Stature and body mass estimation from skeletal remains in the European Holocene.

Techniques that are currently available for estimating stature and body mass from European skeletal remains are all subject to various limitations. Here, we develop new prediction equations based on large skeletal samples representing much of the continent and temporal periods ranging from the Mesolithic to the 20th century. Anatomical reconstruction of stature is carried out for 501 individuals, and body mass is calculated from estimated stature and biiliac breadth in 1,145 individuals. These data are used to derive stature estimation formulae based on long bone lengths and body mass estimation formulae based on femoral head breadth. Prediction accuracy is superior to that of previously available methods. No systematic geographic or temporal variation in prediction errors is apparent, except in tibial estimation of stature, where northern and southern European formulae are necessary because of the presence of relatively longer tibiae in southern samples. Thus, these equations should bebroadly applicable to European Holocene skeletal samples.

Development and characterization of mouse monoclonal antibodies reactive with chicken CD83.

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein containing the extracellular region of chCD83 was expressed in Chinese Hamster Ovary (CHO) cells and isolated from the spent cell culture medium by protein G affinity chromatography. The extracellular region of the chCD83 protein was purified and used to immunize mice. A cell fusion was performed, from which 342 hybridomas were screened for mAbs to chCD83. Two mAbs, chCD83-159 and chCD83-227, stained the greatest percentage of chCD83-transfected CHO cells and were selected for further characterization. By flow cytometry, both mAbs reacted with a chicken macrophage cell line, HD11. Both mAbs also recognized a single 53 kDa protein on Western blots of lysates from lipopolysaccharide-stimulated spleen mononuclear cells or unstimulated HD11 cells. Immunostaining of chicken secondary lymphoid organs identified chCD83(+) cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen mononuclear cells with concanavalin A (Con A) decreased the percentage of chCD83(+) cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII(+) cells compared with cells treated with an isotype-matched negative control mAb. These chCD83 mAbs may be useful for future investigations of chicken immune cell maturation and mechanisms of action.

Development and characterization of mouse monoclonal antibodies reactive with chicken interleukin-2 receptor αlpha chain (CD25).

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 (chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25(+) spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25(+) cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.

Expressed gene sequence of the IFNγ-response chemokine CXCL9 of cattle, horses, and swine.

This report describes the cloning and characterization of expressed gene sequences of bovine, equine, and swine CXCL9 from RNA obtained from peripheral blood mononuclear cells (PBMC) and other tissues. The bovine coding region was 378 nucleotides in length, while the equine and swine coding regions were 381 nucleotides. Mapping showed that all three sequences were coded for in four exons in the genome, as are the human and mouse genes. The bovine, equine, and swine coding regions shared 83%, 86%, and 84% homology with human CXCL9, respectively, and all three were 74% homologous with mouse CXCL9. Cladogram comparison of the nucleotide sequences of CXCL9 showed that the bovine, equine and swine sequences were more closely related to one another than to either the human or the mouse sequences. However, the human sequence was more closely related to them than it was to the mouse sequence. These relationships were preserved when the deduced amino acid sequences were evaluated and all sequences showed conservation of the characteristic four cysteines. This work sets the stage for further work with these molecules; an integral goal of the U.S. Veterinary Immune Reagent Network is to develop reagents for investigating diseases in livestock species, poultry, and fish.

Development and characterization of mouse monoclonal antibodies reactive with chicken CD80.

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD80 (chCD80). A recombinant plasmid containing a chCD80/horse IgG4 fusion gene was constructed and expressed in CHO cells to produce recombinant chCD80/IgG4 protein. Chicken CD80 was purified from the chCD80/IgG4 fusion protein following enterokinase digestion, and used to immunize BALB/c mice, resulting in 158 hybridomas that produced mAbs against chCD80. Three mAbs with high binding specificity for recombinant chCD80/IgG4-transfected CHO cells were identified by flow cytometry, and one of these (#112) was selected for further characterization. Immunoprecipitation of CD80/IgG4-CHO cell extract, or lipopolysaccharide (LPS)-treated monocytes identified 35.0kDa proteins. Immunohistochemical analysis revealed chCD80-expressing cells exclusively in the bursal follicles at the outer portion of the cortex, and throughout the red pulp and the outer boundary of the white pulp in the spleen. By immunofluorescence microscopy, chCD80 was observed on intestinal dendritic cells. LPS treatment of bursa or spleen monocytes for 24 or 48h increased chCD80 expression. Finally, addition of chCD80 mAb to Con A-stimulated spleen cells inhibited the expression of major histocompatibility complex class II antigens and IL-2-driven proliferation of lymphoblast cells. In summary, these chCD80 mAbs will serve as valuable immunological reagents for basic and applied poultry immunology research.

Development and characterization of mouse monoclonal antibodies specific for chicken interleukin 18.

Four mouse monoclonal antibodies (mAbs) which are specific for chicken interleukin 18 (chIL18) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative real-time PCR and neutralization assays. Using Western blot analysis, monoclonal antibodies specific for chIL18 identified a 23 kDa Pichia pastoris-expressed chIL18 and 66 kDa E. coli-derived MBP fusion protein of chIL18. Bioassays for chIL18 using primary chicken spleen cells showed dose-dependent IFN-γ mRNA expression and induction of IFN-γ from primary splenocytes, and triggered nitric oxide (NO) production in the HD11 macrophage cell line. These mAbs showed neutralizing chIL18 activity. Taken together, these mouse mAbs which detect chicken IL-18 will be significant new immune reagents and useful tools for basic and applied research in poultry.

Expressed gene sequence and bioactivity of the IFNgamma-response chemokine CXCL11 of swine and cattle.

This report describes the cloning and characterization of expressed gene sequences of the swine and bovine interferon-gamma inducible chemokine CXCL11, or I-TAC, associated with type 1 T-helper immune responses, and affirmation of bioactivity of their yeast-expressed protein products. The coding regions of both cDNA sequences were 303 nucleotides in length; each is coded for four exons in the genome. The bovine coding region shared 82% and 70% homology with human and mouse CXCL11, respectively, and the swine coding region 84% and 72% homology, respectively. As expected the swine and bovine CXCL11 sequences showed less homology with other human and mouse C-X-C motif chemokine sequences. Each cDNA was cloned into plasmids and transfected into Pichia pastoris (yeast) and the resultant expressed protein purified. Biological activity of each purified chemokine was affirmed by chemotaxis assays. Both swine and bovine CXCL11 were chemotactic for mitogen and IL-2 stimulated peripheral blood mononuclear cells. This is the first report for bioactivity of this chemokine in livestock species. This work provides valuable new reagents for investigating basic immunity as well as vaccine and disease responses in swine and cattle, goals of the U.S. Veterinary Immune Reagent Network which supported this effort.

Characterization of anti-channel catfish IgL sigma monoclonal antibodies.

This study characterizes three monoclonal antibodies (mAbs) developed against the constant (C) region of the immunoglobulin light (IgL) sigma chain isotype of the channel catfish, Ictalurus punctatus. Microsphere bead assays and Western blot analyses utilizing different recombinant (r) proteins show that these anti-catfish IgL sigma chain mAbs each specifically recognize the denatured form of IgL sigma. Importantly, Western blotting of catfish sera using the anti-IgL sigma mAbs also identified an IgL chain-sized immunoreactive band(s) of approximately 27kDa. It is anticipated that these mAbs in combination with the already existing anti-catfish Ig heavy (H) and IgL chain mAbs will be useful in future studies examining the functional roles of the different catfish IgL isotypes.

Expressed gene sequences of the equine cytokines interleukin-17 and interleukin-23.

This report describes the initial cloning and characterization of the equine interleukin-17 (IL-17) expressed gene sequence from mRNA obtained from equine intestinal tissue and interleukin-23 (IL-23) expressed gene sequence from mRNA obtained from equine peripheral blood mononuclear cells. Equine IL-17 has 462 nucleotides in the translated region, determined by homology with known human and mouse sequences, and shares 84% and 75% identity, respectively. For the deduced amino acid sequences, the identity with human and mouse is 76% and 70%. Equine IL-23 has 579 nucleotides in the translated region. Homology with known human and mouse sequences was determined to be 89% and 77%. Deduced amino acid identities are 89% with the human sequence and 70% with the mouse sequence. The gene sequences were identified as part of the U.S. Veterinary Immune Reagent Network with a goal of developing reagents in order to aid veterinary researchers in the investigation of diseases in livestock species.