Functional characterization of aquaporin-4 specific T cells: towards a model for neuromyelitis optica

Background

Antibodies to the water channel protein aquaporin-4 (AQP4), which is expressed in astrocytic endfeet at the blood brain barrier, have been identified in the serum of Neuromyelitis optica (NMO) patients and are believed to induce damage to astrocytes. However, AQP4 specific T helper cell responses that are required for the generation of anti-AQP4 antibodies and most likely also for the formation of intraparenchymal CNS lesions have not been characterized.

Methodology/Principal Findings

Using overlapping 15-meric peptides of AQP4, we identified the immunogenic T cell epitopes of AQP4 that are restricted to murine major histocompatibility complex (MHC) I-A^b. The N-terminal region of AQP4 was highly immunogenic. More precisely, the intracellular epitope AQP4_22–36 was detected as major immunogenic determinant. AQP4_82–108 (located in the second transmembrane domain), AQP4_139–153 (located in the second extracellular loop), AQP4_211–225 (located in the fifth transmembrane domain), AQP4_235–249 (located in the sixth transmembrane domain), as well as AQP4_289–306 in the intracellular C-terminal region were also immunogenic epitopes. AQP4_22–36 and AQP4_289–303 specific T cells were present in the natural T cell repertoire of wild type C57BL/6 mice and T cell lines were raised. However, active immunization with these AQP4 peptides did not induce signs of spinal cord disease. Rather, sensitization with AQP4 peptides resulted in production of IFN-γ, but also IL-5 and IL-10 by antigen-specific T cells. Consistent with this cytokine profile, the AQP4 specific antibody response upon immunization with full length AQP4 included IgG1 and IgG2, which are associated with a mixed Th2/Th1 T cell response.

Conclusions and Significance

AQP4 is able to induce an autoreactive T cell response. The identification of I-A^b restricted AQP4 specific T cell epitopes will allow us to investigate how AQP4 specific autoimmune reactions are regulated and to establish faithful mouse models of NMO that include both cellular and humoral responses against AQP4.     

Structural and mechanical analysis of tectorial membrane Tecta mutants

The tectorial membrane (TM) is an extracellular matrix of the cochlea whose prominent role in hearing has been demonstrated through mutation studies. The C1509G mutation of the Tecta gene, which encodes for the α-tectorin protein, leads to hearing loss. The heterozygote TM only attaches to the first row of outer hair cells (OHCs), and the homozygote TM does not attach to any OHCs. Here we measured the morphology and mechanical properties of wild-type, heterozygous, and homozygous Tecta TMs. Morphological analyses conducted with second- and third-harmonic imaging, scanning electron microscopy, and immunolabeling revealed marked changes in the collagen architecture and stereocilin-labeling patterns of the mutant TMs. The mechanical properties of the mutant TM were measured by force spectroscopy. Whereas the axial Young's modulus of the low-frequency (apical) region of Tecta mutant TM samples was similar to that of wild-type TMs, it significantly decreased in the basal region to a value approaching that found at the apex. Modeling simulations suggest that a reduced TM Young's modulus is likely to reduce OHC stereociliary deflection. These findings argue that the heterozygote C1509G mutation results in a lack of attachment of the TM to the OHCs, which in turn reduces both the overall number of OHCs that are involved in mechanotransduction and the degree of mechanotransduction exhibited by the OHCs that remain attached to the TM.     

Encephalopathy caused by ablation of very long acyl chain ceramide synthesis may be largely due to reduced galactosylceramide levels

Sphingolipids (SLs) act as signaling molecules and as structural components in both neuronal cells and myelin. We now characterize the biochemical, histological, and behavioral abnormalities in the brain of a mouse lacking very long acyl (C22-C24) chain SLs. This mouse, which is defective in the ability to synthesize C22-C24-SLs due to ablation of ceramide synthase 2, has reduced levels of galactosylceramide (GalCer), a major component of myelin, and in particular reduced levels of non-hydroxy-C22-C24-GalCer and 2-hydroxy-C22-C24- GalCer. Noteworthy brain lesions develop with a time course consistent with a vital role for C22-C24-GalCer in myelin stability. Myelin degeneration and detachment was observed as was abnormal motor behavior originating from a subcortical region. Additional abnormalities included bilateral and symmetrical vacuolization and gliosis in specific brain areas, which corresponded to some extent to the pattern of ceramide synthase 2 expression, with astrogliosis considerably more pronounced than microglial activation. Unexpectedly, unidentified storage materials were detected in lysosomes of astrocytes, reminiscent of the accumulation that occurs in lysosomal storage disorders. Together, our data demonstrate a key role in the brain for SLs containing very long acyl chains and in particular GalCer with a reduction in their levels leading to distinctive morphological abnormalities in defined brain regions.     

From organelle to organ: ZRIZI MATE-Type transporter is an organelle transporter that enhances organ initiation

Plant architecture is a predictable but flexible trait. The timing and position of organ initiation from the shoot apical meristem (SAM) contribute to the final plant form. While much progress has been made recently in understanding how the site of leaf initiation is determined, the mechanism underlying the temporal interval between leaf primordia is still largely unknown. The Arabidopsis ZRIZI (ZRZ) gene belongs to a large gene family encoding multidrug and toxic compound extrusion (MATE) transporters. Unique among plant MATE transporters identified so far, ZRZ is localized to the membrane of a small organelle, possibly the mitochondria. Plants overexpressing ZRZ in initiating leaves are short, produce leaves much faster than wild-type plants and show enhanced growth of axillary buds. These results suggest that ZRZ is involved in communicating a leaf-borne signal that determines the rate of organ initiation.     

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background

DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.

Aim

To investigate the effect of red blood cells on H₂O₂-induced DNA damage and repair in human peripheral blood mononuclear cells.

Methods

DNA breaks were induced in peripheral blood mononuclear cells by H₂O₂ in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by ³H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.

Results

Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.

Conclusion

Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.     

Chemokines in hepatitis C virus infection: pathogenesis, prognosis and therapeutics

Hepatitis C virus infection and its associated liver inflammatory disease is a major global health problem affecting over 170 million people worldwide. Following viral infection, multiple pro-inflammatory mediators contribute to recruitment of immune cells to the liver and to the generation of an anti-viral immune response. However, when this vigorous immune response fails to eliminate the virus, chronic infection is established. This in turn, results in an ongoing process of inflammation, regeneration and fibrosis that in many cases leads to the development of cirrhosis and of hepatocellular carcinoma. Multiple recent publications mark chemokines and their receptors as key players in leukocyte recirculation through the inflamed liver. Furthermore, chemokines may also be involved in liver regeneration, fibrosis, and in malignant transformation, which is induced by the persistence of inflammation. Accumulating data indicates that distinct chemokines and chemokine receptors may be associated with different stages of the chronic hepatitis C virus infection-associated liver disease. Multiple small molecules and peptide antagonizing chemokines and their receptors are in advanced phase 3 and phase 2 clinical trials. In the near future, such drugs are expected to enter clinical use raising the question whether they may be applicable for the treatment of chronic viral infection-associated liver disease. In this review, recent advances in understanding the role of chemokines and their receptors in the pathogenesis of chronic viral infection-associated liver disease are presented. Furthermore, the clinical implications of these novel findings, which mark chemokines as prognostic markers and therapeutic targets for immune-modulation during chronic liver viral infection, are documented.     

Viral and developmental cell fusion mechanisms: conservation and divergence

Membrane fusion is a fundamental requirement in numerous developmental, physiological, and pathological processes in eukaryotes. So far, only a limited number of viral and cellular fusogens, proteins that fuse membranes, have been isolated and characterized. Despite the diversity in structures and functions of known fusogens, some common principles of action apply to all fusion reactions. These can serve as guidelines in the search for new fusogens, and may allow the formulation of a cross-species, unified theory to explain divergent and convergent evolutionary principles of membrane fusion.