Structural basis for CD1d presentation of a sulfatide derived from myelin and its implications for autoimmunity

Sulfatide derived from the myelin stimulates a distinct population of CD1d-restricted natural killer T (NKT) cells. Cis-tetracosenoyl sulfatide is one of the immunodominant species in myelin as identified by proliferation, cytokine secretion, and CD1d tetramer staining. The crystal structure of mouse CD1d in complex with cis-tetracosenoyl sulfatide at 1.9 Å resolution reveals that the longer cis-tetracosenoyl fatty acid chain fully occupies the A′ pocket of the CD1d binding groove, whereas the sphingosine chain fills up the F′ pocket. A precise hydrogen bond network in the center of the binding groove orients and positions the ceramide backbone for insertion of the lipid tails in their respective pockets. The 3′-sulfated galactose headgroup is highly exposed for presentation to the T cell receptor and projects up and away from the binding pocket due to its β linkage, compared with the more intimate binding of the α-glactosyl ceramide headgroup to CD1d. These structure and binding data on sulfatide presentation by CD1d have important implications for the design of therapeutics that target T cells reactive for myelin glycolipids in autoimmune diseases of the central nervous system

A long-term study of stable isotopes as tracers of processes governing water flow and quality in a lowland river basin: the upper Thames, UK

A long-term study of O, H and C stable isotopes has been undertaken on river waters across the 7000 km2 upper Thames lowland river basin in the southern UK. During the period, flow conditions ranged from drought to flood. A 10-year monthly record (2003–2012) of the main River Thames showed a maximum variation of 3‰ (δ18O) and 20‰ (δ2H), though inter-annual average values varied little around a mean of –6.5‰ (δ18O) and –44‰ (δ2H). The δ2H/δ18O slope of 5.3 suggested a degree of evaporative enrichment, consistent with derivation from local rainfall with a weighted mean of –7.2‰ (δ18O) and –48‰ (δ2H) for the period. A tendency towards isotopic depletion of the river with increasing flowrate was noted, but at very high flows (>100 m3/s) a reversion to the mean was interpreted as the displacement of bank storage by rising groundwater levels (corroborated by measurements of specific electrical conductivity). A shorter quarterly study (October 2011 – April 2013) of isotope variations in 15 tributaries with varying geology revealed different responses to evaporation, with a high inverse correlation between Δ18O and baseflow index (BFI) for most of the rivers. A comparison with aquifer waters in the basin showed that even at low flow, rivers rarely consist solely of isotopically unmodified groundwater. Long-term monitoring (2003–2007) of carbon stable isotopes in dissolved inorganic carbon (DIC) in the Thames revealed a complex interplay between respiration, photosynthesis and evasion, but with a mean inter-annual δ13C-DIC value of –14.8 ± 0.5‰, exchange with atmospheric carbon could be ruled out. Quarterly monitoring of the tributaries (October 2011 – April 2013) indicated that in addition to the above factors, river flow variations and catchment characteristics were likely to affect δ13C-DIC. Comparison with basin groundwaters of different alkalinity and δ13C-DIC values showed that the origin of river baseflow is usually obscured. The findings show how long-term monitoring of environmental tracers can help to improve the understanding of how lowland river catchments function

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Male-like external genitalia with epididymis in a case of 46, XX disorder of sex development due to congenital adrenal hyperplasia

<ul> <li>A case of five and half-year-old 46,XX phenotypic male with hyper pigmented empty scrotum, penile urethra, epididymis along with fallopian tubes, uterus and upper vagina as well as ovary is described. Hormonal studies were consistent with the diagnosis of congenital adrenal hyperplasia. The case represents the first documented case of 46,XX disorder of sex development due to virilizing CAH associated with differentiation of Wolffian ducts into epididymis.</li> <li><strong>KEY WORDS</strong>: 46,XX, virilization, external genitalia, epididymis, congenital adrenal hyperplasia</li> </ul&gt

Germline deletion of beta2 microglobulin or CD1d reduces anti-phospholipid antibody, but increases autoantibodies against non-phospholipid antigens, in NZB/W F1 model of lupus

Abstract Introduction β2-microglobulin (β2m) is required for the surface expression of MHC class I and class I-like proteins such as CD1d, Qa1 and neonatal Fc receptor (FcRn), all of which may impact the development of autoimmunity. Since CD1d is known to bind and present phospholipid antigens to T cells, we asked if the deficiency of β2m or CD1d will impact the development of anti-phospholipid antibodies as compared to other aspects of lupus autoimmunity. Methods We introgressed the β2m-null genotype onto the NZB and NZW backgrounds for 12 to 14 generations to generate genetically lupus-susceptible (NZB/NZW)F1 (BWF1) mice that are β2m-deficient (β2m°). Circulating immunoglobulins (Ig), rheumatoid factor (RF), anti-DNA and anti-cardiolipin (anti-CL) antibodies, and renal disease were analyzed in these and CD1d-deficient (CD1d°) BWF1 mice that we had previously generated. Results Whereas β2m° BWF1 mice had reduced serum IgG, they had increased mortality, nephritis, serum IgG anti-DNA antibody and RF as compared to heterozygous and wild-type littermates. These effects were recapitulated in CD1d° BWF1 mice, except that they also had increased serum IgG as compared to control littermates. Intriguingly, both β2m° and CD1d° mice had lower serum anti-CL antibody levels than in control littermates. Such CD1d dependence of anti-CL antibody production is not mediated by CD1d/glycolipid-reactive iNKT cells, as these cells reduced the production of RF and anti-DNA antibodies but had no effect on anti-CL antibodies. Conclusions We report a novel dichotomous role of β2m and CD1d, whereby these molecules differently regulate autoimmunity against phospholipid versus non-phospholipid autoantigens

Prevention of Autoimmunity by Targeting a Distinct, Noninvariant CD1d-reactive T Cell Population Reactive to Sulfatide

Class I and class II MHC-restricted T cells specific for proteins present in myelin have been shown to be involved in autoimmunity in the central nervous system (CNS). It is not yet known whether CD1d-restricted T cells reactive to myelin-derived lipids are present in the CNS and might be targeted to influence the course of autoimmune demyelination. Using specific glycolipid-CD1d tetramers and cloned T cells we have characterized a T cell population reactive to a myelin-derived glycolipid, sulfatide, presented by CD1d. This population is distinct from the invariant Vα14(+) NK T cells, and a panel of Vα3/Vα8(+) CD1d-restricted NK T cell hybridomas is unable to recognize sulfatide in the presence of CD1d(+) antigen-presenting cells. Interestingly, during experimental autoimmune encephalomyelitis a model for human multiple sclerosis, sulfatide-reactive T cells but not invariant NK T cells are increased severalfold in CNS tissue. Moreover, treatment of mice with sulfatide prevents antigen-induced experimental autoimmune encephalomyelitis in wild-type but not in CD1d-deficient mice. Disease prevention correlates with the ability of sulfatide to suppress both interferon-γ and interleukin-4 production by pathogenic myelin oligodendrocyte glycoprotein-reactive T cells. Since recognition of sulfatide by CD1d-restricted T cells has now been shown both in mice and humans, study of murine myelin lipid-reactive T cells may form a basis for the development of intervention strategies in human autoimmune demyelinating diseases