Repeated domestication of melon (Cucumis melo) in Africa and Asia and a new close relative from India.

PREMISE OF THE STUDY: The domestication history of melon is still unclear. An African or Asian origin has been suggested, but its closest wild relative was recently revealed to be an Australian species. The complicated taxonomic history of melon has resulted in additional confusion, with a high number of misidentified germplasm collections currently used by breeders and in genomics research. METHODS: Using seven DNA regions sequenced for 90% of the genus and the major cultivar groups, we sort out described names and infer evolutionary origins and domestication centers. KEY RESULTS: We found that modern melon cultivars go back to two lineages, which diverged ca. 2 million years ago. One is restricted to Asia (Cucumis melo subsp. melo), and the second, here described as C. melo subsp. meloides, is restricted to Africa. The Asian lineage has given rise to the widely commercialized cultivar groups and their market types, while the African lineage gave rise to cultivars still grown in the Sudanian region. We show that C. trigonus, an overlooked perennial and drought-tolerant species from India is among the closest living relatives of C. melo. CONCLUSIONS: Melon was domesticated at least twice: in Africa and Asia. The African lineage and the Indian C. trigonus are exciting new resources for breeding of melons tolerant to climate change.

Coexpression of CD25 and OX40 (CD134) receptors delineates autoreactive T-cells in type 1 diabetes.

T-cell-mediated loss of pancreatic beta-cells is the crucial event in the development of type 1 diabetes. The phenotypic characteristics of disease-associated T-cells in type 1 diabetes have not yet been defined. The negative results from two intervention trials (the Diabetes Prevention Trial-Type 1 Diabetes and the European Nicotinamide Diabetes Intervention Trial) illustrate the need for technologies to specifically monitor ongoing autoimmune reactions. We used fluorescence-activated cell sorter analysis to study surface marker expression on T-cell lines specific for two major type 1 diabetes autoantigens, GAD65 and proinsulin. We then applied this knowledge in a cross-sectional approach to delineate the phenotype of circulating memory T-cells. The autoreactive T-cells of patients could be distinguished from those of control subjects by their coexpression of CD25 and CD134. Autoantigen-specific T-cells that recognized multiple GAD65- and preproinsulin-derived peptides and coexpressed CD25(+)CD134(+) were confined to patients (n = 32) and pre-diabetic probands (n = 5). Autoantigen-reactive T-cells in control subjects (n = 21) were CD25(+)CD134(-) and recognized fewer autoantigen-derived peptides. Insulin therapy did not induce CD25(+)CD134(+) T-cells in type 2 diabetic patients. The coexpression of CD25 and the costimulatory molecule CD134 on memory T-cells provides a novel marker for type 1 diabetes-associated T-cell immunity. The CD134 costimulatory molecule may also provide a novel therapeutic target in type 1 diabetes.

Activation of cytosolic phospholipase A2 in human T-lymphocytes involves inhibitor-kappaB and mitogen-activated protein kinases.

The group IV 85 kDa cytosolic phospholipase A(2) regulates many aspects of innate immunity. However, the function of this enzyme in T-cells remains controversial. We show here that human peripheral blood lymphocytes and Jurkat cells express cytosolic phospholipase A(2) and produce prostaglandin A(2) and leukotriene B(4). Selective inhibitors of this enzyme suppressed Ca(2+)-ionophore-, mitogen- and T-cell receptor-mediated expression of interleukin-2 at the level of transcription from the promoter. Activation of mitogen-activated protein kinases (MAPK), degradation of inhibitor-kappaBalpha and transactivation by nuclear factor-kappaB (NFkappaB) were impaired as was the antigen-, lectin- and interleukin-2-driven proliferation of T-cells in vitro. Ligands of peroxisome proliferator-activated receptor-gamma (PPARgamma) induced rapid phosphorylation of MAPK in human monocytic but not in Jurkat cells. These data indicated that in T-cells, eicosanoids generated upon signal-activated cytosolic phospholipase A(2) promote NFkappaB-dependent interleukin-2 transcription via a PPARgamma-independent mechanism involving the MAPK-pathway.

A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood.

We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.

Strongly enhanced antitumor activity of trastuzumab and pertuzumab combination treatment on HER2-positive human xenograft tumor models.

The human epidermal growth factor receptor (HER) family plays an important role in cell survival and proliferation, and is implicated in oncogenesis. Overexpression of HER2 is associated with aggressive disease and poor prognosis. Trastuzumab is a humanized monoclonal antibody targeting HER2 and has proven survival benefit for women with HER2-positive early and metastatic breast cancer. Pertuzumab, another monoclonal antibody, is a HER2 dimerization inhibitor that binds to a different epitope on HER2 than trastuzumab and inhibits HER2 dimer formation with other HER family members such as HER3 and HER1. We investigated the antitumor activity of these agents alone and in combination in HER2-positive breast and non-small cell lung cancer xenografts. Our data show that the combination of trastuzumab and pertuzumab has a strongly enhanced antitumor effect and induces tumor regression in both xenograft models, something that cannot be achieved by either monotherapy. The enhanced efficacy of the combination was also observed after tumor progression during trastuzumab monotherapy. Near-IR fluorescence imaging experiments confirm that pertuzumab binding to tumors is not impaired by trastuzumab pretreatment. Furthermore, we show by in vitro assay that both trastuzumab and pertuzumab potently activate antibody-dependent cellular cytotoxicity. However, our data suggest that the strongly enhanced antitumor activity is mainly due to the differing but complementary mechanisms of action of trastuzumab and pertuzumab, namely inhibition of HER2 dimerization and prevention of p95HER2 formation.

Mapping of epitopes for autoantibodies to the type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modeling: overlap of antibody and T cell determinants.

IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787-817 and 841-869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment of peptides obtained after screening phage-displayed peptide libraries with purified 96/3 identified a consensus binding sequence of Asn-x-Glu-x-x-(aromatic)-x-x-Gly. The predicted surface on a three-dimensional homology model of the tyrosine phosphatase domain of IA-2 was analyzed for clusters of Asn, Glu, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn(858), Glu(836), and Trp(799) reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1 diabetic patients. This study identifies a region commonly recognized by autoantibodies in type 1 diabetes that overlaps with dominant T cell determinants.