Characterization of the human homolog of the IL-4 induced gene-1 (Fig1)

Mouse interleukin-four induced gene-1 (mFig1) maps to a region of susceptibility for systemic lupus erythematosus (SLE) that includes the Sle3 locus. To begin examining this relationship in humans, we have isolated and characterized the human homolog of mFig1. Human Fig1 (hFig1) has the same eight exon genomic structure as mFig1. The predicted 63-kDa protein, like mFig1, contains a signal peptide, a large internal sequence that is most similar (43% identical over 484 amino acids) to L-amino acid oxidase (LAAO), and a carboxy terminal domain with no similarity to known genes. When compared to the LAAO crystal structure, hFig1 conserves key residues thought to be involved in catalysis and binding of the flavin adenine dinucleotide cofactor. Surprisingly, the carboxy terminal domains of hFig1 and mFig1 have little similarity (<11% identity), different lengths and amino acid composition. Like mFig1, hFig1 RNA is induced by interleukin-4 (IL-4) in B lymphocytes, and is primarily found in immune tissues. Finally, hFig1 maps to the predicted mFig1 syntenic region on human chromosome 19q13.3-19q13.4, a hot spot for susceptibility to several autoimmune diseases, including SLE.     

Dissecting the genetic complexity of the association between human leukocyte antigens and rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory disease with a complex genetic component. An association between RA and the human leukocyte antigen (HLA) complex has long been observed in many different populations, and most studies have focused on a direct role for the HLA-DRB1 "shared epitope" in disease susceptibility. We have performed an extensive haplotype analysis, using 54 markers distributed across the entire HLA complex, in a set of 469 multicase families with RA. The results show that, in addition to associations with the DRB1 alleles, at least two additional genetic effects are present within the major histocompatibility complex. One of these lies within a 497-kb region in the central portion of the HLA complex, an interval that excludes DRB1. This genetic risk factor is present on a segment of a highly conserved ancestral A1-B8-DRB1*03 (8.1) haplotype. Additional risk genes may also be present in the HLA class I region in a subset of DRB1*0404 haplotypes. These data emphasize the importance of defining haplotypes when trying to understand the HLA associations with disease, and they clearly demonstrate that such associations with RA are complex and cannot be completely explained by the DRB1 locus.     

Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules

The development of novel approaches that allow accurate targeting of therapeutics to the intestinal mucosa is a major task in the research on intestinal inflammation. For the first time, a live genetically modified bacterial strain has been approved by Dutch authorities as a therapeutic agent for experimental therapy of intestinal bowel disease (IBD) in humans. Genetically modified probiotics can very well be used as carriers for localized antigen delivery into the intestine. Therapeutic safety, however, of such a carrier organism, is crucial, especially when a specific probiotic strain has to be used under diseased conditions. In this study, we tested the potential of Escherichia coli NISSLE 1917 to serve as a safe carrier for targeted delivery of recombinant proteins to the intestinal mucosa. In a well-defined and very sensitive immunological system, we demonstrate that intestinal recombinant E. coli NISSLE 1917 has no effect on migration, clonal expansion and activation status of specific CD4+ T cells, neither in healthy mice nor in animals with acute colitis. Furthermore, recombinant E. coli NISSLE 1917 has no effect on the induction or breakdown of peripheral T-cell tolerance in an autoimmune environment. The excellent colonization properties of E. coli NISSLE 1917 render this strain an ideal candidate as carrier organism for gut-focused in situ synthesis of therapeutic molecules.     

Cytotoxic T-lymphocyte elicited therapeutic vaccine candidate targeting cancer against MAGE-A11 carcinogenic protein

Immunotherapy is a breakthrough approach for cancer treatment and prevention. By exploiting the fact that cancer cells have overexpression of tumor antigens responsible for its growth and progression, which can be identified and removed by boosting the immune system. In silico techniques have provided efficient ways for developing preventive measures to ward off cancer. Herein, we have designed a potent cytotoxic T-lymphocyte epitope to elicit a desirable immune response against carcinogenic melanoma-associated antigen-A11. Potent epitope was predicted using reliable algorithms and characterized by advanced computational avenue CABS molecular dynamics simulation, for full flexible binding with HLA-A*0201 and androgen receptor to large-scale rearrangements of the complex system. Results showed the potent immunogenic construct (KIIDLVHLL), from top epitopes using five algorithms. Molecular docking analyses showed the strong binding of epitope with HLA-A*0201 and androgen receptor with docking score of −780.6 and −641.06 kcal/mol, respectively. Molecular dynamics simulation analysis revealed strong binding of lead epitope with androgen receptor by involvement of 127 elements through atomic-model study. Full flexibility study showed stable binding of epitope with an average root mean square deviation (RMSD) 2.21 Å and maximum RMSD value of 6.48 Å in optimal cluster density area. The epitope also showed remarkable results with radius of gyration 23.0777 Å, world population coverage of 39.08% by immune epitope database, and transporter associated with antigen processing (TAP) affinity IC₅₀ value of 2039.65 nm. Moreover, in silico cloning approach confirmed the expression and translation capacity of the construct within a suitable expression vector. The present study paves way for a potential immunogenic construct for prevention of cancer.     

Immobilization of glucansucrase for the production of gluco-oligosaccharides from Leuconostoc mesenteroides

Glucansucrase from Leuconostoc mesenteroides was immobilized in 1?% (w/v) with sodium alginate to produce oligosaccharides. Glucansucrase gave three activity bands of approx. 240, 178, and 165?kDa after periodic acid-Schiff staining with sucrose. The immobilized enzyme had 40?% activity after ten batch reactions at 30?°C and 75?% activity after a month of storage at 4?°C, which is six times more stable than the free enzyme. Immobilized enzyme was more stable at lower (3.5?4.5) and higher (6.5?7.0) pH ranges and higher temperatures (35?40?°C) compared with the free enzyme. Immobilized and free glucansucrase were employed in the acceptor reaction with maltose and each produced gluco-oligosaccharide ranging from trisaccharides to homologous pentasaccharides.     

Genome-wide meta-analysis for rheumatoid arthritis

Meta-analysis is being increasingly used as a tool for integrating data from different studies of complex phenotypes, because the power of any one study to identify causal loci is limited. We applied a novel meta-analytical approach (Loesgen et al. in Genet Epidemiol 21(Suppl 1):S142–S147, 2001) in compiling results from four studies of rheumatoid arthritis in Caucasians including two studies from NARAC (Jawaheer et al. in Am J Hum Genet 68:927–936, 2001; Jawaheer et al. in Arthritis Rheum 48:906–916, 2003), one study from the UK (MacKay et al. in Arthritis Rheum 46:632–639, 2001) and one from France (Cornelis et al. in Proc Natl Acad Sci USA 95:10746–10750, 1998). For each study, we obtained NPL scores by performing interval mapping (2 cM intervals) using GeneHunter2 (Kruglyak et al. in Am J Hum Genet 58:1347–1363, 1996; Markianos et al. in Am J Hum Genet 68:963–977, 2001). The marker maps differed among the three consortium groups, therefore, the marker maps were aligned after the interval mapping was completed and the NPL scores that were within 1 cM of each other were combined using the method of Loesgen et al. (Genet Epidemiol 21(Suppl 1):S142–S147, 2001) by calculating the weighted average of the NPL score. This approach avoids some problems in analysis encountered by using GeneHunter2 when some markers in the sample are not genotyped. This procedure provided marginal evidence (P<0.05) of linkage on chromosome 1, 2, 5 and 18, strong evidence (P<0.01) on chromosomes 8 and 16, and overwhelming evidence in the HLA region of chromosome 6.     

Visualizing human leukocyte antigen class II risk haplotypes in human systemic lupus erythematosus

Human leukocyte antigen (HLA) class I and class II alleles are implicated as genetic risk factors for many autoimmune diseases. However, the role of the HLA loci in human systemic lupus erythematosus (SLE) remains unclear. Using a dense map of polymorphic microsatellites across the HLA region in a large collection of families with SLE, we identified three distinct haplotypes that encompassed the class II region and exhibited transmission distortion. DRB1 and DQB1 typing of founders showed that the three haplotypes contained DRB1*1501/ DQB1*0602, DRB1*0801/ DQB1*0402, and DRB1*0301/DQB1*0201 alleles, respectively. By visualizing ancestral recombinants, we narrowed the disease-associated haplotypes containing DRB1*1501 and DRB1*0801 to an approximately 500-kb region. We conclude that HLA class II haplotypes containing DRB1 and DQB1 alleles are strong risk factors for human SLE.