The heat shock protein gp96: a receptor-targeted cross-priming carrier and activator of dendritic cells

Heat shock proteins like gp96 (grp94) are able to induce specific cytotoxic T-cell (CTL) responses against cells from which they originate and are currently studied in clinical trials for use in immunotherapy of tumors. We have recently demonstrated that gp96 binds to at least one yet unidentified receptor restricted to antigen-presenting cells (APCs) like dendritic cells (DCs) but not to T cells. Moreover we have shown, that for CTL activation by gp96-chaperoned peptides receptor-mediated uptake of gp96 by APCs is required. Lately, we have discovered a second function of gp96 when interacting with professional APCs. Gp96 is able to mediate maturation of DCs as determined by upregulation of MHC class II, CD86 and CD83 molecules, secretion of pro-inflammatory cytokines IL-12 and TNF-alpha and enhanced T-cell simulatory capacity. Furthermore, the gp96 receptor(s) are down-regulated on mature DCs, suggesting that the gp96 receptor(s) behave similar to other endocytic receptors like CD36, mannose receptor etc. Our findings now provide additional evidence for the remarkable immunogenicity of gp96: first, the existence of specific gp96 receptors on APCs and second, the capacity to activate dendritic cells which is strictly required to enable these highly sophisticated APCs to prime CTL responses.     

Genomic sequencing reveals the structure of the Kcnk6 and map3k11 genes and their close vicinity to the sipa1 gene on mouse chromosome 19

In this report we present the analysis of two overlapping mouse cosmid clones that contain the entire Kcnk6, Map3k11 and Pcnxl3 genes, as well as part of the Sipa1 gene. The sequence and genomic organisation of the Kcnk6 and Map3k11 genes are described in detail. Sipa1 and Map3k11, which have independently been mapped with low resolution to the centromeric region of mouse chromosome 19, are shown here to lie close to each other and to the Kcnk6 gene, which has not previously been mapped. This gene cluster maps to the vicinity of the Dancer (Dc) mutation, which involves inner ear abnormalities and circling phenotypes. Since potassium channels have been implicated in deafness disorders, we have analysed the Kcnk6 gene, which encodes a two-P domain potassium channel, in the Dc mutant. No Dc-causing mutation in the Kcnk6 coding region could be identified. However, we detected a polymorphism in the Kcnk6 gene that leads to a C-terminal extension of the encoded protein by eight amino acids.     

The lipopolysaccharide-activated toll-like receptor (TLR)-4 induces synthesis of the closely related receptor TLR-2 in adipocytes

The central regulatory role of the adipocyte in whole body energy homeostasis is well established. However, recent findings suggest that preadipocytes and adipocytes may play an important physiological role in the regulation of both the innate and adaptive immune response. To systematically characterize the molecular machinery of the adipocyte that mediates the recognition of pathogens, we have focused our analysis on the recently identified Toll-like receptors (TLRs). These receptors have been implicated as mediators of the cellular response to bacterial lipopolysacharides (LPSs). Here, we report the cloning and functional characterization of mouse TLR-2 from 3T3-L1 adipocytes. TLR-2 synthesis is strongly induced in the adipocyte by LPS, TNFalpha, and the yeast cell wall extract zymosan. TLR-2 undergoes a lengthy intracellular maturation process with a half-life of exit from the ER of approximately 3 h. Furthermore, LPS treatment of adipocytes results in dramatic changes at the level of gene expression, including the synthesis of a distinct set of secretory proteins such as interleukin-6. Our studies demonstrate the presence of a fully intact pathway of innate immunity in the adipocyte that can be activated by LPS binding to the cell surface and results in the secretion of immunomodulatory molecules.     

Identification of a novel gene on chromosome 7q31 that is interrupted by a translocation breakpoint in an autistic individual

The results of genetic linkage studies for autism have suggested that a susceptibility locus for the disease is located on the long arm of chromosome 7 (7q). An autistic individual carrying a translocation, t(7;13)(q31.3;q21), with the chromosome 7 breakpoint located in the region of 7q implicated by genetic studies was identified. A novel gene known as "RAY1" (or "FAM4A1") was found to be directly interrupted by the translocation breakpoint. The gene, which was found to be encoded by 16 exons with evidence of alternative splicing, spanned > or =220 kb of DNA at 7q31.3. Mutation screening of the entire coding region in a set of 27 unrelated autistic individuals failed to identify phenotype-specific variants, suggesting that coding region mutations are unlikely to be involved in the etiology of autism. Apparent homologues of RAY1 have also been identified in mouse, rat, pig, chicken, fruit fly, and nematode. The human and mouse genes share similar splicing patterns, and their predicted protein products are 98% identical.     

Clustering of Human Chromosome Fragments on the Mouse Genome by Chromosome-Mediated Gene Transfer

Pieces of metaphase chromosomes prepared from mouse cells containingneo-tagged human chromosome 7 were transferred to mouse cellswith calcium phosphate to isolate G418-resistant clones. FISHanalysis revealed that the majority of them contained humanDNA at a single site on their genome. These transformants containedSTS markers mapped to various regions of chromosome 7. It isthus suggested that pieces of human chromosomes tend to assembleand integrate on the mouse genome.     

A Computer-Assisted 3D Model for Analyzing the Aggregation of Tumorigenic Cells Reveals Specialized Behaviors and Unique Cell Types that Facilitate Aggregate Coalescence

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named “facilitators” and “probes.” A third cell type, the “dervish”, is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.