HLA class II haplotype DRB1*04-DQB1*0301 contributes to risk of familial generalized vitiligo and early disease onset

Generalized vitiligo is a common autoimmune disorder characterized by white patches of skin and overlying hair caused by loss of pigment-forming melanocytes from involved areas. Familial clustering of vitiligo is not uncommon, and patients and their relatives are at increased risk for a specific complex of other autoimmune diseases. Compared with sporadic vitiligo, familial vitiligo is characterized by earlier disease onset and greater risk and broader repertoire of autoimmunity, suggesting a stronger genetic component, and perhaps stronger associations with specific alleles. To determine whether the major histocompatibility complex (MHC) contributes to the familial clustering of vitiligo and vitiligo-associated autoimmune/autoinflammatory diseases, we performed case-control and family-based association analyses of HLA class II-DRB1 and -DQB1 alleles and haplotypes in affected probands and their parents from 76 European-American Caucasian families with familial vitiligo. Affected probands showed a significantly increased frequency of DRB1*04-DQB1*0301 and a significantly decreased frequency of DRB1*15-DQB1*0602 compared with a large sample of reference chromosomes. Family-based association analyses confirmed these results. Probands with DRB1*04-DQB1*0301 developed vitiligo an average of 13.32 yr earlier than probands with DRB1*15-DQB1*0602. Overall, our results indicate that specific MHC-linked genetic variation contributes to risk of familial vitiligo, although HLA does not completely explain familial clustering of vitiligo-associated autoimmune/autoinflammatory diseases.     

Intra- or intercomplex binding to the gamma-secretase enzyme. A model to differentiate inhibitor classes

Gamma-secretase is one of the critical enzymes required for the generation of amyloid-beta peptides from the beta-amyloid precursor protein. Because amyloid-beta peptides are generally accepted to play a key role in Alzheimer disease, gamma-secretase inhibition holds the promise for a disease-modifying therapy for this neurodegenerative condition. Although recent progress has enhanced the understanding of the biology and composition of the gamma-secretase enzyme complex, less information is available on the actual interaction of various inhibitor classes with the enzyme. Here we show that the two principal classes of inhibitor described in the scientific and patent literature, aspartyl protease transition state analogue and small molecule non-transition state inhibitors, display fundamental differences in the way they interact with the enzyme. Taking advantage of a gamma-secretase enzyme overexpressing cellular system and different radiolabeled gamma-secretase inhibitors, we observed that the maximal binding of non-transition state gamma-secretase inhibitors accounts only for half the number of catalytic sites of the recombinant enzyme complex. This characteristic stoichiometry can be best accommodated with a model whereby the non-transition state inhibitors bind to a unique site at the interface of a dimeric enzyme. Subsequent competition studies confirm that this site appears to be targeted by the main classes of small molecule gamma-secretase inhibitor. In contrast, the non-steroidal anti-inflammatory drug gamma-secretase modulator sulindac sulfide displayed noncompetitive antagonism for all types of inhibitor. This finding suggests that non-steroidal anti-inflammatory drug-type gamma-secretase modulators target an alternative site on the enzyme, thereby changing the conformation of the binding sites for gamma-secretase inhibitors.     

Exploring interactions by second-stage community analyses

Many biological data sets, from field observations and manipulative experiments, involve crossed factor designs, analysed in a univariate context by higher-way analyses of variance which partition out ‘main’ and ‘interaction’ effects. Indeed, tests for significance of interactions among factors, such as differing Before-After responses at Control and Impact sites, are the basis of the widely used BACI strategy for detecting impacts in the environment. There are difficulties, however, in generalising simple univariate definitions of interaction, from classic linear models, to the robust, non-parametric multivariate methods that are commonly required in handling assemblage data. The size of an interaction term, and even its existence at all, depends crucially on the measurement scale, so it is fundamentally a parametric construct. Despite this, certain forms of interaction can be examined using non-parametric methods, namely those evidenced by changing assemblage patterns over many time periods, for replicate sites from different experimental conditions (types of ‘Beyond BACI’ design) - or changing multivariate structure over space, at many observed times. Second-stage MDS, which can be thought of as an MDS plot of the pairwise similarities between MDS plots (e.g. of assemblage time trajectories), can be used to illustrate such interactions, and they can be formally tested by second-stage ANOSIM permutation tests. Similarities between (first-stage) multivariate patterns are assessed by rank-based matrix correlations, preserving the fully non-parametric approach common in marine community studies. The method is exemplified using time-series data on corals from Thailand, macrobenthos from Tees Bay, UK, and macroalgae from a complex recolonisation experiment carried out in the Ligurian Sea, Italy. The latter data set is also used to demonstrate how the analysis copes straightforwardly with certain repeated-measures designs.     

Differential selection pressure exerted on HIV by CTL targeting identical epitopes but restricted by distinct HLA alleles from the same HLA supertype

HLA diversity is seen as a major challenge to CTL vaccines against HIV. One current approach focuses on "promiscuous" epitopes, presented by multiple HLA alleles from within the same HLA supertype. However, the effectiveness of such supertype vaccines depends upon the functional equivalence of CTL targeting a particular epitope, irrespective of the restricting HLA. In this study, we describe the promiscuous HIV-specific CTL epitopes presented by alleles within the B7 supertype. Substantial differences were observed in the ability of CTL to select for escape mutation when targeting the same epitope but restricted by different HLA. This observation was common to all six promiscuous B7 epitopes identified. Moreover, with one exception, there were no significant differences in the frequency, magnitude, or immunodominance of the CTL responses restricted by different HLA alleles to explain these discrepancies. This suggests that the unique peptide/MHC complexes generated by even closely related HLA induce CTL responses that are qualitatively different. This hypothesis is supported by additional differences observed between CTL targeting identical epitopes but restricted by different HLA: first, the occurrence of distinct, HLA-specific escape mutation; second, the recruitment of distinct TCR repertoires by particular peptide/MHC complexes; and, third, significant differences in the functional avidity of CTL. Taken together, these data indicate that significant functional differences exist between CTL targeting identical epitopes but restricted by different, albeit closely related HLA. These findings are of relevance to vaccine approaches that seek to exploit HLA supertypes to overcome the problem of HLA diversity.     

Characterization of native and reconstituted exosome complexes from the hyperthermophilic archaeon Sulfolobus solfataricus

The eukaryotic exosome is a protein complex with essential functions in processing and degradation of RNA. Exosome-like complexes were recently found in Archaea. Here we characterize the exosome of Sulfolobus solfataricus. Two exosome fractions can be discriminated by density gradient centrifugation. We show that the Cdc48 protein is associated with the exosome from the 30S-50S fraction but not with the exosome of the 11.3S fraction. While only some complexes contain Cdc48, the archaeal DnaG-like protein was found to be a core exosome subunit in addition to Rrp4, Rrp41, Rrp42 and Csl4. Assays with depleted extracts revealed that the exosome is responsible for major ribonucleolytic activity in S. solfataricus. Various complexes consisting of the Rrp41-Rrp42 hexameric ring and Rrp4, Csl4 and DnaG were reconstituted. Dependent on their composition, different complexes showed variations in RNase activity indicating functional interdependence of the subunits. The catalytic activity of these complexes and of the native exosome can be ascribed to the Rrp41-Rrp42 ring, which degrades RNA phosphorolytically. Rrp4 and Csl4 do not exhibit any hydrolytic RNase activity, either when assayed alone or in context of the complex, but influence the activity of the archaeal exosome.     

GTP drives myosin light chain 1 interaction with the class V myosin Myo2 IQ motifs via a Sec2 RabGEF-mediated pathway

The yeast myosin light chain 1 (Mlc1p) belongs to a branch of the calmodulin superfamily and is essential for vesicle delivery at the mother-bud neck during cytokinesis due to is ability to bind to the IQ motifs of the class V myosin Myo2p. While calcium binding to calmodulin promotes binding/release from the MyoV IQ motifs, Mlc1p is unable to bind calcium and the mechanism of its interaction with target motifs has not been clarified. The presence of Mlc1p in a complex with the Rab/Ypt Sec4p and with Myo2p suggests a role for Mlc1p in regulating Myo2p cargo binding/release by responding to the activation of Rab/Ypt proteins. Here we show that GTP or GTPgammaS potently stimulate Mlc1p interaction with Myo2p IQ motifs. The C-terminus of the Rab/Ypt GEF Sec2p, but not Sec4p activation, is essential for this interaction. Interestingly, overexpression of constitutively activated Ypt32p, a Rab/Ypt protein that acts upstream of Sec4p, stimulates Mlc1p/Myo2p interaction similarly to GTP although a block of Ypt32 GTP binding does not completely abolish the GTP-mediated Mlc1p/Myo2p interaction. We propose that Mlc1p/Myo2p interaction is stimulated by a signal that requires Sec2p and activation of Ypt32p.     

Combination of cytosine deaminase with uracil phosphoribosyl transferase leads to local and distant bystander effects against RM1 prostate cancer in mice

BACKGROUND: We aimed to evaluate the efficacy of gene-directed enzyme-prodrug therapy (GDEPT) using cytosine deaminase in combination with uracil phosphoribosyl transferase (CDUPRT) against intraprostatic mouse androgen-refractory prostate (RM1) tumors in immunocompetent mice. The product of the fusion gene, CDUPRT, converts the prodrug, 5-fluorocytosine (5FC), into 5-fluorouracil (5FU) and other cytotoxic metabolites that kill both CDUPRT-expressing and surrounding cells, via a 'bystander effect'. METHODS: Stably transformed andogen-independent mouse prostate cancer (PC) cells, RM1-CDUPRT, -GFP or GFP/LacZ cells were used. To assess the local bystander effects of CDUPRT-GDEPT, immunocompetent C57BL/6 mice implanted with cell mixtures of RM1-GFP/CDUPRT and RM1-GFP cells in different proportions intraprostatically were treated with 5FC. Pseudo-metastases in the lungs were established by a tail vein injection of untransfected RM1 cells. At necropsy, prostate weight/volume and lung colony counts were assessed. Tumors, lymph nodes, spleens and lungs were frozen or fixed for immunohistochemistry. RESULTS: CDUPRT expression in RM1-GFP/CDUPRT cells or tumors was confirmed by enzymic conversion of 5FC into 5FU, using HPLC. Treatment of mice bearing intraprostatic RM1-GFP/CDUPRT tumors with 5FC resulted in complete regression of the tumors. A 'local bystander effect' was seen, even though only 20% of the cells expressed CDUPRT. More importantly a significant reduction in pseudo-metastases of RM1 cells in lungs indicated a 'distant bystander effect'. Immunohistochemical evaluation of the treated tumors showed increased necrosis and apoptosis, with decreased tumor vascularity. There was also a significant increase in tumour-infiltration by macrophages, CD4+ T and natural killer cells. CONCLUSIONS: We conclude that CDUPRT-GDEPT significantly suppressed the aggressive growth of RM1 prostate tumors and lung pseudo-metastases via immune mechanisms involving necrosis and apoptosis.     

The immunobiology of Acanthamoeba keratitis

Acanthamoeba spp. are free-living amoebae that cause Acanthamoeba keratitis, a blinding corneal infection. The innate immune apparatus is crucial for the resolution of the disease. With the exception of mucosal antibody, elements of the adaptive immune system fail to prevent infection or contribute to its resolution in experimental animals.