A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency

Alagille syndrome is a human autosomal dominant developmental disorder characterized by liver, heart, eye, skeletal, craniofacial and kidney abnormalities. Alagille syndrome is caused by mutations in the Jagged 1 (JAG1) gene, which encodes a ligand for Notch family receptors. The majority of JAG1 mutations seen in Alagille syndrome patients are null alleles, suggesting JAG1 haploinsufficiency as a primary cause of this disorder. Mice homozygous for a Jag1 null mutation die during embryogenesis and Jag1/+ heterozygous mice exhibit eye defects but do not exhibit other phenotypes characteristic of Alagille syndrome patients ( Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B., Hicks, C., Gendron-Maguire, M., Rand, E. B., Weinmaster, G. and Gridley, T. (1999) HUM: Mol. Genet. 8, 723-730). Here we report that mice doubly heterozygous for the Jag1 null allele and a Notch2 hypomorphic allele exhibit developmental abnormalities characteristic of Alagille syndrome. Double heterozygous mice exhibit jaundice, growth retardation, impaired differentiation of intrahepatic bile ducts and defects in heart, eye and kidney development. The defects in bile duct epithelial cell differentiation and morphogenesis in the double heterozygous mice are similar to defects in epithelial morphogenesis of Notch pathway mutants in Drosophila, suggesting that a role for the Notch signaling pathway in regulating epithelial morphogenesis has been conserved between insects and mammals. This work also demonstrates that the Notch2 and Jag1 mutations interact to create a more representative mouse model of Alagille syndrome and provides a possible explanation of the variable phenotypic expression observed in Alagille syndrome patients.     

Fragmentation of Golgi complex and Golgi autoantigens during apoptosis and necrosis

Anti-Golgi complex autoantibodies are found primarily in patients with Sjögren's syndrome and systemic lupus erythematosus, although they are not restricted to these diseases. Several Golgi autoantigens have been identified that represent a small family of proteins. Common features of all Golgi autoantigens appear to be their distinct structural organization of multiple α-helical coiled-coil rods in the central domains flanked by non-coiled-coil N-termini and C-termini, and their localization to the cytoplasmic face of Golgi cisternae. Many autoantigens in systemic autoimmune diseases have distinct cleavage products in apoptosis or necrosis and this has raised the possibility that cell death may play a role in the generation of potentially immunostimulatory forms of autoantigens. In the present study, we examined changes in the Golgi complex and associated autoantigens during apoptosis and necrosis. Immunofluorescence analysis showed that the Golgi complex was altered and developed distinctive characteristics during apoptosis and necrosis. In addition, immunoblotting analysis showed the generation of antigenic fragments of each Golgi autoantigen, suggesting that they may play a role in sustaining autoantibody production. Further studies are needed to determine whether the differences observed in the Golgi complex during apoptosis or necrosis may account for the production of anti-Golgi complex autoantibodies.     

Hypergravity-induced immunomodulation in a rodent model: lymphocytes and lymphoid organs

The major goal of this study was to quantify changes in lymphoid organs and cells over time due to centrifugation-induced hypergravity. C57BL/6 mice were exposed to 1, 2 and 3 G and the following assays were performed on days 1, 4, 7, 10, and 21: spleen, thymus, lung, and liver masses; total leukocyte, lymphocyte, monocyte/macrophage, and granulocyte counts; level of splenocyte apoptosis; enumeration of CD3+ T, CD3+/CD4+ T helper, CD3+/CD8+ T cytotoxic, B220+ B, and NK1.1+ natural killer cells; and quantification of cells expressing CD25, CD69, and CD71 activation markers. The data show that increased gravity resulted in decreased body, spleen, thymus, and liver, but not lung, mass. Significant reductions were noted in all three major leukocyte populations (lymphocytes, granulocytes, monocyte/macrophages) [correction of macrphages] with increased gravity; persistent depletion was noted in blood but not spleen. Among the various lymphocyte populations, the CD3+/CD8+ T cells and B220+ B cells were the most affected and NK1.1+ NK cells the least affected. Overall, the changes were most evident during the first week, with a greater influence noted for cells in the spleen. A linear relationship was found between some of the measurements and the level of gravity, especially on day 4. These findings indicate that hypergravity profoundly alters leukocyte number and distribution in a mammalian model and that some aberrations persisted throughout the three weeks of the study. In certain cases, the detected changes were similar to those observed after whole-body irradiation. In future investigations we hope to combine hypergravity with low-dose rate irradiation and immune challenge.     

gamma-secretase functions through Notch signaling to maintain skin appendages but is not required for their patterning or initial morphogenesis

The role of Notch signaling during skin development was analyzed using Msx2-Cre to create mosaic loss-of-function alleles with precise temporal and spatial resolution. We find that gamma-secretase is not involved in skin patterning or cell fate acquisition within the hair follicle. In its absence, however, inner root sheath cells fail to maintain their fates and by the end of the first growth phase, the epidermal differentiation program is activated in outer root sheath cells. This results in complete conversion of hair follicles to epidermal cysts that bears a striking resemblance to Nevus Comedonicus. Sebaceous glands also fail to form in gamma-secretase-deficient mice. Importantly, mice with compound loss of Notch genes in their skin phenocopy loss of gamma-secretase in all three lineages, demonstrating that Notch proteolysis accounts for the major signaling function of this enzyme in this organ and that both autonomous and nonautonomous Notch-dependent signals are involved.     

Low-dose total-body γ irradiation modulates immune response to acute proton radiation

Health risks due to exposure to low-dose/low-dose-rate radiation alone or when combined with acute irradiation are not yet clearly defined. This study quantified the effects of protracted exposure to low-dose/low-dose-rate γ rays with and without acute exposure to protons on the response of immune and other cell populations. C57BL/6 mice were irradiated with ??Co (0.05 Gy at 0.025 cGy/h); subsets were subsequently exposed to high-dose/high-dose-rate proton radiation (250 MeV; 2 or 3 Gy at 0.5 Gy/min). Analyses were performed at 4 and 17 days postexposure. Spleen and thymus masses relative to body mass were decreased on day 4 after proton irradiation with or without pre-exposure to γ rays; by day 17, however, the decrease was attenuated by the priming dose. Proton dose-dependent decreases, either with or without pre-exposure to γ rays, occurred in white blood cell, lymphocyte and granulocyte counts in blood but not in spleen. A similar pattern was found for lymphocyte subpopulations, including CD3+ T, CD19+ B, CD4+ T, CD8+ T and NK1.1+ natural killer (NK) cells. Spontaneous DNA synthesis by leukocytes after proton irradiation was high in blood on day 4 and high in spleen on day 17; priming with γ radiation attenuated the effect of 3 Gy in both body compartments. Some differences were also noted among groups in erythrocyte and thrombocyte characteristics. Analysis of splenocytes activated with anti-CD3/anti-CD28 antibodies showed changes in T-helper 1 (Th1) and Th2 cytokines. Overall, the data demonstrate that pre-exposure of an intact mammal to low-dose/low-dose-rate γ rays can attenuate the response to acute exposure to proton radiation with respect to at least some cell populations.     

Signature whistles in free‐ranging populations of Indo‐Pacific bottlenose dolphins,Tursiops aduncus

Common bottlenose dolphins (Tursiops truncatus) use individually distinctive signature whistles which are highly stereotyped and function as contact calls. Here we investigate whether Indo‐Pacific bottlenose dolphins (T. aduncus) use signature whistles. The frequency trace of whistle contours recorded from three genetically distinct free‐ranging populations was extracted and sorted into whistle types of similar shape using automated categorization. A signature whistle identification method based on the temporal patterns in signature whistle sequences of T. truncatus was used to identify signature whistle types (SWTs). We then compared the degree of variability in SWTs for several whistle parameters to determine which parameters are likely to encode identity information. Additional recordings from two temporarily isolated T. aduncus made during natural entrapment events in 2008 and 2009 were analyzed for the occurrence of SWTs. All populations were found to produce SWTs; 34 SWTs were identified from recordings of free‐ranging T. aduncus and one SWT was prevalent in each recording of the two temporarily isolated individuals. Of the parameters considered, mean frequency and maximum frequency were the least variable and therefore most likely to reflect identity information encoded in frequency modulation patterns. Our results suggest that signature whistles are commonly used by T. aduncus.     

Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseases

Replication protein A (RPA), a heterotrimer with subunits of molecular masses 70, 32, and 14 kDa, is a single-stranded-DNA-binding factor involved in DNA replication, repair, and recombination. There have been only three reported cases of anti-RPA in systemic lupus erythematosus (SLE) and Sjögren syndrome (SjS). This study sought to clarify the clinical significance of autoantibodies against RPA. Sera from 1,119 patients enrolled during the period 2000 to 2005 were screened by immunoprecipitation (IP) of ³⁵S-labeled K562 cell extract. Antigen-capture ELISA with anti-RPA32 mAb, immunofluorescent antinuclear antibodies (ANA) and western blot analysis with purified RPA were also performed. Our results show that nine sera immunoprecipitated the RPA70–RPA32–RPA14 complex and all were strongly positive by ELISA (titers 1:62,500 to 1:312,500). No additional sera were positive by ELISA and subsequently confirmed by IP or western blotting. All sera showed fine speckled/homogeneous nuclear staining. Anti-RPA was found in 1.4% (4/276) of SLE and 2.5% (1/40) of SjS sera, but not in rheumatoid arthritis (0/35), systemic sclerosis (0/47), or polymyositis/dermatomyositis (0/43). Eight of nine patients were female and there was no racial predilection. Other positive patients had interstitial lung disease, autoimmune thyroiditis/hepatitis C virus/pernicious anemia, or an unknown diagnosis. Autoantibody specificities found in up to 40% of SLE and other diseases, such as anti-nRNP, anti-Sm, anti-Ro, and anti-La, were unusual in anti-RPA-positive sera. Only one of nine had anti-Ro, and zero of nine had anti-nRNP, anti-Sm, anti-La, or anti-ribosomal P antibodies. In summary, high titers of anti-RPA antibodies were found in nine patients (1.4% of SLE and other diseases). Other autoantibodies found in SLE were rare in this subset, suggesting that patients with anti-RPA may form a unique clinical and immunological subset.     

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

Background

To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH).

Methods

Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.

Results

We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.

Conclusion

Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.