Resolution of an immunodiagnostic dilemma: heavy chain chimeric antibodies for species in which plasmocytomas are unknown.

The immunoglobulin (Ig) genes of many vertebrates have been characterized but IgG subclasses, IgD and IgE proteins are only available for three species in which plasmacytomas occur. This creates a major problem in the production and specificity verification of diagnostic anti-Ig reagents for the vast majority of mammals. We describe a novel solution using the swine system with its eleven different variants of IgG. It involves the in vitro synthesis of chimeric porcine-camelid heavy chain antibodies (HCAbs) that do not require light chains and therefore only a single transfection vector. The expressed chimeric HCAbs are comprised of the camelid VHH domain encoding specificity for lysozyme and the hinge, CH2 and CH3 domains of the various porcine IgGs. These HCAb retain their antigenic integrity and their ability to recognize lysozyme. The engineered specificity assures that these HCAb can be immobilized in native configuration when used for testing the specificity of anti-swine IgG antibodies. Comparative data to illustrate the importance of this point are provided. These are now available for use in hybridoma selection and as reference standards for evaluating the specificity of currently available anti-swine IgG antibodies.

Antibody repertoire development in fetal and neonatal piglets. XXIII: fetal piglets infected with a vaccine strain of PRRS Virus display the same immune dysregulation seen in isolator piglets.

The Ig levels and antibody repertoire diversification in fetal piglets infected with an attenuated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) were measured. Serum Ig levels were greatly elevated in PRRSV-infected fetuses; IgG was elevated >50-fold, IgM>5-15-fold and IgA>2-fold compared to control fetuses. Their IgM to IgG to IgA profile was the same as that in isolator piglets infected for the same period with wild-type PRRSV. Fetal animals showed less repertoire diversification than even isolator piglets that were maintained germfree (GF) while the repertoire diversification index (RDI) for PRRSV-infected isolator piglets was 10-fold higher and comparable to littermates infected with swine influenza (S-FLU). However, when expressed as the RDI:Ig ratio, infected fetuses appeared 10-fold less capable of repertoire diversification than uninfected littermates and GF isolator piglets. Compared to S-FLU isolator piglets that resolve the infection, the RDI:Ig of PRRSV-infected isolator piglets was 100-fold lower. Overall, infection of fetuses with an attenuated virus shows the same immune dysregulation seen postnatally in wild type infected isolator piglets, indicating that: (a) attenuation did not alter the ability of the virus to cause dysregulation and (b) the isolator infectious model reflects the fetal disease.

Linkage haplotype for allotypic variants of porcine IgA and IgG subclass genes.

Six putative subclasses of expressed porcine IgG have been described from gene sequences and allotypic variants for five of these have been proposed. We tested this hypothesis by studying the transcription of these 11 variants in outbred hemizygous farm pigs. Since Cγ subclass genes are closely linked, they are most likely inherited as a haplotype. Since hemizygous pigs can only express genes encoded on one chromosome, identifying the expressed genes can indicate which allelic variants are linked as well as testing whether the putative alleles are indeed alleles or separate subclass genes. The procedure for producing B cell knockout pigs has recently been described; our study examines transcripts from the hemizygous parents and offspring generated by this technology. More than 570 Cγ gene clones from hemizygous animals were identified according to subclass and allotype by a combination of clone hybridization and sequencing. IgG3 accounted for 80% in newborn animals but <5% in adults. IgG1 accounted for ~50% of all clones recovered from adults and IgG4 was the least frequently recovered (4%). Results indicate that IgG1(b), IgG2(a), IgG3, IgG4(a), IgG5(a), and IgG6(a) are linked and also linked to IgA(a). This comprises a haplotype for domesticated swine. For simplicity, we propose that the current nomenclature for the allotypes of IgG1 be reversed so that all genes in the Cγ(a)-Cα(a) haplotype are designated "a".

Targeted disruption of the porcine immunoglobulin kappa light chain locus.

Inactivation of the endogenous pig immunoglobulin (Ig) loci, and replacement with their human counterparts, would produce animals that could alleviate both the supply and specificity issues of therapeutic human polyclonal antibodies (PAbs). Platform genetics are being developed in pigs that have all endogenous Ig loci inactivated and replaced by human counterparts, in order to address this unmet clinical need. This report describes the deletion of the porcine kappa (κ) light chain constant (Cκ) region in pig primary fetal fibroblasts (PPFFs) using gene targeting technology, and the generation of live animals from these cells via somatic cell nuclear transfer (SCNT) cloning. There are only two other targeted loci previously published in swine, and this is the first report of a targeted disruption of an Ig light chain locus in a livestock species. Pigs with one targeted Cκ allele (heterozygous knockout or ±) were bred together to generate Cκ homozygous knockout (-/-) animals. Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph nodes (MLNs) from Cκ -/- pigs were devoid of κ-containing Igs. Furthermore, there was an increase in lambda (λ) light chain expression when compared to that of wild-type littermates (Cκ +/+). Targeted inactivation of the Ig heavy chain locus has also been achieved and work is underway to inactivate the pig lambda light chain locus.

Generation of antibody- and B cell-deficient pigs by targeted disruption of the J-region gene segment of the heavy chain locus.

A poly(A)-trap gene targeting strategy was used to disrupt the single functional heavy chain (HC) joining region (J(H)) of swine in primary fibroblasts. Genetically modified piglets were then generated via somatic cell nuclear transfer (SCNT) and bred to yield litters comprising J(H) wild-type littermate (+/+), J(H) heterozygous knockout (±) and J(H) homozygous knockout (-/-) piglets in the expected Mendelian ratio of 1:2:1. There are only two other targeted loci previously published in swine, and this is the first successful poly(A)-trap strategy ever published in a livestock species. In either blood or secondary lymphoid tissues, flow cytometry, RT-PCR and ELISA detected no circulating IgM(+) B cells, and no transcription or secretion of immunoglobulin (Ig) isotypes, respectively in J(H) -/- pigs. Histochemical and immunohistochemical (IHC) studies failed to detect lymph node (LN) follicles or CD79α(+) B cells, respectively in J(H) -/- pigs. T cell receptor (TCR)(ß) transcription and T cells were detected in J(H) -/- pigs. When reared conventionally, J(H) -/- pigs succumbed to bacterial infections after weaning. These antibody (Ab)- and B cell-deficient pigs have significant value as models for both veterinary and human research to discriminate cellular and humoral protective immunity to infectious agents. Thus, these pigs may aid in vaccine development for infectious agents such as the pandemic porcine reproductive and respiratory syndrome virus (PRRSV) and H1N1 swine flu. These pigs are also a first significant step towards generating a pig that expresses fully human, antigen-specific polyclonal Ab to target numerous incurable infectious diseases with high unmet clinical need.

Immunoglobulins, antibody repertoire and B cell development.

Swine share with most placental mammals the same five antibody isotypes and same two light chain types. Loci encoding lambda, kappa and Ig heavy chains appear to be organized as they are in other mammals. Swine differ from rodents and primates, but are similar to rabbits in using a single VH family (VH3) to encode their variable heavy chain domain, but not the family used by cattle, another artiodactyl. Distinct from other hoofed mammals and rodents, Ckappa:Clambda usage resembles the 1:1 ratio seen in primates. Since IgG subclasses diversified after speciation, same name subclass homologs do not exist among swine and other mammals unless very closely related. Swine possess six putative IgG subclasses that appear to have diversified by gene duplication and exon shuffle while retaining motifs that can bind to FcgammaRs, FcRn, C1q, protein A and protein G. The epithelial chorial placenta of swine and the precosial nature of their offspring have made piglets excellent models for studies on fetal antibody repertoire development and on the postnatal role of gut colonization, maternal colostrum and neonatal infection on the development of adaptive immunity during the "critical window" of immunological development. This chapter traces the study of the humoral immune system of this species through its various eras of discovery and compiles the results in tables and figures that should be a useful reference for educators and investigators.

The piglet as a model for B cell and immune system development.

The ability to identify factors responsible for disease in all species depends on the ability to separate those factors which are environmental from those that are intrinsic. This is particularly important for studies on the development of the adaptive immune response of neonates. Studies on laboratory rodents or primates have been ambiguous because neither the effect of environmental nor maternal factors on the newborn can be controlled in mammals that: (i) transmit potential maternal immunoregulatory factors in utero and (ii) are altricial and cannot be reared after birth without their mothers. Employing the newborn piglet model can address each of these concerns. However, it comes at the price of having first to characterize the immune system of swine and its development. This review focuses on the porcine B cell system, especially on the methods used for its characterization in fetal studies and neonatal piglets. Understanding these procedures is important in the interpretation of the data obtained. Studies on neonatal piglets have (a) provided valuable information on the development of the adaptive immune system, (b) lead to important advances in evolutionary biology, (c) aided our understanding of passive immunity and (d) provided opportunities to use swine to address specific issues in veterinary and biomedical research and immunotherapy. This review summarizes the history of the development of the piglet as a model for antibody repertoire development, thus providing a framework to guide future investigators.

The pre-immune variable kappa repertoire of swine is selectively generated from certain subfamilies of Vkappa2 and one Jkappa gene.

Combinatorial diversity is highly restricted during formation of the pre-immune heavy chain repertoire of swine, raising the question of whether the same is true for the pre-immune light chain repertoire. Before addressing this question, we first used competitive PCR to show that kappa and lambda light chains in swine are equally expressed in mature B cells similar to the situation in humans but alike that in other studied Ungulates. This justified efforts to examine the repertoire of both light chain types. These studies also revealed that lambda is preferentially expressed at sites of B cells lymphogenesis, perhaps because of the use of a surrogate light chain containing lambda5. Data are presented here on >100 VkappaJkappa-containing transcripts and approximately 180 genomic Vkappa genes to show that >90% of the pre-immune repertoire is generated from three subfamilies of IGKV2 genes and one of five Jkappa segments. The kappa locus contains >or=50 IGKV2 genes belonging to at least five subfamilies and an undetermined but perhaps equal number of IGKV1 genes. The porcine IGKV1 and IGKV2 genes share 87% sequence similarity with their human counterparts and Jkappa1 through Jkappa5 share sequence and organizational homology with those in sheep, horse, human and mouse. Swine have a single Ckappa gene. These findings contrast with those from rodents and primates but are reminiscent of those on the pre-immune heavy chain repertoire of swine in that it is generated using a relatively restricted number of gene segments. These restricted pre-immune repertoires may reflect the minimal exposure of the fetus to maternal factors and environmental antigens. The significance for swine immunology of characterizing the pre-immune repertoire is discussed.

Regulation of pituitary growth and prolactin gene expression by estrogen.

We presented evidence that primary cultures of rat pituitary cells respond to estradiol by increased incorporation of precursors into prolactin. The response is specific for estrogenic hormones and is maximal at physiological concentrations of estradiol. The time course and magnitude of the response in cultured cells is in agreement with that observed under in vivo conditions, suggesting that estrogen exerts its effect mainly through a direct action on the pituitary gland. The data presented indicate that estrogen stimulates prolactin synthesis predominantly through increased prolactin mRNA accumulation, and to a lesser extent, through mammotroph cell proliferation. Chronic treatment with DES caused sustained proliferation of pituitary cells leading to prolactin producing pituitary tumors in the Fischer 344 rat, but not in the Holtzman rat. The genetic basis for these differences are currently under investigation.

Purification and characterization of the estrogen-induced protein (IP) of the rat uterus.

One of the earliest responses of the rat uterus to estrogen is increased synthesis of a specific cytosol protein (IP). This synthesis is detectable within 40 min and is dependent on an even earlier actinomycin D-sensitive function. IP has now been purified to homogeneity as determined by SDS polyacrylamide gel electrophoresis (SDS PAGE). The procedure was complicated by a tendency of the more homogeneous and concentrated material to aggregate. Purification consisted of sequential chromatography, in the presence of 0.1% triton X-100, via DEAE-cellulose (2x), hydroxylapatite and agarose-acrylamide. These were followed by preparative PAGE and finally SDS PAGE. Molecular weight determination by Ferguson plot analysis yielded an apparent molecular weight of 45 000. On final SDS PAGE, the material consisted of two major bands: the 45 000 molecular weight IP band and a band with an estimated molecular weight of 80 000. This second band displayed an elevated synthesis in E2-stimulated uteri similar to IP and appeared to consist of some form of aggregated IP. Carbohydrate determination on SDS gels using periodic acid-Schiff (PAS) stain was negative. Co-purification of labeled cytosol proteins from uteri of control and E2-stimulated rats revealed that IP is synthesized to some extent in the unstimulated animal as well as the stimulated.

Hormone regulation of growth: stimulatory and inhibitory influences of estrogens on DNA synthesis.

Estrogenic hormones first stimulate and then inhibit DNA synthesis in the uterus of the immature rat. Both the stimulatory and the inhibitory effects depend on the sustained presence of estrogen. Thus, estriol, which is equal in effectiveness to estradiol on early (up to 6 hr) responses, has only a partial stimulatory effect on DNA synthesis. Estradiol initially stimulates DNA synthesis, but the sustained presence of this steroid inhibits further synthesis of this macromolecule and cell division. These observations are discussed in terms of their relationship to current models of estrogen action and to estrogen dependency in some types of cancer.

Stimulatory and inhibitory effects of estrogen on uterine DNA synthesis.

The induction of long-term responses in the uterus following estrogen treatment is discussed, with special reference to DNA synthesis. Immature female rats injected daily with estradiol-17beta or estriol (0.01 to 1 mug) or a combination of the two steroids for one, two or three consecutive days were sacrificed at intervals from 12 to 24 h after the last injection of vehicle or steroid. In vitro incorporation of [3H]thymidine into DNA [14C]leucine into protein, and oxidation of [14C)glucose to 14CO2 were determined. Nuclear-bound estradiol was determined by use of exchange assay or following incubation of intact uteri with 1 X 10(-8)M ([3H]estradiol for 1 h at 37 C. Injection of estriol only partially stimulated DNA synthesis by 18 to 24 h post-treatment. However, injection of estriol followed by injection of estradiol 6 h later resulted in increased DNA synthesis, suggesting that estrogen must be present for up to 6 h to induce subsequent DNA synthesis. Maximal DNA and protein synthesis and oxidation of glucose occurred at 24 h after injection of estradiol (0.1 or 1 mug) but was depressed to control levels by 24 h after the last of three daily injections. Daily injections of 0.01 mug of estradiol resulted in a similar pattern of DNA synthesis, although of lesser magnitude than that observed after injection of 0.1 or 1 mug of estradiol. However, if rats receiving daily injections of 0.01 mug estradiol were challenged with a higher dose of estradiol (1 mug), uterine DNA synthesis was markedly increased. The data suggest that prolonged exposure to estrogen causes uterine cells to become metabolically "refractory" to further estrogen stimulation. Sequential injections of estriol (1 mug) or intermittent injections of estradiol (1 mug) were ineffective in causing this uterine "refractoriness". Receptor binding of estrogen, translocation to the nucleus, and retention of receptor in the nucleus were not affected by sequential estrogen treatment. The accumulation of an inhibitory product is suggested as a possible explanation for this phenomenon.