Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-kappaB. Bone marrow-derived macrophages from TLR4- and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2- and TLR9-deficient mice. In vivo THP-driven TNF-alpha production was evident in WT but not in Tlr4-/- mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4-/- mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.

The NK gene complex is a region on human chromosome 12 containing several families of lectin‐like genes including the CD94 and NKG2 NK receptor genes. We report here that the region telomeric of CD94 contains in addition to the LOX‐1 gene the novel human DECTIN‐1 and the CLEC‐1 and CLEC‐2 genes within about 100 kb. Sequence similarities and chromosomal arrangement suggest that these genes form a separate subfamily of lectin‐like genes within the NK gene complex. DECTIN‐1 is selectively expressed in dendritic cells and to a lowerextent in monocytes and macrophages. mRNA forms with and without a stalk exon are observed. During functional maturation of dendritic cells the level of DECTIN‐1 mRNA is down‐regulated several‐fold. CLEC‐1 is found to be not only expressed in dendritic cells, but also in endothelial cells and in the latter aspect resembles the LOX‐1 gene. Whereas recombinant full‐length DECTIN‐1 and LOX‐1 are transported to the cell surface, CLEC‐1 proteins accumulate in perinuclear compartments. We propose that this family of lectin‐like genes encodes receptors with important immune and/or scavenger functions in monocytic, dendritic and endothelial cells.

Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-κB. Bone marrow-derived macrophages from TLR4-and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2-and TLR9-deficient mice. In vivo THP-driven TNF-α production was evident in WT but not in Tlr4 -/-mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4 -/-mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.

Interactions of natural killer cell receptors with their cognate ligands play a major role in regulating NK cell function. The NKG2 gene family encodes several highly similar proteins, which are known to form heterodimers with the CD94 receptor. These dimers play a role in the inhibition as well as the activation of NK cells. We have analyzed the gene structures of the NKG2C, D, E, and F genes, and determined their genomic organization. Restriction mapping and sequencing revealed the four genes to be closely linked to one another, and of the same transcriptional orientation. An exon duplication within the NKG2C and E genes was identified, although the duplicated version of this exon has not yet been found in mRNA sequences. The NKG2C, E, and F genes, despite being highly similar, are variable at their 3' ends. We show that NKG2C consists of six exons, whereas NKG2E has seven, and the splice acceptor site for the seventh exon occurs in an Alu repeat. NKG2F consists of only four exons and part of exon IV is in some cases spliced to the 5' end of the NKG2D transcript. NKG2D has only a low similarity to the other NKG2 genes.

Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a "caninized" version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer.