Recombinant single domain antibody fragments (VHHs) that derive from the unusual camelid heavy chain only IgG class (HCAbs) have many favourable properties compared with single-chain antibodies prepared from conventional IgG. As a result, VHHs have become widely used as binding reagents and are beginning to show potential as therapeutic agents. To date, the source of VHH genetic material has been camels and llamas despite their large size and limited availability. Here we demonstrate that the smaller, more tractable and widely available alpaca is an excellent source of VHH coding DNA. Alpaca sera IgG consists of about 50% HCAbs, mostly of the short-hinge variety. Sequencing of DNA encoding more than 50 random VHH and hinge domains permitted the design of PCR primers that will amplify virtually all alpaca VHH coding DNAs for phage display library construction. Alpacas were immunized with ovine tumour necrosis factor alpha (TNFalpha) and a VHH phage display library was prepared from a lymph node that drains the sites of immunizations and successfully employed in the isolation of VHHs that bind and neutralize ovine TNFalpha.
Cytokine gene expression in cells migrating in afferent and efferent intestinal lymph was monitored for extended time periods in individual sheep experimentally infected with the nematode Trichostrongylus colubriformis. Animals from stable selection lines with increased levels of either genetic resistance (R) or susceptibility (S) to nematode infection were used. Genes for interleukin-5 (IL-5), IL-13, and tumor necrosis factor alpha (TNF-␣), but not for IL-4, IL-10, or gamma interferon (IFN-␥), were consistently expressed at higher levels in both afferent and efferent lymph cells of R sheep than in S sheep. However, only minor differences were observed in the surface phenotypes and antigenic and mitogenic responsiveness of cells in intestinal lymph between animals from the two selection lines. The IL-4 and IL-10 genes were expressed at higher levels in afferent lymph cells than in efferent lymph cells throughout the course of the nematode infection in animals of both genotypes, while the proinflammatory TNF-␣ gene was relatively highly expressed in both lymph types. These relationships notwithstanding, expression of the IL-10 and TNF-␣ genes declined significantly in afferent lymph cells but not in efferent lymph cells during infection. Collectively, the results showed that R-line sheep developed a strong polarization toward a Th2-type cytokine profile in immune cells migrating in lymph from sites where the immune response to nematodes was initiated, although the IFN-␥ gene was also expressed at moderate levels. Genes or alleles that predispose an animal to develop this type of response appear to have segregated with the R selection line and may contribute to the increased resistance of these animals.
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