAT-354 inhibited airways hyperresponsiveness and bronchoalveolar lavage eosinophilia. CONCLUSIONS AND IMPLICATIONS: CAT-354 is a potent and selective IL-13-neutralizing IgG4 mAb. The preclinical data presented here support the trialling of this mAb in patients with moderate to severe uncontrolled asthma.

The small muscle-specific protein Csl modifies cell shape and promotes myocyte fusion in an insulin-like growth factor 1-dependent manner.

We have isolated a murine cDNA encoding a 9-kD protein, Chisel (Csl), in a screen for transcriptional targets of the cardiac homeodomain factor Nkx2-5. Csl transcripts were detected in atria and ventricles of the heart and in all skeletal muscles and smooth muscles of the stomach and pulmonary veins. Csl protein was distributed throughout the cytoplasm in fetal muscles, although costameric and M-line localization to the muscle cytoskeleton became obvious after further maturation. Targeted disruption of Csl showed no overt muscle phenotype. However, ectopic expression in C2C12 myoblasts induced formation of lamellipodia in which Csl protein became tethered to membrane ruffles. Migration of these cells was retarded in a monolayer wound repair assay. Csl-expressing myoblasts differentiated and fused normally, although in the presence of insulin-like growth factor (IGF)-1 they showed dramatically enhanced fusion, leading to formation of large dysmorphogenic "myosacs." The activities of transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer-binding factor (MEF)2, were also enhanced in an IGF-1 signaling-dependent manner. The dynamic cytoskeletal localization of Csl and its dominant effects on cell shape and behavior and transcription factor activity suggest that Csl plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair.

Cardiac septal and valvular dysmorphogenesis in mice heterozygous for mutations in the homeobox gene Nkx2-5.

Heterozygous mutations in the cardiac homeobox gene, NKX2-5, underlie familial cases of atrial septal defect (ASD) with severe atrioventricular conduction block. In this study, mice heterozygous for Nkx2-5-null alleles were assessed for analogous defects. Although ASD occurred only rarely, atrial septal dysmorphogenesis was evident as increased frequencies of patent foramen ovale and septal aneurysm, and decreased length of the septum primum flap valve. These parameters were compounded by genetic background effects, and in the 129/Sv strain, septal dysmorphogenesis bordered on ASD in 17% of Nkx2-5 heterozygotes. In a proportion of neonatal heterozygotes, as well as in adults with ASD, we found that the size of the foramen ovale was significantly enlarged and altered in shape, potentially exposing the normally thin septum primum to excessive hemodynamic forces. Therefore, defective morphogenesis of the septum secundum may be one contributing factor in the generation of patent foramen ovale, septal aneurysm, and certain ASDs. Mild prolongation of P-R interval in females and an increased frequency of stenotic bicuspid aortic valves were also features of the Nkx2-5 heterozygous phenotype. Our data demonstrate that the complex effects of Nkx2-5 haploinsufficiency in mice are weaker but convergent with those in humans. As in the mouse, the phenotype of human NKX2-5 mutations may be modulated by interacting alleles.

Homeodomain factor Nkx2-3 controls regional expression of leukocyte homing coreceptor MAdCAM-1 in specialized endothelial cells of the viscera.

Regulated emigration of blood-borne leukocytes plays a defining role in lymphoid organ development, immune surveillance, and inflammatory responses. We report here that mice deficient in the homeobox gene Nkx2-3, expressed in developing visceral mesoderm, show a complex intestinal malabsorption phenotype and striking abnormalities of gut-associated lymphoid tissue and spleen suggestive of deranged leukocyte homing. Mutant Peyer's patches were reduced in number and size, intestinal villi contained few IgA(+) plasma cells, and mutant spleens were small and often atrophic, showing fused periarterial lymphoid sheaths, partially merged T and B cell zones, an absent marginal zone, and a dearth of macrophages in red pulp. Semiquantitative RT-PCR analysis and immunohistochemistry revealed down-regulation of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in endothelial cells in which Nkx2-3 is normally expressed. MAdCAM-1 is a member of the immunoglobulin superfamily, acting as an endothelial cell ligand for leukocyte homing receptors L-selectin and alpha4beta7 integrin. Our data suggest a role for a homeodomain factor in establishing the developmental and positional cues in endothelia that regulate leukocyte homing through local control of cellular adhesion and identify MAdCAM-1 as a candidate target gene of Nkx2-3.

Targeted insertion of a lacZ reporter gene into the mouse Cer1 locus reveals complex and dynamic expression during embryogenesis.

The mouse Cer1 (mCer1, Cer-l, Cerr1) gene encodes one member of a family of cytokines structurally and functionally related to the Xenopus head-inducing factor, Cerberus (xCer). We generated a mouse line in which the Cer1 gene was inactivated by replacing the first coding exon with a lacZ reporter gene. Mice homozygous for this allele (Cer1(lacZ)) showed no apparent perturbation of embryogenesis or later development. However, the lacZ reporter revealed a number of hitherto uncharacterised sites of Cer1 expression in late fetal and adult tissues. Preliminary analysis suggests that Cer1 is not essential for their morphogenesis, differentiation, or homeostasis.

A direct method for the formation of peptide and carbohydrate dendrimers.

Two new methods for the modification of PAMAM dendrimers have been developed which allow the covergent synthesis of either peptide or carbohydrate-bearing dendrimer molecules. Both methods involve condensation between hydroxylamino nucleophiles and appropriate carbonyl-bearing reaction partners.

Infertility in female mice lacking the receptor for interleukin 11 is due to a defective uterine response to implantation.

During early pregnancy, in response to the implanting embryo, the surrounding uterine stroma undergoes a dramatic transformation into a specialized tissue known as the decidua. The decidua encapsulates the developing embryo, facilitating nutrient transfer and limiting trophoblast invasion. Here we show that female mice with a null mutation of the interleukin-11 receptor alpha chain are infertile because of defective decidualization. A temporal analysis revealed IL-11 expression is maximal in the normal pregnant uterus at the time of decidualization, and in situ hybridization studies showed expression of the IL-11 and the IL-11 receptor alpha chain in the developing decidual cells. These observations reveal a previously unrecognized critical role for IL-11 signaling in female reproduction.

The role of leukemia inhibitory factor in skeletal muscle regeneration.

Although a number of cytokines have been implicated in tissue regeneration, it is unknown which ones actually function in vivo. Here, we use mice with a targeted mutation in the leukemia inhibitory factor (LIF) gene to examine the role of LIF in muscle regeneration. Using a muscle crush model, we show that muscle regeneration in LIF knockout mice is significantly, reduced compared to control littermates. Further, targeted infusion of LIF in both normal and LIF knockout animals stimulated muscle regeneration, but the stimulation observed was much greater in the mutant animals than in controls. In contrast, interleukin-6 and transforming growth factor-alpha, which also stimulate myoblast proliferation in vitro, had no effect on regeneration. These findings demonstrate directly that LIF is involved in regeneration of injured muscle and points to the use of LIF as a therapeutic agent in the treatment of neuromuscular disease.