PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome

Usher syndrome is a genetically heterogeneous recessive disease characterized by hearing loss and retinitis pigmentosa (RP). It frequently presents with unexplained, often intrafamilial, variability of the visual phenotype. Although 9 genes have been linked with Usher syndrome, many patients do not have mutations in any of these genes, suggesting that there are still unidentified genes involved in the syndrome. Here, we have determined that mutations in PDZ domain–containing 7 (PDZD7), which encodes a homolog of proteins mutated in Usher syndrome subtype 1C (USH1C) and USH2D, contribute to Usher syndrome. Mutations in PDZD7 were identified only in patients with mutations in other known Usher genes. In a set of sisters, each with a homozygous mutation in USH2A, a frame-shift mutation in PDZD7 was present in the sister with more severe RP and earlier disease onset. Further, heterozygous PDZD7 mutations were present in patients with truncating mutations in USH2A, G protein–coupled receptor 98 (GPR98; also known as USH2C), and an unidentified locus. We validated the human genotypes using zebrafish, and our findings were consistent with digenic inheritance of PDZD7 and GPR98, and with PDZD7 as a retinal disease modifier in patients with USH2A. Pdzd7 knockdown produced an Usher-like phenotype in zebrafish, exacerbated retinal cell death in combination with ush2a or gpr98, and reduced Gpr98 localization in the region of the photoreceptor connecting cilium. Our data challenge the view of Usher syndrome as a traditional Mendelian disorder and support the reclassification of Usher syndrome as an oligogenic disease.     

Analysis of the skin of mice humanized for the immune system

Development of new immunotherapeutic strategies relies on the ability to activate the right cells at the right place and at the right moment and on the capacity of these cells to home to the right organ(s). Skin delivery has shown high potency for immunotherapeutic administration. However, an adequate in vivo model of human skin immunity is still a critical bottleneck. We demonstrated here that the skin of human immune system mice is colonized by human hematopoietic cells, mainly human T cells and that complementation with human antigen‐presenting cells at the vaccination site allowed the induction of an immune response.     

Biomonitoring of ciguatoxin exposure in mice using blood collection cards.

Ciguatera is a human food poisoning caused by consumption of tropical and subtropical fish that have, through their diet, accumulated ciguatoxins in their tissues. This study used laboratory mice to investigate the potential to apply blood collection cards to biomonitor ciguatoxin exposure. Quantitation by the neuroblastoma cytotoxicity assay of Caribbean ciguatoxin (C-CTX-1) spiked into mice blood was made with good precision and recovery. The blood collected from mice exposed to a sublethal dose of Caribbean ciguatoxic extract (0.59 ng/g C-CTX-1 equivalents) was analyzed and found to contain detectable toxin levels at least 12 h post-exposure. Calculated concentration varied from 0.25 ng/ml at 30 min post-exposure to 0.12 ng/ml at 12 h. A dose response mice exposure revealed a linear dose-dependent increase of ciguatoxin activity in mice blood, with more polar ciguatoxin congeners contributing to 89% of the total toxicity. Finally, the toxin measurement in mice blood exposed to toxic extracts from the Indian Ocean or from the Pacific Ocean showed that the blood collection card method could be extended to each of the three known ciguatoxin families (C-CTX, I-CTX and P-CTX). The low matrix effect of extracted dried-blood samples (used at 1:10 or 1:20 dilution) and the high sensitivity of the neuroblastoma assay (limit of detection 0.006 ng/ml C-CTX-1), determined that the blood collection card method is suitable to monitor ciguatoxin at sublethal doses in mice and opens the potential to be a useful procedure for fish screening, environmental risk assessment or clinical diagnosis of ciguatera fish poisoning in humans or marine mammals.     

Characterization of Thymic Nurse-Cell Lymphocytes,Using an Improved Procedure for Nurse-Cell Isolation

Thymic nurse cells (TNC), multicellular complexes consisting of lymphoid cells enclosed within cortical epithelial cells, were isolated from mouse thymus by a modified procedure allowing immunofluorescent labeling and flow cytometric analysis of their lymphoid contents (TNC-L). Collagenase was the only protease used for tissue digestion, to ensure that surface antigen markers remained intact. Zonal unit-gravity elutriation was used to enrich the TNC on the basis of their high sedimentation rate, followed by immunomagnetic bead depletion to remove residual mononuclear cell contaminants and a density separation to remove debris. The TNC-L were then released from inside TNC by a short period of culture. The measured contamination of TNC-L with exogenous thymocytes was around 0.5%. Three-color immunofluorescent labeling revealed that TNC-L included, as well as a maiority of immature CD4⁺8⁺3^low thymocytes, about 12% of apparently mature CD4⁺8^-3^high and CD4^-8⁺3^high thymocytes. TNC are located in the cortex, where mature cells are rare; the occurrence of mature phenotype cells within these structures suggests that they represent a microenvironment for the selection and generation of mature T cells.     

Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen

OBJECTIVE: Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoid arthritis (RA). This study was undertaken to test whether RA-associated HLA-DR alleles are associated with anti-citrullinated fibrinogen in RA patient sera and whether replacement of arginyl by citrullyl residues on fibrinogen peptides modifies their binding to HLA-DR molecules and their recognition by T cells. METHODS: Antikeratin, antifilaggrin, and anti-citrullinated fibrinogen antibodies were assayed in RA patients who had undergone HLA-DR typing. Direct assays were performed to investigate binding of citrullinated or native fibrinogen peptides (encompassing the entire alpha- and beta-chains of fibrinogen) to purified HLA-DR molecules. T cell proliferative responses to citrullinated or native fibrinogen peptides were measured in RA patients and controls. RESULTS: HLA-DRB1*0404 was associated with anti-citrullinated fibrinogen in RA sera (P = 0.002). For the RA-associated alleles HLA-DRB1*0401 and HLA-DR1, there was a nonsignificant trend toward association (P = 0.07). Multiple peptides from the alpha- and beta-chains of fibrinogen bound many HLA-DR alleles; DRB1*0404 was the best fibrinogen peptide binder. Citrullination did not influence fibrinogen peptide binding to HLA-DR or fibrinogen peptide recognition by T cells. Peripheral blood T cells that recognized native or citrullinated fibrinogen peptides were common in RA patients but not in healthy controls. CONCLUSION: The RA-associated HLA-DRB1*0404 allele is also associated with production of antibodies to citrullinated fibrinogen. DRB1*0401 and DRB1*01 tend to be associated with anti-citrullinated fibrinogen, but this is not statistically significant. Citrullination of fibrinogen peptide does not influence peptide-DR-T cell interaction. Finally, T cell proliferation in response to citrullinated or uncitrullinated fibrinogen peptides is frequent in RA patients and very infrequent in controls.     

Studies on thymus products: I. Modification of rosette-forming cells by thymic extracts. Determination of the target RFC sub-population

Thymic extracts confer on normal bone marrow rosette-forming cells (RFC) in vitro a high sensitivity to anti-theta serum (AθS) and azathioprine (AZ) which they usually lack. Thymic extracts can also confer high sensitivity to AθS and AZ to spleen RFC from adult thymectomized, neonatally thymectomized `thymus-deprived' and nude mice. However, the amount of thymic extracts necessary to get the effect is significantly higher on thymus-deprived and nude mouse spleen RFC than on RFC from spleens of adult thymectomized mice or normal mouse bone marrow. Thymic extracts are also active in vivo and there is a good correlation between the in vitro minimum concentration giving AθS and AZ sensitivity to spleen RFC from adult thymectomized mice and the in vivo minimum dose giving such an effect after intravenous injection. The effect of extract in vivo does not appear before the fourth hour after the injection and is transient, disappearing after 48 hours. Injection of thymic extracts induces the appearance of a `thymic activity' (TA) in the serum with a short half-life (2 hours). Injection of thymic extracts into normal mice does not modify sRFC characteristics in spleen, lymph nodes and bone marrow. It is suggested that thymic extracts act reversibly on a population of T-RFC precursors.