Overexpression of Fibrinogen-Like Protein 2 Promotes Tolerance in a Fully Mismatched Murine Model of Heart Transplantation.

Fibrinogen-like protein 2 (FGL2) is an immunomodulatory protein that is expressed by regulatory T cells (Tregs). The objective of this study was to determine if recombinant FGL2 (rFGL2) treatment or constitutive FGL2 overexpression could promote transplant tolerance in mice. Although rFGL2 treatment prevented rejection of fully mismatched cardiac allografts, all grafts were rejected after stopping treatment. Next, we generated FGL2 transgenic mice (fgl2(Tg) ) that ubiquitously overexpressed FGL2. These mice developed normally and had no evidence of the autoimmune glomerulonephritis seen in fgl2(-/-) mice. Immune characterization showed fgl2(Tg) T cells were hypoproliferative to stimulation with alloantigens or anti-CD3 and anti-CD28 stimulation, and fgl2(Tg) Tregs had increased immunosuppressive activity compared with fgl2(+/+) Tregs. To determine if FGL2 overexpression can promote tolerance, we transplanted fully mismatched cardiac allografts into fgl2(Tg) recipients. Fifty percent of cardiac grafts were accepted indefinitely in fgl2(Tg) recipients without any immunosuppression. Tolerant fgl2(Tg) grafts had increased numbers and proportions of Tregs and tolerant fgl2(Tg) mice had reduced proliferation to donor but not third party antigens. These data show that tolerance in fgl2(Tg) recipients involves changes in Treg and T cell activity that contribute to a higher intragraft Treg-to-T cell ratio and acceptance of fully mismatched allografts.

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.

Long-term surgical and visual outcomes in primary congenital glaucoma: 360 degrees trabeculotomy versus goniotomy.

PURPOSE: To compare the visual outcome and refractive status of children with primary congenital glaucoma who underwent 360 degrees trabeculotomy or goniotomy as an initial surgical procedure. METHODS: This retrospective study describes 24 eyes (15 patients) with primary congenital glaucoma that underwent 360 degrees trabeculotomy as the initial procedure and 40 eyes (23 patients) that underwent goniotomy as the initial procedure. Inclusion criteria were: (1) diagnosis of primary congenital glaucoma and initial angle surgery before 1 year of age, (2) no other ocular or systemic diseases, (3) 360 degrees trabeculotomy or goniotomy as the first surgical procedure, and (4) ability to obtain an Allen or Snellen visual acuity. A postoperative vision of 20/50 or better was considered good. Surgical success was defined as an intraocular pressure (IOP) less than 22 mm Hg with or without medication and without evidence of a progressive optic neuropathy. RESULTS: The IOP was successfully controlled in 92% of eyes in the trabeculotomy group and in 58% of eyes in the goniotomy group (P =.004). Of eyes in the trabeculotomy group, 79% had vision of 20/50 or better compared with 53% in the goniotomy group (P =.03). High myopia was more prevalent in the goniotomy group, but this difference was not statistically significant (P =.16). A poor visual outcome was associated with failure of the angle surgery or poor compliance with follow-up and amblyopia therapy. CONCLUSION: For primary congenital glaucoma, 360 degrees trabeculotomy is a highly effective procedure that results in excellent pressure control and is at least as successful as multiple standard procedures. In this study, 360 degrees trabeculotomy resulted in better vision than what is reported in the literature for standard angle procedures.

The ophthalmology of intracranial vascular abnormalities.

PURPOSE: To provide a practical review of the ophthalmologic manifestations of intracranial vascular abnormalities. METHODS: We reviewed ocular manifestations of the most common intracranial vascular abnormalities: intracranial aneurysms, carotid-cavernous fistulas, arteriovenous malformations, and cavernous malformations. RESULTS: Unruptured aneurysms can compress the third cranial nerve and the anterior visual pathways. Ruptured aneurysms and subarachnoid hemorrhage can result in Terson syndrome and papilledema. Direct and indirect carotid-cavernous fistulas most commonly cause the classic triad of proptosis, conjunctival chemosis, and cranial bruit but can masquerade as chronic conjunctivitis. Arteriovenous malformations, with or without hemorrhage, may compress portions of the retrochiasmal pathways, causing visual field loss. Cavernous malformations, when in the brainstem, commonly cause abnormalities of supranuclear, nuclear, and fascicular ocular motility. CONCLUSIONS: The ophthalmologist may be the first physician to encounter clinical manifestations of intracranial vascular abnormalities that may herald devastating neurologic complications. Prompt diagnosis facilitates appropriate management and therapy.