Thomas C. Brodnicki

Proapoptotic BH3-Only Bcl-2 Family Member Bik/Blk/Nbk Is Expressed in Hemopoietic and Endothelial Cells but Is Redundant for Their Programmed Death

ABSTRACT The BH3-only members of the Bcl-2 protein family are essential for initiation of programmed cell death and stress-induced apoptosis. We have determined the expression pattern in mice of the BH3-only protein Bik, also called Blk or Nbk, and examined its physiological function by gene targeting. We found that Bik is expressed widely in the hematopoietic compartment and in endothelial cells of the venous but not arterial lineages. Nevertheless, its loss did not increase the numbers of such cells in mice or protect hematopoietic cells in vitro from apoptosis induced by cytokine withdrawal or diverse other cytotoxic stimuli. Moreover, whereas loss of the BH3-only protein Bim rescued mice lacking the prosurvival protein Bcl-2 from fatal polycystic kidney disease and lymphopenia, loss of Bik did not. These results indicate that any function of Bik in programmed cell death and stress-induced apoptosis must overlap that of other BH3-only proteins.

Cloning, expression analysis, and chromosomal localization of murine and human homologues of a Xenopus Mix gene

We report the cloning and chromosomal localization of murine and human Mix genes, members of a subclass of paired‐like homeobox genes of which the Xenopus laevis Mix.1 gene is the founding member. The murine Mix gene was mapped to the distal region of chromosome 1 and the human region to the syntenic region 1q41‐42. Northern analysis and RT‐PCR of murine adult and embryonic tissues demonstrated that Mix expression was restricted to the early embryo. Whole‐mount in situ hybridization revealed patchy but symmetrical Mix expression in visceral endoderm of embryonic day (E)5.5 embryos. In slightly older embryos, the expression was skewed to one side of the embryo and by E6.5, at the onset of gastrulation, expression was seen in the epiblast, visceral endoderm, nascent mesoderm, and the primitive streak. This expression pattern was maintained in mid‐ and late‐streak embryos. In early bud‐stage embryos, expression was strongest in the proximal two thirds of the streak, extending to the base of the allantois. By the headfold‐stage, expression was confined to the remnant of the primitive streak in the caudal region of the embryo and, after E8.0, in the caudal notochord and tail bud mesoderm. Mix transcripts were no longer detectable after embryonic day 9.5.

Proinsulin-Specific, HLA-DQ8, and HLA-DQ8-Transdimer–Restricted CD4+ T Cells Infiltrate Islets in Type 1 Diabetes

Type 1 diabetes (T1D) develops when insulin-secreting β-cells, found in the pancreatic islets of Langerhans, are destroyed by infiltrating T cells. How human T cells recognize β-cell-derived antigens remains unclear. Genetic studies have shown that HLA and insulin alleles are the most strongly associated with risk of T1D. These long-standing observations implicate CD4+ T-cell responses against (pro)insulin in the pathogenesis of T1D. To dissect the autoimmune T-cell response against human β-cells, we isolated and characterized 53 CD4+ T-cell clones from within the residual pancreatic islets of a deceased organ donor who had T1D. These 53 clones expressed 47 unique clonotypes, 8 of which encoded proinsulin-specific T-cell receptors. On an individual clone basis, 14 of 53 CD4+ T-cell clones (26%) recognized 6 distinct but overlapping epitopes in the C-peptide of proinsulin. These clones recognized C-peptide epitopes presented by HLA-DQ8 and, notably, HLA-DQ8 transdimers that form in HLA-DQ2/-DQ8 heterozygous individuals. Responses to these epitopes were detected in the peripheral blood mononuclear cells of some people with recent-onset T1D but not in HLA-matched control subjects. Hence, proinsulin-specific, HLA-DQ8, and HLA-DQ8-transdimer–restricted CD4+ T cells are strongly implicated in the autoimmune pathogenesis of human T1D.

Molecular cloning of a C-type lectin superfamily protein differentially expressed by CD8α− splenic dendritic cells

Dendritic cells (DC) are potent antigen presenting cells that activate naive T cells. It is becoming increasingly clear that DC are not a homogeneous cell population, but comprise different subpopulations that differ in ontogeny and function. To further the molecular characterisation of DC, we screened for genes that were differentially expressed amongst DC subsets and could therefore give insight into their varying biological functions. Using Representational Difference Analysis (RDA) we identified a gene (CIRE) that is expressed at higher levels in the myeloid-related CD8alpha(-) DC than in the lymphoid-related CD8alpha(+) DC. CIRE is a 238 amino acid type II membrane protein, of approximately 33 kDa in size, whose extracellular region contains a C-type lectin domain. Northern blot analysis revealed that CIRE is almost exclusively expressed in DC and was not detected in organs such as heart, brain, kidney, liver, and thymus. T cells failed to express message for CIRE, whilst B cells expressed very low levels. These data here further substantiated by Northern blot analysis of 18 cell lines of various origins (myeloid, macrophage, B and T cell) where only one cell line, which was of myeloid origin and could give rise to DC, expressed mRNA for CIRE. Semi-quantitative RT-PCR suggested that CIRE is down-regulated upon activation. CIRE shares 57% identity with human DC-SIGN, a molecule that has been shown to be the ligand of ICAM-3 and that is also a receptor that binds HIV and facilitates trans-infection of T cells.

Cloning and characterization of the genes encoding the ankyrin repeat and SOCS box-containing proteins Asb-1, Asb-2, Asb-3 and Asb-4.

Members of the suppressor of cytokine signalling (SOCS) family of proteins have been shown to inhibit cytokine signalling via direct interactions with JAK kinases or activated cytokine receptors. In addition to their novel amino-terminal regions and SH2 domains that mediate these interactions, the SOCS proteins also contain carboxy-terminal regions of homology called the SOCS box. The SOCS box serves to couple SOCS proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. Several other families of proteins also contain SOCS boxes but differ from the SOCS proteins in the type of domain or motif they contain upstream of the SOCS box. We report here the cloning, characterization, mapping and expression analysis of four members of the ankyrin repeat and SOCS box-containing (Asb) protein family.

Localization of Idd11 using NOD congenic mouse strains: elimination of Slc9a1 as a candidate gene.

Abstract Type 1 diabetes is a multigenic autoimmune disease, the genetic basis for which is perhaps best characterized in the nonobese diabetic (NOD) mouse model. We previously located a NOD diabetes susceptibility locus, designated Idd11, on mouse Chromosome (Chr) 4 by analyzing diabetic backcross mice produced after crossing NOD/Lt with the nondiabetic resistant strain C57BL/6 (B6) strain. In order to confirm Idd11 and further refine its location, three NOD congenic mouse strains with different B6 derived intervals within Chr 4 were generated. Two of the congenic strains had a significant decrease in the cumulative incidence of diabetes compared with NOD/Lt control mice. The third NOD congenic strain, containing a B6 interval surrounding the Slc9a1 locus, was not protected against diabetes. These results define a new distal boundary for Idd11 and eliminate the Slc9a1 gene as a candidate. The Idd11 locus has now been definitively mapped to a 13cM interval on mouse Chr 4.

Mind the gap: analysis of marker-assisted breeding strategies for inbred mouse strains

The development of congenic mouse strains is the principal approach for confirming and fine mapping quantitative trait loci, as well as for comparing the phenotypic effect of a transgene or gene-targeted disruption between different inbred mouse strains. The traditional breeding scheme calls for at least nine consecutive backcrosses before establishing a congenic mouse strain. Recent availability of genome sequence and high-throughput genotyping now permit the use of polymorphic DNA markers to reduce this number of backcrosses, and empirical data suggest that marker-assisted breeding may require as few as four backcrosses. We used simulation studies to investigate the efficiency of different marker-assisted breeding schemes by examining the trade-off between the number of backcrosses, the number of mice produced per generation, and the number of genotypes per mouse required to achieve a quality congenic mouse strain. An established model of crossover interference was also incorporated into these simulations. The quality of the strain produced was assessed by the probability of an undetected region of heterozygosity (i.e., “gaps”) in the recipient genetic background, while maintaining the desired donor-derived interval. Somewhat surprisingly, we found that there is a relatively high probability for undetected gaps in potential breeders for establishing a congenic mouse strain. Marker-assisted breeding may decrease the number of backcross generations required to generate a congenic strain, but only additional backcrossing will guarantee a reduction in the number and length of undetected gaps harboring contaminating donor alleles.

Deficiency in Type I Interferon Signaling Prevents the Early Interferon–Induced Gene Signature in Pancreatic Islets but Not Type 1 Diabetes in NOD Mice

Type I interferons (IFNs) have been implicated in the initiation of islet autoimmunity and development of type 1 diabetes. To directly test their involvement, we generated NOD mice deficient in type I IFN receptors (NOD.IFNAR1−/−). Expression of the type I IFN-induced genes Mx1, Isg15, Ifit1, Oas1a, and Cxcr4 was detectable in NOD islets as early as 1 week of age. Of these five genes, expression of Isg15, Ifit1, Oas1a, and Mx1 peaked at 3–4 weeks of age, corresponding with an increase in Ifnα mRNA, declined at 5–6 weeks of age, and increased again at 10–14 weeks of age. Increased IFN-induced gene expression was ablated in NOD.IFNAR1−/− islets. Loss of Toll-like receptor 2 (TLR2) resulted in reduced islet expression of Mx1 at 2 weeks of age, but TLR2 or TLR9 deficiency did not change the expression of other IFN-induced genes in islets compared with wild-type NOD islets. We observed increased β-cell major histocompatibility complex class I expression with age in NOD and NOD.IFNAR1−/− mice. NOD.IFNAR1−/− mice developed insulitis and diabetes at a similar rate to NOD controls. These results indicate type I IFN is produced within islets in young mice but is not essential for the initiation and progression of diabetes in NOD mice.

Harp (harmonin-interacting, ankyrin repeat-containing protein), a novel protein that interacts with harmonin in epithelial tissues

Mutations in the triple PDZ domain‐containing protein harmonin have been identified as the cause of Usher deafness syndrome type 1C. Independently, we identified harmonin in a screen for genes expressed in pancreatic β cells. Using a yeast two‐hybrid assay, we show that the first PDZ domain of harmonin interacts with a novel protein, designated harp for harmonin‐interacting, ankyrin repeat‐containing protein. This interaction was confirmed in an over‐expression system and in mammalian cells, and shown to be mediated by the three C‐terminal amino acids of harp. Harp is expressed in many of the same epithelia as harmonin and co‐localization of native harp and harmonin was demonstrated by confocal microscopy in pancreatic duct epithelium and in a pancreatic β‐cell line. Harp, predicted molecular mass 48 kDa, has a domain structure which includes three ankyrin repeats and a sterile alpha motif. Human harp maps to chromosome 16, and its mouse homologue to chromosome 7. Sequences with similarity to harp include the sans gene, mutations of which are responsible for deafness in the Jackson shaker 2 (js) mutant mouse and in human Usher syndrome type 1G. The functional domain structures of harp and harmonin, their interaction under native conditions and their co‐localization suggest they constitute a scaffolding complex to facilitate signal transduction in epithelia.

Measuring bacterial load and immune responses in mice infected with Listeria monocytogenes.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8α+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6. Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6 . There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design. We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to β-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.