Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy.

Whether host DNA receptors have any capacity to distinguish self from nonself at the molecular level is an outstanding question in the innate immunity of mammals. Here, by using quantitative assays and electron microscopy, we show that cooperatively assembling into filaments on dsDNA may serve as an integral mechanism by which human IFN-inducible protein-16 (IFI16) engages foreign DNA. IFI16 is essential for defense against a number of different pathogens, and its aberrant activity is also implicated in several autoimmune disorders, such as Sjögren syndrome. IFI16 cooperatively binds dsDNA in a length-dependent manner and clusters into distinct protein filaments even in the presence of excess dsDNA. Consequently, the assembled IFI16⋅dsDNA oligomers are clearly different from the conventional noninteracting entities resembling beads on a string. The isolated DNA-binding domains of IFI16 engage dsDNA without forming filaments and with weak affinity, and it is the non-DNA-binding pyrin domain of IFI16 that drives the cooperative filament assembly. The surface residues on the pyrin domain that mediate the cooperative DNA binding are conserved, suggesting that related receptors use a common mechanism. These results suggest that IFI16 clusters into signaling foci in a switch-like manner and that it is capable of using the size of naked dsDNA as a molecular ruler to distinguish self from nonself.

Semaphorin 3A contributes to distal pulmonary epithelial cell differentiation and lung morphogenesis.

RATIONALE: Semaphorin 3A (Sema3A) is a neural guidance cue that also mediates cell migration, proliferation and apoptosis, and inhibits branching morphogenesis. Because we have shown that genetic deletion of neuropilin-1, which encodes an obligatory Sema3A co-receptor, influences airspace remodeling in the smoke-exposed adult lung, we sought to determine whether genetic deletion of Sema3A altered distal lung structure. METHODS: To determine whether loss of Sema3A signaling influenced distal lung morphology, we compared pulmonary histology, distal epithelial cell morphology and maturation, and the balance between lung cell proliferation and death, in lungs from mice with a targeted genetic deletion of Sema3A (Sema3A(-/-)) and wild-type (Sema3A(+/+)) littermate controls. RESULTS: Genetic deletion of Sema3A resulted in significant perinatal lethality. At E17.5, lungs from Sema3A(-/-) mice had thickened septae and reduced airspace size. Distal lung epithelial cells had increased intracellular glycogen pools and small multivesicular and lamellar bodies with atypical ultrastructure, as well as reduced expression of type I alveolar epithelial cell markers. Alveolarization was markedly attenuated in lungs from the rare Sema3A(-/-) mice that survived the immediate perinatal period. Furthermore, Sema3A deletion was linked with enhanced postnatal alveolar septal cell death. CONCLUSIONS: These data suggest that Sema3A modulates distal pulmonary epithelial cell development and alveolar septation. Defining how Sema3A influences structural plasticity of the developing lung is a critical first step for determining if this pathway can be exploited to develop innovative strategies for repair after acute or chronic lung injury.

B lymphocyte activation by human papillomavirus-like particles directly induces Ig class switch recombination via TLR4-MyD88.

Vaccination with human papillomavirus type 16 (HPV16) L1 virus-like particles (VLP) induces both high titer neutralizing IgG and protective immunity. Because protection from experimental infection by papillomavirus is mediated by neutralizing IgG, we sought the mechanisms that trigger humoral immunity to HPV16 L1 VLP. We find that HPV16 L1 VLP bind to murine B lymphocytes thereby inducing activation-induced cytidine deaminase expression and Ig class switch recombination to cause the generation of IgG. HPV16 L1 VLP also activate production of proinflammatory factors IFN-alpha, IL-6, MIP-1alpha, RANTES, and KC, up-regulate the expression of costimulatory molecules by naive B cells, and increase the B1 B cell subpopulation. These B cell responses to HPV16 L1 VLP are dependent upon MyD88. Although MyD88(-/-) B cells produce only mu transcript after exposure to HPV16 L1 VLP, MyD88(+/+) B cells express alpha, gamma, and mu Ig H chain and activation-induced cytidine deaminase transcripts. Notably, TLR4 mutant C3H/HeJ mice exhibited significantly reduced HPV16 VLP-specific IgG1, IgG2a, IgG2b, and IgG3 titers after vaccination as compared with the control C3H/HeOuJ mice. HPV16 L1 VLP directly activated class switch recombination and costimulatory molecule expression by B cells of C3H/HeOuJ mice but not C3H/HeJ mice. Thus HPV16 L1 VLP directly activate B cells to induce CD4(+) T cell independent humoral immune responses via TLR4- and MyD88-dependent signaling.

African trypanosome interactions with an in vitro model of the human blood-brain barrier.

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood-brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly-infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstreamform T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.

Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis.

Chromosomal instability appears to be key to the pathogenesis of malignant transformation in human cancers, yet the precise molecular mechanisms underlying chromosomal rearrangements remain largely unknown. Telomeres stabilize and protect the ends of chromosomes, but shorten because of cell division and/or oxidative damage. Critically short telomeres, in the setting of abrogated DNA damage checkpoints, have been shown to cause chromosomal instability in vitro and in animal models, leading to an increased cancer incidence as a result of chromosome fusions, subsequent breakage, and rearrangement. We present results from a quantitative, high-resolution, in situ method for telomere length assessment used to test the hypothesis that telomere shortening is an early contributor to human tumorigenesis. High-grade prostatic intraepithelial neoplasia (HGPIN) is a putative preinvasive precursor of prostatic adenocarcinoma, the most common noncutaneous malignancy in Western men. The telomere lengths of epithelial cells within HGPIN lesions were strikingly shorter than those of adjacent normal appearing epithelial cells in 93% (28 of 30) of lesions examined. This shortening is similar to what has been shown in fully invasive prostate adenocarcinomas. Interestingly, telomere shortening was restricted to the luminal epithelial cells of HGPIN and was not present in the underlying basal epithelial cells; this provides strong evidence that basal cells are most likely not the direct targets of neoplastic transformation. These findings reveal that telomere shortening is a defining somatic DNA alteration characterizing HGPIN. The implications of this are that the earliest phase of human prostate carcinogenesis may proceed as a consequence of chromosomal instability mediated by shortened, dysfunctional telomeres.