T-cell receptor BV gene usage in colorectal carcinoma patients immunised with recombinant Ep-CAM protein or anti-idiotypic antibody

The tumour-associated antigen, Ep-CAM, is over-expressed in colorectal carcinoma (CRC). In the present study, a recombinant Ep-CAM protein or a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM, either alone or in combination, was used for vaccination of CRC patients (n=9). GM-CSF was given as an adjuvant cytokine. A cellular immune response was assessed by measuring anti-Ep-CAM lymphoproliferation, IFN- production (ELISPOT) and by analysing the TCR BV gene usage within the CD4+ and CD8+ T-cell subsets followed by CDR3 fragment analysis. A proliferative and/or IFN- T-cell response was induced against the Ep-CAM protein in eight out of nine patients, and against Ep-CAM-derived peptides in nine out of nine patients. Analysis of the TCR BV gene usage showed a significantly higher usage of BV12 family in CD4+ T cells of patients both before and after immunisation than in those of healthy control donors (p<0.05). In the CD8+ T-cell subset, a significant (p<0.05) increase in the BV19 usage was noted in patients after immunisation. In individual patients, a number of TCR BV gene families in both CD4+ and CD8+ T cells were over-expressed mainly in post-immunisation samples. Analysis of the CDR3 length polymorphism revealed a higher degree of clonality in post-immunisation samples than in pre-immunisation samples. In vitro stimulation with Ep-CAM protein confirmed the expansion of anti-Ep-CAM T-cell clones. The results indicate that immunisation with the Ep-CAM protein and/or anti-Id entails the induction of an anti-Ep-CAM T-cell response in CRC patients, and suggest that BV19+ CD8+ T cells might be involved in a vaccine-induced immune response.     

Global-scale genetic identification of hammerhead sharks: Application to assessment of the international fin trade and law enforcement

The future status of sharks is an issue of widespread conservation concern due to declines in many species in the face of high levels of exploitation to satisfy market demands for products, especially fins. Substantial declines in the large-bodied hammerhead sharks, Sphyrna lewini, S. mokarran and S. zygaena, even in regions where some management occurs, indicate that informed conservation measures are warranted for these circumglobally distributed species. Despite the importance of assessing shark catch and trade on a species-specific basis to detect potential overexploitation of individual species, achieving this goal for hammerheads has proven elusive due to difficulties in identification of their products. Here, we present the development and application of a diagnostic, streamlined, five-primer multiplex polymerase chain reaction assay utilizing species-specific primers based on nuclear ribosomal ITS2 for the three hammerhead species throughout their global distribution. Application of this assay to investigations of the fin market confirmed the presence of hammerhead fins in the international trade. A study of the world’s largest fin market in Hong Kong revealed a high concordance between specific Chinese-name trade categories and fins from these three species (“Bai Chun” with S. lewini, “Gui Chun” with S. zygaena and “Gu Pian” with S.␣mokarran), and clear species preferences. This concordance information allows the use of market records for monitoring species-specific trends in trade and exploitation rates. The assay is also proving useful for identification of shark body parts in U.S. fisheries law-enforcement activities. Screening of morphologically identified “ S. lewini” from globally distributed areas using this assay with subsequent whole ITS2 sequencing suggests a cryptic species closely related to S. lewini occurs off the SE USA coast.     

Genetic profiling reveals illegal international trade in fins of the great white shark, <Emphasis Type="Italic">Carcharodon carcharias</Emphasis>

Great white sharks are protected by national legislation in several countries, making this species the most widely protected elasmobranch in the world. Although the market demand for shark fins in general has continued to grow, the value and extent of utilization of white shark fins in trade has been controversial. We combine law enforcement with genetic profiling to demonstrate that illegal trade in fins of this species is occurring in the contemporary international market. Furthermore, we document the presence of fins from very young white sharks in the trade, suggesting a multiple-use market (food to trophies) exists for fins of this species. The presence of small fins in the trade contradicts the view that white shark fins have market value only as large display trophies, and not as food. Our findings indicate that effective conservation of protected shark species will require international management regimes that include monitoring of the shark fishery and trade on a species-specific basis.     

Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.     

CD4+CD25+ cells controlling a pathogenic CD4 response inhibit cytokine differentiation, CXCR-3 expression, and tissue invasion

It is well established that CD4(+)CD25(+) regulatory T cells (Tregs) inhibit autoimmune pathology. However, precisely how the behavior of disease-inducing T cells is altered by Tregs remains unclear. In this study we use a TCR transgenic model of diabetes to pinpoint how pathogenic CD4 T cells are modified by Tregs in vivo. We show that although Tregs only modestly inhibit CD4 cell expansion, they potently suppress tissue infiltration. This is associated with a failure of CD4 cells to differentiate into effector cells and to up-regulate the IFN-gamma-dependent chemokine receptor CXCR-3, which confers the ability to respond to pancreatic islet-derived CXCL10. Our data support a model in which Tregs permit T cell activation, yet prohibit T cell differentiation and migration into Ag-bearing tissues.     

Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice

Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.